WO2022094174A1 - Compositions and methods for the treatment of esophageal conditions - Google Patents

Compositions and methods for the treatment of esophageal conditions Download PDF

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Publication number
WO2022094174A1
WO2022094174A1 PCT/US2021/057189 US2021057189W WO2022094174A1 WO 2022094174 A1 WO2022094174 A1 WO 2022094174A1 US 2021057189 W US2021057189 W US 2021057189W WO 2022094174 A1 WO2022094174 A1 WO 2022094174A1
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indole
composition
ovol1
ahr
spink7
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PCT/US2021/057189
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French (fr)
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Nurit Azouz
Marc E. Rothenberg
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Children's Hospital Medical Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Eosinophilic esophagitis is an inflammatory process leading to eosinophil migration to esophageal tissue and is considered the leading cause of dysphagia and food impaction in adults and children.
  • the incidence of EoE is increasing, in part due to the increasing frequency of allergies and better diagnostic tools, but remains underdiagnosed and undertreated.
  • Patients with EoE suffer from physical symptoms such as heartburn, abdominal pain, dysphagia.
  • the prevalence of EoE ranges from 5 to more than 80 cases per 100,000 inhabitants depending on the assessment method; in the United States it is estimated that prevalence of EoE occurs in 56.7/100,000 persons. (See, e.g., Kanikowska et al., Int J Mol Sci.
  • the instant disclosure relates to composition and methods for the treatment of eosinophilic esophagitis (EoE) in an individual in need thereof.
  • the methods include treatment of eosinophilic esophagitis by administration of one or more aryl hydrocarbon receptor (AHR) agonists to the individual.
  • AHR aryl hydrocarbon receptor
  • compositions containing aryl hydrocarbon receptor (AHR) agonists are disclosed.
  • FIGS 1A-1F SPINK7 expression is induced by calcium and cell confluency.
  • 1A Quantitative polymerase chain reaction (qPCR) of SPINK7 expression in EPC2 cells that were plated at 24 wells (50,000 cells/well or 300,000 cells/well) that were incubated with either 0.045 or 1.8 rnM of calcium for 48 hours.
  • IB levels of H3K27Ac in the promoters of SPINK7 in EPC2 cells in the indicated conditions.
  • 1C is qPCR
  • Promoter activity was determined by nLUC measurements relative to firefly measurements and presented as relative luminescence units (RLU). ID.
  • Promoter activity was determined by nLUC measurements relative to firefly measurements and normalized according to the promoter less nLUC measurements. Promoter activity is presented as relative luminescence units (RLU).
  • Promoter activity was determined by nLUC measurements relative to firefly measurements and normalized according to the promoter less nLUC measurements. Then, the values of the 1.8 mM of CaCh lysates were divided to the values of the cells cultured in 0.09 mM of CaCh.
  • FIG 2A-2G OVOL1 binds to SPINK7 promoter and promotes SPINK7 expression.
  • 2A Nano luciferase activity in lysates transfected with SPINK7 promoter, firefly plasmid and plasmids encoding for the indicated transcription factors compared with control. Cells were either left untreated or were treated with 1 pm FICZ.
  • 2B Western blot analysis of OVOL1 in control or OVOL1 overexpressing EPC2 cells. P-actin was used as a loading control.
  • 2C Proximal human SPINK7 promoter sequence identified 4 potential OVOL1 binding sites. Transcription Start Site +1. 2D.
  • Nano luciferase activity in lysates co-transfected with either OVOL1 or a control plasmid and with SPINK7 promoter deletion constructs were either left untreated or treated with FICZ (1 pM).
  • 2F Representative results from EMSA experiment using recombinant human OVOL1 protein. Fluorescent IRDye 700 labeled probe sequence, Un-labeled (cold) wt competitor sequence, cold mutant sequence.
  • FIG 3A-3F Loss of OVOL1 impairs the barrier function and promote innate response.
  • 3A qPCR analysis of OVOL1 expression from NSC- treated and OVOL1- silenced EPC2 cells.
  • 3B TSLP release from NSC- treated and OVOL1 -silenced EPC2 cells that were grown in high-calcium media for 64 hours and then stimulated for 8 hours with the indicated concentrations of polyinosinic-polycytidylic acid (polyLC). Cell supernatants were assessed for TSLP levels from three independent experiments. Data are the means ⁇ SD. 3C.
  • TEER ohm/cm2 measurement from CRISPR/Cas9 OVOL1 KO and control EPC2 cells at day 7 of ALI differentiation. Data are the means ⁇ SD from three independent experiments performed in triplicate. All P values were calculated by t test (unpaired, two-tailed).
  • FIG 4A-4D Loss of OVOL1 in EoE biopsies.
  • 4A mRNA expression of OVOL1 in EoE biopsies compared with control biopsies.
  • 4B Representative image of immunofluorescence staining of OVOL1 (pink) and DAPI staining in control biopsy. White line separates the lumen from the epithelium and the lumen side is marked by the letter “L”.
  • 4C Representative images of immunofluorescence staining of OVOL1 (pink) and DAPI staining in control and EoE biopsies. White line separates the lumen from the epithelium and the lumen side is marked by the letter “L”.
  • 4D Western blot analysis of OVOL1 expression in control and EoE biopsies. The graph on the right showed the OVOL1 expression relative to HSP90.
  • FIG 5A-5F Environmental cues affect SPINK7 expression in an AHR dependent and independent manner.
  • 5C Representative images of co-immunofluorescence of desmogleinl (DSG1, green), OVOL1 (pink) and DAPI after 0, 1 or 18 hrs of stimulations with FICZ (1 pm), ITE (1 pm), or omeprazole (10 pm).
  • 5D Representative western blot with quantitation of 3 independent experiments.
  • 5E Heatmap representing the fold change of genes that are significantly altered by FICZ treatment (Padj ⁇ 0.05).
  • 5F Gene ontology (GO) analyses of genes that are dysregulated by FICZ treatment.
  • FIG 6A-6J IL-13 and IL-4 prevents OVOLl-dependent SPINK7 expression.
  • 6A Representative images of co-immunofluorescence of desmogleinl (DSG1, green), OVOL1 (pink) and DAPI stain in OVOL1 overexpression cells that were either left untreated or treated over night with IL-4 or IL-13 (100 ng/mL) with or without FICZ (1 pm). Promoter activity in lysates triple- transfected with either SPINK7-nLUC or nLUC and firefly vector and either OVOL1 or a control plasmid.
  • Cells were either left untreated or treated with 1 pm FICZ, with or without IL-4 (6B), or IL- 13 (6C). 6D. Representative images of coimmunofluorescence of DSG1 (pink), OVOL1 (Cyan) and DAPI stain in cells that were differentiated in the ALI model (ALI). Cells were either left untreated or treated with IL-4 or IL-13 (100 ng/mL) with or without FICZ (1 pm).
  • FIG 7A-7H OVOL1 undergoes post-translational modifications.
  • 7A Western blot analysis of OVOL1, DSG1 and GAPDH expression in differentiated EPC2 cells that were either left untreated or stimulated with IL- 13 (100 ng/mL) for 48 hrs. The graphs on the right show quantification of OVOL1 and DSG1 relative to GAPDH with or without IL- 13 treatment.
  • 7B qPCR analysis of OVOL1 and DSG1 mRNA expression in differentiated EPC2 cells that were either left un-treated or stimulated with IL- 13 (100 ng/mL) for 48 hrs.
  • 7C Western blot analysis of OVOL1, DSG1 and GAPDH expression in differentiated EPC2 cells that were either left untreated or stimulated with IL- 13 (100 ng/mL) for 48 hrs.
  • Heatmap represent the relative expression of the indicated genes in epithelial clusters based on single cell RNA-sequencing data of dispersed cells from esophageal control biopsies.
  • 7D Western blot analysis of OVOL1 and Calpain-14 expression in differentiated EPC2 cells with inducible expression of CAPN14 expression.
  • CAPN14 is fused to a flag tag and is induced by doxycycline (Dox) treatment.
  • GAPDH was used as a loading control.
  • Anti-GFP was used for detection of GFP and CAPN14-GFP.
  • 7H Western blot analysis of recombinant human OVOL1 (100 ng) that was either left untreated or incubated with cytoplasmic protein fractions (C), or nuclear protein fractions (N) for the indicated times. The graph on the right is a quantification of OVOL1 band intensity (O.D).
  • FIG 8A-9B Involvement of AHR in EoE pathogenicity in a murine model.
  • FIG 9A-9B Regulation of SPINK7 promoter.
  • 9A Promoter activity in lysates cotransfected with nLUC and firefly vector that were grown in the indicated concentrations of CaC12. Promoter activity was determined by nLUC measurements relative to firefly measurements and normalized according to the promoter less nLUC measurements. Promoter activity is presented as relative luminescence units (RLU).
  • 9B Promoter activity in lysates of cells that were grown in 0.09 mM of CaC12 and co-transfected with nLUC constructs that contain either 0, 1, 2, 3, 4 or 4.5 kb of the SPINK7 promoter sequence and firefly vector. Promoter activity was determined by nLuc measurements relative to firefly measurements and normalized according to the promoter less nLuc measurements.
  • FIG 10. Generation of OVOL1 KO EPC2 cells.
  • 10A A chromatogram depicting the genomic DNA sequence of EPC2 cells in the vicinity of the sequence targeted for CRISPR/Cas9-mediated editing. The box indicates the location of the PAM sequence.
  • 10B Prediction of the protein sequences of OVOL1 KO cells and control cells according to their genomic sequence. Black text indicates amino acids that match WT protein sequence. Blue text indicates amino acids that deviate from WT protein sequence.
  • FIG 11A-11B Overlap between FICZ- induced response and the EoE transcriptome.
  • B Gene ontology analysis depicting cellular component of the overlapping genes. P value for GO analysis was calculated by ANOVA test.
  • FIG 12. A regulatory network controls SPINK7 expression which is influenced by the exposome.
  • AHR is activated and influenced by diet nutrients, environmental toxicant, microbiome composition, tryptophan metabolites and drugs. When AHR is activated, it promotes translocation of OVOL1 to the nucleus which in turn promotes SPINK7 expression.
  • SPINK7 expression promotes epithelial differentiation, barrier function, decreased proteolytic activity and decreased TSLP production.
  • IL-4 and IL- 13 inhibit OVOL1 nuclear translocation and therefore, repress SPINK7 expression.
  • IL- 13 -stimulated CAPN14 expression decreases OVOL1 protein expression and SPINK7 transcription.
  • FIG 13. Dysregulation of phase II enzymes in EoE. Expression of AHR, NQO1, HM0X1 and HM0X2 in biopsies from 10 EoE patients compared with 6 control patients.
  • Quercetin data slide 2 depicts Cyplal/Hprt expression in liver in control and quercetin enriched diet.
  • FIG 15 Quercetin data slide 3 depicts Cyplal/Hprt expression in esophageal tissue in control and quercetin enriched diet.
  • FIG 16 Quercetin data slide 4 depicts Cyplal/Hprt expression in skin in control and quercetin enriched diet.
  • FIG 17 Quercetin data slide 5 depicts Cyplal/Hprt expression in tongue in control and quercetin enriched diet.
  • FIG 18 Quercetin data slide 6 depicts Spink7 expression in esophageal tissue in control and quercetin enriched diet.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • the terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.
  • the active agent may form salts, which are also within the scope of the preferred embodiments.
  • Reference to a compound of the active agent herein is understood to include reference to salts thereof, unless otherwise indicated.
  • an active agent contains both a basic moiety, such as, but not limited to an amine or a pyridine or imidazole ring, and an acidic moiety, such as, but not limited to a carboxylic acid
  • zwitterions inner salts
  • Salts of the compounds of the active agent may be formed, for example, by reacting a compound of the active agent with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
  • the compounds may comprise pharmaceutically acceptable salts.
  • Such salts may include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts.
  • Acid addition salts include salts of inorganic acids as well as organic acids.
  • suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like.
  • suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p- aminobenzoic, glutamic, benzenesulfonic, p-toluenesul
  • metal salts include lithium, sodium, potassium, magnesium salts and the like.
  • ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like.
  • organic bases include lysine, arginine, guanidine, diethanolamine, choline and the like.
  • the method may comprise administering a composition comprising an aryl hydrocarbon receptor (AHR) agonist to an individual in need thereof.
  • AHR aryl hydrocarbon receptor
  • the AHR agonist may be selected from one or more of quercetin, tapinarof, 6-formylindolo[3,2-b]carbazole (FICZ), 6,12-diformylindolo[3,2-b] carbazole (dFICZ), B[a]P , TCDD, apigenin, and luteolin , urolithin A (3,8-dihydroxy-6H- dibenzo[b,d]pyran-6-on), urolithin A03 (6H-benzo[c]chromene-3,8-diol), bilirubin, biliverdin, butyrate, indirubin, 2-(10-H-indole-3-carbonyl)thiazole-4-carboxylic acid methyl ester (“ITE,” a metabolite derived from glucobrassicins), L-kynurenine, derived from tryptophan metabolism, indole-3 -carbinol (I
  • the AHR agonist may be a derivative of a disclosed AHR agonist, an analogue of a disclosed AHR agonist, a derivative of a disclosed AHR agonist, a salt of a disclosed AHR agonist, an ion of a disclosed AHR agonist, or a complex of a disclosed AHR agonist.
  • the AHR agonist is quercetin.
  • the AHR agonist is tapinarof.
  • the AHR agonist is 6-Formylindolo[3,2-b]carbazole (FICZ).
  • the said AHR agonist may be administered in a dose effective to reduce inflammation of the esophagus.
  • the AHR ligand may be administered in an amount of from about 50 microgram/kilogram to about 50,000 microgram/kilogram, or from about 100 microgram/kilogram to about 25,000 microgram/kilogram, or from about 200 microgram/kilogram to about 20,000 microgram/kilogram, or from about 300 microgram/kilogram to about 15,000 microgram/kilogram, or from about 400 microgram/kilogram to about 10,000 microgram/kilogram, or from about 500 microgram/kilogram to about 7,500 microgram/kilogram, or from about 1000 to about 5000 microgram/kilogram.
  • the composition employed in the disclosed methods may be a unit dose comprising from about 1 mg to about 500 mg, or from about 2 mg to about 250 mg, or from about 3 mg to about 200 mg, or from about 5 mg to about 150 mg, or from about 6 mg to about 100 mg, or from about 7 mg to about 75 mg, or from about 8 mg to about 70 mg, or about 8 mg to about 65 mg, or from about 9 mg to about 60 mg, or from about 10 mg to about 50 mg of an AHR agonist.
  • the AHR agonist may be administered in a food product, wherein the AHR agonist may be present in the food product in an amount of from about 100 mg/kg of food product to about 10 g/kg of food product.
  • the AHR agonist of the disclosed methods may be administered as a product of a bacteria, wherein said AHR agonist is produced by said bacteria.
  • the bacteria producing the AHR agonist may be administered to the individual in need of AHR agonist treatment.
  • the bacteria may be selected from one or more of L.
  • the bacteria may be a genetically modified bacteria, wherein said genetically modified bacteria has increased production of an AHR agonist caused by said genetic modification.
  • the administration may be via oral administration.
  • the administration may be topical administration to one or more of the esophagus, stomach, and small intestine of said individual.
  • Topical administration of the small intestine, stomach, and/or esophagus may be carried out by oral administration of a composition containing an AHR agonist, as described herein, and may advantageously employ formulations of particular viscosities to deliver the AHR agonist topically to the esophagus.
  • the method may include treating EoE by administering to an individual in need thereof (e.g., one diagnosed with or suspected of suffering from eosinophilic esophagitis), a composition comprising a corticosteroid and a liquid vehicle, wherein the composition has a volume sufficient to coat (or at least coat in a effective amount) of a targeted portion of the gastrointestinal tract (e.g. esophagus).
  • a volume sufficient to coat the esophagus is a volume that provides a bolus when orally administered to an individual.
  • a volume sufficient to coat the esophagus is a volume that provides a bolus along the entire length of the esophagus (i.e., from immediately after passing the upper esophageal sphincter through the distal end of the esophagus, e.g., immediately prior to entering or passing the lower esophageal sphincter.
  • a coating volume is optionally utilized instead of or in addition to a coating agent described herein in order to coat the targeted portion of the gastrointestinal tract (e.g., esophagus), as described herein.
  • the individual may be an adult individual. In other aspects, the individual may be a pediatric individual. For example, in certain aspects, the individual is less than 18 years of age, or less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 year of age.
  • compositions comprising an AHR agonist.
  • the composition may comprise a therapeutically effective amount of an AHR agonist to prevent or alleviate esophageal inflammation in an individual having EoE.
  • the composition may be provided in a unit dose containing a therapeutically effective amount of an AHR agonist to prevent or alleviate esophageal inflammation in an individual having EoE.
  • the AHR agonist containing compositions may take a variety of different forms.
  • the composition may be in a form selected from a liquid, an emulsion, a solution, a suspension, a syrup, a slurry, a dispersion, a colloid, a dissolving tablet, a dissolving wafer, a capsule, a gel capsule, a semi-solid, a solid forma gel, a gel matrix a cream, or a paste.
  • the composition may comprise a viscosity-increasing excipient, for example, a viscosity-increasing excipient that improves the topical administration to the deired area, which may include the esophagus, stomach, and/or small intestine.
  • a viscosity-increasing excipient for example, a viscosity-increasing excipient that improves the topical administration to the deired area, which may include the esophagus, stomach, and/or small intestine.
  • the composition may comprise a viscosity increasing excipient selected from one or more of lactose, sucrose, sucralose (Splenda®), mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethyl-cellulose (CMC), and polyvinylpyrrolidone (PVP: povidone), acacia, agar, bentonite, carbomers, carboxymethylcellulose calcium, ceratonia, cetostearyl alcohol, colloidal silicon dioxide, cyclomethicone, glyceryl behenate, guar gum, hectorite, hydrogenated vegetable oil type I, hydroxypropyl starch, hydroxypropylmethylcellulose, hydroxyethylcellulose, magnesium aluminum silicate, maltodextrin, polycarbophil, polydextrose, poly(methylvinyl ether/maleic
  • the viscosity increasing excipient may comprise one or more of a crosslinked poly(acrylic acid) (e.g., Carbopol 974P), glycerine, a carbomer homopolymer, a carbomer copolymer, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, cellulose, ceratonia, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, polyethylene glycol (e.g.
  • a crosslinked poly(acrylic acid) e.g., Carbopol 974P
  • glycerine e.g.
  • PEG 200-4500 gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxy ethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxy ethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly (methoxy ethyl methacrylate), poly (methoxy ethoxy ethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethylcellulose, carboxymethyl-cellulose (CMC) (including, e.g., sodium carboxymethyl-cellulose (NaCMC)), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® or combinations thereof.
  • CMC carboxymethyl-cellulose
  • the composition may further comprise one or more mucoadhesive agents, for example, a soluble polyvinylpyrrolidone polymer (PVP), a carbopol, a crosslinked poly(acrylic acid) (e.g., Carbopol 974P), a carbomer homopolymer, a carbomer copolymer, a water-swellable, but water-insoluble, fibrous, cross-linked carboxy-functional polymer, a hydrophilic polysaccharide gum, one or more maltodextrin, alginate, a cross-linked aliginate gum gel, thiomers (e.g., thiolated chitosan, thiolated polycarbophil, thiolated alginate, thiolated cellulose derivatives, thiolated carboxymethyl cellulose, thiolated polyacrylic acid, or thiolated poly acrylates), PEGylated polymers (e.g., PEGylated polyacrylic acid
  • the mucoadhesive agent may be a carbopol.
  • the mucadhesive agent may be selected from one or more of Carbopol 974P, Carbopol Ultrez 10, sodium alginate LF120 and sodium alginate H120L.
  • mucoadhesive agents that may be used in certain embodiments of the compositions and methods described herein are described, for example, in U.S. Pat. Nos. 6,638,521, 6,562,363, 6,509,028, 6,348,502, 6,306,789, 5,814,330, and 4,900,552, each of which is hereby incorporated by reference in its entirety.
  • the mucoadhesive agent may be at least one or at least two particulate components selected from titanium dioxide, silicon dioxide, and clay.
  • the level of silicon dioxide may be from about 3% to about 15%, by weight of the composition.
  • silicon dioxide may be selected from one or more of fumed silicon dioxide, precipitated silicon dioxide, coacervated silicon dioxide, gel silicon dioxide, and mixtures thereof.
  • clay may be selected from, by way of non- limiting example, kaolin minerals, serpentine minerals, smectites, illite or mixtures thereof.
  • clay may be selected from one or more of laponite, bentonite, hectorite, saponite, montmorillonites or mixtures thereof.
  • compositions may comprise maltodextrin, for example, about 0.05 g of maltodextrin per mL of liquid vehicle to about 0.6 g of maltodextrin per mL of liquid vehicle, or about 0.1 g of maltodextrin per mL of liquid vehicle to about 0.6 g of maltodextrin per mL of liquid vehicle, or about 0.2 g of maltodextrin per mL of liquid vehicle to about 0.5 g of maltodextrin per mL of liquid vehicle, or about 0.1 g of maltodextrin per mL of liquid vehicle to about 0.4 g of maltodextrin per mL of liquid vehicle, or about 0.2 g of maltodextrin per mL of liquid vehicle to about 0.4 g of maltodextrin per mL of liquid vehicle, or about 0.2 g of maltodextrin per mL of liquid vehicle to about 0.4 g of maltodextrin per m
  • a mucoadhesive agent used in an oral pharmaceutical composition described herein imparts an increased viscosity upon the oral pharmaceutical composition (e.g., compared to an otherwise identical composition lacking the mucoadhesive agent).
