Use of extracellular matrix (ECM) hydrogels as a fluid cushion in the esophageal mucosa

Abstract

Methods for dissociating mucosa and submucosa from the muscularis propria from a region of the esophagus of a subject are disclosed. These methods include submucosally injecting into the esophagus 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 esophagus, 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 the esophagus, and c) a stiffness of about 10 to about 400 Pascals (Pa). The ECM hydrogel is not a bladder ECM hydrogel.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 62 / 688,195, filed on June 21, 2018, which is incorporated herein by reference in its entirety.

[0002] This relates to the use of an extracellular matrix (ECM) hydrogel as a submucosal cushion for endoscopic resection, specifically for separating the mucosa and submucosa from the muscularis propria in the esophagus, where the ECM hydrogel is not bladder ECM hydrogel. [Background technology]

[0003] Endoscopy is a procedure that uses an instrument called an endoscope to allow examination of the inside of hollow organs or cavities in the body without the use of invasive surgery. Endoscopy can be used for surgical procedures such as cauterizing bleeding blood vessels, dilating narrow esophagi, removing polyps, adenomas, and small tumors, performing biopsies, or removing foreign bodies. Endoscopic procedures can be performed in the gastrointestinal tract, airways, ears, urinary tract, female reproductive system, and in body cavities that are normally closed, such as the abdominal or pelvic cavity (laparoscopy), inside joints (arthroscopy), and organs of the chest (thoracoscopy and mediastinoscopy), through small incisions. An endoscope is a flexible or rigid tubular instrument, usually made of fiber optics and equipped with a light source, for visualizing the inside of hollow organs or hollow portions (e.g., the esophagus) for diagnostic or therapeutic purposes, and typically has one or more working channels that allow the passage of instruments (e.g., forceps, electrosurgical units, endoscopic needles, or scissors) or facilitate the removal of biopsy specimens. The endoscope is equipped with a suitable lamp and imaging device at the distal end and can be inserted through naturally occurring openings in the body such as the mouth, anus, ears, and nose, or through small surgical incisions.

[0004] Endoscopic procedures are widely applied in the gastrointestinal tract. For example, endoscopic procedures can be used to examine the mucosa extending into the gastrointestinal lumen, and to detect small and large pathological lesions, such as inflammatory tissue, polyps, pseudopolyps, serrated lesions, adenomas, ulcers, dysplasia, preneoplasms and neoplasms, and tumors. Endoscopic procedures can be used for biopsy and removal of pathological lesions (polyps, adenomas, dysplasia, Barrett's dysplasia, preneoplasms and neoplasms, tumors). Surgical interventions include two types of endoscopic resection techniques commonly used to remove pathological lesions in gastrointestinal endoscopy: endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD). These two techniques allow for minimally invasive treatment of gastrointestinal polyps, adenomas, dysplasia, Barrett's dysplasia, and early-stage cancers with minimal risk of lymph node metastasis. There is still a need for useful drugs in endoscopic procedures in the esophagus. [Overview of the project] [Means for solving the problem]

[0005] Methods for separating the mucosa and submucosa from the muscularis propria in a region of the esophagus of a subject are disclosed herein. These methods include the steps of submucosally injecting a pharmaceutical composition containing an extracellular matrix (ECM) hydrogel into a region of the esophagus of a subject to form a cushion between the submucosa and the underlying muscularis propria in the esophagus, 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 fluid viscosity suitable for injection into the esophagus, and c) a stiffness of about 10 to about 400 Pascals (Pa), and thereby separating the mucosa and submucosa in the region of the esophagus from the underlying muscularis propria. In some embodiments, the methods also suppress inflammation of the esophagus in the subject. ECM hydrogel is not bladder ECM hydrogel.

[0006] In other embodiments, these methods also include submucosal injection of a pharmaceutical composition containing an extracellular matrix (ECM) hydrogel into a region of the esophagus of a subject to form a cushion between the submucosa and the underlying muscularis propria in the esophagus, thereby separating the mucosa and submucosa in the region of the esophagus from the underlying muscularis propria. In some embodiments, the methods also suppress inflammation of the esophagus in the subject. The ECM hydrogel is not bladder ECM hydrogel.

[0007] In some embodiments, the dissection method includes endoscopic mucosal resection or endoscopic mucosal dissection.

[0008] The aforementioned and other objects, features, and advantages of the present invention will become more apparent from the following detailed description, which is made with reference to the accompanying drawings. [Brief explanation of the drawing]

[0009] [Figure 1-1] Figures 1A-1E show the viscoelastic properties. The viscosity profiles of ELEVIEW® and esophageal ECM (eECM) 12 mg / mL were tested at 10°C while increasing the shear rate (0.1 to 1000 1 / s) (A). The temperature was rapidly increased to 37°C to induce gelation, and the maximum storage (G') and loss (G") modulus 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 it did not gel (G'' > G' during the time sweep test) (E). [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0010] [Figure 2]Figures 2A - 2B are diagrams showing mucosal adhesion strength. Mucosal adhesion strength to the muscular layer of the porcine esophagus (A) or esophageal mucosa (B) of ELEVIEW (trademark) and eECM at 12 mg / mL.

[0011] [Figure 3] Figure 3 is a diagram showing macrophage activation. Macrophage expression of anti - inflammatory and pro - inflammatory markers after exposure to eECM and ELEVIEW (trademark).

[0012] [Figure 4-1] Figures 4A - 4C are diagrams showing the submucosal fluid cushion - esophagus. Time - dependent measurements of the height of the submucosal fluid cushion compared to ELEVIEW (trademark) after injection of ECM (eECM) into the esophageal submucosal tissue (A). Appearance of the tissue after injection of 2 mL of the test agent, after injection and 75 minutes later (B). Dissociation and exposure of the test agent 75 minutes later (C). [Figure 4-2] Same as above.

Mode for Carrying Out the Invention

[0013] For example, the use of an extracellular matrix (ECM) hydrogel as a submucosal cushion for dissociating mucosa and submucosal tissue from the muscularis propria in the esophagus in any endoscopic resection technique commonly used for removing pathological lesions in esophageal endoscopy, such as EMR and ESD, is disclosed herein. The ECM hydrogel is not a bladder ECM hydrogel.

[0014] Term Unless otherwise noted, technical terms will be used according to conventional terminology. 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). To facilitate consideration of the various embodiments of this disclosure, the following explanations of specific terms are provided.

[0015] Acid protease: An enzyme that cleaves peptide bonds and has increased activity in cleaving peptide bonds at an acidic pH. Examples of acid proteases, though not limited to them, include pepsin and trypsin.

[0016] Barrett's esophagus: Abnormal changes (metaplasia or dysplasia) in the cells of the esophagus, typically the lower (distal) part of the esophagus. Barrett's esophagus is diagnosed when multiple portions of the normal stratified squamous epithelium lining of the esophagus are replaced by simple columnar epithelium containing goblet cells. Barrett's esophagus is found in 5-15% of patients seeking medical treatment for gastroesophageal reflux disease (GERD), although a large subgroup of patients with Barrett's esophagus are asymptomatic. Barrett's esophagus is strongly associated with esophageal adenocarcinoma and is considered a premalignant condition. The main cause of Barrett's esophagus is thought to be an adaptation to chronic acid exposure from gastric reflux. After biopsy, the cells of Barrett's esophagus are classified into four general categories: non-dysplasia, low-grade dysplasia, high-grade dysplasia, and obvious carcinoma.

[0017] Base: A compound or solution of a compound having 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.

[0018] Grinding (grinding and crushing): The process of reducing larger particles to smaller particles by, for example, grinding, blending, crushing, slicing, milling, cutting, or shredding. ECM can be ground in any form, including but not limited to moistened form, frozen sheet form, air-dried sheet form, freeze-dried sheet form, and powder sheet form.

[0019] Diagnosis: The process of identifying a disease based on its signs, symptoms, and the results of various tests. The conclusion reached through this process is also called a "diagnosis." Common tests include blood tests, medical imaging, and biopsies.

[0020] Dissociation: The process of separating or detaching tissue, for example, during a surgical procedure.

[0021] Endoscopic injection needle or endoscopic injection needle catheter: A generally long (e.g., up to approximately 230 cm) device comprising a long catheter in which an internal injection tube with a distal injection needle is slidably positioned inside. Generally, a proximal operating handle is connected to the catheter and injection tube for moving one side relative to the other. The needle may be retractable. Access to the injection tube for fluid is typically achieved via a Luer connector on the handle.

[0022] Endoscopic injection needles are typically delivered to the injection site by an endoscopic catheter. To protect the lumen from damage, the device is inserted into the endoscope after manipulating the handle of the injection needle device to retract the distal injection needle into the catheter lumen. Once the distal end of the endoscopic injection needle device is positioned at the injection site, its handle is manipulated to move the injection needle distally from the catheter lumen. In some embodiments, when advanced to its most distal position, the exposed portion of the injection needle may be approximately 4–6 mm in length.

