Ascaroside treatment of eosinophilic esophagitis

A method of preventing or treating eosinophilic esophagitis (EoE) comprising administering a composition comprising ascr#7 is described.

BACKGROUND

Eosinophilic esophagitis (EoE) is a chronic immune and antigen-mediated disease that is characterized by eosinophilic infiltration into the tissue of the esophagus, resulting in difficulty swallowing, food impaction, regurgitation or vomiting, and decreased appetite. It is defined by the symptoms as well as the finding of a high number of eosinophils found in the esophageal tissue through endoscopic biopsy (eosinophils are not normally found in the esophagus). Currently, there are no approved drugs for the treatment of EoE. Rather, there is a trial-and-error approach of different classes of treatment, ranging from food elimination, off-label steroid use, and surgical intervention. Between 40-50% of patients do not respond to first line treatments, lending great need for new therapeutic strategies.

SUMMARY

EoE is a multifaceted disease that manifests characteristics of both autoimmune disease and allergic inflammation. In performing biopsies on both EoE and control patients, and looking for significant differences in signaling molecules between these groups, researchers have found that IL-6 is among the most elevated signaling molecules in EoE biopsies. IL-1β levels are also increased in EoE patients.

The inventors hereof have found that one of the ascarosides, ascr#7, markedly reduces IL-6 and IL-1β levels in an in-vitro model of immune activation. Presumably, at least in part by modulating these signaling molecules, ascr#7 also inhibits hallmark pathologies in a mouse model of the gastrointestinal autoimmune disease (inflammatory bowel disease) and a mouse model of eosinophilic allergic disease (asthma). These findings support the use of ascr#7 for treating EoE by virtue of its therapeutic effect in diseases where elevated levels of IL-6 and/or IL-1β contribute to the diseases' pathogeneses.

In some aspects, the invention provides a method of preventing, alleviating, or treating eosinophilic esophagitis (EoE) in a subject. The method comprises administering to the subject a composition comprising ascr#7 or a pharmaceutically acceptable salt or prodrug thereof.

Numerous embodiments are described that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in some embodiments, the subject has an elevated level of IL-6 and/or IL-1β. In some other embodiments, IL-6 and/or IL-1β contribute to one or more symptoms of the disease. In such embodiments, the method may comprise assessing the level of IL-6 and/or IL-1β in an affected tissue of the subject and, if the level exceeds 200% of a normal level in the affected tissue, administering ascr#7 or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments, the method further comprises administering an agent with an anti-inflammatory effect, such as a corticosteroid or a proton pump inhibitor. In some embodiments, the method further comprises administering a corticosteroid to the subject. In some embodiments, the corticosteroid is selected from aldosterone, betamethasone, budesonide, corticosterone, cortisol, cortisone, dexamethasone, fluticasone (e.g., fluticasone propionate), hydrocortisone, methylprednisolone, prednisolone, and prednisone.

In some embodiments, the proton pump inhibitor is selected from dexlanoproazole, esomeprazole, ilaprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole. In some other embodiments, the method further comprises administering losartan to the subject. Losartan has the following structural formula:

In some embodiments, the method further comprises a dietary therapy, e.g., elemental diets, directed elimination diets, or non-directed, empirical elimination diets. In some embodiments, the elimination diet is a six-food elimination diet. In some embodiments, the elimination diet eliminates milk, soy, eggs, wheat, peanuts/tree nuts, and seafood from the subject's diet.

In some embodiments, the method further comprises dilation of the esophagus. Esophageal dilation can be performed using either through-the-scope balloons or wire-guided bougies.

In some embodiments, the method comprises administering to the subject a composition comprising ascr#7, and further comprises one or more of administering a corticosteroid to the subject, administering losartan to the subject, a dietary therapy, and/or dilation of the esophagus.

In some embodiments, the subject is a mammal. In certain embodiments, the mammal is a mouse or a human. In certain preferred embodiments, the mammal is a human.

DETAILED DESCRIPTION

For convenience, certain terms employed in the specification, and appended claims are collected here.

