Anti-dissolution flowable chitosan bioadhesive hemostatic composition

Abstract

The present invention relates to biocompatible, tissue-adhesive, chitosan flowable dressings optionally modified with catechol and suitable for the treatment of bleeding in physiological environments such as the gastrointestinal tract, bladder, particularly associated with TURP surgery. Characteristics and structures of the chitosan dressings are provided. Methods of making and using the chitosan dressings are also provided.

Description

Technical Field

[0001] The present disclosure relates to fluidized, flowable, liquid, hemostatic dispersions (or particle dispersions, dispersed particles, suspensions, suspended particles, or particle suspensions) of chitosan materials. The dispersions of the present disclosure provide the ability to accurately deliver safe, effective, controlled amounts of tissue-adherent, dissolution-resistant chitosan materials in vivo via minimally invasive techniques. The flowable chitosan materials of the present invention are capable of rapidly adhering to mucosal and various tissue surfaces, remaining in place, and maintaining adherence in inhospitable, internal, biological wet tissue environments to maintain and provide prolonged hemostatic, protective, antimicrobial, and wound healing efficacy, in environments where no other flowable device or material currently exhibits similar durability and efficacy.

[0002] Flowable, liquid hemostats for in vivo use that do not rely on a mechanical compression mode of action for hemostasis are limited to two types of hemostats: (1) tissue sealing hemostats; and (2) dispersed particulate hemostats (alternatively dispersed hemostats or liquid dispersed hemostats).

[0003] Flowable tissue-sealing hemostats comprise continuous, liquid adhesive compositions (>98% w / w) that are designed to adhere directly to the site of injury and promote wound healing through a non-biological mechanical process. Tissue-sealing hemostats are generally contraindicated for direct use on actively bleeding wounds. Because tissue sealants are non-porous, continuous-phase adhesive materials, their mode of action is easily compromised by the presence of liquids (including blood) that interfere with the adhesion process. Tissue-sealing hemostats do not effectively clot blood because they present a low surface area to blood. If tissue-sealing hemostats do not completely seal the wound, they quickly become ineffective.

[0004] In contrast, flowable, liquid dispersed particulate hemostat consists of a continuous diluent carrier fluid (<95% w / w) phase and a dispersed discrete (particulate) phase (≥5% w / w) with a pasty but flowable consistency. Dispersed particulate hemostat consisting of a low viscosity diluent and dispersed solid or semisolid particles ideally has an interconnected porous structure that enables the hemostat to present a high specific surface area to the blood.

[0005] There is little information in the scientific and patent literature regarding flowable, liquid-dispersible particulate hemostats, except that they typically consist of micron-sized solid or semisolid cross-linked gelatin particles dispersed at ≥5% w / w in a carrier water or saline solution. The procoagulant bioactive thrombin is added to the gelatin particle dispersion to provide a high specific surface area of ​​rapidly coagulant gelatin particles. SURGIFLO (Ethicon) and FLOSEAL (Baxter) are commercially available examples of thrombin-based bioflowable liquid-dispersible hemostats. Bioflowable liquid-dispersible hemostats are those that include one or more active therapeutic biologically derived agents that promote clot formation through an active therapeutic chemical pathway such as thrombin or fibrinogen. SURGIFLO and FLOSEAL are homogenously mixed into their liquid dispersions at the point of care in 5 ml or 10 ml sizes by mixing the dry powder components with a carrier liquid and thrombin in two connected syringes. In biofluidizable liquid-dispersible hemostats, blood flow between the hemostat particles results in a high surface area of ​​blood-particle contact and rapid clot formation in a biopharmaceutical-rich environment. As a substantially passive hydrophilic material, the cross-linked gelatin particles of the biofluidized particulate hemostat provide mechanical support, partial swelling in the presence of a liquid diluent, and adsorption of the bioactive hemostatic drug. Biofluidizable liquid-dispersible hemostats can be applied directly to actively bleeding wounds to achieve rapid, emergency hemorrhage control. Some issues with the use of biofluidizable liquid-dispersible hemostats include: (i) their hemostatic efficacy relies on the use of expensive, limited-shelf-life biopharmaceuticals such as thrombin or other biopharmaceuticals, including fibrinogen, and their use is known to cause immune reactions in some patients; (ii) biofluidizable liquid-dispersible hemostats cannot be effectively used to control bleeding in situations where prolonged tissue adhesion to the surface (>6 hours) is necessary, such as in the gastrointestinal tract, in the bladder, and in inverted resected prostates; and iii) adverse scarring has been reported with the use of biofluidizable liquid-dispersible hemostats. There is no address in the literature or by currently available hemostatic products for non-biological, tissue-adhesive, fluid-type, liquid-dispersible hemostatic agents for direct application to actively bleeding wounds and for prolonged application (>6 hours) in general and difficult settings to control moderate to severe bleeding. The compositions and methods described herein attempt to address these deficiencies.

[0006] The preparation of non-biological, liquid-dispersible material compositions whose mode of action relies on tissue adhesion and safe, reliable, and effective delivery of bioactive materials in vivo remains a major problem. The non-biological compositions do not include one or more active therapeutic biologically derived agents that promote clot formation through active therapeutic chemical pathways (e.g., thrombin or fibrinogen). In use, the non-biological, liquid-dispersible hemostatic materials are intended to remain intact in the presence of biological fluids such as gastric fluid, blood, bile, and urine for a period of at least 12 hours and to remain adhered to tissues including the gastrointestinal mucosa and the internal elastic lamina of the vascular system for a period exceeding 12 hours while promoting local hemostasis and wound healing with a reduced risk of scarring. The non-biological, liquid-dispersible hemostatic materials of the present invention that meet these stringent requirements are particle dispersions having a radius of gyration ranging from 10 to 350 microns. The dispersed particles of the present invention are formed from a composition comprising a bioactive chitosan material that provides local hemostasis without the risk of distal embolism or thrombosis and promotes normal healing without the risk of scarring.

[0007] Bioactive chitosan has many applications in vivo, not limited to drug delivery, hemostasis, wound healing, tissue regeneration and transarterial embolization therapy. "Bioactive" materials are materials that have non-drug or therapeutic activity and are therefore not subject to the same regulations as active drugs or biotherapeutics. The bioactivity may be related to the interaction of an insoluble material surface (usually a polymeric material) with the cellular environment and produce a measurable change or modification to the cellular environment. The use of chitosan materials and compositions of chitosan materials currently used on or in the body is limited because they cannot resist dissolution and rapid loss in harsh environments such as the stomach and bladder, and they cannot remain in place by their adhesion to tissue. As an example of this limitation in chitosan materials, Table 4 in Subramanian et al. 2022 [1] details the lack of commercial chitosan-based materials for therapeutic applications in the stomach that require mucosal adhesion and dissolution resistance. There are no recognized hemostatic therapies for the bladder and urinary tract, except for the provision of last resort hemostatic solutions to control systemic and localized bleeding. Prolonged exudative bleeding, which remains the most frequent adverse event after transurethral resection of the prostate (TURP), still relies on the use of painful 1959 Foley balloon catheter traction that can last up to 72 hours.

[0008] Liquid-dispersed hemostatic material delivery has advantages over other delivery modes, such as gas delivery. Unlike liquid-borne particle dispersions, gas-borne particle dispersions must be dry when delivered and have a number of delivery issues, including i) risk of tissue damage from the velocity and momentum of the pressurized flow; ii) blockage of the delivery catheter lumen tip if wet; iii) unwanted gas expansion in the body during delivery; iv) long-term loss of visibility of the injury site due to turbidity of the suspended particles; iv) inability to deliver the diverging aerosolized particle stream locally to the injury site; v) up to 100 times the required dose of hemostatic agent due to poor targeting and loss of visibility; vi) delivery of aerosolized airborne particles to undesirable locations, such as the articulation joints of an endoscope, where they can subsequently become stuck and potentially make removal of the endoscope in a bent-back position a serious adverse event; and vi) lack of immediate adhesion / cohesion due to the dryness of the delivered particles (dry particles need to be wetted before they stick together and adhere to the wound surface). This drying and initial poor adhesion results in migration of the topically delivered substance from the target area, further exacerbating the problem of adequate hemostatic delivery.

[0009] The present disclosure relates to the field of flowable, liquid, dispersible, dissolution-resistant, tissue-adherent, and persistently adherent chitosan hemostatic materials that can be effectively applied in challenging wet tissue environments. Typical challenging wet tissue environments include the highly digestive wet environment of the stomach and the urine-saturated environment of the bladder and urethra. The flowable chitosan material systems and methods of the present invention preferably include catechol-modified chitosan and uses thereof. Background Art

[0010] In many surgical procedures involving vascularized organs and tissues (including but not limited to the heart, liver, pancreas, stomach, intestines, colon, prostate, tonsils, ears, nose, throat, and brain), the vascularized organs and tissues typically bleed continuously after injury and throughout the wound healing process. Unless standard hemostasis is applied, the initial injury may continue to bleed for several days, and bleeding may also recur later. The initial hemostatic care standard for postoperative bleeding management varies depending on the type of surgery. Ligation, gauze packing, biological dressings, cauterization, and bandages can be used to locally address this problem. In cases of persistent bleeding, packing can be applied for up to 24 hours. If bleeding cannot be controlled after a period of conservative treatment, the patient may have to return to the surgical ward immediately for open or endoscopic hemostasis. Although advances have been made in controlling bleeding using advanced dressings, none of these advances has been translated into a reliable treatment option under unique surgical conditions where delivery, tissue adhesion, and continuous bleeding mixed with other biological fluid concerns are very challenging. Rapid control of bleeding is highly desirable in all cases.

[0011] Minimally invasive surgical procedures are becoming the preferred means of interventional access due to the ability to access body areas with significantly reduced risk (compared to open surgery), lower morbidity, lower hospital costs and lower patient discomfort. Advanced biomaterials are at the forefront of addressing current limitations and enabling improved safety, reliability and increased practice and application of minimally invasive surgery. Current limitations in minimally invasive surgical practice include control of bleeding (especially major bleeding), seamless closure of delicate soft tissue areas, local promotion of healing, local treatment of pathological conditions and local placement by anchoring or adhesion to the surgical / intervention site. Recently, minimally invasive interventions have been used to address vascular pathogenesis including vascular malformations, aneurysms and vascular tumors. The preferred method for addressing vascular malformations, aneurysms and vascular tumors is to occlude the abnormal blood vessels using transarterial embolization therapy. Transarterial embolization therapy includes local delivery of an occluding device, such as a stent or material roll or anchored biomaterial, which remains in place for an extended period of time to promote the formation of a local clot, which then permanently seals the vessel.

[0012] A topically applied and delivered biocompatible, tissue-adherent chitosan material that remains intact over extended periods, promotes local clot formation without the risk of distal embolization, and promotes normal tissue healing with reduced risk of scarring presents advanced material properties that will enable significant advancements in surgical practice.

[0013] Furthermore, prolonged bleeding, with its associated mortality and morbidity risks, remains a serious problem in the gastrointestinal (GI) tract. Techniques and devices that can provide rapid bleeding control for both upper and lower gastrointestinal bleeding (UGIB) are needed. Current bleeding control during and after transurethral resection of the prostate (TURP) relies on cautery for small vessel arterial bleeding and the application of balloon pressure to address venous oozing. The bladder neck and prostate are highly vascularized tissues, and bleeding often persists after injury and during wound healing. The initial site of injury may continue to bleed for several days unless standard hemostasis is administered, and bleeding may recur in the first or second week after TURP when the scab in the prostatic cavity sloughs. The current standard initial hemostasis for post-TURP bleeding involves manual traction with a balloon catheter, followed by continuous bladder irrigation with saline. Typically, balloon pressure is applied for up to 24 hours in cases of persistent bleeding. If bleeding is uncontrollable after a period of conservative management, the patient may have to return to the surgical ward immediately for open or endoscopic hemostasis.

[0014] Although there have been advances in bleeding control using advanced dressings for applications other than GIB or TURP bleeding control, none of the advances using chitosan materials [1] have been translated to the unique conditions of the gastrointestinal tract or bladder, particularly the upper gastrointestinal tract and prostate, where delivery, adhesion, enzyme activity, persistent exudative bleeding, acidity, and urine-related considerations are very challenging. Rapid bleeding control in TURP, open prostatectomy, and cystectomy is highly desirable.

[0015] Gastrointestinal bleeding (GIB) is a common presentation to the emergency department. According to the U.S. Department of Health and Human Services, an average of over 350,000 patients were discharged annually with major GI bleeding from 2000 to 2014. In the United States, direct hospital costs due to GIB exceeded $1.1 billion in 2010 [2]. Upper GIB (UGIB), defined as bleeding in the gastrointestinal tract proximal to the ligament of Treitz, is approximately five times more common than lower GIB (LGIB) [3]. Acute UGIB is a potentially life-threatening emergency requiring prompt evaluation, resuscitation, and appropriate medical and endoscopic management. Despite recent advances in the treatment of GIB in Western countries, the mortality rate of acute UGIB has not improved significantly and remains as high as 10–14% [4,5]. The leading cause of death after GIB is death secondary to cardiopulmonary complications, which is not surprising given the comorbidity burden of such patients; death due to uncontrolled massive bleeding has been reported to range between 20% and 25% of cases [6,7]. Although comorbidities can rarely be corrected urgently, more effective and rapid bleeding control will significantly reduce the incidence of UGIB-related morbidity and mortality. In summary, the most common causes of acute UGIB are peptic ulcers, gastroesophageal varices, Mallory-Weiss tears, and erosive esophagogastritis [8]. Nonvariceal upper gastrointestinal bleeding (NVUGIB) encompasses all causes of UGIB except bleeding from esophageal or gastric varices. The incidence of peptic ulcer disease has decreased due to the development and use of proton pump inhibitors and the identification, treatment, and eradication of Helicobacter pylori in individual patients [9]. Despite the decrease in the incidence of peptic ulcers, the mortality rate among patients with NVUGIB is 3% to 4%

[10] . Although rarely life-threatening, gastric malignancies can result in friable tissue with diffuse bleeding that is difficult to resolve with conventional physical hemostasis methods (clamps, bandages, ligatures) or cauterization

[11] .

[0016] Current endoscopic treatments for patients with acute UGIB include thermal therapy (eg, bipolar electrocoagulation, heater probe, monopolar electrocoagulation, argon plasma coagulation, and laser), injections (epinephrine, sclerosants (eg, anhydrous ethanol, polidocanol, and ethanolamine)), thrombin or fibrin glue (thrombin plus fibrinogen), and clips [12,13].

[0017] In summary, hemostasis is achieved in most patients with peptic ulcer bleeding by a combination of the above endoscopic treatment modalities. However, there is still a proportion of patients, approximately 5%, in whom endoscopic treatment is insufficient to achieve hemostasis and therefore requires interventional radiology or surgical intervention [14,15].

[0018] Endoscopic therapy can fail for a variety of reasons, including poor lesion visualization due to active, pulsatile bleeding, difficulty anatomically localizing the lesion endoscopically, maximal treatment with available tools, and severe coagulopathy. Three different gas-propelled, gas-dispersed, spray-based hemostatic powder devices, ENDOCLOT

[16] , HEMOSPRAY[17-21], and NEXPOWDER[18,22,23], have been developed to help control NVUGIB. HEMOSPRAY was approved for sale in the United States in 2018, but despite demonstrating improved control of acute upper gastrointestinal bleeding, it has not improved the incidence of rebleeding

[20] . In moderate to high flow bleeding, poorly viscous fluids or particles are easily washed away from the wound. This problem of delivering sufficiently loose hemostatic material to the vicinity of moderate to high flow bleeding lesions to achieve hemostasis can lead to excessive material application, resulting in adverse events such as endoscope retention

[21] .

[0019] Benign prostatic hyperplasia (BPH) and prostate cancer are the two most common urological conditions treated with surgical intervention in older men. An estimated 50% of men have histological evidence of BPH by age 50, and 75% are thought to have such evidence by age 80. In 40-50% of these patients, BPH becomes clinically significant. Although the incidence of uncontrolled bleeding caused by surgical intervention involving the prostate and urethra is relatively low, it is still a significant risk that must be addressed by a hospital stay of at least two to three nights. According to a statistical analysis by the U.S. Department of Health and Human Services from 2005 to 2010, an average of 150,000 men were discharged from the hospital each year in the United States after open or transurethral prostatectomy, with direct surgical costs averaging over $4.5 billion annually. Among these patients, the average length of stay for open or transurethral prostatectomy was 3.1 days and 2.4 days, respectively. Among patients who required a blood transfusion due to significant intraoperative blood loss (4-5%), the average hospital stay was extended to five or six days, with an average additional cost of $15,700 per case, compared to the average cost of prostatectomy in 2010 ($29,300). Prostatectomy is expensive due to operating room time, surgeon time, and hospital stay.

[0020] TURP is considered the benchmark treatment for BPH. In TURP, partial removal (resection) of the prostate is performed through a minimally invasive transurethral procedure using a cystoscope (an endoscope inserted through the urethra into the bladder) and electrocautery. The fine-loop electrocautery used in TURP results in less tissue necrosis than in other less common minimally invasive prostatectomy procedures, but intraoperative bleeding is greater with TURP. As with other forms of prostatectomy, adequate prostate resection and bleeding control are fundamental challenges in TURP. The amount of intraoperative bleeding during prostatectomy depends on the size of the prostate, the duration of prostatectomy, and the surgeon's skill. Excessive or massive bleeding after prostatectomy often leads to unwanted clot retention in the bladder and urethra (and consequent urinary retention), which can prolong hospital stays and even require repeat surgery. While arterial bleeding is generally easy to identify and control with electrocoagulation, venous bleeding, common in TURP, is more difficult to control. This is because highly vascularized organs and glands, such as the prostate, present a large surface area for continuous exudative bleeding from the resected injury, rather than just the needle-tip arterial vessels, which are easily controlled with point cautery. In a significant number of patients, the resected surface of the prostate will continue to display problematic exudative bleeding for up to 72 hours. Attempts to control venous bleeding with electrocautery and irrigation may result in undesirable consequences such as TURP syndrome. Standard of care management involves filling the bladder with irrigation fluid and applying an inflated transurethral balloon catheter to compress the bleeding prostatic cavity. TURP-related postoperative morbidity has been reported as high as 18%, with an operative mortality rate of 0.3%. Morbidity and mortality associated with blood loss related to coagulopathy and cardiovascular abnormalities are significantly increased in elderly patients. Uncontrolled bleeding during TURP remains one of the major complications of prostatectomy and often leads to conversion to less desirable open surgery. Despite significant advances in the treatment of BPH, the incidence of uncontrolled severe bleeding remains approximately 6%, with a transfusion rate of 4% to 5% to address this bleeding.

[0021] In a typical TURP, the hospital stay is two to four days, and the patient has an inflated allantoic catheter in place until the bleeding stops and the urine becomes clear. Any significant reduction in postoperative bleeding after TURP will shorten the catheterization time and hospital bed requirements. It will also reduce the incidence of urinary tract infections, catheter-related patient discomfort and related complications. Severe hematuria (blood in the urine) caused by transurethral or open surgery can lead to hemodynamic instability and blood clot retention, requiring immediate medical care for hemostasis, clot removal, blood transfusion and coagulation assessment. Due to limited surgical vision and space constraints, it is difficult to treat severe hematuria through the transurethral approach. Most commonly, the patient has to return to the operating room for open bladder surgery to achieve hemostasis and remove the cystic clot. A complicating factor in prostatectomy is that TURP patients are often anticoagulated due to the presence of other chronic conditions such as cardiovascular disease. Although it is preferred that these patients stop their anticoagulant medications, such as Coumadin and Clopidogrel, prior to TURP surgery due to the risk of bleeding, it would be more preferable to be able to perform the surgery while the patient remains on their medication to reduce the likelihood of a stroke or myocardial infarction during the procedure. There is a pressing need for reliable and sustainable hemostatic techniques (preferably effective in individuals who are anticoagulated) for transurethral application to control severe bleeding after prostatectomy.

[0022] Chitosan materials have been used in the art to address bleeding problems in various applications with varying degrees of success, but advances and improvements are urgently needed to unlock the potential of chitosan materials (such as catechol-modified chitosan) to provide alternative and better chitosan-based solutions for the safe, reliable and effective delivery of bioactive chitosan materials in vivo, especially in challenging in vivo biological environments.

[0023] Summary of the invention

[0024] The present disclosure relates to readily deliverable, flowable, fluidized (the carrier fluid is a liquid), dispersed particulate, tissue-adherent chitosan materials and compositions thereof that resist dissolution, provide durable adhesion to tissue, and are highly effective in promoting rapid hemostasis. These chitosan materials, including catechol-modified chitosan, are generally referred to herein as "flowable dressings," a term used to refer to the fluidized, dissolution-resistant, tissue-adherent particles formed from the chitosan materials of the present invention. The flowable dressings described herein are easily delivered topically, for example, via a catheter, and are suitable for minimally invasive surgery.

[0025] It should be understood that the flowable chitosan materials described herein have many beneficial properties that originate from the chitosan material itself, and that these chitosan materials can take many potential pulverized particle forms. Such pulverized particle forms can be produced by methods including, but not limited to, granulation (forming larger solid forms into coarse particles with a diameter >1 mm), milling (converting particles into powders with a diameter <0.5 mm), grinding (reducing powders to a diameter <0.2 mm), sieving (to select particle size), spray drying (forming small solid particles directly from a liquid without milling or grinding), and chopping (micron and submicron diameter fibers to produce short, low aspect ratio fibers with a length: diameter <200:1). It should be understood that the reduced particle forms of the present invention remain solid materials in both their dry and wet flowable configurations. In the presence of liquid, the particles can exist as solid and semi-solid materials. The solid particle forms of the present disclosure can expand in volume (<50%) but remain solid in the presence of liquid. These solid materials remain as discrete individual particles that retain their essential solid properties by resisting dissolution and deformation. The volume of the semi-solid particulate forms of the present disclosure can expand (≥50%). These semi-solid materials remain as discrete individual particles, and they maintain their basic semi-solid properties by resisting dissolution while exhibiting expansion deformation in shape. The dry semi-solid particles can have an original appearance of rough, sharp-edged particles that, when exposed to liquids, become larger and expand to a more rounded, possibly spherical shape. The preferred form of milling and grinding is at or below -40°C, such as in the presence of dry ice. A more preferred form of milling and grinding is at or below -180°C, such as in the presence of liquid argon or liquid nitrogen.

[0026] In one embodiment, chitosan particles, including catechol-modified chitosan particles having their chitosan Schiff base crosslinked, remain largely insoluble as solid or semi-solid discrete particles in the presence of a diluent carrier liquid and in the presence of biological fluids such as blood, gastrointestinal fluids, and urine, providing a high specific surface area for suspended solids in the diluent carrier liquid. Even in the presence of a certain level of agglomeration and aggregation of the chitosan particles, the essentially discrete nature of the particles is retained.