[0023] 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): An endoscopic technique developed specifically for the removal of larger lesions, such as those from the gastrointestinal tract, in which the mucosa and submucosa are separated from other layers of the gastrointestinal tract. Both EMR and EMD typically involve the injection of a substance that acts as a cushion, lifting the submucosa and the mucosa above it, beneath the targeted lesion, i.e., between the submucosa and the underlying muscularis propria. In the case of EMR, the lifted lesion is then removed using a snare and separated into a small cup by suction. In the case of ESD, the submucosa beneath the lesion is dissected using a specialized knife, causing separation of the submucosa and the mucosa above it. For therapeutic purposes, ESD allows for the removal of larger lesions and potentially deeper lesions than is possible with EMR. Both EMR and ESD are facilitated by injecting a substance into the submucosal surface of the esophagus, which effectively separates the superior mucosa from the underlying muscularis propria and simultaneously elevates the mucosa above the adjacent esophageal mucosa. This separation of the layer and elevation of the affected tissue helps the surgeon isolate, grasp, and remove the tissue of interest.

[0024] Extracellular matrix (ECM): Noncellular components of tissues and organs. Natural ECM (ECM found in mammals and multicellular organisms such as humans) is a complex mixture of structural and nonstructural biomolecules, including but not limited to collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobial agents, chemoattractants, cytokines, and growth factors, and typically its specific composition differs 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 the ECM. The composition and structure of ECM vary depending on the anatomical source of the tissue. For example, the submucosal tissue of the small intestine (SIS), bladder matrix (UBM), submucosal tissue of the bladder (UBS), esophagus (E), and liver stromal ECM each differ in overall structure and composition due to the unique cellular niches required by each tissue. The bioscaffolds of intact "extracellular matrix" and "intact ECM" consist of an extracellular matrix, for example, pulverized ECM as described herein, which is not solubilized, 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, antimicrobial agents, chemoattractants, cytokines, and growth factors.

[0025] The activity of biomolecules within the ECM can be altered chemically or mechanically, for example, by chemical or enzymatic crosslinking and / or by dialysis of the ECM. Intact ECM is essentially not enzymatically digested, crosslinked, and / or dialyzed, meaning that intact ECM has not been subjected to digestion, dialysis, and / or crosslinking processes, nor to conditions other than those naturally occurring during the storage and handling of the ECM before solubilization. Therefore, ECM that is dialyzed (by any method other than trivial methods that do not substantially affect the gelation and functional characteristics of the ECM in the use described herein) cannot be considered “intact.”

[0026] Esophagogastroduodenoscopy (EGD) or upper gastrointestinal endoscopy: A diagnostic endoscopic procedure that visualizes any upper part of the gastrointestinal tract up to the duodenum. "Esophageal endoscopy" refers to any endoscopic procedure that visualizes the esophagus. Esophageal endoscopy may, in some cases, be performed as part of EGD or upper gastrointestinal endoscopy. These terms are not mutually exclusive unless explicitly stated otherwise.

[0027] Gelation: Formation of a gel from a sol.

[0028] Gastroesophageal reflux disease (GERD): A chronic condition of mucosal damage caused by the reflux of stomach acid contents from the stomach into the esophagus. GERD is usually caused by abnormal relaxation of the lower esophageal sphincter, which normally closes the uppermost (proximal) part of the stomach, obstruction of the esophageal expulsion of gastric reflux, or changes in the barrier between the stomach and esophagus, including hiatal hernia. These changes may be permanent or temporary.

[0029] Fluid viscosity: A measure of a fluid's resistance to gradual deformation due to shear or tensile stress. Viscosity is a property of a fluid that resists the relative motion between two surfaces of the fluid moving at different velocities. When a fluid flows through a tube, the particles that make up the fluid generally move faster near the tube's axis and slower near the tube's walls. Stress (e.g., a pressure difference between the two ends of the tube) is required to overcome friction between the particle layers and keep the fluid moving. For a given velocity pattern, the required stress is proportional to the fluid's viscosity. Viscosity is measured using a viscometer and a rheometer. Viscosity is expressed in Pascal-seconds (Pa). * It can be measured as s). Water at 20℃ has a pressure of 1.002 mPa. * It has a viscosity of s.

[0030] Hydrogel: A network of hydrophilic polymer chains, sometimes found as a colloidal gel with water as the dispersion medium. Hydrogels are highly absorbent natural or synthetic polymer networks. Hydrogels also possess a degree of flexibility similar to that of natural tissues. The term "bladder ECM hydrogel" includes UBM and UBS hydrogels.

[0031] Inflammation: A localized response induced by injury to tissue. Inflammation is characterized by the appearance or migration of more leukocytes of any class than the number of such cells found in any area of ​​tissue under normal (healthy) conditions, in any tissue space, unit, or region. Inflammation is formed by a complex biological response of vascular tissue to harmful stimuli such as pathogens, damaged cells, or irritants.

[0032] Isotonic buffer solution: A solution buffered to a pH between 7.2 and 7.8, containing a salt at equilibrium concentration, and promoting an isotonic environment.

[0033] Low-grade and high-grade dysplasia and metaplasia (of the esophagus): Pathological conditions of the esophagus characterized by abnormal cell morphology, but where the cell type is still recognizable as squamous epithelium. Generally, in esophageal dysplasia, apical mucin is absent in the lining cells of the esophagus. At low detection power, these areas may appear more hyperpigmented compared to unrelated areas.

[0034] In cases of high-grade dysplasia, the changes in cell morphology are more pronounced, but the cells are technically still a type of squamous epithelium.

[0035] When cells change from squamous epithelial cells to another cell type, such as glandular cells, which are often cuboidal or columnar in shape, this process is called metaplasia. In relation to metaplasia, distortions of the esophageal glandular structure are usually present and may be prominent, consisting of branching and lateral extension of the crypts, a villous structure on the mucosal surface, or intraglandular crosslinks of the epithelium that form a "continuous" cribriform pattern of glands. Abnormal epithelium with loss of nuclear polarity is present on the mucosal surface, and this epithelium is characterized by the "roundness" of the nuclei and the absence of consistent relationships between the nuclei.

[0036] Prevention or treatment: Inhibiting a disease means preventing the partial or complete onset of a disease in a person at risk of a disease, such as one caused by inflammation. An example of a person at risk of esophageal adenocarcinoma is someone with Barrett's esophagus or GERD. Inhibiting a disease process includes preventing the onset of the disease. Treatment refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop.

[0037] Shear stress: The stress component that lies coplane with the cross-section of the material. Shear stress arises from force vector components parallel to the cross-section. The formula for calculating the mean shear stress is the force per unit area.

number

[0038] Stenosis: A narrowing or constriction of the esophagus that causes difficulty swallowing. Symptoms of esophageal stricture include heartburn, a bitter or sour taste in the mouth, choking, coughing, shortness of breath, frequent belching or hiccups, painful or dysphagia, vomiting blood, and / or weight loss. Esophageal stricture can be caused by gastroesophageal reflux disease, esophagitis, lower esophageal sphincter dysfunction, motility disorders, alkaline fluid intake (lye ingestion), or hiatal hernia. Stenosis can form after esophageal surgery and other procedures, such as laser therapy or photodynamic therapy. As the area heals, scarring occurs, which causes the tissue to be pulled and constricted, resulting in difficulty swallowing. Stenosis can also be a result of inflammation. Barium swallowing tests or upper gastrointestinal endoscopy can be used to diagnose esophageal stricture.

[0039] Stiffness: The rigidity of an object or fluid. The stiffness of the extracellular matrix is ​​important for guiding cell migration in durotaxis motion. Stiffness can be measured in Pascals (Pa), where 1 Newton per square meter is the unit of stiffness.

[0040] Therapeutic agents: When used in a general sense, therapeutic agents include treatment agents, prophylactic agents, and alternative agents. "Treatment" or "to treat" means providing a patient with a substance, such as ECM hydrogel, in an amount sufficient to reduce, suppress or alleviate any disease symptoms to a measurable degree, slow disease progression, or cause disease regression. In certain embodiments, treatment of a disease may be initiated before the patient exhibits symptoms of the disease. The disclosed methods suppress inflammation of the esophagus and / or mitigate the effects of inflammation of the esophagus.

[0041] Therapeutic Effective Dose: The "therapeutic effective dose" of a composition such as ECM hydrogel refers to the amount that, when administered to a patient, is effective in providing therapeutic benefits such as symptom improvement. The amount of ECM hydrogel is therapeutically effective if it is sufficient to achieve the desired effect in the treated subject, for example, by forming a gel when injected into the submucosal tissue of the esophagus, thereby separating the upper mucosa from the lower muscularis propria. The amount effective in forming a submucosal cushion depends on the preparation applied, the treated subject, the severity and type of pain, and the method of administration.

[0042] The ECM hydrogels useful in the methods disclosed herein have applications in both medical and veterinary settings. Therefore, the general terms “subject” or “patient” are understood to include all animals, including but not limited to human or veterinary subjects, such as other primates, dogs, cats, horses, and cattle.

