ECM hydrogels for treating esophageal inflammation

Abstract

To provide an ECM hydrogel for treating esophageal inflammation. [Solution] Methods for inhibiting esophageal inflammation in a subject are disclosed, comprising administering a therapeutically effective amount of an extracellular matrix (ECM) hydrogel to the esophagus of a subject with esophageal inflammation. Methods for reducing esophageal strictures are also disclosed. Compositions comprising esophageal extracellular matrix (ECM) hydrogels are disclosed. These methods comprise administering a therapeutically effective amount of an extracellular matrix (ECM) hydrogel having predetermined characteristics to the esophagus of a subject, for example, a subject with esophageal inflammation or stricture or at risk for stricture.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 465,985, filed March 2, 2017, which is incorporated herein by reference in its entirety.

[0002] Field This relates to the field of hydrogels, and in particular to the use of extracellular matrix (ECM) hydrogels for the treatment of inflammation of the esophagus, such as Barrett's esophagus. [Background technology]

[0003] background The incidence of esophageal adenocarcinoma (EAC) is rising rapidly, outpacing the increase in all other cancers. EAC is associated with a poor prognosis, with a 5-year survival rate of less than 15%. The number of affected patients is up to 600% higher than in the 1970s (Dubecz et al., J Gastrointest Surg. 2013 Nov. 15; Prasad et al., Amer. J. Gastroentero. 105(7):1490-502, 2010).

[0004] Barrett's esophagus is characterized by the replacement of the normal stratified squamous epithelial lining of the esophagus with simple columnar epithelium with goblet cells, involving cell metaplasia in the lower (distal) part of the esophagus. Barrett's esophagus is closely associated with esophageal adenocarcinoma. The main cause of Barrett's esophagus is thought to be an adaptation and response to chronic acid exposure due to reflux esophagitis. Barrett's esophagus cells are classified into four general categories after biopsy: non-dysplastic, low-grade dysplasia, high-grade dysplasia, and overt carcinoma. Early stages of high-grade dysplasia and adenocarcinoma are usually treated by endoscopic therapy, such as endoscopic resection and radiofrequency ablation, while non-dysplastic and low-grade dysplasia patients are generally advised to undergo annual endoscopic observation. There remains a need for methods and compositions that can be used to treat esophagitis and Barrett's esophagus. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Dubecz et al., J Gastrointest Surg. 2013-11-15 [Non-Patent Document 2] Prasad et al., Amer. J. Gastroentero. 105(7):1490-502, 2010 Summary of the Invention [Means for solving the problem]

[0006] Abstract Methods are disclosed for inhibiting esophagitis inflammation and / or alleviating the effects of esophagitis in a subject. Methods are also disclosed for reducing esophageal stricture. These methods include administering a therapeutically effective amount of an extracellular matrix (ECM) hydrogel to the esophagus of a subject, for example, a subject having or at risk for esophageal inflammation or stricture, wherein the ECM hydrogel has the following characteristics: a) time to 50% gelation at a temperature of about 37° C. is less than 30 minutes; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of 10-600 Pascals (Pa), for example, but not limited to, 10-70 Pa. In a specific, non-limiting example, the hydrogel can be an esophageal ECM hydrogel. In another specific, non-limiting example, the subject can have Barrett's esophagus.

[0007] In additional embodiments, compositions are disclosed that include an esophageal extracellular matrix (ECM) hydrogel, the esophageal ECM hydrogel having the following characteristics: a) a time to 50% gelation at about 37° C. of less than 10 minutes; b) a flow viscosity sufficient for injection into the esophagus; and c) a stiffness of 10-70 Pascals (Pa), and the composition is formulated for administration to the esophagus. These compositions are useful in the methods disclosed herein.

[0008] The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for inhibiting inflammation or reducing esophageal stricture in a subject, comprising administering to a subject an esophageal stenosis comprising administering to said subject an esophageal stenosis comprising: a) time to 50% gelation at a temperature of about 37° C. is less than 30 minutes; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of about 10 to about 300 Pascals (Pa) to the esophagus of said subject having esophageal inflammation; thereby inhibiting inflammation or reducing esophageal stricture in said subject. (Item 2) 2. The method of claim 1, wherein the time to 50% gelation is from about 3 to about 30 minutes at about 37° C. (Item 3) 2. The method of claim 1, wherein the time to 50% gelation is from about 3 to about 10 minutes at about 37° C. (Item 4) 3. The method of claim 2, wherein the time to 50% gelation is from about 4 to about 10 minutes. (Item 5) The flow viscosity is from about 1 to about 40 Pa at a shear rate of about 0.1 / s. * s, and at a shear rate of 1000 / s, from about 0.01 to about 0.2 Pa * 5. The method according to any one of items 1 to 4, wherein (Item 6) The flow viscosity is from about 0.1 to about 25 Pa at a shear rate of 1 / s. * s, and at a shear rate of about 100 / s, from about 0.02 to about 0.8 Pa * 5. The method according to any one of items 1 to 4, wherein (Item 7) 7. The method of any one of items 1 to 6, wherein the ECM hydrogel has a stiffness of 10 to 70 Pa. (Item 8) 8. The method of any one of items 1 to 7, wherein the ECM hydrogel is an esophageal ECM hydrogel. (Item 9) 9. The method of any one of items 1 to 8, wherein the ECM concentration of the hydrogel is from 2 mg / ml to about 16 mg / ml. (Item 10) 10. The method of any one of items 1 to 9, wherein the ECM hydrogel is administered orally, endoscopically or via a catheter. (Item 11) The ECM hydrogel comprises: (a) To solubilize a decellularized extracellular matrix (ECM) by digestion of tissue with an acidic protease in an acidic solution to generate digested esophageal ECM; and (b) to raise the pH of the digested esophageal ECM to a pH between 7.2 and 7.8 to produce a neutralized digestion solution The method according to any one of items 1 to 10, produced by (Item 12) The method according to item 11, wherein raising the pH of the digested ECM (b) comprises adding a base or an isotonic buffer to raise the pH of the digested ECM. (Item 13) The method according to item 10 or item 11, wherein the acidic protease is pepsin, trypsin, or a combination thereof. (Item 14) The method according to any one of items 1 to 13, wherein the ECM hydrogel is maintained at 25°C or below before administration to the subject. (Item 15) The method according to any one of items 1 to 14, wherein the subject has Barrett's esophagus or is at risk of Barrett's esophagus. (Item 16) The method according to item 15, which inhibits the development of esophageal neoplasms in the subject. (Item 17) The method according to any one of items 1 to 16, wherein the ECM hydrogel restores the epithelial barrier in the subject's esophagus. (Item 18) The method according to any one of items 1 to 17, wherein the ECM hydrogel increases the chemotaxis of both epithelial cells and / or stem cells to the site of injury in the subject's esophagus. (Item 19) The method according to any one of items 1 to 18, wherein the ECM hydrogel reduces esophageal stricture in the subject. (Item 20) A composition comprising an extracellular matrix (ECM) hydrogel, wherein the ECM hydrogel has the following characteristics: a) time to 50% gelation at about 37°C is less than 10 minutes; b) sufficient flow viscosity for injection into the esophagus; c) a stiffness of about 10 to about 300 Pascals (Pa); and d) The hydrogel is an esophageal hydrogel. wherein the composition is formulated for administration to the esophagus. (Item 21) 21. The composition of claim 20, wherein the time to 50% gelation is, at about 37° C., a) about 3 to about 30 minutes; b) about 4 to about 10 minutes; or c) about 3 to about 10 minutes. (Item 22) 22. The composition according to claim 20 or 21, wherein the hydrogel has a stiffness of about 10 to about 70 Pa. (Item 23) 23. The composition of any one of items 20 to 22, comprising about 2 mg / ml to about 16 mg / ml of said ECM hydrogel. (Item 24) The ECM hydrogel comprises: (a) solubilizing decellularized extracellular matrix (ECM) by digestion of esophageal tissue with acid proteases in an acidic solution to generate digested esophageal ECM; (b) increasing the pH of the digested esophageal ECM to a pH between 7.2 and 7.8 to produce a neutral digestion solution; and (c) diluting the digested esophageal ECM to a concentration of about 8 mg / ml to about 12 mg / ml of the ECM hydrogel. 24. The composition according to any one of items 21 to 23, produced by (Item 25) 25. The composition of claim 24, wherein (b) increasing the pH of the digested ECM comprises adding a base or an isotonic buffer to increase the pH of the digested esophageal ECM. (Item 26) 26. The composition of claim 24 or 25, wherein the acid protease is pepsin, trypsin or a combination thereof. (Item 27) 27. The composition of any one of items 20 to 26, wherein the ECM hydrogel is maintained at or below 25° C. (Item 28) 28. The composition of any one of items 20 to 27 for use in inhibiting inflammation of the esophagus in a subject. (Item 29) 28. The composition of any one of items 20 to 27 for use in restoring the epithelial barrier in the esophagus of a subject. (Item 30) 30. The composition of claim 28 or 29, wherein the subject has Barrett's Esophagus. (Item 31) 28. The composition according to any one of items 20 to 27 for reducing esophageal stricture in a subject. (Item 32) 32. A kit comprising: a) a container containing the composition according to any one of items 20 to 31, or a lyophilized form thereof; and b) instructions for using said composition. [Brief description of the drawings]

[0009] [Figure 1] Viscosity profile of esophageal ECM hydrogel.