[0027] The chitosan materials described herein and the beneficial properties derived therefrom include, but are not limited to, the ability to tune the material by controlling the degree of substitution and oxidation level. By tuning the material by controlling the degree of substitution and oxidation level, the chitosan material can resist rapid degradation and dissolution in difficult biological environments (such as the GI tract, urethra, lower GI tract, body cavities such as the abdominal cavity and thoracic cavity) at temperatures approaching 37°C, yet remain sufficiently soluble to disappear in less than about seven (7) days. It should be understood that the anti-dissolution materials described herein impart a degree of degradation resistance. This anti-dissolution protective material will not degrade when exposed to harsh wet environments such as the acid and enzyme-rich environment of the upper gastrointestinal tract and the enzyme and urea-rich environment of the bladder. Other beneficial properties of the chitosan material are that it is also biocompatible, hemostatic, and tissue-adhesive. In addition, the hemostatic mode of action of the chitosan material avoids the risk of embolism and thrombosis. The chitosan material can also promote the normal healing of injuries and reduce the risk of scar formation.

[0028] The flowable dressing chitosan material described herein can be accurately delivered to a distant injury site in the form of a final fluidized dispersion, for example, by catheter delivery, without relying on gravity to adhere to the tissue of the distant site, so as to quickly achieve bleeding control, close the injury site and remain in situ to resist dissolution for more than 6 hours. The flowable dressing chitosan material disclosed herein adheres to mucosal tissue and tissue injury site upon contact and lasts for more than 6 hours. The flowable chitosan material disclosed herein can be applied upside down under normal gravity and adhered to the injury site by endoscopic application without losing coverage or flowing away from its application site. After being applied to the injury site for 6 hours or more, the top of the applied flowable dressing chitosan material may be eroded or biodegraded, but the flowable dressing material closest to the injury remains adhered for at least 12 hours in the form of a thin, uniform layer covering the injury site to protect the injury site and reduce the chance of rebleeding. The flowable dressings of the present disclosure can be used with minimally invasive techniques for remote dressing delivery to rapidly deploy dressings to achieve hemostasis, to fill and seal resection and biopsy sites, narrow recesses, and defects and openings around hemostatic clips, non-metallic sutures, clamps, staplers, metallic sutures, and fixation pins.

[0029] The flowable dressing materials and compositions described herein are provided as non-limiting examples of the final form of the present invention. The flowable dressings described and exemplified herein provide examples of flowable catechol-modified chitosan material compositions and their properties. In addition, flowable chitosan compositions include, but are not limited to, fluidized solid and semi-solid particles. The solid particulate flowable materials of the present invention are dispersed in a diluent carrier liquid to form a fluidized dispersion of solid particles that can be swollen by the diluent liquid but still resist dissolution of the particles. It should be understood that the dispersion (sometimes referred to as "suspension") of particles in a fluid results in dispersed (sometimes referred to as "suspended") particles. In such dispersed particle liquid systems, the liquid phase is described as the continuous phase, while the dispersed particle phase is referred to as the discrete phase. It should be understood that the fluidized system of the present disclosure is a combination of greater than 75% w / w continuous liquid carrier and no more than 25% w / w discrete particles. For the discrete particle phase, the liquid carrier in the dispersion is a non-solvent, or "diluent."

[0030] In a preferred embodiment, the flowable material of the present disclosure, upon mixing the discrete particles and the liquid component, immediately has a viscosity greater than the viscosity of the carrier liquid, which in the case of water at 20°C has a viscosity of 1 mPa.s. In a preferred embodiment, the discrete particles and the liquid component can be mixed together in the presence of atmospheric gases without any foam formation, and substantially all of the gas is absorbed by the liquid and the discrete particle dispersion. Preferably, uniform mixing of the liquid and discrete particle components is achieved in no more than 180 seconds, more preferably, mixing of the liquid and discrete particle components is achieved in no more than 60 seconds, and most preferably, mixing of the liquid and discrete particle components is achieved in no more than 30 seconds. Furthermore, the viscosity of the mixed flowable material is no greater than the viscosity of a fluid that can be delivered through a 1.5 meter long tube or channel having an inner diameter of 1.5 mm, connected to a 5 ml syringe having a 12 mm inner diameter barrel, and delivered at greater than 5 ml / min at a maximum barrel load of 50 kgf. Once delivered to the site of injury at 37°C, the flowable dressing adheres to the wound tissue and has a resistance to flow, allowing it to remain adhered to the wound and not flow away from its application site under the influence of gravity. In one embodiment, the flowable composition of the dressing can be mixed up to 2 hours before its intended use without losing any of its desired flowable and hemostatic properties. In one embodiment, the flowable composition of the dressing can be mixed up to 1 hour before its intended use without losing any of its desired flowable and hemostatic properties. In one embodiment, the flowable composition of the dressing can be mixed up to 45 minutes before its intended use without losing any of its desired flowable and hemostatic properties. In one embodiment, the flowable composition of the dressing can be mixed up to 30 minutes before its intended use without losing any of its desired flowable and hemostatic properties. In one embodiment, the flowable composition of the dressing can be mixed up to 15 minutes before its intended use without losing any of its desired flowable and hemostatic properties. In one embodiment, the flowable composition of the dressing can be mixed up to 10 minutes before its intended use without losing any of its desired flowable and hemostatic properties. In one embodiment, the flowable composition of the dressing may be mixed up to 5 minutes prior to its intended use without losing any of its desired flowable and hemostatic properties.

[0031] In a preferred embodiment of the flowable dressing, the viscosity of the diluent carrier liquid in which the dissolution-resistant particles are suspended is equal to or less than the viscosity of blood at 37°C (which is between 3.5 mPa.s and 5.5 mPa.s). The lower viscosity of the carrier liquid (relative to the viscosity of blood at 37°C) provides the ability of the blood to dilute and displace the carrier liquid as the continuous phase supporting the solid particles of the flowable dressing, thereby providing a high specific surface area of ​​blood to flowable dressing (e.g., >100 cm). 2 / g) ability to interact.

[0032] In one embodiment, the flowable composition comprises a particulate chitosan material and a diluent carrier liquid. In one embodiment, the flowable composition comprises greater than or equal to about 5% of the particulate chitosan material by weight of the total flowable composition. In one embodiment, the flowable composition comprises the particulate chitosan material provided in solid or semi-solid form, and optionally, wherein the semi-solid particulate chitosan material is swollen. In one embodiment, the flowable composition comprises the particulate chitosan material provided in the form of a powder, granules, pellets, fibers, or any combination thereof. In one embodiment, the flowable composition comprises a particulate chitosan material comprising regularly or irregularly shaped particles having a radius of gyration in the range of about 10 to 350 microns. In one embodiment, the flowable composition comprises a particulate chitosan material comprising one or both of catechol-modified chitosan and cross-linked chitosan gelatin. In one embodiment, the flowable composition comprises a granular chitosan material comprising one or more of a densified chitosan material, a frozen phase-separated and dried chitosan material, a densified frozen phase-separated and dried chitosan material, a spray-dried chitosan material, a dried cast film chitosan material, a frozen phase-separated chitosan material dried by freeze-substitution, a sublimed frozen separated chitosan material, a dried freeze-thaw chitosan material, and a dried asymmetric centrifuge mixed material. In one embodiment, the flowable composition comprises a diluent carrier liquid which may be selected from the group consisting of one or more of water, a standard 0.9% saline solution, and autologous plasma. In one embodiment, the flowable composition comprises a diluent carrier liquid which may comprise at least one of about 85% by weight of the total flowable composition, about 90% by weight of the total flowable composition, or about 95% by weight of the total flowable composition. In one embodiment, the flowable composition comprises a diluent carrier fluid which may comprise a thermoresponsive fluid capable of being delivered through a 23 gauge needle or a 24 gauge needle at about 18-25°C and capable of gelling at about 37°C. In one embodiment, the flowable composition is hemostatic. In one embodiment, the flowable composition comprises a diluent carrier fluid that resists dissolution. In one embodiment, the flowable composition comprises a granular chitosan material that is substantially insoluble and remains solid or semi-solid. In one embodiment, the flowable composition is capable of resisting dissolution in at least one of urine, water, saline solution, blood, or gastrointestinal (GI) fluid at about 37°C for at least about 6 hours. In one embodiment, the flowable composition comprises a granular chitosan material further characterized by exhibiting a viscosity greater than about 100 cm 2 / g of the specific surface area of ​​the flowable dressing. In one embodiment, the flowable composition comprises at least a first outer layer and a second tissue adhesion layer, wherein the first outer layer resists dissolution for at least about 6 hours and the second tissue adhesion layer resists dissolution for at least about 12 hours. In one embodiment, the flowable composition is tissue adhesive. In one embodiment, the flowable composition is tissue adhesive and adheres to at least one of mucosal tissue and tissue injury site after contact for a period of time greater than about 6 hours. In one embodiment, the flowable composition can be inverted under normal gravity for application endoscope and adhere to tissue. In one embodiment, the flowable composition is biocompatible. In one embodiment, the flowable composition can be delivered to the tissue site by a channel having a diameter of less than about 7mm, less than about 4.5mm, less than about 4.0mm, less than about 3.2mm, less than about 2.8mm and about 0.5mm.

[0033] In one embodiment, the flowable composition comprising a particulate chitosan material and a diluent carrier liquid is an intraluminal hemostatic dressing, as well as methods of making and applying the flowable composition.

[0034] In one embodiment, the flowable composition comprising a particulate chitosan material and a diluent carrier liquid is a gastrointestinal hemostatic dressing, and methods of preparing and applying the flowable composition. In one such embodiment, the present disclosure provides a method of applying the flowable composition comprising sealing a tissue site with the gastrointestinal hemostatic dressing in an acidic environment of about pH 3 for at least 6 hours. In one such embodiment, the present disclosure provides a method of applying the flowable composition comprising providing for dissolution of the gastrointestinal hemostatic dressing from the tissue site over a period of time less than or equal to about 7 days.

[0035] Another embodiment of the present disclosure relates to a method for preparing a flowable composition as described herein. In one embodiment, this method includes preparing a chitosan material for a flowable composition. In one embodiment, this method includes preparing one or both of a catechol-modified chitosan and a chitosan-gelatin cross-linked chitosan material. In one embodiment, this method includes preparing a chitosan material that is one or more of a densified chitosan material, a frozen phase-separated and dried chitosan material, a densified frozen phase-separated and dried chitosan material, a spray-dried chitosan material, a dried cast film chitosan material, a frozen phase-separated chitosan material dried by freeze-substitution, a sublimed frozen separated chitosan material, a dried freeze-thaw chitosan material, and a dried asymmetric centrifugal mixed material. In one embodiment, this method includes preparing a chitosan material and grinding the chitosan material to form a granular chitosan material.

[0036] One embodiment of the present disclosure provides a method for delivering a flowable composition to a tissue site of a subject in need, comprising mixing a granular chitosan material and a diluent carrier liquid before delivering to the subject. In one such embodiment, the method further comprises providing a granular chitosan material and a diluent carrier liquid as separate components for combination, and optionally, sterilizing the separate components separately. In one such embodiment, the method comprises delivering a flowable composition in an amount sufficient to achieve hemostasis at a bleeding tissue site of the subject. In one such embodiment, the method further comprises delivering the flowable composition to the tissue site through a channel having a diameter of at least one of less than about 7 mm, less than about 4.5 mm, less than about 4.0 mm, less than about 3.2 mm, less than about 2.8 mm, and about 0.5 mm. In one such embodiment, the method further comprises delivering the flowable composition to the tissue site with one or more layers, and optionally, wherein the flowable composition comprises at least a first outer layer and a second tissue adhesion layer, and wherein the first outer layer resists dissolution for at least about 6 hours, and the second tissue adhesion layer resists dissolution for at least about 12 hours. In one such embodiment, the method further comprises adhering the flowable composition to the tissue site, and optionally, wherein the flowable composition adheres to the tissue site upon contact and for a period of time greater than about 6 hours, and wherein the tissue site comprises at least one of mucosal tissue and a tissue injury. In one such embodiment, the flowable composition can be administered endoscopy upside down under normal gravity and adhered to the tissue site.

[0037] In one embodiment, the present disclosure provides a method for delivering a flowable composition comprising a particulate chitosan material and a diluent carrier to a tissue site of a subject in need thereof, comprising: mixing the particulate chitosan material and the diluent carrier prior to delivery to the subject; applying the flowable composition; and adhering the flowable composition to the tissue site upon contact. In one such embodiment, the diluent carrier has a viscosity less than or equal to 3.5 mPa.s and 5.5 mPa.s. In one such embodiment, the method further comprises applying the flowable composition using a minimally invasive technique, and optionally, wherein the minimally invasive technique provides remote flowable composition delivery. In one such embodiment, the method further comprises applying the flowable composition for resection, biopsy sites, stenotic recesses, and one or both of filling and sealing of defects and openings around hemostatic clips, non-metallic sutures, clamps, staplers, metallic sutures, and fixation pins.

[0038] The present disclosure generally relates to flowable chitosan catechol-modified dressing compositions that, due to their properties, can be applied in various physiological environments to stop bleeding and provide long-term wound protection. Embodiments include, among others, chitosan gastrointestinal hemostatic flowable dressings (CGHFD) and chitosan intraluminal hemostatic flowable dressings (CEHFD).

[0039] The flowable dressing material described herein has one or more or all of the following properties in combination: it (1) is capable of rapid and accurate delivery of an excipient from a syringe or other delivery vehicle through a catheter or other conduit to a remote location; (2) can be applied at about 37°C in the presence of actively flowing blood and other biological fluids without significant dimensional changes in length, width, and height to achieve rapid hemostatic control of bleeding; (3) is capable of delivery in the presence of biological fluids and blood; (4) is capable of being delivered to the surgical site manually or by a minimally invasive delivery device; (5) is capable of adhering to its application site in an inverted orientation (upside down) and maintaining its original shape without loss of material due to flow or dripping; and (6) is capable of being applied as a non-occlusive, flowable material to maintain a specific surface area of ​​dispersed solids and semi-solids greater than 100 cm 2 / g, thereby promoting blood cell interaction and adsorption of hydrophilic and hydrophobic biological fluids that can interfere with adhesion; (7) maintaining interconnected porosity of radius ≥ 5 microns between individual flowable dressing chitosan particles; (8) adhering to mucosa, excised mucosa and excised tissue when applied; (9) being able to adhere uniformly to tissue and rapidly promote local blood clot formation without the risk of distal embolism or thrombosis; (10) being able to be released from the delivery catheter to allow the catheter to be withdrawn from the surgical site; (11) being exposed to a medium containing a pH of about 3.0 to about pH at about 37°C. 8.0, resist dissolution and provide prolonged (persistent) adhesion to tissue for up to 48 hours when placed in an enzymatic wet biological environment of biological fluids in the range of 8.0; (12) enable ostomy access passages (such as those in the gastrointestinal tract and urinary tract) to remain open (with or without a delivery device in place) where material or material residues are present and fecal matter passes unimpeded; (13) protect the site of injury and promote healing while reducing the risk of scarring; (14) provide controlled, slow degradation and / or dissolution from the site of attachment to allow removal without surgical assistance in less than 7 days in ostomy applications; (15) the flowable material may comprise one or more a plurality of dispersed milled powders, dispersed milled powders, dispersed spray dried powders, dispersed chopped fibers, and combinations of these different dispersions; (16) the tissue-adherent flowable material of the present invention may include, but is not limited to, one or more uses as an adhesive dressing, an adhesive hemostatic dressing, an adhesive patch for local controlled release of an active agent, a material for promoting coagulation, a tissue-adherent matrix material for tissue regeneration, and a tissue-adherent matrix material for vascular malformation closure; (17) the tissue-adherent flowable material of the present invention may include, but is not limited to, use in combination with sutures, staples, hemostatic clips, hemostatic forceps, orthopedic bone fixation devices, or occlusive suture-less patches; (18) the tissue-adherent flowable material of the present invention may be used as part of a combined treatment for definitive control of difficult bleeding, wherein the flowable dressing may be initially used to stop bleeding, providing sufficient visibility of the injury site for the application of other hemostatic therapies, including but not limited to one of injection of epinephrine, clamping, cauterization, and suturing. In one embodiment, the porosity in the flowable dressing is continuous, with interconnected pore sizes ranging from 10 to 100 microns, with a substantial majority of pores approximately 10 to 50 microns. The continuous pore structure in flowable dressings is reflected in their ability to absorb biological fluids such as blood. Uncontrolled bleeding commonly occurs in patients receiving anticoagulant medications and those with bleeding disorders. It can occur during surgery and is caused by injuries associated with disease, tissue failure, and surgical errors.This is a serious problem in minimally invasive surgical procedures when standard bleeding control and hemostasis techniques fail, transfusion of blood products becomes necessary, and the only option to control bleeding becomes high-risk open surgery.

[0040] While the majority of UGIB is easily controlled with existing tools in the United States, there remains an unmet need for safe flowable dressing materials that provide rapid control of bleeding, as disclosed herein. Widespread application of the subject flowable dressing materials described herein could significantly reduce morbidity and mortality in the treatment of gastrointestinal bleeding, along with associated reductions in health care costs.

[0041] The subject flowable dressing material disclosed herein is suitable for all gastrointestinal bleeding applications and can be delivered as a chitosan gastrointestinal hemostatic flowable dressing (CGHFD) through, for example, a catheter passed through a standard endoscope working channel (≤3.8 mm in diameter). The subject material of the present invention will provide an opportunity to address or alleviate the shortcomings of existing approaches such as clamping, thermal coagulation, and injection, which require precise positioning accuracy, which is challenging under the impaired visibility of rapid bleeding conditions. Thermal coagulation is also problematic because it tends to induce undesirable scar tissue formation.

[0042] The subject flowable dressing material described herein is suitable for all transurethral prostatectomy bleeding applications and can be delivered as a chitosan intraluminal hemostatic flowable dressing (CEHFD) through, for example, a transurethral balloon catheter channel. The subject material will provide an opportunity to address or alleviate the shortcomings of existing approaches such as applying traction to the bleeding site through a Foley catheter or using a biological flowable system such as FLOSEAL, which does not remain in place in the presence of urine.

[0043] The subject flowable dressing materials described herein may be used for procedures other than gastrointestinal and transurethral bleeding control, such as bleeding control in surgical procedures including, but not limited to, maxillofacial surgery, otolaryngology (ENT) / head and neck surgery, bladder surgery, oral surgery, and may be delivered, for example, through a catheter channel as a chitosan hemostatic flowable dressing (CHFD). The subject materials of the present invention will provide an opportunity to address or mitigate the shortcomings of existing approaches, such as applying traction to the bleeding site or using biological flowable systems that do not remain in place in a wet or inverted environment.

[0044] The present invention includes flowable dressing materials and compositions, methods of using the compositions, and methods of making the compositions.

[0045] In a preferred embodiment, the flowable dressing material comprises a tissue-adherent fluidized particulate composition, wherein the fluidized tissue-adherent particulates comprise dissolution-resistant cross-linked chitosan particles.

[0046] In a preferred embodiment, the flowable dressing material comprises a tissue-adherent fluidized particulate composition, wherein the fluidized tissue-adherent particulates include dissolution-resistant cross-linked chitosan particles that provide a high surface area interaction with the injury site, wherein the particles adhere to the site for greater than 6 hours.

[0047] In a preferred embodiment, the flowable dressing material comprises a tissue-adherent fluidized particulate composition, wherein the fluidized tissue-adherent particulates include dissolution-resistant cross-linked chitosan particles that provide a high surface area interaction with the injury site, wherein the particles remain attached to the site for more than 6 hours, which results in rapid and prolonged hemostasis at the injury site and prevents rebleeding and re-injury.

[0048] In a preferred embodiment, the flowable dressing material composition has a sufficient volume of material, preferably 3 ml, more preferably 5 ml, and most preferably 7 ml, such that in one delivery there is enough material to cover a typical peptic ulcer bed of 20 mm in diameter in a gastrointestinal tract lesion to a depth of 2 to 5 mm. In a preferred embodiment, the flowable dressing material composition has a sufficient volume of material, preferably 5 ml, more preferably 7 ml, and most preferably 10 ml, such that in one delivery there is enough material to cover a depth of 7 to 15 mm to balloon pack a resected prostatic fossa lesion of 30 to 50 mm in length, 15 mm in depth, and 10 mm in width. It will be understood that if the lesion is larger than typical lesion sizes, or if the bleeding field proves difficult to control with a single application, the flowable dressing of the present disclosure may be applied multiple times to the same lesion.

[0049] In a preferred embodiment, the flowable dressing material composition can be delivered from a distal end of a conduit, such as a catheter, in a continuous stream at near-constant pressure to fill cavities or, alternatively, provide a uniform coating on a bleeding wound. In an alternative embodiment, the flowable dressing material can be delivered from a distal end of a delivery conduit in regular and irregular discrete volumes under pressure pulses in a discontinuous manner to provide increased momentum for applying the flowable dressing to the target tissue surface. The resulting discrete volume sputtering application can be used to build a three-dimensional dressing structure through repeated, discrete, flowable, print-like delivery to a deposited dressing body.

[0050] In a preferred embodiment, the flowable dressing material comprises catechol-modified chitosan, wherein the catechol-modified chitosan material is preferably at least 25% w / w of the dry solids dispersed in the flowable composition, more preferably at least 50% w / w, and most preferably at least 75% w / w. The catechol-modified chitosan flowable dressing may adhere immediately to the site of injury to which it is applied upon application. The flowable dressing may form a quaternary ammonium cation on the chitosan glucosamine C-2 amine at the site of tissue injury. The flowable dressing may comprise catechol that is oxidized to o-quinone and cross-linked in the chitosan. In one embodiment, the flowable chitosan dressing may have a brown coloration, including a dark brown to black coloration. In one embodiment, the fluidized dressing particles may comprise catechol having a low oxidation level, and wherein the flowable chitosan dressing has a brown to pink colored appearance.

[0051] The particles of the flowable dressing may include, but are not limited to, powders and fibers.

[0052] In a preferred embodiment, the dry particles of the flowable dressing can be formed from one or more of spray-dried, dried cast films, sublimed freeze-fractionated, solvent-substituted freeze-fractionated, dried freeze-thawed, dried asymmetric centrifugally mixed, extruded, and continuous fibrous compositions containing catechol-modified chitosan. Solid powders can be formed from solid films, sheets, rods, granules, and chips of the compositions containing catechol-modified chitosan by grinding and milling processes. The preferred grinding and milling process is cryogenic grinding at temperatures below -40°C.

[0053] In a preferred embodiment, the raw dry particles, prior to forming the tissue-adherent fluidized particles of the flowable dressing, may be regular or irregular shaped particles having a radius of gyration in the range of 10 to 350 microns.

[0054] In a more preferred embodiment, the raw dry particles, prior to forming the tissue-adherent fluidized particles of the flowable dressing, may be irregularly or regularly shaped particles having a radius of gyration in the range of 10 to 200 microns.

[0055] In the most preferred embodiment, the raw dry particles, prior to forming the tissue-adherent fluidized particles of the flowable dressing, may be irregularly or regularly shaped particles having a radius of gyration in the range of 10 to 100 microns.