[0043] Bladder ECM: Extracellular matrix derived from the bladder of any mammal. This term includes bladder matrix (UBM) ECM and bladder submucosa (UBS) ECM. The bladder wall consists of the following layers: mucous layer (tunica mucosa) (including the transitional epithelium and lamina propria), submucosa layer, and up to three muscular and adventitia layers (loose connective tissue layer) - enumerated from luminal to antiluminal in a thickness cross-section. UBS is prepared from a tissue composition including submucosa tissue of the bladder peeled from the antiluminal muscular layer, and the luminal portion of the mucous layer of at least a segment of the 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 directly beneath the basement membrane (see U.S. Patent No. 6,576,265 incorporated herein by reference). The disclosed method does not use a hydrogel made from bladder ECM.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural subjects unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It should be further understood that all base size or amino acid size and all molecular weight or molecular mass values ​​given with respect to nucleic acids or polypeptides are approximations and are provided for illustrative purposes only. Methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. The term “comprises” means “includes.” The term “about” means within 5 percent. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including the definitions of terms, shall prevail. Furthermore, the materials, methods, and examples provided are illustrative only and not intended to be limiting.

[0045] Extracellular matrix (ECM) hydrogel 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 prepare hydrogels that can be used in the methods disclosed herein (U.S. Patents No. 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, and 5,762,963 relating to ECM). See also No. 6, No. 5,866,414, No. 6,099,567, No. 6,485,723, No. 6,576,265, No. 6,579,538, No. 6,696,270, No. 6,783,776, No. 6,793,939, No. 6,849,273, No. 6,852,339, No. 6,861,074, No. 6,887,495, No. 6,890,562, No. 6,890,563, No. 6,890,564, and No. 6,893,666). In certain embodiments, the ECM is isolated from vertebrates, including but not limited to warm-blooded mammalian vertebrates such as humans, monkeys, horses, pigs, cattle, and sheep. In non-limiting specific examples, the ECM is porcine or human ECM.

[0046] ECM may be derived from any organ or tissue other than the bladder, including but not limited to the intestines, liver, esophagus, and dermis. ECM may be derived from the kidneys, heart, uterus, brain, blood vessels, lungs, bones, muscles, pancreas, stomach, spleen, or colon. In one embodiment, ECM is derived from the esophagus. In another embodiment, ECM is derived from the small intestine or dermis. ECM may or may not include the basement membrane portion of the ECM. In certain embodiments, ECM includes at least a portion of the basement membrane. In other embodiments, ECM is harvested from a cell culture. ECM hydrogels can be prepared from a combination of two or more tissue sources.

[0047] U.S. Patent No. 6,893,666, incorporated herein by reference, also discloses the preparation of ECMs from skin, esophagus, and small intestine. The preparation of hydrogels from decellularized dermal ECMs is disclosed in Wolf et al., Biomaterials 33: 7028-7038, 2012, incorporated herein by reference. The preparation of ECMs from esophageal tissues is disclosed, for example, in Badylak et al., J Pediatr Surg. 35(7):1097-103, 2000 and Badylak et al., J. Surg. Res. 2005 September; 128(1):87-97, 2005, both incorporated herein by reference. Any of these methods can be used, however, ECMs are not prepared from the bladder.

[0048] Commercially available ECM preparations can also be used in the methods, devices, and compositions described herein. In one embodiment, the ECM is derived from the submucosa of the small intestine, i.e., the SIS. Commercially available preparations include, but are not limited to, SURGISIS®, SURGISIS-ES®, STRATASIS®, and STRATASIS-ES® (Cook Urological Inc., Indianapolis, Ind.), and GRAFTPATCH® (Organogenesis Inc., Canton Mass.). In another embodiment, the ECM is derived from the dermis. Commercially available preparations include, but are not limited to, PELVICOL® (sold as PERMACOL® in Europe, Bard, Covington, Ga.), REPLIFORM® (Microvasive, Boston, Mass.), and ALLODERM® (LifeCell, Branchburg, NJ).

[0049] Source tissues used for the preparation of ECM can be collected in a wide variety of ways, and various parts of the collected tissue may be used immediately after collection. 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 exostata, digesting the mucosa in a trypsin-containing buffer, 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 mucous layer, serosal membrane, and surface layer of the muscularis exostata from an intact small intestine, leaving the submucosa, muscularis mucosa, and basal compacta intact. The SIS is then treated with peracetic acid. An exemplary protocol is provided in Keane et al.

[0050] In some embodiments, epithelial cells can be exfoliated by first immersing the tissue in a deepithelializing solution, such as hypertonic saline, for a period ranging from 10 minutes to 4 hours, but not limited to 1.0 N saline. Exposure to hypertonic saline effectively removes the epithelial cells from the underlying basement membrane. The tissue remaining after the initial exfoliation procedure includes the epithelial basement membrane and the tissue layer antiluminal relative to the epithelial basement membrane. This tissue is then subjected to further processing to remove most of the antiluminal tissue rather than the epithelial basement membrane.

[0051] ECM can be sterilized or disinfected by any number of standard techniques, including but not limited to exposure to peracetic acid, low-dose gamma-ray radiation, gas plasma sterilization, ethylene oxide treatment, supercritical CO2, or electron beam treatment. More typically, ECM sterilization is achieved by immersion for 2 hours in 0.1% (v / v) peracetic acid, 4% (v / v) ethanol, and 95.9% (v / v) sterile water. The peracetic acid residue is removed by washing twice for 15 minutes with PBS (pH=7.4) and twice for 15 minutes with sterile water. ECM materials can be sterilized by propylene oxide or ethylene oxide treatment, gamma-ray irradiation (0.05-4 mRad), gas plasma sterilization, supercritical CO2, or electron beam treatment. ECM can also be sterilized by treatment with glutaraldehyde, which induces crosslinking of protein materials, but this treatment substantially alters the material so that it is slowly reabsorbed or not reabsorbed at all, promoting a different type of host remodeling that is more similar to scar tissue formation or encapsulation than to constructive remodeling. Crosslinking of protein materials can also be induced using carbodiimide, or by dehydrothermal or photo-oxidation methods. As disclosed in U.S. Patent No. 8,361,503, ECM is sterilized by immersion for 2 hours in 0.1% (v / v) peracetic acid (a), 4% (v / v) ethanol, and 96% (v / v) sterile water. The ECM material is then washed twice for 15 minutes with PBS (pH=7.4) and twice for 15 minutes with deionized water.

[0052] After isolation of the target tissue, decellularization is carried out by various methods, such as exposure to hypertonic saline, peracetic acid, TRITON®-X, or other surfactants, though not limited to these. Sterilization and decellularization may be performed simultaneously. For example, sterilization using peracetic acid as described above may also contribute to the decellularization of the ECM, though not limited to these methods. The decellularized ECM can then be dried, i.e., freeze-dried or air-dried. The dried ECM can be pulverized by methods including, but not limited to, smashing, grinding, cutting, crushing, and shearing. The pulverized ECM can be further processed into a powder form by methods such as grinding or grinding in a frozen or freeze-dried state, though not limited to these methods.

[0053] To prepare solubilized ECM tissue for use in preparing ECM hydrogels, pulverized ECM is digested in an acidic solution using an acidic protease to form a digest solution. The ECM digest solution is typically kept at room temperature for a certain amount of time with constant stirring. The ECM digest may be used immediately, stored at -20°C, or frozen at -20°C or -80°C, for example, but not limited to these.

[0054] 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 7.4. Bases, such as bases containing hydroxide ions including NaOH, can be used to raise the pH of the solution. Similarly, buffers, such as isotonic buffers 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 target levels, such as physiological pH and ionic conditions. This forms a “pregel” solution, which is in liquid form as a viscous solution at room temperature. The neutralized digested solution (pregel) can gel at temperatures approaching physiological temperature, approaching 37°C. In some embodiments, the method does not involve a dialysis step before gelation and produces a more complete ECM-like matrix that gels at a characteristic rate, typically at 37°C (see below).

[0055] Therefore, the extracorporeal membrane complex (ECM) can typically originate from mammalian tissue, such as, but not limited to, the dermis, esophagus, small intestine, kidney, liver, heart, uterus, brain, blood vessels, lungs, bone, muscle, pancreas, stomach, spleen, or colon. The ECM is not bladder ECM, and therefore neither UBM nor UBS.

[0056] ECM hydrogels can be prepared from two or more tissue sources, e.g., two, three, or four tissue sources. In one non-limiting embodiment, the ECM is freeze-dried and pulverized. The ECM is then solubilized in an acidic solution using an acidic protease to prepare digestive ECM, such as esophageal ECM. The acidic protease may be pepsin, trypsin, or a combination thereof, but is not limited. The ECM can then be solubilized in a solution, for example, 0.01 M HCl, at an acidic pH suitable or optimal for the protease, e.g., a pH greater than about 2 or up to 4. The solution is typically solubilized for about 12 to about 48 hours, depending on the tissue type (see, for example, the examples below), while being mixed (stirring, mixing, blending, rotating, and tilting, etc.). ECM hydrogels are prepared by (i) pulverizing the extracellular matrix, (ii) solubilizing the intact, undialysis-free, or uncrosslinked extracellular matrix in an acidic solution using an acidic protease to prepare a digested solution, (iii) raising the pH of the digested solution to between 7.2 and 7.8 to prepare a neutralized digested solution (pregel solution), and (iv) gelling the solution in the esophagus of the subject at a temperature of approximately 37°C. When an acidic protease is used to digest the ECM, the pregel solution and the resulting hydrogel may contain inactivated proteases.