The term “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

The singular forms “a,” “an,” and “the” include the plurals unless the context clearly dictates otherwise.

The terms “subject” refer to either a human or a non-human animal. This term includes mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice, rabbits and rats).

“About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.

The term “prodrug” is intended to encompass compounds which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., ascr#7). A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the subject. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the compounds of the present invention (e.g., ascr#7) in a formulation represented above can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester.

As used herein, a therapeutic that “prevents” a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature and techniques relating to chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

Ascr#7 is an ascaroside produced by a nematode, e.g.,Nippostrongylus brasiliensis.

Ascr#7 has the structure:

The compositions and methods disclosed herein may use any suitable form, prodrug, and/or salt of ascr#7 that exhibits the desired therapeutic effects in subjects.

Pharmaceutical Compositions

In certain embodiments, the present invention provides pharmaceutical compositions comprising ascr#7 or a pharmaceutically acceptable salt or prodrug thereof and a pharmaceutically acceptable carrier.

The compositions and methods of the present invention may be utilized to treat a subject in need thereof. In certain embodiments, the subject is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an eye drop.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment.

Alternatively or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. Exemplary ophthalmic formulations are described in U.S. Publication Nos. 2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Pat. No. 6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic formulations have properties similar to that of lacrimal fluids, aqueous humor or vitreous humor or are compatable with such fluids. A preferred route of administration is local administration (e.g., topical administration, such as eye drops, or administration via an implant).

For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

The subject receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the subject, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic compounds.

This invention includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. The term “pharmaceutically acceptable salt” as used herein includes salts derived from inorganic or organic acids including, for example, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic, salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic, trifluoroacetic, trichloroacetic, naphthalene-2-sulfonic, and other acids.

In further embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. Pharmaceutically acceptable salt forms can include forms wherein the ratio of molecules comprising the salt is not 1:1. For example, the salt may comprise more than one ascr#7 molecule per molecule of base, such as two ascr#7 molecules per molecule of compound of base. As another example, the salt may comprise less than one ascr#7 molecule per molecule of base, such as two molecules of ascr#7 per molecule of lysine, or per atom of magnesium or zinc.

The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

In certain aspects, the invention provides methods of preventing, alleviating, or treating EoE in a subject, comprising administering to the subject a composition comprising ascr#7 or a pharmaceutically acceptable salt and/or prodrug thereof.

In certain aspects, the invention provides use of ascr#7 in the manufacture of a medicament for the treatment of EoE.

Elevated levels of IL-6 and/or IL-1β have been found in patients with various diseases, and studies have shown that IL-6 and/or IL-1β contribute to these diseases' pathogeneses. For example, IL-6 level was significantly elevated (281.5 pg/ml in the new/active EoE patient group compared to 16.2 pg/ml in the control group) in biopsy culture supernatant from esophageal samples from patients with eosinophilic esophagitis (Sayej et al. Clinical & Translational Immunology 2016; 5:25-36). The average serum IL-6 level in the EoE patients is also at least two-fold higher than the level in the healthy controls (Blanchard et al. J Allergy Clin Immunol. 2011; 127(1):208-17). In addition, the IL-1β gene was also significantly elevated (about 7-fold using microarray analysis and about 5.5-fold using real-time PCR) in biopsies from esophageal samples from patients with eosinophilic esophagitis (Blanchard et al. J Allergy Clin Immunol. 2011; 127(1):208-17).

In some embodiments, the IL-6- and/or IL-1β-mediated disease is EoE.

The subject may be a mammal. The subject may be a rodent, lagomorph, feline, canine, porcine, ovine, bovine, equine, or primate. In some embodiments, the subject is a human. The subject may be a female or male. The subject may be an infant, child, adolescent, or adult. In some embodiments, the subject has an elevated level of IL-6 and/or IL-1β.

V. Routes of Administration

A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compound may also be formulated for inhalation.