[0010] [Diagram 2] Esophageal ECM hydrogel stiffness.

[0011] [Diagram 3] Esophageal ECM gelation time.

[0012] [Figure 4A] The viscosity profile is tissue specific. [Figure 4B] The viscosity profile is tissue specific. [Figure 4C] The viscosity profile is tissue specific.

[0013] [Figure 5A] The stiffness of the gel is tissue specific. [Figure 5B] The stiffness of the gel is tissue specific. [Figure 5C] The stiffness of the gel is tissue specific.

[0014] [Figure 6A] ECM gelation time is tissue specific. [Figure 6B] ECM gelation time is tissue specific. [Figure 6C] ECM gelation time is tissue specific.

[0015] [Figure 7] The ECM hydrogel promotes the secretion of anti-inflammatory cytokines.

[0016] [Figure 8A-8B] The ECM promotes the chemotaxis of epithelial and stem cells.

[0017] [Figure 9] Dynamic reciprocity in the clinical treatment of EAC.

[0018] [Figure 10A-10B] Effect of hydrogel administration.

[0019] [Figure 11] Effect of hydrogel administration after 30 days.

[0020] [Figure 12A] eEvaluation of the safety of ECM hydrogels. [Figure 12B] eEvaluation of the safety of ECM hydrogels.

[0021] [Figure 13A-13D] Histological examination.

[0022] [Figure 14] Use for treating stenosis.

[0023] [Figure 15] Histological analysis, control dogs.

[0024] [Figure 16] Histological analysis, ECM hydrogel treated dogs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description When fabricated as hydrogels, extracellular matrix (ECM) bioscaffolds are bioactive (Freytes et al., Biomaterials 29:1630-1640). 7, 2008). However, ECM hydrogels have unique physical and mechanical properties. These unique properties expand the application of ECM beyond what is possible in sheet form. Compared to sheet forms, ECM hydrogels provide irregularly shaped and irregularly sized surface areas, ensuring contact with tissues with the irregular morphology of the ECM, without the need for fixation devices (e.g., sutures, stents), and without adversely imparting stiffness to the implanted area. Hydrogels are characterized by being liquid at room temperature (pre-gel), but become gels when exposed to body temperature (37° C.), making them easily deliverable via devices such as syringes, catheters, irrigators, and probes, among others.

[0026] ECM in sheet form has been used to treat late stage esophageal dysplasia and neoplastic disease, but the need for fixation devices (i.e., stents), the rigidity of the sheet form, and the relative invasiveness of implantation have prevented its use from being expanded. The unique properties of ECM hydrogels make them ideal for application to the surface of the esophagus and / or for treating esophageal diseases that are not treatable in sheet form. The viscoelastic and mucoadhesive properties of ECM hydrogels support their potential for treating esophageal diseases. Additionally, the hydrogels can be used to reverse cancerous and precancerous lesions of the esophagus.

[0027] Recently, the sheet form has been successfully used to treat late stage cancer and precancerous disease of the esophagus. For use in the esophagus, the sheet is deployed circumferentially and held in place by a stent. There are limitations to the use of the sheet form, including the need for fixation devices (i.e., sutures or stents), the inability to fill irregularly sized defects, and limited coverage area (relative to the size of the sheet). In the esophagus, these limitations limit the use of ECM sheets to the treatment of late stage esophageal disease, which is one of the few situations where the use of temporary stents and advanced procedures are justified.

[0028] The hydrogels of the present disclosure can be administered locally to the esophagus. The hydrogels can be administered to the cavity of the esophagus to coat the surface. This is a non-invasive application. In some embodiments, the application is oral, e.g., by swallowing. In other embodiments, the application can be by tube feeding. The hydrogels are formed on the surface of the esophageal tissue. In some embodiments, the hydrogels coat the mucosa and do not invade the underlying submucosa or muscle tissue.

[0029] It is disclosed herein that hydrogels, such as those derived from the ECM of the esophagus, can be used to treat Barrett's esophagus and inhibit the development of adenocarcinoma. The ECM hydrogels can inhibit inflammation and mitigate the effects of inflammation. The ECM hydrogels can reduce stenosis. In some embodiments, the ECM hydrogels are effective when utilized at a concentration of about 2 mg / ml to about 20 mg / ml, for example, about 8 mg / ml to about 12 mg / ml.

[0030] term Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology. Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (Eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) can be found in.

[0031] In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[0032] Acid protease: An enzyme that cleaves peptide bonds and whose activity in cleaving peptide bonds is increased at an acidic pH. For example, but not limited to, acid proteases can include pepsin and trypsin.

[0033] Barrett's Esophagus: Abnormal changes (metaplasia or dysplasia) in the cells of the lower (terminal) esophagus. Barrett's Esophagus is a diagnosis when the normal stratified squamous epithelial lining of the esophagus is replaced by simple columnar epithelium with goblet cells. Barrett's Esophagus is seen in 5-15% of patients who seek medical care for gastroesophageal reflux disease (GERD), although a large subgroup of patients with Barrett's Esophagus do not have symptoms. Barrett's Esophagus is closely related to esophageal adenocarcinoma and is thought to be a premalignant condition. The primary cause of Barrett's Esophagus is thought to be an adaptation to chronic acid exposure due to reflux esophagitis. Cells in Barrett's Esophagus are classified into four general categories after biopsy: non-dysplastic, low-grade dysplasia, high-grade dysplasia, and overt carcinoma.

[0034] Base: A compound or a solution of a compound with a pH greater than 7. For example, but not limited to, the base is an alkaline hydroxide or an aqueous solution of an alkaline hydroxide. In certain embodiments, the base is NaOH or NaOH in PBS.

[0035] Comminuting (grinding and grinding): A process of reducing large particles to smaller particles, including but not limited to grinding, blending, crushing, slicing, milling, cutting, crushing. The ECM can be milled in any form, including but not limited to hydrated form, frozen, air-dried, lyophilized, powdered sheet form.

[0036] Diagnosis: The process of identifying a disease by its signs, symptoms, and the results of various tests. The conclusion reached through that process is also called a "diagnosis." Commonly performed forms of testing include blood tests, medical imaging, and biopsies.

[0037] Extracellular matrix (ECM): non-cellular components of tissues and organs. Native ECM (ECM found in multicellular organisms, e.g., mammals and humans) is a complex mixture of structural and non-structural biomolecules, including, but not limited to, collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors. In mammals, ECM often contains about 90% collagen by dry weight mass in various forms. Biological scaffolds can be created by removing cells from a given tissue or organ. The composition and structure of the ECM varies depending on the source of the tissue. For example, the ECM of the small intestinal submucosa (SIS), urinary bladder matrix (UBM), esophagus (E), and liver interstitium each differ in their overall structure and composition due to the unique cellular niches required for each tissue. An intact "extracellular matrix" and "intact ECM" biological scaffold is an extracellular matrix that ideally retains the activity of its structural and non-structural biomolecules, including but not limited to collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors, such as, but not limited to, pulverized ECM as described herein.

[0038] The activity of biomolecules in the ECM can be removed chemically or mechanically, for example, by chemical or enzymatic cross-linking and / or by dialyzing the ECM. Intact ECM is essentially not enzymatically digested, cross-linked and / or dialyzed, meaning that the ECM is not exposed to conditions other than the digestion, dialysis and / or cross-linking processes, or processes that naturally occur during storage and handling of the ECM before solubilization. Thus, ECM that is substantially cross-linked and / or dialyzed (in any manner other than the usual manner that does not substantially affect the gelation and functional characteristics of the ECM in its use as described herein) is not considered "intact".

[0039] Esophagogastroduodenoscopy (EGD) or upper GI endoscopy: A diagnostic endoscopic procedure that visualizes any portion of the digestive tract up to the duodenum. An "esophageal endoscopy" is any endoscopic procedure that visualizes the esophagus. Esophageal endoscopy may also be performed as part of an EGD or upper GI endoscopy. The terms are not mutually exclusive unless specifically stated to be so.

[0040] Gelation: The formation of a gel from a sol.

[0041] Gastroesophageal reflux disease (GERD): A chronic condition of mucosal damage caused by stomach acid refluxing from the stomach into the esophagus. GERD is usually caused by changes in the barrier between the stomach and esophagus, including abnormal relaxation of the lower esophageal sphincter, which normally holds the upper (proximal) part of the stomach closed, impaired clearance of gastric reflux from the esophagus, or a hiatal hernia. These changes may be permanent or temporary.

[0042] Flow Viscosity: A measure of a fluid's resistance to gradual deformation due to shear or tensile stress. Viscosity is the property of a fluid that opposes the relative motion between two surfaces in the fluid that are moving at different speeds. When a fluid is forced through a tube, the particles that make up the fluid generally move faster near the axis of the tube and slower near its walls. A stress (such as a pressure difference between the two ends of the tube) is required to overcome the friction between the particle layers and keep the fluid moving. For a given velocity pattern, the stress required is proportional to the viscosity of the fluid. Viscosity is measured with viscometers and rheometers. Viscosity is measured in Pascal seconds (Pa * The viscosity of water at 20°C is 1.002 mPa * It has a viscosity of s.

