Use of bladder ECM hydrogel as a submucosal fluid cushion for the esophagus

Abstract

Providing the use of bladder ECM hydrogel as a submucosal fluid cushion for the esophagus. [Solution] A method is disclosed for dissociating the mucosa and submucosa from the muscularis propria derived from a region of the esophagus of a subject. These methods include the step of submucosally injecting a pharmaceutical composition comprising a bladder 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 esophageal region, wherein 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 rigidity of about 10 to about 400 Pascals (Pa).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 62 / 688,191, filed on 21 June 2018, which is incorporated herein by reference in its entirety.

[0002] This relates to endoscopic resection, specifically the use of bladder matrix extracellular matrix (ECM) hydrogel as a submucosal cushion for separating the mucosa and submucosa from the muscularis propria in the esophagus. [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] The aforementioned and other features and advantages of the present invention will become more apparent from the following detailed description of some embodiments, made with reference to the accompanying drawings.

[0006] 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 step of submucosally injecting a pharmaceutical composition containing a bladder 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 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 methods separate the mucosa and submucosa in the region of the esophagus from the underlying muscularis propria. The methods can also suppress inflammation in the region of the esophagus in the subject.

[0007] Methods for separating the mucosa and submucosa from the muscularis propria derived from a region of the esophagus of a subject are also disclosed herein. These methods include submucosal injection of a pharmaceutical composition containing bladder 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. The methods separate the mucosa and submucosa in the esophageal region from the underlying muscularis propria. The methods can also suppress inflammation in the esophageal region of the subject.

[0008] In some embodiments, the dissection method includes endoscopic mucosal resection or endoscopic mucosal dissection. [Brief explanation of the drawing]

[0009] [Figure 1] Figures 1A-1C illustrate the in vivo use of ECM as a submucosal fluid cushion. (A) Use of bladder extracellular matrix (UBM-ECM) hydrogel as a submucosal fluid cushion, including a visible area where EMR has already been performed. (B) Esophageal tissue removed with the submucosal fluid cushion (SFC) shows the gel present in the area of ​​the removed esophageal mucosa. (C) Results of circumferential EMR, where the entire circumference of the mucosa is excised.

[0010] [Figure 2-1] Figures 2A and 2B are cross-sectional views of the bladder. Bladder submucosal tissue (UBS) ECM hydrogel is prepared from bladder submucosal tissue, including D - submucosal layer (including muscularis mucosa and submucosal layer) of the bladder (A). Bladder matrix (UBM) ECM hydrogel is prepared from UBM, including B - epithelial basement membrane and C - lamina propria of the bladder (B). L indicates the location of the lumen. These figures are cited from U.S. Patent No. 6,576,265, which is incorporated herein by reference. [Figure 2-2] Same as above.

[0011] [Figure 3-1]Figures 3A to 3C are diagrams showing rheology data including the kinematic viscosity (A) of UBM, the time sweep (B) of UBM, and the time to 50% gelation of UBM (C). [Figure 3-2] Same as above. [Figure 3-3] Same as above.

Mode for Carrying Out the Invention

[0012] For example, in any endoscopic resection technique commonly used to remove pathological lesions in esophageal endoscopy, such as in EMR and ESD, the use of bladder extracellular matrix (ECM) hydrogel as a submucosal cushion for dissociating the mucosa and submucosal tissue from the muscularis propria in the esophagus is disclosed herein. The bladder ECM hydrogel is UBM It can be an ECM hydrogel or a UBS ECM hydrogel.

[0013] Term Unless otherwise indicated, technical terms are used according to conventional grammar. 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 the discussion of various embodiments of the present disclosure, the following explanations regarding specific terms are provided.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] Dissociation: The process of separating or detaching tissue, for example, during a surgical procedure.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.”

[0025] 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.

[0026] Gelation: Formation of a gel from a sol.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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

[0037] 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.

[0038] 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.

[0039] Therapeutic agents: When used in a comprehensive sense, therapeutic agents include treatment agents, prophylactic agents, and alternative agents. "Treatment" or "to treat" means providing a patient with a substance such as bladder ECM hydrogel, e.g., UBM or UBS 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. Disclosed methods suppress esophageal inflammation and / or mitigate the effects of esophageal inflammation.