In some embodiments, the compound may be delivered orally in a slurry to coat the throat. In some embodiments, the slurry is formed by mixing the compound with a pharmaceutically acceptable excipient. In some embodiments, the slurry comprises the compound and a pharmaceutically acceptable excipient, for example, sucralose, syrup, honey, applesauce, RINCINOL® (topical adherent), Neocate Nutra, or a mixture thereof. In some embodiments, the slurry comprises the compound and sucralose. In some embodiments, the slurry further comprises an agent with an anti-inflammatory effect. In some embodiments of the slurries disclosed herein, the agent with an anti-inflammatory effect is a corticosteroid (e.g., aldosterone, betamethasone, budesonide, corticosterone, cortisol, cortisone, dexamethasone, fluticasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone). In some embodiments of the slurries disclosed herein, the agent with an anti-inflammatory effect is a proton pump inhibitor (e.g., dexlanoproazole, esomeprazole, ilaprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole). In some embodiments, the slurry further comprises losartan.

In certain embodiments, a compound may be simply dissolved or suspended in sterile water.

Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

One of skill in the art would appreciate that a method of administering a therapeutically effective substance formulation or composition of the disclosure would depend on factors such as the age, weight, and physical condition of the subject being treated, and the disease or condition being treated. The skilled worker would, thus, be able to select a method of administration optimal for a subject on a case-by-case basis.

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. As such, it will be readily apparent that any of the disclosed beneficial substances and therapies can be substituted within the scope of the present disclosure.

EXAMPLES

As described above, EoE is a multifaceted disease that manifests characteristics of both autoimmune disease and allergic disease. Like patients with the gastrointestinal autoimmune disease, IBD, EoE patients experience dysregulation of the gastrointestinal lumen and mucosal barrier. The signaling molecules IL-6 and IL-1β both promote intestinal pathology in IBD. EoE also shares features with the allergic disorder, asthma, in that patients have increased eosinophils in affected tissues and have increased mucus production. IL-6 and IL-1β also both play an active role in the pathogenesis of asthma.

The Examples demonstrate that ascr#7 significantly reduced levels of both IL-6 and IL-1β in an in-vitro model of inflammation. They further demonstrate that ascr#7 profoundly diminished disease progression in both a mouse model of inflammatory bowel disease (IBD) as well as a mouse model of asthma.

Ascr#7 significantly reduces both IL-6 and IL-1β secretion in an in vitro model of inflammation. Specifically, ascr#7 reduces IL-6 and IL-1β in a stimulated culture of bone marrow-derived mouse dendritic cells.

a. Generation of Bone Marrow Dendritic Cells

Bone marrow was extracted from femurs of C57BL/6 mice (6-12 weeks of age). These mice were euthanized by CO2inhalation and their femurs were removed. After removing the remaining flesh from the bone, they were cut at the metaphysis. Bone marrow was flushed out using a syringe filled with RPMI. The extracted marrow was further broken down by resuspension with syringe. The mixture was centrifuged and the pellet was re-suspended in RPMI. Cells from the bone marrow were seeded in 6 wells such that each well had a density of 106cells/ml. 20 ng of GM-CSF was added to each well. At day 3, and every 3 days thereafter, the media was replaced. Cells were used for experiments between day 7-9.

b. Stimulation of Dendritic Cells: Testing the Effect of Ascr#7

Bone marrow dendritic cells were collected between day 7-9 of culture. Cells were washed twice and counted. Ascr#7 in water or RPMI were placed in corresponding wells, and are allowed to dry. 106cells were then plated in each well in a 24 well culture plate. Cells were left to incubate with ascr#7 for 5 minutes. Following the brief incubation, different TNFα was added to the wells. These bone marrow dendritic cells were then cultured at 37° C. for 18-72 hours. Cells were collected via the pellet generated by centrifuge. The supernatant was also preserved. EBioscience ELISA kits was used to determine the concentration of different cytokines present in each well. Results are shown inFIG. 1andFIG. 2.

