Extracellular matrix (ECM) hydrogels as submucosal fluid cushions

Abstract

To provide methods for dissecting a mucosa and a submucosa from a muscularis propria from a region of an organ of a subject.SOLUTION: Methods are disclosed for dissecting a mucosa and a submucosa from a muscularis propria from a region of an organ of a subject, wherein the organ is not the esophagus. In some embodiments, the organ is in the gastrointestinal tract. These methods include injecting submucosally into the organ of the subject a pharmaceutical composition comprising an extracellular matrix (ECM) hydrogel to form a cushion between the submucosa and the underlying muscularis propria at the region of the organ, wherein the ECM hydrogel has the following characteristics: a) a time to 50% gelation of less than 30 minutes at a temperature of about 37°C; b) a flow viscosity suitable for infusion into the organ; and c) a stiffness of about 10 to about 400 Pascal (Pa).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 688,198, filed June 21, 2018, which is incorporated herein by reference in its entirety.

[0002] This relates to endoscopic resection, specifically the use of extracellular matrix (ECM) hydrogel as a submucosal cushion to dissociate mucosa and submucosa from the muscularis propria from regions of the organ other than the esophagus. [Background technology]

[0003] Endoscopy is a procedure that allows the examination of the interior or cavity of hollow organs in the body without invasive surgery using an instrument called an endoscope. Endoscopy can be used for surgical procedures such as cauterizing bleeding blood vessels, removing polyps, adenomas, and small tumors, performing biopsies, or removing foreign bodies. Endoscopic procedures can be performed in the gastrointestinal tract, respiratory tract, ear, urinary tract, and female reproductive system, as well as in normally closed body cavities through small incisions, such as the abdominal or pelvic cavity (laparoscopy), the inside of joints (arthroscopy), and the organs of the chest (thoracoscopy and mediastinoscopy). Endoscopy can be performed in the upper or lower gastrointestinal tract. An endoscope is a lighted, usually fiber-optic, flexible or rigid tubular instrument for visualizing the interior of hollow organs or hollow parts (e.g., the bladder, esophagus, stomach, or intestines) for diagnostic or therapeutic purposes, and typically has one or more working channels that allow the passage of instruments (e.g., forceps, electrocautery, endoscopic needles, or scissors) or facilitate the removal of biopsy samples. Endoscopes are equipped with a suitable lamp and imaging device at their distal end and can be inserted through natural body openings, such as the mouth, anus, ear, or nose, or through small surgical incisions. Given the wide variety of body organs or cavities that can be examined by endoscopic procedures, several specialized endoscopes exist, including laryngoscopes, thoracoscopes, angioscopes, colonoscopes, enteroscopes, sigmoidoscopes, rectoscopes, proctoscopes, anoscopes, arthroscopes, nasoscopes, laparoscopes, hysteroscopes, encephaloscopes, nephroscopes, esophagoscopes, bronchoscopes, gastroscopes, amnioscopes, and cystoscopes.

[0004] Endoscopic procedures are widely applied in the gastrointestinal tract, including the upper and lower gastrointestinal tract. For example, endoscopic procedures can be used to examine the mucosa spanning the gastrointestinal cavity and to detect small and large pathological lesions, such as inflammatory tissue, polyps, pseudopolyps, serrated lesions, adenomas, ulcers, dysplasia, preneoplasia and neoplasia, and tumors. Endoscopic procedures can also be used for biopsy and removal of pathological lesions (polyps, adenomas, dysplasia, preneoplasia and neoplasia, tumors). Surgical interventions include two types of endoscopic resection procedures commonly used to remove pathological lesions during gastrointestinal endoscopy: endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD). These two techniques enable minimally invasive treatment of gastrointestinal polyps, adenomas, dysplasia, and early-stage cancers with minimal risk of lymph node metastasis. There remains a need for drugs useful in these procedures. Summary of the Invention [Means for solving the problem]

[0005] Disclosed herein are methods for dissociating mucosa and submucosa from the muscularis propria from a region of an organ of a subject, where the organ is not the esophagus. These methods include submucosally injecting into the organ of the subject a pharmaceutical composition comprising an extracellular matrix (ECM) hydrogel to form a cushion between the submucosa and the underlying muscularis propria in the region of the organ, the ECM hydrogel having the following characteristics: 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 organs, and c) Stiffness of about 10 to about 400 Pascals (Pa) and thereby dissociating the mucosa and submucosa from the underlying muscularis propria and reducing inflammation in the region of the organ.

[0006] In other embodiments, the methods include submucosally injecting a pharmaceutical composition comprising an extracellular matrix (ECM) hydrogel into an organ of a subject to form a cushion between the submucosa and the underlying muscularis propria in a region of the organ, thereby dissociating the mucosa and submucosa from the underlying muscularis propria and suppressing inflammation in the region of the organ.

[0007] The organ can be an organ of the gastrointestinal tract, including the upper or lower gastrointestinal tract. Exemplary organs include, but are not limited to, the stomach, small intestine, large intestine (including the transverse, ascending, or descending colon), or rectum.

[0008] In some embodiments, the method of dissection comprises endoscopic mucosal resection or endoscopic mucosal dissection.

[0009] The foregoing and other features and advantages of the present invention will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0010] [Figure 1-1] Figures 1A-1E show viscoelastic properties. The viscosity profiles of ELEVIEW™ and esophageal (eECM) 12 mg / mL were tested at 10°C with increasing shear rates (0.1-1000 1 / s) (A). The temperature was rapidly raised to 37°C to induce gelation, and the maximum storage (G') and loss moduli (G") were measured (B). Representative graphs of the time sweep are shown for ELEVIEW™ (C) and 12 mg / mL eECM hydrogel (D). The time to 50% gelation was measured for eECM 12 mg / mL, but could not be measured for ELEVIEW™ because ELEVIEW™ did not gel (G" > G' during the time sweep test) (E). [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0011] [Figure 2] 2A and 2B are diagrams showing the mucoadhesion strength of ELEVIEW™ and eECM 12 mg / mL to porcine muscle layer (A) or mucosa (B).

[0012] [Figure 3] Figure 3 shows macrophage activation. Macrophage expression of anti- and pro-inflammatory markers after exposure to eECM and ELEVIEW™.

[0013] [Figure 4-1] 4A-4C show submucosal fluid cushion-colon. Measurement of submucosal fluid cushion elevation over time comparing ECM (eECM) with ELEVIEW™ (A). Tissue appearance after injection and 75 minutes after injection of 2 mL of test agent (B). Dissection and exposure of test agent after 75 minutes (C). [Figure 4-2] Same as above.

[0014] [Figure 5-1] 5A-5C show the submucosal fluid cushion-stomach. Measurement of submucosal fluid cushion elevation over time using eECM or ELEVIEW™ (A). Tissue appearance after injection and 75 minutes after injection of 2 mL of test agent (B). Dissociation and exposure of test agent after 75 minutes. [Figure 5-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0015] Disclosed herein are methods for dissociating mucosa and submucosa from the muscularis propria from a region of an organ of a subject, wherein the organ is not the esophagus. In some embodiments, the organ is in the gastrointestinal tract. These methods include submucosally injecting a pharmaceutical composition comprising an extracellular matrix (ECM) hydrogel into the organ of the subject to form a cushion between the submucosa and the underlying muscularis propria in the region of the organ. The organ may be, for example, the stomach, small intestine, large intestine (including the transverse, ascending, or descending colon), or rectum.

[0016] term Unless otherwise indicated, 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, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.

[0017] Acid protease: An enzyme that cleaves peptide bonds and has increased activity at acidic pH. For example, but not limited to, acid proteases include pepsin and trypsin.

[0018] 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 alkali hydroxide or an aqueous solution of an alkali hydroxide. In certain embodiments, the base is NaOH or NaOH in PBS.

[0019] Comminuting (grinding and grinding): The process of reducing large particles to smaller particles, for example, but not limited to, by grinding, blending, crushing, slicing, milling, cutting, or crushing. The ECM can be milled in any form, including, but not limited to, a rehydrated form, a frozen sheet form, an air-dried sheet form, a freeze-dried sheet form, or a powdered sheet form.

[0020] Colon cancer: Cancer of the large intestine. Tubular adenoma is a type of colon polyp and a precursor to colorectal cancer. Colon cancer may be, for example, colon carcinoid or adenocarcinoma. Colorectal cancer diagnosis is typically performed by sampling areas of the colon suspected of harboring a tumor during colonoscopy or sigmoidoscopy, depending on the location of the lesion.

[0021] Diagnosis: The process of identifying a disease through its signs, symptoms, and the results of various tests. The conclusion reached by this process is also called a "diagnosis." Commonly performed forms of testing include blood tests, medical imaging, and biopsies.

[0022] Dissection: The process of separating or detaching tissue, for example during a surgical procedure.

[0023] Endoscopic Injection Needle or Endoscopic Injection Needle Catheter: A generally long (e.g., up to about 230 cm) device comprising a long catheter with an inner injection tube having a distal injection needle slidably disposed therein. A proximal actuation handle generally connects the catheter and the injection tube for moving one relative to the other. The needle may be retractable. Access to the injection tube for fluid is typically achieved through a Luer connector on the handle.