[0056] In a preferred embodiment, the dry solid particulate fraction of the flowable dressing composition is sterilized separately from the carrier liquid diluent fraction. Immediately before use, the sterilized dry solid and liquid fractions are aseptically mixed to form the flowable fluidized dressing of the present invention.

[0057] In a preferred embodiment, the sterile dry solid particulate fraction of the flowable dressing is present in a first closed syringe and the sterile liquid carrier fraction is present in a second syringe, wherein both the first and second syringes are capable of being aseptically connected together to combine and mix the liquid and solid particulate components into the final flowable form upon delivery and use.

[0058] Sterilization of the solid fraction may include, but is not limited to, gamma-irradiation, electron beam irradiation, x-ray irradiation, and ethylene oxide gas exposure.

[0059] The carrier liquid diluent fraction of the flowable dressing can be sterilized separately from the solid fraction using sterilization methods including, but not limited to, sterile filtration, gamma irradiation, electron beam irradiation, and x-ray irradiation. Alternatively, the fluid fraction can be prepared aseptically from a sterile source.

[0060] The carrier liquid diluent of the flowable dressing may include, but is not limited to, one or more of water, standard 0.9% saline solution, and autologous plasma.

[0061] The carrier fluid can be a thermosensitive fluid that provides fluidized particle delivery through a catheter and a narrow gauge needle (e.g., a 24 to 23 gauge needle) at room temperature (18-25° C.) and provides gelation (near solidification) in vivo at 37° C. Such delivery is particularly desirable in esophageal submucosal dissection procedures where it is necessary to lift the submucosal layer with a flowable lifting agent but it is desirable to hold the fluid in place with a flowable hemostatic agent after lifting.

[0062] In a preferred embodiment, the carrier liquid fraction of the flowable composition preferably does not exceed 95% w / w, more preferably does not exceed 90% w / w, and most preferably does not exceed 85% w / w of the total weight of the flowable dressing (total weight = solid weight fraction + liquid weight fraction).

[0063] The dressing adhesion strength may be greater than or equal to about 1 kPa. The dressing resists dissolution in urine, water, saline solution, blood, or GI fluid at about 37°C for at least about 6 hours. The angle of delivery and the rate of delivery (ml / min) combined with the inner diameter of the delivery catheter and the shape of the tip will determine the shape and volume of the flowable dressing delivered. The tip shape used to achieve release of the flowable dressing from the delivery catheter (or tube, channel, etc.) can vary and include, but are not limited to, tips with narrow ends, narrow flared ends, driven release, printhead sputtering type systems that can be used at controlled speeds to deliver deposits of different sizes at different times, with large deposits covering large bleeds, smaller deposits covering (or printing) large areas, and the like.

[0064] Flowable dressings can be applied as a thin, uniform coating that adheres to tissue and can be applied in one layer or over a wide area of ​​tissue, or in one or more deposits to a target site or wound coverage area. Flowable dressings can be applied as tissue-adherent dressing beads that can be used to fill and approximate wound openings. In one embodiment, the flowable dressing can be "applied" in a layer about 1-2 mm thick, or layered to about 5-7 mm thick, or used to fill cavities that are, for example, 10-35 mm deep.

[0065] Flowable dressings can be applied in combination with other materials, including application into, around, and through previously applied patch-type dressings, clamps, and forceps to immediately stop all exudative bleeding. As a preferred non-ablative hemostatic treatment, flowable dressings can be widely applied to bleeding surfaces to provide the desired wound healing with minimal to no scarring compared to the high scarring and poor wound healing effects of standard hemostatic thermal cauterization treatments. After adhering to the target tissue site, the dressing is easily released from its delivery catheter by squeezing the flowable dressing beads at the distal delivery tube to separate the dressing from the delivery catheter because the flowable dressing tissue adhesion is greater than the cohesive strength of the flowable dressing beads at the end of the delivery tube. During application, alternative means of separating the dressing from its delivery device may include squeezing the tip of the catheter against the wound or other tissue, flicking the end of the catheter, cutting off or gating the flowable dressing flow, and / or applying the dressing in discrete, discontinuous volume pulses.

[0066] The dressing resists dissolution after being adhered to the site of injury in the presence of corrosive enzymes and an acidic environment of approximately pH 3 for at least six hours. The dressing seals and protects the target tissue site for at least six hours. The dressing is capable of achieving a controlled, slow dissolution from the site of attachment over a period of no more than seven (7) days. The dressing is not readily soluble in water, saline solution, blood, or GI fluids at approximately 37°C for at least six hours after application. The dressing is not readily soluble in water, saline solution, blood, or GI fluids at approximately 37°C for at least 12 hours after application. The dressing does not adhere to or accumulate residue on the delivery device due to the non-stick material surface of the delivery device and the shearing of the material against itself during delivery.

[0067] In one embodiment, the flowable dressing is like thick ketchup and as it flows down the delivery tube it simply pushes everything in front of it forward and it is non-settling (i.e. no chemical reactions occur) so it remains wet and once delivered it is just sticky enough to adhere to tissue and itself and it reacts with blood to produce a clot so when added to the blood environment the dressing exhibits and is characterized by increased cohesion and adhesion to the wound.

[0068] In a preferred embodiment, the flowable dressing includes an optical contrast material that provides enhanced endoscopic visualization of the deployed flowable dressing for improved wound placement and post-placement observation of the wound and dressing. The material can be easily mixed with the composition of the flowable dressing and delivered with the flowable dressing to address bleeding issues and provide enhanced visualization of the dressing edge, the dressing body, and successful hemostasis. While the flowable dressing provides hemostasis, the enhanced visibility material remains incorporated and uniformly present in the flowable dressing composition without any significant leakage of material into the biological environment. The enhanced visualization material may include, but is not limited to, fluorescent agents, nanoparticles containing fluorescent agents, chitosan covalently modified with fluorescent agents, quantum dots, gold nanoparticles, organically modified dye-doped silica, upconversion phosphors, and lanthanide-based contrast agents.

[0069] In a preferred embodiment, once the flowable dressing has been deployed over the bleeding site, hemostasis of the flowable dressing can be enhanced by endoscopically placing a supportive solid mesh or dressing over the flowable dressing. In cases of intense arterial bleeding (which may prove too high a pressure for the cohesive strength of the flowable dressing to immediately control the bleeding due to arterial bleeding pressure tunneling through or otherwise penetrating the flowable dressing), a gentle packing pressure is briefly applied by a balloon or similar endoscopically applied basket device through an intermediate dressing or mesh material over the flowable dressing. Applying the support dressing or mesh with gentle pressure seals any tunnels or other penetrations within the flowable dressing and provides prolonged hemostasis in difficult bleeding situations.

[0070] In the presence of water, saline solution, blood, or GI fluid at a wound site at about 37° C., the dressing does not increase or decrease in size by more than about 25% in length and width, or by more than about 50% in thickness. The individual flowable dressing components can be stored in their packaging at or below room temperature (25° C.) for 2 years or longer without affecting the dressing properties.

[0071] The lytically resistant, tissue-adherent, flowable hemostatic dressing of the present invention can be used alone or as an adjunctive hemostatic dressing to control all bleeding in patients with normal coagulation function as well as in patients receiving anticoagulant therapy, antiplatelet therapy, and those suffering from bleeding disorders.

[0072] The anti-lytic, tissue-adhesive, flowable hemostatic dressing of the present invention can be applied to an injured area to remain in place, control bleeding and promote rapid tissue regeneration at the injured area while reducing the risk of scar formation.

[0073] The UGIB bleeding rate or blood flow rate in ml / min suitable for treatment by the flowable hemostatic dressing described herein can be in the range of about 1 ml / min to about 100 ml / min. In a preferred embodiment, the bleeding rate addressed by the device ranges from about 1 ml / min to about 40 ml / min. Forrest 1a UGIB is about 25 ml / min. For subjects with bleeding rates much greater than Forrest 1a, survival is unlikely unless they are already in the operating room. UGIB bleeding rates between about 20 ml / min and 25 ml / min are considered "fast" bleeding. Exudative bleeding is typically greater than about 1 ml / min, as it is noted that low bleeding rates such as 1 ml / min will typically clot and stop on their own, unless the subject is receiving anticoagulant therapy or has a coagulation cascade disorder for reasons other than taking anticoagulant medication. For subjects receiving irreversible anticoagulant therapy or suffering from bleeding disorders, exudative bleeding of 1 ml / min remains a concern and needs to be addressed, such as by the flowable dressing disclosed herein. In some embodiments, the flowable dressings described herein are used to address UGIB bleeding rates between about 1 ml / min and about 25 ml / min, or between about 1 ml / min and about 20 ml / min, or between about 1 ml / min and about 15 ml / min, or between about 1 ml / min and about 10 ml / min, or between about 1 ml / min and about 5 ml / min. In some embodiments, the dressing can be used to treat a disease, illness, condition, wound, or injury. For example, the use of the dressing in treating a disease, illness, condition, wound, or injury includes adhering the dressing directly to the injury site. Dressings for treating a disease, illness, condition, wound, or injury can remove (through high surface area adsorption) anti-adhesion hydrophilic and hydrophobic biological fluids that would normally interfere with adhesion. Dressings for treating a disease, illness, condition, wound, or injury can include leaving the dressing in place at the target tissue site, and the dressing can remain at the target tissue site for at least 12 hours. A dressing for treating a disease, illness, condition, wound, or injury may be capable of slowly dissolving at the target tissue site and completely dissolving in less than or equal to 7 days without human intervention.

[0074] In some embodiments, the invention disclosed herein includes a method for producing a flowable chitosan dressing. In one embodiment, the method includes: synthesizing chitosan and catechol in an aqueous reaction solution to produce a modified catechol chitosan; maintaining the pH of the reaction solution at or below pH 5.5; increasing the pH of the reaction solution and controlling the oxygen exposure to the reaction solution to provide catechol oxidation and cross-linking; and drying the reaction solution. Another embodiment of the method for producing a freeze-dried modified catechol chitosan sheet includes: freeze-drying an aqueous solution containing a modified catechol chitosan material; and obtaining a freeze-dried chitosan solid having an interconnected porous structure from each of the above steps. Another embodiment of the method for producing a freeze-dried modified catechol chitosan sheet includes compressing the freeze-dried modified catechol chitosan material to a density greater than 0.5 g / cm 3 In certain embodiments, the compression step can be performed at a temperature of about 20° C. to about 150° C. In certain embodiments, the dry chitosan solids are dried to a moisture content of less than about 15% (w / w).

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) will prevail.

[0076] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims.

[0077] BRIEF DESCRIPTION OF THE DRAWINGS Several views

[0078] Figure 1 .FTIR spectrum of dried material of CSGelatin1.

[0079] Figure 2 .FTIR spectrum of Z-type freeze-dried material.

[0080] Figure 3 .FTIR spectrum of type Y freeze-dried material.

[0081] Figure 4 Image of cross-linked chitosan gelatin (1:4) powder.

[0082] Figure 5 Image of Z-type catechol-modified powder.

[0083] Figure 6Image of the first syringe of Example 3 with a male Luer connector end, with powder loaded in the syringe (without cap).

[0084] Figure 7 An image of a first syringe and a second syringe with a female Luer connector (containing liquid on the left) of Example 3 before the syringes are connected to each other.

[0085] Figure 8 .Image of the first syringe and the second syringe (containing liquid on the left) of Example 3, wherein the syringes are connected to each other by a female Luer connector end (on the left) and a male Luer thread connector end (on the right).

[0086] Figure 9 An image of the first and second syringes of Example 3, wherein the syringes are connected to one another by male and female Luer thread ends, and the flowable particles are substantially uniformly dispersed (mixed) into the liquid without foaming or other evidence of bubbles.

[0087] Figure 10 . Image of a first syringe with a male luer connector containing a uniformly dispersed particle-liquid mixture ready for delivery.

[0088] Figure 11 . Image of a first syringe containing a uniformly dispersed particle-liquid mixture ready for delivery and connected to a catheter delivery tube for precise, localized, minimally invasive application to a lesion.

[0089] Figure 12 Image of a flowable dressing bead being deployed from the tip of a first syringe delivery catheter onto a horizontal clear PVC plate having a central hole with a diameter of 4.0 mm.

[0090] Figure 13 Image of a flowable dressing bead being deployed from the tip of a first syringe delivery catheter onto a horizontal clear PVC plate having a central hole with a diameter of 4.0 mm.

[0091] Figure 14 Image of adhered flowable dressing beads deployed from the tip of a first syringe delivery catheter onto an upright clear PVC plate with a 4.0 mm diameter central hole.

[0092] Figure 15 Image of all flowable dressing (approximately 3.5 ml) transferred from the first syringe delivery catheter to partially cover the bottom of a thermoformed polystyrene dish (10 cm x 10 cm).

[0093] Figure 16. Chitosan flowable dressing adheres to GI tissue located in the horizontal plane ( Figure 16A , Z-type; Figure 16B , 1 to 1; Figure 16C , CsGelatin2) and the same dressing upside down ( Figure 16D , Z-type; Figure 16E , 1 to 1; Figure 16F , CsGelatin2) images.

[0094] FIG. 17. Chitosan flowable dressing adhered to GI tissue during ex vivo treatment at time = 0 ( Figure 17A , Z-type; Figure 17B , 1 to 1; Figure 17C , CsGelatin2); the same dressing at 7.8 hours ( Figure 17D , Z-type; Figure 17E , 1 to 1; Figure 17F , CsGelatin2) and the dressing at 19.9 hours ( Figure 17G , Z-type; Figure 17H , 1 to 1; Figure 17I , CsGelatin2)

[0095] Figure 18. Chitosan flowable dressing adhered to liver tissue located on a horizontal plane ( Figure 18A , Z-type; Figure 18B , 1 to 1; Figure 18C , CsGelatin2) and the same dressing upside down ( Figure 18D , Z-type; Figure 18E , 1 to 1; Figure 18F , CsGelatin2)

[0096] FIG19. Chitosan flowable dressing adhered to liver tissue during ex vivo testing at time = 0 ( Figure 19A , Z-type; Figure 19B , 1 to 1; Figure 19C , CsGelatin2); the same dressing at 7.8 hours ( Figure 19D , Z-type; Figure 19E , 1 to 1; Figure 19F , CsGelatin2); the dressing at 19.9 hours ( Figure 19G , Z-type; Figure 19H , 1 to 1; Figure 19I , CsGelatin2); and the remaining dressing adhered at 33 hours ( Figure 19J , Z-type; Figure 19K , 1:1) image.

[0097] Figure 20. Chitosan flowable dressing adhered to TURP bladder tissue in a horizontal plane ( Figure 20A , Z-type; Figure 20B , 1 to 1; Figure 20C , CsGelatin2) and the same dressing upside down ( Figure 20D , Z-type; Figure 20E , 1 to 1; Figure 20F , CsGelatin2) images.

[0098] FIG21. Chitosan flowable dressing adhered to TURP bladder tissue during ex vivo testing at time = 0 ( Figure 21A , Z-type; Figure 21B , 1 to 1; Figure 21C , CsGelatin2); the same dressing at 2.25 hours ( Figure 21D , Z-type; Figure 21E , 1 to 1; Figure 21F , CsGelatin2); the dressing at 6.2 hours ( Figure 21G , Z-type; Figure 21H , 1 to 1; Figure 21I , CsGelatin2); at 13.9 hours, the dressing ( Figure 21J , Z-type; Figure 21K , 1 to 1; Figure 21L , CsGelatin2); the dressing at 24 hours ( Figure 21M , Z-type; Figure 21N , 1 to 1); the dressing at 29.5 hours and 48.7 hours ( Figure 21O and Figure 21P , Z-type) images.

[0099] Figure 22. Chitosan flowable dressing adhered to esophageal tissue in a horizontal plane ( Figure 22A , Z-type; Figure 22B , 1 to 1; Figure 22C , CsGelatin2) and the same dressing upside down ( Figure 22D , Z-type; Figure 22E , 1 to 1; Figure 22F , CsGelatin2) images.

[0100] FIG23. Chitosan flowable dressing adhered to esophageal tissue during ex vivo testing at time = 0 ( Figure 23A , Z-type; Figure 23B , 1 to 1; Figure 23C , CsGelatin2); the same dressing at 2.25 hours ( Figure 23D , Z-type; Figure 23E , 1 to 1; Figure 23F , CsGelatin2); the dressing at 6.2 hours ( Figure 23G , Z-type; Figure 23H , 1 to 1; Figure 23I, CsGelatin2); the dressing at 13.9 hours ( Figure 23J , Z-type; Figure 23K , 1 to 1; Figure 23L , CsGelatin2); dressing at 24 hours ( Figure 23M , Z-type; Figure 23N , 1 to 1; Figure 23O , CsGelatin2) images.

[0101] Figure 24. Image of Z-type powder ( Figure 24A and Figure 24B : dry; Figure 24C and Figure 24D : moistened by water; Figure 24E and Figure 24F : moistened with blood and water).

[0102] Figure 25. Image of CsGelatin2 ( Figure 25A and Figure 25B : dry; Figure 25C and Figure 25D : moistened with water; Figure 25E and Figure 25F : moistened with blood and water).

[0103] Figure 26.1:1 images of Z-type and CsGelatin2 ( Figure 26A and Figure 26B : dry; Figure 26C and Figure 26D : moistened by water; Figure 26E and Figure 26F : moistened with blood and water).

[0104] Figure 27. Image of Y-type powder soaked in blood and water ( Figure 27A and Figure 27B ).

[0105] Figures 28A-28D .FLOSEAL prepared image.

[0106] Figures 29A-29D Images of FLOSEAL on the stomach, bladder, liver, and esophagus.

[0107] Figure 30. Image of FLOSEAL powder taken using an Amscope T490-DK microscope ( Figure 30A dry; Figure 30B moistened by water; Figure 30C moistened with blood).

[0108] Figure 31. Histogram box plots of mean post-treatment bleeding scores and their standard deviations for the catechol-modified chitosan test material and HEMOSPRAY (HS) control for porcine parenchymal lesions used to study heparinized bleeding. The plots provide Z-type, Y-type, Y1-type and Y1-type resid The hemostatic properties, expressed as bleeding scores after treatment (low bleeding scores indicating better hemostasis), of the catechol-modified chitosan test samples of the type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 5

[0109] Figure 32 Box plots of mean hemostasis times and standard deviations for catechol-modified chitosan test materials and the HEMOSPRAY (HS) control used to study hemostasis in heparinized, bleeding porcine parenchymal lesions. The best-performing catechol-chitosan dressings were Y and Y1. Based on a two-tailed Student's t-test, Y and Y1 were statistically equivalent in terms of hemostasis time in parenchymal lesions. Hemospray demonstrated statistically significantly longer hemostasis times than Y and Y1.

[0110] Figure 33 . Histogram box plots of pre-treatment bleeding rates for catechol-modified chitosan (Y and Y1) and HEMOSPRAY (HS) administration.

[0111] Figure 34 Box plots of hemostasis time in gastroepiploic artery injury treated with catechol-modified chitosan dressings (Y and Y1) and control HEMOSPRAY (HS).

[0112] Figure 35 Viability of HEE tissue treated with sample extracts including catechol-modified chitosan dressing Y3 and HEMOSPRAY control relative to negative control.

[0113] Detailed description of the invention

[0114] As used herein, chitosan intracavitary hemostatic flowable dressing (CEHFD) refers to a flowable chitosan dressing that is hemostatic and can be used in intracavitary areas (e.g., within the resected prostate fossa to control bleeding) or the bladder neck of a resected prostate. CEHFD is not limited to its application site and includes chitosan dressings applied to any site in the human body (including but not limited to the bladder mucosa).

[0115] The bleeding rate or blood flow rate in ml / min that are applicable to be treated by device described herein can be in the scope of about 1ml / min to about 200ml / min.In a preferred embodiment, the bleeding rate scope solved by this device is about 1ml / min to about 150ml / min.The bleeding rate between about 20ml / min and 25ml / min is considered to be " fast " bleeding.Exudative bleeding is usually greater than about 1ml / min, because it is noted that low bleeding rate such as 1ml / min will coagulate and stop voluntarily usually, unless the experimenter is receiving anticoagulant therapy or suffers from coagulation cascade disorder due to the reason other than taking anticoagulant drugs.For the experimenter who accepts irreversible anticoagulant drug treatment or suffers from hemorrhagic disease, the exudative bleeding of 1ml / min is still worrying, needs such as by the device being formed by material of the present invention to solve. In some embodiments, the devices described herein are used to address TURP bleeding rates between about 1 ml / min and about 25 ml / min, or between about 1 ml / min and about 20 ml / min, or between about 1 ml / min and about 15 ml / min, or between about 1 ml / min and about 10 ml / min, or between about 1 ml / min and about 5 ml / min.

[0116] In one embodiment, the presently disclosed materials, compositions, and methods are characterized by one or more of the following features: (1) the ability to rapidly control massive bleeding during prostatectomy using a noninvasive approach; (2) the ability to control bleeding in anticoagulated patients; (3) a significant reduction in pain and discomfort for the patient by controlling bleeding without the need for prolonged catheterization; (4) a significant reduction in hospital stays; (5) a significant reduction in medical costs; (6) a significant reduction in morbidity; and (7) results that trend toward reduced mortality.

[0117] As used herein, bladder mucosa is broadly defined to include any exposed tissue surface in the bladder, including any tissue surface exposed by surgery (e.g., surgical procedure). Thus, bladder mucosa includes bladder mucosa naturally present in the bladder, excised bladder mucosa, and excised prostate, among others.

[0118] A TURP delivery device can include any device used in or in conjunction with a TURP procedure.

[0119] Solid chitosan material

[0120] Solid chitosan materials can refer to compositions containing varying amounts of chitosan. The general contents, general chemical composition, and different forms of chitosan dressings are described, for example, in U.S. Patents Nos. 7,820,872, 7,482,503, 7,371,403, 8,313,474, 7,897,832, 9,004,918, 8,920,514, 9,204,957, 8,741,335, 8,269,058, 9,205,170, 10,086,105, and U.S. Patent Applications Nos. 16 / 958,301, 16 / 958,304, 16 / 958,311, 16 / 958,307, and 16 / 958,309. Such solid chitosan materials have been used for hemostasis due to their chemical and physical properties as described above.

[0121] The chitosan used preferably comprises the non-mammalian material poly-β-(1-4)-2-amino-2-deoxy-glucopyranose, also known as poly-β-(1-4)-N-acetyl-D-glucosamine. Chitosan can be processed in a conventional manner from chitin obtained from sources including, but not limited to, fungi, diatoms, and crustaceans such as shrimp. Chitosan can be biocompatible and biodegradable in vivo and can be broken down into glucosamine, a benign substance. Catechol-modified chitosan used herein may include chitosan to which catechol has been added.