[0043] Hydrogel: a network of hydrophilic polymer chains that can be viewed as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent natural or synthetic polymer networks. Hydrogels also have flexibility similar to natural tissue.

[0044] Inflammation: A localized response induced by injury to tissue. Inflammation is characterized by the appearance or migration of any class of leukocytes into any tissue space, unit or area in numbers that exceed the number of such cells found in such an area of ​​tissue under normal (healthy) circumstances. Inflammation is orchestrated by a complex biological response of vascular tissues to harmful stimuli such as pathogens, damaged cells, or irritants.

[0045] Isotonic Buffered Solution: A solution that is buffered to a pH between 7.2 and 7.8 and has a balanced salt concentration to promote an isotonic environment.

[0046] Low-grade and high-grade dysplasia (of the esophagus): Pathologies of the esophagus. Generally, in esophageal dysplasia, apical mucin is absent from the lining of the esophagus. Both absence of goblet cells and depletion of mucin in non-goblet columnar cells are frequently seen in dysplastic epithelium. At low power, these areas appear to be more hyperpigmented compared to uninvolved areas.

[0047] In high-grade dysplasia, distortion of the esophageal glandular architecture is usually present and may be noted; this consists of branching and lateral budding of crypts, villus-like structures at the mucosal surface, or intraglandular bridging of epithelium forming a cribriform pattern of "back-to-back" glands. Dysplastic epithelium is present at the mucosal surface with loss of nuclear polarity, characterized by nuclear "rounding" and the absence of a consistent relationship of nuclei to each other.

[0048] Preventing or Treating: Inhibiting disease refers to partially or completely inhibiting the onset of disease in a person who is at risk for disease, such as those caused by inflammation. An example of a person who is at risk for esophageal adenocarcinoma is one who has Barrett's esophagus or GERD. Inhibiting disease process includes preventing the onset of disease. "Treatment" refers to a therapeutic intervention that ameliorates the signs or symptoms or pathology of disease, for example, after the disease begins to develop.

[0049] Shear Stress: The component of stress that is coplanar with the cross-section of a material. Shear stress results from force vector components that are parallel to the cross-section. The formula for calculating average shear stress is force per unit area,

number

[0050] Stricture: A narrowing or pinching of the esophagus that makes swallowing difficult. Symptoms of esophageal stricture include heartburn, a bitter or sour taste in the mouth, choking, coughing, shortness of breath, frequent burping or hiccups, pain or difficulty swallowing, vomiting blood and / or weight loss. Esophageal strictures can be caused by gastroesophageal reflux disease, esophagitis, lower esophageal sphincter dysfunction, motility disorders, alkaline digestion, or hiatal hernia. Strictures can form after esophageal surgery and other procedures such as laser therapy or photodynamic therapy. While the area heals, scars form, causing tissue to pull and pinch, making swallowing difficult. Strictures can be the result of inflammation. A barium swallow study or upper gastrointestinal endoscopy can be used to diagnose esophageal strictures.

[0051] Stiffness: The hardness of an object or fluid. The stiffness of the extracellular matrix is ​​important for guiding the movement of cells in durotaxis. Stiffness can be measured in Pascals (Pa), which is one Newton per square meter.

[0052] Therapeutic Agent: When used in a general sense, this includes therapeutic agents, prophylactic agents, and replacement agents. "Treatment" or "treating" means providing a substance, such as an ECM hydrogel, to a patient in an amount sufficient to measurably reduce, inhibit, or alleviate any disease symptoms, slow the progression of the disease, or cause regression of the disease. In certain embodiments, treatment of a disease may begin before the patient exhibits symptoms of the disease. The methods of the present disclosure inhibit esophageal inflammation and / or alleviate the effects of esophageal inflammation.

[0053] Therapeutically effective amount: A "therapeutically effective amount" of a composition, such as an ECM hydrogel, refers to an amount that, when administered to a patient, is effective to provide a therapeutic benefit, such as alleviating symptoms, reducing the slowing of progression, or regressing a disease. The amount of ECM hydrogel specified is sufficient to achieve a desired effect in the subject being treated, such as inhibiting inflammation and / or alleviating the effects of such inflammation, e.g., stricture. The therapeutically effective amount can be administered systemically or locally, e.g., to the esophagus. Furthermore, an effective amount of ECM hydrogel can be administered in a single dose or in multiple doses over time. However, the effective amount will vary depending on the preparation applied, the subject being treated, the severity and type of affliction, and the manner in which the compound is administered. The use of ECM hydrogel in the methods disclosed herein has application in both medical and veterinary settings. Thus, it is understood that the general term "subject" or "patient" includes all animals, including, but not limited to, humans or veterinary subjects, e.g., other primates, dogs, cats, horses, and cows.

[0054] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an" and "the" include plural referents unless expressly stated otherwise. Similarly, the word "or" is intended to include "and" unless expressly stated otherwise. It should be further understood that all base or amino acid sizes and all molecular weights or masses given for nucleic acids or polypeptides are approximate values ​​and are provided for illustration purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes". "About" indicates within 5 percent. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0055] Extracellular Matrix (ECM) Hydrogels Methods for preparing ECM hydrogels are disclosed, for example, in U.S. Patent No. 8,361,503. Any type of extracellular matrix tissue can be used to generate hydrogels that can be used in the methods disclosed herein (see U.S. Patent Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463; 5,372,821; 5,554,389; 5,573,784; 5,645,860; 5,771,969; 5,753,267; 5,762,966; 5,777,969 ... (See Nos. 5,866,414; 6,099,567; 6,485,723; 6,576,265; 6,579,538; 6,696,270; 6,783,776; 6,793,939; 6,849,273; 6,852,339; 6,861,074; 6,887,495; 6,890,562; 6,890,563; 6,890,564; and 6,893,666). In certain embodiments, the ECM is isolated from a vertebrate, for example, but not limited to, a warm-blooded mammalian vertebrate, including, but not limited to, a human, monkey, horse, pig, cow, and sheep. In specific, non-limiting examples, the ECM is porcine or human.

[0056] The ECM can be derived from any organ or tissue, including, but not limited to, the bladder, intestine, liver, esophagus and dermis. In one embodiment, the ECM is isolated from the bladder. In another embodiment, the ECM is derived from the esophagus. The ECM may or may not include the basement membrane portion of the ECM. In certain embodiments, the ECM includes at least a portion of the basement membrane. In other embodiments, the ECM is harvested from cell culture. The ECM hydrogel can be produced by combining two or more tissue sources.

[0057] As disclosed in U.S. Pat. No. 8,361,503 (incorporated herein by reference), bladder ECM, e.g., porcine bladder ECM, is prepared by scraping bladder tissue and removing the outer (abluminal) layers, including both the serosa and muscularis, using a longitudinal wiping motion with a scalpel handle and moistened gauze. After inverting the tissue fragment, the luminal portion of the mucosa is delaminated from the underlying tissue using the same wiping motion. In some embodiments, perforation of the submucosa is prevented. After these tissues are removed, the resulting ECM consists primarily of the submucosa. Generation of hydrogels from decellularized dermal ECM has been disclosed by Wolf et al., Biomaterials 33:7028-7038, 2012, incorporated herein by reference. The generation of ECM from esophageal tissue has been disclosed in Badylak et al., J Pediatr Surg. 35(7):1097-103, 2000 and Badylak et al., J Surg Res. 2005 Sep;128(1):87-97, 2005, both of which are incorporated herein by reference. U.S. Patent No. 6,893,666, which is incorporated herein by reference, discloses the generation of ECM from the bladder, skin, esophagus and small intestine.

[0058] Commercially available ECM preparations can also be used in the methods, devices and compositions described herein. In one embodiment, the ECM is derived from the small intestinal submucosa or SIS. Commercially available preparations include, but are not limited to, SURGISIS™, SURGISIS-ES™, STRATASIS™, and STRATASIS-ES™ (Cook Urological Inc.; Indianapolis, Ind.) and GRAFTPATCH™ (Organogenesis Inc.; Canton Mass.). In another embodiment, the ECM is derived from the dermis. Commercially available preparations include, but are not limited to, PELVICOL™ (sold as PERMACOL™ in Europe; Bard, Covington, Ga.), REPLIFORM™ (Microvasive; Boston, Mass.) and ALLODERM™ (LifeCell; Branchburg, NJ). In another embodiment, the ECM is derived from the urinary bladder. Commercially available preparations include, but are not limited to, UBM (Acell Corporation; Jessup, Md.).

[0059] The source tissue used for preparation of the ECM can be harvested in a variety of ways, and once harvested, various portions of the harvested tissue may be used. ECM has also been prepared from the esophagus and small intestine, and hydrogels have been prepared from this ECM. See, for example, Keane et al., Tissue Eng. Part A, Vol. 21 (1999), incorporated herein by reference. 7-18):2293-2300, 2015. Esophageal ECM can be prepared by mechanically separating the mucosa and submucosa from the muscularis externa, digesting the mucosa in a buffer containing trypsin, followed by exposure to sucrose, TRITON-X100®, deoxycholic acid, peracetic acid, and DNAse. Small intestinal submucosa (SIS) can be prepared from intact small intestine by mechanically removing the mucosa, serosa, and superficial layers of the muscularis externa, leaving the submucosa, muscularis mucosa, and basal cell layer (basilar stratum compactum) intact. The SIS is then treated with peracetic acid. An exemplary protocol is provided in Keane et al.