[0024] The endoscopic injection needle is typically delivered to the injection site through the catheter of the endoscope. To protect the lumen from damage, the handle of the injection needle device is manipulated to retract the distal injection needle into the lumen of the catheter before inserting the device into the endoscope. When the distal end of the endoscopic injection needle device is positioned at the injection site, the handle is manipulated to move the injection needle distally from the lumen of the catheter. In some embodiments, when advanced to its distalmost position, the exposed portion of the injection needle can be approximately 4-6 mm long.

[0025] Endoscopic mucosal resection (EMR): An endoscopic technique developed for the removal of sessile or flat neoplasms confined to the superficial layers (mucosa and submucosa) of the gastrointestinal (GI) tract. The mucosa and submucosa are resected from the underlying muscularis propria. Endoscopic mucosal dissection (ESD) refers to an endoscopic technique developed specifically for the removal of larger lesions, e.g., from the GI tract, in which the mucosa and submucosa are dissected from the other layers of the GI tract. Both EMR and EMD typically involve the injection of a substance beneath the targeted lesion, i.e., between the submucosa and the underlying muscularis propria, that acts as a cushion to elevate the submucosa and overlying mucosa. In EMR, the elevated lesion is then removed using a snare and pulled apart into a small cup by suction. In ESD, the submucosa beneath the lesion is dissected using a specialized knife, causing separation of the submucosa and overlying mucosa. ESD allows for the removal of larger and potentially deeper lesions, with curative intent, than is possible with EMR. Both EMR and ESD are facilitated by the injection of a substance into the submucosal surface of the organ, which effectively separates the overlying mucosa from the underlying muscularis propria and simultaneously elevates the mucosa above the adjacent esophageal mucosa. This separation of layers and elevation of the affected tissue aids the surgeon in isolating, grasping, and removing the tissue of interest.

[0026] Extracellular matrix (ECM): The noncellular component of tissues and organs. Native ECM (ECM found in multicellular organisms such as mammals and humans) is a complex mixture of structural and nonstructural biomolecules, including but not limited to collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors. The specific composition typically varies between different tissues and organs. In mammals, ECM often contains approximately 90% collagen by dry weight in various forms. Biological scaffolds composed of ECM can be created by removing cells from a given tissue or organ, leaving behind the ECM. The composition and structure of ECM vary depending on the anatomical source of the tissue. For example, small intestinal submucosa (SIS), urinary bladder matrix (UBM), esophageal (E), and liver interstitial ECM each differ in overall structure and composition due to the unique cellular niches required for each tissue. Intact "extracellular matrix" and "intact ECM" biological scaffolds consist of extracellular matrix, such as, but not limited to, pulverized ECM as described herein, that is not solubilized and retains its three-dimensional ultrastructure and, ideally, retains the activity of structural and non-structural biomolecules, including, but not limited to, collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors.

[0027] The activity of biomolecules within the ECM can be altered 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 it has not been subjected to digestion, dialysis, and / or cross-linking processes or conditions other than those naturally occurring during storage and handling of the ECM prior to solubilization. Thus, ECM that is dialyzed (in any manner other than insignificantly, which does not substantially affect the gelation and functional characteristics of the ECM in the uses described herein) is not considered "intact."

[0028] Esophagogastroduodenoscopy (EGD) or upper gastrointestinal endoscopy: A diagnostic endoscopic procedure that visualizes any portion of the gastrointestinal tract up to the duodenum. Endoscopy may be performed as part of an EGD or upper gastrointestinal endoscopy, as the case may be. These terms are not mutually exclusive unless expressly stated to be so.

[0029] Gastrointestinal tract: the organ system in mammals that takes in food, digests it, and expels remaining waste products. The oral cavity, pharynx, esophagus, stomach, and duodenum form the upper gastrointestinal tract. The lower gastrointestinal tract includes the small intestine, large intestine (colon), and rectum.

[0030] Gelation: The formation of a gel from a sol.

[0031] 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 relative motion between two surfaces of the fluid that are moving at different speeds. When a fluid passes through a pipe, the particles that make up the fluid generally move faster near the axis of the pipe and slower near the wall of the pipe. Stress (e.g., a pressure difference between the two ends of the pipe) 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 using viscometers and rheometers. Viscosity is measured in Pascal-seconds (Pa * s) Water at 20°C has a viscosity of 1.002 mPa * It has a viscosity of s.

[0032] Hydrogel: A network of hydrophilic polymer chains that may be found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent natural or synthetic polymer networks. Hydrogels also have a degree of flexibility similar to natural tissue. The term "bladder ECM hydrogel" includes UBM and UBS hydrogels.

[0033] Inflammation: A localized response induced by injury to tissue. Inflammation is characterized by the appearance in or migration into any tissue space, unit, or area of ​​white blood cells in numbers that exceed the number of such cells of any class found in that area under normal (healthy) circumstances. Inflammation is formed by the complex biological response of vascular tissues to harmful stimuli such as pathogens, damaged cells, or irritants.

[0034] Isotonic buffered solution: a solution buffered to a pH between 7.2 and 7.8 and having an equilibrium concentration of salt to promote an isotonic environment.

[0035] Low-grade and high-grade dysplasia and metaplasia: Pathological conditions characterized by abnormal cell morphology, but the cell type is still recognizable as squamous epithelium. Generally, in dysplasia, apical mucin is absent from the lining cells of portions of the gastrointestinal tract. With low power detection, these areas may appear more hyperpigmented compared to uninvolved areas.

[0036] In high-grade dysplasia, the changes in cell morphology are more pronounced, but the cells are still technically a type of squamous epithelium.

[0037] When cells change from squamous cells to another cell type, such as glandular cells, which are often cuboidal or columnar in shape, the process is called metaplasia. With metaplasia, distortion of the glandular architecture of the tissue is typically present and may be pronounced, consisting of branching and lateral extension of crypts, a villus-like organization of the mucosal surface, or intraglandular bridging of the epithelium that forms a cribriform pattern of "continuous" glands. Abnormal epithelium with loss of nuclear polarity is present at the mucosal surface, characterized by nuclear "rounding" and the absence of a consistent relationship of nuclei to one another.

[0038] Preventing or Treating: Inhibiting a disease refers to inhibiting the partial or complete development of a disease in a person at risk of the disease, e.g., caused by inflammation. Inhibiting a disease process includes preventing the onset of the disease. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, e.g., after the disease or pathological condition has begun to develop.

[0039] Shear Stress: The component of stress that is in the same plane as the cross section of a material. Shear stress results from the component of the force vector that is parallel to the cross section. The formula for calculating average shear stress is force per unit area

number

[0040] Stiffness: The hardness of an object or fluid. The stiffness of the extracellular matrix is ​​important for guiding cell migration in durotaxis. Stiffness can be measured in Pascals (Pa), which is one Newton per square meter.

[0041] Therapeutic Agent: When used in a general sense, therapeutic agents include treatment agents, preventative 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 disease progression, or cause disease regression. In certain embodiments, disease treatment may begin before a patient exhibits disease symptoms. The disclosed methods suppress esophageal inflammation and / or alleviate the effects of esophageal inflammation.

[0042] 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 symptomatic improvement, reduced progression of attenuation, or to cause disease regression. An amount of ECM hydrogel is therapeutically effective if it is sufficient to achieve the desired effect in a treated subject, for example, to form a gel when injected into the submucosal tissue of an organ, causing the overlying mucosa to dissociate from the underlying muscularis propria.

[0043] The amount effective to form a submucosal cushion depends on the preparation applied, the subject being treated, the severity and type of affliction, and the method of administration. The ECM hydrogels useful in the methods disclosed herein have applications in both medical and veterinary settings. Thus, the general term "subject" or "patient" is understood to include all animals, including, but not limited to, humans or veterinary subjects, such as other primates, dogs, cats, horses, and cattle.

[0044] Bladder ECM: Extracellular matrix derived from any mammalian bladder. This term includes urinary bladder matrix (UBM) ECM and bladder submucosa (UBS) ECM. The bladder wall is composed of the following layers: the mucus layer (including the transitional epithelial layer and the lamina propria), the submucosa layer, and up to three muscle and adventitia layers (loose connective tissue layers)—listed from luminal to abluminal in a thickness section. UBS is prepared from a tissue composition containing bladder submucosa tissue stripped from the abluminal muscle layer and at least the luminal portion of the mucus layer of a segment of a vertebrate bladder. (See U.S. Patent No. 5,554,389, incorporated herein by reference.) UBM ECM is prepared from the bladder epithelial basement membrane and the lamina propria immediately below the basement membrane. See U.S. Patent No. 6,576,265, incorporated herein by reference.

[0045] 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 forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. It should be further understood that all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximations and are provided for illustrative 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." The term "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.

[0046] 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 make 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, 5,762,968, 5,866,869 ... (See Nos. 6, 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, such as 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 ECM.

[0047] The ECM can be derived from any organ or tissue, including, but not limited to, the bladder, intestine, large intestine (colon), liver, esophagus, and dermis. The ECM may also be derived from the kidney, heart, uterus, brain, blood vessels, lung, bone, muscle, pancreas, stomach, spleen, or colon. 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 contain the basement membrane portion of the ECM. In certain embodiments, the ECM comprises at least a portion of the basement membrane. In other embodiments, the ECM is harvested from cell culture. The ECM hydrogel can be made by combining two or more tissue sources.