[0057] ECM hydrogels form a gel when exposed to a temperature of approximately 37°C. ECM hydrogels in "pre-gel" form can be frozen and stored at, for example, -20°C or -80°C, though not limited to these temperatures. ECM hydrogels in "pre-gel" form can be stored at room temperature, for example, approximately 25°C. Therefore, ECM hydrogels are in pre-gel form below 37°C, for example, at 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, and 4°C. ECM hydrogels can be frozen for storage and therefore stored below 0°C. As used herein, the terms "pre-gel form" or "pre-gel" refer to ECM hydrogels that have increased pH but have not gelled. For example, pre-gel ECM hydrogels have a pH between 7.2 and 7.8, though not limited to these temperatures. ECM hydrogel can be delivered to the subject in pregel form using an endoscope.

[0058] ECM hydrogel in pregel form is suitable for introduction into the patient's esophagus. When introduced into the submucosa of the esophagus, which is approximately 37°C, the ECM hydrogel gels, creating a cushion between the muscularis propria and submucosa of the esophagus, lifting the submucosa for surgical resection. While not bound by theory, ECM hydrogel contains many innate soluble factors, such as cytokines, for example, but not limited to these. Specific characteristics of undialysis-free (whole ECM) preparations prepared from diverse tissues are disclosed herein.

[0059] In some embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation in less than 30 minutes at a temperature of about 37°C, b) a flow viscosity suitable for esophageal infusion, 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 about 1,000 Pa, or vi) about 10 to about 70 Pa.

[0060] In several embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation in less than 30 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, 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 in less than 30 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, 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 in less than 30 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, and c) a stiffness of about 10 to about 600 Pascals (Pa).

[0061] In other embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation in less than 30 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, 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 in less than 30 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, 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 in less than 30 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, and c) a stiffness of 10 to 70 Pascals (Pa).

[0062] In some embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation in less than 30 minutes at a temperature of about 37°C, b) a flow viscosity suitable for esophageal infusion, 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 about 1,000 Pa, or vi) about 10 to about 70 Pa.

[0063] In some embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation in less than 20 minutes at a temperature of about 37°C, b) a flow viscosity suitable for esophageal infusion, 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 about 1,000 Pa, or vi) about 10 to about 70 Pa.

[0064] In several embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation in less than 20 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, 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 in less than 20 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, 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 in less than 20 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, and c) a stiffness of about 10 to about 600 Pascals (Pa).

[0065] In other embodiments, the ECM hydrogel has the following characteristics: a) a time to 50% gelation in less than 20 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, 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 in less than 20 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, 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 in less than 20 minutes at a temperature of about 37°C, b) a fluid viscosity suitable for esophageal infusion, and c) a stiffness of 10 to 70 Pascals (Pa).

[0066] 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 fluid viscosity sufficient for injection into the esophagus, c) a stiffness of about 10 to about 300 Pascals (Pa), and d) the hydrogel is an esophageal hydrogel.

[0067] In a non-limiting example, ECM hydrogel is esophageal hydrogel. In another non-limiting example, ECM hydrogel can be prepared from two or more tissue sources. In a further non-limiting example, ECM hydrogel can be prepared from the dermis, esophagus, or small intestine. ECM hydrogel is not bladder ECM hydrogel.

[0068] In additional, non-limiting examples, an ECM hydrogel may be prepared by (a) solubilizing cell-free extracellular matrix (ECM) by tissue digestion using an acidic protease in an acidic solution to produce digested ECM; (b) raising 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 at a concentration of approximately 2 mg / ml to 16 mg / ml, for example, approximately 8 mg / ml to 12 mg / ml. This hydrogel is then introduced into the esophagus of the subject, where it gels. In a non-limiting example, the ECM may be esophageal ECM.

[0069] In the methods disclosed herein, useful ECM hydrogels have a time to 50% gelation of less than 30 minutes, such as less than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 minutes at a temperature of about 37°C. In some embodiments, the ECM hydrogel has a time to 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.

[0070] The disclosed ECM hydrogels can have a flow viscosity suitable for injection into the esophagus. In some embodiments, the ECM hydrogel has a flow viscosity of about 10 to about 100 Pa * s, such as about 10, 20, 30, 40, 50, 60, 70, 80, or 90 Pa * s at a shear rate of 0.2 / s. In further embodiments, the flow viscosity is about 0.1 to about 100 Pa * s at a shear rate of about 0.1 / s and about 0.01 to about 0.2 Pa * s at a shear rate of 1000 / s. In further embodiments, the flow viscosity is about 0.1 to about 30 Pa * s at a shear rate of 1 / s and about 0.02 to about 0.8 Pa * s at a shear rate of about 100 / s.

[0071] In some embodiments, the ECM hydrogel has a flow viscosity of about 0.1 to about 30 Pa *It has a flow viscosity of s. In a further embodiment, the ECM hydrogel has a shear rate of about 0.1 to about 100 Pa at about 0.1 / s. * It has a fluid viscosity of s. In non-limiting specific examples, ECM hydrogels have a viscosity of 0.5 to about 50 Pa. * The ECM hydrogel has a flow viscosity of s, or approximately 1 to approximately 40 Pa at a shear rate of 0.1 / s. * It has a fluid viscosity of s. Exemplary fluid viscosities are approximately 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.

[0072] In other embodiments, the ECM hydrogel has a shear rate of approximately 0.01 to approximately 0.20 Pa at 1000 / s. * At shear rates of s or 1000 / s, approximately 0.01 to approximately 0.10 Pa * The fluid viscosity is approximately 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 at a shear rate of, for example, 1000 / s.

[0073] In further embodiments, the ECM hydrogel is subjected to approximately 0.02 to approximately 0.8 Pa at a shear rate of 100 / s. * At shear rates of s or 100 / s, approximately 0.1 to approximately 0.8 Pa * s, for example, about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.08 Pa * It has s.

[0074] In other embodiments, the ECM hydrogel exhibits a shear rate of approximately 0.1 to approximately 30 Pa at 1 / s. * For example, s, approximately 1 to approximately 20 Pa * s, or 1 to approximately 10 Pa * s, or 0.5-25 Pa *At a shear rate of s, for example 1 / s, the pressures are approximately 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. * It has a fluid viscosity of s. The shear rate is, for example, 5, 10, 20, or 30 Pa at a shear rate of 1 / s. * It may be 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, for example, 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 is about 0.1 to about 30 Pa at a shear rate of 1 / s. * It may also be s, and at a shear rate of 100 / s, it is approximately 0.02 to approximately 0.8 Pa. * s. In additional embodiments, the fluid viscosity is about 1 to about 10 Pa at a shear rate of 1 / s. * It is s, and at a shear rate of 100 / s, it is approximately 0.02 to approximately 0.5.

[0075] In further embodiments, the ECM hydrogel is subjected to a shear rate of 0.1 / s at approximately 10 to approximately 100 Pa. * It has a flow viscosity of s. In other embodiments, the ECM hydrogel has a viscosity of about 0.01 to about 0.2 Pa at a shear rate of 1000 / s. * It has a flow viscosity of s. In other embodiments, the ECM hydrogel has a shear rate of 0.1 / s of about 1 to about 40 Pa * It has a fluid viscosity of s and a shear rate of 0.01-0.2 Pa at 1000 / s. * It is s.

[0076] 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 may have a stiffness of about 10 to about 300 Pascals (Pa), for example, 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 hydrogel has 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 hydrogel may 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.

[0077] In some embodiments, the ECM concentration in the hydrogel is about 2 mg / ml to about 20 mg / ml, for example, about 8 mg / ml to about 12 mg / ml or about 2 mg / ml to about 16 mg / ml. In other embodiments, the ECM concentration in the hydrogel is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 mg / ml. Useful exemplary concentrations, though not limited to these, include about 9 mg / ml to about 11 mg / ml and about 10 mg / ml to about 12 mg / ml. Additional exemplary concentrations include about 8 mg / ml to about 10 mg / ml, about 8 mg / ml to about 11 mg / ml, about 8 mg / ml to about 13 mg / ml, about 8 mg / ml to about 14 mg / ml, about 8 mg / ml to about 15 mg / ml, and about 8 mg / ml to about 16 mg / ml. Further exemplary concentrations that may be useful include approximately 6 mg / ml to 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, or 16 mg / ml.

[0078] The disclosed ECM hydrogel can be provided as a component of a kit. The ECM hydrogel can be provided in a frozen or lyophilized form. In some embodiments, the kit may include components necessary to form the hydrogel, e.g., one container containing the hydrogel, e.g., in a lyophilized form; one container containing a solution for solubilizing the lyophilized hydrogel; and a container containing a neutralizing solution for neutralizing the solubilized form as needed. In other embodiments, the kit may include a container containing the solubilized hydrogel and a second container containing a neutralizing agent.