[0060] In one embodiment, ECM is isolated from harvested porcine bladders to prepare urinary bladder matrix (UBM). Excess connective tissue and residual urine are removed from the bladder. Most of the serous membrane, muscularis externa, submucosa, and muscularis mucosa can be removed by mechanical abrasion or by a combination of enzymatic treatment, hydration, and abrasion. Mechanical removal of these tissues can be achieved by abrasion using a longitudinal wiping motion that removes the outer layer (particularly the abluminal smooth muscle layer) as well as the luminal portion of the mucosa (epithelial layer). Mechanical removal of these tissues can be achieved, for example, by removal of the mesenteric tissue using Adson-Brown forceps and Metzenbaum scissors and wiping off the muscularis and submucosa using a longitudinal wiping motion with a scalpel handle or other hard object wrapped in moist gauze. Mucosal epithelial cells can also be isolated by immersing the tissue in a de-epithelializing solution, such as, but not limited to, hypertonic saline. The resulting UBM, which contains the basement membrane of the mucosa and adjacent lamina propria, is further treated with peracetic acid, lyophilized and powdered. See U.S. Patent No. 8,361,503.

[0061] In some embodiments, epithelial cells may be first delaminated by immersing the tissue in a de-epithelializing solution such as hypertonic saline, for example, but not limited to, 1.0N saline, for a period ranging from 10 minutes to 4 hours. Exposure to hypertonic saline effectively removes the epithelial cells from the underlying basement membrane. The tissue remaining after the initial delamination procedure includes the epithelial basement membrane and the tissue layer abluminal to the epithelial basement membrane. This tissue is then subjected to further processing to remove most of the abluminal tissue but not the epithelial basement membrane. Most of the outer serosa, adventitia smooth muscle tissue, submucosa and muscularis mucosa are removed from the remaining de-epithelialized tissue by mechanical abrasion or by a combination of enzymatic treatment, hydration and abrasion.

[0062] ECM can be sterilized using a variety of methods, including, but not limited to, exposure to peracetic acid, low-dose gamma irradiation, gas plasma sterilization, ethylene oxide treatment, and supercritical CO sterilization. 2 Sterilization can be achieved by a number of standard techniques, including 0.1% (v / v) peracetic acid, 4% (v / v) ethanol, and 95.9% (v / v) sterile water for 2 h. Residual peracetic acid is removed by washing twice for 15 min with PBS (pH=7.4) and twice for 15 min with sterile water. ECM materials can also be sterilized by a number of standard techniques, including 0.1% (v / v) peracetic acid, 0.1% (v / v) ethanol, or electron beam treatment. More typically, sterilization of the ECM is achieved by immersion in 0.1% (v / v) peracetic acid, 4% (v / v) ethanol, and 95.9% (v / v) sterile water for 2 h. Residual peracetic acid is removed by washing twice for 15 min with PBS (pH=7.4) and twice for 15 min with sterile water. ECM materials can also be sterilized by propylene oxide or ethylene oxide treatment, gamma irradiation treatment (0.05 to 4 mRad), gas plasma sterilization, peracetic acid sterilization, supercritical CO sterilization, and sterilization using 0.1% (v / v) peracetic acid. 2, or electron beam treatment. The ECM can also be sterilized by treatment with glutaraldehyde, which causes cross-linking of the protein material, but this treatment substantially alters the material so that the ECM is slowly or not resorbed at all, inducing various types of host remodeling that more closely resemble scar tissue formation or encapsulation than architectural remodeling. Cross-linking of the protein material can also be induced by carbodiimide or dehydrothermal or photooxidative methods. As disclosed in U.S. Pat. No. 8,361,503, the ECM is sterilized by immersion in 0.1% (v / v) peracetic acid (a), 4% (v / v) ethanol, and 96% (v / v) sterile water for 2 hours. The ECM material is then washed twice for 15 minutes in PBS (pH=7.4) and twice for 15 minutes in deionized water.

[0063] After isolation of the tissue of interest, decellularization is performed by various methods, including but not limited to exposure to hypertonic saline, peracetic acid, TRITON-X® or other detergents. Sterilization and decellularization may be simultaneous. For example, but not limited to, sterilization with peracetic acid as described above can also serve to decellularize the ECM. The decellularized ECM is then dried, lyophilized (freeze-dried) or air-dried. The dried ECM can be pulverized by methods including, but not limited to, breaking, milling, cutting, grinding, and shearing. The pulverized ECM can be further processed into a powdered form, including but not limited to, grinding or milling in a frozen or freeze-dried state. To prepare solubilized ECM tissue, the pulverized ECM is digested with acid proteases in an acidic solution to form a digestion solution.

[0064] The ECM digestion solution is typically kept under constant agitation at room temperature for a certain amount of time. The ECM digest may be used immediately or stored at -20°C or frozen, for example, but not limited to, at -20°C or -80°C.

[0065] Once the ECM is solubilized (typically substantially completely), the pH of the solution is raised to between 7.2 and 7.8, according to one embodiment to pH 7.4. A base, such as a base containing hydroxide ions, including NaOH, can be used to raise the pH of the solution. Similarly, a buffer, such as an isotonic buffer, including but not limited to phosphate buffered saline (PBS), can be used to bring the solution to a target pH or to help maintain the pH and ionic strength of the gel at a target level, e.g., physiological pH and ionic conditions. This forms a "pre-gel" solution. The neutralized digestion solution (pre-gel) can be gelled at a temperature approaching physiological temperature, approaching 37°C. This method typically does not include a dialysis step prior to gelation, and typically results in a more intact ECM-like matrix that gels at a specific rate at 37°C (see below).

[0066] Thus, the ECM can typically be derived from one of mammalian tissues, such as, but not limited to, the bladder, esophagus, or small intestine. The ECM hydrogel can be produced from two or more tissue sources, such as, for example, two, three, or four tissue sources. In a non-limiting embodiment, the ECM is freeze-dried and pulverized. The ECM is then solubilized by an acidic protease in an acidic solution to produce digested ECM, such as, for example, esophageal ECM. The acidic protease can be, but is not limited to, pepsin or trypsin, or a combination thereof. The ECM can then be solubilized at an appropriate or optimal acidic pH for the protease, such as, for example, greater than about pH 2 or between pH and 4, for example, in a 0.01M HCl solution. The solution is typically solubilized by mixing (stirring, swirling, blending, rolling, tilting, etc.) for about 12 to about 48 hours, depending on the tissue type (see, e.g., Examples below). The ECM hydrogel is prepared by (i) comminuting the extracellular matrix, (ii) solubilizing the intact, undialyzed or uncrosslinked extracellular matrix by digestion with an acidic protease in an acidic solution to produce a digested solution, (iii) increasing the pH of the digested solution to a pH between 7.2 and 7.8 to produce a neutralized digested solution (pre-gel solution), and (iv) gelling the solution within the esophagus of a subject of interest at a temperature of approximately 37° C.

[0067] The ECM hydrogel forms a gel when exposed to a temperature of about 37°C. The ECM hydrogel in the "pregel" form can be frozen and stored, for example, but not limited to, at -20°C or -80°C. The ECM hydrogel in the "pregel" form can be stored at room temperature, for example, about 25°C. Thus, the ECM hydrogel is in the pregel form at temperatures below 37°C, for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4°C. The ECM hydrogel can be frozen for storage and thus stored below 0°C. As used herein, the term "pregel form" or "pregel" refers to an ECM hydrogel that has an elevated pH but has not gelled. For example, but not limited to, the ECM hydrogel in the pregel form has a pH between 7.2 and 7.8. The ECM hydrogel can be delivered in a pregel form orally, via a catheter, or endoscopically to a subject with esophageal inflammation.

[0068] The ECM hydrogel in pregel form is suitable for introduction into the esophagus of a patient. When introduced into the esophagus, which is approximately 37°C, the ECM hydrogel gels and coats the esophagus. Without being bound by theory, the ECM hydrogel contains many native soluble factors, including but not limited to cytokines. Specific characteristics of non-dialyzed (whole ECM) preparations prepared from various tissues, such as the esophagus, are disclosed herein. The hydrogel gels with kinetics such that the ECM hydrogel can be administered orally, endoscopically, or via a catheter to the esophagus, and then the hydrogel gels in the esophagus and coats the esophagus.

[0069] In some embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation at a temperature of about 37° C. of less than 30 minutes; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of i) about 10 to about 300 Pascals (Pa), ii) about 10 to about 450 Pa, iii) about 10 to about 600 Pa, iv) about 5 to about 1,000 Pa, v) about 10 to 1,000 Pa, or vi) about 10 to about 70 Pa.

[0070] In embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation at a temperature of about 37° C. is less than 30 minutes; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of about 10 to about 300 Pascals (Pa). In other embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation at a temperature of about 37° C. is less than 30 minutes; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of about 10 to about 450 Pascals (Pa). In other embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation at a temperature of about 37° C. is less than 30 minutes; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of about 10 to about 600 Pascals (Pa).