[0048] As disclosed in U.S. Patent No. 8,361,503 (incorporated herein by reference), bladder ECM, such as porcine bladder ECM, is prepared by abrading bladder tissue using a longitudinal wiping motion with a scalpel handle and moistened gauze to remove the outer (abluminal) layers, including the serosa, muscularis externa, and submucosal layer. After eversion of the tissue segment, the luminal portion of the mucus layer is peeled away from the underlying tissue using the same wiping motion. The serosa, muscularis externa, submucosal layer, and most of the muscularis mucosae can be removed by a combination of enzymatic treatment, hydration, and abrasion. In some embodiments, mechanical removal of these tissues is accomplished by removing mesenteric tissue using, for example, Adson-Brown forceps and Metzenbaum scissors, and wiping away the muscularis and submucosal layers using a longitudinal wiping motion with a scalpel handle or other rigid object wrapped in moistened gauze. In other embodiments, the epithelial cells of the mucus layer can be dissociated by immersing the tissue in a de-epithelialization solution, such as, but not limited to, hypertonic saline. The resulting UBM, which contains the basement membrane of the mucus layer and the adjacent lamina propria, is further treated with peracetic acid, lyophilized, and powdered. See U.S. Patent No. 8,361,503.

[0049] In another embodiment, the ECM is derived from the urinary bladder. A method for producing urinary bladder matrix (UBM) ECM is disclosed in U.S. Patent No. 6,576,265, which is incorporated herein by reference. A method for producing urinary bladder submucosa (UBS) ECM is disclosed in U.S. Patent No. 5,554,389, which is incorporated herein by reference. Both of these types of bladder ECM are useful in the methods disclosed herein. Commercially available preparations that do not contain UBM may also be used (Acell Corporation, Jessup, Md.).

[0050] U.S. Patent No. 6,893,666, incorporated herein by reference, also discloses the preparation of ECM from the bladder, skin, esophagus, and small intestine. The preparation of hydrogels from decellularized dermal ECM is disclosed in Wolf et al., Biomaterials 33: 7028-7038, 2012, incorporated herein by reference. The preparation of ECM from esophageal tissue is described, for example, in Badylak et al., J. Pediatr. Surg. 35(7):1097-103, 2000, and Badylak et al., J. Surg., both of which are incorporated herein by reference. Res. 2005 September; 128(1):87-97, 2005.

[0051] Commercially available ECM preparations can also be used in the methods, devices, and compositions described herein. In one embodiment, the ECM is derived from 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 in Europe as PERMACOL™, Bard, Covington, Ga.), REPLIFORM™ (Microvasive, Boston, Mass.), and ALLODERM™ (LifeCell, Branchburg, NJ). A commercially available UBM ECM is MATRISTEM UBM™ (Acell, Layfayette, IN).

[0052] The source tissue used for ECM preparation can be harvested in a wide variety of ways, and various portions of the harvested tissue may be used immediately after harvest. 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, 21(17-18): 2293-2300, 2015, incorporated herein by reference. Esophageal ECM can be prepared by mechanically separating the mucosa and submucosa from the muscularis externa, digesting the mucosa in a buffer containing trypsin, and subsequently exposing it to sucrose, TRITON®-X100®, deoxycholic acid, peracetic acid, and DNAse. Small intestinal submucosa (SIS) can be prepared by mechanically removing the mucus layer, serosa, and superficial layer of the muscularis externa from intact small intestine, leaving the submucosa, muscularis mucosa, and basal compact layer intact. The SIS is then treated with peracetic acid. An exemplary protocol is provided in Keane et al.

[0053] In some embodiments, epithelial cells can be stripped by first immersing the tissue in a de-epithelializing solution, such as hypertonic saline, for example, but not limited to, 1.0 N saline, for a period ranging from 10 minutes to 4 hours. Exposure to the hypertonic saline solution effectively removes the epithelial cells from their underlying basement membrane. The tissue remaining after the initial de-epithelializing procedure includes the epithelial basement membrane and the abluminal tissue layer relative 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. The outer serosal tissue, adventitia tissue, smooth muscle tissue, submucosal layer, and most of the muscularis mucosa are removed from the remaining de-epithelialized tissue by mechanical abrasion or a combination of enzymatic treatment, hydration, and abrasion.

[0054] ECM can be disinfected or sterilized by any number of standard techniques, including, but not limited to, exposure to peracetic acid, low-dose gamma radiation, gas plasma sterilization, ethylene oxide treatment, supercritical CO2, or electron beam treatment. More typically, sterilization of 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 hours. Peracetic acid residue is removed by washing twice with PBS (pH = 7.4) for 15 minutes and twice with sterile water for 15 minutes. ECM materials can be sterilized by propylene oxide or ethylene oxide treatment, gamma irradiation (0.05-4 mRad), gas plasma sterilization, supercritical CO2, or electron beam treatment. ECM can also be sterilized by treatment with glutaraldehyde, which causes crosslinking of the protein material; however, this treatment effectively alters the material so that it is resorbed slowly or not at all, promoting a different type of host remodeling that more closely resembles scar tissue formation or encapsulation rather than architectural remodeling. Crosslinking of the protein material can also be induced using carbodiimides, or dehydrothermal or photooxidation methods. As disclosed in U.S. Patent No. 8,361,503, 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 with PBS (pH=7.4) and twice for 15 minutes with deionized water.

[0055] 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 can be simultaneous. For example, but not limited to, sterilization using peracetic acid, as described above, can also contribute to decellularizing the ECM. The decellularized ECM can then be dried, i.e., 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 also be further processed into a powdered form by methods such as, but not limited to, grinding or milling in a frozen or freeze-dried state.

[0056] To prepare solubilized ECM tissue for use in preparing ECM hydrogels, ground ECM is digested with an acidic protease in an acidic solution to form a digestion solution. The ECM digestion solution is typically kept at room temperature with constant agitation for a specific amount of time. The ECM digest can be used immediately, stored at -20°C, or frozen at, for example, but not limited to, -20°C or -80°C.

[0057] Once the ECM is solubilized (typically substantially completely), the pH of the solution is raised to between 7.2 and 7.8, and in one embodiment, to a pH of 7.4. A base, such as a hydroxide ion-containing base 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 "pregel" solution. The pregel is in liquid form as a viscous solution at room temperature. The neutralized digestion solution (pregel) can gel at temperatures approaching physiological temperatures, approaching 37°C. The 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 characteristic rate at 37°C (see below).

[0058] Thus, the ECM can typically be derived from one of mammalian tissues, such as, but not limited to, the bladder, dermis, esophagus, small intestine, kidney, liver, heart, uterus, brain, blood vessels, lung, bone, muscle, pancreas, stomach, spleen, or colon. The ECM hydrogel can be made from two or more tissue sources, for example, two, three, or four tissue sources. In one non-limiting embodiment, the ECM is freeze-dried and pulverized. The ECM is then solubilized using an acidic protease in an acidic solution to produce digested ECM, such as 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 in, for example, a 0.01 M HCl solution at an acidic pH suitable or optimal for the protease, for example, a pH greater than about 2 or up to 4. The solution is typically solubilized with mixing (such as stirring, agitating, mixing, blending, rotating, and tilting) for about 12 to about 48 hours, depending on the tissue type (see, e.g., examples below). ECM hydrogels are prepared by (i) disrupting the extracellular matrix, (ii) solubilizing intact, undialyzed, or uncrosslinked extracellular matrix by digestion with an acidic protease in an acidic solution to create a digestion solution, (iii) increasing the pH of the digestion solution to between 7.2 and 7.8 to create a neutralized digestion solution (pregel solution), and (iv) gelling the solution at approximately 37°C within the organ of interest. When an acidic protease is used to digest the ECM, the pregel solution and the resulting hydrogel may contain inactivated proteases.

[0059] ECM hydrogel forms a gel when exposed to temperatures of approximately 37°C. ECM hydrogel in "pregel" form can be frozen and stored, for example, but not limited to, at -20°C or -80°C. ECM hydrogel in "pregel" form can be stored at room temperature, for example, approximately 25°C. Therefore, ECM hydrogel is in 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, or 4°C. ECM hydrogel can be frozen for storage and therefore stored below 0°C. As used herein, the term "pregel form" or "pregel" refers to an ECM hydrogel that has an increased pH but has not yet gelled. For example, but not limited to, an ECM hydrogel in pregel form has a pH between 7.2 and 7.8. The ECM hydrogel can be delivered to a subject in a pre-gel form using an endoscope.

[0060] The ECM hydrogel in pregel form is suitable for introduction into a patient's organ, such as an organ of the gastrointestinal tract other than the esophagus. When introduced into the submucosa of the organ at approximately 37°C, the ECM hydrogel gels, creating an ECM hydrogel cushion between the organ's muscularis propria and submucosa, elevating the submucosa for surgical resection. 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 are disclosed herein.

[0061] In some embodiments, the ECM hydrogel has the following characteristics: 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 an organ, and c) a stiffness of i) about 10 to about 400 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.

[0062] In some embodiments, the ECM hydrogel has the following characteristics: 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 an organ, 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 of less than 30 minutes at a temperature of about 37°C, b) a flow viscosity suitable for injection into an organ, 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 of less than 30 minutes at a temperature of about 37°C, b) a flow viscosity suitable for injection into an organ, and c) a stiffness of about 10 to about 600 Pascals (Pa).