[0079] If necessary, such a kit may include additional components, such as packaging materials, instructions, and various other reagents, e.g., buffers, supports, or other therapeutic components. The kit may include a container and a label or accompanying information on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Containers typically contain a composition, such as an ECM hydrogel in a freeze-dried or lyophilized form, which is effective in suppressing esophageal inflammation and / or mitigating the effects of esophageal inflammation in a subject. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a subcutaneous needle). The label or accompanying information indicates that the composition is used for endoscopic procedures related to a specific condition, such as colorectal cancer.

[0080] Labels or accompanying documents typically further include instructions for use. Accompanying documents typically include instructions customarily included on the commercial packaging of such therapeutic products, containing information about the indications, use, dosage, administration, contraindications, and / or warnings regarding the use of the therapeutic product. Instructional materials may be written in electronic format (e.g., computer diskette or compact disk) or visual (e.g., video file). Kits may also include additional components, such as needles or catheters, to facilitate the specific application for which the kit is designed. In addition, kits may include buffers and other reagents customarily used for the implementation of a particular method. Kits and suitable contents are well known to those skilled in the art.

[0081] Extracellular matrix (ECM) hydrogel Treatment method Methods for dissociating the mucosa and submucosa from the muscularis propria in the esophagus of a subject are disclosed herein, the methods comprising the step of submucosally injecting into the esophagus a pharmaceutical composition comprising an ECM hydrogel other than bladder extracellular matrix (ECM) hydrogel (e.g., UBS or UBM) to form a cushion between the submucosa and the underlying muscularis propria in a region of the esophagus. The method may be EMR or EMD.

[0082] Endoscopic mucosal resection (EMR) is an endoscopic technique developed for the removal of sessile or flat neoplasms confined to the superficial layers (mucosa and submucosa) of the GI duct. EMR is typically used for the removal of lesions smaller than 2 cm or for the gradual removal of larger lesions. EMR also plays an important role in evaluating the excised specimen for accurate pathological staging. Unlike polypectomy, EMR involves lifting the lesion from the muscle layer by injecting a fluid, generally 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 the complete removal of the affected mucosa by excising the central or deeper portion of the submucosa of the intestinal wall. Various EMR techniques have been described, with four commonly used methods involving snare resection: (1) injection and dissection method, (2) injection, lift, and dissection method, (3) cap-assisted EMR (EMRC), and (4) EMR with ligation (EMRL). In injection and cutting techniques, the affected mucosa is lifted from the muscle layer by creating a submucosal fluid cushion, captured, constricted using an electrosurgical snare, and then excised. However, injection into the thin submucosa is a delicate process, the injected solution tends to dissipate quickly, squamous and depressed lesions are more difficult to capture with a snare compared to protruding lesions, and large or awkwardly located lesions may be difficult to remove (Uraoka et al., Drug Design, Development and Therapy 2008:2 131-138). Injection-assisted EMR is often used for large squamous colon polyps.

[0083] Endoscopic submucosal dissection (ESD) was developed specifically to remove larger lesions. Lesions are directly dissected along the submucosa using an electrosurgical unit, resulting in en bloc resection of even large lesions. While ESD is predicted to replace conventional surgery in treating certain cancer stages, it requires more advanced endoscopic skills and experience than conventional EMR due to its higher rate of perforation and hemorrhagic complications. ESD can utilize a variety of electrosurgical units, such as insulated-tip diathermy knives, needle knives, hook knives, flex knives, triangle-tipped knives, flush knives, splash needles, and small-diameter transparent hoods. These knives can be used in conjunction with high-frequency electrosurgical current (HFEC) generators. ESD is characterized by three steps: (1) injecting a fluid to form a submucosal cushion and elevate the lesion from the muscle layer, (2) circular cutting of the mucosa surrounding the lesion, and (3) dissociation of the connective tissue of the submucosa beneath the lesion (see Kakushima et al., Wold J. Gastroenterol. 14(9): 2962-2967, 2008, incorporated herein by reference). While various submucosal injection solutions have been developed and shown to be sufficient for use during EMR, the introduction of longer-duration ESD procedures has required longer-lasting solutions that help identify the cutting line during submucosal dissociation (Uraoka et al., Drug Design, Development and Therapy 2008:2 131-138). The method of this disclosure satisfies this need.

[0084] Submucosal injection is used in EMR because it facilitates the isolation of tissue to be removed, for example, immediately before capturing the target lesion using a snare, thereby reducing the risk of burns, as well as perforation and bleeding, while also facilitating excision. Submucosal injection plays a crucial role in EMR procedures because the solution must be held in place for a sufficient duration and form a hemispherical shape to facilitate snareing. In addition, achieving sufficiently high elevation of the submucosa leads to safe submucosal dissection during ESD procedures (Uraoka et al., Drug Design, Development and Therapy 2008:2 131-138). Furthermore, since inflammation is caused by the procedure, any cushion held at the procedure site should have anti-inflammatory properties. ECM hydrogel reduces stenosis and promotes re-epithelialization. The method of this disclosure also satisfies this need.

[0085] In some embodiments, the disclosed method utilizes an ECM hydrogel that has anti-inflammatory properties, is inexpensive, non-toxic, easy to inject, and provides a long-lasting, high submucosal cushion. The ECM hydrogel is administered in a pre-gel form and then gels at the injection site to form a cushion. The cushion can be dissociated during the procedure so that some of the hydrogel remains in the underlying muscularis propria, thereby aiding healing. The disclosed ECM hydrogel facilitates the closure of wounds created by the removal of excised mucosa / submucosa tissue. In some embodiments, the procedure is ESD. In other embodiments, the procedure is EMR.

[0086] Physiological saline (NS) and more dilute solutions (e.g., ELEVIEW®, see U.S. Patent No. 9,226,996 incorporated herein by reference) are used as submucosal cushions for endoscopic resection; however, 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 tissue is removed, these agents are no longer retained in the underlying muscularis propria. Moreover, these agents do not aid the healing process by reducing inflammation, etc. The use of ECM hydrogels fulfills these needs.

[0087] The ECM hydrogels disclosed herein can be used in any ESD or ESR. As disclosed in U.S. Patent No. 9,364,580, incorporated herein by reference, an endoscopic injection needle is a device that may be long (up to about 230) cm, comprising a relatively long catheter in which an internal injection tube with a distal injection needle is slidably positioned inside. A proximal working handle is connected to the catheter and injection tube to move one relative to the other when necessary. Access to the injection tube is typically achieved via a Luer connector on the handle. The endoscopic injection needle device is typically delivered to the injection site through the working channel of the endoscope. To protect the lumen of the endoscopic working channel from damage, the device is inserted into the endoscope after operating the handle of the injection needle device to retract the distal injection needle into the lumen of the catheter. As the device moves through the lumen of the endoscope, this prevents exposure of the sharp tip of the injection needle. Once the distal end of the endoscopic injection needle device is positioned at the injection site, the handle is operated again to move the injection needle distally from the catheter lumen. When advanced to its most distal position, the exposed portion of the injection needle is approximately 4-6 mm long.

[0088] After the injection site has been penetrated, ECM in pregel form, typically contained in a 5 mL to 10 mL syringe equipped with a Luer lock fitting attached to the handle of the injection needle, can be delivered through the injection tube and needle between the injection site, for example, the submucosa and the underlying muscularis propria.

[0089] Injection needles and other accessories commonly used during endoscopic procedures, such as snares, clip devices, and biopsy forceps for polypectomy, typically pass through one or more specific channels in the endoscope, usually called working channels or operating channels. Depending on the type of endoscope used, the inner diameter of the working channel may vary. However, the most common endoscopes used in GI endoscopy have working channels with inner diameters ranging from approximately 2 mm to approximately 5 mm. In general, manufacturers of endoscopic accessories produce accessories with outer diameters that allow the accessories to fit into all working channels. In some embodiments of endoscopic injection needles, the outer diameter of the catheter is in the range of 1.9 mm to 2.3 mm, for example, approximately 1.9, 2.0, 2.1, 2.2, or 2.3 cm. Therefore, considering that the internal injection tube is contained within the external catheter, its inner diameter is typically 1 mm or less. ECM hydrogels disclosed in pregel form can easily pass through these catheters.

[0090] ECM hydrogel in pregel form can be used in endoscopic resection procedures by aspirating a certain volume of emulsion from its primary container with a syringe, and injecting a suitable volume of the emulsion just below the surface mucosal layer using an endoscopic injection needle inserted into the working channel of the endoscope, thereby placing a cushioning liquid volume in the submucosa when in a predetermined position. The elevation of the mucosal surface allows for easy resection of mucosal lesions discovered by the endoscopist during the performance of the endoscopic procedure, even when the lesion is flat and therefore does not protrude into the lumen, such as the esophageal lumen.