[0071] In other embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation at a temperature of about 37° C. is less than 30 minutes; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of about 5 to about 1,000 Pascals (Pa). In other embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation at a temperature of about 37° C. is less than 30 minutes; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of about 10 to about 1,000 Pascals (Pa). In more embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation at a temperature of about 37° C. is less than 30 minutes; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of 10 to 70 Pascals (Pa).

[0072] In a specific, non-limiting example, the ECM hydrogel is an esophageal hydrogel. In another specific, non-limiting example, the ECM hydrogel can be generated from two or more tissue sources. In a further non-limiting example, the ECM hydrogel can be generated from the bladder or small intestine.

[0073] In a further specific non-limiting example, the ECM hydrogel is produced by (a) solubilizing the acellular extracellular matrix (ECM) by digestion of tissue with acidic proteases in an acidic solution to produce digested esophageal ECM; (b) increasing the pH of the digested ECM to a pH between 7.2 and 7.8 to produce a neutralized digestion solution; (c) diluting the digested ECM to an ECM hydrogel at a concentration of about 2 mg / ml to about 16 mg / ml, for example, about 8 mg / ml to about 12 mg / ml. This hydrogel is then introduced into the subject's esophagus and allowed to gel. The ECM may be esophageal ECM.

[0074] The ECM hydrogels used in the methods disclosed herein have a time to 50% gelation of less than 30 minutes, e.g., less than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1, 10, 9, 8, 7, 6, 5, 4, 3 minutes, at a temperature of about 37° C. In some embodiments, the ECM hydrogel has a time to gelation of less than 10 minutes at a temperature of about 37° C. In other embodiments, the time to 50% gelation is about 3 to about 30 minutes at a temperature of about 37° C. In further embodiments, the time to 50% gelation is about 4 to about 10 minutes at a temperature of about 37° C. In yet other embodiments, the time to 50% gelation is about 5 to about 10 minutes or about 10 to about 20 minutes at a temperature of about 37° C.

[0075] The ECM hydrogels of the present disclosure can have a flow viscosity suitable for injection into the esophagus. In some embodiments, the ECM hydrogel has a flow viscosity of about 10 to about 100 Pa at a shear rate of 0.2 / s. * s, e.g., about 10, 20, 30, 40, 50, 60, 70, 80, or 90 Pa at a shear rate of 0.2 / s* In a further embodiment, the ECM hydrogel has a flow viscosity of about 1 to about 40 Pa at a shear rate of 0.1 / s. * s, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 Pa at a shear rate of 0.1 / s * It has a flow viscosity of s.

[0076] In other embodiments, the ECM hydrogel has a shear strength of about 0.01 to about 0.20 Pa at a shear rate of 1000 / s. * s, or 1000 / s shear rate, from about 0.01 to about 0.10 Pa * s, for example, at a shear rate of 1000 / s, the flow viscosity is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.19 or 0.2.

[0077] In more embodiments, the ECM hydrogel has a shear strength of about 0.02 to about 0.8 Pa at a shear rate of 100 / s. * s, or a shear rate of 100 / s, from about 0.1 to about 0.8 Pa * s, for example, about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.08 Pa * Has s.

[0078] In a further embodiment, the ECM hydrogel has a shear strength of about 10 to about 100 Pa at a shear rate of 0.2 / s. * s and a shear rate of 1000 / s, from about 0.01 to about 0.10 Pa * In more embodiments, the ECM hydrogel has a flow viscosity of 1 to 40 Pa at a shear rate of 0.1 / s. * s and 1000 / s shear rates from 0.01 to 0.2 Pa * It has a flow viscosity of s.

[0079] In other embodiments, the ECM hydrogel has a shear rate of about 1 to about 40 Pa at a shear rate of 1 / s. * s, for example, 1 to about 30 Pa * s, or 1 to about 20 Pa * s, or 1 to about 10 Pa * s, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, or 9 Pa at a shear rate of 1 / s * The shear rate may be, for example, 10, 20, 30 or 40 Pa at a shear rate of 1 / s. * In other embodiments, the ECM hydrogel has a flow viscosity of about 0.05 to about 0.20 at a shear rate of 100 / s, e.g., about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, or 0.2 at a shear rate of 100 / s. The flow viscosity can be about 0.1 to about 25 Pa at a shear rate of 1 / s. * s, and at a shear rate of 100 / s, from about 0.02 to about 0.8 Pa * In additional embodiments, the flow viscosity is from about 1 to about 10 Pa at a shear rate of 1 / s. * s, from about 0.05 to about 0.20 at a shear rate of 100 / s.

[0080] In a further embodiment, the ECM hydrogel has a shear strength of about 10 to about 100 Pa at a shear rate of 0.2 / s. * In other embodiments, the ECM hydrogel has a flow viscosity of about 0.01 to about 0.10 Pa at a shear rate of 1000 / s. * In other embodiments, the ECM hydrogel has a flow viscosity of about 1 to about 40 Pa at a shear rate of 0.1 / s. * s and a flow viscosity of 0.01 to 0.2 Pa at a shear rate of 1000 / s * It is s.

[0081] The ECM hydrogels of the present disclosure have a stiffness of i) about 10 to about 300 Pascals (Pa), ii) about 10 to about 600 Pa, iii) about 5 to about 1,000 Pa, iv) about 10 to 1,000 Pa, or v) about 10 to about 70 Pa. The ECM hydrogels can have a stiffness of about 10 to about 300 Pascals (Pa), e.g., about 10 to about 70 Pa, about 10 to about 100 Pascals (Pa), or about 10 to about 150 Pa, about 10 to about 200 Pa, or about 10 to about 250 Pa. In some embodiments, the ECM hydrogels of the present disclosure have a stiffness of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 Pa. In other embodiments, the ECM hydrogels of the present disclosure have a stiffness of about 10 to about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 Pa. In further embodiments, the ECM hydrogels of the present disclosure can have a stiffness of about 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 Pa.

[0082] In some embodiments, the ECM concentration in the hydrogel is about 2 mg / ml to about 20 mg / ml, for example, about 8 mg / ml to about 12 mg / ml or about 2 mg / ml to about 16 mg / ml. In other embodiments, the ECM concentration in the hydrogel is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 mg / ml. Exemplary concentrations of use include, but are not limited to, about 9 mg / ml to about 11 mg / ml, and about 10 mg / ml to about 12 mg / ml. Additional exemplary concentrations include about 8 mg / ml to about 10 mg / ml, about 8 mg / ml to about 11 mg / ml, about 8 mg / ml to about 13 mg / ml, about 8 mg / ml to about 14 mg / ml, about 8 mg / ml to about 15 mg / ml, and about 8 mg / ml to about 16 mg / ml. Further exemplary concentrations of use include about 6 mg / ml to about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml or about 16 mg / ml.

[0083] The ECM hydrogel of the present disclosure may be provided as a component of a kit. The ECM hydrogel may be provided in frozen or lyophilized form. In some embodiments, the kit may include components required to form a hydrogel, such as one container containing the hydrogel in lyophilized form, one container containing a solution to solubilize the lyophilized hydrogel, and optionally a container containing a neutralizing solution to neutralize the solubilized form. In other embodiments, the kit may include a container containing the solubilized hydrogel and a second container containing a neutralizing agent.

[0084] Optionally, such kits include additional components, including packaging, instructions, and various other reagents, such as buffers, substrates, or other therapeutic ingredients. The kits can include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers may be formed from a variety of materials, such as glass or plastic. The containers typically hold a composition comprising an ECM hydrogel, such as a frozen or lyophilized form, that is effective for inhibiting esophageal inflammation and / or alleviating the effects of esophageal inflammation in a subject. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a particular condition, such as Barrett's esophagus.

[0085] The label or package insert will typically further include instructions for use. Package inserts typically include instructions that are customarily included in the commercial packaging of such therapeutic products, including directions, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. Instruction materials may be written in electronic form (such as computer diskette or compact disk) or may be visual (such as video file). Kits may also include additional components, such as needles or catheters, that facilitate the particular application for which the kit is designed. Kits may further include buffers and other reagents routinely used in the practice of a particular method. Kits and suitable contents are well known to those skilled in the art.

[0086] Treatment Methods for treating esophageal inflammation are disclosed herein. Methods for reducing esophageal strictures are also disclosed. Without being bound by theory, ECM hydrogels allow for the treatment of early stage neoplastic and dysplastic esophageal diseases. The hydrogels of the present disclosure can be used to treat both pre-neoplastic and neoplastic esophageal diseases. The versatility of the hydrogels of the present disclosure allows them to be used to treat esophageal burns, ulcerations, and other pathologies that may extend throughout multiple unconnected areas of the esophagus. The hydrogels of the present disclosure are also useful for treating long fragments or scattered esophageal wounds without the need for the use of stents or invasive techniques. In these conditions, the use of sheet-form ECM is not feasible, as multiple stents and ECM sheets would be required across the esophageal surface. Furthermore, the sheet and hydrogel forms of ECM have more significant differences with respect to the active components. Any of the hydrogels of the present disclosure are useful in these treatment methods. A person skilled in the art, e.g., a skilled physician, can easily identify the therapeutic efficacy.

[0087] Thus, subjects with esophageal inflammation can be selected. Subjects with or at risk of having esophageal stricture can also be selected.