[0040] Therapeutic Effective Dose: The "therapeutic effective dose" of a composition such as bladder ECM hydrogel refers to the amount that, when administered to a patient, is effective in providing therapeutic benefits such as symptom improvement, reduced attenuation and progression, or in causing disease regression. The amount of bladder 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 subject being treated, the severity and type of pain, and the method of administration.

[0041] The bladder 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.

[0042] 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 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).

[0043] 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.

[0044] Bladder extracellular matrix (ECM) hydrogel Methods for preparing bladder ECM hydrogels are disclosed, for example, in U.S. Patents 8,361,503, 6,576,265, and 5,554,389. Any method for preparing hydrogels can be used to prepare bladder ECM hydrogels useful in the methods disclosed herein. Additional methods include, for example, U.S. Patent Nos. 4,902,508, 4,956,178, 5,281,422, 5,352,463, 5,372,821, 5,554,389, 5,573,784, 5,645,860, 5,771,969, 5,753,267, 5,762,966, 5,866,414, 6,099,567, and the same. Disclosed in Patent Nos. 6,485,723, 6,576,265, 6,579,538, 6,696,270, 6,783,776, 6,793,939, 6,849,273, 6,852,339, 6,861,074, 6,887,495, 6,890,562, 6,890,563, 6,890,564, and 6,893,666. The ECM is derived from the bladder. In certain embodiments, the bladder ECM is isolated from vertebrates, mammals, including but not limited to humans, monkeys, horses, pigs, cattle, and sheep. The mammals may be veterinary animals. In non-limiting specific examples, the bladder ECM is a pig or human bladder ECM. In some embodiments, the ECM includes a basement membrane portion of the bladder ECM. In certain embodiments, the bladder ECM includes at least a portion of the basement membrane. In other embodiments, the ECM is harvested from a cell culture. The source tissue used for the preparation of the bladder ECM can be harvested in a wide variety of ways, and various portions of the harvested tissue may be used immediately after harvesting. The bladder ECM hydrogel may be a UBM or UBS ECM hydrogel. See Figures 2A and 2B.

[0045] As disclosed in U.S. Patent No. 8,361,503 (incorporated herein by reference), bladder ECMs, such as porcine bladder ECMs, are prepared by abrading bladder tissue using a longitudinal swabbing motion with a scalpel handle and moistened gauze to remove the outer (anti-luminal) layers, including the serosal, muscular, and submucosal layers. After abduction of the tissue segments, the luminal portion of the mucosal layer is separated from the underlying tissue using the same swabbing motion. After these tissues are removed, the resulting ECM consists of the lamina propria and the basement membrane above it. U.S. Patent No. 6,893,666 (incorporated herein by reference) also discloses the preparation of ECMs derived from bladder, skin, esophagus, and small intestine.

[0046] In one embodiment, the extracellular matrix (ECM) is isolated from harvested pig bladders to prepare the bladder matrix (UBM). Excess connective tissue and residual urine are removed from the bladder. The serosa, muscularis crust, submucosa, and most of the muscularis mucosa can be removed by mechanical abrasion (see above) or by a combination of enzymatic treatment, hydration, and abrasion. Mechanical removal of these tissues can be carried out by abrasion using a longitudinal wiping motion, which removes the outer layers (particularly the anti-luminal smooth muscle layer) and even the luminal portion of the mucosa (epithelial layer). Mechanical removal of these tissues is carried out, for example, by removing the mesenteric tissue using Adson-Brown forceps and Metzenbaum scissors, and by wiping away the muscularis and submucosa using a longitudinal wiping motion with a scalpel handle or other hard object wrapped in moistened gauze. The epithelial cells of the mucosa can also be dissociated by immersing the tissue in a deepithelializing solution, e.g., hypertonic saline, but not limited to these. The resulting UBM, comprising a basement membrane of the mucous layer and an adjacent lamina propria, is further treated with peracetic acid, freeze-dried, and powdered. See U.S. Patent No. 8,361,503.