[0122] Solid chitosan material can be dry or wet. If the moisture content in the chitosan is less than about 15 wt %, preferably about 10 wt %, more preferably about 5 wt %, then solid chitosan material is " dry ". When the chitosan dressing contacts with a water source, the chitosan dressing is " wet ", and the water source includes the water in the physiological environment and biological fluid, or the water in the aqueous solution. For example, when solid chitosan as described in the present disclosure is mixed in its flowable composition, when contacting with gastrointestinal fluid, urine or blood or the tissue surface (bladder mucosa or GI mucosa) of the gastrointestinal tract or bladder subsequently, solid chitosan material of the present disclosure begins to be wetted. The solid chitosan material that substantially maintains solid form in the flowable dressing absorbs, displaces, redirects or guides the water / moisture in the physiological environment of the gastrointestinal tract or bladder mucosa with the amount that is enough to allow the solid chitosan material to adhere to the tissue surface. The chitosan material of adhesion can be used for sealing wound surface and slowing down or preventing further bleeding.

[0123] In a preferred embodiment, the solid chitosan material is a reduced particulate component of a chitosan gastrointestinal flowable hemostatic dressing (CGHFD) or a chitosan endoluminal hemostatic flowable dressing (CEHFD) formed from the materials described herein. In a preferred embodiment, the solid chitosan material preferably comprises greater than or equal to 25% chitosan by weight; more preferably greater than or equal to 50% chitosan by weight, and most preferably greater than or equal to 75% chitosan by weight. Chitosan is a general term used to describe linear polypeptides composed of glucosamine and N-acetylglucosamine residues linked by β-(1-4) glycosidic linkages (generally the amount of glucosamine is ≥ the amount of N-acetylglucosamine), and the composition of which is soluble in dilute aqueous acid solutions (Roberts 1992

[24] ). The chitosan family encompasses poly-β-(1-4)-N-acetyl-glucosamine and poly-β-(1-4)-ND-glucosamine, where the acetyl residue moiety and its motif modification (random or block) influence chitosan chemistry. The C-2 amino group on the glucosamine ring in chitosan allows protonation, so chitosan is soluble in water (pKa ≈ 6.5) (Roberts 1992

[25] ).

[0124] In preferred embodiments of CGHFD, the solid particulate component of the flowable dressing formed from the material of the present invention is polymeric, biocompatible, tissue-adhesive, and hemostatic.

[0125] In preferred embodiments of CEEFD, the solid particulate component of the flowable dressing formed from the materials of the present disclosure is polymeric, biocompatible, tissue-adhesive, and hemostatic.

[0126] In some CGHFD embodiments, the most common gastroscope channel diameter is 0.28 cm (2.8 mm), and therefore this is the most preferred size for flowable dressing delivery. Alternatively, a more preferred size is 0.32 cm diameter, which is a standard gastroscope channel diameter, but less common than a 0.28 cm channel. Another preferred gastroscope channel diameter size is between 0.45 cm and 0.32 cm, which is a more customized gastroscope channel size and therefore less common than the 0.32 cm or 0.28 cm gastroscope channel diameter sizes.

[0127] In a preferred embodiment of the CEFD, the flowable dressing is delivered to the resected prostate fossa via a balloon catheter having a delivery port located between the distal and proximal balloons. In a preferred embodiment of the delivery catheter, there is an expandable region of the catheter between the proximal and distal balloons that expands to apply pressure on the volume of flowable dressing that has been delivered into the cavity of the resected fossa. The purpose of the distal and proximal balloons is to position and isolate the flowable dressing port over the cavity of the resected fossa and possible damage in the bladder neck so that the delivered volume of flowable dressing will fill the cavity of the resected fossa up to the area of ​​the bladder neck to provide local control of bleeding. In one embodiment, the channel size used for the CEFD is not limited by endoscopic use, but rather by the typical urological catheter size (16-24 Fr. 1 Fr = 0.33 mm) and the number of connecting channels available in the catheter (up to 5, with the main channel used for irrigation of approximately 12 Fr).

[0128] In a preferred embodiment of the CEFD balloon catheter, the delivery catheter has an inner diameter greater than 1.5 mm. Alternatively, a more preferred delivery catheter has an inner diameter greater than 1.8 mm. Alternatively, a most preferred delivery catheter has an inner diameter greater than 2.4 mm.

[0129] The flowable dressings described herein are readily deliverable through an endoscopic channel via a catheter, do not readily dissolve in blood or body fluids (such as gastrointestinal fluid or urine) at about 37°C, preferably within the first 6 hours of administration, more preferably within the first 12 hours of administration, and most preferably within the first 24 hours of administration, and completely degrade and / or dissolve within about 7 days upon contact with gastrointestinal fluid or bladder fluid at about 37°C.

[0130] The flowable dressings described herein do not adhere to the delivery device or clog the delivery catheter, and they do not significantly expand or contract, i.e., they do not increase or decrease in size by more than about 25% at about 37°C in the presence of blood and body fluids (GI fluid or urine or bladder fluid or a mixture thereof).

[0131] In a preferred embodiment, the dressing can be terminally sterilized without affecting its properties. When stored in its packaging under controlled conditions at room temperature of about 21°C to about 25°C, its tissue adhesion properties, mechanical properties, dissolution properties in GI or bladder fluid, swelling properties, and hemostatic properties are stable and do not change significantly over time (e.g., about ≤ 2 years).

[0132] A preferred embodiment of the flowable dressing is formed from a substantially dry chitosan powder composition and a separate (before mixing) carrier fluid immediately before use and mixing. The water content of the dry chitosan powder composition before mixing is about ≤15 wt %, or about ≤8 wt %. The dry chitosan composition is preferably a powder formed by a spray drying process or by pulverizing a solid dry chitosan material. The solid dry chitosan material can include, but is not limited to, coarse particles, sheets, films, membranes, tapes, nets, rods, fiber mats, and fibers.

[0133] In a preferred embodiment of the solid dry chitosan material, the flowable dressing preparation process may include grinding the material resulting from a compression process that reduces the density of the solid chitosan material from about 0.005 g / cm 3 to about 0.05g / cm 3 The initial preferred range was changed to about 0.03 g / cm 3 to about 0.7g / cm 3 The final preferred range is 0.08 g / cm2; however, about 0.08 g / cm2 is also envisioned. 3 to about 1.2g / cm 3 Note that about 1.5g / cm 3 The density is the density of a solid chitosan material without voids. The compression process may include applying a temperature in the range of about 20°C to about 150°C. To avoid significant expansion of the dry compression dressing upon contact with biological fluids, the compression temperature is preferably applied by methods including, but not limited to, convection, conduction, and radiation. The temperature of the compressed chitosan material should preferably be maintained at at least about 80°C for at least about 15 seconds.

[0134] Heating during compression allows the dry chitosan solid material to be plasticized and formed without cracking or tearing the chitosan (non-destructive forming) tools. Pure dry chitosan has a first glass transition temperature (Tg) close to 80°C, which, if processed near 80°C in the case of pure dry chitosan, allows for rapid non-destructive forming of the chitosan and some crystalline annealing of its structure. The Tg can be lowered by adding plasticizers such as water or glycerol to the chitosan, providing similar levels of non-destructive forming at lower temperatures. It is noted here that non-destructive forming of chitosan is possible within a range of 20°C to 150°C. Outside this range, non-destructive forming of chitosan is still possible, but much more difficult. Above 150°C, chitosan begins to thermally degrade, while below 20°C, the addition of plasticizers can lead to an undesirable loss of chitosan crystallinity, which provides resistance to dissolution and degradation processes such as those that occur during sterilization.

[0135] Preferably, compression prevents the dry compressed chitosan solid from significantly expanding upon contact with biological fluids and is performed with a moisture content of the dry compress of about ≤ 15% w / w during compression.Compression can be by double or multiple roller compression and / or uniaxial application between adjacent platens.

[0136] The compression may be against a flat surface.

[0137] Alternatively, compression may be applied against etching, machining, ablation or other types of surface treatments that impart a depleted or enhanced surface texture.The surface texture may be random or in a regularly repeating pattern.

[0138] Preferred embodiments of the biocompatible, biodissolvable, tissue-adherent chitosan flowable dressing are capable of resisting dissolution in gastrointestinal (GI) fluids and blood for at least about 6 hours at about 37° C., are tissue-adherent, and include materials and material structures that promote resistance to rapid dissolution and degradation in the low pH and strong enzymatic digestive fluids of the upper GI tract. This is a significant advantage of the chitosan flowable dressings disclosed herein because the upper GI tract has evolved to rapidly digest most organic substances, including chitosan, cellulose, and starch.

[0139] Preferred embodiments of the biocompatible, biodissolvable, tissue-adherent chitosan flowable dressing are capable of resisting dissolution in bladder fluid and blood for at least about 6 hours at about 37° C., are tissue-adherent, and include materials and material structures that promote resistance to rapid dissolution and degradation in bladder urine. This is a significant advantage of the chitosan dressing disclosed herein.

[0140] The chitosan flowable dressings provided herein can be applied in contact on a tissue surface or by light contact to a mucus surface, such as in the GI or bladder, to interact to promote adhesion to the site of injury, thereby stopping bleeding.

[0141] Production of Chitosan Flowable Dressing Granules

[0142] The solid powder of the chitosan dressing of the present invention can be produced using a variety of comminution methods and processes. Such comminution methods and processes may include, but are not limited to, granulation (forming larger solid forms into coarse particles with a diameter >1 mm), milling (converting particles into powders with a diameter <0.5 mm), grinding (reducing powders to a diameter <0.2 mm), sieving (to select particle size), spray drying (forming small solid particles directly from a liquid without milling or grinding), and chopping (micron and submicron diameter fibers to produce short-cut, low aspect ratio fibers with a length: diameter <200:1). It should be understood that the reduced particle forms of the present disclosure remain solid materials in their dry or wet flowable configurations. In the presence of liquid, the particles can exist as solid and semi-solid materials. The solid particle forms of the present disclosure can expand in volume (<50%), but remain solid in the presence of liquid. These solid materials remain as discrete individual particles that retain their essential solid properties by resisting dissolution and deformation. The volume of the semi-solid particle forms of the present disclosure can expand (≥50%). These semisolid materials remain as discrete individual particles, and they maintain their essential semisolid properties by resisting dissolution while exhibiting expansion deformation in shape. Dry semisolid particles may have an initial appearance of rough, sharp-edged particles that, when exposed to liquid, enlarge and expand to a more rounded, possibly spherical shape.

[0143] A preferred form of milling and grinding is at -40°C or below, such as in the presence of dry ice. A more preferred form of milling and grinding is at temperatures of -180°C or below, such as in the presence of liquid argon or liquid nitrogen. A preferred cryogenically cooled milling system is one that incorporates a screen within the mill that selects particles with a preferred larger particle radius during the milling process and minimizes / eliminates the presence of fine particles (any particles with a radius of gyration less than <10 microns). Alternatively, fine particles generated during the milling process can be removed by sieving the milled powder, but this reduces yield compared to sieving milling processes. Once the flowable dressing is deposited on the lesion, the presence of significant levels of fine particles in the dispersion of flowable dressing particles directly interferes with the ability of blood and its constituent cells to permeate through the interconnected pore structure of the flowable dressing, thereby significantly impairing the flowable dressing's ability to achieve rapid and reliable hemostasis. In one embodiment, the porosity in the flowable dressing is uninterrupted, with interconnected pore sizes ranging from 10 to 100 microns, with a substantial majority of pores approximately 10 to 50 microns. The continuous pore structure in flowable dressings demonstrates their ability to absorb biological fluids such as blood.

[0144] In some embodiments of CGHFD, due to its composition structure and characteristic, chitosan flowable dressing provided herein can be delivered to stop bleeding. Therefore, in some embodiments, chitosan dressing provided herein can be delivered by narrow working channel. Chitosan dressing provided herein can include an exemplary diameter of about 3.2mm or less of the narrow working channel that can be delivered by it, including but not limited to 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1.0mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2.0mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3.0mm, 3.1mm and 3.2mm diameter.

[0145] In some embodiments of CEFD, due to its composition structure and characteristics, the chitosan flowable dressing provided herein can be delivered to stop bleeding. Therefore, in some embodiments, the chitosan dressing provided herein can be delivered to one or more injury sites via a balloon catheter through one or more delivery channels. Exemplary diameters of the delivery channel through which the chitosan flowable dressing provided herein can be delivered include diameters of about 3.2 mm or less, including but not limited to diameters of 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, and 3.2 mm.

[0146] Catechol-modified chitosan; and its production

[0147] The chitosan solid dry chitosan material described herein relates to chitosan comprising catechol-modified chitosan and / or a hydrophilic polymer. Other aspects of the chitosan dressing comprising catechol-modified chitosan will be described in more detail below.

[0148] Preferred embodiments of the CGHFD or CEFFD of the present disclosure include compositions having catechol-modified chitosan and / or optionally other hydrophilic polymers. Preferably, the catechol-modified chitosan in the dressing provides prolonged adhesion to moist tissue, with a tissue adhesion of ≥ about 1 kPa and resistance to dissolution in water, saline solution, blood, and / or GI or bladder fluid for ≥ about 6 hours at about 37°C.

[0149] A preferred embodiment of the present invention is that catechol-modified chitosan can be formed by N-acylation of the C-2 amine on the chitosan glucosamine with 3,4-dihydroxyhydrocinnamic acid (alternatively known as 3-(3,4-dihydroxyphenyl)propionic acid, hydrogenated caffeic acid). Alternatively, N-acylation of chitosan to produce catechol-modified chitosan can include, but is not limited to, modification with one of 3,4-dihydroxycinnamic acid (caffeic acid), trans-3,4-dihydroxycinnamic acid (trans-caffeic acid), and 3,4-dihydroxyphenylacetic acid (DOPAC, plateau catechin).

[0150] A preferred embodiment of the present invention is that catechol-modified chitosan can be formed by N-acylation of the C-2 amine on the chitosan glucosamine and the amine on 3,4-dihydroxyphenylethylamine (or dopamine) with carboxylic acid groups on tricarboxylic acids including citric acid, isocitric acid and aconitic acid.

[0151] A preferred embodiment of the present invention is that catechol-modified chitosan can be formed by reductive alkylation of the C-2 amine on the chitosan glucosamine by adding 3,4-dihydroxybenzaldehyde to the chitosan and reducing the intermediate imminium functional group using sodium cyanoborohydride or sodium borohydride.

[0152] The presence of catechol in the composition provides some polyconjugated structure when catechol is oxidized to o-quinone. This results in a visible difference between unmodified chitosan and catechol-modified chitosan compositions, which can range in color from beige or pink to dark brown and black, respectively. It is noted that the catechol-modified chitosan composition changes from pink to black as oxidation occurs in the catechol.

[0153] By maintaining the pH of the reaction solution at or below 5.5, a pink coloration in the catechol-modified chitosan is provided in aqueous synthesis, indicating a substantial absence of crosslinking. Pink coloration can also be provided in aqueous synthesis by conducting the modification and subsequent processing steps in the substantial absence of oxygen (such as by using an aqueous system purged with an inert gas, which may include, but is not limited to, argon or nitrogen). Although pink coloration is undesirable in the final solution or catechol-modified product, it may be desirable to perform it at an intermediate processing stage (such as immediately after derivatization of chitosan with catechol and / or dialysis and / or washing of the subsequent catechol-chitosan solution to remove residual unreacted material) because it allows for stable dry product polymer storage and dry product weight determination, and then the pure dry catechol-modified product can be substantially redissolved in water at a later time to the desired dry weight. This water-soluble chitosan catechol material (having a brown or darker coloration) is subsequently oxidized and crosslinked.

[0154] In a preferred embodiment, the catechol-modified chitosan is not removed from the solution by an intermediate drying step to allow storage, but is instead kept in an aqueous solution and oxidized in the aqueous solution by exposure to an environment above about pH 5.5 in the presence of atmospheric oxygen. Preferably, pH control is achieved by adjusting the partial pressure of water-soluble carbon dioxide (an increase in partial pressure lowers the pH, while a decrease in partial pressure raises the pH closer to pH 7). An alternative preferred method of controlling pH is by gradually adding a strong acid to lower the pH and a strong base to raise the pH. Examples of strong acids may include, but are not limited to, hydrochloric acid, sulfuric acid, and nitric acid. Examples of strong bases may include, but are not limited to, sodium hydroxide and potassium hydroxide. Subsequent drying of this aqueous, water-soluble, oxidized catechol-modified chitosan results in a preferred level of catechol-chitosan crosslinking that has good resistance to dissolution and degradation in the upper gastrointestinal tract and bladder. The catechol-chitosan solution may be diluted with water or concentrated by removing water. Water may be removed by techniques including, but not limited to, ultrafiltration, reverse dialysis, and centrifugation. The solids fraction of a solution can be determined by taking a sample of known volume from the solution and performing analyses including, but not limited to, gravimetry, Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, refractometry, and pycnometry.

[0155] In preferred embodiments, the catechol-modified chitosan composition is brown to darker in color, resulting from the oxidation of catechol to o-quinone. Quinones are produced by auto-oxidation of catechol hydroxyl groups in the presence of oxygen and at a pH above about 5.5. The Schiff base reaction of the quinone with the chitosan C-2 amine produces crosslinks in the modified chitosan. The color of the catechol-modified chitosan composition is controlled by controlling pH and oxygen exposure during synthesis. Maintaining the pH at or below about pH 5.5 inhibits the production of o-quinone. The dialysis solution is then adjusted within the preferred pH range of 5.8 to 6.2, and the final washed or dialyzed catechol polysaccharide solution provides a more resistant, darker, and more oxidized catechol. In some embodiments, the coloration of the catechol-modified chitosan characterizes one aspect of the catechol-modified chitosan dressing. In some embodiments, the coloration reflects the degree of substitution of the chitosan with catechol. In some embodiments, the coloration, ranging from pink to brown to darker colors, correlates with the degree of substitution.

[0156] In a preferred embodiment, the solid particles of the flowable chitosan dressing of the present invention are formed from an iron-enhanced catechol chitosan composition. The addition of an Fe(III) salt, such as FeCl3, to the catechol-modified synthesis of chitosan at a molar ratio of Fe(III) to catechol-reactive species (such as 3,4-dihydroxyhydrocinnamic acid) of 1:1 to 1:5 results in a significant increase in reaction efficiency, enabling the achievement of a fractional degree of substitution of the catechol-reactive species covalently attached to the chitosan of up to about 0.7.

[0157] To prepare a dry powder for a flowable dressing from catechol chitosan, a preferably light brown to dark brown to black aqueous solution of catechol chitosan is prepared, which can be used alone or mixed with aqueous solutions of other hydrophilic polymers, including but not limited to solutions of chitosan and / or optional hydrophilic polymers. Preferably, the dry phase-separated catechol chitosan particles and the powder for the flowable dressing are prepared from a dense dry structure.

[0158] The preferred cross-linked catechol-modified chitosan compositions of the present invention provide good tissue adhesion and increased resistance to dissolution in the upper gastrointestinal tract or bladder by 10 to 100 times compared to dressings formed essentially of unmodified chitosan. The catechol-modified chitosan compositions described herein provide flowability, longevity, biocompatibility, and the ability to ultimately dissolve.

[0159] By promoting the formation of quaternary ammonium cations at the C-2 amine of chitosan glucosamine in the presence of acid in the dry dressing composition or in the presence of acid naturally present in a biological environment such as the upper gastrointestinal tract, CGHFD or CEFFD formed from the materials of the present invention is provided in a flowable chitosan dressing for preferred rapid adhesion (≤ 1 minute) to the gastrointestinal mucosa or bladder mucosa. Preferred chitosan acid salts in the dressing may include salts of acetic acid, lactic acid, glycolic acid, citric acid, succinic acid, malic acid, hydrochloric acid, glutamic acid, ascorbic acid, malonic acid, glutaric acid, adipic acid, pimelic acid, and tartaric acid, and combinations thereof. Preferably, the weight percent of the acid salt of chitosan is greater than about 2% and less than about 15%. To achieve rapid adhesion to wet tissue (e.g., ≤ 1 minute), the water content of the dry gastrointestinal or bladder solid chitosan particles prior to mixing with the carrier diluent is preferably less than about 15% by weight; more preferably, less than about 10% by weight, and most preferably, less than about 5% by weight.

[0160] In the case where a solidified, frozen phase-separated, and sublimed (to remove ice) chitosan material is used as a precursor solid flake material for preparing a dry powder, the chitosan solution is poured into a frozen phase-separated mold (typically in the shape of a pan with a horizontal flat bottom), preferably having a hydrophilic polymer chitosan solution of about 0.25% w / w, more preferably about 0.5% w / w, and most preferably 1.0% w / w. The hydrophilic polymer solution is preferably added to the horizontal pan to a vertical depth of preferably about 5 mm, more preferably 8 mm, and most preferably 12 mm. The solution in the mold is then frozen and dried to remove water by sublimation or frozen phase substitution (extraction of ice into the polymer with a non-solvent), resulting in a low density (>99% void volume) open or porous dry sponge with a dry density of <about 0.01 g / cm 3 (Or, for example, about 0.005 g / cm for a catechol chitosan uncompressed dressing from a 0.5% solution 3, which is about 1 / 5 or 20% of the density of the uncompressed HemCon bandage chitosan sponge, which has a density of about 0.025 g / cm 3 Lyophilization is typically performed at pressures below 300 mTorr, while freeze-drying involving dry, cold (e.g., <-20°C) solvents such as ethanol is performed at atmospheric pressure. Dry sponges can be compressed to greater than about 0.4 g / cm 3 The compression may include, but is not limited to, uniaxial compression between aligned flat platens, wherein the platens are heated to between 18° C. and 150° C., and a pressure load of up to 10,000 bar.

[0161] The compression produces thin (eg, ranging from about ≤ 100 microns to about ≤ 500 microns), strong (eg, 5 MPa to 25 MPa UTS) chitosan flakes that can be ground and / or milled to form the final dried chitosan powder component of the flowable dressing.

[0162] Frozen phase separation of a dilute aqueous polymer solution results in the phase separation of micron- and submicron-thin polymeric chitosan flakes, which are regularly interspersed between ice flakes approximately 200 microns in width. Ice is removed by sublimation (freeze drying) or, alternatively, by solvent extraction, leaving a dry sponge composed of nearly aligned thin (≤1 micron) polymeric chitosan flakes. Compressing the polymeric chitosan flakes at temperatures near or above their glass transition temperature (Tg of dry chitosan is approximately 80°C) compresses them into a thin (approximately 100 micron) dense polymeric structure formed from hundreds of layers of strong, compliant polymeric chitosan leaves (lamellae) that are not susceptible to crack propagation and can be repeatedly folded without failure. This multi-lobed stacking achieves remarkable strength. Prior to the present invention, frozen phase-separated chitosan sheets had not been previously investigated for manufacture and use as described herein and are intended to address the key issues addressed by the present invention, such as the ability to easily deliver flowable materials for bleeding control by removing adhesion that interferes with fluid flow (by absorbing, channeling, displacing, and / or redirecting).