  • the composition is a formulation used to treat a targeted portion of the gastrointestinal tract (e.g., the esophagus).
  • the composition may comprise (or is administered in) a volume used to coat a targeted portion of the gastrointestinal tract (e.g., the esophagus).
  • the volume used to coat a targeted portion of the gastrointestinal tract is a volume that is sufficient to coat the targeted portion.
  • an appropriate palatable dosage is in a volume that coats or at least partially coats the esophagus, and in one aspect, the volume coats or at least partially coats the esophagus and delivers the AHR agonist to the affected areas, for example, the esophagus, a portion of the esophagus, the upper esophagus, or the lower esophagus.
  • the volume of a composition administered can provide a desired coating characteristic of a composition.
  • a composition comprising an AHR agonist wherein the composition comprises (or is administered in) a volume sufficient to coat a targeted portion of the gastrointestinal tract (e.g., the esophagus).
  • Excipients such as, for example, those listed herein, may be included in the composition to increase the viscosity of the delivered composition.
  • the liquid viscosity may be increased in the oral form, or the excipient may increase the viscosity of the dissolved form of a tablet.
  • the viscosity should be at a level that is sufficient to deliver an effective amount of the composition to the esophagus, for example, in an amount that may coat the esophagus.
  • the viscosity should be at a level that may be given orally, thus not so thick that it is either too difficult to swallow, causes gagging, or is unpalatable.
  • compositions may determine appropriate ranges.
  • One method of determining whether the composition is sufficiently viscous is by determining whether the inflammation, or eosinophilic infiltration, of the esophagus is reduced after treatment with the AHR agonist.
  • Viscosity may be determined by any method that will measure the resistance to shear offered by the substance or preparation. Many viscometers are available to those in the pharmaceutical field, and include those built by, for example, Brookfield. Viscosity may be, for example, measured at room temperature, at about 20-25 degrees Celsius, or at about 37 degrees Celsius to mimic body temperature. The viscosity of a liquid generally decreases as the temperature is raised.
  • the viscosity is about the viscosity of about 1 grams, about 2 grams, about 3 grams, about 4 grams, about 5 grams, about 6 grams, about 7 grams, about 8 grams, about 9 grams, about 10 grams, about 11 grams, about 12 grams, about 13 grams, about 14 grams, about 15 grams, about 1 to about 5 grams, about 1 to about 50 grams, or about 5 to about 25 grams of sucralose (Splenda®, Distributed By: McNeil Nutritionals, LLC, Fort Washington, Pa.
  • the viscosity is about the viscosity of 10 grams of sucralose (Splenda®) added to 4 ml of water, at 25 degrees Celsius.
  • the viscosity is about the viscosity of 5 to 20 grams of sucralose (Splenda®) in 8 ml total liquid volume, at 25 degrees Celsius. In other embodiments, the viscosity is about the viscosity of 5 to 15 grams of sucralose (Splenda®) in an 8 ml total liquid volume, at room temperature. In other aspects, the viscosity is about the viscosity of 8 to 12 grams of sucralose (Splenda®) in an 8 ml total liquid volume at 25 degrees Celsius. In some aspects, the viscosity is between that of about a fruit nectar and commercial honey, where the viscosity is measured at 25 degrees Celsius.
  • the viscosity of a composition provided herein is at least 2 centipoise (cP), at least 5 cP, at least 10 cP, at least about 25 cP, at least about 30 cP, at least about 35 cP, at least about 40 cP, at least about 50 cP, at least about 200 cP, at least about 225 cP, about 2 cP to about 10 cP, about 2 cP to about 25 cP, about 2 cP to about 50 cP, about 20 cP to about 50 cP, about 20 cP to about 100 cP, or about 50 cP to about 100 cP.
  • cP centipoise
  • the viscosity of the composition is at least about 100 cP. In certain embodiments, the viscosity of the composition, measured at about 25 degrees Celsius, is about 50 cP to about 250,000 cP, about 50 cP to about 70,000 cP, about 50 cP to about 25,000 cP, about 50 cP to about 10,000 cP, about 50 cP to about 3,000 cP, or about 50 cP to about 2,000 cP.
  • the viscosity of the composition is from about 25 centipoise (cP) to about 800 cP, about 50 cP to about 800, or about 300 cP to about 800 cP (e.g., measured by a Brookfield viscometer).
  • the viscosity of the composition may range from about 100 cP to about 200 cP, about 200 cP to about 300 cP, about 250 cP to about 600 cP or about 400 cP to about 600 cP.
  • the viscosity of the formulation is about 30 cP, about 100 cP, about 200 cP, about 300 cP, about 400 cP, about 500 cP, or about 250,000 cP (e.g., as measured with a Brookfield viscometer at about 25 degrees Celsius equipped with an ultra low adapter).
  • the viscosity of a composition provided herein may be measured at room temperature (about 25 degrees C.) with a shear rate of about 13.2 sec -1 (e.g., with gap between the spindle and sample chamber wall of about 6.0 mm).
  • composition having a viscosity under such conditions that is at least 2 centipoise (cP), at least 5 cP, at least 10 cP, at least about 25 cP, at least about 30 cP, at least about 35 cP, at least about 40 cP, at least about 50 cP, at least about 200 cP, at least about 225 cP, at least about 250 cP, at least about 300 cP, or at least about 400 cP.
  • cP centipoise
  • the viscosity of the composition under such conditions is about 50 cP to about 250,000 cP, about 50 cP to about 70,000 cP, about 50 cP to about 25,000 cP, about 50 cP to about 10,000 cP, about 50 cP to about 3,000 cP, about 50 cP to about 2,000 cP, about 250 cP to about 250,000 cP, about 250 cP to about 70,000 cP, about 250 cP to about 25,000 cP, about 250 cP to about 10,000 cP, about 250 cP to about 3,000 cP, or about 250 cP to about 2,000 cP.
  • the viscosity of the composition is from about 25 centipoise (cP) to about 800 cP, about 50 cP to about 800, or about 300 cP to about 800 cP (e.g., measured by a Brookfield viscometer).
  • the viscosity of the composition under such conditions may range from about 100 cP to about 200 cP, about 200 cP to about 300 cP, about 250 cP to about 600 cP or about 400 cP to about 600 cP.
  • the viscosity of the formulation measured under such conditions is about 30 cP, about 40 cP, about 100 cP, about 200 cP, about 300 cP, about 400 cP, about 500 cP, or about 250,000 cP.
  • a pharmaceutical composition described herein is a non- newtonian fluid.
  • the non-newtonian fluid is thixotropic.
  • the non-newtonian fluid composition thins with shear, and thickens upon the absence of shear.
  • compositions may further comprise a cytokine inhibitor.
  • cytokine inhibitors may include one or more of benralizumab (as described in US Patent Publication 20200362027A1), mepolizumab (as described in US Patent 6,129,913), reslizumab (as described in US Patent 10,577,414), dectrekumab (QAX576), monoclonal antibody cendakimab (RPC4046) (as described in Hirano I, Collins MH, Assouline-Dayan Y, et al.
  • RPC4046 a Monoclonal Antibody against IL13, Reduces Histologic and Endoscopic Activity in Patients With Eosinophilic), dupilumab (as described in US Patent 7,608,693), tezepelumab (“AMG157”)(as described in US Patent 10,828,365), firentelumab, and itepekimab.
  • compositions may comprise a corticosteroid.
  • corticosteroids include, for example, one or more of budesonide, hydrocortisone, hydroxyltriamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethsone dipropionate, clobetasol valemate, ciclesonide, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenol
  • the composition may further comprise a bacteria as described above.
  • the food additive composition may comprise an AHR agonist and a food-safe carrier.
  • the AHR agonist may be present in the food additive in an amount of from about 100 mg/kg of food additive to about lOg/kg of food additive.
  • the food additive may comprise at least 150 mg, or at least 200 mg, or at least 300 mg, or at least 400 mg, or at least 500 mg, or at least 600 mg, or at least 700 mg, or at least 800 mg, or at least 900 mg, or at least 1 g, or at least 5 g, or at least 10 g quercetin per 100 g food product or food additive.
  • the food additive may contain an AHR agonist-producing bacteria as described herein.
  • the food additive may comprise a flavoring agent.
  • the flavoring agent may be selected from one or more of vanilla, cocoa, vanillin, salt, coffee, chocolate, berry flavors, and fruit flavors, acids (lactic, malic, etc.), caramel, mint, natural and/or artificial sweeteners, sodium sources such as sodium chloride, hydrocolloids, and combinations thereof.
  • the food additive may comprise a masking agent.
  • the masking agent may comprise one or more of natural and artificial sweeteners; sodium sources such as sodium chloride; hydrocolloids such as guar gum, xanthan gum, carrageenan, gellan gum, other suitable gums; emulsifiers; encapsulating agents such as starches and modified starch products; and combinations thereof.
  • active agents provided herein may be administered orally, and active agents provided herein may be formulated into liquid preparations, suspensions, syrups, elixirs, and the like.
  • a unit dosage form for oral administration may include tablets and capsules. Unit dosage forms may be configured for administration once a day, twice a day, or more.
  • the compositions may be isotonic with a body fluid of the recipient.
  • the isotonicity of the compositions may be attained using sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • An example includes sodium chloride.
  • Buffering agents may be employed, such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts.
  • Preservatives may be employed to increase the shelf life of the composition.
  • Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed.
  • a suitable concentration of the preservative may include from about 0.02% to about 2% based on the total weight of the composition, although larger or smaller amounts may be desirable depending upon the agent selected. Reducing agents may be used to maintain good shelf life of the formulation.
  • active agents provided herein may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like, and may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
  • a suitable carrier such as sterile water, physiological saline, glucose, or the like
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
  • Such preparations may include complexing agents, metal ions, polymeric compounds such as polyacetic acid, poly glycolic acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. The presence of such additional components may influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration.
  • compositions may be provided as a tablet, aqueous or oil suspension, dispersible powder or granule, emulsion, hard or soft capsule, syrup or elixir.
  • Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and may include one or more of the following agents: sweeteners, flavoring agents, coloring agents and preservatives.
  • Aqueous suspensions may contain the active ingredient in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • Formulations for oral use may also be provided as hard gelatin capsules, wherein the active ingredient(s) are mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules.
  • the active agents may be dissolved or suspended in suitable liquids, such as water or an oil medium, such as peanut oil, olive oil, fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • Stabilizers and microspheres formulated for oral administration may also be used.
  • Capsules may include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredient in admixture with fillers such as lactose, binders such as starches, and/or lubricants, such as talc or magnesium stearate and, optionally, stabilizers.
  • a composition may contain from about 1 mg or less to about 1,000 mg or more of a active agent provided herein, for example, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg to about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900 mg.
  • unit dose formulations may be provided in a range of dosages to permit divided dosages to be administered. A dosage appropriate to the patient and the number of doses to be administered daily may thus be conveniently selected.
  • two or more of the therapeutic agents may be incorporated to be administered into a single tablet or other dosage form (e.g., in a combination therapy); however, in other embodiments the therapeutic agents may be provided in separate dosage forms.
  • compositions may comprise inert materials such as diluents, such as carbohydrates, mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, starch, and the like, or inorganic salts such as calcium triphosphate, calcium phosphate, sodium phosphate, calcium carbonate, sodium carbonate, magnesium carbonate, and sodium chloride.
  • inert materials such as carbohydrates, mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, starch, and the like
  • inorganic salts such as calcium triphosphate, calcium phosphate, sodium phosphate, calcium carbonate, sodium carbonate, magnesium carbonate, and sodium chloride.
  • Disintegrants or granulating agents may be included in the formulation, for example, starches such as com starch, alginic acid, sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite, insoluble cationic exchange resins, powdered gums such as agar, or karaya, or alginic acid or salts thereof.
  • Binders may be used.
  • Binders may include materials from natural products such as acacia, starch and gelatin, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, and the like.
  • Lubricants such as stearic acid or magnesium or calcium salts thereof, polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes, sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol, starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like, may be used in the compositions.
  • Controlled release formulations may be employed wherein the active agent or analog(s) thereof is incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms. Slowly degenerating matrices may also be incorporated into the formulation. Other delivery systems may include timed release, delayed release, or sustained release delivery systems.
  • Coatings may be used, for example, nonenteric materials such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols, or enteric materials such as phthalic acid esters.
  • Dyestuffs or pigments may be added for identification or to characterize different combinations of active agent doses.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added to the active ingredient(s).
  • Physiological saline solution, dextrose, or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol are also suitable liquid carriers.
  • the pharmaceutical compositions may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil, such as olive or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof.
  • Suitable emulsifying agents include naturally-occurring gums such as gum acacia and gum tragamayth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Suspensions may be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The preparation of acceptable aqueous solutions with suitable pH, isotonicity, stability, and the like, is within the skill in the art.
  • the active agents provided herein may be provided to an administering physician or other health care professional in the form of a kit.
  • the kit may be in the form of a package that houses a container which contains the active agent(s) in a suitable pharmaceutical composition, and instructions for administering the pharmaceutical composition to a subject.
  • the kit may optionally also contain one or more additional therapeutic agents currently employed for treating a disease state as described herein.
  • a kit containing one or more compositions comprising active agents provided herein in combination with one or more additional active agents may be provided, or separate compositions containing an active agent as provided herein and additional therapeutic agents may be provided.
  • the kit may also contain separate doses of an active agent provided herein for serial or sequential administration.
  • the kit may optionally contain one or more diagnostic tools and instructions for use.
  • the kit may contain suitable delivery devices, e.g., syringes, and the like, along with instructions for administering the active agent(s) and any other therapeutic agent.
  • the kit may optionally contain instructions for storage, reconstitution (if applicable), and administration of any or all therapeutic agents included.
  • the kits may include a plurality of containers reflecting the number of administrations to be given to a subject.
  • the epithelium is at the forefront of the protective innate immune system.
  • expression of antiserine proteases of the kazal type provide homeostatic control of inflammation.
  • SPINK5 or acquired loss of SPINK7 has profound pro-inflammatory consequences, yet there is a limited understanding of the factors that regulate their basal expression direct responsiveness to inflammatory stimuli.
  • Applicant has identified the transcription factor, Ovo Like Transcriptional Repressor 1 (OVOL1) as an esophageal selective gene that regulates SPINK7 promoter activity and expression.
  • OVOL1 Ovo Like Transcriptional Repressor 1
  • AHR antagonists inhibited SPINK7 expression induced by a variety of stimuli including dietary compounds, microbiota metabolites and pharmacological agents.
  • Interleukin (IL)-4 and IL-13 abolished AHR ligand induced OVOL1 nuclear translocation and SPINK7 expression.
  • Stimulation with IL- 13 abrogated the nuclear translocation of OVOL1 and promoted enhanced degradation of OVOL1 protein in the cytoplasm. This effect of IL- 13 was dependent on the cysteine protease calpain-14.
  • the epithelium is at the forefront of the protective innate immune system.
  • expression of the anti-serine proteases of the kazal type provide homeostatic control of inflammation.
  • Loss of SPINK5 and/or SPINK7, the two main SPINK family members expressed in the squamous epithelium leads to profound consequences including impaired epithelial barrier function and elicitation of allergic inflammation in the skin and/or esophagus.
  • Epithelial cells use sensors to monitor the external and internal environment. For example, pattern recognition receptors including Toll-like receptors (TLRs) and protease- activated receptors (PARs) in the epidermis sense pathogenic insults. Once the sensor received an input (i.e., recognition of molecules from pathogens, microbiota dysbiosis, proteolytic activity), a cellular decision is made accordingly. Distinct signals will promote diverse cellular responses which result in sealing the barrier tightly against toxins or loosen the barrier to enable immune cells to infiltrate and fight pathogens. Despite the importance of investigating such sensor molecules, a master epithelial sensor in the esophagus has not yet been identified.
  • TLRs Toll-like receptors
  • PARs protease- activated receptors
  • SPINK7 expression was elevated during epithelial cell differentiation induced by air liquid interface (ALI) culture and/or modification of calcium concentrations.
  • Histone 3 acetylation marks suggested the importance of a putative binding motif for the C2H2 zinc finger transcription factor, Ovo Like Transcriptional Repressor 1 (OVOL1), an esophageal enriched gene. Indeed, overexpression and repression of OVOL1 regulated SPINK7 promoter activity.
  • OVOL1 Ovo Like Transcriptional Repressor 1
  • AHR ligands including proton-pump inhibitors, dietary compounds, metabolites produced by bacteria and particles found in the air, modulated OVOL1 activation, nuclear localization and subsequent SPINK7 expression.
  • type 2 cytokines IL-4 and IL-13 inducers of allergic responses
  • IL-13 inducers of allergic responses
  • OVOL1 activation via an AHR-dependent mechanism repressed OVOL1 activation via an AHR-dependent mechanism.
  • AHR may therefore serves as a sensor for environmental signals and has potential to rapidly control the esophageal epithelium fate by controlling SPINK7 levels via OVOL1 activation.
  • the disclosed dataset highlights a potential role of the AHR/OVOL1/SPINK7 pathway in pathoetiology of EoE. As such, modulation of this pathway (e.g., supplementing AHR ligands) may be therapeutic in EoE and related allergic diseases.
  • SPINK7 expression is induced during epithelial differentiation
  • Applicant analyzed SPINK7 expression in high confluent cultures of esophageal epithelial progenitor cell line (EPC2) cells, a condition that induce cellular differentiation. SPINK7 expression was induced under high confluency conditions compared to low confluency conditions (FIG 1A). SPINK7 expression was further increased in a high calcium (1.8 mM) media compared to a low calcium media (0.09 mM) (FIG 1A).
  • EPC2 esophageal epithelial progenitor cell line
  • Applicant cloned the 4.5 kb region upstream to the TSS of SPINK7 into a vector that contain nano-luciferase reporter (SPINK7).
  • SPINK7 nano-luciferase reporter
  • control a promoterless nano-luciferase vector (control).
  • the 4.5 kb sequence was selected based on transcriptional and epigenetic data from FIG IB and from ENCODE and CisBP datasets (Yanez-Cuna, 2012 and Weirauch, 2014).
  • EPC2 cells were grown at high density and in high calcium media to induce cell differentiation and then were transiently co-transfected with firefly luciferase vector (to control for transfection efficiency) and either SPINK7 or control vectors.
  • the SPINK7-transfected cells had on average about 340-fold increase in the luminescence signal compared to cells transfected with the empty vector (p ⁇ 0.0001; FIG 1C). Indicating that this region of the SPINK7 gene has promoter activity under these conditions.
  • Applicant subsequently considered the minimal sequence required for promoter induction.
  • Applicant tested various construct lengths (FIG IE).
  • the promoter activity of all reporter constructs was sufficient to drive promoter activity including the shortest construct with the first 1 kb sequence from the 5’ TSS of SPINK7, which was sufficient to drive promoter activity in the high calcium condition compared to the empty vector (80-fold increase, p ⁇ 0.0002; FIG IE).
  • the luciferase activity of the cells transfected with the 2 and 3 kb constructs were not significantly different compared to the luciferase activity of the cells that were transfected with the 1 kb construct (FIG IE).
  • FOG IE luciferase activity of the cells transfected with the 2 and 3 kb constructs
  • a low calcium media low promoter activity was observed in all the constructs with no difference between the 1 kb and the 4 or 4.5 kb (FIG 9B).
  • the 1, 2, and 3 kb constructs were not affected by high calcium (FIG IF).
  • TFs transcription factors
  • FICZ 6-formylindolo(3,2-b)carbazole
  • OVOE1 binds to SPINK7 promoter
  • SPINK7 is a direct OVOE1 target gene in esophageal epithelial cells.
  • Applicant predicted 4 OVOE1 binding sites up upstream of the SPINK7 TSS (FIG 2C).
  • Applicant tested the specificity of this response by subjecting the SPINK7 deletion constructs to FICZ activation in the presence or absent of OVOE1 overexpression (FIG 2C). Consisting with previous results, overexpression of OVOE1 induced promoter activity in cells that were transfected with the 4.5 kb construct of SPINK7. The promoter activity was further induced in the OVOE1 overexpressing cells after FICZ stimulation (FIG 2D).
  • OVOE1 site mutant in -4139 bp when TGTTACA sequence was replaced with GTGGCAC, did not affect the SPINK7-nLUC activation (FIG 2E).
  • ESA Electrophoretic Mobility- Shift Assay
  • Applicant examined OVOL1 capacity to bind to the SPINK7 gene promoter.
  • Applicant analyzed the binding of recombinant human OVOL1 protein to a fluorescent DNA probe corresponds to the -4139, -3379, -2078, and -208 bp of the SPINK7 promoter.
  • OVOL1 shifted the mobility of the fluorescent probe in all probes, except the -3379 probe (FIG 2F).
  • Un-labelled (cold) wt competitors that contains the predicted binding site at 4139, -2078, and -208 bp inhibited the mobility shift (FIG 2F).
  • a mutant cold competitor that contains the predicted binding site at -4139 bp (GTGGCAC) failed to inhibit the mobility shift (FIG 2F).