[0047] 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 epithelial cells from the underlying basement membrane. The tissue remaining after the initial exfoliation procedure includes the epithelial basement membrane and the tissue layers antiluminal 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. The outer serosal tissue, exocarpum tissue, smooth muscle tissue, submucosal layer, and most of the muscularis mucosa are removed from the remaining deepithelialized tissue by mechanical abrasion or by a combination of enzymatic treatment, hydration, and abrasion.

[0048] Commercially available bladder ECM preparations can also be used in the methods, devices, and compositions described herein. Examples of commercially available preparations include, but are not limited to, MATRISTEM UBM® (Acell Corporation, Jessup, Md.).

[0049] Bladder ECM can be sterilized by any number of standard techniques, including but not limited to exposure to peracetic acid. Bladder ECM can be sterilized by any number of standard techniques, including but not limited to low-dose gamma radiation, gas plasma sterilization, ethylene oxide treatment, supercritical CO2, or electron beam treatment. More typically, sterilization of the ECM 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. Subsequently, the ECM material can be sterilized by propylene oxide or ethylene oxide treatment, gamma irradiation (0.05-4 mRad), gas plasma sterilization, supercritical CO2, or electron beam treatment. ECM can also be sterilized by treatment with glutaraldehyde, which 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.

[0050] 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.

[0051] 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.

[0052] 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, e.g., physiological pH and ionic conditions. This forms a “pregel” solution, which at room temperature is in liquid form as a viscous solution. The neutralized digested solution (pregel) can gel at temperatures approaching physiological temperature, approaching 37°C. The method typically 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).

[0053] Therefore, the ECM is derived from the bladder. Bladder ECM can be prepared from one subject / animal, or from more than one subject / animal, e.g., two, three, or four subjects / animal, or from a bladder cell line. The ECM may be commercially available bladder ECM. 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 digested 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., 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, agitating, 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 of interest 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 an inactivated protease. Any of these bladder ECM hydrogels are useful in the methods disclosed herein.

[0054] Bladder ECM hydrogels form a gel when exposed to a temperature of approximately 37°C. Bladder ECM hydrogels in "pre-gel" form can be frozen and stored at, for example, -20°C or -80°C, but are not limited to these. Bladder ECM hydrogels in "pre-gel" form can be stored at room temperature, for example, approximately 25°C. Therefore, bladder ECM hydrogels are in pre-gel form at temperatures below 37°C, for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, and 4°C. Bladder ECM hydrogels can be frozen for storage and therefore can be stored below 0°C. As used herein, the terms "pre-gel form" or "pre-gel" refer to bladder ECM hydrogels that have increased pH but have not gelled. For example, but not limited to, bladder ECM hydrogels in pregel form have a pH between 7.2 and 7.8. Bladder ECM hydrogels can be delivered to subjects with esophageal inflammation in pregel form, for example, orally, via a catheter, or endoscopically.

[0055] Pregel-formed bladder ECM hydrogel is suitable for introduction into the patient's esophagus. When introduced into the submucosa of the esophagus, which is approximately 37°C, the bladder ECM hydrogel gels, creating a cushion of ECM hydrogel between the muscularis propria and submucosa of the esophagus, lifting the submucosa for surgical resection.

[0056] In some embodiments, the bladder 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 infusion into the esophagus, 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.

[0057] In several embodiments, the bladder 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 400 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 bladder 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).

[0058] In other embodiments, the bladder 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 bladder 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 bladder 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).

[0059] In additional, non-limiting specific examples, a bladder ECM hydrogel is prepared by (a) solubilizing a cell-free extracellular matrix by bladder digestion using an acidic protease in an acidic solution to produce a digested ECM; (b) raising the pH of the digested ECM to between 7.2 and 7.8 to produce a neutralized digested solution; and (c) diluting the digested ECM to a bladder 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 bladder ECM hydrogel is then introduced into the esophagus of the subject, where it gels.

[0060] The bladder ECM hydrogels useful in the methods disclosed herein have a time to 50% gelation of less than 20 minutes at a temperature of about 37°C, for example, less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, and 2 minutes. In some embodiments, the bladder ECM hydrogel has a time to 50% gelation of less than 10 minutes at a temperature of about 37°C. In other embodiments, the time to 50% gelation is about 2 to about 20 minutes at a temperature of about 37°C. In further embodiments, the time to 50% gelation is about 2 to about 10 minutes at a temperature of about 37°C. In yet another embodiment, the time to 50% gelation is about 3 to about 8 minutes at a temperature of about 37°C.