[0063] In another embodiment, the ECM hydrogel has the following characteristics: 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 an organ, and c) a stiffness of about 5 to about 1,000 Pascals (Pa). In another embodiment, the ECM hydrogel has the following characteristics: 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 an organ, and c) a stiffness of about 10 to about 1,000 Pascals (Pa). In a further embodiment, the ECM hydrogel has the following characteristics: 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 an organ, and c) a stiffness of 10 to 70 Pascals (Pa). The organ may be any organ of the gastrointestinal tract, except for the esophagus.

[0064] In some embodiments, the ECM hydrogel has the following characteristics: 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 an organ, and c) a stiffness of i) about 10 to about 400 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.

[0065] In some embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation of less than 20 minutes at a temperature of about 37°C, b) a flow viscosity suitable for injection into an organ, and c) a stiffness of i) about 10 to about 400 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.

[0066] In some embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation of less than 20 minutes at a temperature of about 37°C, b) a flow viscosity suitable for injection into an organ, 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 of less than 20 minutes at a temperature of about 37°C, b) a flow viscosity suitable for injection into an organ, 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 of less than 20 minutes at a temperature of about 37°C, b) a flow viscosity suitable for injection into an organ, and c) a stiffness of about 10 to about 600 Pascals (Pa).

[0067] In other embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation of less than 20 minutes at a temperature of about 37° C., b) a flow viscosity suitable for injection into an organ, 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 of less than 20 minutes at a temperature of about 37° C., b) a flow viscosity suitable for injection into an organ, and c) a stiffness of about 10 to about 1,000 Pascals (Pa). In further embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation of less than 20 minutes at a temperature of about 37° C., b) a flow viscosity suitable for injection into an organ, and c) a stiffness of 10 to 70 Pascals (Pa).

[0068] In another embodiment, the ECM hydrogel has the following characteristics: a) a time to 50% gelation of less than 10 minutes at about 37°C, b) a flow viscosity sufficient for injection into an organ, c) a stiffness of about 10 to about 300 Pascals (Pa), and d) the hydrogel is an esophageal hydrogel.

[0069] In a specific, non-limiting example, the ECM hydrogel is an esophageal hydrogel. In another non-limiting example, the ECM hydrogel can be made from two or more tissue sources. In a further non-limiting example, the ECM hydrogel can be made from the urinary bladder or small intestine. The ECM hydrogel can be UBM ECM or UBS ECM.

[0070] In additional non-limiting examples, ECM hydrogels are prepared by (a) solubilizing acellular extracellular matrix (ECM) by digesting tissue with an acidic protease in an acidic solution to produce digested esophageal ECM, (b) increasing the pH of the digested ECM to between 7.2 and 7.8 to produce a neutralized digestion solution, and (c) diluting the digested ECM to an ECM hydrogel concentration of about 2 mg / ml to about 16 mg / ml, e.g., about 8 mg / ml to about 12 mg / ml. This hydrogel is then introduced into a subject's organ, where it gels. The ECM can be esophageal ECM.

[0071] ECM hydrogels useful 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, or 3 minutes, at a temperature of about 37°C. In some embodiments, the ECM hydrogel has a time to 50% gelation of less than 10 minutes at a temperature of about 37°C. In some embodiments, the time to 50% gelation is about 2 to about 30 minutes at about 37°C. In additional embodiments, the time to 50% gelation is about 2 to about 10 minutes at about 37°C. In further embodiments, the time to 50% gelation is about 3 to about 10 minutes. 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 still 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.

[0072] The disclosed ECM hydrogels can have a flow viscosity suitable for injection into organs. 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−1. * 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 flow viscosity is from about 0.1 to about 100 Pa at a shear rate of about 0.1 / s. * s, and about 0.01 to about 0.2 Pa at a shear rate of 1000 / s * In a further embodiment, the flow viscosity is from about 0.1 to about 30 Pa at a shear rate of 1 / s. * s, and about 0.02 to about 0.8 Pa at a shear rate of about 100 / s * It is s.

[0073] In some embodiments, the ECM hydrogel has a shear strength of about 0.1 to about 30 Pa at a shear rate of 1 / s. *In a further embodiment, the ECM hydrogel has a flow viscosity of about 0.1 to about 100 Pa at a shear rate of about 0.1 / s. * In a specific, non-limiting example, the ECM hydrogel has a flow viscosity of 0.5 to about 50 Pa. * s, or the ECM hydrogel has a flow viscosity of about 1 to about 40 Pa at a shear rate of 0.1 / s. * Exemplary flow viscosities are about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 Pa at a shear rate of 0.1 / s. * It is s.

[0074] 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 about 0.01 to about 0.10 Pa at a shear rate of 1000 / s * 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.

[0075] In a further embodiment, 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 about 0.1 to about 0.8 Pa at a shear rate of 100 / s * 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 * It has s.

[0076] In other embodiments, the ECM hydrogel has a shear strength of about 0.1 to about 30 Pa at a shear rate of 1 / s. * s, for example, about 1 to about 20 Pa * s, or 1 to approximately 10 Pa * s, or 0.5 to 25 Pa *s, e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 Pa at a shear rate of 1 / s * The shear rate may be, for example, 5, 10, 20, or 30 Pa at a shear rate of 1 / s. * In other embodiments, the ECM hydrogel has a flow viscosity of about 0.02 to about 0.8 at a shear rate of 100 s, e.g., about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 at a shear rate of 100 s. The flow viscosity may be about 0.1 to about 30 Pa at a shear rate of 1 s. * s, and about 0.02 to about 0.8 Pa at a shear rate of 100 / s * In additional embodiments, the flow viscosity is from about 1 to about 10 Pa at a shear rate of 1 / s. * s, and is about 0.02 to about 0.5 at a shear rate of 100 / s.

[0077] In a further embodiment, the ECM hydrogel has a shear strength of about 10 to about 100 Pa at a shear rate of 0.1 / s. * In other embodiments, the ECM hydrogel has a flow viscosity of about 0.01 to about 0.2 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. * It has a flow viscosity of 0.01 to 0.2 Pa at a shear rate of 1000 / s. * It is s.

[0078] The disclosed ECM hydrogels have a stiffness of i) about 10 to about 400 Pascals (Pa), ii) about 10 to about 600 Pa, iii) about 5 to about 1,000 Pa, iv) about 10 to about 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 300 Pa. In some embodiments, the disclosed ECM hydrogels have a stiffness of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 Pa. In other embodiments, the disclosed ECM hydrogels 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, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 Pa. In further embodiments, the disclosed ECM hydrogels 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.

[0079] In some embodiments, the ECM concentration in the hydrogel is about 2 mg / ml to about 20 mg / ml, e.g., 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. Useful exemplary concentrations 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 useful concentrations include about 6 mg / ml to about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, or even about 16 mg / ml.

[0080] The disclosed ECM hydrogels can be provided as components of a kit. The ECM hydrogel can be provided in frozen or lyophilized form. In some embodiments, the kit can include components necessary to form the hydrogel, such as one container containing the hydrogel, e.g., in lyophilized form, one container containing a solution for solubilizing the lyophilized hydrogel, and, optionally, a container containing a neutralizing solution for neutralizing the solubilized form. In other embodiments, the kit can include a container containing the solubilized hydrogel and a second container containing a neutralizing agent.

[0081] Optionally, such kits include additional components, including packaging, instructions, and various other reagents, e.g., buffers, supports, or other therapeutic ingredients. The kit may 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 container may be formed from a variety of materials, such as glass or plastic. The container typically contains a composition effective for suppressing esophageal inflammation and / or alleviating the effects of esophageal inflammation in a subject, including, for example, an ECM hydrogel in frozen or lyophilized form. 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 pierceable by a hypodermic needle). The label or package insert indicates that the composition is for use in endoscopic procedures for a particular condition, such as colorectal cancer.

[0082] The label or package insert typically further includes instructions for use. Package inserts typically include instructions customarily included in the commercial packaging of therapeutic products, containing information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. The instructional materials may be written in electronic form (e.g., computer diskette or compact disk) or visual (e.g., video file). The kit may also include additional components, such as needles or catheters, that facilitate the specific application for which the kit is designed. In addition, the kit may include buffers and other reagents routinely used for the implementation of a particular method. Kits and suitable contents are well known to those skilled in the art.

[0083] Treatment method Disclosed herein is a method for dissecting mucosa and submucosa from the muscularis propria from a region of a subject's organ, wherein the organ is not the esophagus. The organ can be in the gastrointestinal tract, such as the duodenum, stomach, small intestine, large intestine (colon), or rectum. The organ can be the bladder, organs of the respiratory system from the mouth (lungs, throat (pharynx), tongue, nasal cavity, sinuses), skin, or the uterus and vaginal canal. Specific examples of tissues are respiratory epithelium, nasal epithelium, dermal or epidermal tissue, and uterine epithelium. The method is useful in any organ having mucosa and submucosa where superficial lesions, such as malignant or premalignant lesions, can form. The organ is not the esophagus.