[0163] To prepare a dressing from two sheets (wherein the dressing has a catechol chitosan tissue adhesive surface layer and an unmodified chitosan surface layer), the two sheets (one catechol modified chitosan and the other chitosan acid salt) are bonded together by, for example, placing one sheet on top of the other and applying sufficient uniform pressure on the dressing to compress them to a higher density. In a preferred process, the density is ≤ about 0.03 g / cm 3 The original density of each sheet is increased to ≥ about 0.30 g / cm 3 In a more preferred process, the final dressing density is ≤ about 0.015 g / cm3 The original density of each sheet is increased to ≥ about 0.4 g / cm 3 In the most preferred process, the final dressing density is ≤ about 0.01 g / cm 3 The original density of each sheet is increased to ≥ about 0.5 g / cm 3 At the end of compression, the two compressed sheets are bonded together so that one cannot be easily peeled off from the other and the dressing can be manipulated by folding and rolling it up without any separation.

[0164] Physical bonding of materials by compressing two or more low density porous materials together to form a final two or more layers of higher density porous material solves the problem of how to bond such materials together without changing the physical or chemical properties of the individual materials and without adding additional binders or adhesives. The bonding is expected to be attributable to micro-surface collisions and penetration of the dressing through its pores, with physical interlocking due to pore compression. The physical interlocking of such low density, frozen phase separated dry sheets is not limited to two materials of the same thickness or only two layers, as the interlocking effect is neither dependent on sidedness nor thickness. Thus, multi-layer sheet structures of separate frozen phase separated and dried sheets of the same or different materials of the same or different thicknesses can be obtained by combining a low density sheet (preferably having a density of ≤ 0.05 g / cm 3 density) and compress the components together to ≥ 0.3 g / cm 3 Such a final physically bonded assembly would be expected to provide the advantages of thin top and bottom surface layers, including but not limited to adhesive or anti-adhesive materials, wherein the inner layers include but are not limited to structural, physical and chemical essential parts. Preferred powders of the flowable hemostat of the present invention can be formed by grinding and / or milling the multilayer sheet.

[0165] In one mechanism, the flowable chitosan dressings provided herein can stop bleeding by absorbing, directing, and / or redirecting hydrophilic and hydrophobic fluids at the site of injury. The absorption removes sufficient moisture from the site of injury to allow a subsequent hemostatic reaction to occur between the chitosan dressing and the tissue at the site of injury, which in turn stops bleeding and allows the chitosan dressing to remain attached, thereby sealing the site of injury. The porous, dense, and multilayered structure of the flowable chitosan dressing particles provided herein facilitates the absorption, directing, and / or redirecting of moisture at the site of injury, as well as the attachment or adhesion of the flowable chitosan dressing to the site of injury.

[0166] The chitosan dressings disclosed herein are biocompatible. In some embodiments, dissolved residues from a chitosan dressing applied to an injured site in the body safely pass through the digestive or urinary tract and are excreted along with other bodily wastes.

[0167] More than one or more flowable chitosan dressings may be used, or applied in a continuous manner to a tissue treatment site or injury site. When more than one chitosan dressing is used, such dressings may be adhered to adjacent tissue sites or injury sites, or may overlap to varying degrees. Due to the economical use of materials for the flowable chitosan dressings described herein, it is contemplated that multiple chitosan flowable dressings may be used as needed to promote or achieve hemostasis at an injury site, depending on the application.

[0168] In one embodiment, the chitosan flowable dressings overlap each other when applied. In this case, it is desirable that the moistened adhesive side of the subsequent dressing has some adhesion to the moistened top dressing surface of the previous dressing. Thus, the chitosan flowable dressing has a top surface that provides sufficient adhesion for the placement of a subsequent overlapping chitosan flowable dressing.

[0169] Delivery device

[0170] The delivery device used herein is a device for delivering chitosan dressings. A delivery device delivers flowable chitosan dressings to injured sites at different locations in the body of an animal (including but not limited to humans, pigs, dogs, etc.).

[0171] In some embodiments, the delivery device is a minimally invasive device that can deliver a dressing (e.g., a chitosan flowable dressing) to a physiological site in an animal's body in a non-invasive or minimally invasive manner. In some embodiments, the delivery device is a catheter. In some embodiments, the non-invasive or minimally invasive feature of the delivery device is achieved by delivering a flowable dressing, such as a chitosan flowable dressing, through a narrow catheter or equivalent working channel. In some embodiments of CGHFD, the catheter or equivalent working channel has a diameter of less than 3.2 mm. In other embodiments of CGHFD, the diameter size of the gastroscope channel can be in the range of 2.8 mm to 4.5 mm. In some embodiments of CEFD, the balloon catheter or equivalent working channel has a diameter of less than about 7 mm. In other CEFD embodiments, the diameter size of the channel of the TURP delivery device can be in the range of 0.5 mm to 4.0 mm.

[0172] In some CGHFD embodiments, the catheter or balloon catheter or equivalent working channel has a diameter of less than 3.2 mm. In other CGHFD embodiments, the diameter size of the gastroscope channel may range from 2.8 mm to 4.5 mm.

[0173] In some CEEHFD embodiments, the catheter or balloon catheter or equivalent working channel has a diameter of less than about 7 mm. In other embodiments, the diameter size of the channel of the TURP delivery device can range from 0.5 mm to 4.0 mm.

[0174] Exemplary delivery devices include, but are not limited to, balloon devices, balloon catheters, indwelling catheters, urethral or suprapubic catheters, external catheters, short-term catheters, and intermittent catheters.

[0175] The delivery device can also be an endoscopic device used in various aspects of medical procedures.In some embodiments, the endoscopic device is non-invasive or minimally invasive due to the narrow catheter or tube / pipe or similar narrow diameter portion of the device.

[0176] The delivery device may also be a transluminal or transurethral delivery device.In some embodiments, the transurethral delivery device is non-invasive or minimally invasive due to the narrow catheter or tube / conduit or similar narrow diameter portion of the device.

[0177] Delivery devices include other devices having narrow diameter tubes, channels or conduits or similar structures.

[0178] The flowable dressing chitosan material described herein can be accurately delivered to a distant injury site in the form of a final fluidized dispersion, for example, by catheter delivery, thereby adhering to the tissue of the distant site independently of gravity to quickly achieve bleeding control, close the injury site and remain in situ to resist dissolution for more than 6 hours. The flowable dressing chitosan material of the present disclosure adheres to mucosal tissue and tissue injury site upon contact and lasts for more than 6 hours. The flowable chitosan material of the present disclosure can be applied upside down under normal gravity to adhere to the injury site applied endoscopically without losing coverage or flowing away from its application site. After being applied to the injury site for 6 hours or more, the top of the applied flowable dressing chitosan material may be eroded or biodegraded, but the flowable dressing material closest to the injury remains adhered for at least 12 hours in the form of a thin, uniform layer covering the injury site to protect the injury site and reduce the chance of rebleeding. The flowable dressings of the present disclosure can be used with minimally invasive techniques for remote dressing delivery to rapidly deploy dressings to achieve hemostasis, fill and seal resections, biopsy sites, narrow recesses, and defects and openings around hemostatic clips, non-metallic sutures, clamps, staplers, metallic sutures, and fixation pins.

[0179] Applications and treatment methods

[0180] Chitosan flowable dressing embodiments formed from the materials of the present invention provided in this disclosure can be used to stop bleeding in suitable diseases, illnesses, conditions, or emergency trauma or injuries. In some embodiments, the dry solid chitosan materials of the present invention can be used to stop bleeding from any wet physiological surface, such as mucus. Exemplary applications include, but are not limited to, gastrointestinal or bladder bleeding, other intracavitary applications, including vascular applications, internal surgical bleeding, internal biopsy bleeding, internal bleeding after solid organ resection, and oral, eye, ear, or nasal bleeding. Other applications that may require the addition of water or fluid to promote adhesion of the chitosan dressing to a tissue surface or site of injury are also envisioned, for example, the use of the chitosan dressing on an external surface.

[0181] The chitosan flowable material of the present invention can be used to treat gastrointestinal bleeding, including but not limited to treating esophageal variceal bleeding, peptic ulcer bleeding, duodenal ulcer bleeding, bleeding associated with upper and lower gastrointestinal biopsies, upper and lower gastrointestinal resections, and upper and lower gastrointestinal tract tears or ruptures. Other diseases, illnesses, conditions, or acute trauma or injuries may include but are not limited to internal arterial injury; intrahepatic bleeding, intracaval bleeding; thoracic injury, including perforation of the heart, lungs, and their blood vessels; and abdominal injury.

[0182] The chitosan flowable material of the present invention may also be used for the treatment of transurethral prostatectomy and / or bladder neck bleeding, which may include but is not limited to the treatment of bleeding.

[0183] The chitosan material of the present invention can also be used after acute internal injuries (such as occur in UGIB, TURP or other minimally invasive or open surgery) to protect the injured site by sealing the injured site and providing an environment conducive to cell regeneration before dissolving or degrading within 7 days.

[0184] In a preferred embodiment, the flowable dressing includes an optical contrast material that provides enhanced endoscopic visualization of the deployed flowable dressing for improved wound placement and post-placement observation of the wound and dressing. The material can be easily mixed with the composition of the flowable dressing and delivered with the flowable dressing to address bleeding issues and provide enhanced visualization of the dressing edge, the dressing body, and successful hemostasis. While the flowable dressing provides hemostasis, the enhanced visibility material remains incorporated and uniformly present in the flowable dressing composition without any significant leakage of material into the biological environment. The enhanced visualization material may include, but is not limited to, fluorescent agents, quantum dots, nanoparticles containing fluorescent agents, chitosan covalently modified with fluorescent agents, gold nanoparticles, organically modified dye-doped silica, upconversion phosphors, and lanthanide-based contrast agents.

[0185] In a preferred embodiment, once the flowable dressing has been deployed over the bleeding site, hemostasis of the flowable dressing can be enhanced by endoscopically placing a supportive solid mesh or dressing over the flowable dressing. In cases of intense arterial bleeding (which may prove too high a pressure for the cohesive strength of the flowable dressing to immediately control the bleeding due to the arterial bleeding pressure tunneling through or otherwise penetrating the flowable dressing), a gentle packing pressure is then applied briefly by a balloon or similar endoscopically applied basket device through the intermediate dressing or mesh material over the flowable dressing. Applying the support dressing or mesh with gentle pressure seals any tunnels or other penetrations within the flowable dressing and provides prolonged hemostasis in difficult bleeding situations.

[0186] The chitosan flowable materials of the present invention can also be used for delivery and local adhesion to specific target sites for general therapeutic purposes, including the delivery of active pharmaceutical agents and / or biologics. Such target sites include, but are not limited to, anastomosis, esophageal varices, peptic ulcers, excised prostate fossa, resection and biopsy of the liver, resection and biopsy of the kidney, resection and biopsy of the bladder, resection and biopsy of the pharynx, resection and biopsy of the pancreas, resection and biopsy of the stomach, resection and biopsy of the lower gastrointestinal tract, resection and biopsy of the lung, and resection and biopsy of the heart.

[0187] The resistant, tissue-adherent, flowable hemostatic dressing of the present invention can be applied to an injury site to remain in place, control bleeding, and close openings such as fistulas by promoting tissue growth over the opening.

[0188] The anti-lytic, tissue-adherent, flowable hemostatic dressing of the present invention can be used for transarterial embolization therapy by intravascular delivery to achieve local occlusion of blood vessels. Transarterial embolization therapy is a local treatment that blocks blood flow to a tumor to slow and / or eliminate its growth.

[0189] The various embodiments described above can be combined to provide additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited in this specification and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments may be modified if necessary to provide further embodiments using concepts from various patents, applications, and publications.

[0190] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments and the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure. DETAILED DESCRIPTION

[0191] Examples 1-12 relate to chitosan intraluminal hemostatic flowable dressing (CEHFD) and chitosan gastrointestinal hemostatic flowable dressing (CGHFD) devices for flowable, hemostatic, anti-lytic, tissue-adherent, and durably tissue-adherent applications.

[0192] The following materials and formulations are contemplated in the inventive methods of the chitosan intraluminal hemostatic flowable dressing and the chitosan gastrointestinal hemostatic flowable dressing.

[0193] Chitosan A: Primex CHITOCLEAR 65010, TM 4375, MW = 250-300 kDa, Brookfield

[0194] Chitosan B: Primex CHITOCLEAR 43000,™ 4167, MW = 110-150 kDa, Brookfield viscosity of 1.0% w / w chitosan solution in 1.0% acetic acid at 25°C, spindle LV1 = 9 cPs, DDA = 95% (by colloid titration).

[0195] Glacial acetic acid: Fisher Scientific, cat. no. A38-212.

[0196] Hydrochloric acid: 1.0 M in water, Sigma Aldrich, catalog number H9892.

[0197] L-lactic acid: JT Baker, cat. no. 0196-01.

[0198] Glycolic acid: JT Baker, Cat. No. M821-05.

[0199] Sodium hydroxide: 5.0 M sodium hydroxide in water, Sigma Aldrich, catalog number S8263-150 ml.

[0200] Potassium hydroxide: 0.1 M potassium hydroxide (BDH) in methanol.

[0201] Ethanol: 200° Proof, Sigma Aldrich, catalog number 459844-1L.

[0202] Acetic anhydride: ACS reagent grade obtained from Sigman Aldrich, catalog number 320102-1L.

[0203] Sodium cyanoborohydride: Sigma Aldrich catalog number 8180530250

[0204] Sodium borohydride: Sigma Aldrich catalog number 452882-500G

[0205] N,N-Dimethylformamide: Sigma Aldrich catalog number 227056-2L

[0206] Deionized water: Sigma Aldrich catalog number 8483339010

[0207] 3,4-Dihydroxyhydrocinnamic acid (hydrocaffeic acid): 98% Sigma Aldrich, catalog number 102601.

[0208] 3,4-Dihydroxycinnamic acid (caffeic acid): 98% Sigma Aldrich Cat. No. C0625

[0209] Trans-3,4-dihydroxycinnamic acid (trans-caffeic acid): Sigma Aldrich catalog number 51868

[0210] 3,4-Dihydroxyphenylacetic acid (DOPAC, plateau catechin): 98% Sigma Aldrich Cat. No. 850217

[0211] 3,4-Dihydroxybenzaldehyde: Sigma Aldrich catalog number 8204750100

[0212] 3,4-Dihydroxyphenylethylamine (DOPA, dopamine): Sigma Aldrich catalog number H8502-100G

[0213] 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide: (or N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, often abbreviated as EDC) Sigma Aldrich Cat. No. E7750

[0214] N-Hydroxysuccinimide (NHS): Sigma Aldrich Cat. No. 8045180100

[0215] Citric acid: Sigma Aldrich, catalog number C0759-1KG

[0216] Sodium chloride, Sigma Aldrich, catalog number 793566-500g.

[0217] Poloxamer 407: Spectrum, catalog number P1166.

[0218] Synthetic urine formulation: Calcium chloride dihydrate (Sigma catalog number C5080) (1.30 g), magnesium chloride hexahydrate (Sigma catalog number M2670) (1.30 g), sodium chloride (Sigma catalog number 793566) (9.20 g), sodium sulfate (Aldrich catalog number 238597) (4.60 g), sodium citrate dihydrate (Sigma catalog number S4641) (1.30 g), sodium oxalate (Sigma catalog number 71800) (0.04 g), potassium dihydrogen orthophosphate (Sigma catalog number P5655) (5.60 g), potassium chloride (Fisher catalog number BP366) (3.20 g), ammonium chloride (Sigma catalog number 09718) (2.00 g), urea (Sigma catalog number U1250) (50.00 g), and creatinine (Acros catalog number 228940500) (2.20 g) were added to a 2.0 L volumetric flask and dissolved in 1.5 L of deionized water. Each component was dissolved in water and the solution was allowed to equilibrate to room temperature, and then the volume was increased to 2.0 L with deionized water.

[0219] Porcine bladder with urethra, Animal Biotech Industries Inc., (Danboro, PA 18916)

[0220] Citrated bovine whole blood: Lampire Biological Laboratory Bovine CPD, catalog number 7720010.

[0221] Cyanoacrylate A: Permabond 910 Tissue Adhesive, catalog number 72590.

[0222] Cyanoacrylate B: Loctite 4902 instant adhesive, catalog number 1875841

[0223] Dialysis tubing: 3,500 Da MWCO Snakeskin dialysis tubing (Fisher Scientific), catalog number PI88244.

[0224] Sealing film: “M” laboratory film, Pechiney Plastic Packaging (Chicago, IL 60631)

[0225] FeCl3: Sigma Aldrich anhydrous grade Cat. No. 8039450500 500g

[0226] Gelatin: Sigma, porcine gelatin Bloom type 300A, catalog number G2500

[0227] FLOSEAL Hemostatic Matrix 5ml, Baxter Product No. ADS201844, Lot No. (10)HA220132, EXP 11 / 29 / 23

[0228] HEMOSPRAY powder, Cook Medical, PN G56572, Batch W4337018, EXP 04 / 02 / 23

[0229] HEMOSPRAY powder, Cook Medical, PN G56572, Batch W4529951, EXP 10 / 26 / 24

[0230] Quartz UV test cell, 1 cm path length: HACH Co., catalog number 48228-00 Example 1

[0231] Preparation and Characterization of Catechol Chitosan

[0232] This article describes methods 1 and 2 for the preferred cross-linking of chitosan gelatin materials (for use as additives) in terms of chemical formulation and preparation, as well as methods 3 to 8 for the synthesis of catechol-modified chitosan materials by a preferred N-acylation reaction between catechol molecules containing terminal carboxylic acids and the C-2 amine of the glucosamine polymers of chitosan.

[0233] Although not presented here in an exemplified form, the present disclosure also encompasses the synthesis of a preferred catechol-modified chitosan, which is the N-acylation of the C-2 amine on the chitosan glucosamine and the amine on 3,4-dihydroxyphenylethylamine (or dopamine) with a carboxylic acid group on a tricarboxylic acid, including citric acid, isocitric acid, and aconitic acid.

[0234] Although not presented herein as an example, the present disclosure also encompasses the synthesis of a preferred catechol-modified chitosan by reductive alkylation of the C-2 amine on the chitosan glucosamine by addition of 3,4-dihydroxybenzaldehyde to chitosan and reduction of the intermediate iminium cation functionality using sodium cyanoborohydride or sodium borohydride.

[0235] Examples of N-acylated catechol-modified chitosans are provided below in Methods 3 to 8. Following covalent chemical attachment (modification) of catechol at the chitosan C-2 amine, the degree of substitution of the chitosans of Methods 3 to 8 was determined as follows:

[0236] Quartz UV test cells, 1 cm path length, x2 were used to obtain UV / Vis spectra at room temperature. The UV / Vis spectrophotometer was a Varian Cary Bio 100.

[0237] A standard solution of 3,4-dihydroxyhydrocinnamic acid was prepared in water and the absorbance at 280 nm was plotted against the concentration. The extinction coefficient (ε) was determined to provide quantitative use of the absorbance in dilute solution in the Beer Lambert relation (shown below).

[0238] A=ε·c·l

[0239] A is the absorbance (dimensionless), l / cm is the path length, ε / L.mol -1 .cm -1 is the extinction coefficient.

[0240] The extinction coefficient of aromatic catechol at peak absorbance at 280 nm (absorbance < 0.5) was determined to be 2540 ± 50 liters / (mol.cm). This value was used to determine the degree of substitution of modified chitosan in dilute aqueous solutions with known masses of modified chitosan, known volumes of solution, and peak absorbance measured at 280 nm.

[0241] The chitosan-catechol solution is diluted to an absorbance of less than 0.5 at 280 nm (typically about 1:50 or 1:100). The absorbance, the weight of the solution used in the dilution, and the percent solids (CS-catechol) are used to calculate the fractional substitution (f) of HCA relative to the free amines on the chitosan backbone according to the following equation: DS ):

[0242]

[0243] Where A is the UV / visible absorbance of the modified chitosan at 280 nm; V is the volume (liters) of the modified chitosan solution dried to a constant dry mass; m CC is the measured dry mass of catechol-modified chitosan (g); f DDA is the fractional deacetylation degree of chitosan.

[0244] Examples of two cross-linked chitosan gelatin materials (without catechol modification) and five catechol-modified chitosan materials are provided in the eight methods provided below.

[0245] Method 1

[0246] Chitosan-gelatin cross-linked matrix 1:4 chitosan to gelatin (CsGelatin1):

[0247] Prepare a 25% gelatin solution (Sigma, porcine gelatin Bloom 300A) in DI water. For example, add 16 g of gelatin to 48 mL of DI water and stir to dissolve.

[0248] Prepare a 2% chitosan solution (Chitosan 43000 Primex) in 2% acetic acid (glacial acetic acid), with the mass of chitosan equal to 1 / 4 the mass of gelatin (a final chitosan to gelatin ratio of 1:4). The chitosan solids percentage is indicated. For example, add 4.4 grams of chitosan to 191 mL of DI water and 3.8 mL of acetic acid. Stir to dissolve.

[0249] The chitosan solution was slowly added to the gelatin solution while stirring.

[0250] The solution was heated at 40-50 °C for 0.5-2 hours with occasional stirring.

[0251] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added with stirring to a final concentration of 30 mM.

[0252] The gel was spread in 75-100 g portions in 150 mm Petri dishes and left outside to dry until hard.

[0253] Alternatively, the gel was spread into Petri dishes at 50-75 g per dish and lyophilized for 48 hours.

[0254] Once the gel is dry, it is cryogenically ground into a powder using a cryogenic hammer mill such as a SPEX 6775 mill. Prior to grinding, the membrane is cut into smaller pieces. The membrane material and its container are pre-cooled under liquid nitrogen for approximately 15 minutes. The SPEX grinder run time is approximately 2.0-1.5 minutes per cycle, with a cool-down time of 1 minute, 4 cycles, and a hammer cycle rate of 6.0-5.0 Hz. After grinding, the powder is sieved to remove particles with a gyration radius of <12.5 microns. The final dry powder (% moisture content ≤15% w / w) is stored in a sealed container in a cool, dry place.

[0255] Method 2

[0256] Chitosan-gelatin cross-linked matrix 1:24 chitosan to gelatin (CsGelatin2)

[0257] Prepare a 12% gelatin solution (Sigma, porcine gelatin Bloom 300A) in DI water. For example, add 24 g of gelatin to 176 mL of DI water and stir to dissolve.

[0258] Prepare a 2% chitosan solution (Chitosan 43000 PRIMEX) in 2% acetic acid (glacial acetic acid). The chitosan mass is 1 / 24 of the gelatin mass (the final chitosan to gelatin ratio is 1:24), and the volume of the chitosan solution is 1 / 4 of the volume of the gelatin solution (the volume ratio of the chitosan solution to the gelatin solution is 1:4). The percentage of solids of the chitosan is indicated. For example, add 1.11 grams of chitosan to 50 mL of DI water and 0.95 mL of acetic acid. Stir to dissolve.

[0259] The chitosan solution was slowly added to the gelatin solution while stirring.

[0260] The solution was heated at 40-50 °C for 0.5-2 hours with occasional stirring.

[0261] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added with stirring to a final concentration of 30 mM.