[0061] The disclosed bladder ECM hydrogel may have a flow viscosity suitable for infusion into the submucosal space of the esophagus. In some embodiments, the bladder ECM hydrogel has a flow viscosity of about 0.1 to about 100 Pa at a shear rate of 0.1 / s. * For example, at a shear rate of 0.1 / s, the pressure is approximately 0.2, 1, 10, 20, 30, 40, 50, 60, 70, 80, or 90 Pa. * It has a flow viscosity of s. In a further embodiment, the bladder ECM hydrogel has a shear rate of 0.1 / s of about 1 to about 40 Pa * For example, at a shear rate of 0.1 / s, the pressures are approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 Pa. * It has a fluid viscosity of s.

[0062] In other embodiments, the bladder ECM hydrogel is subjected to 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.

[0063] In a further embodiment, the bladder ECM hydrogel has a shear rate of about 0.02 to about 0.8 Pa * s, or a shear rate of about 0.1 to about 0.8 Pa * s, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 Pa * s.

[0064] In a further embodiment, the bladder ECM hydrogel has a dynamic viscosity of about 10 to about 100 Pa * s at a shear rate of 0.2 / s, and a dynamic viscosity of about 0.01 to about 0.10 Pa * s at a shear rate of 1000 / s. In a further embodiment, the ECM hydrogel has a shear rate of 1 to 40 Pa * s at a shear rate of 0.1 / s, and a dynamic viscosity of 0.01 to 0.2 Pa * s at a shear rate of 1000 / s.

[0065] In other embodiments, the bladder ECM hydrogel has a shear rate of about 0.1 to about 25 Pa * s, for example, 1 to about 25 Pa * s, or 1 to about 20 Pa * s, or 1 to about 10 Pa * s, for example 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, 11, 12, 13, 14, 15, 20, 25 Pa at a shear rate of 1 / s * s. The shear rate can be, for example, 5, 10, 15, or 20 Pa * s at a shear rate of 1 / s. In other embodiments, the bladder ECM hydrogel has a dynamic viscosity of about 0.025 to about 0.6, for example about 0.025, 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, or 0.060 at a shear rate of 100 / s. The dynamic viscosity is about 0.1 to about 25 Pa * s at a shear rate of 1 / s, and about 0.02 to about 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 0.1 to approximately 0.3.

[0066] In further embodiments, the bladder ECM hydrogel is subjected to approximately 10 to approximately 100 Pa at a shear rate of 0.2 / s. * It has a flow viscosity of s. In other embodiments, the bladder ECM hydrogel has a viscosity of about 0.01 to about 0.10 Pa at a shear rate of 1000 / s. * It has a flow viscosity of s. In other embodiments, the bladder 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.

[0067] The disclosed bladder 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 bladder ECM hydrogels may have a stiffness of about 10 to about 400 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 250 Pa. In some embodiments, the disclosed bladder 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 bladder 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 bladder ECM hydrogel may have a stiffness of approximately 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, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400 Pa.

[0068] In some embodiments, the bladder 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 bladder 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.

[0069] Treatment method Methods for dissociating the mucosa and submucosa from the muscularis propria in the esophagus of a subject are disclosed herein, which include the step of submucosally injecting into the esophagus a pharmaceutical composition comprising a bladder extracellular matrix (ECM) hydrogel, such as UBS or UBM ECM hydrogel, to form a cushion between the submucosa and the underlying muscularis propria in the area of ​​the organ. The method may be EMR or EMD.

[0070] 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.

[0071] 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 has a higher rate of perforation and hemorrhagic complications than conventional EMR, thus requiring more advanced endoscopic skills and experience. ESD utilizes a variety of electrosurgical units, such as insulated-tip diathermy knives, needle knives, hook knives, flex knives, and triangular-tipped knives. A knife, flash knife, splash needle, and small-diameter transparent tip hood can be used. These knives can be used with a high-frequency electrosurgical current (HFEC) generator. 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. Gstroenterol. 14(9): 2962-2967, 2008, incorporated herein by reference). Various submucosal injection solutions have been developed to date and have been shown to be sufficient for use during EMR, but the introduction of longer-duration ESD procedures has required longer-lasting solutions that help identify the cutting line during submucosal dissociation (see Uraoka et al., Drug Design, Development and (Therapy 2008:2 131-138). The method described herein satisfies this need.