  • rabbit administration of an antihuman OVOL1 antibody resulted in a supershift of the -2078 probe.
  • Applicant generated nuclear extracts of HEK-293T cells overexpressing OVOL1.
  • Applicant then depleted OVOL1 expression in EPC2 cells by stably transduction with a vector expressing either shRNA targeting OVOL1 or non-silencing control (NSC) shRNA.
  • OVOL1 silenced cells had > 2-fold decrease in OVOL1 mRNA expression compared to NSC-treated cells (FIG 3A).
  • OVOL1 silenced cells had increased TSLP release compared to NSC- treated cells after PolyLC stimulation, (FIG 3B).
  • the increased TSLP production of OVOL1 silenced cells may be partially mediated by decreased expression of SPINK7.
  • OVOL1 silenced cells that were differentiated in ALI culture system had 3-fold decrease in SPINK7 expression compared to differentiated NSC-treated cells (FIG 3C) and revealed barrier impairment as asses by Trans Epithelial Electrical Resistance (TEER; FIG 3D). These data suggest that OVOL1 expression is critical for maintaining SPINK7 expression, barrier integrity and controlling innate cytokine production by epithelial cells.
  • Applicant subsequently generated OVOL1 gene deleted cells using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genomic editing (FIG 10A,B).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • FIG. 4A Analysis of OVOL1 protein revealed that OVOL1 is expressed in epithelial cells (FIG 4B). Applicant noted that OVOL1 intracellular localization was changed in different clusters of the epithelium. In basal and suprabasal layers of the epithelium, OVOL1 was localized to the cells’ nuclei while in the squamous epithelium, OVOL1 intracellular localization was transformed to cytoplasmic expression (FIG 4B). Analysis of OVOL1 expression in EoE biopsies revealed a marked decrease in the protein expression compared to control biopsies (FIG 4C).
  • AHR is activated in response to a variety of ligands such as environmental toxicants including vehicle exhaust and cigarette smoke, dietary compounds (i.e., flavonoids, indole-3-carbinol derivatives, extracts from fruits, vegetables especially cruciferous), products from commensal bacteria (such as FICZ, kynurenine and Butyrate), tryptophan metabolism and drugs including proton pump inhibitors, which are interesting used to treat EoE. Applicant then asked which inducers of the AHR pathway might regulate SPINK7 expression via OVOL1.
  • environmental toxicants including vehicle exhaust and cigarette smoke, dietary compounds (i.e., flavonoids, indole-3-carbinol derivatives, extracts from fruits, vegetables especially cruciferous), products from commensal bacteria (such as FICZ, kynurenine and Butyrate), tryptophan metabolism and drugs including proton pump inhibitors, which are interesting used to treat EoE.
  • Applicant then asked which inducers of
  • Applicant stimulated EPC2 cells with either FICZ, 2-(F H-indole-3'- carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), indole-3-carbinol (I3C), Omeprazole, Benzo [a] Pyrene (B[a]P), Urolithin A (UroA) and Quercitin.
  • Omeprazole, B [a]P, Quercitin and ITE efficiently stimulated the expression of the AHR target gene CYP1A1 and SPINK7 expression (FIG 5A,B).
  • Applicant next aimed to determine if SPINK7 up-regulation was AHR-dependent.
  • OVOL1 was redistributed between the cytoplasmic fraction and the nuclear fraction (FIG 5D).
  • Applicant then performed transcriptomic analysis of FICZ- stimulated cells. This analysis revealed 842 dysregulated genes (FIG 5E).
  • the up-regulated genes were enriched for functional pathways involved in epithelial differentiation such as IVL (encoding for the barrier gene and differentiation marker, involucrin), late cornified envelope protein family including LCE3E and LCE3D, and small proline rich proteins including SPRR2G, SPRR1A, SPRR2A (FIG 5F).
  • FICZ-up regulated genes were also enriched for inflammatory responses, cell proliferation, membrane assembly, response to lipids and cell motility (FIG 5F).
  • FICZ dysregulated genes More than 18% of the FICZ dysregulated genes, overlapped with the EoE transcriptome (156 genes out of 842 FICZ-regulated genes; FIG 11A). Most genes that were up-regulated in the EoE transcriptome were down-regulated by FICZ and vice versa (FIG 11 A). The overlapped genes were enriched for cornified envelope proteins (FIG 11B). This suggests that loss of epithelial differentiation as observed in EoE biopsies, can be partially reversed by AHR activation.
  • IL-4 and IL- 13 which are Th2 cytokines with established roles in atopic diseases inducing EoE pathology (Blanchard, 2010, Leung, 2015, Hirano, 2020) on OVOL1 intracellular localization.
  • OVOL1 was primarily localized to cytoplasmic vesicles which were bordered by the membranal protein desmoglein 1 (DSG1) (FIG 6A).
  • DSG1 membranal protein desmoglein 1
  • FICZ stimulation induced nuclear mobilization of OVOL1
  • IL-4 or IL- 13 stimulation prevented the FICZ-induced OVOL1 mobilization to the nucleus in OVOL1 overexpressing cells (FIG 6A).
  • IL-4 and IL- 13 significantly decreased the SPINK7 promoter activity in OVOL1 overexpressing cells that were stimulated with FICZ (FIG 6B,C). Because cells that were differentiated in the ALI culture system express high endogenous levels of OVOL1, Applicant analyzed the effect of IL-4 and IL-13 on differentiated cells. In unstimulated cells, OVOL1 was mostly nuclear and remained nuclear after FICZ stimulation (FIG 6D). IL-4 or IL- 13 stimulation promoted OVOL1 translocation from the nucleus to the cytoplasm (FIG 3F).
  • FICZ stimulation increased the endogenous SPINK7 expression (FIG 6E) and IL- 13 decreased SPINK7 expression when the cells were stimulated with FICZ (FIG 6E).
  • FICZ and IL-13 stimulations As a control for FICZ and IL-13 stimulations, CYP1A1 and CCL26 expression were analyzed respectively (FIG 6F,G).
  • FICZ stimulation was able to decrease the IL-13- dependent CCL26 expression by 2.7-fold (p ⁇ 0.0001; FIG 6G).
  • Transcriptomic analysis of FICZ, IL- 13 and FICZ+IL-13 treated cells compared with untreated cells revealed that the 4 groups of treatment were significantly different (FIG 6H).
  • the majority of the IL- 13 dysregulated genes (55%) overlapped with the IL-13 + FICZ transcriptome (FIG 61). While only less than 10% of the IL- 13 + FICZ transcriptome overlapped with the IL- 13 transcriptome (FIG 61).
  • IL- 13 can regulate OVOL1 expression.
  • OVOL1 mRNA expression was comparable between IL-13 stimulation and control cells (FIG 7B). Consistent with the protein level, DSG1 mRNA was decreased after IL- 13 stimulation compared with untreated cells (FIG 7B). Suggesting that nuclear OVOL1 is a stable protein, while IL- 13 mediated nuclear transport inhibition promotes loss of cytoplasmic OVOL1 protein expression.
  • IL-13 may stimulate cytoplasmic retention of OVOL1 and loss of OVOL1 in EoE patients compared with controls (FIG 4 C,D).
  • the EoE transcriptome is enriched with proteases and shows dysregulation between proteases and protease inhibitors (Azouz, 2018, Rochman, 2018 Rochman, 2017) Therefore, Applicant hypothesized that OVOL1 may be post-transcriptionally modified by proteases in the esophagus of EoE patients. Applicant then asked which genes are co-expressed with OVOL1 in the same esophageal clusters.
  • RNAseq analysis revealed that CAPN14 and OVOL1 are co-expressed in the same cellular cluster in the esophagus (FIG 7C).
  • the epithelial clusters that co-expressed OVOL1 and CAPN14 were enriched with SPINK7 and correspond to differentiated epithelial cells that express differentiation markers (i.e., FLG, MUC22) and esophageal enriched genes (i.e., MUC22, MAL, KLK13; FIG 7C).
  • differentiation markers i.e., FLG, MUC22
  • esophageal enriched genes i.e., MUC22, MAL, KLK13; FIG 7C.
  • calpain-14 is an esophageal epithelial specific cysteine protease that is overexpressed in esophageal biopsies from EoE patients compared to controls and is regulated by IL-13 ⁇ Davis, 2016 #121;Kottyan, 2014 #123 ⁇ .
  • Inducible CAPN14 expression in differentiated esophageal epithelial cells revealed a marked reduction in OVOL1 protein expression (FIG 7D).
  • constitutive expression of CAPN14 by CAPN14-GFP vector transduction, decreased the expression of OVOL1 compared with control GFP vector transduction (FIG 7G), indicating that the reduction in OVOL1 protein expression resulted from CAPN14 expression and not as a result of dox treatment.
  • IL- 13 treatment decreased OVOL1 expression in the CAPN14-GFP overexpressing cells (FIG 7G).
  • IL-13 stimulation and CAPN14 expression decrease OVOL1 protein expression.
  • Applicant then hypothesized that nuclear mobilization of OVOL1 by AHR protects OVOL1, while IL-13- mediated cytoplasmic retention of OVOL1, promotes degradation of OVOL1 by cytoplasmic proteases such as calpin-14.
  • Applicant performed a biochemical fractionation of cellular proteins that differentiated the nuclear proteins from the rest of the cellular proteins which contain the cytosol and other organelles (here refer to cytosol).
  • Applicant then incubated recombinant OVOL1 protein with cytosolic or nuclear proteins.
  • Applicant then investigated if AHR ligands can stimulate esophageal SPINK7 expression ex vivo in a murine model. Esophagi were collected from C57BL/6 mice and were stimulated with AHR ligands. Six hrs of stimulation, were sufficient for detection of CYP1A1 induction, indicating that AHR pathway was activated (FIG 8A). At the same time, SPINK7 mRNA was induced by 10-fold (FIG 8B).
  • the data presented here identify a complexed regulatory network that controls SPINK7 expression in esophageal epithelial cells. Applicant demonstrated that AHR regulates the expression of SPINK7 via OVOL1 and that IL-4 and IL- 13 inhibit this pathway by inhibiting the OVOL1 nuclear mobilization (FIG 12). The cysteine protease calpain-14 inhibits SPINK7 expression by post-translational modifications of OVOL1.
  • IL-13 can potentially inhibit SPINK7 expression by 2 mechanisms; first, by inhibiting OVOL1 nuclear translocation which prevents OVOL1 from binding to its target genes and decreasing OVOL1 stability, and second, by inducing calpain-14 expression, which in turn degrades OVOL1 and prevents SPINK7 expression.
  • IL- 13 and calpain-14 are overexpressed in the esophagus of EoE patients compared with control individuals and were suggested to be major drivers in EoE pathogenesis; IL- 13 induces epithelial cell transcriptional changes that overlap with the EoE transcriptome (the list of genes that are altered in EoE esophageal biopsies compared to control biopsies). Its importance in disease pathogenesis is implicated by the positive effects of anti-IL-13 treatment (QAX576 and RPC4046) in EoE.
  • Calpain-14 is an esophageal- specific protease, encoded by the CAPN14 gene which is located in the strongest associated EoE risk locus (i.e., 2p23). Calpain-14 is up regulated by exposure of esophageal epithelial cells to IL- 13 and has been shown to regulate epithelial barrier homeostasis and repair. Therefore, Applicant’ s findings reveal an altered molecular pathway which is relevant in disease state. OVOL1 protein expression was lost in esophageal biopsies from EoE patients compared to controls. However, OVOL1 mRNA expression levels were comparable between EoE patients and controls.
  • OVOL1 controls SPINK7 expression.
  • OVOL1 is an enriched esophageal transcription factor that is induced during esophageal epithelial differentiation (Uhlen, 2015, Tsuji, 2017).
  • OVOL1 regulates expression of barrier genes such as FLG and LOR in the skin (Tsuji, 2018).
  • variants in OVOL1 gene associate with atopic dermatitis, a type 2 allergic disease that is characterized by barrier impairment of the skin (Marenholz, 2015, Hirota, 2012, Paternoster, 2011).
  • Applicant’s data demonstrates that OVOL1 depletion decreases SPINK7 expression and promotes impaired esophageal barrier function and cytokine production in vitro. Therefore, it is suggested that OVOL1 has a key role in regulating esophageal homeostasis and immune tolerance.
  • AHR as an esophageal epithelial sensor that promotes the activation of OVOL1 which then induce transcription program of differentiation and barrier genes including SPINK7 and FLG.
  • AHR is activated in response to many ligands such as vehicle exhaust and cigarette smoke, dietary compounds (i.e. flavonoids, indole-3 -carbinol derivatives, extracts from fruits and vegetables especially cruciferous), products from commensal bacteria and tryptophan metabolism (Moura-Alves, 2014, Rothhammer, 2019).
  • AHR is capable of initiating distinct signaling pathways in response to different ligands (Quintana, 2008). In this way, AHR senses host/microbiome dysbiosis, dietary compounds, drugs and environmental toxicant and initiates an appropriate cellular response.
  • AHR is a member of the basic helix-loop-helix per-Amt-sim (bHLH/PAS) protein family.
  • AHR is trapped in a cytosolic multiprotein complex consisting of heat shock protein 90, tyrosine kinase c-src, and other co-chaperones.
  • AHR translocates from the cytoplasm into the nucleus upon ligand binding and dimerizes with AHR nuclear translocator (ARNT).
  • the AHR /AHR ligand/ ARNT complex recognizes promoters containing specific enhancer sequences termed xenobiotic responsive elements (XRE) and then activates the transcription of target genes such as phase I and phase II detoxification enzymes (cytochrome P450 (CYP1A1)) (Rothhammer, 2019).
  • XRE xenobiotic responsive elements
  • cytochrome P450 CYP1A1
  • Applicant’s data suggests that the AHR pathway is dysregulated in the esophagus of EoE patients compared to control patients.
  • phase II enzymes were markedly decreased (FIG 13).
  • Applicant’ s data reveals a cross talk between the AHR/OVOL1/SPINK7 pathway, the type 2 cytokines, IL-4 and IL-13 and calpain-14.
  • Applicant propose that local fluctuations in SPINK7, that curtails inflammatory responses in the squamous epithelium, particularly in the esophagus, can be controlled by environmental cues that are converged by AHR.
  • Applicant have demonstrated that esophageal SPINK7 expression can be modulated by food supplements that induce AHR signaling. Therefore, Applicant propose that modulation of SPINK7 expression by AHR ligand supplement may be useful for a new avenue to reconsider AHR as a pharmacological target for mechanism-based drugs for food allergic diseases.
  • the 4.5kb region was chosen based on bioinformatics analysis of transcriptional and epigenetic data from the following databases: ENCODE (Encyclopedia of DNA Elements), CIS-BP (Catalog of Inferred Sequence Binding Preferences) and BioWardrobe (corresponds to ENCODE hg 19-2009; Cincinnati Children's Epigenetic Database). Cross analysis of these databases has shown that the 4.5kb region consists of highly-conserved sites enriched with histone acetylation marks (H3K27ac) and overlapped with DNase clusters.
  • ENCODE Encyclopedia of DNA Elements
  • CIS-BP Catalog of Inferred Sequence Binding Preferences
  • BioWardrobe corresponds to ENCODE hg 19-2009; Cincinnati Children's Epigenetic Database.
  • the BioWardrobe database (internal unpublished data) has shown that a region of 1.8kb is enriched with H3K27ac marks at 2kb upstream of the transcription start site (TSS).
  • TSS transcription start site
  • the 4.5kb non-coding putative promoter sequence was obtained from the ENCODE UCSC Genome Browser of the Human genome 2013 database (hg38_dna range) and the coordinates are chromosome 5:148307922-148312422.
  • Promoter constructs were created by cloning the immediate 4.5kb region adjacent to the 5’ TSS of SPINK7 into the promoter-less Nano-luciferase reporter vector PNL1.1-NL (Promega).
  • the 4.5kb sequence and subsequent constructs were created by using primers with the restriction enzyme sites KpnI-HF and Xhol.
  • Applicant utilized SnapGene software that employed In-Fusion cloning techniques. Cloning was performed with In-Fusion HD methods (Clonteck, Takara Bio Company). PNL1.1-NL is defined as the empty vector (EV).
  • Post-cloning with the sequence of interest is termed as SPINK7 [4.5kb].
  • the full-length SPINK7 consists of 4.5kb and short lengths were defined as SPINK7 lkb-3kb from TSS.
  • EPC2 Human esophageal epithelial progenitor cells
  • KSFM Keratinocyte serum-free medium
  • EGF epidermal growth factor
  • BPE bovine pituitary extract
  • IX penicillin/streptomycin Invitrogen
  • EPC2 cells were grown for 3-4 days until they reached 80-90% confluent. Cells were then harvested by addition of trypsin/EDTA (Invitrogen) and incubated for 3-5 min, at 37°C.
  • soybean trypsin inhibitor 250 mg/L in IX DPBS was added, and cells were pelleted at 300G/5min.
  • EPC2 cells were seeded on day-0 at 200k/48well plate in two conditions: low-calcium (KSFM medium alone -CaC120.09 mM) and high calcium (KSFM+CaC12 1.8 mM). 24 hours later (day-1) cells were transiently transfected at >95% density with Opti-MEM (ThermoFisher) and Mirus TransIT-2020 (Mirusbio, Madison, WI) according to the manufacturer instructions. Applicant used 3ul of TransIT-2020 per 1000 ng of construct DNA and 50 ng Firefly DNA (1:20 dilution).
  • Nano-Luciferase activity was normalized to Firefly-Luciferase, then, the activity was normalized to control promoterless transfected cells for each sample transfection variance per well. All assays were conducted in triplicates.
  • EPC2 cells were plated in a high density (250,000 cells/well in a 48 well plate) in KSFM media with 1.8 mM CaC12. For low density, 250,000 cells/well were grown in a 6 well plate in KSFM media with 1.8 mM CaC12.
  • RNA was isolated with Quick-RNA Micro-prep (Zymo; Irvine, CA).
  • ProtoScript First Strand cDNA Synthesis kit (NEB; Ipswich, MA) was employed according to the manufacturer instructions to obtain RT-PCR data.
  • AEI differentiation cells were grown as previously described (ref). Briefly, 150,000 cells/well were plated in a transwell system with 24 well plate. After 48 hrs, media was replaced to a high calcium media (1.8 mM CaC12). On day 8, media was aspirated from the top chambers, on day 12, cells were stimulated and on day 14 cells were harvested.
  • RNA was treated with On-Column DNase Digestion kit (Qiagen) according to the supplied protocol.
  • qPCR was performed using a 7900HT Fast Real-Time PCR system from Applied Biosystems (Fife Technologies) with FastStart Universal SYBR Green Master mix (Roche Diagnostics Corporation) by using primer sets.
  • Next-generation RNA sequencing was performed by the CCHMC Genetic Variation and Gene Discovery Core Facility using Illumina TruSeq kits and sequenced on the Illumina HiSeq2000.
  • RNA sequencing analysis For RNA sequencing analysis, Fastq files from the Illumina pipeline were aligned by BioWardrobe (42). Gene ontology enrichment analysis, which uses statistical methods to determine functional pathways and cellular processes associated with a given set of genes, was performed with the ToppGene suite (43).
  • Lentiviral shRNA vectors against OVOE1 (MISSION shRNA, Sigma- Aldrich, clone NM_004561, TRCN00000257410, TRCN0000229665, and TRCN0000229664) and a control vector that targets no known mammalian genes (SHC002 SIGMA MISSION® pLKO.l-puro Non-Mammalian shRNA Control) were used.
  • EPC2 cells grown in KSFM media were transduced. Twenty-four hours after transduction, cells were selected for stable integration using puromycin (1 pg/mL). After ALI differentiation, gene silencing efficiency of target vectors in transduced cells was assessed by quantitative PCR relative to that of cells transduced with NSC shRNA.
  • gRNA guide RNA complementary to OVOL1 and CAPN14 open reading frame sequence and located directly 5’ of a protospacer adjacent motif (PAM) was identified (http://tools.genome-engineering.org; (Ran, 2013), and oligonucleotides were annealed and ligated into the BbsI restriction site of plasmid pX459M2 (obtained from CCHMC Transgenic Mouse and Gene Editing Core Facility) to produce pX459M2-SPINK7gl.
  • EPC2 cells were transfected with pX459M2 or pX459M2-SPINK7gl using Viromer (Origen) according to the manufacturer’s protocol. Transfected cells were selected, grown and sequenced as previously described (Azouz, 2018).
  • OVOL1 and calpain-14 protein expression were determined by rabbit anti human OVOL1 antibody and rabbit anti human calpain-14 antibodies (Sigma Aldrich).
  • Proteins from cell cultures were extracted with RIPA buffer (Pierce) with protease and phosphatase inhibitors. Loading buffer (Life Technologies) was added, and samples were heated to 95°C for 5 min, subjected to electrophoresis on 12% NuPAGE BisTris gels (Life Technologies), transferred to nitrocellulose membranes (Life Technologies), and visualized using the Odyssey CLx system (LI-COR Biosciences) with IRDye 800RD goat anti-rabbit (LLCOR Biosciences), and IRDye 680RD goat anti-mouse (LLCOR Biosciences) secondary antibodies.