[0084] These methods include submucosally injecting a pharmaceutical composition comprising an extracellular matrix (ECM) hydrogel into an organ of a subject to form a cushion between the submucosa and the underlying muscularis propria in a region of the organ, where the organ is not the esophagus. The method can be endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD).

[0085] EMR is an endoscopic technique developed for the removal of sessile or flat neoplasms confined to the superficial layers (mucosa and submucosa) of the gastrointestinal (GI) tract. EMR is typically used for the removal of lesions smaller than 2 cm or for the incremental removal of larger lesions. EMR also plays an important role in the evaluation of the resected specimen for accurate pathological staging. Unlike polypectomy, EMR involves lifting the lesion from the muscular layer by injecting a fluid agent, typically saline (NS) solution, into the submucosa. EMR is also useful for obtaining specimens for accurate histopathological staging and determining the risk of lymph node metastasis. EMR facilitates complete removal of the diseased mucosa by excising the central or deeper portions of the intestinal wall submucosa. Various EMR techniques have been described, and four commonly used methods involve snare resection: (1) injection and cutting, (2) injection, lifting, and cutting, (3) cap-assisted EMR (EMRC), and (4) EMR with ligation (EMRL). In the injection and cutting technique, the affected mucosa is elevated from the muscle layer by creating a submucosal fluid cushion, captured, strangulated using an electrosurgical snare, and then excised. However, injection into the thin submucosal layer is a delicate process, the injected solution tends to dissipate within a short time, flat and depressed lesions are more difficult to capture with a snare than elevated lesions, and large or awkwardly located lesions can be difficult to remove (Uraoka et al. al., Drug Design, Development and Therapy 2008:2 131-138). Injection-assisted EMR is often used for large flat colon polyps.

[0086] Endoscopic submucosal dissection (ESD) was developed specifically for removing larger lesions. The lesion is dissected directly along the submucosal layer using an electrocautery knife, resulting in en bloc resection of even large lesions. ESD is predicted to replace conventional surgery in treating certain cancer stages, but it has a higher rate of perforation and bleeding complications than conventional EMR, and therefore requires more advanced endoscopic skills and experience than EMR. ESD can be performed with a number of electrocauteries, including the insulated-tip diathermy knife, needle knife, hook knife, flex knife, and triangle-tipped knife. Knife), flush knife, splash needle, and small-bore tip transparent hood can be used. These knives can be used with high-frequency electrosurgical current (HFEC) generators. ESD is characterized by three steps: (1) injecting fluid to form a submucosal cushion and elevate the lesion from the muscle layer, (2) circumferential incision of the mucosa around the lesion, and (3) dissection of the connective tissue of the submucosa below the lesion (see Kakushima et al., Wold J. Gstroenterol. 14(9): 2962-2967, 2008, incorporated herein by reference). Various submucosal injection solutions have been developed and shown to be sufficient for use during EMR, but the introduction of longer-lasting ESD procedures required longer-lasting solutions to aid in identifying the cut line during submucosal dissection (Uraoka et al., Drug Design, Development and Therapy 2008:2 131-138). The methods of the present disclosure fulfill this need.

[0087] Submucosal injection is used in EMR because injection of fluid into the submucosal cushion facilitates isolation of the tissue to be removed, for example, immediately prior to capture of the target lesion using a snare, thereby reducing the risk of burns and perforation and bleeding while also facilitating resection. Submucosal injection plays an important role in EMR procedures because the solution must be held in place for a sufficient duration and form a hemispherical shape that facilitates snaring. Additionally, achieving a sufficiently high elevation of the submucosal tissue results in safe submucosal cutting during ESD procedures (Uraoka et al., Drug Design, Development and Therapy 2008:2 131-138). Furthermore, because inflammation results from the procedure, any cushion retained at the procedure site should have anti-inflammatory properties. ECM hydrogels reduce strictures and promote reepithelialization. The disclosed method also meets this need.

[0088] In some embodiments, the disclosed methods utilize ECM hydrogels that have anti-inflammatory properties, are inexpensive, non-toxic, and easy to inject, and provide a long-lasting, enhanced submucosal cushion. The ECM hydrogel is administered in a pregel form and then gels at the site of injection to form a cushion. The cushion can dissociate during the procedure so that some of the hydrogel remains in the underlying muscularis propria, thereby aiding healing. The disclosed ECM hydrogels facilitate closure of wounds created by removal of excised mucosa / submucosa. In some embodiments, the procedure is ESD. In other embodiments, the procedure is EMR.

[0089] While saline solution (NS) and more dilute solutions (e.g., ELEVIEW™, see U.S. Pat. No. 9,226,996, incorporated herein by reference) have been used as submucosal cushions for endoscopic resection, the inherent characteristics of these solutions make it difficult to generate a suitable submucosal fluid cushion, maintain the desired height, and retain the cushion in the desired location due to the rapid dispersion of the solution. Furthermore, in ESD, once the mucosa / submucosa is removed, these agents are believed to no longer be retained in the underlying muscularis propria. Furthermore, these agents do not aid in the healing process by, for example, reducing inflammation. The use of ECM hydrogels fulfills these needs.

[0090] The ECM hydrogels disclosed herein can be used in any ESD or ESR procedure. As disclosed in U.S. Patent No. 9,364,580, incorporated herein by reference, an endoscopic injection needle is a potentially long (up to approximately 230 cm) device comprising a relatively long catheter with an internal injection tube with a distal injection needle slidably disposed therein. A proximal actuation handle connects the catheter and the injection tube to move one relative to the other as needed. Fluid access to the injection tube is typically achieved through a luer connector on the handle. Endoscopic injection needle devices are typically delivered to the injection site through the working channel of an endoscope. To protect the lumen of the endoscope working channel from damage, the handle of the injection needle device is manipulated to retract the distal injection needle into the lumen of the catheter before the device is inserted into the endoscope. This prevents the sharp tip of the injection needle from being exposed as the device moves through the lumen of the endoscope. When the distal end of the endoscopic needle device is positioned at the injection site, the handle is again manipulated to move the needle distally out of the catheter lumen. When advanced to its most distal position, the exposed portion of the needle is approximately 4-6 mm long.

[0091] After the injection site has been penetrated, the ECM in pre-gel form, typically contained in a 5 ml to 10 ml syringe with a Luer lock fitting attached to the handle of the injection needle, can be delivered through the injection tube and needle to the injection site, e.g., between the submucosa and the underlying muscularis propria.

[0092] Injection needles and other accessories commonly used during endoscopic procedures, such as polypectomy snares, clip devices, and biopsy forceps, pass through one or more specific channels of the endoscope, commonly referred to as the working or operating channel. Depending on the type of endoscope used in GI endoscopy (e.g., gastroscope, enteroscope, colonoscope, duodenoscope, sigmoidoscope, etc.), the inner diameter of the working channel can vary significantly. However, the most common endoscopes used in GI endoscopy have working channels with inner diameters ranging from about 2 mm to about 5 mm. Endoscope accessory manufacturers generally create accessories with outer diameters that allow the accessory to fit into all working channels. In some embodiments of endoscopic injection needles, the catheter outer diameter ranges from 1.9 mm to 2.3 mm, e.g., about 1.9, 2.0, 2.1, 2.2, or 2.3 cm. Therefore, considering that the inner injection tube is contained within the outer catheter, its inner diameter is typically 1 mm or smaller. The disclosed ECM hydrogels in pre-gel form can easily pass through these catheters.

[0093] The ECM hydrogel in pre-gel form can be used in endoscopic resection procedures by aspirating a volume of emulsion from its primary container with a syringe and injecting a suitable volume of said emulsion just below the superficial mucosal layer with an endoscopic needle inserted into the working channel of an endoscope, depositing a cushioning liquid volume into the submucosal layer when in place; the elevation of the mucosal surface allows the endoscopist to perform easy resection of mucosal lesions discovered during the performance of an endoscopic procedure, even if the lesion is flat and therefore not protruding into a lumen, such as the intestinal or gastric lumen.

[0094] The presence of at least one dye in the cushion can aid the endoscopist in visualizing structures beneath the mucosa (e.g., the submucosa and external muscle wall), thereby reducing the risk of causing damage to such structures when performing a resection procedure. The use of dyes can enable visualization of the cushion cavity and mucosal base. Removal of a lesion from the mucosal surface results in a mucosal wound. The retention of the cushion caused by the injected volume of pharmaceutical composition allows the endoscopic resection procedure to be performed without the need for reinjection. The ECM hydrogel in pregel form is injected submucosally into a target area in a subject's organ, such as the area of ​​a lesion or tumor, to form a cushion between the submucosa and the underlying muscularis propria in that area of ​​the organ. The cushion can dissociate, allowing a portion of the ECM hydrogel to remain in the underlying muscularis propria and aid in the healing process.

[0095] The organ can be any organ of interest, for example, an organ of the gastrointestinal tract. The organ is not the esophagus. The organ can be in the upper gastrointestinal tract, such as the pharynx, tongue, or mouth. The organ can be the bladder, vaginal canal, or uterus. In some embodiments, the organ is the colon, duodenum, stomach, cecum, colon, sigmoid colon, rectum, small intestine, or large intestine. In one non-limiting example, the organ is the stomach, small intestine, or large intestine, and the method includes dissociating carcinoma or adenocarcinoma from the stomach. In a further non-limiting example, the organ is the colon, and the method includes dissociating polyps or carcinomas from the colon.