[0262] The gel was spread in 75-100 g portions in 150 mm Petri dishes and left outside to dry until hard.

[0263] Once the gel is dry, it is cryogenically ground into a powder using a low-temperature hammer mill such as a SPEX 6775 mill. Prior to grinding, the membrane is cut into smaller pieces. The membrane material and its container are pre-cooled under liquid nitrogen for approximately 15 minutes. The SPEX grinder run time is approximately 2.0-1.5 minutes per cycle, with a cool-down time of 1 minute, 4 cycles, and a hammer cycle rate of 6 Hz. After grinding, the powder is sieved to remove particles with a gyration radius of <12.5 microns. The final dry powder (moisture % ≤ 15% w / w) is stored in a sealed container in a cool, dry place.

[0264] Method 3

[0265] Z-type catechol-modified chitosan

[0266] In one embodiment, the method comprises preparing a 1% w / w chitosan aqueous solution at pH 5.5, adding Fe(III) salt (in molar equivalents to catechol in the following steps), and then synthesizing chitosan with chitosan, iron, and catechol in the aqueous reaction solution to achieve a 0.5% w / w chitosan solution. The pH of the reaction solution is maintained between 5.3 and 5.7, with a target pH of 5.5, and then raised while dialyzing against DI water to a target pH of 6.0-6.3 to provide for oxidation and crosslinking of the catechol. The fractional substitution (f) of the Z-type catechol-modified chitosan is shown in FIG. DS ) is in the range of 0.50 to 0.90.

[0267] The Z-type powder was prepared as follows. The solidification method of the Z-type catechol-modified chitosan solid film was to freeze the catechol-modified chitosan solution at -40°C for phase separation, and then remove the water by sublimation until the moisture content of the final freeze-dried film was less than 5% w / w. The final freeze-dried film was close to 0.005 g / cm 3 The final membrane is cryogenically ground to a powder using a cryogenic hammer mill such as a SPEX6775 mill. Prior to grinding, the membrane is cut into smaller pieces. The membrane material and its container are pre-cooled under liquid nitrogen for approximately 15 minutes. The SPEX grinder run time is approximately 2.0-1.5 minutes per cycle, with a cool-down time of 1 minute, 4 cycles, and a hammer cycle rate of 6.0-5.0 Hz. After grinding, the powder is sieved to remove particles with a gyration radius of <12.5 microns. The final dry powder (% moisture ≤ 15% w / w) is stored in a cool, dry, sealed container.

[0268] Method 4

[0269] Y-type catechol-modified chitosan

[0270] In one embodiment, the method comprises preparing a 1% w / w chitosan aqueous solution at pH 5.5 and synthesizing chitosan with chitosan and catechol in the reaction aqueous solution to a chitosan solution of 0.5% w / w. Iron salt is then added in a molar amount of 1 / 5 of the molar amount of catechol. The pH of the reaction solution is maintained between 5.3 and 5.7 with a target pH of 5.5, which is then raised while dialyzing against DI water to a target pH of 6.0-6.3 to provide for oxidation and crosslinking of the catechol. The fractional degree of substitution (f) of the Y-type catechol-modified chitosan is determined by HPLC. DS ) is in the range of 0.30-0.70.

[0271] The Y-type powder is prepared as follows. In a preferred embodiment, the method for solidifying the Y-type catechol-modified chitosan solid film from solution is to freeze-phase separate the catechol-modified chitosan solution at approximately -40°C, followed by sublimation to remove water to a final freeze-dried film with a moisture content of less than 5% w / w. The final freeze-dried film has a moisture content of approximately 0.005 g / cm 3 The final membrane is cryogenically ground to a powder using a cryogenic hammer mill such as a SPEX 6775 mill. Prior to grinding, the membrane is cut into smaller pieces. The membrane material and its container are pre-cooled under liquid nitrogen for approximately 15 minutes. The SPEX grinder run time is approximately 2.0-1.5 minutes per cycle, with a cool-down time of 1 minute, 4 cycles, and a hammer cycle rate of 6.0-5.0 Hz. After grinding, the powder is sieved to remove particles with a gyration radius of <12.5 microns and >125 microns. The final dry powder (% moisture ≤ 15% w / w) is stored in a cool, dry, sealed container.

[0272] Method 5

[0273] Y1-type catechol-modified chitosan

[0274] In one embodiment, the method comprises preparing a 1% w / w chitosan aqueous solution at pH 5.5 and synthesizing chitosan with catechol in the reaction aqueous solution to a chitosan solution of 0.5% w / w. Iron salt is then added in a molar amount of 1 / 5 of the molar amount of catechol. The pH of the reaction solution is maintained between 5.3 and 5.7 with a target pH of 5.5, which is then raised while being dialyzed against DI water to a higher target pH of 6.0-6.3 to provide for oxidation and crosslinking of the catechol. The fractional degree of substitution (f) of the Y-type catechol-modified chitosan is shown in FIG. DS ) is in the range of 0.30-0.70.

[0275] The Y1 type powder is prepared as follows. In a preferred embodiment, the method for solidifying the Y type catechol-modified chitosan solid film from solution is to freeze-phase separate the catechol-modified chitosan solution at approximately -40°C, followed by sublimation to remove water to a final freeze-dried film with a moisture content of less than 5% w / w. The final freeze-dried film has a moisture content of approximately 0.005 g / cm 3 The final membrane is cryogenically ground to a powder using a cryogenic hammer mill such as a SPEX 6775 mill. Prior to grinding, the membrane is cut into smaller pieces. The membrane material and its container are pre-cooled under liquid nitrogen for approximately 15 minutes. The SPEX grinder run time is approximately 2.0-1.5 minutes per cycle, with a cool-down time of 1 minute, 4 cycles, and a hammer cycle rate of 6.0-5.0 Hz. After grinding, the powder is sieved to remove particles with a gyration radius of <31.5 microns and >125 microns. The final dry powder (% moisture ≤15% w / w) is stored in a cool, dry, sealed container.

[0276] Method 6

[0277] Y2-type catechol-modified chitosan

[0278] In one embodiment, the method comprises preparing a 2% w / w chitosan aqueous solution at pH 5.5 and synthesizing chitosan with chitosan and catechol in the reaction aqueous solution to a chitosan solution of 1% w / w. Iron salt is then added in a molar amount of 1 / 5 of the molar amount of catechol. The pH of the reaction solution is maintained between 5.3 and 5.7 with a target value of 5.5, which is then raised by dialysis (x4) against DI water to a higher target pH of 6.0-6.3 to provide oxidation and cross-linking of the catechol. The fractional degree of substitution (f) of the Y2-type catechol-modified chitosan is shown in FIG. DS) was determined to be in the range of 0.30-0.70.

[0279] The Y2 powder is prepared as follows. In a preferred embodiment, the method for solidifying the Y2 catechol-modified chitosan solid film from solution is to freeze-phase separate the catechol-modified chitosan solution at approximately -40°C, followed by sublimation to remove water to a final freeze-dried film with a moisture content of less than 5% w / w. The final freeze-dried film has a moisture content of approximately 0.01 g / cm 3 The final membrane is cryogenically ground into a powder using a cryogenic hammer mill such as a universal micro-pulverizer hammer mill. Prior to grinding, the membrane is cut into smaller pieces. The membrane material and its container are pre-cooled under liquid nitrogen for nearly 20 minutes. The micro-pulverizer is operated at a speed greater than 1200 rpm under liquid nitrogen and has an extraction sieve in an appropriate position. After grinding, the powder is further sieved to remove particles with a gyration radius of <12.5 microns and >125 microns. The final dry powder (moisture content % ≤15% w / w) is stored in a cool, dry, sealed container.

[0280] Method 7

[0281] Y3-type catechol-modified chitosan

[0282] In one embodiment, the method comprises preparing a 2% w / w chitosan aqueous solution at pH 5.5 and synthesizing chitosan with chitosan and catechol in the reaction aqueous solution to a chitosan solution of 1% w / w. Iron salt is then added in a molar amount of 1 / 5 of the molar amount of catechol. The pH of the reaction solution is maintained between 5.3 and 5.7 with a target value of 5.5, which is then raised by dialysis (x4) against DI water to a higher target pH of 6.0-6.3 to provide oxidation and cross-linking of the catechol. The fractional degree of substitution (f) of the Y2-type catechol-modified chitosan is shown in FIG. DS ) was determined to be in the range of 0.30-0.70.

[0283] The Y3 powder is prepared as follows. In a preferred embodiment, the method for solidifying the Y3 catechol-modified chitosan solid film from solution is to freeze-phase separate the catechol-modified chitosan solution at approximately -40°C, followed by sublimation to remove water to a final freeze-dried film with a moisture content of less than 5% w / w. The final freeze-dried film has a moisture content of approximately 0.01 g / cm 3The final membrane is cryogenically ground to a powder using a cryogenic hammer mill such as a SPEX 6775 mill. Prior to grinding, the membrane is cut into smaller pieces. The membrane material and its container are pre-cooled under liquid nitrogen for approximately 15 minutes. The SPEX grinder run time is approximately 2.0-1.5 minutes per cycle, with a cool-down time of 1 minute, 4 cycles, and a hammer cycle rate of 6.0-5.0 Hz. After grinding, the powder is sieved to remove particles with a gyration radius of <31.5 microns and >125 microns. The final dry powder (% moisture ≤15% w / w) is stored in a cool, dry, sealed container.

[0284] Method 8

[0285] Catechol-modified chitosan backed by chitosan acetate

[0286] In one embodiment, the method comprises preparing a first aqueous solution of 0.5% w / w catechol-modified chitosan according to methods 3 to 5 (Type Z, Y, or Y2). A second aqueous solution of 0.5% w / w chitosan acetate is prepared by dissolving chitosan powder in an aqueous solution of acetic acid while maintaining the solution pH below 4 and stirring.

[0287] A combination of catechol-modified chitosan and chitosan acetate powders was prepared as follows. In a preferred embodiment, the solidification method for the catechol-modified chitosan and chitosan acetate solid films is through two-layer frozen phase separation. A transparent, flat-bottomed polystyrene Petri dish with a diameter of 150 mm and a wall height of 15 mm was used to contain the combined solution. The catechol-modified solution was first poured to fill the mold to a height of approximately 4 mm. The mold and its solution were then placed on a freezing plate at approximately -40°C to freeze the aqueous solution. A 0.5% w / w solution of chitosan acetate was then poured, and an additional 2 mm of solution was added on top of the frozen catechol-modified chitosan. The mold and its contents were then placed on a heat transfer rack in a freeze dryer at approximately -40°C to freeze the two layers by inducing frozen phase separation of the solute components. The freeze-drying cycle was completed to achieve sublimation removal of water until the final freeze-dried film had a moisture content of less than 5% w / w. The final freeze-dried film had a moisture content of approximately 0.005 g / cm 3The freeze-dried film comprises a base layer of catechol-modified chitosan and a top layer of chitosan acetate, wherein the weight ratio of catechol-modified chitosan to chitosan acetate is close to 2:1. The final film is cryogenically ground into a powder using a cryogenic hammer mill such as a SPEX 6775 mill. Prior to grinding, the film is cut into smaller pieces. The film material and its container are pre-cooled under liquid nitrogen for approximately 15 minutes. The SPEX grinder operating time is approximately 2.0-1.5 minutes per cycle, with a cool-down time of 1 minute, 4 cycles, and a hammer cycle rate of 6.0-5.0 Hz. After grinding, the powder is sieved to remove particles with a gyration radius of <12.5 microns. The final dry powder (moisture content % ≤ 15% w / w) is stored in a sealed container in a cool, dry place.

[0288] Example 2

[0289] Characterization of cryogenically ground powders

[0290] Through the midfield (4000-600cm -1 ) Fourier transform infrared (FTIR) attenuated total reflectance (ATR) spectroscopy was used to characterize the chemical composition of the cryogenically ground powders of Methods 1, 3, and 4 of Example 1. The powder density was characterized by measuring bulk density, compactness, and compressibility. Density measurements were performed manually on the individual powder components, rather than on the combined final powder to be mixed for the flowable hemostat, without specialized equipment.

[0291] Fourier transform infrared spectroscopy

[0292] The ZnSe ATR accessory of the ThermoNicolet Avatar 380 FTIR spectrophotometer was used to analyze the -1 The 4000 cm-1 of the powders of methods 1, 3, and 4 of Example 1 was obtained using 32 scans. -1 with 600cm -1 Representative Fourier transform infrared (FTIR) absorption spectra of Figure 1-3 ).

[0293] Powder bulk density

[0294] The bulk density of a powder is the ratio of the mass of an untapped powder sample to its volume (including the contribution of the interparticle void volume) (bulk density and compactness of the powder).

[0295] program:

[0296] Pass the powder through a sieve with a pore size greater than or equal to 1.0 mm. Weigh the amount of powder required to complete the test (depending on how much powder is available). The target weight is 5 g. Add the powder to a graduated cylinder that can be read to within 1 to 2 ml.

[0297] The unsettled volume of the powder was recorded and the bulk density was calculated in g / ml.

[0298] Repeat this operation 3 times.

[0299] Powder density

[0300] Compactness is the increased bulk density obtained after mechanical compaction of a container containing a powder sample.

[0301] program:

[0302] The compactness is performed manually as follows. A graduated cylinder containing the powder prepared in the bulk density determination using the recorded volume and mass is raised by 3 ± 0.2 mm and allowed to fall under its own weight. This is done ten times, with the volume recorded at the completion of the tenth tap. Another 10 taps are performed, for a total of 20 taps, with the volume of the 20th tap recorded. Multiple sets of 10 taps are performed until the difference between the measured values ​​is less than or equal to 2 mL. Once this point is reached, the final compactness (g / mL) and the change in volume are recorded. This process is repeated three times for each powder type.

[0303] Powder compressibility

[0304] Comparing bulk density to compactness provides a measure of a powder's interparticle interactions. These interactions influence a powder's ability to flow and are expressed using the compressibility index or Hausner ratio. The compressibility index and Hausner ratio reflect a powder's ability to be compressed. In free-flowing powders, these interactions are less pronounced, and the bulk density and compactness values ​​are closer. In less-flowing powders, interparticle interactions are generally greater, and a larger difference between bulk density and compactness is observed. These differences are reflected in the compressibility index and Hausner ratio.

[0305] Where V0 = apparent volume without sedimentation

[0306] V f = Final tapped volume

[0307]

[0308] FTIR spectrum

[0309] For the FTIR spectrum of CsGelatin1 dry material, please see Figure 1 .

[0310] For the FTIR spectrum of Z-type freeze-dried material, please see Figure 2 .

[0311] For the FTIR spectrum of Y-type freeze-dried material, please see Figure 3 .

[0312] Bulk density and compactness of powder

[0313] Table 1: Average bulk density and compactness of cryogenically ground powders

[0314] Description of powder Average bulk density (g / mL) Average density (g / mL) CsGelatin1 powder 0.607±0.006 0.805±0.019 CsGelatin2 powder 0.548±0.006 0.803±0.002 Z-type powder 0.365±0.002 0.566±0.026 Y-type powder 0.166±0.01 0.331±0.02

[0315] Powder compressibility

[0316] Table 2: Average compressibility index and Hausner ratio of powders

[0317] batch Average compression index Average Housena ratio CsGelatin1 powder 24.5±2.1 1.3±0.0 CsGelatin2 powder 31.7±0.6 1.4±0.0 Z-type powder 35.4±3.1 1.6±0.0 Y-type powder 50.0±2.8 2.0±0.1

[0318] Example 3

[0319] Preparation of flowable chitosan dressing from powder and water

[0320] A selected mass of powder is weighed and added to a first syringe having a mating end connector with a cap, such as a screw-type or Luer-type connector. If two types of powder (such as Z-type and Cs-gelatin) are to be filled and mixed into the syringe, the selected amounts (typically a 1:1 mass ratio) are weighed into the syringes one by one. When loading the powder, the syringe piston (plunger) is partially placed within the powder filling barrel of the first syringe, sealing the large open end of the syringe and taking care not to lose any powder in the process. The syringe is inverted so that the powder falls onto the syringe piston end. If more than one powder type is to be loaded, the powders can be mixed by shaking the syringe until the mixture appears homogeneous. Keeping the syringe inverted with the powder resting against the plunger under gravity, the capped end of the syringe is opened to allow the plunger to be slowly depressed, expelling air from the syringe without losing any powder or substantially compacting the powder. After the air is expelled, the cap is reattached to the end of the syringe to secure the contents of the first syringe.

[0321] In a second syringe having a mating connector opposite to the first syringe and a similar volume to the first syringe, a selected volume of water is drawn into the syringe while avoiding the inhalation of air. After the selected volume is drawn, the syringe ends are capped. For Z-type, Y-type and Z / Cs gelatin dressings, the typical volume of water is 6 to 8 times the volume (ml) per mass (g) of dry powder. Therefore, for 0.5g of powder mixture, 3.5ml of water is usually added to the second syringe. For Y2-type dressings, the typical volume of water is 15 to 20 times the volume (ml) per mass (g) of dry powder. Therefore, for 0.5g of powder mixture, 8.5ml of water is usually added to the second syringe. The preferred syringe volume is 2.5 to 3.5 times the volume of liquid added to the second syringe. Check the syringes to ensure that both syringe pistons are fixed and both syringe ends are securely capped.

[0322] In the case of sterilization by high energy radiation such as electron beam, x-ray or gamma irradiation at a sterilization assurance level (SAL) of 10 -6 The dual syringes can be packaged with their delivery catheters as a medical device in a single closed package (both syringes together) prior to terminal sterilization, or they can be packaged individually in their own closed portion of the device packaging. The flowable dressing can be stored long-term between 25°C and 4°C prior to use.

[0323] At the time of care, the syringe is removed from its packaging and the syringe end cap is removed. The mating ends of the syringes are connected together to provide a closed connection between the two syringes. The piston of the second syringe containing the liquid medium is depressed, causing the liquid to fill the first syringe and mix with the powder in the first syringe, and the piston of the first syringe is pressed upward. After the piston of the second syringe is fully depressed, the raised piston of the first syringe is fully depressed to force the entire partially mixed volume of the first syringe back into the second syringe. The pistons of the two syringes are pressed back and forth at a rate of approximately one full depression per second to mix the liquid and solid powder components of the flowable dressing. After approximately 30 seconds, a viscous and consistent fluid dispersion is formed. The ideal fluid properties of the flowable dressing make it suitable for delivery and use at least 15 minutes to one hour after mixing.

[0324] The final syringe, used to connect to the delivery catheter (a tube with a matching end connector), is the syringe filled with the flowable mixture after the final mixing plunger is depressed. The final syringe is then connected to the proximal end of a single-lumen delivery tube or catheter. The flowable dressing is compressed by the delivery device (e.g., by depressing the syringe plunger) and delivered to the wound site or affected area through the distal end of the delivery tube or catheter. If the flowable dressing composition cannot be completely expelled from the delivery catheter volume, a liquid, including but not limited to sterile water and 0.9% w / w saline solution, can be added to the syringe-connected end (proximal end) of the delivery catheter to completely expel the flowable dressing material from the distal end of the catheter. An alternative method to expel excess flowable dressing from the catheter is to pressurize the syringe-connected end of the delivery catheter with a gas, including but not limited to atmospheric air, nitrogen, and argon. A small, removable solid plug with a diameter close to the inner diameter of the catheter and a plug length greater than the diameter can be used within the catheter, close to the proximal end of the flowable dressing, to prevent interfacial mixing of the flowable dressing with the liquid or gas used to expel excess flowable dressing from the catheter. Preferably, the embolic material is formed by an insoluble chitosan material. In a preferred embodiment, the flowable dressing is applied to the mucosal wound site from the distal end of the catheter in a layered, smeared manner. In a preferred embodiment, the flowable dressing can be applied from the distal end of the catheter to fill the bleeding cavity. When a thick layer (2-5 mm thick) is applied to the bleeding area, blood penetrates through the catechol-modified chitosan matrix, triggering a firm adhesive clot that quickly stops bleeding in all patients, including those undergoing anticoagulant and antiplatelet therapy. The treatment area can be rinsed within 5-10 minutes of hemostasis to study the robustness of hemostasis and, if necessary, allow additional flowable dressings to be accurately placed on locations where further application is required.

[0325] Table 3 provides recommended water-to-powder ratios for different tube sizes.

[0326] Table 3. Powder to water ratio of chitosan gelatin Z-type flowable dressing

[0327] Working channel size (mm) Powder components Water component 2.8 Z type: Cs-gelatin; Z type; Y type 7 times the mass of powder 3.7 Z type: Cs-gelatin; Z type; Y type 6-6.5 times the mass of powder

[0328] Example 4

[0329] Image of flowable chitosan hemostatic powder, mixing, and delivery

[0330] For images of CsGelatin1 powder, please see Figure 4 .

[0331] For pictures of Z-type catechol-modified powders, please see Figure 5 .

[0332] For an image of the first syringe (without cap) of Example 3 having a male Luer thread end and containing powder, see Figure 6 .

[0333] For an image of the first syringe of Example 3 before the syringes are connected to each other and the second syringe with a female Luer connector (containing liquid on the left), see Figure 7 .

[0334] For an image of the first and second syringes (containing liquid on the left) of Example 3, where the syringes are connected to each other via female (left) and male (right) threaded connector ends, see Figure 8 .

[0335] For images of the first and second syringes of Example 3, wherein the syringes are connected to each other by male and female threaded connector ends, and the flowable particles are substantially uniformly dispersed (mixed) into the liquid without foaming or other evidence of bubbles, see Figure 9 .

[0336] For an image of the first syringe with a male luer connector containing a uniformly dispersed particle-liquid mixture ready for delivery, see Figure 10 .

[0337] For an image of the first injection containing a uniformly dispersed particle-liquid mixture ready for delivery and connected to a catheter delivery tube for precise, localized, minimally invasive application to the lesion, see Figure 11 .

[0338] For images of deploying a flowable dressing bead from the tip of a first syringe delivery catheter onto a horizontal clear PVC plate (with a 4.0 mm diameter center hole), see Figure 12 .

[0339] For images of deploying a flowable dressing bead from the tip of a first syringe delivery catheter onto a horizontal clear PVC plate (with a 4.0 mm diameter center hole), see Figure 13 .

[0340] For images of the deployment of adhered flowable dressing beads from the tip of a first syringe delivery catheter onto an upright clear PVC plate (with a 4.0 mm diameter center hole), see Figure 14 .

[0341] See Figure 15 For an image of all the flowable dressing (approximately 3.5 ml) transferred from the first syringe delivery catheter to partially cover the bottom of a thermoformed polystyrene dish (10 cm x 10 cm), see Figure 15 .