[0088] In some embodiments, the subject does not show symptoms of esophageal adenocarcinoma (EAC) (e.g., does not have EAC and / or does not have previous gastroesophageal reflux disease (GERD) or Barrett's esophagus), but is apparently healthy, such as a subject with esophageal inflammation. In some examples, a healthy subject is a subject that, when examined by a medical professional, is characterized as healthy and does not have symptoms such as GERD. However, the subject has esophageal inflammation, as determined, for example, by endoscopic administration. In some embodiments, the method of the present disclosure inhibits this inflammation.

[0089] In other embodiments, the subject has GERD and / or Barrett's esophagus. In a specific non-limiting example, the subject can use acid reducing drugs such as proton pump inhibitors or histamine antagonists to suppress gastroesophageal discomfort. The subject may increase risk by smoking and / or alcohol use. The subject may have low-grade dysplasia or high-grade dysplasia of the esophagus. In some embodiments, the subject does not have esophageal adenocarcinoma. However, the subject may be at risk for esophageal adenocarcinoma.

[0090] In some embodiments, the method inhibits or reverses the development of esophageal neoplasia in a subject. In other embodiments, the method restores the epithelial barrier of the esophagus of a subject. In further embodiments, the method increases the chemotaxis of both epithelial cells and / or stem cells to the site of injury in the esophagus of a subject. In further embodiments, the method inhibits the development of esophageal adenocarcinoma. In other embodiments, the treatment allows the subject to reduce or avoid the use of proton pump inhibitors and / or histamine antagonists. However, the methods of the present disclosure can be used in conjunction with proton pump inhibitors and / or histamine antagonists.

[0091] In further embodiments, the treatment reduces stenosis as compared to a control, such as a subject not treated with ECM hydrogel. The treatment can increase the circumference of the esophagus as compared to a control, such as a subject not treated with ECM hydrogel.

[0092] The ECM hydrogels disclosed herein are maintained at or below gelation, for example, at room temperature (e.g., about 25° C.) or below room temperature. The ECM hydrogel can be maintained, for example, at 25° C. or 4° C. prior to administration. An effective amount of the ECM hydrogel in pregel form is then administered to the subject's esophagus. The ECM hydrogel can be administered orally, such that the hydrogel is swallowed by the subject and gels upon delivery to the esophagus. The ECM hydrogel can be administered directly to the esophagus using either a catheter or by endoscopic administration. The ECM hydrogel gels in the subject's esophagus, which is at a temperature of approximately 37° C. In some embodiments, about 5 to about 60 ml of ECM hydrogel, for example, about 10 ml to about 30 ml of ECM hydrogel, for example, about 10, 15, 20, 25, or 20 ml of ECM hydrogel, is administered to the subject. The ECM hydrogel can be provided in a lyophilized or frozen form and reconstituted immediately prior to administration to the subject.

[0093] The disclosed method includes administering a therapeutically effective amount of an ECM hydrogel in pre-gel form, as disclosed herein, to the esophagus of a subject, such as, but not limited to, a subject having esophageal inflammation or a subject having or at risk for stenosis, and allowing the hydrogel to gel within the subject's esophagus. In some embodiments, the ECM hydrogel has a) a time to 50% gelation of less than 30 minutes at a temperature of about 37° C.; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of 10-70 Pascals (Pa). However, any of the above disclosed hydrogels can also be utilized. The ECM hydrogel, in some embodiments, can be derived from any mammalian tissue, such as, but not limited to, porcine or human tissue, and in some non-limiting examples, from the bladder, small intestine, or esophagus. Any of the above disclosed hydrogels can be used for the treatment of inflammation of the esophagus of a subject and / or to restore the epithelial barrier within the esophagus. Any of the above disclosed hydrogels can also be used to treat stenosis. In some embodiments, localized delivery to the surface avoids any undesirable side effects. In a specific, non-limiting example, the subject has or is at risk for having Barrett's esophagus.

[0094] The hydrogels of the present disclosure can be administered locally to the esophageal mucosa in pregel form. The hydrogels can be administered to the lumen of the esophagus and coat the surface using non-invasive application methods. In some embodiments, application is oral, such as by swallowing the hydrogel in pregel form. In other embodiments, application can be by tube feeding, where the hydrogel in pregel form is placed at the desired location. In some embodiments, the hydrogel coats the mucosa and does not invade the underlying submucosa or muscle tissue.

[0095] Those skilled in the art can easily formulate the hydrogel so that the pregel form is swallowed by the subject being treated. In yet another embodiment, the hydrogel is provided in pregel form by endoscopy to ensure that the medicament is delivered locally, for example, specifically to the area of ​​the esophagus that requires treatment. For example, local delivery of the hydrogel may be via endoscope / gastroscope. Generally, delivery is local to the esophageal mucosa to non-invasively deliver the pregel form of the hydrogel. The hydrogel gels at the surface and covers the desired area of ​​the mucosa. In some embodiments, the ECM hydrogel gels and provides a protective barrier that protects the mucosa.

[0096] In some embodiments, esophagogastroduodenoscopy (EGD) or upper gastrointestinal endoscopy can be performed on the subject of interest. These procedures can be performed before applying hydrogel to select the subject of interest. These procedures can also be performed according to the use of the method of the present disclosure to evaluate the effect on the subject and to determine whether additional application is required.

[0097] Exemplary embodiments Item 1. A method for inhibiting inflammation of the esophagus or reducing esophageal stricture in a subject, comprising administering to the esophagus of a subject having esophageal inflammation a therapeutically effective amount of an extracellular matrix (ECM) hydrogel having the following characteristics: a) a time to 50% gelation at a temperature of about 37° C. is less than 30 minutes; b) a flow viscosity suitable for injection into the esophagus; and c) a stiffness of i) about 10 to about 300 Pascals (Pa), ii) about 10 to about 450 Pa, iii) about 10 to about 600 Pa, iv) about 5 to about 1,000 Pa, v) about 10 to 1,000 Pa, or vi) about 10 to about 70 Pa, thereby inhibiting inflammation of the esophagus in the subject or reducing esophageal stricture.

[0098] Item 2. The method according to item 1, wherein the time to 50% gelation is about 3 to about 30 minutes at about 37°C.

[0099] Item 3. The method according to item 1, wherein the time to 50% gelation is about 3 to about 10 minutes at about 37°C.

[0100] Item 4. The method according to item 2, wherein the time to 50% gelation is from about 4 to about 10 minutes.

[0101] Item 5. The flow viscosity is about 1 to about 40 Pa at a shear rate of about 0.1 / s. * s, and at a shear rate of 1000 / s, from about 0.01 to about 0.2 Pa * 5. The method according to any one of items 1 to 4, wherein

[0102] Item 6. The flow viscosity is about 0.1 to about 25 Pa at a shear rate of 1 / s. * s, and at a shear rate of about 100 / s, from about 0.02 to about 0.8 Pa * 5. The method according to any one of items 1 to 4, wherein

[0103] Item 7. The method of any one of items 1 to 6, wherein the ECM hydrogel has a stiffness of 10 to 70 Pa.

[0104] Item 8. The method of any one of items 1 to 7, wherein the ECM hydrogel is an esophageal ECM hydrogel.

[0105] Clause 9. The method of any one of clauses 1 to 8, wherein the ECM concentration of the hydrogel is from 2 mg / ml to about 16 mg / ml.

[0106] Clause 10. The method of any one of clauses 1 to 9, wherein the ECM hydrogel is administered orally, endoscopically or via a catheter.

[0107] Item 11. The ECM hydrogel, (a) solubilizing a decellularized extracellular matrix (ECM) by digestion of the tissue with an acid protease in an acidic solution to generate a digested esophageal ECM; and (b) increasing the pH of the digested esophageal ECM to a pH between 7.2 and 7.8 to generate a neutralized digestion solution. 11. The method of any one of items 1 to 10, wherein the compound is produced by

[0108] Clause 12. The method of clause 11, wherein (b) increasing the pH of the digested ECM comprises adding a base or an isotonic buffer to increase the pH of the digested ECM.

[0109] Item 13. The method of item 10 or 11, wherein the acid protease is pepsin, trypsin, or a combination thereof.

[0110] Clause 14. The method of any one of clauses 1 to 13, wherein the ECM hydrogel is maintained at or below 25° C. prior to administration to the subject.

[0111] Clause 15. The method of any one of clauses 1 to 14, wherein the subject has or is at risk for Barrett's Esophagus.

[0112] Clause 16. The method of clause 15, wherein development of an esophageal neoplasm in said subject is inhibited.

[0113] Clause 17. The method of any one of clauses 1 to 16, wherein the ECM hydrogel restores an epithelial barrier in the esophagus of the subject.

[0114] Clause 18. The method of any one of clauses 1 to 17, wherein the ECM hydrogel increases chemotaxis of both epithelial cells and / or stem cells to a site of injury in the esophagus of the subject.

[0115] Clause 19. The method of any one of clauses 1 to 18, wherein the ECM hydrogel reduces esophageal stricture in the subject.