[0091] The presence of at least one dye in the cushion can visualize the underlying structures (e.g., the submucosa and the outer muscular wall), thereby assisting the endoscopist in reducing the risk of causing damage to these structures during the resection procedure. The use of dyes can enable visualization of the cushion cavity and the mucosal base. Removal of the lesion from the mucosal surface results in mucosal wounds. The retention of the cushion created by the injected volume of the 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 the esophagus of a subject, for example, in the area of ​​a lesion or tumor, to form a cushion between the submucosa and the underlying muscularis propria in the area of ​​the esophagus. The cushion can be dissociated so that a portion of the ECM hydrogel is maintained in the underlying muscularis propria and assists the healing process.

[0092] The disclosed method is useful in the esophagus. In one non-limiting example, the method includes a method for dissecting esophageal cancer or esophageal adenocarcinoma from the esophagus. In another non-limiting example, the method includes dissecting the mucosa and submucosa from the esophagus of a subject having Barrett's esophagus.

[0093] 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 hydrogels may be maintained at, for example, 25°C or 4°C before administration. An effective amount of the ECM hydrogel in pre-gel form is then administered into the esophagus of the subject. The ECM hydrogels are provided in lyophilized or frozen form and may be reconstituted immediately before administration to the subject.

[0094] The disclosed method is useful in any subject, including human and veterinary subjects. Subjects may be of any age. In one embodiment, a composition comprising an ECM hydrogel in pregel form is injected into human target tissue to form a cushion which is provided as needed after an endoscopic surgical procedure such as an excision procedure. The ECM may be of the same species as the subject being treated, or of a different species. In some embodiments, the subject is human, and the ECM hydrogel is derived from human and / or porcine tissue. In other embodiments, the ECM hydrogel is derived from a non-human primate, dog, cat, horse, or cattle. The ECM may also be derived from a commercially available source. The ECM hydrogel is not a bladder ECM hydrogel such as UBM or UBS.

[0095] The disclosed methods are invasive because they require injection to dissociate the mucosa and submucosa from the muscularis propria originating from the esophageal region. Any of the methods disclosed herein may include the step of submucosally injecting a pharmaceutical composition containing ECM hydrogel into the esophagus of a subject to form a cushion between the submucosa and the underlying muscularis propria in the esophageal region. 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 fluid viscosity suitable for injection into the esophagus, and c) a stiffness of about 10 to about 400 Pascals (Pa). The ECM hydrogel gels, dissociating the mucosa and submucosa from the underlying muscularis propria, thereby suppressing inflammation in the esophageal region of the subject. The ECM hydrogel in pregel form may be administered endoscopically or via catheter.

[0096] In some embodiments, the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection of the esophagus, and the method includes a method for dissecting esophageal cancer or esophageal adenocarcinoma from the esophagus. In further embodiments, the method includes dissecting the mucosa and submucosa from the esophagus of a patient with dysplasia. In further embodiments, the method includes dissecting the mucosa and submucosa from the esophagus of a subject with Barrett's esophagus.

[0097] The method may also include performing an endoscopic resection procedure on the cushion. In some embodiments, the method involves dividing the cushion so that the hydrogel is retained on the underlying muscularis propria of the esophagus and the mucosa and submucosa are removed from the area of ​​the esophagus. In some non-limiting examples, a portion of the hydrogel cushion retained on the underlying muscularis propria downmodulates pro-inflammatory macrophage activation in the esophagus.

[0098] ECM can originate from any tissue other than the bladder. Therefore, the hydrogel is neither UBM nor UBS. In some embodiments, the time to 50% gelation of the hydrogel is less than 30 minutes at a temperature of approximately 37°C. In some non-limiting specific examples, the time to 50% gelation is approximately 2 to approximately 30 minutes at approximately 37°C. In other non-limiting specific examples, the time to 50% gelation is approximately 2 to approximately 10 minutes at approximately 37°C. In further non-limiting examples, the time to 50% gelation is approximately 3 to approximately 10 minutes.

[0099] In additional embodiments, the flow viscosity of the pregel form is sufficient for injection into the esophagus. In some embodiments, the flow viscosity of the ECM hydrogel is about 0.1 to about 100 Pa at a shear rate of about 0.1 / s. * The pressure is approximately 0.01 to 0.2 Pa at a shear rate of 1000 / s. * It is s. In some non-limiting examples, the flow viscosity is about 30 Pa at a shear rate of 1 / s. * The pressure is approximately 0.02 to 0.8 Pa at a shear rate of approximately 100 / s. * It is s.

[0100] In further embodiments, the ECM hydrogel has a stiffness of about 10 to about 300 Pascals (Pa) when introduced into tissue, and the ECM hydrogel has a stiffness of 10 to 70 Pa. In further embodiments, the ECM concentration in the hydrogel is 2 mg / ml to about 16 mg / ml.

[0101] ECM hydrogels can be prepared by any of the methods disclosed herein. In some embodiments, ECM hydrogels are prepared by (a) solubilizing decellularized extracellular matrix (ECM) by tissue digestion using an acidic protease in an acidic solution to prepare digested ECM, and (b) raising the pH of the digested ECM to a pH between 7.2 and 7.8 to prepare a neutralized digested solution. In further embodiments, step (b) of raising the pH of the digested ECM includes raising the pH of the digested ECM by adding a base or isotonic buffer. In further embodiments, acidic proteases such as pepsin, trypsin, or a combination thereof are used. The ECM hydrogel may be an esophageal ECM hydrogel.

[0102] In some embodiments, the ECM hydrogel is maintained at 25°C or below 25°C before administration to the subject. In some embodiments, the ECM hydrogel is maintained at approximately 4°C to approximately 28°C, for example, approximately 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 approximately 4°C and used at approximately 4°C to approximately 25°C, or it may be warmed to approximately 25°C immediately before use. In some embodiments, controlling the temperature ensures that the ECM hydrogel is maintained in a pre-gel form and is therefore suitable for injection between the submucosa and the underlying muscularis propria. In further embodiments, the hydrogel gels when administered to the subject, for example, when it reaches a temperature of 37°C. [Examples]

[0103] 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 lifting for EMR and ESD procedures. It is disclosed herein that ELEVIEW® does not form a hydrogel that is stably formed at 37°C, but rather remains liquid for more than one hour at 37°C. In contrast, 12 mg / mL of esophageal ECM hydrogel (eECM) (prepared according to the method disclosed herein, i.e., by protease digestion of ECM) forms a hydrogel that is stably formed at 37°C (body temperature). ELEVIEW® and eECM can be injected submucosa. An ideal biomaterial would adhere to both layers. For muscle, there was no difference in mucosal adhesion strength between ELEVIEW® and eECM 12 mg / mL. Surprisingly, eECM exhibited stronger mucosal adhesion to mucosa than ELEVIEW®. Furthermore, eECM demonstrated bioactivity by macrophage polarization to a remodeling phenotype. This demonstrates that extracellular matrix hydrogels can be effectively used as submucosal cushions and therefore can be used to dissociate submucosal tissue from the underlying muscularis propria.

[0104] (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). The samples were kept at 4°C and loaded into a rheometer with a parallel-plate shape that had been 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 sequentially for each sample. Steady-state flow curves at 10°C were obtained for various shear rates (0.1 to 1000 s). -1The viscosity profile of the sample was determined. The plate temperature was rapidly increased from 10°C to 37°C, and a vibration time sweep was performed at 37°C by applying a small vibration strain of 0.5% at a frequency of 1 rad / s to measure the maximum storage modulus (G'), maximum loss modulus (G"), and gelation dynamics. The data were extracted for statistical analysis and analyzed in Prism (version 6, GraphPad) (n=3).

[0105] Mucosal attachment to the muscular layer Porcine mucosa and muscularis were mechanically isolated by peeling the mucosa and submucosa from the underlying muscularis layer. ELEVIEW® and eECM (12 mg / mL) were pipettered into a 6-well plate. The mucosa or muscularis was attached to the bottom of a hemisphere (40 mm in diameter) on top of ELEVIEW® or eECM, ensuring that the surface area of ​​the mucosa or muscularis in contact with ELEVIEW® or eECM remained constant for all tests. The construct was incubated at 37°C for 1 hour to allow adhesion to the mucosa or muscularis. After 1 hour, the construct was placed on an MTS Insight tensile testing machine set to a measurement frequency of 10 Hz, equipped with a 10 N load cell and a ball-bursting attachment. 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 taken only when peeling occurred between the mucosa or muscle layer and the hydrogel (n=3).

[0106] Performance of submucosal fluid cushions in ex vivo Pork esophagus was placed in a 37°C incubator, and the 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 submucosa using a 23G needle. The eECM was kept on ice during the procedure. The tissue was evaluated at 15-minute intervals for up to 75 minutes and photographed alongside evidence of the measurement criteria. The tissue was kept incubated at 37°C throughout the procedure. After 75 minutes, the injection site was dissected and evaluated. ImageJ was used to quantify mucosal elevation after drug injection throughout the experiment.