[0342] Example 5

[0343] Z type and C S - In vitro testing of gelatin flowable chitosan dressing

[0344] The flowable hemostatic dressing compositions of the present invention were tested against FLOSEAL controls for their ability to adhere to and remain adhered to different types of ex vivo freshly harvested porcine tissue upon delivery. The flowable hemostatic agents of the present invention were first tested for cohesiveness and tissue adhesion at the site of delivery to ex vivo tissue to investigate their ability to remain adhered and resist the effects of gravity (e.g., inversion) without flowing from the site of application. The flowable hemostatic dressing compositions were also investigated for their ability to remain adhered to different tissue types in difficult, wet environments (e.g., urine in the bladder; in gastrointestinal fluids; in congested airways or nasal passages or other passages) without significantly losing adhesion and cohesion over extended periods of time. The importance of identifying a catechol-modified chitosan composition that reliably adheres to and remains adhered to these critical tissue types is evident, as there are no known hemostatic compositions for treating localized bleeding in the bladder and its urinary tract; and currently available hemostatics for the gastrointestinal tract offer no benefit other than acute bleeding control for difficult bleeding in the stomach (which has not demonstrated a reduction in the incidence of rebleeding beyond 12 hours). The ex vivo beaker assay allows the study of the persistence and extent to which test samples remain attached to different tissue types under wet conditions that simulate in vivo wet conditions.

[0345] In a benchtop study, flowable hemostatic compositions of i) the Z-form, ii) a 1:1 ratio of the Z-form and CsGelatin 2, and iii) CsGelatin 2 alone were investigated. Freshly harvested porcine tissues included: 1) gastric mucosal tissue, 2) liver, 3) esophagus, and 3) bladder. Testing was performed at 37°C in a fully submerged, wet environment containing: 1) synthetic gastric fluid for gastric tissue, 2) synthetic urine for bladder tissue, and 3) 0.9% isotonic saline solution for liver and esophagus.

[0346] Under the same test conditions used for the chitosan flowable dressing of the present invention, a FLOSEAL hemostatic matrix (Baxter) flowable dressing (5 ml) was used as a control flowable dressing.

[0347] definition

[0348] the term definition CEHFD Chitosan intracavitary hemostatic flowable dressing CGHFD Chitosan gastrointestinal hemostatic dressing GI Gastrointestinal TURP Transurethral prostatectomy Z-type <![CDATA[Catechol-modified iron-enhanced chitosan material (f DS = 0.5 - 0.9)]]> Y-type <![CDATA[Catechol-modified iron-enhanced chitosan material (f DS = 0.3 - 0.7)]]> Cs-gelatin Chitosan gelatin cross-linked materials (including CsGelatin1 and CsGelatin2 examples) CsGelatin1 1:4w / w chitosan gelatin cross-linked material CsGelatin2 1:24w / w chitosan gelatin cross-linked material 1 to 1 1:1 w / w Z-type and CsGelatin2 FLOSEAL FLOSEAL Hemostatic Matrix, Baxter

[0349] Table 4: Mass of Z-form and CsGelatin 2 used in the flowable composition for in vitro testing

[0350]

[0351] Table 5: Volume of water used in the flowable compositions for ex vivo testing

[0352]

[0353] method

[0354] Table 6: Tissue Preparation

[0355]

[0356] The base of the tissue was secured to the bottom of a 150 mL polystyrene beaker using cyanoacrylate adhesive. The top surface of the adhered tissue was moistened with one or two drops of test system solution (synthetic urine, synthetic gastrointestinal fluid, or 0.9% saline).

[0357] Prepare a chitosan flowable dressing as follows: One male Luer lock 5 mL syringe contains the weighed dry powder components. Fill the corresponding female Luer lock syringe with the volume of water specified in Table 5. Connect the syringes and mix for 30 seconds. Use the male Luer lock syringe with the flowable mixture to deliver the flowable dressing to the tissue of the beaker test replicates.

[0358] Prepare FLOSEAL according to the manufacturer's instructions.

[0359] For each test tissue preparation, 3-4 drops of bovine blood were applied to the center of the top surface of the tissue. Using one syringe of chitosan flowable hemostatic dressing per replicate, chitosan flowable hemostatic dressing was applied to their tissue surface, with each replicate receiving an equal portion. For FLOSEAL samples, a 5 mL volume was evenly distributed among the four samples (approximately 1.2 ml per cup).

[0360] The flowable dressings were then allowed to sit on their respective wet tissues for 30 minutes before their test solution systems were added to the polystyrene beakers to completely submerge the flowable dressings in either synthetic urine, synthetic gastrointestinal fluid, or 0.9% saline.

[0361] The test beaker with its tissue and test flowable dressing was placed on a shelf in a 37°C incubator and placed under regular observation for tissue adhesion and the flowable composition's resistance to a wet environment in which it was completely immersed. Water evaporation from the beaker was prevented by sealing the top of the test beaker with parafilm.

[0362] The chitosan flowable test samples and FLOSEAL flowable controls were then monitored for detachment and degree of detachment from the tissue surface. Typically, upon detachment of the catechol-modified chitosan material, the less cohesive upper layer or "piles" of the bulk sample will separate from the tissue, leaving a more firmly adhered lower layer in place. In the catechol-modified chitosan material, this lower layer typically appears as a tough, strongly adhered, and uniform protective surface layer. The beaker test investigated the persistence of the applied test and control materials on their respective tissues under different wet immersion conditions.

[0363] The observation time for pile detachment was recorded, where the time provided was the average time between when the flowable dressing pile was observed to be detached and when it was last observed to be attached. The time when the adherent lower layer of the flowable sample was observed to be attached to the tissue surface was also monitored. When the layer was observed to be absent or depleted, the observation time for absence / significant depletion of surface residue was recorded, where the time provided was the average time between when the adherent dressing surface layer was observed to be substantially absent and when it was last observed to be present.

[0364] The dissolution rate of the test and control flowable dressing samples was also monitored. Chitosan gelatin flowable dressings generally completely dissolved in the fluid. Z-type or hybrid chitosan flowable dressings did not completely dissolve but tended to break down into small particles. The dissolution time of the Z-type or hybrid chitosan flowable dressing was the time it took for it to completely break down and pass through or out of the body. This time was recorded as the first time the flowable dressing was observed to completely disintegrate or dissolve.

[0365] result

[0366] Tables 7 to 9 below provide the results of the ex vivo tissue adhesion test:

[0367] Table 7: Average detachment time (hours) for bulk (piles) of sample material to detach from tissue

[0368] preparation Stomach liver bladder esophagus Z-type 3.03±0.84 17.47±3.60 16.26±11.18 1.26±0.0 1 to 1 2.18±0.0 4.62±1.22 3.12±0.0 1.26±0.0 CsGelatin2 4.01±1.83 0.87±0.0 1.26±0.0 1.26±0.0 FLOSEAL 0.25±0.0 3.83±0.0 0.25±0.0 0.496±0.25

[0369] Table 8: Average time (hours) without adherence of sample material to tissue

[0370] preparation Stomach liver bladder esophagus Z-type 13.87±0.0 26.49±6.58 48.75±0.0 1.26±0.0 1 to 1 8.63±5.23 18.23±14.83 12.21±0.0 1.26±0.0 CsGelatin2 4.01±1.83 0.87±0.0 1.26±0.0 1.26±0.0 FLOSEAL 0.74±0.0 13.2±0.0 1.81±1.07 0.50±0.25

[0371] Table 9: Average sample material dissolution times in test liquids

[0372] preparation Stomach liver bladder esophagus Z-type 19.92±0.0 26.49±6.58 48.75 24.00 1 to 1 19.92±0.0 26.49±6.58 24.00 24.00 CsGelatin2 19.92±0.0 9.96±0.0 6.17 6.17 FLOSEAL 1.23±0.0 21.17±0.0 25.37±0.0 25.37±0.0

[0373] Regarding chitosan flowable dressings that adhere to GI tissues located in the horizontal plane ( Figure 16A , Z-type; Figure 16B , 1 to 1; Figure 16C , CsGelatin2) and the same dressing upside down ( Figure 16DZ-type; Figure 16E , 1 to 1; Figure 16F , CsGelatin2) images, see Figure 16.

[0374] Regarding chitosan flowable dressing adhered to GI tissue during ex vivo testing at time = 0 ( Figure 17A , Z-type; Figure 17B , 1 to 1; Figure 17C , CsGelatin2); the same dressing at 7.8 hours ( Figure 17D , Z-type; Figure 17E , 1 to 1; Figure 17F , CsGelatin2) and the dressing at 19.9 hours ( Figure 17G , Z-type; Figure 17H , 1 to 1; Figure 17I , CsGelatin2) images, see Figure 17.

[0375] Regarding chitosan flowable dressings that adhere to liver tissue located on a horizontal plane ( Figure 18A , Z-type; Figure 18B , 1 to 1; Figure 18C , CsGelatin2) and the same dressing upside down ( Figure 18D , Z-type; Figure 18E , 1 to 1; Figure 18F , CsGelatin2) images, see Figure 18.

[0376] Regarding chitosan flowable dressing adhered to liver tissue during ex vivo testing at time = 0 ( Figure 19A , Z-type; Figure 19B , 1 to 1; Figure 19C , CsGelatin2); the same dressing at 7.8 hours ( Figure 19D , Z-type; Figure 19E , 1 to 1; Figure 19F , CsGelatin2); the dressing at 19.9 hours ( Figure 19G , Z-type; Figure 19H , 1 to 1; Figure 19I , CsGelatin2); and the remaining dressing adhered at 33 hours ( Figure 19J , Z-type; Figure 19K , 1:1) image, please see Figure 19.

[0377] Regarding chitosan flowable dressing adhered to TURP bladder tissue located in the horizontal plane ( Figure 20A , Z-type; Figure 20B , 1 to 1; Figure 20C , CsGelatin2) and the same dressing upside down ( Figure 20D , Z-type; Figure 20E, 1 to 1; Figure 20F , CsGelatin2) image, see Figure 20.

[0378] Regarding chitosan flowable dressing adhered to TURP bladder tissue during ex vivo testing at time = 0 ( Figure 21A , Z-type; Figure 21B , 1 to 1; Figure 21C , CsGelatin2); the same dressing at 2.25 hours ( Figure 21D , Z-type; Figure 21E , 1 to 1; Figure 21F , CsGelatin2); the dressing at 6.2 hours ( Figure 21G , Z-type; Figure 21H , 1 to 1; Figure 21I , CsGelatin2); the dressing at 13.9 hours ( Figure 21J , Z-type; Figure 21K , 1 to 1; Figure 21L , CsGelatin2); the dressing at 24 hours ( Figure 21M , Z-type; Figure 21N , 1 to 1); the dressing at 29.5.7 hours and 48.7 hours respectively ( Figure 21O , Z-type; Figure 21P , 1:1) image, please see Figure 21.

[0379] Regarding chitosan flowable dressings that adhere to esophageal tissue located in the horizontal plane ( Figure 22A , Z-type; Figure 22B , 1 to 1; Figure 22C , CsGelatin2) and the same dressing upside down ( Figure 22D , Z-type; Figure 22E , 1 to 1; Figure 22F , CsGelatin2) image, see Figure 22.

[0380] Regarding chitosan flowable dressing adhered to esophageal tissue during ex vivo testing at time = 0 ( Figure 23A , Z-type; Figure 23B , 1 to 1; Figure 23C , CsGelatin2); the same dressing at 2.25 hours ( Figure 23D , Z-type; Figure 23E , 1 to 1; Figure 23F , CsGelatin2); the dressing at 6.2 hours ( Figure 23G , Z-type; Figure 23H , 1 to 1; Figure 23I , CsGelatin2); the dressing at 13.9 hours ( Figure 23J , Z-type; Figure 23K , 1 to 1; Figure 23L , CsGelatin2); dressing at 24 hours ( Figure 23M , Z-type; Figure 23N , 1 to 1; Figure 23A O, image of CsGelatin2), see Figure 23.

[0381] in conclusion:

[0382] The catechol-modified chitosan flowable dressing of the present invention exhibited persistence as an adherent mass and protective material on tissue for over 12 hours under difficult wet conditions, and significantly longer persistence as an adherent mass and material layer compared to FLOSEAL. The wet conditions tested simulate clinical use in wet natural orifice minimally invasive applications, such as in the gastrointestinal tract, urethra, and bladder. The flowable chitosan material of the present disclosure remained substantially adhered to tissue as a protective layer under difficult wet conditions for up to 49 hours, then substantially degraded and dissolved after 49 hours, being removed from the body by excretion.

[0383] Example 6

[0384] Optical microscopy images of flowable dressing powder and its wetting behavior

[0385] Images of the powder composition of the present invention were collected using an Amscope T490-DK optical trinocular microscope at approximately 40x, 100x, and 400x magnification (further magnified 1.75x when printed). Note that Figures 24, 25, and 26 are shown at 40x and 100x magnifications, while Figure 27 is shown at 100x and 400x magnifications.

[0386] About Z-type powder ( Figure 24A and Figure 24B : dry; Figure 24A C and Figure 24D : moistened by water; Figure 24E and Figure 24F : Image wetted by blood and water), see Figure 24.

[0387] About CsGelatin2( Figure 25A and Figure 25B : dry; Figure 25C and Figure 25D : moistened by water; Figure 25E and Figure 25F : Image wetted by blood and water), see Figure 25.

[0388] Regarding the 1:1 Z-type and CsGelatin2 ( Figure 26A and Figure 26B : dry; Figure 26C and Figure 26D : moistened by water; Figure 26E and Figure 26F : Image wetted by blood and water), see Figure 26.

[0389] Image of Y-type powder moistened with blood and water ( Figure 27A and Figure 27B :), please see Figure 27.

[0390] in conclusion:

[0391] Optical microscopy images 24 to 27 demonstrate that the micron-sized particle composition of the chitosan flowable dressing remains as microparticles when wetted by water and biological fluids such as blood. Furthermore, optical microscopy of blood flow through the deposited wet flowable matrix demonstrates the rapid and permeable travel of red blood cells and platelets through the porous particle matrix and, after traveling along the interconnected porous channels, the subsequent binding and attachment of the same red blood cells and platelets to the surface of the suspended particles.

[0392] Example 7

[0393] FLOSEAL control test image

[0394] For images of FLOSEAL preparation, see Figures 28A-28D .

[0395] For images of FLOSEAL on the stomach, bladder, liver, and esophagus, see Figures 29A-29D .

[0396] Image of FLOSEAL powder observed using Amscope T490-DK microscope ( Figure 30A , dry; Figure 30B , moistened by water; Figure 30C moistened with blood), see Figure 30.

[0397] Example 8

[0398] In vitro testing of flowable chitosan Y and Y2 dressings with HEMOSPRAY powder control

[0399] The Y- and Y2-type catechol-modified chitosan flowable dressings were tested against a HEMOSPRAY powder control (lot W4529951) in an in vitro benchtop beaker test similar to the in vitro benchtop beaker test described in Example 5. There were two differences in the beaker test method described here compared to Example 5: i) beaker studies for the Y and Y2 flowable dressings and Hemospray powder involved shaking at 60 rpm using an IKA KS260 shaker in a controlled environment at 37°C; and ii) small 0.5" x 0.5" holes / depressions were created in the test tissue to better simulate the environment of lesions such as peptic ulcers. The tissues tested during these studies were freshly harvested from gastric mucosal tissue and bladders of porcine animals. The tests were conducted at 37°C in a fully immersed wet environment containing: i) synthetic gastric fluid for gastric tissue (Y-type, Y2-type, and HEMOSPRAY); and ii) synthetic urine for bladder tissue (Y-type only).

[0400] Table 10: Chitosan flowable hemostatic dressing formulation

[0401]

[0402] method:

[0403] Tissue preparation

[0404] Stomach and bladder tissues were cut into approximately 1.5 x 1.5 inch pieces without removing the lining. A concave surface was created in the test tissue sample surface by lifting the mucosa and cutting a small wound hole (approximately 0.5 inch x 0.5 inch) in the middle to simulate an ulcer site.

[0405] The tissue substrate was secured to the bottom of a 150 mL polystyrene beaker using cyanoacrylate adhesive. The top surface of the adhered tissue was wetted with 1 to 2 drops of test system solution (synthetic urine, synthetic gastrointestinal fluid), and then 2 to 3 drops of bovine blood were added to the wound hole.

[0406] HEMOSPRAY (0.6 g) was scooped into a puff and sprayed onto the stomach and blood. Four replicates were tested, each with 0.6 g of HEMOSPRAY.

[0407] Chitosan flowable dressings were prepared as follows: One 10 mL syringe contained the powder components. The corresponding female Luer lock syringe was filled with the volume of water listed in Table 10. The syringes were connected and mixed by alternately inserting the syringe plungers at approximately one insertion per second for 30 seconds. Once mixed, all the flowable dressing was pushed into one syringe and connected to a 220 mm x 2.7 mm (ID 2.1 mm) catheter. The flowable dressing was poured into the catheter so that the flowable dressing reached near the distal end of the catheter. Test replicates were prepared using one syringe containing approximately 0.4 g of dry powder.

[0408] The flowable dressing and HEMOSPRAY powder were delivered to their pre-wet tissue test surfaces and allowed to sit for 30 minutes before the test solution system was added to the polystyrene beaker to completely immerse the flowable dressing in the synthetic gastrointestinal fluid or synthetic urine.

[0409] The test beakers containing their tissues, test flowable dressings and control HEMOSPRAY were placed on a rack within a 37°C incubator and IKA KS260 shaker and allowed to shake at 60 rpm with periodic observations of tissue adhesion and resistance of the flowable composition to a fully submerged wet environment.

[0410] The time (hours) for bulk (piles) of sample material to detach from the tissue was monitored for the chitosan flowable test sample and the HEMOSPRAY control sample. The observation time for bulk detachment was recorded, where the time provided was the average time between when the flowable dressing pile was observed to detach and when it was last seen attached.

[0411] The chitosan flowable dressing test and HEMOSPRAY control samples were also monitored for the time at which no bound surface layer was observed on the tissue surface. Typically, a large amount of the flowable hemostatic agent dissociates, leaving a layer of adhered sample on the tissue surface. In the case of the chitosan catechol material, this adhered layer on the tissue helps prevent rebleeding and also aids tissue healing. The time at which the surface residue disappears is described herein as the time at which no adhered sample material is present on the tissue, and this time is recorded, wherein the time provided is the average time between when a substantially non-adherent dressing layer is observed and when it was last observed to be adhered.

[0412] result:

[0413] Tables 7 through 9 above provide the results of the ex vivo tissue adhesion tests.

[0414] Table 11: Average detachment time (hours) for bulk (piles) of sample material to detach from tissue

[0415] preparation Stomach / GI TURP Y-type 30.02±14.17 31.05±21.14 Y2 type 14.30±0.0 NA Hemospray 10.36±6.09 NA

[0416] Table 12: Average time (hours) without adherence of sample material to tissue

[0417] preparation GI TURP Y-type 32.08±16.25 37.7±11.74 Y2 type 14.30±0.0 NA Hemospray 13.88±0.00 NA

[0418] in conclusion:

[0419] The catechol-modified chitosan flowable dressing of the present invention exhibited persistence as an adherent mass and protective material on tissue for over 12 hours under difficult wet conditions, and exhibited significantly longer persistence as an adherent mass and material layer compared to HEMOSPRAY. The wet conditions tested simulate clinical use in wet natural orifice minimally invasive applications, such as in the gastrointestinal tract, urethra, and bladder.

[0420] Example 9

[0421] Porcine liver capsule dissection model of acute in vivo hemostasis

[0422] Acute in vivo testing was conducted in female domestic pigs weighing 40-50 kg. Treatment was performed with a 1:1 Z-type CsGelatin2CEHFD prototype. All experiments were conducted in accordance with the 2011 National Research Council Guide for the Care and Use of Laboratory Animals and applicable federal regulations. The experimental protocol for animals complied with the NIH Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee. All procedures and animal care were performed in an approved animal research facility. The animals were anesthetized, and a laparotomy was performed to expose the liver. To induce a coagulopathy, 5000 units of heparin were administered intravenously (IV). A continuous infusion of 50 units / kg of heparin was maintained during the procedure to maintain anticoagulation. Activated clotting time (ACT) levels were measured after 10 minutes and then every 20 minutes during the procedure, with additional IV heparin (50% of the initial dose, 2500 units) administered as needed to maintain anticoagulation with an ACT > 250 seconds. A rotary cutting instrument was used to create a depressed lesion 2.5 cm in diameter and 1 cm deep in the liver lobe. The bleeding rate at the time of treatment is determined by absorbing blood flow in a pre-weighed gauze for 15 seconds and measuring the weight change of the gauze. A test flowable hemostat volume (4 ml) is applied directly to the bleeding injury site to cover and fill the wound with a 10 ml balloon catheter, which applies a total load of 35 g load on the injury site for 3 minutes, after which it is deflated and removed. The decision of success or failure in hemostasis is at the discretion of the surgeon applying the flowable hemostat. The bleeding rate immediately after the flowable dressing is applied and the balloon catheter is removed is measured.

[0423] result

[0424] The bleeding rate before treatment was 12.72 g / min. The activated clotting time (ACT) was greater than 198 seconds. The surgeon evaluated the application as a hemostatic agent, with the final bleeding rate determined to be <0.9 ml / min (>90% reduction in bleeding).

[0425] Example 10

[0426] Acute in vivo hemostatic pig spleen and liver capsule dissection model using HEMOSPRAY control

[0427] The purpose of animal experiments was to evaluate the Z-type, Y-type, Y1-type and Y1 resid Type (Y1 resid Four different catechol chitosan flowable dressings (containing residual fine particles with a radius of gyration of 12.5-31.5 μm removed from Y1 by sieving) were compared with a positive control, HEMOSPRAY (lot W4337018), hemostatic powder for the control of difficult bleeding. Control tests were performed in acute bleeding in heparinized porcine lesions of the liver and spleen parenchyma.

[0428] All experiments were performed in accordance with the 2011 National Research Council Guide for the Care and Use of Laboratory Animals and applicable federal regulations. Experimental protocols involving animals were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. All procedures and animal care were performed in an approved animal research facility.

[0429] method:

[0430] Hemostatic efficacy was assessed by visual scoring (or grading) of the bleeding around the application using a bleeding score of 0 to 5 from "Comparison of two gelatin and thrombin combination hemostatics in a porcine liver abrasion model" by KM et al. 2013

[26] . Lower bleeding scores (0-2) indicate that the bleeding was controlled (successful hemostasis), while higher scores (3-5) indicate that the bleeding was barely controlled or not controlled (failed hemostasis). The scoring system is outlined in the iv below:

[0431] i.0 = no bleeding, no condition

[0432] ii.1-2 = No active bleeding, some residual blood may be present

[0433] iii.3 = Slow active bleeding

[0434] iv.4 = Rapid active bleeding

[0435] v.5 = uncontrolled bleeding

[0436] The treated lesion hemostasis score was assessed for parenchymal lesions at 1.5 and 3 minutes, with a 3-minute observation period after the last application to confirm success or failure. Bleeding scores were determined at the surgeon's discretion. Although the study was open-label, the surgeon was unaware of which catechol chitosan test product was being administered. All catechol-modified chitosan dressings tested were prepared at room temperature with a solid powder:water ratio (g / ml) ranging from 1:7.0 to 10.0, mixed together in a 10 ml male / female Luer-connected syringe, and delivered from a male Luer syringe containing approximately 4 ml of the catechol-modified chitosan dressing.