[0116] Item 20. A composition comprising an extracellular matrix (ECM) hydrogel, the ECM hydrogel having the following characteristics: a) a time to 50% gelation at about 37°C of less than 10 minutes; b) a flow viscosity sufficient for injection into the esophagus; and c) a stiffness of i) about 10 to about 300 Pascals (Pa), ii) about 10 to about 450 Pa, iii) about 10 to about 600 Pa, iv) about 5 to about 1,000 Pa, v) about 10 to 1,000 Pa, or vi) about 10 to about 70 Pa, wherein the composition is formulated for administration to the esophagus.

[0117] Item 21. The composition according to item 20, wherein the time to 50% gelation is, at about 37° C., a) about 3 to about 30 minutes; b) about 4 to about 10 minutes; or c) about 3 to about 10 minutes.

[0118] Item 22. The composition of item 20 or 21, wherein the hydrogel has a stiffness of about 10 to about 70 Pa.

[0119] Clause 23. The composition of any one of clauses 20 to 22, comprising about 2 mg / ml to about 16 mg / ml of the ECM hydrogel.

[0120] Item 24. The ECM hydrogel, (a) solubilizing decellularized extracellular matrix (ECM) by digestion of esophageal tissue with acid proteases in an acidic solution to generate digested esophageal ECM; (b) increasing the pH of the digested esophageal ECM to a pH between 7.2 and 7.8 to produce a neutral digestion solution; and (c) diluting the digested esophageal ECM to a concentration of about 8 mg / ml to about 12 mg / ml of the ECM hydrogel. 24. The composition according to any one of claims 21 to 23, produced by

[0121] Clause 25. The composition of clause 24, wherein (b) increasing the pH of the digested ECM comprises adding a base or an isotonic buffer to increase the pH of the digested esophageal ECM.

[0122] Item 26. The composition of item 24 or 25, wherein the acid protease is pepsin, trypsin, or a combination thereof.

[0123] Clause 27. The composition of any one of clauses 20 to 26, wherein the ECM hydrogel is maintained at or below 25°C.

[0124] Clause 28. The composition of any one of clauses 20 to 27 for use in inhibiting inflammation of the esophagus in a subject.

[0125] Clause 29. The composition of any one of clauses 20 to 27 for use in restoring an epithelial barrier in the esophagus of a subject.

[0126] Item 30. The composition of item 28 or 29, wherein the subject has Barrett's esophagus.

[0127] Clause 31. The composition of any one of clauses 20 to 27 for reducing esophageal stricture in a subject.

[0128] Item 32. A kit comprising: a) a container containing the composition according to any one of items 20 to 31, or a lyophilized form thereof; and b) instructions for using the composition.

[0129] Item 33. A composition for use in any one of the methods of items 1 to 19.

[0130] The present disclosure is illustrated by the following non-limiting examples. EXAMPLES

[0131] Mucosal inflammation, or mucositis, is an inflammatory condition characterized by swelling, irritation, and discomfort of the mucosal lining of the digestive tract. Mucositis can result in erosions or ulcers that can be present throughout the digestive tract. As an inflammation of the mucosal lining, often accompanied by infection and / or ulceration, mucositis is often a severe and painful condition. It is disclosed herein that extracellular matrix (ECM) hydrogels are effective therapeutic agents for treating mucosal inflammation, such as inflammation of the esophagus. ECM hydrogels can provide a protective barrier against continued trauma to the mucosa, promote an anti-inflammatory environment, and / or facilitate the repair of damaged and inflamed mucosa.

[0132] Example 1 Viscoelastic properties of hydrogels Rheology was performed on homogeneous esophageal ECM (eECM) hydrogels with various ECM concentrations (4–16 mg / mL). Samples were placed in the rheometer at 10 °C, well below the gelation temperature, and steady-state flow tests (shear rates from 0.1 to 1000 1 / s) were performed to determine the viscosity profile of the ECM pregel (Figure 1).

[0133] At each shear rate, the viscosity increases with increasing ECM concentration, and the ECM pregel is shear thinning (viscosity decreases with increasing shear rate). Shear thinning is a good property for an ECM pregel that can be injected through a catheter, which can experience a range of shear rates from 10 to 1000 1 / s. Further evidence of injectability was obtained with video, which showed that the ECM pregels (esophageal ECM and UBM, 8-12 mg / mL tested stained blue) were all injectable (5fr size, approximately 15.9 G) by oral gavage. The temperature was then rapidly increased to 37 °C to induce gelation, and a time sweep (oscillating strain of 0.5%) was performed at 37 °C to measure gel stiffness (Figure 2) and gelation time (Figure 3). Figure 2 shows that the storage modulus (G') or "stiffness" of the formed ECM hydrogel increases as the ECM concentration increases. A similar trend was observed for the loss modulus (G''), or viscous component, of the formed ECM hydrogels. The time to 50% gelation was measured during the time sweep study (Figure 3). eECM exhibits a concentration-dependent gelation time, i.e., gelation time decreases with increasing ECM concentration.

[0134] Rheology was performed on two homogenous ECM hydrogels: urinary bladder matrix ECM (UBM) and skin ECM (MIRM5). Figures 4-6 show the viscoelastic properties compared to a homogenous esophageal ECM hydrogel (eECM). [Table 1]

[0135] Steady shear testing was performed similarly as described for Figure 1. Skin ECM (Figure 4B) and UBM (Figure 4C) show a concentration-dependent increase in viscosity with increasing ECM concentration and a shear-thinning profile for the ECM hydrogels, i.e., viscosity decreases with increasing shear rate for each ECM concentration. Viscosity ranges were specific for each tissue type.

[0136] The UBM ECM (Figure 5C) showed an increase in storage modulus (stiffness) with increasing ECM concentration, similar to the esophageal ECM (Figure 5A). The skin ECM (Figure 5B) did not form a hydrogel at concentrations as low as 4 mg / mL, demonstrating that not all ECM hydrogels derived from different tissue sources behave similarly. The stiffness ranges of the three ECM hydrogels were unique.

[0137] Skin ECM (FIG. 6B) exhibited a concentration-dependent gelation time, i.e., gelation time decreased as ECM concentration increased, similar to esophageal ECM (FIG. 6A), whereas UBM exhibited a concentration-independent gelation time, i.e., gelation time remained constant for all ECM concentrations (FIG. 6C). The unique gelation profiles further demonstrate the variability of ECM hydrogels derived from different tissue sources. Thus, esophageal hydrogels offer unique properties and are useful at a variety of concentrations, including but not limited to, 8 mg / ml to about 12 mg / ml.

[0138] Example 2 Treating mucosal inflammation with ECM hydrogel Mucosal inflammation, or mucositis, is an inflammatory condition characterized by swelling, irritation, and discomfort of the mucosal lining of the digestive tract. Mucositis can result in ulcers that may be present throughout the digestive tract. As an inflammation of the mucosal lining, often accompanied by infection and / or ulceration, mucositis is often a severe and painful condition.

[0139] Mucositis often occurs as a complication of chemotherapy or radiation therapy for cancer, for example. The purpose of radiation and chemotherapy in cancer treatment, i.e., killing rapidly dividing cancer cells, also affects epithelial cells in mucosal lining areas, such as the digestive tract, causing mucositis. Exposure to radiation and / or chemotherapy significantly disrupts the cellular integrity of the mucosal epithelium and underlying connective tissue, often causing inflammation, infection and / or ulceration at mucosal sites, such as in the esophagus and other parts of the Gl tract.

[0140] Extracellular matrix (ECM) hydrogels are therapeutic agents for treating mucosal inflammation. Without being bound by theory, the mechanism by which ECM supports mucosal healing is (1) by forming a hydrogel that provides a protective barrier against continued trauma to the mucosa, (2) by promoting an anti-inflammatory environment, and / or (3) by facilitating the repair of damaged and inflamed mucosa. The properties of the esophageal hydrogel were investigated as disclosed in Example 1.

[0141] Macrophages, when exposed to ECM hydrogels, induce a secreted cytokine profile that is primarily anti-inflammatory, including elevated levels of PGE2 (Figure 7). The levels of anti-inflammatory cytokines vary depending on the tissue from which the ECM is derived. Esophageal hydrogels produce a potent anti-inflammatory effect.

[0142] Mucosal repair requires not only the reduction of inflammation but also the re-establishment of epithelial and / or physical barriers. ECM hydrogels can promote the restoration of epithelial barriers by increasing the chemotaxis of both epithelial cells and stem cells. Secretory products of macrophages exposed to ECM facilitate the migration of epithelial cells (Figure 8A). Furthermore, ECM hydrogels directly promote the chemotaxis of esophageal stem cells, and these effects vary depending on the source tissue from which the ECM is derived (Figure 8B). Esophageal stem cells preferentially migrated to the ECM of the esophagus and the ECM of the small intestine. Figure 9 shows an exemplary treatment of Barrett's esophagus.

[0143] Figure 10A shows the effect 40 minutes after oral administration, where the hydrogel remains coated on the mucosa and can be identified despite normal swallowing. This was done to test the mucoadhesiveness of the gel in vivo to confirm that oral swallowing was effective. As shown in Figure 10B, a catheter and an endoscope were used to deliver the hydrogel to a specific location (in this case, in the shape of a ring) in the esophagus. Thus, the hydrogel was delivered to a specific local location in the esophagus.