  • the primary antibodies were Rabbit anti-OVOLl (Sigma Aldrich) or Rabbit anti-OVOLl (LifeSpan Biosciences), mouse anti-HSP90 (Cell Signaling Technology Inc), mouse anti-desmoglein-1 (Sigma Aldrich) and anti-Histon H3 (Abeam). Blots were quantified using the Image Studio software (LLCOR Biosciences). [00135] Statistical Analysis
  • Raw data was measured in RLU and it is defined by the ratio of NanoLuciferase reporter activity (NL) to the Firefly (FF) activity [NL/FF]. Normalized data is defined by the ratio of raw data of the promoter activity [NL/FF] to the average of the EV activity [NL/FF].
  • Statistical analysis was completed with GraphPad PRISM. One-way and Two-way ANOVA and t-test were performed.
  • EXAMPLE 2 Quercetin diet induced SPINK7 expression in control mice (ED-L2) but not in mice deficient of AHR in esophageal epithelial
  • mice were fed with control diet or Quercetin enriched diet for 30 days.
  • AIN 93 Vitamin Mix contains Sucrose, DL- Alpha Tocopheryl Acetate (Form of Vitamin E), Phylliquinone (Form of Vitamin K), Nicotinic Acid, Calcium Pantothenate, Vitamin A Palmitate, Pyridoxine Hydrochloride, Thiamin, Hydrochloride, Riboflavin, Vitamin B-12 Supplement, Folic Acid, Cholecalciferol, Biotin.)
  • Mice were harvested and the tongue, esophagus, liver and skin were collected. Then, RNA was extracted from the tissues and the expression of Cyplal (AHR target gene) was analyzed. In addition, protein lysates were generated to analyze CYP1A1 activity.
  • esophageal explants were stimulated ex vivo with FICZ for 6 hrs. Quercetin diet induced SPINK7 expression in control mice (ED-L2) but not in mice deficient of AHR in esophageal epithelial.

Abstract

The instant disclosure relates to composition and methods for the treatment of eosinophilic esophagitis (EoE) in an individual in need thereof. The methods include treatment of eosinophilic esophagitis by administration of one or more aryl hydrocarbon receptor (AHR) agonists to the individual. Further disclosed are compositions containing aryl hydrocarbon receptor (AHR) agonists.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF ESOPHAGEAL CONDITIONS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of U.S. Provisional Patent Serial No. 63/107,571, entitled “Aryl Hydrocarbon Receptor (AHR) Activation for the Treatment of Eosinophilic Esophagitis,” filed October 30, 2020. The contents of each are incorporated in their entirety for all purposes.
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH
[0002] This invention was made with government support under Al 070235 awarded by National Institutes of Health. The government has certain rights in the invention.”
SEQUENCE LISTING
[0003] The contents of the file named “Sequence_Listing_ST25.txt”, which was created on October 28, 2021, and is 6,248 bytes in size, are hereby incorporated by reference in their entirety.
BACKGROUND
[0004] Eosinophilic esophagitis (EoE) is an inflammatory process leading to eosinophil migration to esophageal tissue and is considered the leading cause of dysphagia and food impaction in adults and children. The incidence of EoE is increasing, in part due to the increasing frequency of allergies and better diagnostic tools, but remains underdiagnosed and undertreated. Patients with EoE suffer from physical symptoms such as heartburn, abdominal pain, dysphagia. The prevalence of EoE ranges from 5 to more than 80 cases per 100,000 inhabitants depending on the assessment method; in the United States it is estimated that prevalence of EoE occurs in 56.7/100,000 persons. (See, e.g., Kanikowska et al., Int J Mol Sci. 2021 ;22(19): 10830. doi:10.3390/ijms221910830) Accordingly, there is a need for improved methods and compositions for the treatment of EoE. The instant disclosure addresses one or more of the aforementioned needs in the art. BRIEF SUMMARY
[0005] The instant disclosure relates to composition and methods for the treatment of eosinophilic esophagitis (EoE) in an individual in need thereof. The methods include treatment of eosinophilic esophagitis by administration of one or more aryl hydrocarbon receptor (AHR) agonists to the individual. Further disclosed are compositions containing aryl hydrocarbon receptor (AHR) agonists.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0007] FIGS 1A-1F. SPINK7 expression is induced by calcium and cell confluency. 1A. Quantitative polymerase chain reaction (qPCR) of SPINK7 expression in EPC2 cells that were plated at 24 wells (50,000 cells/well or 300,000 cells/well) that were incubated with either 0.045 or 1.8 rnM of calcium for 48 hours. IB. levels of H3K27Ac in the promoters of SPINK7 in EPC2 cells in the indicated conditions. 1C. Promoter activity in lysates cotransfected with either nano luciferase (nLUC) vector containing SPINK7 promoter (SPINK7-nLUC) or promoter less nLUC vector and with firefly vector to control for transfection efficiency. Promoter activity was determined by nLUC measurements relative to firefly measurements and presented as relative luminescence units (RLU). ID. Promoter activity in lysates co-transfected with either SPINK7-nLUC or nLUC and firefly vector that were grown in the indicated concentrations of CaCh. Promoter activity was determined by nLUC measurements relative to firefly measurements and normalized according to the promoter less nLUC measurements. Promoter activity is presented as relative luminescence units (RLU). IE. Promoter activity in lysates of cells that were grown in 1.8 mM of CaCh and co-transfected with nLUC constructs that contain either 0, 1, 2, 3, 4 or 4.5 kb of the SPINK7 promoter sequence and firefly vector. Promoter activity was determined by nLUC measurements relative to firefly measurements and normalized according to the promoter less nLUC measurements. IF. Promoter activity in lysates co-transfected with nLUC constructs that contain either 0, 1, 2, 3, 4 or 4.5 kb of the SPINK7 promoter sequence and firefly vector, that were grown in either 0.09 or 1.8 mM of CaCh. Promoter activity was determined by nLUC measurements relative to firefly measurements and normalized according to the promoter less nLUC measurements. Then, the values of the 1.8 mM of CaCh lysates were divided to the values of the cells cultured in 0.09 mM of CaCh.
[0008] FIG 2A-2G. OVOL1 binds to SPINK7 promoter and promotes SPINK7 expression. 2A. Nano luciferase activity in lysates transfected with SPINK7 promoter, firefly plasmid and plasmids encoding for the indicated transcription factors compared with control. Cells were either left untreated or were treated with 1 pm FICZ. 2B. Western blot analysis of OVOL1 in control or OVOL1 overexpressing EPC2 cells. P-actin was used as a loading control. 2C. Proximal human SPINK7 promoter sequence identified 4 potential OVOL1 binding sites. Transcription Start Site +1. 2D. Nano luciferase activity in lysates co-transfected with either OVOL1 or a control plasmid and with SPINK7 promoter deletion constructs. Cells were either left untreated or treated with FICZ (1 pM). 2E. Nano luciferase activity in lysates cotransfected OVOL1 or a control plasmid and with SPINK7 promoter with either mutated OVOL1 binding site 1, or mutated OVOL1 binding site 2, or wild-type SPINK7 promoter. Cells were either left untreated or treated with FICZ (1 pM). 2F. Representative results from EMSA experiment using recombinant human OVOL1 protein. Fluorescent IRDye 700 labeled probe sequence, Un-labeled (cold) wt competitor sequence, cold mutant sequence.
2G. Representative results from EMSA experiment using nuclear extracts from HEK-293T cells transfected with OVOL1 plasmid or a control plasmid.
[0009] FIG 3A-3F. Loss of OVOL1 impairs the barrier function and promote innate response. 3A. qPCR analysis of OVOL1 expression from NSC- treated and OVOL1- silenced EPC2 cells. 3B. TSLP release from NSC- treated and OVOL1 -silenced EPC2 cells that were grown in high-calcium media for 64 hours and then stimulated for 8 hours with the indicated concentrations of polyinosinic-polycytidylic acid (polyLC). Cell supernatants were assessed for TSLP levels from three independent experiments. Data are the means ± SD. 3C. qPCR analysis of SPINK7 expression from NSC- treated and OVOL1- silenced EPC2 cells at day 14 of ALI differentiation. 3D. TEER (ohm/cm2) measurement from NSC- treated, OVOL1- silenced EPC2 cells at day 7 of ALI differentiation. Data are the means ± SD from three independent experiments performed in triplicate. All P values were calculated by t test (unpaired, two-tailed). 3E. qPCR analysis of SPINK7 expression from CRISPR/Cas9 OVOL1 KO and control EPC2 cells at day 14 of ALI differentiation. 3F. TEER (ohm/cm2) measurement from CRISPR/Cas9 OVOL1 KO and control EPC2 cells at day 7 of ALI differentiation. Data are the means ± SD from three independent experiments performed in triplicate. All P values were calculated by t test (unpaired, two-tailed).
[0010] FIG 4A-4D. Loss of OVOL1 in EoE biopsies. 4A. mRNA expression of OVOL1 in EoE biopsies compared with control biopsies. 4B. Representative image of immunofluorescence staining of OVOL1 (pink) and DAPI staining in control biopsy. White line separates the lumen from the epithelium and the lumen side is marked by the letter “L”. 4C. Representative images of immunofluorescence staining of OVOL1 (pink) and DAPI staining in control and EoE biopsies. White line separates the lumen from the epithelium and the lumen side is marked by the letter “L”. 4D. Western blot analysis of OVOL1 expression in control and EoE biopsies. The graph on the right showed the OVOL1 expression relative to HSP90.
[0011] FIG 5A-5F. Environmental cues affect SPINK7 expression in an AHR dependent and independent manner. qPCR analysis of CYP1A1 (5 A) and SPINK7 (5B) expression after stimulation with the indicated stimuli with or without GNF351. 5C. Representative images of co-immunofluorescence of desmogleinl (DSG1, green), OVOL1 (pink) and DAPI after 0, 1 or 18 hrs of stimulations with FICZ (1 pm), ITE (1 pm), or omeprazole (10 pm). 5D. Representative western blot with quantitation of 3 independent experiments. 5E. Heatmap representing the fold change of genes that are significantly altered by FICZ treatment (Padj < 0.05). 5F. Gene ontology (GO) analyses of genes that are dysregulated by FICZ treatment.
[0012] FIG 6A-6J. IL-13 and IL-4 prevents OVOLl-dependent SPINK7 expression. 6A. Representative images of co-immunofluorescence of desmogleinl (DSG1, green), OVOL1 (pink) and DAPI stain in OVOL1 overexpression cells that were either left untreated or treated over night with IL-4 or IL-13 (100 ng/mL) with or without FICZ (1 pm). Promoter activity in lysates triple- transfected with either SPINK7-nLUC or nLUC and firefly vector and either OVOL1 or a control plasmid. Cells were either left untreated or treated with 1 pm FICZ, with or without IL-4 (6B), or IL- 13 (6C). 6D. Representative images of coimmunofluorescence of DSG1 (pink), OVOL1 (Cyan) and DAPI stain in cells that were differentiated in the ALI model (ALI). Cells were either left untreated or treated with IL-4 or IL-13 (100 ng/mL) with or without FICZ (1 pm). mRNA expression of SPINK7 (6E), or CYP1A1 (6F), or CCL26 (6G), in cells that were either left untreated, or stimulated with IL- 13 (100 ng/mL), or FICZ (1 pm), or IL-13 (100 ng/mL) and FICZ (1 |im). 6H. Principle component analysis using a permanova weighted test of dysregulated genes from cells that were either left untreated, or stimulated with IL-13 (100 ng/mL), or FICZ (1 pm), or IL-13 (100 ng/mL) and FICZ (1 pm) according to RNAseq data. 61. Overlap between IL-13 transcriptome (IL- 13 compared to UT) and IL- 13 and FICZ transcriptome (IL- 13 + FICZ comared to UT). J. The fold change of IL- 13 treatment compared to UT and the fold change of IL- 13 + FICZ treatment compared to UT of most upregulated genes in the IL- 13 transcriptome.
[0013] FIG 7A-7H. OVOL1 undergoes post-translational modifications. 7A. Western blot analysis of OVOL1, DSG1 and GAPDH expression in differentiated EPC2 cells that were either left untreated or stimulated with IL- 13 (100 ng/mL) for 48 hrs. The graphs on the right show quantification of OVOL1 and DSG1 relative to GAPDH with or without IL- 13 treatment. 7B. qPCR analysis of OVOL1 and DSG1 mRNA expression in differentiated EPC2 cells that were either left un-treated or stimulated with IL- 13 (100 ng/mL) for 48 hrs. 7C. Heatmap represent the relative expression of the indicated genes in epithelial clusters based on single cell RNA-sequencing data of dispersed cells from esophageal control biopsies. 7D. Western blot analysis of OVOL1 and Calpain-14 expression in differentiated EPC2 cells with inducible expression of CAPN14 expression. CAPN14 is fused to a flag tag and is induced by doxycycline (Dox) treatment. qPCR analysis of OVOL1 (7E) and SPINK7 (7F) in differentiated EPC2 with inducible expression of CAPN14 expression of CAPN14. 1. Western blot analysis of OVOL1 in GFP or CAPN14-GFP overexpressing cells with or without IL- 13 treatment. GAPDH was used as a loading control. Anti-GFP was used for detection of GFP and CAPN14-GFP. 7H. Western blot analysis of recombinant human OVOL1 (100 ng) that was either left untreated or incubated with cytoplasmic protein fractions (C), or nuclear protein fractions (N) for the indicated times. The graph on the right is a quantification of OVOL1 band intensity (O.D).
[0014] FIG 8A-9B. Involvement of AHR in EoE pathogenicity in a murine model. Ex vivo mRNA expression of Cyplal (8A) and Spink7 (8B) in murine esophageal explants that were untreated (UT) or stimulated with FICZ (IpM) for 6 hours.
[0015] FIG 9A-9B. Regulation of SPINK7 promoter. 9A. Promoter activity in lysates cotransfected with nLUC and firefly vector that were grown in the indicated concentrations of CaC12. Promoter activity was determined by nLUC measurements relative to firefly measurements and normalized according to the promoter less nLUC measurements. Promoter activity is presented as relative luminescence units (RLU). 9B. Promoter activity in lysates of cells that were grown in 0.09 mM of CaC12 and co-transfected with nLUC constructs that contain either 0, 1, 2, 3, 4 or 4.5 kb of the SPINK7 promoter sequence and firefly vector. Promoter activity was determined by nLuc measurements relative to firefly measurements and normalized according to the promoter less nLuc measurements.
[0016] FIG 10. Generation of OVOL1 KO EPC2 cells. 10A. A chromatogram depicting the genomic DNA sequence of EPC2 cells in the vicinity of the sequence targeted for CRISPR/Cas9-mediated editing. The box indicates the location of the PAM sequence. 10B. Prediction of the protein sequences of OVOL1 KO cells and control cells according to their genomic sequence. Black text indicates amino acids that match WT protein sequence. Blue text indicates amino acids that deviate from WT protein sequence.
[0017] FIG 11A-11B. Overlap between FICZ- induced response and the EoE transcriptome. A. Analysis of genes differentially expressed (-2 > fold change > 2 , p < 0.05) in EPC2 cells following 18 hrs of FICZ stimulation compared with untreated cells and EoE transcriptome was identified from RNA sequencing of esophageal biopsies (n = 6 control patients [Control] and n = 10 patients with active EoE [EoE] as described in (Sherrill, 2014). Venn diagrams depicting genes overlapping between FICZ transcriptome and the EoE transcriptome. A heatmap represents the different fold change of genes that overlapped between the FICZ transcriptome and EoE transcriptome. B. Gene ontology analysis depicting cellular component of the overlapping genes. P value for GO analysis was calculated by ANOVA test.
[0018] FIG 12. A regulatory network controls SPINK7 expression which is influenced by the exposome. AHR is activated and influenced by diet nutrients, environmental toxicant, microbiome composition, tryptophan metabolites and drugs. When AHR is activated, it promotes translocation of OVOL1 to the nucleus which in turn promotes SPINK7 expression. SPINK7 expression promotes epithelial differentiation, barrier function, decreased proteolytic activity and decreased TSLP production. IL-4 and IL- 13, inhibit OVOL1 nuclear translocation and therefore, repress SPINK7 expression. IL- 13 -stimulated CAPN14 expression decreases OVOL1 protein expression and SPINK7 transcription. [0019] FIG 13. Dysregulation of phase II enzymes in EoE. Expression of AHR, NQO1, HM0X1 and HM0X2 in biopsies from 10 EoE patients compared with 6 control patients.
[0020] FIG 14. Quercetin data slide 2 depicts Cyplal/Hprt expression in liver in control and quercetin enriched diet.
[0021] FIG 15 Quercetin data slide 3 depicts Cyplal/Hprt expression in esophageal tissue in control and quercetin enriched diet.
[0022] FIG 16 Quercetin data slide 4 depicts Cyplal/Hprt expression in skin in control and quercetin enriched diet.
[0023] FIG 17 Quercetin data slide 5 depicts Cyplal/Hprt expression in tongue in control and quercetin enriched diet.
[0024] FIG 18 Quercetin data slide 6 depicts Spink7 expression in esophageal tissue in control and quercetin enriched diet.
[0025] FIG 19. Tapinarof increased SP1NK7 expression in vitro similar to FICZ.
[0026] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
DETAILED DESCRIPTION
[0027] DEFINITIONS
[0028] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. [0029] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.
[0030] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
[0031] As used herein, the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
[0032] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.
[0033] The active agent may form salts, which are also within the scope of the preferred embodiments. Reference to a compound of the active agent herein is understood to include reference to salts thereof, unless otherwise indicated. The term “salt(s)”, as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when an active agent contains both a basic moiety, such as, but not limited to an amine or a pyridine or imidazole ring, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the term “salt(s)” as used herein. Pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps, which may be employed during preparation. Salts of the compounds of the active agent may be formed, for example, by reacting a compound of the active agent with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. When the compounds are in the forms of salts, they may comprise pharmaceutically acceptable salts. Such salts may include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p- aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates, phosphates, perchlorates, borates, acetates, benzoates, hydroxy naphthoates, glycerophosphates, ketoglutarates and the like. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like. Examples of organic bases include lysine, arginine, guanidine, diethanolamine, choline and the like.
[0034] Disclosed herein are methods of treating eosinophilic esophagitis (EoE) in an individual in need thereof. In one aspect, the method may comprise administering a composition comprising an aryl hydrocarbon receptor (AHR) agonist to an individual in need thereof. In one aspect, the AHR agonist may be selected from one or more of quercetin, tapinarof, 6-formylindolo[3,2-b]carbazole (FICZ), 6,12-diformylindolo[3,2-b] carbazole (dFICZ), B[a]P , TCDD, apigenin, and luteolin , urolithin A (3,8-dihydroxy-6H- dibenzo[b,d]pyran-6-on), urolithin A03 (6H-benzo[c]chromene-3,8-diol), bilirubin, biliverdin, butyrate, indirubin, 2-(10-H-indole-3-carbonyl)thiazole-4-carboxylic acid methyl ester (“ITE,” a metabolite derived from glucobrassicins), L-kynurenine, derived from tryptophan metabolism, indole-3 -carbinol (I3C), cinnabarinic acid, kynurenin, kynurenin acid, xanthrenic acid, 3,3’-diindoylmethane, indole-3 -acetonitrile, indole[3,2]carbazole, Indole-3 -acetaldehyde, LTr-1, indoxyl sulfate, indole-3 -acetic acid, flavanoid, flavanol, isoflovanones, carotinoid, indole, 3-methyl indole (Skatole), 2-oxindole, Tryptamine, Indirubin, Indigo, 3-hydroxyl-indole, trypanthrin, malassezin, diosmin, tangeritin, tamarixetin, luteolin, myricetin, canthaxanthin, tryptanthrin, DIM (3,3'diindolylmethane, diindolylmethane), 3, 6-formylindolo[3,2-b]carbazole, IAA indole-3 -acetic acid, lAld, indole-3-aldehyde, lAAld, indole[3,2-b]carbazole; I3S, indoxyl-3 -sulfate, 2-(l’H-indole-3’ - carbonyl)-thiazole-4-carboxylic acid, I3AC, 2-(indol-3-ylmethyl)-3,3'diindolylmethane, indolo[3,2-b]carbazole (ICZ), sulindac, leflunomide, lipoxin A4 (LXA4), alprostadil, nimodipine, leflunomide, flutamide, omeprazole, mexiletine, atorvastatin , esomeprazole, berberine (an isoquinoline alkaloid present in many medicinal herbs), sinomenine, resveratrol, VAF347, tetrandrine, and tryptophan metabolites (such as kynurenine). In a further aspect, the AHR agonist may be a derivative of a disclosed AHR agonist, an analogue of a disclosed AHR agonist, a derivative of a disclosed AHR agonist, a salt of a disclosed AHR agonist, an ion of a disclosed AHR agonist, or a complex of a disclosed AHR agonist. In one aspect, the AHR agonist is quercetin. In one aspect, the AHR agonist is tapinarof. In one aspect, the AHR agonist is 6-Formylindolo[3,2-b]carbazole (FICZ).
[0035] Exemplary structures of AHR agonists are provided as follows:
Figure imgf000012_0001
[0036] In one aspect, the said AHR agonist may be administered in a dose effective to reduce inflammation of the esophagus. In certain aspects, the AHR ligand may be administered in an amount of from about 50 microgram/kilogram to about 50,000 microgram/kilogram, or from about 100 microgram/kilogram to about 25,000 microgram/kilogram, or from about 200 microgram/kilogram to about 20,000 microgram/kilogram, or from about 300 microgram/kilogram to about 15,000 microgram/kilogram, or from about 400 microgram/kilogram to about 10,000 microgram/kilogram, or from about 500 microgram/kilogram to about 7,500 microgram/kilogram, or from about 1000 to about 5000 microgram/kilogram.