[0107] Isolation and activation of macrophages Mouse bone marrow was collected as previously described [1, 2]. Briefly, 6-8 week old female C57bl / 6 mice (Jackson Laboratories, Bar Harbor, ME) were euthanized by CO2 inhalation and cervical dislocation. Aseptically, the skin from the proximal hindlimb to the foot was removed, the tarsal and posterior knee joints were dissected, and the tibia was isolated. The coxafemoral joint was dissected for femoral isolation. After removing excess tissue, the bones were kept on ice and rinsed in a sterile dish containing macrophage complete 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 transversely incised, and the medullary cavity was washed with complete medium to collect the bone marrow. The cells were washed, plated at 2 × 10⁶ cells / ml, and differentiated into macrophages for 7 days at 37°C, 5% CO₂, with complete medium changes every 48 hours, as previously described[3]. Seven days later, the resulting naive macrophages were treated for 24 hours at 37°C and 5% CO2 with a basal medium consisting of 10% FBS in DMEM, 100 ug / ml streptomycin, and 100 U / ml penicillin, as well as one of the following conditions previously described: (1) 20 ng / ml IFNγ and 100 ng / ml LPS to promote the M1-like phenotype, (2) 20 ng / ml IL-4 to promote the M2-like phenotype, (3) 250 ug / ml pepsin control buffer, (4) 250 ug / ml esophageal ECM, or (5) the same volume of ELEVIEW®.[4]

[0108] Immunolabeling of macrophages After 24 hours, macrophages were washed and fixed with 2% paraformaldehyde. After washing with PBS, the cells were incubated at room temperature for 1 hour in a blocking solution consisting of 0.1% Triton®-X100, 0.1% Tween® 20, 4% normal goat serum, and 2% bovine serum albumin (BSA) to prevent nonspecific antibody binding. The following primary antibodies were diluted in blocking solutions: (1) monoclonal anti-F4 / 80 (Abcam, Cambridge, MA) at a 1:100 dilution for a pan-macrophage marker, (2) polyclonal anti-iNOS (Abcam, Cambridge, MA) at a 1:100 dilution for an M2 marker, (3) polyclonal anti-Fizz1 (Peprotech, Rocky Hill, NJ) at a 1:100 dilution for an M2 marker, and (4) polyclonal anti-liver arginase (Abcam, Cambridge, MA) at a 1:100 dilution for an M2-like marker [5-7]. Cells were incubated in primary antibodies at 4°C for 16 hours. After washing with PBS, cells were incubated in fluorophore-conjugated secondary antibodies (Alexa Fluor goat anti-rat 488 or goat anti-rabbit 488, Invitrogen) at room temperature for 1 hour. After PBS washing, the nuclei were counterstained with 4'6'-diamidino-2-phenylindole (DAPI), and three 200x fields were imaged using a live cell microscope. Exposure time was standardized for negative isotype controls and kept constant throughout the image. Images were quantified using CellProfiler image analysis software to obtain percentages of positive F4 / 80, iNOS, Fizz1, and arginase 1.

[0109] In vivo use of the ECM as a submucosal fluid cushion for EMR. Anesthesia is induced using acepromazine (0.01 mg / kg, SC) and ketamine (5-11 mg / kg), and surgical-level anesthesia is maintained via endotracheal tube with 1-5% isoflurane. Animals are administered IV at a dose of 2 ml / kg / hour of Ringer's lactate solution during the procedure and immediately afterward. Temperature is controlled by a warm water recirculation pad placed beneath the animal. Physiological parameters, such as heart rate, respiratory rate, body temperature, and responsiveness, are monitored during the procedure. The procedure is initiated after administering antibiotic prophylaxis with 25 mg / kg cefazolin.

[0110] The animal is placed in a supine position, and the organs are evaluated using a Pentax EG3430K endoscope. After identifying reference points in the organs, the mucosa and submucosa at the excision site are separated at 4°C using an Olympus Injectorforce 4mm 23G needle with 8 mg / ml of blue-stained hydrogel for injection. This temperature is maintained throughout to prevent gelation and potential needle occlusion. Approximately 2-5 ml of blue gel is injected at each site. The entire circumference (100%) of a 5 cm length of mucosa is removed using the band ligation EMR technique. For EMR, a Cook Duette kit including ligation bands is used. The mucosa is then excised using a snare.

[0111] statistics Two-way ANOVA was used to compare the effects of the independent variables, shear rate and sample, on the dependent variable, viscosity, and also to compare the effects of the independent variables, sample and type of elastic modulus, on the dependent variable, elastic modulus. Sidac post-hoc multiple comparison tests were used to determine significance using 95% confidence intervals, and p-values ​​were adjusted for multiple comparisons. A t-test was performed for mucosal adhesion strength, comparing ELEVIEW® with eECM 12 mg / mL.

[0112] (Example 2) Viscoelastic properties ELEVIEW® exhibited significantly lower viscosity than eECM 12 mg / mL at a shear rate of 0.1 1 / s (p<0.0001), and this trend was no longer significant at 1 1 / s (p=0.054) (Figure 1A). ELEVIEW® did not form a stably formed hydrogel because its average loss modulus (G”) (0.09±0.04 Pa) was greater than its average storage modulus (G') (0.05±0.01 Pa). However, eECM 12 mg / mL, according to the definition of a stably formed ECM hydrogel (Freytes et al., Biomaterials, 2008. 29(11): p. 1630-7), had a storage modulus (G') (56.95±66.72 Pa) that was approximately an order of magnitude larger than its loss modulus (G”) (7.62±6.30 Pa) (Figure 1B). A representative graph of the time sweep of ELEVIEW® further demonstrates that ELEVIEW® does not form a hydrogel (Figure 1C), and the storage modulus of eECM 12 mg / mL increases in an S-shape and plateaus over time (Figure 1D). Therefore, the gelation time to 50% gelation could be calculated for eECM 12 mg / mL (4.5 ± 3.5 minutes), but could not be calculated for ELEVIEW® (Figure 1E).

[0113] (Example 3) Mucosal adhesion to the muscle layer ELEVIEW® (0.16±0.05N) and eECM® (0.21±0.08N) did not show significantly different mucosal adhesion to the muscular layer (Figure 2A). eECM® had a higher mucosal adhesion strength to the mucosa (0.37±0.02N) than ELEVIEW® (0.15±0.06N) (p=0.0053) (Figure 2B).

[0114] (Example 4) Macrophage activation Macrophages exposed to eECM showed activation of the anti-inflammatory marker FIZZ1, along with minimal iNOS expression (a pro-inflammatory marker). iNOS expression was comparable among ELEVIEW®, eECM, and carrier (pepsin) controls (Figure 3). ELEVIEW® showed no biological activity.

[0115] (Example 5) Performance of submucosal fluid cushions in ex vivo ELEVIEW® and eECM successfully created a fluid cushion upon injection of 2 mL of the test agent into the esophagus. Both test agents were easily injected using a 23G needle. ELEVIEW® appeared to diffuse from the moment of injection. Elevation measurements and macroscopic appearance confirmed this finding (Figure 4). The esophagus experienced a loss of elevation from 0 to 15 minutes (Figure 4). The loss of elevation was greater with ELEVIEW® than with eECM. The decrease in elevation continued for both test agents, but the loss was clearly greater in the esophagus with ELEVIEW®.

[0116] Dissociation after 75 minutes showed a difference between ELEVIEW® and eECM in the esophagus. The area injected with ELEVIEW® showed a viscous fluid with no clear adhesion to the mucosa or the underlying muscle layer. The area previously injected with eECM showed a clear and defined gel mass that remained and adhered to the mucosa and the underlying muscle (Figure 4). This is consistent with the previous results demonstrating that ELEVIEW® cannot form a gel (see Example 2) (Figure 1D).

[0117] (Example 6) In vivo use of the ECM as a submucosal fluid cushion for EMR. ECM hydrogel can be delivered via 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 dissection was created for removal. The tissue was removed using a snare. Macroscopic observation revealed that the removed mucosal tissue contained a portion of the gel. The blue dye in the hydrogel appeared to diffuse to the periphery of the esophagus after the removal of the mucosa and the entire circumference was achieved by the use of the hydrogel.