[0437] Heparinized pig parenchymal capsule injury of the liver and spleen is prepared using surgical scissors, tweezers and a biopsy punch (6mm diameter x3mm deep injury) of 6mm diameter. The parenchymal injury model provides a standard model for difficult-to-control anticoagulation bleeding, which has enough repetitions to study statistical significance. A catechol-modified chitosan dressing is applied directly from its delivery syringe to the bleeding wound, with 1-2ml dressing applied at each time. After the first 1.5 minutes, if the bleeding grade is higher than 2, then reapplication of the dressing is allowed. By pouring HEMOSPRAY from a weighing boat sufficient to fill and cover the injury onto the center of the bleeding injury, the application containing HEMOSPRAY (0.1 to 0.2g) is carried out. Both test and control applications do not accept tamponade. In the parenchymal injury test, catechol chitosan dressing and HEMOSPRAY are used as random pairs. Two animals were tested over a two-day period, and 2x27 injuries were applied in total over these two days.

[0438] result:

[0439] The mean activated clotting time (ACT) of parenchymal lesions was 525 seconds, and the mean pre-treatment bleeding rate was 3.97 g / min, with no significant differences between HEMOSPRAY and catechol-modified chitosan dressings.

[0440] Tables 13 and 14 provide the results of the four catechol-modified chitosan test materials (Z, Y, Y1 and Y1 resid ) and control HEMOSPRAY materials after treatment with in vivo injury tests. Figure 31 and Figure 32The average post-treatment bleeding score and the average hemostasis time of four kinds of test materials and HEMOSPRAY contrast are provided respectively.After 3 minutes observation time in total, catechol chitosan flowable hemostatic dressing Y and Y1 show the final bleeding score that equates statistically, and Y and Y1 all show and compare significantly lower final bleeding score (referring to Table 14) with contrast HEMOSPRAY (HS).Y and Y1 show the final average bleeding score of 1.5, and HEMOSPRAY has the final average score of 3.08 (p<0.05).

[0441] Y1 resid The minimum effective hemostatic performance of fine particles (sieved to 25-63 microns) and Y1 (with the same resid The importance of flowable matrix pore size in a flowable matrix composition is demonstrated by the most effective hemostatic performance of Y (same composition, but with fine particles removed). This has been little or no evaluated in the scientific and patent literature. The substantially similar hemostatic performance between Y and Y1 (mean post-treatment bleeding scores of 1.50 and 1.50; mean hemostasis times of 3.21 ± 0.94 and 2.71 ± 1.29, respectively) suggests that sieving to remove particles of 25 microns (radius of gyration of 12.5 microns) provides sufficient pore size to allow near-optimal hemostatic performance.

[0442] Table 13: Hemostasis test results after treatment

[0443]

[0444] Table 14: Hemostasis test statistical indicators

[0445]

[0446] in conclusion:

[0447] The catechol-modified chitosan flowable dressing of the present invention can precisely deliver to inaccessible anticoagulated bleeding lesions with minimal preparation to quickly and effectively control bleeding. The porosity of the flowable dressing matrix is ​​a key factor in its hemostatic efficacy. In porcine parenchymal injury trials, the catechol-modified chitosan dressing was found to outperform HEMOSPRAY in quickly and effectively controlling anticoagulated bleeding.

[0448] Example 11

[0449] Acute in vivo porcine F ORREST 1A gastroepiploic artery injury model test

[0450] The purpose of the animal study was to evaluate the effect of Y-type and Y1-type catechol chitosan flowable dressings compared to the positive control HEMOSPRAY (lot W4337018) hemostatic powder on the control of gastrointestinal bleeding due to Forrest 1a. The acute bleeding control test was performed in a heparinized porcine laceration of the gastroepiploic bundle placed in the stomach [27,28].

[0451] All experiments were performed in accordance with the 2011 National Research Council Guide for the Care and Use of Laboratory Animals and applicable federal regulations. Experimental protocols involving animals were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. All procedures and animal care were performed in an approved animal research facility.

[0452] method:

[0453] The hemostatic efficacy is assessed by determining the hemostatic time after the full syringe delivery of the chitosan dressing modified by catechol. The hemostatic time of the chitosan flowable dressing modified by catechol is compared with the hemostatic time of the HEMOSPRAY control powder of the delivery poured onto similar hemorrhage Forrest 1a hemorrhage injury in large quantities (0.6g). The bleeding rate before treatment is measured by keeping 15 seconds with a folded 2x2 surgical gauze dressing against the injury site. The activated clotting time during the test is determined to be>250 seconds.

[0454] Treated lesions were assessed every 2.5 minutes, and further hemostatic therapy was applied if bleeding remained uncontrolled. Hemostatic success was determined at the surgeon's discretion. Although the study was open-label, the surgeon was unaware of the catechol chitosan test product being administered. All experimental catechol-modified chitosan dressings were prepared at room temperature with a solid powder:water ratio (g / ml) ranging from 1:7.0 to 10.0, mixed together in a 10 ml or 20 ml male / female Luer-connected syringe, and delivered from a male Luer syringe containing approximately 4 ml of catechol-modified chitosan dressing. The catechol-modified chitosan samples were delivered through a preheated 220 mm × 2.7 mm outer diameter × 2.1 mm inner diameter catheter (at 37°C for 1 minute) and backfilled with saline using a balloon inflation device to allow for delivery of a larger portion of the dressing within the catheter. HEMOSPRAY powder was delivered dry from a modified pipette. A maximum of two lacerations were performed on each bundle. The injuries were not randomly selected. The application was used to study the hemostatic efficacy, hemostasis time and dressing delivery capacity. No dressing received tamponade and the wound site was reused by irrigating with 0.9% isotonic saline solution to remove the previous dressing and removing any clots present in the injury site with forceps.

[0455] The porcine gastroepiploic artery bundle injury model [27, 28] is a well-established model of clinically relevant upper gastrointestinal Forrest-1a bleeding. The catechol-modified chitosan dressing was applied directly from its delivery syringe to the bleeding wound, with 3-4 ml of dressing applied at each time. After the initial 2.5 minutes, additional hemostatic application was allowed for up to 10 minutes after the first application. HEMOSPRAY (0.6 g) was applied by pouring HEMOSPRAY from a drip pipette into the center of the bleeding injury. No sample application received tamponade. The catechol-chitosan dressing and HEMOSPRAY were used in random pairs in the test. Two animals were tested over a two-day period, with a total of 2 x 3 injury applications over these two days.

[0456] result:

[0457] The delivery of catechol chitosan Y and Y1 dressings and HEMOSPRAY to hemorrhagic gastroepiploic bleeding was found to be 100% effective in controlling Forrest 1a bleeding within 15 minutes, and there was no recurrence of bleeding after successful hemostasis was achieved. The mean hemostasis time for the catechol-modified dressings (Y and Y1) and HEMOSPRAY was 9.75 and 9.5 minutes, respectively. Figure 33 Shown are histogram box plots of pre-treatment bleeding rates for catechol-modified chitosan (Y and Y1) and HEMOSPRAY (HS) administration. Figure 34 Shown are histogram box plots of hemostasis time for gastroepiploic artery injuries treated by application of catechol-modified chitosan dressings (Y and Y1) and control HEMOSPRAY (HS).

[0458] One of the C1 administrations used two syringes of dose, while the other used only one syringe. Both C2 administrations used only one syringe. One HEMOSPRAY administration used only a single 0.6g dose, while the other used 1.2g during the first dose, followed by another 0.6g dose after an observation period. Upon delivery to the injury site, both Y and Y1 dressings adhered immediately to the injury surface and resisted blood splatter. Delivery of the Y flowable dressing exhibited a formed rod or "noodle" appearance at the beginning of delivery, which disappeared during delivery. Flowable dressing Y1 also exhibited the appearance of formed or otherwise extruded rods, which disappeared more rapidly than in the C1 prototype delivery. Y and Y1 dressing deliveries were similar in terms of the appearance of formed rods upon delivery and their degree of mixing with the saline backfill in the catheter. No dressing, including HEMOSPRAY, experienced any tamponade. Delivery of HEMOSPRAY powder by pouring / puffing is precise and localized, unlike the dispersed powder stream delivered by the HEMOSPRAY gas jet delivery device.

[0459] in conclusion:

[0460] The catechol-modified flowable dressing system and its delivery are accurate and effective in controlling anticoagulated gastrointestinal bleeding.The catechol-modified chitosan flowable dressing of the present disclosure is non-inferior to the FDA-approved HEMOSPRAY powder in controlling gastrointestinal bleeding.

[0461] Example 12

[0462] Human Epidermal Equivalence (HEE) testing to assess irritation

[0463] Human Epidermal Equivalence (HEE) testing was performed to assess irritation (iFyber LLC, Ithaca, NY). Test samples were catechol-modified chitosan prepared as a Y flowable dressing with water (0.40 g Y powder dispersed in 5.0 ml water) and compressed catechol-modified lyophilized sheets (Y compressed sheets). The milled catechol-modified chitosan sheets for the Y powder were uncompressed, with the Y sheet film density approaching 0.005 g / cm. 3 (>99% void space). Y sheet test samples (pure Y catechol modified chitosan) were tested in the stimulation test described herein as Y compressed sheets to help maintain the integrity of the sheet during the extraction process. The sheets were compressed between parallel heated platens at 60°C to a density of approximately 0.4 g / cm 3 The chitosan material was compressed from its original, dry, freeze-dried, low-density, uncompressed thickness of approximately 7 mm (<0.75% void space) to a Y-compressed sheet approximately 100 microns thick. The catechol-modified chitosan material was sterilized by gamma irradiation at 25-40 kGy. HEMOSPRAY (lot W4337018) powder was included in this study as a positive control. This study was conducted according to ISO 10993-23, a standard for evaluating the irritation potential of medical devices.

[0464] method:

[0465] Human epidermal equivalent (HEE) tissue was established according to the following protocol: after thawing and expanding keratinocytes (ATCC PCS-201-012), the density (2x10 5 Keratinocytes (100 cells / insert) were seeded in 12-well cell culture inserts. The cells were submerged for five days and then transferred to the air / liquid interface and cultured for an additional eight days, with medium changes every 2-3 days.

[0466] Extracts of the test samples (Y flowable dressing, Y compressed sheet) and control HEMOSPRAY samples were prepared according to ISO 10993-12. Before preparing the extracts for the study, an absorption evaluation was performed on each test sample type using DI water to determine the appropriate extraction volume. The selected extraction volume was 6 cm for the Y compressed sheet. 2 / mL, and 0.2 g / mL for Y flowable dressing and HEMOSPRAY powder. Each sample was weighed or measured, excess water was added, the sample was incubated overnight at 37°C, and the remaining water was measured. The assay volume used to achieve the extraction yield was 1 mL for 0.2 g of Y flowable dressing, 2.5 mL for 0.2 g of HEMOSPRAY powder, and 0.2 g / mL for 6 cm 2 -Sized Y compressed tablets are 3.5 mL.

[0467] Each test sample was prepared in triplicate for extract preparation for stimulation testing. 0.2 g / well of HEMOSPRAY powder was transferred to three wells of a 12-well plate and then sterilized under UV light for 30 minutes. A Y flowable dressing was prepared and 0.2 g / well was aseptically added to the 12-well plate. Uniformly sized disc fragments were cut from the Y compressed tablet using a 28 mm diameter punch and placed in a 6-well plate. Hematoxylin and eosin (HEE) medium was used as the extraction medium. This medium is typically serum-free; however, to extract polar and non-polar components from the test article, fetal bovine serum (FBS) was added at 5% v / v. Appropriate volumes of medium were then added to three replicates of each test sample. Plates were wrapped with paraffin film and incubated in a shaking incubator at 37°C and 28 rpm for 72 h. Controls consisting of 1X PBS (negative control) and HEE medium containing FBS (vehicle control) were incubated under the same conditions as the test and control samples.

[0468] At the end of the extraction time point, 200 μL of extract was taken directly from each sample and immediately added to the surface of the HEE tissue. Once all tissues were processed, they were placed in a humidified incubator at 37°C and 5% CO2. After 20 hours, all HEE samples were removed from the incubator and each sample was rinsed 3 times with sterile 1X PBS. The inserts were blotted dry and then placed in a temporary storage plate (i.e., a 12-well plate containing HEE culture medium) until all samples were washed. Next, the samples were transferred to a 12-well plate containing 600 μL of 1 mg / mL of MTT in DMEM without phenol red and incubated for 3 hours in a humidified incubator at 37°C and 5% CO2. Subsequently, the samples were transferred to a 12-well plate containing 3 mL of isopropanol per well to extract formazan (the end product of the MTT reaction). The plate was incubated on a shaker at room temperature at 120 rpm for 2 hours. Next, each cell culture insert was pierced and the solution was pipetted up and down several times for homogenization. Three 200 μL aliquots from each well were transferred to a 96-well plate and the absorbance at 570 nm was measured. Isopropanol was included as a blank.

[0469] Data analysis was performed by first subtracting the average background OD value of the isopropanol blank from each OD value. The average background-corrected OD of the negative control tissue was determined, which represented 100% viability. The viability of each tissue relative to the control was then determined, and the three replicates were averaged to determine the viability of each test article. Finally, statistical analysis was performed using GraphPad Prism software, and data were analyzed using a one-way ANOVA with Tukey's post hoc test. The significance level was set at 5%.

[0470] Results and discussion:

[0471] This study was conducted to determine the irritation potential of Y flowable dressing, Y compressed sheet, and HEMOSPRAY control. The test involved preparing extracts of each material and then adding them to HEE tissue. After a 20-hour incubation, the test articles were removed and tissue viability was determined using the MTT assay.

[0472] Figure 35 The results depicted in Figure 4 show that the viability of the control group, Y flowable dressing, Y compressed sheet, and HEMOSPRAY were similar, with no significant differences (p>0.05). The Y flowable dressing showed a slight decrease in cell viability relative to the negative control, Y compressed sheet, and HEMOSPRAY (p<0.05). However, the cell viability percentage of all samples was above 50%.

[0473] Table 15 shows the criteria for determining irritation potential, as modified from ISO 10993-23. Figure 35As shown, all tested samples resulted in tissue viability >50%; therefore, based on the findings of this study, catechol-modified chitosan Y flowable dressing, catechol-modified chitosan Y compressed tablets, and HEMOSPRAY can all be classified as non-irritating.

[0474] Table 15. Classification of the irritation potential of the test samples

[0475] Criteria for in vitro interpretation Classification Cell viability after exposure <50% Irritation Cell viability after exposure >50% Non-irritating

[0476] in conclusion:

[0477] The catechol-modified chitosan Y material and flowable dressing of the present invention are non-irritating according to ISO-10993-23 and are therefore suitable for use on mucosal tissue at natural body orifices.

[0478] in conclusion

[0479] The present disclosure demonstrates highly effective non-biological, tissue-adhesive, fluid-type, flowable hemostats for direct application to actively bleeding wounds and for prolonged application (>6 hours) to control moderate to severe bleeding in both routine and challenging settings.

[0480] The various embodiments described above can be combined to provide additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited in this specification and / or listed in the application data sheet, including U.S. Provisional Patent Application No. 63 / 386,312 filed on December 6, 2022, U.S. Provisional Patent Application No. 63 / 386,313 filed on December 6, 2022, and U.S. Provisional Patent Application No. 63 / 386,314 filed on December 6, 2022, are incorporated herein by reference in their entirety. If it is necessary to adopt the concepts of various patents, applications, and publications to provide further embodiments, aspects of the embodiments can be modified.

[0481] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments and the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure.

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[0511] 28.Giday,S.,et al.,Safety Analysis of a Hemostatic Powder in aPorcine Model of Acute Severe Gastric Bleeding.Digestive Diseases andSciences,2013.58(12):p.3422-3428.

Claims

1. A flowable composition comprising a particulate chitosan material and a diluent carrier fluid.

2. The flowable composition of claim 1, wherein the particulate chitosan material comprises an amount greater than or equal to about 5% of the total weight of the flowable composition.

3. The flowable composition of claim 1, wherein the particulate chitosan material is provided in solid or semi-solid form.

4. The flowable composition of claim 3, wherein the semi-solid particulate chitosan material is swollen.

5. The flowable composition of claim 1, wherein the particulate chitosan material is provided in the form of a powder, granules, particles, fibers, or any combination thereof.

6. The flowable composition of claim 5, wherein the particulate chitosan material comprises regularly or irregularly shaped particles having a radius of gyration in the range of about 10 to 350 microns.

7. The flowable composition of claim 1, wherein the particulate chitosan material comprises one or both of catechol-modified chitosan and cross-linked chitosan gelatin.

8. The flowable composition of claim 1 , wherein the granular chitosan material comprises one or more of a densified chitosan material, a frozen phase separated and dried chitosan material, a densified frozen phase separated and dried chitosan material, a spray dried chitosan material, a dried cast film chitosan material, a sublimed frozen separated chitosan material, a dried freeze-thaw chitosan material, and a dried asymmetric centrifuge mixed material.

9. The flowable composition of claim 1, wherein the diluent carrier fluid is one or more of water, standard 0.9% saline solution, and autologous plasma.

10. The flowable composition of claim 1, wherein the diluent carrier liquid constitutes at least one of about 85% of the total weight of the flowable composition, about 90% of the total weight of the flowable composition, or about 95% of the total weight of the flowable composition.

11. The flowable composition of claim 1 , wherein the diluent carrier liquid is a heat sensitive fluid capable of being delivered through a 23 gauge needle or a 24 gauge needle at about 18 to 25°C and capable of gelling at about 37°C.

12. The flowable composition of claim 1, wherein the flowable composition is a hemostatic agent.

13. The flowable composition of claim 1, wherein the flowable composition is resistant to dissolution.

14. The flowable composition of claim 13, wherein the flowable composition comprises a particulate chitosan material that does not substantially dissolve and remains solid or semi-solid.

15. The flowable composition of claim 13, wherein the flowable composition is capable of resisting dissolution in at least one of urine, water, saline solution, blood, or gastrointestinal (GI) fluid at about 37°C for at least about 6 hours.

16. The flowable composition of claim 15, further characterized by providing a flowable dressing having a volume of greater than about 100 cm2 per gram of flowable dressing. 2 Specific surface area.

17. The flowable composition of claim 13, wherein the flowable composition comprises at least a first outer layer and a second tissue adhesion layer, and wherein the first outer layer resists dissolution for at least about 6 hours and the second tissue adhesion layer resists dissolution for at least about 12 hours.

18. The flowable composition of claim 1, wherein the flowable composition is tissue-adhesive.

19. The flowable composition of claim 18, wherein the tissue-adhesive flowable composition adheres to at least one of mucosal tissue and a site of tissue injury after a period of contact greater than about 6 hours.

20. The flowable composition of claim 19, wherein the flowable composition can be applied upside down through an endoscope and adhered to tissue under normal gravity.

21. The flowable composition of claim 1, wherein the flowable composition is biocompatible.

22. The flowable composition of claim 1, wherein the flowable composition is capable of being delivered to a tissue site via a channel having a diameter of at least one of less than about 7 mm, less than about 4.5 mm, less than about 4.0 mm, less than about 3.2 mm, less than about 2.8 mm, and about 0.5 mm.

23. An intraluminal hemostatic dressing comprising a flowable composition comprising a particulate chitosan material and a diluent carrier liquid.

24. A gastrointestinal hemostatic dressing comprising a flowable composition comprising a particulate chitosan material and a diluent carrier liquid.

25. A method of preparing the flowable composition of claim 1.

26. The method of claim 25, comprising preparing the chitosan material for use in a flowable composition.

27. The method of claim 25, comprising preparing one or both of a catechol-modified chitosan and a chitosan-gelatin cross-linked chitosan material.

28. The method of claim 25, comprising preparing a chitosan material that is one or more of a densified chitosan material, a frozen phase separated and dried chitosan material, a densified frozen phase separated and dried chitosan material, a spray dried chitosan material, a dried cast film chitosan material, a sublimed frozen separated chitosan material, a dried freeze-thaw chitosan material, and a dried asymmetric centrifuge mixed material.

29. The method of any one of claims 26 to 28, comprising grinding the chitosan material to form the particulate chitosan material.

30. A method of delivering the flowable composition of claim 1 to a tissue site in a subject in need thereof, comprising mixing a particulate chitosan material with a diluent carrier fluid prior to delivery to the subject.

31. The method of claim 30, further comprising providing the particulate chitosan material and the diluent carrier fluid as separate components for combination.

32. The method of claim 31, wherein the separate components are sterilized individually.

33. The method of claim 30, further comprising delivering an amount of the flowable composition at a bleeding tissue site in the subject in an amount sufficient to achieve hemostasis.

34. The method of claim 30, further comprising delivering the flowable composition to the tissue site via a channel having a diameter of at least one of less than about 7 mm, less than about 4.5 mm, less than about 4.0 mm, less than about 3.2 mm, less than about 2.8 mm, and about 0.5 mm.

35. The method of claim 30, further comprising delivering the flowable composition to the tissue site in one or more layers.

36. The method of claim 35, wherein the flowable composition comprises at least a first outer layer and a second tissue adhesion layer, and wherein the first outer layer resists dissolution for at least about 6 hours and the second tissue adhesion layer resists dissolution for at least about 12 hours.

37. The method of claim 30, further comprising adhering the flowable composition to the tissue site.

38. The method of claim 37, wherein the flowable composition adheres to the tissue site upon contact for a period of time greater than about 6 hours, and wherein the tissue site comprises at least one of mucosal tissue and a tissue injury.

39. The method of claim 37, wherein the flowable composition can be administered upside down through an endoscope under normal gravity and adhered to the tissue site.

40. The method of claim 30, further comprising delivering the flowable composition as an intraluminal hemostatic dressing.

41. The method of claim 30, further comprising delivering the flowable composition as a gastrointestinal hemostatic dressing.

42. The method of claim 41, further comprising sealing the tissue site with the gastrointestinal hemostatic dressing in an acidic environment of about pH 3 for at least 6 hours.

43. The method of claim 41, further comprising providing for dissolution of the gastrointestinal hemostatic dressing from the tissue site in a period of less than or equal to about seven days.

44. A method of delivering a flowable composition comprising a particulate chitosan material and a diluent carrier fluid to a tissue site in a subject in need thereof, comprising: mixing the particulate chitosan material with a diluent carrier liquid prior to delivery to the subject; applying the flowable composition; and adhering the flowable composition to the tissue site upon contact.

45. The method of claim 44, wherein the diluent carrier liquid has a viscosity less than or equal to 3.5 to 5.5 mPa.s.

46. ​​The method of claim 44, further comprising administering the flowable composition using a minimally invasive technique.

47. The method of claim 46, wherein the minimally invasive technique provides for remote flowable composition delivery.

48. The method of claim 44, further comprising administering the flowable composition for one or both of filling and sealing of excisions, biopsy sites, stenotic recesses, and defects and openings around hemostatic clips, non-metallic sutures, clamps, staplers, metallic sutures, and fixation pins.

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