[0037] In one aspect, the composition employed in the disclosed methods may be a unit dose comprising from about 1 mg to about 500 mg, or from about 2 mg to about 250 mg, or from about 3 mg to about 200 mg, or from about 5 mg to about 150 mg, or from about 6 mg to about 100 mg, or from about 7 mg to about 75 mg, or from about 8 mg to about 70 mg, or about 8 mg to about 65 mg, or from about 9 mg to about 60 mg, or from about 10 mg to about 50 mg of an AHR agonist. [0038] In one aspect, the AHR agonist may be administered in a food product, wherein the AHR agonist may be present in the food product in an amount of from about 100 mg/kg of food product to about 10 g/kg of food product.
[0039] In one aspect, the AHR agonist of the disclosed methods may be administered as a product of a bacteria, wherein said AHR agonist is produced by said bacteria. In this aspect, the bacteria producing the AHR agonist may be administered to the individual in need of AHR agonist treatment. In one aspect, the bacteria may be selected from one or more of L. reuteri, Lactobacillus murinus, Lactobacillus taiwanensis, Bacillus alvei, Clostridium novyi, Clostridium limosum, Clostridium tetani, Corynebacterium acnes, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides sp., Citrobacter sp., E. coli, Flavobacterium sp., Fusobacterium sp., Haemophilus influenza, Kleibsella planticola, Shigella flexneri, Vibrio cholera, Kleibsella pneumonia, Malassezia, Propionibacterium freudenreichii ET-3, Mycobacteria, Lactobacillus reuteri, Allobaculum, Ppeptostrptococcus, and Providencia stuartii. In one aspect, the bacteria may be a genetically modified bacteria, wherein said genetically modified bacteria has increased production of an AHR agonist caused by said genetic modification.
[0040] Exemplary Bacteria that Produce AHR Agonists
Figure imgf000013_0001
Figure imgf000014_0001
[0041] In one aspect, the administration may be via oral administration. In one aspect, the administration may be topical administration to one or more of the esophagus, stomach, and small intestine of said individual. Topical administration of the small intestine, stomach, and/or esophagus may be carried out by oral administration of a composition containing an AHR agonist, as described herein, and may advantageously employ formulations of particular viscosities to deliver the AHR agonist topically to the esophagus.
[0042] In one aspect, the method may include treating EoE by administering to an individual in need thereof (e.g., one diagnosed with or suspected of suffering from eosinophilic esophagitis), a composition comprising a corticosteroid and a liquid vehicle, wherein the composition has a volume sufficient to coat (or at least coat in a effective amount) of a targeted portion of the gastrointestinal tract (e.g. esophagus). In specific embodiments, a volume sufficient to coat the esophagus is a volume that provides a bolus when orally administered to an individual. In more specific embodiments, a volume sufficient to coat the esophagus is a volume that provides a bolus along the entire length of the esophagus (i.e., from immediately after passing the upper esophageal sphincter through the distal end of the esophagus, e.g., immediately prior to entering or passing the lower esophageal sphincter. Thus, in certain embodiments described herein, a coating volume is optionally utilized instead of or in addition to a coating agent described herein in order to coat the targeted portion of the gastrointestinal tract (e.g., esophagus), as described herein.
[0043] In certain aspects, the individual may be an adult individual. In other aspects, the individual may be a pediatric individual. For example, in certain aspects, the individual is less than 18 years of age, or less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 year of age.
[0044] In a further aspect, a composition comprising an AHR agonist is disclosed. In one aspect, the composition may comprise a therapeutically effective amount of an AHR agonist to prevent or alleviate esophageal inflammation in an individual having EoE. In one aspect, the composition may be provided in a unit dose containing a therapeutically effective amount of an AHR agonist to prevent or alleviate esophageal inflammation in an individual having EoE.
[0045] The AHR agonist containing compositions may take a variety of different forms. For example, the composition may be in a form selected from a liquid, an emulsion, a solution, a suspension, a syrup, a slurry, a dispersion, a colloid, a dissolving tablet, a dissolving wafer, a capsule, a gel capsule, a semi-solid, a solid forma gel, a gel matrix a cream, or a paste. In certain aspects, the composition may comprise a viscosity-increasing excipient, for example, a viscosity-increasing excipient that improves the topical administration to the deired area, which may include the esophagus, stomach, and/or small intestine. In one aspect, the composition may comprise a viscosity increasing excipient selected from one or more of lactose, sucrose, sucralose (Splenda®), mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethyl-cellulose (CMC), and polyvinylpyrrolidone (PVP: povidone), acacia, agar, bentonite, carbomers, carboxymethylcellulose calcium, ceratonia, cetostearyl alcohol, colloidal silicon dioxide, cyclomethicone, glyceryl behenate, guar gum, hectorite, hydrogenated vegetable oil type I, hydroxypropyl starch, hydroxypropylmethylcellulose, hydroxyethylcellulose, magnesium aluminum silicate, maltodextrin, polycarbophil, polydextrose, poly(methylvinyl ether/maleic anhydride), polyvinyl acetate phthalate, potassium chloride, propylene glycol alginate, saponite, sodium chloride, stearyl alcohol, sulfobutylether P-cyclodextrin, tragacanth, and mixtures thereof. In further aspects, the viscosity increasing excipient may comprise one or more of a crosslinked poly(acrylic acid) (e.g., Carbopol 974P), glycerine, a carbomer homopolymer, a carbomer copolymer, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, cellulose, ceratonia, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, polyethylene glycol (e.g. PEG 200-4500) gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxy ethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxy ethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly (methoxy ethyl methacrylate), poly (methoxy ethoxy ethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethylcellulose, carboxymethyl-cellulose (CMC) (including, e.g., sodium carboxymethyl-cellulose (NaCMC)), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® or combinations thereof.
[0046] The composition may further comprise one or more mucoadhesive agents, for example, a soluble polyvinylpyrrolidone polymer (PVP), a carbopol, a crosslinked poly(acrylic acid) (e.g., Carbopol 974P), a carbomer homopolymer, a carbomer copolymer, a water-swellable, but water-insoluble, fibrous, cross-linked carboxy-functional polymer, a hydrophilic polysaccharide gum, one or more maltodextrin, alginate, a cross-linked aliginate gum gel, thiomers (e.g., thiolated chitosan, thiolated polycarbophil, thiolated alginate, thiolated cellulose derivatives, thiolated carboxymethyl cellulose, thiolated polyacrylic acid, or thiolated poly acrylates), PEGylated polymers (e.g., PEGylated polyacrylic acid or PEGylated poly acrylates), lectin, hydroxypropyl methyl cellulose (HPMC), cellulose derivatives, HPMA copolymers, a water-dispersible polycarboxylated vinyl polymer, or combinations thereof. In some embodiments, the mucoadhesive agent may be a carbopol. In one aspect, the mucadhesive agent may be selected from one or more of Carbopol 974P, Carbopol Ultrez 10, sodium alginate LF120 and sodium alginate H120L. In some aspects, , mucoadhesive agents that may be used in certain embodiments of the compositions and methods described herein are described, for example, in U.S. Pat. Nos. 6,638,521, 6,562,363, 6,509,028, 6,348,502, 6,306,789, 5,814,330, and 4,900,552, each of which is hereby incorporated by reference in its entirety.
[0047] In one aspect, the mucoadhesive agent may be at least one or at least two particulate components selected from titanium dioxide, silicon dioxide, and clay. In some aspects, where the composition is not further diluted with any liquid prior to administration, the level of silicon dioxide may be from about 3% to about 15%, by weight of the composition. In some aspects, silicon dioxide may be selected from one or more of fumed silicon dioxide, precipitated silicon dioxide, coacervated silicon dioxide, gel silicon dioxide, and mixtures thereof. In some aspects, clay may be selected from, by way of non- limiting example, kaolin minerals, serpentine minerals, smectites, illite or mixtures thereof. In certain aspects, clay may be selected from one or more of laponite, bentonite, hectorite, saponite, montmorillonites or mixtures thereof.
[0048] In one aspect, the, compositions may comprise maltodextrin, for example, about 0.05 g of maltodextrin per mL of liquid vehicle to about 0.6 g of maltodextrin per mL of liquid vehicle, or about 0.1 g of maltodextrin per mL of liquid vehicle to about 0.6 g of maltodextrin per mL of liquid vehicle, or about 0.2 g of maltodextrin per mL of liquid vehicle to about 0.5 g of maltodextrin per mL of liquid vehicle, or about 0.1 g of maltodextrin per mL of liquid vehicle to about 0.4 g of maltodextrin per mL of liquid vehicle, or about 0.2 g of maltodextrin per mL of liquid vehicle to about 0.4 g of maltodextrin per mL of liquid vehicle, or about 0.2 g of maltodextrin per mL of liquid vehicle to about 0.3 g of maltodextrin per mL of liquid vehicle, or about 0.25 g of maltodextrin per mL of liquid vehicle to about 0.28 g of maltodextrin per mL of liquid vehicle, or about 0.1 g of maltodextrin per mL of liquid vehicle, about 0.15 g of maltodextrin per mL of liquid vehicle, about 0.2 g of maltodextrin per mL of liquid vehicle, about 0.25 g of maltodextrin per mL of liquid vehicle, about 0.3 g of maltodextrin per mL of liquid vehicle, about 0.35 g of maltodextrin per mL of liquid vehicle, about 0.4 g of maltodextrin per mL of liquid vehicle, about 0.45 g of maltodextrin per mL of liquid vehicle, about 0.5 g of maltodextrin per mL of liquid vehicle, about 0.55 g of maltodextrin per mL of liquid vehicle, or about 0.6 g of maltodextrin per mL of liquid vehicle.
[0049] In one aspect, a mucoadhesive agent used in an oral pharmaceutical composition described herein imparts an increased viscosity upon the oral pharmaceutical composition (e.g., compared to an otherwise identical composition lacking the mucoadhesive agent).
[0050] In some aspects, the composition is a formulation used to treat a targeted portion of the gastrointestinal tract (e.g., the esophagus). The composition may comprise (or is administered in) a volume used to coat a targeted portion of the gastrointestinal tract (e.g., the esophagus). In certain aspects, the volume used to coat a targeted portion of the gastrointestinal tract (e.g., the esophagus) is a volume that is sufficient to coat the targeted portion. In some aspects, an appropriate palatable dosage is in a volume that coats or at least partially coats the esophagus, and in one aspect, the volume coats or at least partially coats the esophagus and delivers the AHR agonist to the affected areas, for example, the esophagus, a portion of the esophagus, the upper esophagus, or the lower esophagus. In certain aspects, the volume of a composition administered can provide a desired coating characteristic of a composition. As such, in some aspects, provided herein is a composition comprising an AHR agonist wherein the composition comprises (or is administered in) a volume sufficient to coat a targeted portion of the gastrointestinal tract (e.g., the esophagus).
[0051] Viscosity
[0052] Excipients, such as, for example, those listed herein, may be included in the composition to increase the viscosity of the delivered composition. The liquid viscosity may be increased in the oral form, or the excipient may increase the viscosity of the dissolved form of a tablet. Those of ordinary skill in the art will recognize that the viscosity should be at a level that is sufficient to deliver an effective amount of the composition to the esophagus, for example, in an amount that may coat the esophagus. Also, the viscosity should be at a level that may be given orally, thus not so thick that it is either too difficult to swallow, causes gagging, or is unpalatable. Those of ordinary skill in the art may determine the viscosity of the compositions and may thus determine appropriate ranges. One method of determining whether the composition is sufficiently viscous is by determining whether the inflammation, or eosinophilic infiltration, of the esophagus is reduced after treatment with the AHR agonist.
[0053] Viscosity may be determined by any method that will measure the resistance to shear offered by the substance or preparation. Many viscometers are available to those in the pharmaceutical field, and include those built by, for example, Brookfield. Viscosity may be, for example, measured at room temperature, at about 20-25 degrees Celsius, or at about 37 degrees Celsius to mimic body temperature. The viscosity of a liquid generally decreases as the temperature is raised. In some embodiments of the invention, the viscosity is about the viscosity of about 1 grams, about 2 grams, about 3 grams, about 4 grams, about 5 grams, about 6 grams, about 7 grams, about 8 grams, about 9 grams, about 10 grams, about 11 grams, about 12 grams, about 13 grams, about 14 grams, about 15 grams, about 1 to about 5 grams, about 1 to about 50 grams, or about 5 to about 25 grams of sucralose (Splenda®, Distributed By: McNeil Nutritionals, LLC, Fort Washington, Pa. 19034-2299), or about 7 to about 20 grams of sucralose (Splenda®), or about 5 to about 15 grams of sucralose (Splenda®), or about or about 7 to about 15 grams of sucralose (Splenda®), or about 8 to about 12 grams of sucralose (Splenda®), or about 10 to about 11 grams of sucralose (Splenda®), added to 4 ml water, at 25 degrees Celsius. In an illustrative embodiment, the viscosity is about the viscosity of 10 grams of sucralose (Splenda®) added to 4 ml of water, at 25 degrees Celsius. In other embodiments, the viscosity is about the viscosity of 5 to 20 grams of sucralose (Splenda®) in 8 ml total liquid volume, at 25 degrees Celsius. In other embodiments, the viscosity is about the viscosity of 5 to 15 grams of sucralose (Splenda®) in an 8 ml total liquid volume, at room temperature. In other aspects, the viscosity is about the viscosity of 8 to 12 grams of sucralose (Splenda®) in an 8 ml total liquid volume at 25 degrees Celsius. In some aspects, the viscosity is between that of about a fruit nectar and commercial honey, where the viscosity is measured at 25 degrees Celsius.
[0054] In some embodiments, the viscosity of a composition provided herein is at least 2 centipoise (cP), at least 5 cP, at least 10 cP, at least about 25 cP, at least about 30 cP, at least about 35 cP, at least about 40 cP, at least about 50 cP, at least about 200 cP, at least about 225 cP, about 2 cP to about 10 cP, about 2 cP to about 25 cP, about 2 cP to about 50 cP, about 20 cP to about 50 cP, about 20 cP to about 100 cP, or about 50 cP to about 100 cP. In some embodiments, the viscosity of the composition is at least about 100 cP. In certain embodiments, the viscosity of the composition, measured at about 25 degrees Celsius, is about 50 cP to about 250,000 cP, about 50 cP to about 70,000 cP, about 50 cP to about 25,000 cP, about 50 cP to about 10,000 cP, about 50 cP to about 3,000 cP, or about 50 cP to about 2,000 cP. In one aspect, the viscosity of the composition, as measured at about 25 degrees Celsius, is from about 25 centipoise (cP) to about 800 cP, about 50 cP to about 800, or about 300 cP to about 800 cP (e.g., measured by a Brookfield viscometer). In another aspect, the viscosity of the composition may range from about 100 cP to about 200 cP, about 200 cP to about 300 cP, about 250 cP to about 600 cP or about 400 cP to about 600 cP. In specific embodiments, the viscosity of the formulation is about 30 cP, about 100 cP, about 200 cP, about 300 cP, about 400 cP, about 500 cP, or about 250,000 cP (e.g., as measured with a Brookfield viscometer at about 25 degrees Celsius equipped with an ultra low adapter).
[0055] In some aspects, the viscosity of a composition provided herein may be measured at room temperature (about 25 degrees C.) with a shear rate of about 13.2 sec-1 (e.g., with gap between the spindle and sample chamber wall of about 6.0 mm). In certain embodiments, provided herein is a composition having a viscosity under such conditions that is at least 2 centipoise (cP), at least 5 cP, at least 10 cP, at least about 25 cP, at least about 30 cP, at least about 35 cP, at least about 40 cP, at least about 50 cP, at least about 200 cP, at least about 225 cP, at least about 250 cP, at least about 300 cP, or at least about 400 cP. In some embodiments, the viscosity of the composition under such conditions is about 50 cP to about 250,000 cP, about 50 cP to about 70,000 cP, about 50 cP to about 25,000 cP, about 50 cP to about 10,000 cP, about 50 cP to about 3,000 cP, about 50 cP to about 2,000 cP, about 250 cP to about 250,000 cP, about 250 cP to about 70,000 cP, about 250 cP to about 25,000 cP, about 250 cP to about 10,000 cP, about 250 cP to about 3,000 cP, or about 250 cP to about 2,000 cP. In one aspect, the viscosity of the composition, as measured at about 25 degrees Celsius, is from about 25 centipoise (cP) to about 800 cP, about 50 cP to about 800, or about 300 cP to about 800 cP (e.g., measured by a Brookfield viscometer). In another aspect, the viscosity of the composition under such conditions may range from about 100 cP to about 200 cP, about 200 cP to about 300 cP, about 250 cP to about 600 cP or about 400 cP to about 600 cP. In specific embodiments, the viscosity of the formulation measured under such conditions is about 30 cP, about 40 cP, about 100 cP, about 200 cP, about 300 cP, about 400 cP, about 500 cP, or about 250,000 cP.
[0056] In certain embodiments, a pharmaceutical composition described herein is a non- newtonian fluid. In some specific embodiments, the non-newtonian fluid is thixotropic. In certain embodiments, the non-newtonian fluid composition thins with shear, and thickens upon the absence of shear.
[0057] In one aspect, the disclosed compositions may further comprise a cytokine inhibitor. Exemplary cytokine inhibitors may include one or more of benralizumab (as described in US Patent Publication 20200362027A1), mepolizumab (as described in US Patent 6,129,913), reslizumab (as described in US Patent 10,577,414), dectrekumab (QAX576), monoclonal antibody cendakimab (RPC4046) (as described in Hirano I, Collins MH, Assouline-Dayan Y, et al. RPC4046, a Monoclonal Antibody Against IL13, Reduces Histologic and Endoscopic Activity in Patients With Eosinophilic), dupilumab (as described in US Patent 7,608,693), tezepelumab (“AMG157”)(as described in US Patent 10,828,365), lirentelumab, and itepekimab.
[0058] In one aspect, the disclosed compositions may comprise a corticosteroid. Exemplary corticosteroids include, for example, one or more of budesonide, hydrocortisone, hydroxyltriamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethsone dipropionate, clobetasol valemate, ciclesonide, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, fluticasone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortmate, mepreddisone, paramethasone, prednisolone, prednisone, mometasone, beclomethasone dipropionate, triamcinolone, analogs thereof, derivatives thereof, salts thereof, ions and complexes thereof, and combinations thereof. In one aspect, the composition may comprise budesonide. In one aspect, the composition may be in the form of a unit dose comprising from about 0.05 mg to about 50 mg corticosteroid.
[0059] In one aspect, the composition may further comprise a bacteria as described above.
[0060] In one aspect, disclosed herein is a food additive composition. In this aspect, the food additive composition may comprise an AHR agonist and a food-safe carrier. In one aspect, the AHR agonist may be present in the food additive in an amount of from about 100 mg/kg of food additive to about lOg/kg of food additive. In one aspect, the food additive may comprise at least 150 mg, or at least 200 mg, or at least 300 mg, or at least 400 mg, or at least 500 mg, or at least 600 mg, or at least 700 mg, or at least 800 mg, or at least 900 mg, or at least 1 g, or at least 5 g, or at least 10 g quercetin per 100 g food product or food additive.
[0061] In one aspect, the food additive may contain an AHR agonist-producing bacteria as described herein. In one aspect, the food additive may comprise a flavoring agent. For example, the flavoring agent may be selected from one or more of vanilla, cocoa, vanillin, salt, coffee, chocolate, berry flavors, and fruit flavors, acids (lactic, malic, etc.), caramel, mint, natural and/or artificial sweeteners, sodium sources such as sodium chloride, hydrocolloids, and combinations thereof. In one aspect, the food additive may comprise a masking agent. The masking agent may comprise one or more of natural and artificial sweeteners; sodium sources such as sodium chloride; hydrocolloids such as guar gum, xanthan gum, carrageenan, gellan gum, other suitable gums; emulsifiers; encapsulating agents such as starches and modified starch products; and combinations thereof.
[0062] In one aspect, active agents provided herein may be administered orally, and active agents provided herein may be formulated into liquid preparations, suspensions, syrups, elixirs, and the like. A unit dosage form for oral administration may include tablets and capsules. Unit dosage forms may be configured for administration once a day, twice a day, or more.
[0063] In one aspect, the compositions may be isotonic with a body fluid of the recipient. The isotonicity of the compositions may be attained using sodium tartrate, propylene glycol or other inorganic or organic solutes. An example includes sodium chloride. Buffering agents may be employed, such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts.
[0064] Preservatives may be employed to increase the shelf life of the composition. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative may include from about 0.02% to about 2% based on the total weight of the composition, although larger or smaller amounts may be desirable depending upon the agent selected. Reducing agents may be used to maintain good shelf life of the formulation.
[0065] In one aspect, active agents provided herein may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like, and may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. See, e.g., “Remington: The Science and Practice of Pharmacy”, Lippincott Williams & Wilkins; 20th edition (June 1, 2003) and “Remington’s Pharmaceutical Sciences,” Mack Pub. Co.; 18th and 19th editions (December 1985, and June 1990, respectively). Such preparations may include complexing agents, metal ions, polymeric compounds such as polyacetic acid, poly glycolic acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. The presence of such additional components may influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration.