The left side ofFIG. 1shows that the dendritic cells, when not stimulated with TNF-α, have very low levels of IL-6, and that this remains true with administration of different concentrations of ascr#7. The right side ofFIG. 1shows that dendritic cells, when stimulated with TNF-α, has very high levels of secreted IL-6 and that administration of different concentrations of ascr#7 reduces these levels significantly. The left side ofFIG. 2shows that the dendritic cells, when not stimulated with TNF-α, have very low levels of IL-1β, and that this remains true with administration of different concentrations of ascr#7. The right side ofFIG. 2shows that dendritic cells, when stimulated with TNF-α, has very high levels of secreted IL-β and that administration of different concentrations of ascr#7 reduces these levels significantly.

Ascr#7 reduces the trademark disease pathologies in a mouse model of asthma. Specifically, ascr#7 reduces the development of eosinophilia, goblet cell metaplasia, and airway hyper-reactivity in the ovalbumin model of murine asthma.

Adult C57BL/6 mice were sensitized by administering 2 ug OVA (Sigma-Aldrich) and 1 mg alum (Thermo) with 50ng ascr#7 or saline, via intraperitoneal injection, on Day 0 and 14. On day 24, the mice were challenged with intranasal administration of saline containing 100 μg OVA, under isoflurane anesthesia. Histologic sections were stained with H&E or PAS to score for eosinophilia, goblet cell metaplasia, and airway hyper-reactivity. Mice were also placed in a computer controlled small animal ventilator (SCIREQ) to score for airway hyper-reactivity following methacholine challenge. Results are shown inFIG. 3B-FIG. 3G.FIG. 3Ashows a diagram of a mouse in a respiratory chamber, which has been calibrated to calculate the resistance of air flow as the animal breathes. Animals with asthma have higher resistance of flow, which would be represented as higher “RL” values inFIG. 3B-FIG. 3F.FIG. 3B-FIG. 3Fshow that asthmatic mice, here labeled “Asthma”, have higher resistance of air flow, whereas a normal mouse, here labeled “Normal” has low resistance of air flow. Asthmatic mice that have been administered ascr#7 demonstrate low resistance of air flow, with comparable levels to normal mice. Lungs were collected, postmortem, and then sectioned and stained to show the presence of eosinophils and mucus. These findings are shown inFIG. 3G. InFIG. 3G, the normal mouse lung, here labeled “Normal Lung Section” shows thin boundaries between the central bronchioles and the surrounding alveoli, wherein the thin membranes allow for oxygen to easily diffuse. InFIG. 3G, the asthmatic mouse lung, here labeled “Asthma” has a large number of mucus and eosinophils that surround the bronchioles and occupy the alveolar space; wherein the thick barriers would prohibit oxygen from easily diffusing. InFIG. 3G, the lung of asthmatic mice that were administered ascr#7 has thin boundaries between the bronchioles and the alveolar space, comparable to the normal mice. This demonstrates that ascr#7 reduces the trademark pathologies found in asthmatic mice, specifically resistance to air flow and its associated histopathologies.

Ascr#7 reduces the signature disease pathologies in a mouse model of IBD. Specifically, ascr#7 markedly reduces the development of colon shortening, abnormal lumen dilation, and diarrhea in an immune dysregulation model of inflammatory bowel disease.

A T cell transfer model of colitis where 106CD4+ T cells were transferred to adult Rag −/− mice was used, and either saline or 300 ng ascr#7 were administered via intraperitoneal injection every 3 days between day 14 and day 28. On day 28, the mice were euthanized and colon analysis was performed. n=3 mice per group. Results are shown inFIG. 4. The inset shows a diagram of the colon.FIG. 4shows the normal tissue architecture of a dissected caecum and colon. The normal mouse colon is long, skinny, and has discrete fecal pellets. The diseased mouse colon, with IBD, has a shortened, dilated colon, with diarrhea instead of discrete fecal pellets. The colon of a mouse with IBD, which has been administered ascr#7 has a normal appearing colon, which is long, skinny, and contains discrete fecal pellets.

INCORPORATION BY REFERENCE AND VARIATION

All patent applications, publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present specification, including its specific definitions, will control.

While specific aspects of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.