[0096] The ECM hydrogels disclosed herein are maintained at or below the gelling temperature, for example, room temperature (e.g., about 25°C) or below room temperature. The ECM hydrogel can be maintained at, for example, 25°C or 4°C prior to administration. An effective amount of the ECM hydrogel in pregel form is then utilized. The ECM hydrogel gels in the tissue of the subject, which is at a temperature of approximately 37°C. The ECM hydrogel can be provided in lyophilized or frozen form and reconstituted immediately prior to administration to the region of an organ in the subject.

[0097] The disclosed methods are useful in any subject, including human and veterinary subjects. The subject may be of any age. The subject may be adult or juvenile. In one embodiment, a composition comprising an ECM hydrogel in pregel form is injected into a target tissue in an organ to form a cushion that is optionally used following an endoscopic surgical procedure, such as a resection procedure. The ECM may be from the same species as the subject being treated or from a different species. In some embodiments, the subject is human, and the ECM hydrogel is derived from human or porcine ECM. In other embodiments, the ECM hydrogel is derived from a non-human primate, dog, cat, horse, or cow. The ECM may also be derived from commercial sources. In some embodiments, the ECM hydrogel may 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 ECM hydrogels disclosed above can be used as submucosal cushions and / or in any of the disclosed methods. The hydrogel may be an esophageal ECM hydrogel or a bladder hydrogel.

[0098] The disclosed method is invasive because it requires injection to separate mucosa and submucosa from the muscularis propria of the organ of the subject's intestinal tract.Therefore, ECM is not applied to the surface of organ, for example, gastrointestinal tract organ.The disclosed method is not carried out for the esophagus.

[0099] Any of the methods disclosed herein may include submucosally injecting a pharmaceutical composition containing an extracellular matrix (ECM) hydrogel into a subject's organ to form a cushion between the submucosa and the underlying muscularis propria in a region of the organ. The ECM hydrogel has the following characteristics: 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 organ, and c) a stiffness of about 10 to about 400 Pascals (Pa). The method may utilize any of the hydrogels disclosed above. The ECM hydrogel gels, dissociates the mucosa and submucosa from the underlying muscularis propria, and suppresses inflammation in a region of the organ in the subject. The ECM hydrogel in pregel form may be administered endoscopically or via a catheter. In some embodiments, the organ is the colon, stomach, cecum, colon, sigmoid colon, rectum, small intestine, or large intestine.

[0100] In some embodiments, the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection. In further embodiments, the organ is the stomach, small intestine, or large intestine, and the method comprises dissecting polyps, carcinomas, or adenocarcinomas from the colon. In further embodiments, the method comprises dissecting mucosa and submucosa from an organ of a patient with dysplasia. In specific, non-limiting examples, the method comprises dissecting polyps or carcinomas from the colon. Generally, the organ is not the esophagus.

[0101] The method may also include performing an endoscopic resection procedure on the cushion. In some embodiments, the method includes dividing the cushion such that the hydrogel is retained on the muscularis propria of the underlying organ and the mucosa and submucosa are removed from the region of the organ.

[0102] In some embodiments, the time to 50% gelation of the hydrogel is less than 30 minutes at a temperature of about 37° C. In some non-limiting specific examples, the time to 50% gelation is about 2 to about 30 minutes at about 37° C. In other non-limiting specific examples, the time to 50% gelation is about 2 to about 10 minutes at about 37° C. In further non-limiting examples, the time to 50% gelation is about 3 to about 10 minutes.

[0103] In additional embodiments, the flow viscosity of the pre-gel form is sufficient for injection into the organ of interest. In some embodiments, the flow viscosity of the ECM hydrogel is between about 0.1 and about 100 Pa at a shear rate of about 0.1 / s. * s, and about 0.01 to about 0.2 Pa at a shear rate of 1000 / s * In some non-limiting examples, the flow viscosity is about 30 Pa at a shear rate of 1 / s. * s, and about 0.02 to about 0.8 Pa at a shear rate of about 100 / s * It is s.

[0104] In a further embodiment, the ECM hydrogel, when introduced into tissue, has a stiffness of about 10 to about 300 Pascals (Pa), and the ECM hydrogel has a stiffness of 10 to 70 Pa. In a further embodiment, the ECM concentration in the hydrogel is 2 mg / ml to about 16 mg / ml.

[0105] The ECM hydrogel can be produced by any of the methods disclosed herein. In some embodiments, the ECM hydrogel is produced by (a) solubilizing decellularized extracellular matrix (ECM) by digesting the tissue with an acidic protease in an acidic solution to produce digested ECM, and (b) increasing the pH of the digested ECM to between 7.2 and 7.8 to produce a neutralized digestion solution. In further embodiments, step (b) of increasing the pH of the digested ECM comprises adding a base or an isotonic buffer to increase the pH of the digested ECM. In further embodiments, an acidic protease such as pepsin, trypsin, or a combination thereof is used. The ECM hydrogel may be an esophageal ECM hydrogel. The ECM hydrogel may be a bladder ECM hydrogel.

[0106] In some embodiments, the ECM hydrogel is maintained at or below 25°C prior to administration to a subject. In some embodiments, the ECM hydrogel is maintained at about 4°C to about 28°C, e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28°C. The ECM hydrogel may be maintained at about 4°C and used at about 4°C to about 25°C, or may be warmed to approximately 25°C immediately prior to use. In some embodiments, controlling the temperature ensures that the ECM hydrogel remains in a pregel form, and thus is suitable for injection between the submucosa and the underlying muscularis propria. In further embodiments, the hydrogel gels upon administration to a subject, e.g., upon reaching a temperature of 37°C. [Example]

[0107] ELEVIEW™ and ECM hydrogel are different biomaterials. ELEVIEW™ (Aries Pharmaceuticals, Inc., Dublin, Ireland) is a commercially available low-viscosity emulsion of poloxamer 188 used clinically to provide submucosal tissue lift for EMR and ESD procedures. It is disclosed herein that ELEVIEW™ does not form a stably formed hydrogel at 37°C, but rather remains liquid at 37°C for more than 1 hour. In contrast, 12 mg / mL esophageal ECM hydrogel (eECM) (prepared according to the methods disclosed herein, i.e., by protease digestion of ECM) forms a stably formed hydrogel at 37°C (body temperature). ELEVIEW™ and eECM can be injected under the mucosa. An ideal biomaterial would adhere to both layers. There was no difference in mucoadhesion strength between ELEVIEW™ and 12 mg / mL eECM to muscle. Surprisingly, eECM had stronger mucoadhesion to the mucosa than ELEVIEW™. Furthermore, eECM demonstrated bioactivity by polarizing macrophages toward a remodeling phenotype. This demonstrates that extracellular matrix hydrogels can be effectively used as submucosal cushions and thus can be used to dissociate the submucosa from the underlying muscularis propria.

[0108] Example 1 material and method Rheology The viscoelastic properties of ELEVIEW™ and eECM 12 mg / mL were determined using a temperature-controlled 40 mm parallel-plate rheometer (AR2000). Samples were maintained at 4 °C and loaded into a rheometer with a parallel-plate geometry pre-cooled to 10 °C. Mineral oil was used to seal the sample-plate interface and minimize evaporation during testing. A series of rheological tests were performed on each sample in sequence. Steady-state flow curves at 10 °C were performed to measure the shear rate over a range of shear rates (0.1 to 1000 s ). -1The viscosity profile of the sample was determined at 37°C. The plate temperature was rapidly increased from 10°C to 37°C, and an oscillatory time sweep was performed at 37°C by applying a small 0.5% oscillatory strain at a frequency of 1 rad / s to measure the maximum storage modulus (G'), maximum loss modulus (G"), and gelation kinetics. Data were extracted for statistical analysis and analyzed in Prism (version 6, GraphPad) (n=3).

[0109] Mucosal attachment to the muscularis Porcine mucosal and muscularis segments were mechanically isolated by stripping the mucosa and submucosa from the underlying muscularis segment. ELEVIEW™ and eECM (12 mg / mL) were transferred to a 6-well plate using a pipette. The mucosal or muscularis segment was attached to the bottom of a hemisphere (40 mm diameter) on top of the ELEVIEW™ or eECM, ensuring that the surface area of ​​the mucosal or muscularis segment in contact with the ELEVIEW™ or eECM remained constant for all tests. The constructs were incubated at 37°C for 1 hour for attachment to the mucosal or muscularis segment. After 1 hour, the constructs were placed in an MTS Insight tensile tester equipped with a 10 N load cell and a ball-bursting attachment set at a measurement frequency of 10 Hz. The ball-bursting attachment was securely attached to the hemisphere, and the hemisphere was pushed up at 5 mm / min. The maximum force was considered the adhesion force, and the force of the freely hanging construct was subtracted. Measurements were accepted only when delamination occurred between the mucosa or muscularis and the hydrogel (n=3).

[0110] Performance of submucosal fluid cushions ex vivo The porcine colon and stomach were placed in a 37°C incubator, and their temperature was monitored using a thermometer until the tissue reached 37°C. After reaching the target temperature, 2 mL of either ELEVIEW™ or 12 mg / mL neutralized eECM was injected into the submucosal tissue using a 23G needle. The eECM was kept on ice throughout the procedure. The tissue was evaluated at 15-minute intervals for up to 75 minutes and photographed alongside evidence of metrics. The tissue continued to incubate at 37°C throughout the procedure. After 75 minutes, the area injected with the test agent was dissected and evaluated. ImageJ was used to quantitate mucosal elevation after agent injection throughout the experiment.