[0066] For oral administration, the compositions may be provided as a tablet, aqueous or oil suspension, dispersible powder or granule, emulsion, hard or soft capsule, syrup or elixir. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and may include one or more of the following agents: sweeteners, flavoring agents, coloring agents and preservatives. Aqueous suspensions may contain the active ingredient in admixture with excipients suitable for the manufacture of aqueous suspensions.
[0067] Formulations for oral use may also be provided as hard gelatin capsules, wherein the active ingredient(s) are mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as water or an oil medium, such as peanut oil, olive oil, fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers and microspheres formulated for oral administration may also be used. Capsules may include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredient in admixture with fillers such as lactose, binders such as starches, and/or lubricants, such as talc or magnesium stearate and, optionally, stabilizers.
[0068] A composition may contain from about 1 mg or less to about 1,000 mg or more of a active agent provided herein, for example, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg to about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900 mg. In some aspects, unit dose formulations may be provided in a range of dosages to permit divided dosages to be administered. A dosage appropriate to the patient and the number of doses to be administered daily may thus be conveniently selected. In certain embodiments two or more of the therapeutic agents may be incorporated to be administered into a single tablet or other dosage form (e.g., in a combination therapy); however, in other embodiments the therapeutic agents may be provided in separate dosage forms.
[0069] The compositions may comprise inert materials such as diluents, such as carbohydrates, mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, starch, and the like, or inorganic salts such as calcium triphosphate, calcium phosphate, sodium phosphate, calcium carbonate, sodium carbonate, magnesium carbonate, and sodium chloride. Disintegrants or granulating agents may be included in the formulation, for example, starches such as com starch, alginic acid, sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite, insoluble cationic exchange resins, powdered gums such as agar, or karaya, or alginic acid or salts thereof. [0070] Binders may be used. Binders may include materials from natural products such as acacia, starch and gelatin, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, and the like.
[0071] Lubricants, such as stearic acid or magnesium or calcium salts thereof, polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes, sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol, starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like, may be used in the compositions.
[0072] Controlled release formulations may be employed wherein the active agent or analog(s) thereof is incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms. Slowly degenerating matrices may also be incorporated into the formulation. Other delivery systems may include timed release, delayed release, or sustained release delivery systems.
[0073] Coatings may be used, for example, nonenteric materials such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols, or enteric materials such as phthalic acid esters. Dyestuffs or pigments may be added for identification or to characterize different combinations of active agent doses.
[0074] When administered orally in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added to the active ingredient(s). Physiological saline solution, dextrose, or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol are also suitable liquid carriers. The pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums such as gum acacia and gum tragamayth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsions may also contain sweetening and flavoring agents. [0075] Suspensions may be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The preparation of acceptable aqueous solutions with suitable pH, isotonicity, stability, and the like, is within the skill in the art.
[0076] In some embodiments, the active agents provided herein may be provided to an administering physician or other health care professional in the form of a kit. The kit may be in the form of a package that houses a container which contains the active agent(s) in a suitable pharmaceutical composition, and instructions for administering the pharmaceutical composition to a subject. The kit may optionally also contain one or more additional therapeutic agents currently employed for treating a disease state as described herein. For example, a kit containing one or more compositions comprising active agents provided herein in combination with one or more additional active agents may be provided, or separate compositions containing an active agent as provided herein and additional therapeutic agents may be provided. The kit may also contain separate doses of an active agent provided herein for serial or sequential administration. The kit may optionally contain one or more diagnostic tools and instructions for use. The kit may contain suitable delivery devices, e.g., syringes, and the like, along with instructions for administering the active agent(s) and any other therapeutic agent. The kit may optionally contain instructions for storage, reconstitution (if applicable), and administration of any or all therapeutic agents included. The kits may include a plurality of containers reflecting the number of administrations to be given to a subject.
EXAMPLES
[0077] The following non- limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. [0078] EXAMPLE 1. AHR serves as an esophageal sensor which activates OVOL1 and promotes SPINK7 expression in response to environmental cues
[0079] The epithelium is at the forefront of the protective innate immune system. In the squamous epithelium of the skin and esophagus, expression of antiserine proteases of the kazal type (SPINK) provide homeostatic control of inflammation. Genetic loss of SPINK5 or acquired loss of SPINK7 has profound pro-inflammatory consequences, yet there is a limited understanding of the factors that regulate their basal expression direct responsiveness to inflammatory stimuli. Herein, Applicant has identified the transcription factor, Ovo Like Transcriptional Repressor 1 (OVOL1) as an esophageal selective gene that regulates SPINK7 promoter activity and expression. Whereas overexpression of OVOL1 increased SPINK7 expression in a promoter reporter assay (P < 0.0001), its depletion decreased SPINK7 expression (P = 0.0004), impaired epithelial barrier (P < 0.0001) and increased production of the pro-atopy inflammatory cytokine thymic stromal lymphopoietin (TSLP; P = 0.001). Mechanistically, ligands of the aryl hydrocarbon receptor (AHR) induced OVOL1 activation and its nuclear translocation which in turn promoted epithelial cell differentiation, barrier function and SPINK7 expression. Conversely, AHR antagonists inhibited SPINK7 expression induced by a variety of stimuli including dietary compounds, microbiota metabolites and pharmacological agents. Interleukin (IL)-4 and IL-13 abolished AHR ligand induced OVOL1 nuclear translocation and SPINK7 expression. Stimulation with IL- 13 abrogated the nuclear translocation of OVOL1 and promoted enhanced degradation of OVOL1 protein in the cytoplasm. This effect of IL- 13 was dependent on the cysteine protease calpain-14.
Translational studies demonstrated a decrease (P < 0.02) in OVOL1 protein expression in patients with eosinophilic esophagitis (EoE). This data suggests that AHR may serve as an esophageal sensor, mediates its action via OVOLl-induced SPINK7 transcription, and that IL-4 and IL- 13 repress this pathway in EoE. Therefore, activation of the AHR pathway can serve as an intervention strategy for maintaining proper epithelial barrier function by inducing SPINK7 expression, promoting epithelial differentiation and controlling innate immune responses.
[0080] The epithelium is at the forefront of the protective innate immune system. In the squamous epithelium of the skin and esophagus, expression of the anti-serine proteases of the kazal type (SPINK) provide homeostatic control of inflammation. Loss of SPINK5 and/or SPINK7, the two main SPINK family members expressed in the squamous epithelium, leads to profound consequences including impaired epithelial barrier function and elicitation of allergic inflammation in the skin and/or esophagus. For example, experimental depletion of SPINK7 in esophageal epithelial cells is sufficient to induce barrier dysfunction, elicit marked production of pro-inflammatory and pro-atopy cytokines and activate eosinophils by the urokinase plasminogen activator pathway. Similarly, rare homozygous mutations of SPINK5 as well as spink5 experimental gene deletion in mice, are sufficient to elicit loss of epithelial barrier integrity, and pro-inflammatory and pro-atopy responses including atopic dermatitis and EoE in vivo. Yet, there is little data about how these SPINKs are regulated under basal conditions, how it becomes a surveillance constituent of the squamous epithelium, and the mechanism of acquired SPINK7 loss that occurs in allergic inflammation.
[0081] Epithelial cells use sensors to monitor the external and internal environment. For example, pattern recognition receptors including Toll-like receptors (TLRs) and protease- activated receptors (PARs) in the epidermis sense pathogenic insults. Once the sensor received an input (i.e., recognition of molecules from pathogens, microbiota dysbiosis, proteolytic activity), a cellular decision is made accordingly. Distinct signals will promote diverse cellular responses which result in sealing the barrier tightly against toxins or loosen the barrier to enable immune cells to infiltrate and fight pathogens. Despite the importance of investigating such sensor molecules, a master epithelial sensor in the esophagus has not yet been identified.
[0082] Herein, Applicant investigated the regulatory mechanism that controls SPINK7 expression. SPINK7 expression was elevated during epithelial cell differentiation induced by air liquid interface (ALI) culture and/or modification of calcium concentrations. Histone 3 acetylation marks suggested the importance of a putative binding motif for the C2H2 zinc finger transcription factor, Ovo Like Transcriptional Repressor 1 (OVOL1), an esophageal enriched gene. Indeed, overexpression and repression of OVOL1 regulated SPINK7 promoter activity. Mechanistically, a variety of AHR ligands, including proton-pump inhibitors, dietary compounds, metabolites produced by bacteria and particles found in the air, modulated OVOL1 activation, nuclear localization and subsequent SPINK7 expression. Furthermore, the type 2 cytokines IL-4 and IL-13 (inducers of allergic responses) repressed OVOL1 activation via an AHR-dependent mechanism. Additionally, the product of the chief EoE susceptibility locus (2p23), calpain-14, an intracellular regulatory protease induced by IL-13 in esophageal epithelial cells, post-transcriptionally modified OVOL1 levels. Translational studies demonstrated a marked lost expression of OVOL1 protein in esophageal biopsies of EoE patients compared to control patients. AHR may therefore serves as a sensor for environmental signals and has potential to rapidly control the esophageal epithelium fate by controlling SPINK7 levels via OVOL1 activation. The disclosed dataset highlights a potential role of the AHR/OVOL1/SPINK7 pathway in pathoetiology of EoE. As such, modulation of this pathway (e.g., supplementing AHR ligands) may be therapeutic in EoE and related allergic diseases.
[0083] Results
[0084] SPINK7 expression is induced during epithelial differentiation
[0085] Applicant analyzed SPINK7 expression in high confluent cultures of esophageal epithelial progenitor cell line (EPC2) cells, a condition that induce cellular differentiation. SPINK7 expression was induced under high confluency conditions compared to low confluency conditions (FIG 1A). SPINK7 expression was further increased in a high calcium (1.8 mM) media compared to a low calcium media (0.09 mM) (FIG 1A). Epigenetic analysis of the SPINK7 promoter region of EPC2 cells, revealed an increased in K27H3 acetylation mark in the 2-4 kb region from the SPINK7 transcription start site (TSS) in high confluence conditions compared to low confluence conditions (FIG IB). The highest K27 acetylation mark was observed in cells that were grown in high confluency and high calcium (1.8 mM; FIG IB).
[0086] Identification of the SPINK7 promoter region
[0087] Applicant cloned the 4.5 kb region upstream to the TSS of SPINK7 into a vector that contain nano-luciferase reporter (SPINK7). As a negative control, Applicant used a promoterless nano-luciferase vector (control). The 4.5 kb sequence was selected based on transcriptional and epigenetic data from FIG IB and from ENCODE and CisBP datasets (Yanez-Cuna, 2012 and Weirauch, 2014). EPC2 cells were grown at high density and in high calcium media to induce cell differentiation and then were transiently co-transfected with firefly luciferase vector (to control for transfection efficiency) and either SPINK7 or control vectors. The SPINK7-transfected cells had on average about 340-fold increase in the luminescence signal compared to cells transfected with the empty vector (p < 0.0001; FIG 1C). Indicating that this region of the SPINK7 gene has promoter activity under these conditions.
[0088] Applicant then investigated SPINK7 promoter activity in a range of calcium concentrations. Calcium ions increased SPINK7 promoter activity in a dose dependent manner and reached a plateau at 1.8mM of calcium (FIG ID). As expected, no difference was observed in the empty vector activity in different calcium concentrations (FIG 9A).
[0089] Applicant subsequently considered the minimal sequence required for promoter induction. Applicant tested various construct lengths (FIG IE). The promoter activity of all reporter constructs was sufficient to drive promoter activity including the shortest construct with the first 1 kb sequence from the 5’ TSS of SPINK7, which was sufficient to drive promoter activity in the high calcium condition compared to the empty vector (80-fold increase, p < 0.0002; FIG IE). The luciferase activity of the cells transfected with the 4.5 and 4 kb constructs increased by 3 and 2-fold compared to the luciferase activity of the cells that were transfected with the 1 kb construct respectively (p = 0.0004, p = 0.0001 respectively; FIG IE). The luciferase activity of the cells transfected with the 2 and 3 kb constructs were not significantly different compared to the luciferase activity of the cells that were transfected with the 1 kb construct (FIG IE). In a low calcium media, low promoter activity was observed in all the constructs with no difference between the 1 kb and the 4 or 4.5 kb (FIG 9B). In the 4.5 and 4 kb constructs the promoter activities were increased by 7 and 4-fold respectively in the high calcium media compared to the low calcium media (p = 0.05 and p = 0.0003 respectively; FIG IF). The 1, 2, and 3 kb constructs were not affected by high calcium (FIG IF). These collective data suggest that induction of cellular differentiation (i.e., high calcium and high confluency conditions) promotes SPINK7 promoter activity.
[0090] Candidate approach reveals that the transcription factor, OVOL1 regulates SPINK7 expression
[0091] Applicant asked which transcription factors (TFs) regulate SPINK7 promoter activity. Analysis of SPINK7 promoter using CisBP revealed 36 TFs that are predicted to bind to SPINK7 promoter (data not shown). Applicant intersected this list with a list of TFs that are induced during esophageal epithelial differentiation and with a list of esophageal specific genes. Additionally, Applicant analyzed which of these TFs are dysregulated in EoE patients compare to control patients. Based on these analyses, Applicant transiently overexpressed 4 TFs (i.e., OVOL1, VDR, PO2F3 and PRDM1) and assessed their effect on SPINK7 promoter activity compared to control cells that were transiently transfected with an empty vector. Overexpression of VDR (in the presence or absence of the VDR ligand calcipo triol), PO2F3 and PRDM1 did not affect SPINK7 promoter activity (FIG 2A). In contrast, overexpression of OVOE1 increased SPINK7 promoter activity by 2.4-fold (p = 0.0006; FIG 2A). Indeed, western blot analysis confirmed that OVOE1 protein was overexpressed in the OVOE1 transfected cells compared to control cells (FIG 2B). Because it had been shown that activation of AHR induces activation of OVOE1, Applicant stimulated the OVOE1 overexpressing cells with the AHR ligand, 6-formylindolo(3,2-b)carbazole (FICZ). FICZ induced a 5-fold increase in SPINK7 promoter activity in OVOE1 over expressing cells compared to control cells (p = 0.0003) and significantly increased the SPINK7 promoter activity compared to unstimulated OVOE1 overexpressing cells (2-fold increase, p = 0.04; FIG 2A).
[0092] OVOE1 binds to SPINK7 promoter
[0093] Applicant next examined whether SPINK7 is a direct OVOE1 target gene in esophageal epithelial cells. Applicant predicted 4 OVOE1 binding sites up upstream of the SPINK7 TSS (FIG 2C). Applicant tested the specificity of this response by subjecting the SPINK7 deletion constructs to FICZ activation in the presence or absent of OVOE1 overexpression (FIG 2C). Consisting with previous results, overexpression of OVOE1 induced promoter activity in cells that were transfected with the 4.5 kb construct of SPINK7. The promoter activity was further induced in the OVOE1 overexpressing cells after FICZ stimulation (FIG 2D). Cells that were transfected with the 4 kb construct had promoter activity that was comparable with the 4.5 kb construct. In contrast, the promoter activity of cells that were transfected with the 1, 2 or 3 kb of SPINK7 constructs were not markedly induced by OVOE1 overexpression or by OVOE1 overexpression and FICZ stimulation (FIG 2D). Applicant tested the specificity of this response by constructing OVOE1 site mutants and assessing SPINK7-nEUC promoter reporter construct activation (FIG 2E). SPINK7- nEUC activation was significantly decreased in the OVOE1 site mutant in -3379 bp when ACTGTTCCC sequence was replaced with CAGTGGAAA (FIG 2E). In contrast, OVOE1 site mutant in -4139 bp, when TGTTACA sequence was replaced with GTGGCAC, did not affect the SPINK7-nLUC activation (FIG 2E). These data suggests that the -3379 bp region from the SPINK7 TSS is important for OVOL1 binding. Using an Electrophoretic Mobility- Shift Assay (EMSA), Applicant examined OVOL1 capacity to bind to the SPINK7 gene promoter. Applicant analyzed the binding of recombinant human OVOL1 protein to a fluorescent DNA probe corresponds to the -4139, -3379, -2078, and -208 bp of the SPINK7 promoter. Unexpectedly, OVOL1 shifted the mobility of the fluorescent probe in all probes, except the -3379 probe (FIG 2F). Un-labelled (cold) wt competitors that contains the predicted binding site at 4139, -2078, and -208 bp inhibited the mobility shift (FIG 2F). A mutant cold competitor that contains the predicted binding site at -4139 bp (GTGGCAC) failed to inhibit the mobility shift (FIG 2F). In addition, rabbit administration of an antihuman OVOL1 antibody, resulted in a supershift of the -2078 probe. To examine the possibility that more cellular co-factors are required for the binding of OVOL1, Applicant generated nuclear extracts of HEK-293T cells overexpressing OVOL1. The nuclear extracts shifted the -3379 probe, while cold wt probe, dose dependently inhibited the shifted band (FIG 2G). A mutant cold competitor that contains the predicted binding site at -3379 bp (CAGTGGAAA) failed to inhibit the mobility shift (FIG 2G). These collective data suggest that OVOL1 binding to four sites in the SPINK7 promoter may be required for SPINK7 expression.
[0094] Loss of OVOL1 promotes impaired barrier function and cytokine production
[0095] Applicant then depleted OVOL1 expression in EPC2 cells by stably transduction with a vector expressing either shRNA targeting OVOL1 or non-silencing control (NSC) shRNA. OVOL1 silenced cells had > 2-fold decrease in OVOL1 mRNA expression compared to NSC-treated cells (FIG 3A). Consistent with the phenotype of SPINK7 depleted cells which were previously demonstrated by Applicant to produce increased levels of TSLP upon polyLC stimulation, OVOL1 silenced cells had increased TSLP release compared to NSC- treated cells after PolyLC stimulation, (FIG 3B). The increased TSLP production of OVOL1 silenced cells may be partially mediated by decreased expression of SPINK7. OVOL1 silenced cells that were differentiated in ALI culture system had 3-fold decrease in SPINK7 expression compared to differentiated NSC-treated cells (FIG 3C) and revealed barrier impairment as asses by Trans Epithelial Electrical Resistance (TEER; FIG 3D). These data suggest that OVOL1 expression is critical for maintaining SPINK7 expression, barrier integrity and controlling innate cytokine production by epithelial cells.
[0096] Applicant subsequently generated OVOL1 gene deleted cells using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genomic editing (FIG 10A,B). OVOL1 CRISPR/Cas9 deficient cells that were differentiated in ALI culture system had 10-fold decrease in SPINK7 expression compared to differentiated control EPC2 cells (p = 0.0004; FIG 3E) and revealed barrier impairment as asses by TEER (FIG 3F).
[0097] OVOL1 expression is lost in EoE
[0098] Analysis of OVOL1 mRNA expression did not reveal any significant difference between EoE biopsies and controls (FIG 4A). Analysis of OVOL1 protein revealed that OVOL1 is expressed in epithelial cells (FIG 4B). Applicant noted that OVOL1 intracellular localization was changed in different clusters of the epithelium. In basal and suprabasal layers of the epithelium, OVOL1 was localized to the cells’ nuclei while in the squamous epithelium, OVOL1 intracellular localization was transformed to cytoplasmic expression (FIG 4B). Analysis of OVOL1 expression in EoE biopsies revealed a marked decrease in the protein expression compared to control biopsies (FIG 4C). Western blot analysis of OVOL1 confirmed that OVOL1 is decreased by 10-fold in esophageal biopsies from EoE patients compared to control patients (FIG 4D). These collective data suggest that OVOL1 protein is deficient in the esophagus of EoE patients compared to control individual.
[0099] Environmental and internal cues promote SPINK7 expression in vitro
[00100] AHR is activated in response to a variety of ligands such as environmental toxicants including vehicle exhaust and cigarette smoke, dietary compounds (i.e., flavonoids, indole-3-carbinol derivatives, extracts from fruits, vegetables especially cruciferous), products from commensal bacteria (such as FICZ, kynurenine and Butyrate), tryptophan metabolism and drugs including proton pump inhibitors, which are interesting used to treat EoE. Applicant then asked which inducers of the AHR pathway might regulate SPINK7 expression via OVOL1. Applicant stimulated EPC2 cells with either FICZ, 2-(F H-indole-3'- carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), indole-3-carbinol (I3C), Omeprazole, Benzo [a] Pyrene (B[a]P), Urolithin A (UroA) and Quercitin. Omeprazole, B [a]P, Quercitin and ITE efficiently stimulated the expression of the AHR target gene CYP1A1 and SPINK7 expression (FIG 5A,B). Applicant next aimed to determine if SPINK7 up-regulation was AHR-dependent. To test this, cells were pre-treated with the AHR antagonist, N-(2-(3H-Indol-3-yl)ethyl)-9-isopropyl-2-(5-methyl-3-pyridyl)-7H-purin-6-amine (GNF-351).