[0072] 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 dissection of the submucosa 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. Bladder ECM hydrogel reduces stenosis and promotes re-epithelialization. The method of this disclosure also satisfies this need.

[0073] 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.

[0074] 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 bladder ECM hydrogel fulfills these needs.

[0075] The bladder ECM hydrogel 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.

[0076] 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.

[0077] Injection needles and other accessories commonly used during endoscopic procedures, such as snares for polypectomy, clip devices, and biopsy forceps, pass through one or more specific channels of the endoscope, typically 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. Bladder ECM hydrogels disclosed in pregel form can easily pass through these catheters.

[0078] Pregel-formed bladder ECM hydrogel 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 the endoscopist to perform easy resection of mucosal lesions discovered during the endoscopic procedure, even if the lesion is flat and therefore does not protrude into the lumen, such as the esophageal lumen.

[0079] 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. Bladder ECM hydrogel in pregel form is injected submucosally into the area of ​​a subject's organ, e.g., a lesion or tumor, to form a cushion between the submucosa and the underlying muscularis propria in the area of ​​the organ. The cushion can be dissociated so that a portion of the bladder ECM hydrogel is maintained in the underlying muscularis propria and assists the healing process.

[0080] The disclosed method is useful in the esophagus. In one non-limiting example, the organ is the esophagus, and 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.

[0081] The bladder 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 bladder ECM hydrogels may be maintained at, for example, 25°C or 4°C before administration. An effective amount of bladder ECM hydrogel in pre-gel form is then administered into the esophagus of the subject. The bladder ECM hydrogels are provided in lyophilized or frozen form and may be reconstituted immediately before administration to the subject.

[0082] 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 a bladder ECM hydrogel in pregel form is injected into a human target tissue to form a cushion that is provided as needed following an endoscopic surgical procedure such as a resection. The bladder 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 bladder ECM hydrogel is derived from human and / or pig bladders. In other embodiments, the bladder ECM hydrogel is derived from non-human primates, dogs, cats, horses, or cattle. The bladder ECM may also be derived from a commercially available source.

[0083] The disclosed methods are invasive because they require injection to dissociate the mucosa and submucosa from the muscularis propria originating from the organ region of the subject's intestinal tract. Any of the methods disclosed herein may include the step of submucosally injecting a pharmaceutical composition containing bladder ECM hydrogel into the subject's esophagus to form a cushion between the submucosa and the underlying muscularis propria in the organ region. The bladder 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 bladder ECM hydrogel gels, dissociating the mucosa and submucosa from the underlying muscularis propria and suppressing inflammation in the esophageal region of the subject. The bladder ECM hydrogel in pregel form may be administered endoscopically or via catheter.

[0084] 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.

[0085] 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 muscularis propria of the underlying organ and the mucosa and submucosa are removed from the area of ​​the organ. In some non-limiting examples, a portion of the hydrogel cushion retained on the underlying muscularis propria downmodulates pro-inflammatory macrophage activation in this tissue.

[0086] In some embodiments, the time to 50% gelation of the hydrogel is less than 20 minutes at a temperature of approximately 37°C. In some non-limiting specific examples, the time to 50% gelation is approximately 2 to 20 minutes at approximately 37°C. In other non-limiting specific examples, the time to 50% gelation is approximately 2 to 10 minutes at approximately 37°C. In further non-limiting examples, the time to 50% gelation is approximately 3 to 8 minutes.

[0087] In additional embodiments, the fluid viscosity of the pregel form is sufficient for injection into the esophagus. In some embodiments, the fluid viscosity of the bladder 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 0.1 to about 25 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.

[0088] In further embodiments, the bladder ECM hydrogel has a stiffness of about 10 to about 400 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.

[0089] Bladder ECM hydrogels can be prepared by any of the methods disclosed herein. In some embodiments, the ECM hydrogel is 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 esophageal ECM to a pH between 7.2 and 7.8 to prepare a neutralized digestion 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, an acidic protease such as pepsin, trypsin, or a combination thereof is used.