[0111] Macrophage isolation and activation Mouse bone marrow was harvested as previously described [1, 2]. Briefly, 6- to 8-week-old female C57bl / 6 mice (Jackson Laboratories, Bar Harbor, ME) were euthanized via CO2 inhalation and cervical dislocation. The skin from the proximal hind limb down to the foot was aseptically removed, and the tarsal and stifle joints were transected to isolate the tibia. The coxofemoral joint was transected to isolate the femur. After removal of excess tissue, bones were kept on ice and rinsed in a sterile dish containing complete macrophage medium consisting of DMEM (Gibco, Grand Island, NY), 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 10% L929 supernatant [2], 50 μM beta-mercaptoethanol (Gibco), 100 U / ml penicillin, 100 μg / ml streptomycin, 10 mM non-essential amino acids (Gibco), and 10 mM Hepes buffer. The ends of the bones were transected, and the marrow cavity was rinsed with complete medium to collect the bone marrow. Cells were washed, plated at 2 × 10 cells / ml, and differentiated into macrophages at 37°C, 5% CO2 for 7 days, with complete medium changes every 48 hours, as previously described [3]. After 7 days, the resulting naive macrophages were treated with basal medium consisting of 10% FBS, 100 μg / ml streptomycin, 100 U / ml penicillin in DMEM and one of the following conditions as previously described: (1) 20 ng / ml IFNγ and 100 ng / ml LPS to promote an M1-like phenotype, (2) 20 ng / ml IL-4 to promote an M2-like phenotype, (3) 250 μg / ml pepsin control buffer, (4) 250 μg / ml esophageal ECM, or (5) the same volume of ELEVIEW™ for 24 hours at 37°C, 5% CO2.

[0112] Immunolabeling of macrophages After 24 hours, macrophages were washed and fixed with 2% paraformaldehyde. After a PBS wash, cells were incubated in a blocking solution consisting of 0.1% Triton®-X100, 0.1% Tween® 20, 4% normal goat serum, and 2% bovine serum albumin (BSA) at room temperature for 1 hour to prevent nonspecific antibody binding. The following primary antibodies were diluted in blocking solution: (1) monoclonal anti-F4 / 80 (Abcam, Cambridge, MA) at a dilution of 1:100 for the pan-macrophage marker; (2) polyclonal anti-iNOS (Abcam, Cambridge, MA) at a dilution of 1:100 for the M2 marker; (3) polyclonal anti-Fizz1 (Peprotech, Rocky Hill, NJ) at a dilution of 1:100 for the M2 marker; and (4) polyclonal anti-liver arginase (Abcam, Cambridge, MA) at a dilution of 1:100 for the M2-like marker [5-7]. Cells were incubated in the primary antibodies for 16 hours at 4°C. After a PBS wash, cells were incubated in fluorophore-conjugated secondary antibodies (Alexa Fluor goat anti-rat 488 or goat anti-rabbit 488, Invitrogen) for 1 hour at room temperature. After washing with PBS, nuclei were counterstained with 4'6'diamidino-2-phenylindole (DAPI) and then imaged at three 200x fields using a live-cell microscope. Exposure times were normalized to a negative isotype control and kept constant across images. Images were quantified using CellProfiler image analysis software to obtain the percentages of positive F4 / 80, iNOS, Fizz1, and Arginase 1.

[0113] In vivo use of ECM as a submucosal fluid cushion for EMR Anesthesia is induced with acepromazine (0.01 mg / kg, SC) and ketamine (5-11 mg / kg), and surgical-level anesthesia is maintained with 1-5% isoflurane via an endotracheal tube. Animals receive 2 ml / kg / h of lactated Ringer's solution IV immediately throughout the procedure and surgery. Temperature is controlled by a warm-water recirculating heating pad placed under the animal. Physiological parameters, such as heart rate, respiratory rate, body temperature, and responsiveness, are monitored throughout the procedure. Antibiotic prophylaxis with 25 mg / kg cefazolin is administered prior to the start of the procedure.

[0114] The animal was placed in the supine position, and organs were evaluated using a Pentax EG3430K endoscope. After identifying reference points on the organs, the mucosa and submucosa at the site of excision were separated with 8 mg / ml blue-dyed bladder matrix hydrogel at 4°C using an Olympus Injectorforce 4 mm 23G needle for injection. This temperature was maintained at all times to prevent gelation and potential needle blockage. Approximately 2-5 ml of blue gel was injected per site. A 5 cm length of the entire mucosal circumference (100%) was removed using the band ligation EMR technique. For EMR, a Cook Duette kit with ligation bands was used. The mucosa was then excised using a snare.

[0115] statistics Two-way analysis of variance was used to compare the effects of the independent variables shear rate and sample on the dependent variable viscosity, and the effects of the independent variables sample and modulus type on the dependent variable modulus. Sidak post-hoc multiple comparison tests were used to determine significance using 95% confidence intervals, with p-values ​​adjusted for multiple comparisons. A t-test was performed on mucoadhesive strength comparing ELEVIEW™ to eECM 12 mg / mL.

[0116] Example 2 Viscoelastic properties ELEVIEW™ was significantly less viscous than eECM 12 mg / mL at a shear rate of 0.1 1 / s (p<0.0001), trending toward non-significance at 1 1 / s (p=0.054) (Figure 1A). ELEVIEW™ did not form a stably forming hydrogel, as its mean loss modulus (G") (0.09 ± 0.04 Pa) was greater than its mean storage modulus (G') (0.05 ± 0.01 Pa), whereas eECM 12 mg / mL met the Freytes definition of a stably forming ECM hydrogel. et al., Biomaterials, 2008. 29(11): p. 1630-7), has a storage modulus (G') (56.95 ± 66.72 Pa) approximately one order of magnitude greater than its loss modulus (G") (7.62 ± 6.30 Pa) (Figure 1B). A representative graph of a time sweep of ELEVIEW™ further demonstrates that ELEVIEW™ does not form a hydrogel (Figure 1C); the storage modulus of eECM 12 mg / mL increases in a sigmoidal manner and reaches a plateau over time (Figure 1D). Thus, the gelation time to 50% gelation could be calculated for eECM 12 mg / mL (4.5 ± 3.5 min), but not for ELEVIEW™ (Figure 1E).

[0117] Example 3 Mucosal adhesion to the muscular layer ELEVIEW™ (0.16 ± 0.05 N) and eECM (0.21 ± 0.08 N) did not demonstrate significantly different mucoadhesion to the muscularis segment (Figure 2A). eECM had a higher mucoadhesion strength to the mucosa (0.37 ± 0.02 N) than ELEVIEW™ (0.15 ± 0.06 N) (p = 0.0053) (Figure 2B).

[0118] Example 4 Macrophage activation Macrophages exposed to eECM showed minimal iNOS expression (a pro-inflammatory marker) along with activation of the anti-inflammatory marker FIZZ1. iNOS expression was comparable between ELEVIEW™, eECM, and the carrier (pepsin) control (Figure 3). ELEVIEW™ did not exhibit any biological activity.

[0119] Example 5 Performance of submucosal fluid cushions ex vivo ELEVIEW™ and eECM successfully created a fluid cushion upon injection of 2 mL of test agent into the colon (Figure 4) or stomach (Figure 5). Both test agents were easily injectable using a 23G needle. ELEVIEW™ appeared to diffuse from the moment of injection. Cushion height measurements and macroscopic appearance confirmed this finding for the two tissues tested (Figures 4A, 4B, 5A, 5B). The colon and stomach had a greater loss of cushion height from 0 to 15 minutes (Figures 4A, 5A). The loss of cushion height was greater with ELEVIEW™ than with eECM. The decrease in cushion height continued for both test agents, but the loss was clearly greater in the colon with ELEVIEW™. However, the stomach had a similar loss of cushion height for both test agents (Figure 5A).

[0120] Dissociation after 75 minutes showed differences between ELEVIEW™ and eECM in all tissues. The ELEVIEW™-injected area showed a viscous liquid with no clear attachment to either the mucosa or the underlying muscle layer. The eECM-pre-injected area showed a clear, defined mass of gel that remained attached to the mucosa and underlying muscle (Figures 4C, 5C). This is consistent with previous results (see Example 2) demonstrating the inability of ELEVIEW™ to form a gel (Figure ID).

[0121] Example 6 In vivo use of ECM as a submucosal fluid cushion for EMR The ECM hydrogel can be delivered through a long endoscopic needle without any resistance. Mucosal elevation was successfully achieved and maintained, facilitating the EMR procedure, and the blue dye was visible, indicating the location where the dissection was created for removal. The tissue was removed using a snare. Macroscopic observation revealed that the removed mucosal tissue contained some of the gel. The blue dye in the hydrogel appeared to diffuse to the periphery of the organ after the mucosa and entire periphery were removed using the hydrogel.