[00101] Blocking AHR signaling by GNF351, blocked the SPINK7 upregulation induced by all ligands except ITE (FIG 5B). Next, Applicant asked if the AHR-ligands promote OVOL1 nuclear translocation. FICZ and ITE induced OVOL1 nuclear mobilization after 1 hr of stimulation, while 1 hr of omeprazole stimulation was not of sufficient to promote OVOL1 nuclear mobilization as assessed by immunostaining (FIG 5C). By 24 hours, omeprazole induced nuclear mobilization of OVOL1 (FIG 5C). Nuclear fractionation of the cells revealed that OVOL1 was predominantly localized in the cytoplasmic fraction. After FICZ stimulation, OVOL1 was redistributed between the cytoplasmic fraction and the nuclear fraction (FIG 5D). Applicant then performed transcriptomic analysis of FICZ- stimulated cells. This analysis revealed 842 dysregulated genes (FIG 5E). The up-regulated genes were enriched for functional pathways involved in epithelial differentiation such as IVL (encoding for the barrier gene and differentiation marker, involucrin), late cornified envelope protein family including LCE3E and LCE3D, and small proline rich proteins including SPRR2G, SPRR1A, SPRR2A (FIG 5F). FICZ-up regulated genes were also enriched for inflammatory responses, cell proliferation, membrane assembly, response to lipids and cell motility (FIG 5F). More than 18% of the FICZ dysregulated genes, overlapped with the EoE transcriptome (156 genes out of 842 FICZ-regulated genes; FIG 11A). Most genes that were up-regulated in the EoE transcriptome were down-regulated by FICZ and vice versa (FIG 11 A). The overlapped genes were enriched for cornified envelope proteins (FIG 11B). This suggests that loss of epithelial differentiation as observed in EoE biopsies, can be partially reversed by AHR activation.
[00102] IL-13 and IL-4 regulate OVOL1 activation
[00103] Applicant aimed to determine the effect of IL-4 and IL- 13 (which are Th2 cytokines with established roles in atopic diseases inducing EoE pathology (Blanchard, 2010, Leung, 2015, Hirano, 2020) on OVOL1 intracellular localization. In unstimulated cells that overexpress OVOL1, OVOL1 was primarily localized to cytoplasmic vesicles which were bordered by the membranal protein desmoglein 1 (DSG1) (FIG 6A). FICZ stimulation induced nuclear mobilization of OVOL1, while IL-4 or IL- 13 stimulation prevented the FICZ-induced OVOL1 mobilization to the nucleus in OVOL1 overexpressing cells (FIG 6A). IL-4 and IL- 13 significantly decreased the SPINK7 promoter activity in OVOL1 overexpressing cells that were stimulated with FICZ (FIG 6B,C). Because cells that were differentiated in the ALI culture system express high endogenous levels of OVOL1, Applicant analyzed the effect of IL-4 and IL-13 on differentiated cells. In unstimulated cells, OVOL1 was mostly nuclear and remained nuclear after FICZ stimulation (FIG 6D). IL-4 or IL- 13 stimulation promoted OVOL1 translocation from the nucleus to the cytoplasm (FIG 3F). FICZ stimulation increased the endogenous SPINK7 expression (FIG 6E) and IL- 13 decreased SPINK7 expression when the cells were stimulated with FICZ (FIG 6E). As a control for FICZ and IL-13 stimulations, CYP1A1 and CCL26 expression were analyzed respectively (FIG 6F,G). Interestingly, FICZ stimulation was able to decrease the IL-13- dependent CCL26 expression by 2.7-fold (p < 0.0001; FIG 6G). Transcriptomic analysis of FICZ, IL- 13 and FICZ+IL-13 treated cells compared with untreated cells revealed that the 4 groups of treatment were significantly different (FIG 6H). The majority of the IL- 13 dysregulated genes (55%) overlapped with the IL-13 + FICZ transcriptome (FIG 61). While only less than 10% of the IL- 13 + FICZ transcriptome overlapped with the IL- 13 transcriptome (FIG 61). Interestingly, the top overexpressed genes in the IL- 13 transcriptome were shared with the IL- 13 + FICZ transcriptome, however, the fold change of these genes after IL- 13 + FICZ treatment compared to untreated cells was attenuated, compared with IL- 13 treatment alone including key genes such as CAPN14 (encoding for the cysteine protease, calpian-14) and CCL26 (encoding for the eosinophil chemoattractant, eotaxin-3) (FIG 6J). These results suggest that FICZ is able to attenuate some of the genes that are altered by IL- 13.
[00104] IL-13-induced CAPN14 expression depletes OVOL1 protein expression
[00105] Because IL- 13 is a major driver of epithelial transcriptional changes in EoE,
Applicant asked if IL- 13 can regulate OVOL1 expression. IL- 13 stimulation in differentiated esophageal epithelial cells decreased OVOL1 protein expression by 10-fold (P = 0.02; FIG 7A). As a positive control, DSG1 protein expression was decreased by ~ 20% (P = 0.01; FIG 7A). In contrast, OVOL1 mRNA expression was comparable between IL-13 stimulation and control cells (FIG 7B). Consistent with the protein level, DSG1 mRNA was decreased after IL- 13 stimulation compared with untreated cells (FIG 7B). Suggesting that nuclear OVOL1 is a stable protein, while IL- 13 mediated nuclear transport inhibition promotes loss of cytoplasmic OVOL1 protein expression. Therefore, IL-13 may stimulate cytoplasmic retention of OVOL1 and loss of OVOL1 in EoE patients compared with controls (FIG 4 C,D). The EoE transcriptome is enriched with proteases and shows dysregulation between proteases and protease inhibitors (Azouz, 2018, Rochman, 2018 Rochman, 2017) Therefore, Applicant hypothesized that OVOL1 may be post-transcriptionally modified by proteases in the esophagus of EoE patients. Applicant then asked which genes are co-expressed with OVOL1 in the same esophageal clusters. Single cells RNAseq analysis revealed that CAPN14 and OVOL1 are co-expressed in the same cellular cluster in the esophagus (FIG 7C). The epithelial clusters that co-expressed OVOL1 and CAPN14 were enriched with SPINK7 and correspond to differentiated epithelial cells that express differentiation markers (i.e., FLG, MUC22) and esophageal enriched genes (i.e., MUC22, MAL, KLK13; FIG 7C). The expression of CAPN14 and OVOL1 was highly correlated with (r = 0.92) according to spearman correlation test. These findings prompted us to hypothesize that calpain-14 may be involved in OVOL1 post-transcriptional regulation. Notably, calpain-14 is an esophageal epithelial specific cysteine protease that is overexpressed in esophageal biopsies from EoE patients compared to controls and is regulated by IL-13 {Davis, 2016 #121;Kottyan, 2014 #123}. Inducible CAPN14 expression in differentiated esophageal epithelial cells revealed a marked reduction in OVOL1 protein expression (FIG 7D). OVOL1 mRNA expression was not affected by CAPN14 expression and was induced by < 2-fold by AHR ligands (P = 0.009-0.002; FIG 7E). Induction of CAPN14 expression decreased SPINK7 expression by 3.5-fold (P = 0.03; FIG 7F). The expression of SPINK7 was decreased by 3.5-fold to 12-fold after CAPN14 induction even after administration of AHR ligands (P = 0.03-0.002; FIG 7F). In addition, constitutive expression of CAPN14, by CAPN14-GFP vector transduction, decreased the expression of OVOL1 compared with control GFP vector transduction (FIG 7G), indicating that the reduction in OVOL1 protein expression resulted from CAPN14 expression and not as a result of dox treatment. Consistently, IL- 13 treatment decreased OVOL1 expression in the CAPN14-GFP overexpressing cells (FIG 7G). Therefore, IL-13 stimulation and CAPN14 expression decrease OVOL1 protein expression. Applicant then hypothesized that nuclear mobilization of OVOL1 by AHR protects OVOL1, while IL-13- mediated cytoplasmic retention of OVOL1, promotes degradation of OVOL1 by cytoplasmic proteases such as calpin-14. To test this hypothesis, Applicant performed a biochemical fractionation of cellular proteins that differentiated the nuclear proteins from the rest of the cellular proteins which contain the cytosol and other organelles (here refer to cytosol). Applicant then incubated recombinant OVOL1 protein with cytosolic or nuclear proteins. Incubation of OVOL1 with cytoplasmic proteins for 30 minutes resulted in complete loss of OVOL1 protein, indicated by a marked decrease in OVOL1 band intensity (FIG 7H). In contrast, OVOL1 band was still notable after incubation of OVOL1 with nuclear proteins for 30 minutes (FIG 7H). These results indicate that OVOL1 is degraded outside of the nucleus and remains stable in the nucleus.
[00106] Ex vivo activation of AHR
[00107] Applicant then investigated if AHR ligands can stimulate esophageal SPINK7 expression ex vivo in a murine model. Esophagi were collected from C57BL/6 mice and were stimulated with AHR ligands. Six hrs of stimulation, were sufficient for detection of CYP1A1 induction, indicating that AHR pathway was activated (FIG 8A). At the same time, SPINK7 mRNA was induced by 10-fold (FIG 8B).
[00108] Discussion
[00109] The data presented here identify a complexed regulatory network that controls SPINK7 expression in esophageal epithelial cells. Applicant demonstrated that AHR regulates the expression of SPINK7 via OVOL1 and that IL-4 and IL- 13 inhibit this pathway by inhibiting the OVOL1 nuclear mobilization (FIG 12). The cysteine protease calpain-14 inhibits SPINK7 expression by post-translational modifications of OVOL1. Therefore, IL-13 can potentially inhibit SPINK7 expression by 2 mechanisms; first, by inhibiting OVOL1 nuclear translocation which prevents OVOL1 from binding to its target genes and decreasing OVOL1 stability, and second, by inducing calpain-14 expression, which in turn degrades OVOL1 and prevents SPINK7 expression.
[00110] Importantly, IL- 13 and calpain-14 are overexpressed in the esophagus of EoE patients compared with control individuals and were suggested to be major drivers in EoE pathogenesis; IL- 13 induces epithelial cell transcriptional changes that overlap with the EoE transcriptome (the list of genes that are altered in EoE esophageal biopsies compared to control biopsies). Its importance in disease pathogenesis is implicated by the positive effects of anti-IL-13 treatment (QAX576 and RPC4046) in EoE. Calpain-14 is an esophageal- specific protease, encoded by the CAPN14 gene which is located in the strongest associated EoE risk locus (i.e., 2p23). Calpain-14 is up regulated by exposure of esophageal epithelial cells to IL- 13 and has been shown to regulate epithelial barrier homeostasis and repair. Therefore, Applicant’ s findings reveal an altered molecular pathway which is relevant in disease state. OVOL1 protein expression was lost in esophageal biopsies from EoE patients compared to controls. However, OVOL1 mRNA expression levels were comparable between EoE patients and controls. Similar observations were detected in vitro after stimulation of esophageal epithelial cells with IL- 13 or overexpression of CAPN14. This data set prompted Applicant to speculate that the loss of OVOL1 in EoE may be dependent upon IL- 13 and calpain-14 induction. Whether IL-13 and calpain-14 directly regulate OVOL1, may be further investigated.
[00111] A recent publication by Patel et al revealed that SPINK7 expression is induced by DNA damage and mediated by p53 (Patel, 2020). Herein, Applicant revealed that the transcription factor OVOL1 controls SPINK7 expression. OVOL1 is an enriched esophageal transcription factor that is induced during esophageal epithelial differentiation (Uhlen, 2015, Tsuji, 2017). OVOL1 regulates expression of barrier genes such as FLG and LOR in the skin (Tsuji, 2018). In addition, variants in OVOL1 gene associate with atopic dermatitis, a type 2 allergic disease that is characterized by barrier impairment of the skin (Marenholz, 2015, Hirota, 2012, Paternoster, 2011). Applicant’s data demonstrates that OVOL1 depletion decreases SPINK7 expression and promotes impaired esophageal barrier function and cytokine production in vitro. Therefore, it is suggested that OVOL1 has a key role in regulating esophageal homeostasis and immune tolerance.
[00112] Allergic diseases emerge when immune tolerance towards an antigen breaks and a danger signal is produced instead of a tolerogenic signal. Breakdown of immunologic tolerance is influenced by diverse factors such as diet, infections, exposure to antibiotics and chemicals, microbiome composition and stress, collectively term exposome (the sum of external factors that an individual is exposed to throughout their lifetime). In addition, genetic and epigenetic elements have the potential to influence the immunologic tolerance. The exposomal sensors of the esophagus, (which is an organ that most directly interacts with and adapts to the external environment) are yet undefined. Herein, Applicant asked which molecules receive these environmental cues and translate these cues into cellular responses. Applicant identified AHR as an esophageal epithelial sensor that promotes the activation of OVOL1 which then induce transcription program of differentiation and barrier genes including SPINK7 and FLG. AHR is activated in response to many ligands such as vehicle exhaust and cigarette smoke, dietary compounds (i.e. flavonoids, indole-3 -carbinol derivatives, extracts from fruits and vegetables especially cruciferous), products from commensal bacteria and tryptophan metabolism (Moura-Alves, 2014, Rothhammer, 2019). In addition, AHR is capable of initiating distinct signaling pathways in response to different ligands (Quintana, 2008). In this way, AHR senses host/microbiome dysbiosis, dietary compounds, drugs and environmental toxicant and initiates an appropriate cellular response.
[00113] AHR is a member of the basic helix-loop-helix per-Amt-sim (bHLH/PAS) protein family. AHR is trapped in a cytosolic multiprotein complex consisting of heat shock protein 90, tyrosine kinase c-src, and other co-chaperones. AHR translocates from the cytoplasm into the nucleus upon ligand binding and dimerizes with AHR nuclear translocator (ARNT). The AHR /AHR ligand/ ARNT complex recognizes promoters containing specific enhancer sequences termed xenobiotic responsive elements (XRE) and then activates the transcription of target genes such as phase I and phase II detoxification enzymes (cytochrome P450 (CYP1A1)) (Rothhammer, 2019). Applicant’s data suggests that the AHR pathway is dysregulated in the esophagus of EoE patients compared to control patients. Although AHR levels are increased in the esophagus of EoE patients compared to control patients, phase II enzymes were markedly decreased (FIG 13). These data suggest alterations in the AHR pathway in EoE patients that may be stem from environmental exposures and can have downstream implications such as alterations in OVOL1 and its transcription program.
[00114] Interestingly, it was found that Omeprazole, an FDA approved proton pump inhibitor (PPI) used for acid-induced esophageal eosinophilia, can induce SPINK7 expression by promoting nuclear translocation of OVOL1. Recently, it has been shown that in 10-50% of EoE patients, PPI mono-therapy can effectively reverse the histological and clinical features of the disease. It has been suggested that the positive effects of PPI on EoE stem from blockade of acid by PPI and the anti-inflammatory effect of PPI, such as inhibition of eotaxin-3 and STAT6. Rochman et al reported that PPI promote diverse epithelial changes through AHR (Rochman, 2020). The findings that Omeprazole promotes SPINK7 expression makes it tempting to speculate that part of the positive effects of Omeprazole on EoE patients may be because of its effect on SPINK7 expression which promotes barrier function and protects the esophageal homeostasis at least in part. Applicant’ s data also showed that another AHR ligand, FICZ, partially inhibits IL- 13 dependent eotaxin-3 (CCL26) expression. It is therefore interesting to investigate the possibility that PPI drugs inhibit eotaxin-3 and promoting SPINK7 expression in an AHR-dependent manner.
[00115] In conclusion, Applicant’ s data reveals a cross talk between the AHR/OVOL1/SPINK7 pathway, the type 2 cytokines, IL-4 and IL-13 and calpain-14. Given these collective observations, Applicant propose that local fluctuations in SPINK7, that curtails inflammatory responses in the squamous epithelium, particularly in the esophagus, can be controlled by environmental cues that are converged by AHR. Applicant have demonstrated that esophageal SPINK7 expression can be modulated by food supplements that induce AHR signaling. Therefore, Applicant propose that modulation of SPINK7 expression by AHR ligand supplement may be useful for a new avenue to reconsider AHR as a pharmacological target for mechanism-based drugs for food allergic diseases.
[00116] Materials and methods
[00117] Selection of promoter sequence
[00118] The 4.5kb region was chosen based on bioinformatics analysis of transcriptional and epigenetic data from the following databases: ENCODE (Encyclopedia of DNA Elements), CIS-BP (Catalog of Inferred Sequence Binding Preferences) and BioWardrobe (corresponds to ENCODE hg 19-2009; Cincinnati Children's Epigenetic Database). Cross analysis of these databases has shown that the 4.5kb region consists of highly-conserved sites enriched with histone acetylation marks (H3K27ac) and overlapped with DNase clusters. The BioWardrobe database (internal unpublished data) has shown that a region of 1.8kb is enriched with H3K27ac marks at 2kb upstream of the transcription start site (TSS). The 4.5kb non-coding putative promoter sequence (without UTR) was obtained from the ENCODE UCSC Genome Browser of the Human genome 2013 database (hg38_dna range) and the coordinates are chromosome 5:148307922-148312422.
[00119] Plasmid constructions [00120] Promoter constructs were created by cloning the immediate 4.5kb region adjacent to the 5’ TSS of SPINK7 into the promoter-less Nano-luciferase reporter vector PNL1.1-NL (Promega). The 4.5kb sequence and subsequent constructs were created by using primers with the restriction enzyme sites KpnI-HF and Xhol. Applicant utilized SnapGene software that employed In-Fusion cloning techniques. Cloning was performed with In-Fusion HD methods (Clonteck, Takara Bio Company). PNL1.1-NL is defined as the empty vector (EV). Post-cloning with the sequence of interest is termed as SPINK7 [4.5kb]. The full-length SPINK7 consists of 4.5kb and short lengths were defined as SPINK7 lkb-3kb from TSS.
[00121] Transient transfection and luciferase activity assay
[00122] Human esophageal epithelial progenitor cells (EPC2) are immortalized cell lines that were donations from the Dr. Anil Rustgi Lab (University of Pennsylvania, Philadelphia, PA). EPC2 cells were cultured in Keratinocyte serum-free medium (KSFM). The KSFM medium was supplemented with epidermal growth factor (EGF, 1 ng/mL), bovine pituitary extract (BPE 50 mg/mL), and IX penicillin/streptomycin (Invitrogen). EPC2 cells were grown for 3-4 days until they reached 80-90% confluent. Cells were then harvested by addition of trypsin/EDTA (Invitrogen) and incubated for 3-5 min, at 37°C. Next, soybean trypsin inhibitor (250 mg/L in IX DPBS) was added, and cells were pelleted at 300G/5min. EPC2 cells were seeded on day-0 at 200k/48well plate in two conditions: low-calcium (KSFM medium alone -CaC120.09 mM) and high calcium (KSFM+CaC12 1.8 mM). 24 hours later (day-1) cells were transiently transfected at >95% density with Opti-MEM (ThermoFisher) and Mirus TransIT-2020 (Mirusbio, Madison, WI) according to the manufacturer instructions. Applicant used 3ul of TransIT-2020 per 1000 ng of construct DNA and 50 ng Firefly DNA (1:20 dilution). Cells were co-transfected with 1000 ng PNL1.1 containing the SPINK7 promoter sequence, or promoter-less empty vector (EV) PNL1.1-NL with 50 ng Firefly luciferase pGL3 vector (Promega). Firefly, a PGL3 vector (Promega), was employed as an internal control for transfection. On day 2 cells were incubated at 37C and the medium was changed with the appropriate calcium conditions. On day 3 (48hrs posttransfection) Nano and Firefly luciferase activities were measured in relative light units (RLU) by luciferase assay NanoDLR kit (Promega) via Synergy Mx Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT). In order to control for transfection efficiency, Nano-Luciferase activity was normalized to Firefly-Luciferase, then, the activity was normalized to control promoterless transfected cells for each sample transfection variance per well. All assays were conducted in triplicates.
[00123] Endogenous SPINK7 expression
[00124] To determine endogenous SPINK7 expression, EPC2 cells were plated in a high density (250,000 cells/well in a 48 well plate) in KSFM media with 1.8 mM CaC12. For low density, 250,000 cells/well were grown in a 6 well plate in KSFM media with 1.8 mM CaC12. RNA was isolated with Quick-RNA Micro-prep (Zymo; Irvine, CA). ProtoScript First Strand cDNA Synthesis kit (NEB; Ipswich, MA) was employed according to the manufacturer instructions to obtain RT-PCR data.
[00125] For AEI differentiation, cells were grown as previously described (ref). Briefly, 150,000 cells/well were plated in a transwell system with 24 well plate. After 48 hrs, media was replaced to a high calcium media (1.8 mM CaC12). On day 8, media was aspirated from the top chambers, on day 12, cells were stimulated and on day 14 cells were harvested.
[00126] mRNA extraction, quantitative RT-PCR, and RNA sequencing analysis
[00127] Total RNA was isolated from cells with the RNeasy mini kit (Qiagen) according to the manufacturer’s protocol. For RNA sequencing experiments, RNA was treated with On-Column DNase Digestion kit (Qiagen) according to the supplied protocol. cDNA was synthesized with the ProtoScript synthesis kit (New England Biolabs). qPCR was performed using a 7900HT Fast Real-Time PCR system from Applied Biosystems (Fife Technologies) with FastStart Universal SYBR Green Master mix (Roche Diagnostics Corporation) by using primer sets. Next-generation RNA sequencing was performed by the CCHMC Genetic Variation and Gene Discovery Core Facility using Illumina TruSeq kits and sequenced on the Illumina HiSeq2000. For RNA sequencing analysis, Fastq files from the Illumina pipeline were aligned by BioWardrobe (42). Gene ontology enrichment analysis, which uses statistical methods to determine functional pathways and cellular processes associated with a given set of genes, was performed with the ToppGene suite (43).