[0090] In some embodiments, the bladder 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]

[0091] ELEVIEW® and bladder ECM hydrogels are different biomaterials. ELEVIEW® (Aries Pharmaceuticals, Inc., Dublin, Ireland) is a commercially available low-viscosity emulsion of poloxamer 188 used clinically to provide submucosal tissue lifting for EMR and ESD procedures. 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, bladder ECM (prepared according to the methods disclosed herein, i.e., by protease digestion of ECM) forms a hydrogel that is stably formed at 37°C (body temperature). Bladder ECM hydrogels, including ELEVIEW®, UBM, and UBS ECM hydrogels, can be injected submucosa. An ideal biomaterial will adhere to both layers. Bladder ECM hydrogels possess strong mucosal adhesion properties and beneficial biological properties. Bladder ECM hydrogel can be effectively used as a submucosal cushion in vivo and therefore can be used to separate the submucosa from the underlying muscularis propria.

[0092] (Example 1) material and method Preparation of bladder ECM: The surface was sterilized with 70% ethanol and 25% Pyroclean. Pork bladder (Animal Biotech Industries) stored at -20°C was thawed and thoroughly washed with type I water. The bladder was dissected from the apex to the apex and flattened with the luminal side facing downwards. Using an oblique-bladed acrylic scraper and forceps, the smooth muscle tissue (serosa, muscularis exostata, submucosa, and muscularis mucosa) on the non-luminal side was mechanically removed, leaving the lamina propria (plate) and basement membrane (tissue layer containing UBM). The UBM was stored overnight in type I water at 4°C and decellularized using 0.1% peracetic acid (PAA) in 4% ethanol for 4 hours, followed by washing with PBS, type I water, PBS, and type I water for 15 minutes to create the UBM. All decellularization steps were performed at room temperature with stirring at 300 rpm on a shaking plate.

[0093] Formation of bladder ECM hydrogel: UBM was lyophilized and powdered using a Wiley mill. 10 mg / mL of powdered ECM was digested with pepsin (1 mg / mL) in 0.01 M HCl at room temperature for 48 hours. The ECM digest and pepsin control were stored at -20°C until use. On the day of the experiment, the ECM digest and pepsin control were neutralized with 0.1 M NaOH (1 / 10 volume of Pregel solution) and 10×PBS (1 / 9 volume of Pregel solution) at pH 7.4 at 4°C, and diluted to an ECM concentration of 5 mg / mL using ice-cold 1×PBS at 4°C. See Freytes et al., Biomaterials 29(11): p. 1630-7. 2008, incorporated herein by reference.

[0094] Rheology: The viscoelastic properties of the bladder ECM were determined using a temperature-controlled 40 mm parallel-plate rheometer (AR2000). The sample was 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 (0.1~1000 s). -1 The 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).

[0095] In vivo use of ECM as a submucosal fluid cushion for EMR: Anesthesia was induced using acepromazine (0.01 mg / kg, SC) and ketamine (5–11 mg / kg), and surgical-level anesthesia was maintained via endotracheal tube with 1–5% isoflurane. Animals were administered IV at 2 ml / kg / hour of Ringer's lactate solution during the procedure and immediately after the surgical period. Temperature was controlled by a warm water recirculation heating pad placed beneath the animals. Physiological parameters, such as heart rate, respiratory rate, body temperature, and responsiveness, were monitored during the procedure. The procedure was initiated after administering antibiotic prophylaxis with 25 mg / kg cefazolin.

[0096] The animals were placed in a supine position, and the esophagus was evaluated using a Pentax EG3430K endoscope. The distance from the mouth to the GE junction was measured. After identifying reference points in the esophagus, the mucosa and submucosa at the excision site were separated at 4°C using an Olympus Injectorforce 4mm 23G needle with 8 mg / ml of blue-stained bladder matrix hydrogel for injection. This temperature was maintained throughout to prevent gelation and potential needle obstruction. Approximately 2–5 ml of blue gel was injected at each site. The entire circumference (100%) of a 5 cm length of mucosa was removed using the band ligation EMR technique. For EMR, a Cook Duette kit including ligation bands was used. The mucosa was then excised using a snare.