[0144] Figure 11 also shows the effect of hydrogel treatment. The top row shows the results of at least 3 months of constant reflux in animals with esophageal inflammation. On the left, three dogs were treated with omeprazole (a proton pump inhibitor) and esophageal ECM hydrogel. The last animal (right panel) was treated with omeprazole only. After 30 days of treatment, improvement of esophageal inflammation can be seen in the three animals that received ECM hydrogel. Animals that received only omeprazole showed no improvement (grey box, right panel).

[0145] 12A-12B show the safety evaluation of ECM hydrogel. Animals underwent a procedure to increase acid reflux to induce esophagitis and then Barrett's esophagus. None of the eight animals lost weight after surgery and did not induce BE during treatment with hydrogel (FIG. 12A). Physiological parameters were analyzed. After 30 days of twice daily administration of hydrogel, the physiological parameters of the animals were stable and did not exceed the normal range (FIG. 12B).

[0146] In the animal model prior to treatment with ECM, both dogs that would be treated with ECM hydrogel and dogs that were not treated with ECM hydrogel developed columnar metaplasia (Figures 13A and 13B). After treatment with ECM + omeprazole, the treated dogs improved and no columnar metaplasia was observed (Figure 13C). In control animals not treated with ECM hydrogel, columnar metaplasia with compartments of squamous epithelium was present over the same area (Figure 13D).

[0147] Example 3 Treating strictures with ECM hydrogel material and method Ample peripheral mucosal resection with a longitudinal width of 5 cm was performed in two mongrel dogs using a combined EMR and ESD technique (Nieponice, 2009, p. 18657808). Treatments evaluated were administration of UBM hydrogel twice daily and untreated control. Endoscopy was performed if animals showed any clinical signs of stenosis or reached 1 month after the procedure. Dilation was performed if possible and necessary according to the endoscopic findings of the animals. Animals were euthanized if they showed severe stenosis or reached the 2 month time point after balloon dilation. At necropsy, animal tissues were measured to determine stenosis and samples were collected for histological analysis. This animal model allowed for the measurement of the following endpoints: 1. Endoscopic appearance of the cut area 2. Esophageal measurements 3. Final histological assessment

[0148] Surgical procedure and postoperative care Each dog was induced with acepromazine (0.01 mg / kg, SC) and ketamine (5–11 mg / kg), and surgical level anesthesia was maintained with 1–5% isoflurane via an endotracheal tube. After induction, the animals were moved to the operating table and placed in a sterile operating room. Animals were infused with lactated Ringer's solution at 2 ml / kg / h throughout the procedure and observation. Temperature was controlled by a warm water circulating heating pad placed under the animal. Physiological parameters such as heart rate, respiratory rate, temperature, and responsiveness were monitored throughout the procedure. Antibiotic prophylaxis with 25 mg / kg cefazolin was administered before the start of the procedure.

[0149] The animals with the decubitus in supine position were placed and the esophagus was evaluated using a Pentax EG3430K endoscope. The distance from the mouth to the GE junction was measured. After identifying the reference point of the esophagus, the mucosa and submucosa were separated by injecting saline using an Olympus Injectorforce 4mm 23G needle. The entire circumference (100%) of the mucosa with a length of 5 cm was removed using ESD and Loop EMR techniques. The ESD technique was performed by injecting fluid or ECM into the mucosa / submucosa to separate the mucosa from the submucosa, and then the area was excised using the TT knife of the endoscope. To perform EMR, a Cook Duette kit with ligation bands was used. The mucosa was then excised using a snare. A Spot endoscopic marker was used to define the borders of the cut area.

[0150] After removal of the mucosa, 50 mL of 12 mg / mL UBM hydrogel was delivered and applied to the resection area using a MILA EDC190 endoscopic delivery catheter during the procedure. The animals were kept under anesthesia for 5 minutes to allow the hydrogel to gel. After the procedure, the animals were allowed to recover and were kept under observation.

[0151] Following cessation of surgical procedures and inhalation anesthesia, animals were monitored intermittently for 24 hours. Body temperature was determined and recorded every 12 hours. Animals were kept warm and dry to prevent hypothermia and were turned once every 30 minutes until they maintained the sternal position.

[0152] Dogs were kept single case, indoors with other animals, until the animals were stabilized and then placed in regular housing. Buprenorphine (0.005-0.01 mg / kg IM or IV q12h) was administered for pain after each surgical procedure for 5 days and continued if signs of pain were present, and cephalexin (35 mg / kg q12) was administered for 5 days.

[0153] After this procedure, animals were monitored until the end of the study for signs of esophageal stricture such as reduced food consumption, weight loss, and signs of distress as determined by increased breathing patterns, vocalizations, emetic episodes or difficulty swallowing food and / or reduced activity. If these signs were present, animals were evaluated by contrast esophagogram and / or endoscopy.

[0154] Endoscopic monitoring and balloon dilation One month after the initial surgical procedure, animals underwent an endoscopy procedure if any clinical signs of stenosis were present. Additionally, all animals underwent an endoscopy procedure before euthanasia. Anesthesia was induced with acepromazine (0.1–0.5 mg / kg) and maintained with isoflurane (1–5%), and endoscopy was performed.

[0155] If during endoscopy, the animal was diagnosed with mild or moderate stenosis, balloon dilation was performed. An Olympus 20 mm balloon dialator was used to perform the dilation procedure. Under endoscopic guidance, the balloon was inflated with approximately 10 mL of sterile 0.9% NaCl until a moderate or significant amount of resistance could be identified and kept inflated for 30-60 seconds. After dilation, the MILA EDC Using a 190 catheter, 50 mL of ECM was immediately applied to the injured area and allowed to gel for 5 minutes. Following this procedure, animals were denied access to food or water for at least 1 hour.

[0156] Delivery of ECM ECM was delivered orally to animals using a 60 ml catheter-tipped syringe at 15° C. 50 ml was delivered twice daily from day 0 until completion of the study. Animals were not allowed to eat or drink for 1 hour after each delivery of hydrogel.

[0157] autopsy At the time of necropsy, endoscopy was performed as previously described. Euthanasia was performed by administering sodium pentobarbital IV (390 mg / kg BW) under anesthesia. After death was confirmed, the esophagus was retrieved, maintaining the same dimensions it had in the body. Esophageal measurements were obtained at 0.5 cm intervals and recorded.

[0158] The strictures were treated with ECM hydrogel. As shown in FIG. 14, in a circumferential mucosal resection model (different from the Barrett's esophagus model), animals were treated with ECM hydrogel for up to 81 days. Control animals had severe, untreatable strictures and were euthanized after 14 days. ECM-treated animals had esophageal strictures at 21 days and were dilated. Two months after dilation, animals were sacrificed and esophageal measurements were taken. ECM-treated animals had a wider internal circumference and less loss in circumference when compared to controls.

[0159] As shown in Figure 15, control animals on day 14 showed disorganized collagen deposits with high cellular infiltration and erosion in the center where the mucosal resection had been created, and as shown in Figure 16, treated animals showed re-epithelialization with less cellular infiltration and more organized and dense collagen deposits in the center of the defect.

[0160] In view of the many possible embodiments to which the inventive principles of this disclosure may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. 1. An extracellular matrix (ECM) digestion solution for use in a method of inhibiting esophageal inflammation or treating esophageal stricture in the esophagus of a subject, comprising: a digestion solution of hydrated, decellularized, acid protease digested, intact extracellular matrix, said digestion solution having a pH of 7.2-7.8 and forming a gel when heated to greater than 25° C., said method comprising administering said digestion solution to the esophagus of said subject; thereby inhibiting esophageal inflammation or treating esophageal stricture in said subject; The digestive solution, wherein the extracellular matrix is ​​derived from the urinary bladder, small intestinal submucosa, or esophagus.

2. 10. The digestive solution of claim 1, wherein the digestive solution is terminally sterilized.

3. 10. The digestive solution of claim 1, wherein the digestive solution forms a hydrogel when heated to 37°C.

4. 2. The digestive solution of claim 1, wherein the intact extracellular matrix is ​​derived from a monkey, horse, pig, cow or sheep.

5. 10. The digestive solution of claim 1, wherein the digestive solution is administered orally to the subject by swallowing or tube feeding.

6. The digestive solution of claim 1 , wherein the digestive solution is administered to the esophagus by an endoscope or a catheter.

7. 10. The digestive solution of claim 1, wherein upon administration to the subject at 37°C, the digestive solution forms a hydrogel that coats the mucosa of the esophagus.

8. 10. The digestive solution of claim 1, wherein the subject has Barrett's Esophagus.

9. 2. The digestion solution of claim 1, wherein the acid protease digested ECM is present in the digestion solution at a concentration of 2 mg / mL to 16 mg / mL.

10. 2. The digestive solution of claim 1, wherein the digestive solution is administered at 37°C.

11. 10. The digestive solution of claim 1, wherein the digestive solution is maintained below or at 25°C prior to administration to the subject.

12. 10. The digestive solution of claim 1, wherein the digestive solution is maintained below 37°C prior to administration to the subject.

13. 2. The digestive solution of claim 1, wherein the extracellular matrix is ​​derived from the urinary bladder.

14. 2. The digestive solution of claim 1, wherein the extracellular matrix is ​​derived from the small intestinal submucosa.

15. 2. The digestive solution of claim 1, wherein the extracellular matrix is ​​derived from the esophagus.

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