[00128] OVOE1 gene silencing by shRNA
[00129] Lentiviral shRNA vectors against OVOE1 (MISSION shRNA, Sigma- Aldrich, clone NM_004561, TRCN00000257410, TRCN0000229665, and TRCN0000229664) and a control vector that targets no known mammalian genes (SHC002 SIGMA MISSION® pLKO.l-puro Non-Mammalian shRNA Control) were used. EPC2 cells grown in KSFM media were transduced. Twenty-four hours after transduction, cells were selected for stable integration using puromycin (1 pg/mL). After ALI differentiation, gene silencing efficiency of target vectors in transduced cells was assessed by quantitative PCR relative to that of cells transduced with NSC shRNA.
[00130] Generation of CRISPR/Cas9 knocked out EPC2 cells
[00131] A guide RNA (gRNA) complementary to OVOL1 and CAPN14 open reading frame sequence and located directly 5’ of a protospacer adjacent motif (PAM) was identified (http://tools.genome-engineering.org; (Ran, 2013), and oligonucleotides were annealed and ligated into the BbsI restriction site of plasmid pX459M2 (obtained from CCHMC Transgenic Mouse and Gene Editing Core Facility) to produce pX459M2-SPINK7gl. EPC2 cells were transfected with pX459M2 or pX459M2-SPINK7gl using Viromer (Origen) according to the manufacturer’s protocol. Transfected cells were selected, grown and sequenced as previously described (Azouz, 2018). OVOL1 and calpain-14 protein expression were determined by rabbit anti human OVOL1 antibody and rabbit anti human calpain-14 antibodies (Sigma Aldrich).
[00132] Immunofluorescence
[00133] Nuclear and cytoplasmic extraction and Western blotting
[00134] Proteins from cell cultures were extracted with RIPA buffer (Pierce) with protease and phosphatase inhibitors. Loading buffer (Life Technologies) was added, and samples were heated to 95°C for 5 min, subjected to electrophoresis on 12% NuPAGE BisTris gels (Life Technologies), transferred to nitrocellulose membranes (Life Technologies), and visualized using the Odyssey CLx system (LI-COR Biosciences) with IRDye 800RD goat anti-rabbit (LLCOR Biosciences), and IRDye 680RD goat anti-mouse (LLCOR Biosciences) secondary antibodies. The primary antibodies were Rabbit anti-OVOLl (Sigma Aldrich) or Rabbit anti-OVOLl (LifeSpan Biosciences), mouse anti-HSP90 (Cell Signaling Technology Inc), mouse anti-desmoglein-1 (Sigma Aldrich) and anti-Histon H3 (Abeam). Blots were quantified using the Image Studio software (LLCOR Biosciences). [00135] Statistical Analysis
[00136] Raw data was measured in RLU and it is defined by the ratio of NanoLuciferase reporter activity (NL) to the Firefly (FF) activity [NL/FF]. Normalized data is defined by the ratio of raw data of the promoter activity [NL/FF] to the average of the EV activity [NL/FF]. Statistical analysis was completed with GraphPad PRISM. One-way and Two-way ANOVA and t-test were performed.
[00137] EXAMPLE 2. Quercetin diet induced SPINK7 expression in control mice (ED-L2) but not in mice deficient of AHR in esophageal epithelial
[00138] Procedure:
[00139] Mice were fed with control diet or Quercetin enriched diet for 30 days. (AIN- 930 with 300 ppm Quercetin, available from TestDiet, testdiet.com, containing 45.54% com starch, 15.5% maltodextrin, 14% casein, vitamin tested, 10% sucrose, 5% powdered cellulose, 4% soybean oil, 3.5% AIN 93M Mineral Mix, 1% AIN vitamin mix, 0.25% choline biartrate, 0.18% L-cysteine, and 0.03% quercetin; AIN-93M Mineral Mix contains Calcium Carbonate, Monopotassium Phosphate, Sucrose, Sodium Chloride, Potassium Sulfate, Potassium Citrate Monohydrate, Magnesium oxide, Ferric Citrate, Zinc Carbonate, Sodium, Metaslicate, Manganese Carbonate, Copper Carbonate, Chromium Potassium Sulfate, Boric Acid, Sodium Fluoride, Nickel Carbonate, Lithium Chloride, Sodium Selenate, Potassium Iodate, Ammonium Molybdate, and Ammonium Vanadate. AIN 93 Vitamin Mix contains Sucrose, DL- Alpha Tocopheryl Acetate (Form of Vitamin E), Phylliquinone (Form of Vitamin K), Nicotinic Acid, Calcium Pantothenate, Vitamin A Palmitate, Pyridoxine Hydrochloride, Thiamin, Hydrochloride, Riboflavin, Vitamin B-12 Supplement, Folic Acid, Cholecalciferol, Biotin.) Mice were harvested and the tongue, esophagus, liver and skin were collected. Then, RNA was extracted from the tissues and the expression of Cyplal (AHR target gene) was analyzed. In addition, protein lysates were generated to analyze CYP1A1 activity. As a positive control, esophageal explants were stimulated ex vivo with FICZ for 6 hrs. Quercetin diet induced SPINK7 expression in control mice (ED-L2) but not in mice deficient of AHR in esophageal epithelial.
[00140] All percentages and ratios are calculated by weight unless otherwise indicated. [00141] All percentages and ratios are calculated based on the total composition unless otherwise indicated.
[00142] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
[00143] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”
[00144] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUB MED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.
[00145] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

- 46 - CLAIMS What is claimed is:
1. A method of treating eosinophilic esophagitis (EoE) in an individual in need thereof, comprising administering a composition comprising an aryl hydrocarbon receptor (AHR) agonist to said individual.
2. The method of claim 1, wherein said AHR agonist is selected from one or more of quercetin, tapinarof, 6-formylindolo[3,2-b]carbazole (FICZ), 6,12- diformylindolo[3,2-b] carbazole (dFICZ), Benzo [a] Pyrene (B[a]P), TCDD, apigenin, and luteolin , 6,12-diformylindolo[3,2-b]carbazole, urolithin A (3,8-dihydroxy-6H- dibenzo[b,d]pyran-6-on), urolithin A03 (6H-benzo[c]chromene-3,8-diol), bilirubin, biliverdin, butyrate, indirubin, 2-(10-H-indole-3-carbonyl)thiazole-4-carboxylic acid methyl ester (“ITE”), L-kynurenine, indole-3-carbinol (I3C), cinnabarinic acid, kynurenin, kynurenin acid, xanthrenic acid, 3,3’-diindoylmethane, indole-3- acetonitrile, indole[3,2]carbazole, Indole-3-acetaldehyde, LTr-1, indoxyl sulfate, indole-3 -acetic acid, flavanoid, flavanol, isoflovanones, carotinoid, indole, 3 -methyl indole (Skatole), 2-oxindole, Tryptamine, Indirubin, Indigo, 3-hydroxyl-indole, trypanthrin, malassezin, diosmin, tangeritin, tamarixetin, luteolin, myricetin, canthaxanthin, tryptanthrin, DIM (3,3'diindolylmethane, diindolylmethane), 6- formylindolo[3,2-b]carbazole, IAA indole-3 -acetic acid, lAld, indole-3-aldehyde, lAAld, indole[3,2-b]carbazole; I3S, indoxyl- 3 -sulfate, 2-(l’H-indole-3’ -carbonyl)- thiazole-4-carboxylic acid, I3AC, 2-(indol-3-ylmethyl)-3,3'diindolylmethane, indolo[3,2-b]carbazole (ICZ), sulindac, leflunomide, lipoxin A4 (LXA4), alprostadil, nimodipine, leflunomide, flutamide, omeprazole, mexiletine, atorvastatin , esomeprazole, berberine, sinomenine, resveratrol, VAF347, tetrandrine, tryptophan metabolites, derivatives thereof, and analogues thereof.
3. The method of claim 1 or 2, wherein said AHR agonist is quercetin.
4. The method of claim 1 or 2, wherein said AHR agonist is tapinarof.
5. The method of claim 1 or 2, wherein said AHR agonist is 6-Formylindolo[3,2- b]carbazole (FICZ).
6. The method of any preceding claim wherein said AHR agonist is administered in a dose effective to reduce inflammation of one or more of the esophagus, the stomach, and the small intestine. - 47 - The method of any preceding claim wherein said AHR ligand is administered in an amount of from about 50 microgram/kilogram to about 50,000 microgram/kilogram, or from about 100 microgram/kilogram to about 25,000 microgram/kilogram, or from about 200 microgram/kilogram to about 20,000 microgram/kilogram, or from about 300 microgram/kilogram to about 15,000 microgram/kilogram, or from about 400 microgram/kilogram to about 10,000 microgram/kilogram, or from about 500 microgram/kilogram to about 7,500 microgram/kilogram, or from about 1000 to about 5000 microgram/kilogram. The method of any preceding claim wherein said composition is a unit dose comprising from about 1 mg to about 500 mg, or from about 2 mg to about 250 mg, or from about 3 mg to about 200 mg, or from about 5 mg to about 150 mg, or from about 6 mg to about 100 mg, or from about 7 mg to about 75 mg, or from about 8 mg to about 70 mg, or about 8 mg to about 65 mg, or from about 9 mg to about 60 mg, or from about 10 mg to about 50 mg of an AHR agonist. The method of any preceding claim wherein said AHR agonist is administered in a food product, wherein said AHR agonist is present in said food product in an amount of from about 100 mg/kg of food product to about lOg/kg of food product. The method of any preceding claim wherein said AHR agonist is administered as a product of a bacteria, wherein said AHR agonist is produced by said bacteria. The method of claim 10 wherein said bacteria is selected from one or more of L. reuteri, Lactobacillus murinus, Lactobacillus taiwanensis, Bacillus alvei, Clostridium novyi, Clostridium limosum, Clostridium tetani, Corynebacterium acnes, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides sp., Citrobacter sp., E. coli, Flavobacterium sp., Fusobacterium sp., Haemophilus influenza, Kleibsella planticola, Shigella flexneri, Vibrio cholera, Kleibsella pneumonia, Malassezia, Propionibacterium freudenreichii ET-3, Mycobacteria, Lactobacillus reuteri, Allobaculum, Ppeptostrptococcus, and Providencia stuartii. The method of claim 10 or claim 11 wherein said bacteria is genetically modified bacteria, wherein said genetically modified bacteria have increased production of an AHR agonist caused by said genetic modification. The method of any preceding claim wherein said administration is oral administration. - 48 - The method of claim any preceding claim wherein said administration is topical administration to one or more of the esophagus, stomach, and small intestine of said individual. The method of any preceding claim wherein said composition is in a form selected from a liquid, an emulsion, a solution, a suspension, a syrup, a slurry, a dispersion, a colloid, a dissolving tablet, a dissolving wafer, a capsule, a gel capsule, a semi-solid, a solid forma gel, a gel matrix a cream, a paste. The method of any preceding claim wherein said composition comprises a viscosityincreasing excipient. The method of any preceding claim wherein said composition comprises a viscosity increasing excipient selected from one or more of lactose, sucrose, sucralose (Splenda®), mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC), and polyvinylpyrrolidone (PVP: povidone). The method of any preceding claim wherein said composition further comprises a mucoadhesive agent. The method of any preceding claim wherein said composition comprises a mucoadhesive agent is selected from carbopol, alginate, maltodextrin, or a combination thereof The method of any preceding claim, said composition comprising a cytokine inhibitor. The method of any preceding claim, said composition comprising one or more cytokine inhibitor selected from benralizumab, mepolizumab, reslizumab, dectrekumab, monoclonal antibody cendakimab (RPC4046), dupilumab, lirentelumab, itepekimab, and tezepelumab. The method of any preceding claim, said composition comprising a corticosteroid. The method of any preceding claim, said composition comprising a corticosteroid selected from one or more of budesonide, hydrocortisone, hydroxyltriamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethsone dipropionate, clobetasol valemate, ciclesonide, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, fluticasone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortmate, mepreddisone, paramethasone, prednisolone, prednisone, mometasone, beclomethasone dipropionate, triamcinolone, analogs thereof, derivatives thereof, salts thereof, ions and complexes thereof, and combinations thereof. The method of any preceding claim, said composition comprising budesonide. The method of any preceding claim, said composition being a unit dose comprising from about 0.05 mg to about 50 mg corticosteroid. The method of any preceding claim wherein said individual is less than 18 years of age, or less than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year of age. A composition comprising an AHR agonist and an excipient, said excipient comprising a viscosity increasing excipient. The composition of claim 27 wherein said composition comprises a therapeutically effective amount of an AHR agonist to prevent or alleviate esophageal inflammation in an individual having EoE. The composition of claim 27 or 28, said composition provided in a unit dose containing a therapeutically effective amount of an AHR agonist to prevent or alleviate esophageal inflammation in an individual having EoE. The composition of any of claims 27 to 29, said viscosity increasing excipient being one or more of lactose, sucrose, sucralose (Splenda®), mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC), and polyvinylpyrrolidone (PVP: povidone), acacia, agar, bentonite, carbomers, carboxymethylcellulose calcium, ceratonia, cetostearyl alcohol, colloidal silicon dioxide, cyclomethicone, glyceryl behenate, guar gum, hectorite, hydrogenated vegetable oil type I, hydroxypropyl starch, hydroxypropylmethylcellulose, hydroxyethylcellulose, magnesium aluminum silicate, maltodextrin, polycarbophil, poly dextrose, poly(methylvinyl ether/maleic anhydride), polyvinyl acetate phthalate, potassium chloride, propylene glycol alginate, saponite, sodium chloride, stearyl alcohol, sulfobutylether P-cyclodextrin, tragacanth, and mixtures thereof. The composition of any of claims 27 through 30, said composition comprising a corticosteroid. The composition of any of claims 27 through 31, said composition comprising a cytokine inhibitor. The composition of any of claims 27 through 31, wherein said AHR agonist is provided by a bacteria selected from one or more of L. reuteri, Lactobacillus murinus, Lactobacillus taiwanensis, Bacillus alvei, Clostridium novyi, Clostridium limosum, Clostridium tetani, Corynebacterium acnes, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides sp., Citrobacter sp., E. coli, Flavobacterium sp., Fusobacterium sp., Haemophilus influenza, Kleibsella planticola, Shigella flexneri, Vibrio cholera, Kleibsella pneumonia, Malassezia, Propionibacterium freudenreichii ET-3, Mycobacteria, Lactobacillus reuteri, Allobaculum, Ppeptostrptococcus, and Providencia stuartii. A food additive comprising an AHR agonist and a food-safe carrier. The food additive of claim 34, said AHR agonist being selected from one or more of quercetin, tapinarof, 6-formylindolo[3,2-b]carbazole (FICZ), 6,12- diformylindolo[3,2-b] carbazole (dFICZ), B[a]P, TCDD, apigenin, and luteolin , 6,12- diformylindolo[3,2-b]carbazole, urolithin A (3,8-dihydroxy-6H-dibenzo[b,d]pyran-6- on), urolithin A03 (6H-benzo[c]chromene-3,8-diol), bilirubin, biliverdin, butyrate, indirubin, 2-(10-H-indole-3-carbonyl)thiazole-4-carboxylic acid methyl ester (“ITE”), L-kynurenine, derived from tryptophan metabolism, indole-3-carbinol (I3C), cinnabarinic acid, kynurenin, kynurenin acid, xanthrenic acid, 3,3’-diindoylmethane, indole-3 -acetonitrile, indole[3,2]carbazole, Indole-3 -acetaldehyde, LTr-1, indoxyl sulfate, indole-3-acetic acid, flavanoid, flavanol, isoflovanones, carotinoid, indole, 3- methyl indole (Skatole), 2-oxindole, Tryptamine, Indirubin, Indigo, 3-hydroxyl- indole, trypanthrin, malassezin, diosmin, tangeritin, tamarixetin, luteolin, myricetin, canthaxanthin, tryptanthrin, DIM (3,3'diindolylmethane, diindolylmethane), 6- formylindolo[3,2-b]carbazole, IAA indole-3 -acetic acid, lAld, indole-3-aldehyde, lAAld, indole[3,2-b]carbazole; I3S, indoxyl- 3 -sulfate, 2-(l’H-indole-3’ -carbonyl)- thiazole-4-carboxylic acid, 13 AC, 2-(indol-3-ylmethyl)-3,3'diindolylmethane, indolo[3,2-b]carbazole (ICZ), sulindac, leflunomide, lipoxin A4 (LXA4), alprostadil, nimodipine, leflunomide, flutamide, omeprazole, mexiletine, atorvastatin , esomeprazole, berberine, sinomenine, resveratrol, VAF347, tetrandrine, and tryptophan metabolites. The food additive of claim 34, wherein said AHR agonist is quercetin. The food additive of claim 34, wherein said AHR agonist is tapinarof. The food additive of claim 34, wherein said AHR agonist is 6-Formylindolo[3,2- b]carbazole (FICZ). The food additive of any of claims 34 to 38, wherein said AHR agonist is present in an amount of from about 100 mg/kg of food additive to about lOg/kg of food additive. The food additive of any of claims 34 to 39, wherein said AHR agonist is provided by an AHR agonist-producing bacteria selected from one or more of L. reuteri, Lactobacillus murinus, Lactobacillus taiwanensis, Bacillus alvei, Clostridium novyi, Clostridium limosum, Clostridium tetani, Corynebacterium acnes, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides sp., Citrobacter sp., E. coli, Flavobacterium sp., Fusobacterium sp., Haemophilus influenza, Kleibsella planticola, Shigella flexneri, Vibrio cholera, Kleibsella pneumonia, Malassezia, Propionibacterium freudenreichii ET-3, Mycobacteria, Lactobacillus reuteri, Allobaculum, Ppeptostrptococcus, and Providencia stuartii. A quercetin enriched food or food additive comprising at least 150 mg, or at least 200 mg, or at least 300 mg, or at least 400 mg, or at least 500 mg, or at least 600 mg, or at least 700 mg, or at least 800 mg, or at least 900 mg, or at least 1 g, or at least 5 g, or at least 10 g quercetin per 100 g food product or food additive. The food additive of any of claims 34 to 41 comprising a flavoring agent The food additive of any of claims 34 to 42 comprising one or more flavoring agents selected from vanilla, cocoa, vanillin, salt, coffee, chocolate, berry flavors, and fruit flavors, acids, caramel, mint, natural and/or artificial sweeteners, sodium sources such as sodium chloride, hydrocolloids, and combinations thereof The food additive of any of claims 34 to 43 comprising a masking agent - 52 - The food additive of any of claims 34 to 43 comprising one or more masking agents selected from natural and artificial sweeteners; sodium sources such as sodium chloride; hydrocolloids such as guar gum, xanthan gum, carrageenan, gellan gum, other suitable gums; emulsifiers; encapsulating agents such as starches and modified starch products; and combinations thereof. A composition according to any preceding claim for use in a method of treating eosinophilic esophagitis (EoE) in an individual in need thereof.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114903941A (en) * 2022-04-24 2022-08-16 河南中医药大学 Dried orange peel alcohol extract and application thereof in treating eosinophilic esophagitis

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140343051A1 (en) * 2013-05-17 2014-11-20 Rsem Limited Partnership Methods to modulate acute myeloid leukemia stem/progenitor cell expansion and/or differentiation
US20170067065A1 (en) * 2014-12-22 2017-03-09 Synlogic, Inc. Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier
US20190046444A1 (en) * 2015-09-14 2019-02-14 Vgsk Techologies, Inc. A sterically stabilized carrier for subcutaneous, sublingual, and oral therapeutics, compositions and methods for treating a mammal
US20200338073A1 (en) * 2019-04-24 2020-10-29 Theravance Biopharma R&D Ip, Llc Pyrimidine jak inhibitors for the treatment of skin diseases

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140343051A1 (en) * 2013-05-17 2014-11-20 Rsem Limited Partnership Methods to modulate acute myeloid leukemia stem/progenitor cell expansion and/or differentiation
US20170067065A1 (en) * 2014-12-22 2017-03-09 Synlogic, Inc. Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier
US20190046444A1 (en) * 2015-09-14 2019-02-14 Vgsk Techologies, Inc. A sterically stabilized carrier for subcutaneous, sublingual, and oral therapeutics, compositions and methods for treating a mammal
US20200338073A1 (en) * 2019-04-24 2020-10-29 Theravance Biopharma R&D Ip, Llc Pyrimidine jak inhibitors for the treatment of skin diseases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BRISSON JOHN: "What is Eosinophilic Esophagitis and How to Hopefully Find Relief", FIXYOURGUT, 24 June 2020 (2020-06-24), XP055939496, Retrieved from the Internet <URL:https://www.fixyourgut.com/what-is-eosinophilic-esophagitis-and-how-to-hopefully-find-relief/> *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114903941A (en) * 2022-04-24 2022-08-16 河南中医药大学 Dried orange peel alcohol extract and application thereof in treating eosinophilic esophagitis
CN114903941B (en) * 2022-04-24 2024-01-30 河南中医药大学 Dried orange peel alcohol extract and application thereof in treatment of eosinophilic esophagitis

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