[0097] (Example 2) In vivo use of bladder ECM hydrogel as a submucosal fluid cushion for EMR. The bladder ECM hydrogel could 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 (Figure 1A). The tissue was easily removed using a snare. Macroscopic observation revealed that the removed esophageal mucosal tissue contained a portion of the gel (Figure 1B). 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 using the hydrogel (Figure 1C).

[0098] (Example 3) Rheology data The viscoelastic properties of UBM are shown in Figure 3. The steady-state flow curve (Figure 3A), obtained in the same manner as described for UBM, shows a concentration-dependent increase in viscosity with increasing ECM concentration and shear-thinning flow profile of the UBM hydrogel; that is, viscosity decreases with increasing shear rate for each ECM concentration.

[0099] UBM ECM (Figure 3B) showed an increasing storage modulus (stiffness) with increasing ECM. UBM exhibited a storage modulus (G') approximately one order of magnitude larger than the loss modulus (G”) at each ECM concentration, in accordance with the definition of a stably formed ECM hydrogel (Freytes et al., Biomaterials, 2008. 29(11): p. 1630-7). UBM showed a concentration-independent gelation time, i.e., the gelation time remained constant for all ECM concentrations (Figure 3C).

[0100] Given the many possible embodiments to which the principles of our invention may be applied, it should be recognized that the illustrated embodiments are merely examples of the invention and should not be considered limitations on the scope of the invention. Rather, the scope of the invention is defined by the following claims. Accordingly, we claim all things that fall within these claims and their spirit as our inventions. 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 bladder 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 20 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, This process involves separating the mucosa and submucosa in the aforementioned region of the esophagus from the underlying muscularis propria, thereby suppressing inflammation in the aforementioned region of the esophagus in the subject. Methods that include... (Item 2) The method according to item 1, wherein the time to 50% gelation is approximately 2 to approximately 20 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 8 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 25 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 concentration in the hydrogel is 2 mg / ml to approximately 16 mg / ml. (Item 9) The method according to any one of items 1 to 8, wherein the ECM hydrogel is administered endoscopically or via a catheter. (Item 10) 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) Raise the pH of the digested ECM to a pH between 7.2 and 7.8 to prepare a neutralized digested solution. A method described in any one of items 1 to 9, produced by [the specified method]. (Item 11) (b) The method of item 10, wherein raising the pH of the digested ECM includes adding a base or isotonic buffer to raise the pH of the digested ECM. (Item 12) The method according to item 10 or 11, wherein the acid protease is pepsin, trypsin, or a combination thereof. (Item 13) 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 14) The method according to any one of items 1 to 13, wherein the ECM hydrogel is injected endoscopically or via a catheter. (Item 16) The method according to any one of items 1 to 14, wherein the organ is the esophagus, and the method includes a method for dissecting esophageal dysplasia, esophageal adenocarcinoma, or esophageal cancer from the esophagus. (Item 17) The method according to item 16, comprising the step of dissecting the mucosa and submucosa from the esophagus of a subject having Barrett's esophagus. (Item 18) The method according to any one of items 1 to 17, further comprising the step of performing an endoscopic resection procedure on the cushion. (Item 19) The method according to item 18, wherein the resection procedure is an endoscopic mucosal resection or endoscopic submucosal dissection for removing the dissected mucosa and submucosal tissue. (Item 20) The method according to item 19, comprising the step of dividing the cushion so 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. (Item 21) The method according to any one of items 1 to 20, wherein the subject is a human. (Item 22) The method according to any one of items 1 to 21, wherein the method of dissection includes endoscopic mucosal resection or endoscopic mucosal dissection. (Item 23) The method according to any one of items 1 to 22, wherein the bladder ECM hydrogel is a bladder matrix (UBM) ECM hydrogel. (Item 24) The method according to any one of items 1 to 22, wherein the bladder ECM hydrogel is bladder submucosal tissue (UBS) ECM hydrogel. (Item 25) A composition comprising a bladder ECM hydrogel for use in the method described in any one of items 1 to 25, wherein the ECM hydrogel has the following characteristics: a) Time to 50% gelation in less than 20 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.

Claims

[Claim 1] The invention described herein.

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