[0122] In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that the illustrated embodiments are merely examples of the invention and should not be considered limitations on 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. The present invention provides, for example, the following items. (Item 1) 1. A method for dissecting mucosa and submucosa from the muscularis propria from a region of an organ of a subject, comprising: submucosally injecting into the organ of the subject a pharmaceutical composition comprising an extracellular matrix (ECM) hydrogel to form a cushion between the submucosa and the underlying muscularis propria in the region of the organ, wherein the ECM hydrogel has the following characteristics: 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 said organ; and c) Stiffness of about 10 to about 400 Pascals (Pa) and thereby dissociating the mucosa and submucosa from the underlying muscularis propria and suppressing inflammation in the region of the organ in the subject, wherein the organ is not the esophagus. A method comprising: (Item 2) Item 2. The method according to Item 1, wherein the time required for 50% gelation is about 2 to about 30 minutes at about 37°C. (Item 3) Item 2. The method according to Item 1, wherein the time required for 50% gelation is about 2 to about 10 minutes at about 37°C. (Item 4) 3. The method according to item 2, wherein the time required for 50% gelation is about 3 to about 10 minutes. (Item 5) The flow viscosity is about 0.1 to about 100 Pa at a shear rate of about 0.1 / s. * s, and about 0.01 to about 0.2 Pa at a shear rate of 1000 / s * 5. The method according to any one of items 1 to 4, wherein s. (Item 6) The flow viscosity is about 0.1 to about 30 Pa at a shear rate of 1 / s. * s, and about 0.02 to about 0.8 Pa at a shear rate of about 100 / s * 5. The method according to any one of items 1 to 4, wherein s. (Item 7) 7. The method of any one of items 1 to 6, wherein the ECM hydrogel has a stiffness of 10 to 300 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 according to any one of items 1 to 8, wherein the ECM concentration in 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 endoscopically or via a catheter. (Item 11) The ECM hydrogel (a) solubilizing decellularized extracellular matrix (ECM) by digestion of the tissue with an acid protease in an acidic solution to produce digested esophageal ECM; and (b) increasing the pH of the digested ECM to a pH between 7.2 and 7.8 to create a neutralized digestion solution. (Item 12) 12. The method of claim 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. (Item 13) 12. The method according to item 10 or 11, wherein the acid protease is pepsin, trypsin, or a combination thereof. (Item 14) 14. The method of any one of items 1 to 13, wherein the ECM hydrogel is maintained at 25°C or below 25°C prior to administration to the subject. (Item 15) 15. The method of any one of items 1 to 14, wherein the ECM hydrogel is injected endoscopically or via a catheter. (Item 16) 13. The method of any one of items 1 to 12, wherein the ECM hydrogel is maintained at 25°C or below 25°C prior to administration to the subject. (Item 17) 17. The method of any one of items 1 to 16, wherein the organ is the colon, stomach, cecum, colon, sigmoid colon, rectum, small intestine, or large intestine. (Item 18) 18. The method of any one of items 1 to 17, wherein the organ is the stomach, small intestine, or large intestine, and the method comprises dissociating adenocarcinoma or carcinoma from the organ. (Item 19) 20. The method of claim 18, comprising dissecting the mucosa and submucosa from the colon. (Item 20) 18. The method according to any one of items 1 to 17, wherein the organ is the colon and the method comprises dissociating polyps or carcinomas from the colon. (Item 21) 21. The method of any one of items 1 to 20, further comprising performing an endoscopic resection procedure on the cushion to remove the dissected mucosa and submucosa. (Item 22) Item 17. The method according to item 16, wherein the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection. (Item 23) dividing the cushion so that the hydrogel is retained on the muscularis propria of the underlying organ and the mucosa and submucosa are removed from the region of the organ. Item 23. The method according to Item 22, comprising: (Item 24) 24. The method of any one of items 1 to 23, wherein the subject is a human. (Item 25) The method according to any one of items 1 to 24, wherein the organ is in the gastrointestinal tract. 26. The method of any one of items 1 to 25, wherein the organ is selected from the duodenum, stomach, small intestine, colon, or rectum. (Item 27) 27. A composition comprising an extracellular matrix (ECM) hydrogel for use in the method according to any one of items 1 to 26, wherein the ECM hydrogel has the following characteristics: a) a time to 50% gelation of less than 10 minutes at about 37°C; b) sufficient flow viscosity for injection into organs, and c) Stiffness of about 10 to about 300 Pascals (Pa) A composition comprising: (Item 28) 27. The method of any one of items 1 to 26, or the composition of item 27, wherein the method of dissociation comprises endoscopic mucosal resection or endoscopic mucosal dissection.

Claims

1. 1. An extracellular matrix (ECM) pre-gel composition for dissociating mucosal and submucosal tissue from a region of an organ of a subject, comprising: the ECM pre-gel composition is formulated for submucosal injection into the organ of the subject such that, when warmed to about 37°C, it forms a cushion and an ECM hydrogel between the submucosa and the underlying muscularis propria in the region of the organ, thereby dissociating the mucosa and the submucosa from the underlying muscularis propria and reducing inflammation in the region of the organ in the subject; The ECM pregel composition comprises decellularized, acid protease digested, and solubilized ECM, the ECM pregel composition having a pH of 7.2 to 7.8 and forming the ECM hydrogel upon warming to about 37°C, and the ECM hydrogel has the following characteristics: 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 said organ; c) a stiffness of about 10 to about 400 Pascals (Pa); and d) an ECM concentration in the ECM hydrogel of about 2 mg / ml to about 20 mg / ml and An ECM pre-gel composition, wherein the organ is not the esophagus.

2. The ECM pregel composition of claim 1 , wherein the ECM pregel composition is terminally sterilized.

3. 3. The ECM pre-gel composition of claim 1 or 2, wherein the ECM is derived from the bladder, small intestine, colon, liver, esophagus, dermis, kidney, heart, uterus, brain, blood vessels, lung, bone, muscle, pancreas, stomach, or spleen.

4. The ECM pregel composition of claim 1 or 2, wherein the ECM is derived from the esophagus.

5. 3. The ECM pre-gel composition of claim 1, wherein the ECM is derived from a monkey, horse, pig, cow, or sheep.

6. 3. The ECM pre-gel composition of claim 1, wherein the acid protease-digested ECM is present in the ECM pre-gel composition at a concentration of 2 mg / ml to 16 mg / ml.

7. 3. The ECM pregel composition of claim 1 or 2, wherein the ECM pregel composition is maintained at less than 25°C prior to administration to the subject.

8. 3. The ECM pregel composition of claim 1 or 2, wherein the ECM pregel composition is maintained at less than 37°C prior to administration to the subject.

9. The ECM pregel composition of claim 1 or 2, wherein the ECM pregel composition comprises an inactivated protease.

10. The ECM pre-gel composition of claim 9 , wherein the protease is trypsin or pepsin or a combination thereof.

11. 3. The ECM pregel composition of claim 1, wherein the ECM pregel composition is suitable for injection endoscopically or via a catheter.

12. The ECM pregel composition of claim 1 or 2, wherein the organ is the colon, stomach, rectum, or small intestine.

13. 13. The ECM pre-gel composition of claim 12, wherein dissociating the mucosa and submucosa from the muscularis propria from a region of an organ of a subject comprises dissociating the mucosa and submucosa from the colon.

14. The ECM pre-gel composition of claim 1 or 2, wherein the organ is the cecum or sigmoid colon.

15. 3. The ECM pre-gel composition of claim 1 or 2, wherein the organ is the stomach, small intestine, or large intestine, and dissociating the mucosa and submucosa from the muscularis propria from a region of the organ of a subject comprises a method of dissociating carcinoma from the organ.

16. The ECM pre-gel composition of claim 15, wherein the carcinoma is an adenocarcinoma.

17. 3. The ECM pre-gel composition of claim 1 or 2, wherein the organ is the colon, and dissociating the mucosa and submucosa from the muscularis propria from a region of the organ of the subject comprises dissociating polyps or carcinomas from the colon.

18. The ECM pre-gel composition (a) solubilizing the decellularized ECM by digestion of the tissue with an acidic protease in an acidic solution to produce digested ECM; and (b) increasing the pH of the digested ECM to a pH of 7.2 to 7.8 to produce a neutralized digestion solution.

3. The ECM pre-gel composition of claim 1 or 2, produced by

19. 19. The ECM pre-gel composition of claim 18, wherein the acid protease is pepsin, trypsin, or a combination thereof.

20. 3. The ECM pre-gel composition of claim 1 or 2, wherein dissociating mucosal and submucosal tissue from the muscularis propria from a region of an organ of a subject further comprises performing an endoscopic resection procedure on the cushion to remove the dissociated mucosal and submucosal tissue.

21. The ECM pre-gel composition of claim 20 , wherein the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection.

22. 3. The ECM pregel composition of claim 1, wherein dissociating the mucosa and submucosa from the muscularis propria from the region of the organ of the subject comprises dividing the cushion such that the ECM hydrogel formed by the ECM pregel composition is retained on the underlying muscularis propria of the organ and the mucosa and submucosa are removed from the region of the organ.

23. The ECM pregel composition of claim 1 or 2, wherein the subject is a human.

24. The ECM pre-gel composition of claim 1 or 2, wherein the organ is in the gastrointestinal tract.

25. The ECM pregel composition according to claim 1 or 2, wherein the organ is the duodenum.

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