[0118] Given the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the invention and should not be considered to limit the scope of the invention. Rather, the scope of the invention is defined by the following claims. Accordingly, the inventors claim all things that fall within these claims and in spirit as their invention. The present invention provides, for example, the following items: (Item 1) A method for separating the mucosa and submucosa from the muscularis propria derived from the esophageal region of a subject, The step involves submucosal injection of a pharmaceutical composition containing an extracellular matrix (ECM) hydrogel into the region of the esophagus of the subject to form a cushion between the submucosal tissue and the underlying muscularis propria in the esophagus, wherein the ECM hydrogel has the following characteristics: a) Time to 50% gelation in less than 30 minutes at a temperature of approximately 37°C, b) A fluid viscosity suitable for injection into the esophagus, and c) Stiffness of approximately 10 to 400 Pascals (Pa) Steps having, The steps include: separating the mucosa and submucosa from the underlying muscularis propria, thereby suppressing inflammation of the esophagus in the subject, wherein the ECM hydrogel is not bladder ECM hydrogel; Methods that include... (Item 2) The method according to item 1, wherein the time to 50% gelation is approximately 2 to 30 minutes at approximately 37°C. (Item 3) The method according to item 1, wherein the time to 50% gelation is approximately 2 to 10 minutes at approximately 37°C. (Item 4) The method according to item 2, wherein the time until 50% gelation is approximately 3 to approximately 10 minutes. (Item 5) The aforementioned flow viscosity is approximately 0.1 to approximately 100 Pa at a shear rate of approximately 0.1 / s. * The pressure is approximately 0.01 to 0.2 Pa at a shear rate of 1000 / s. * The method described in any one of items 1 to 4, wherein s is the method described in item 1 to 4. (Item 6) The aforementioned flow viscosity is approximately 0.1 to approximately 30 Pa at a shear rate of 1 / s. * The pressure is approximately 0.02 to 0.8 Pa at a shear rate of approximately 100 / s. * The method described in any one of items 1 to 4, wherein s is the method described in item 1 to 4. (Item 7) The method according to any one of items 1 to 6, wherein the ECM hydrogel has a rigidity of 10 to 300 Pa. (Item 8) The method according to any one of items 1 to 7, wherein the ECM hydrogel is esophageal ECM hydrogel. (Item 9) The method according to any one of items 1 to 8, wherein the ECM concentration in the hydrogel is 2 mg / ml to approximately 16 mg / ml. (Item 10) The method according to any one of items 1 to 9, wherein the ECM hydrogel is administered endoscopically or via a catheter. (Item 11) The aforementioned ECM hydrogel (a) Solubilizing decellularized extracellular matrix (ECM) by tissue digestion using acidic proteases in an acidic solution to produce digested ECM, and (b) The method according to any one of items 1 to 10, wherein the pH of the digested ECM is raised to a pH between 7.2 and 7.8 to produce a neutralized digested solution. (Item 12) (b) The method according to item 11, wherein raising the pH of the digested ECM includes adding a base or isotonic buffer to raise the pH of the digested ECM. (Item 13) The method according to item 10 or 11, wherein the acid protease is pepsin, trypsin, or a combination thereof. (Item 14) The method according to any one of items 1 to 13, wherein the ECM hydrogel is maintained at 25°C or below 25°C before administration to the subject. (Item 15) The method according to any one of items 1 to 14, wherein the ECM hydrogel is injected endoscopically or via a catheter. (Item 16) The method according to any one of items 1 to 12, wherein the ECM hydrogel is maintained at 25°C or below 25°C before administration to the subject. (Item 17) The method according to any one of items 1 to 16, comprising a method for dissecting esophageal dysplasia, esophageal adenocarcinoma, or esophageal cancer from the esophagus. (Item 18) The method according to item 17, comprising the step of dissecting the mucosa and submucosa from the esophagus of a subject having Barrett's esophagus. (Item 19) The method according to any one of items 1 to 18, further comprising the step of performing an endoscopic resection procedure on the cushion to remove the dissected mucosa and submucosal tissue. (Item 20) The method according to item 19, wherein the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection. (Item 21) The hydrogel is held on the muscularis propria of the esophagus beneath it, and the cushion is divided so that the mucosa and submucosa are removed from the area of ​​the esophagus. The method described in item 20, including the method described in item 20. (Item 22) The method according to any one of items 1 to 21, wherein the subject is a human. (Item 23) A composition comprising an extracellular matrix (ECM) hydrogel for use in the method described in any one of items 1 to 22, wherein the ECM hydrogel has the following characteristics for use in the method described in any one of items 1 to 24: a) Time to 50% gelation in less than 30 minutes at a temperature of approximately 37°C, b) A fluid viscosity suitable for injection into the esophagus, and c) Stiffness of approximately 10 to 400 Pascals (Pa) A composition having the following characteristics. (Item 24) The aforementioned ECM hydrogel has the following characteristics: a) Time to 50% gelation in less than 10 minutes at approximately 37°C, b) A fluid viscosity sufficient for injection into the esophagus, c) Stiffness of approximately 10 to approximately 300 Pascals (Pa), and d) The hydrogel is an esophageal hydrogel. The composition described in item 23, having the following characteristics. (Item 25) The composition according to item 23 or 24, wherein the method comprises endoscopic mucosal resection or endoscopic mucosal dissection.

Claims

1. A pharmaceutical composition comprising a neutralizing digestion solution (pregel) of an extracellular matrix (ECM) hydrogel for use in a method for dissociating the mucosa and submucosa from the muscularis propria derived from the esophageal region of a subject, wherein the method is The step involves submucosal injection of the pharmaceutical composition into the region of the esophagus of the subject to form a cushion between the submucosal tissue and the underlying muscularis propria in the esophagus, characterized in that the neutralizing digestion solution (pregel) of the ECM hydrogel gels into an ECM hydrogel having a rigidity of about 10 to about 400 Pascals (Pa), and the neutralizing digestion solution (pregel) of the ECM hydrogel has the following characteristics: a) Time to 50% gelation in less than 30 minutes at a temperature of approximately 37°C. b) A fluid viscosity suitable for injection into the esophagus, and c) ECM concentration in the neutralized digestion solution (pregel) of the ECM hydrogel at approximately 8 mg / ml to approximately 20 mg / ml Steps having, The steps include: separating the mucosa and submucosa from the underlying muscularis propria, thereby suppressing inflammation of the esophagus in the subject, wherein the neutralizing digestion solution (pregel) of the ECM hydrogel is not the neutralizing digestion solution (pregel) of the bladder ECM hydrogel; and A pharmaceutical composition containing the above.

2. The pharmaceutical composition according to claim 1, wherein the time to 50% gelation is approximately 2 to approximately 30 minutes at approximately 37°C.

3. The pharmaceutical composition according to claim 1, wherein the time to 50% gelation is approximately 2 to approximately 10 minutes at approximately 37°C.

4. The pharmaceutical composition according to claim 2, wherein the time until 50% gelation is approximately 3 to approximately 10 minutes.

5. The aforementioned flow viscosity is approximately 0.1 to approximately 100 Pa at a shear rate of approximately 0.1 / s. ＊ The pressure is approximately 0.01 to 0.2 Pa at a shear rate of 1000 / s. ＊ A pharmaceutical composition according to any one of claims 1 to 4, wherein s.

6. The aforementioned flow viscosity is approximately 0.1 to approximately 30 Pa at a shear rate of 1 / s. ＊ The pressure is approximately 0.02 to 0.8 Pa at a shear rate of approximately 100 / s. ＊ A pharmaceutical composition according to any one of claims 1 to 4, wherein s.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the ECM hydrogel has a rigidity of 10 to 300 Pa.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the neutralizing digestion solution (pregel) of the ECM hydrogel is a neutralizing digestion solution (pregel) of esophageal ECM hydrogel.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the ECM concentration in the neutralized digestion solution (pregel) of the ECM hydrogel is 8 mg / ml to about 16 mg / ml.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition is administered endoscopically or via a catheter.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the neutralization digestion solution (pregel) of the ECM hydrogel contains an inactivated acidic protease and enzymatically digested ECM, and has a pH between 7.2 and 7.

8.

12. The pharmaceutical composition according to claim 11, wherein the acidic protease is pepsin, trypsin, or a combination thereof.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutical composition is maintained at 25°C or below 25°C before administration to the subject.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the pharmaceutical composition is injected endoscopically or via a catheter.

15. The pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutical composition is maintained at 25°C or below 25°C before administration to the subject.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the method comprises a method for dissecting esophageal dysplasia, esophageal adenocarcinoma, or esophageal cancer from the esophagus.

17. The pharmaceutical composition according to claim 16, wherein the method includes the step of separating the mucosa and submucosa from the esophagus of a subject having Barrett's esophagus.

18. The pharmaceutical composition according to any one of claims 1 to 17, further comprising the step of performing an endoscopic resection procedure on the cushion to remove the dissected mucosa and submucosal tissue.

19. The pharmaceutical composition according to claim 18, wherein the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection.

20. The method involves dividing the cushion such that the hydrogel is held on the muscularis propria of the esophagus beneath it, and the mucosa and submucosa are removed from the area of ​​the esophagus. The pharmaceutical composition according to claim 19, comprising:

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the subject is a human.

22. The neutralized digestion solution (pregel) of the ECM hydrogel has the following characteristics: a) Time to 50% gelation in less than 10 minutes at approximately 37°C, b) A fluid viscosity sufficient for injection into the esophagus, c) The neutralizing digestion solution (pregel) of the ECM hydrogel is a neutralizing digestion solution (pregel) of esophageal ECM hydrogel. The composition according to any one of claims 1 to 21, characterized in that the neutralized digestion solution (pregel) of the ECM hydrogel gels into an ECM hydrogel having a rigidity of about 10 to about 300 Pascals (Pa).

23. The composition according to claim 22, wherein the method comprises endoscopic mucosal resection or endoscopic mucosal dissection.

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