Hemostatic composition and preparation method thereof

Abstract

The invention relates to a hemostatic composition and a preparation method thereof. The present invention relates to a hemostatic composition comprising at least partially integrated agglomerated ORC fibers, fibrinogen and thrombin and a method of forming a powdered hemostatic composition comprising the steps of forming a suspension of particles comprising fibrinogen, thrombin, ORC fibers in a non-aqueous low boiling solvent, stirring and shearing the suspension in a high shear mixing reactor, adding water to agglomerate the particles, evaporating the non-aqueous solvent, drying and screening the composition; and thereby forming the powdered hemostatic composition.

Description

[0001] This application is a divisional application of the invention application filed on March 9, 2018, with Chinese national application number 201810194719.1 and entitled "Hemostatic Composition and Preparation Method Thereof". Technical Field

[0002] The present invention relates generally to reagents and materials for promoting hemostasis and tissue sealing, and more specifically, to absorbable hemostatic particles with improved efficacy, specifically to particle aggregates made of fibrinogen, thrombin and oxidized regenerated cellulose, and to methods for manufacturing such compositions. Background Technology

[0003] Animals, including humans, can suffer bleeding from wounds or during surgery in a wide variety of situations. In some cases, the bleeding is relatively minor, and normal clotting function is all that is needed, in addition to administering simple first aid. In other cases, massive bleeding can occur. These situations typically require specialized equipment and materials, as well as trained personnel to administer appropriate assistance.

[0004] Bleeding during surgery can take many forms. It can be discrete or diffuse over a large surface area. It can be a large or small amount of bleeding originating from large or small blood vessels, arteries (high pressure) or veins (low pressure). It can be easily accessible or originate from hard-to-reach sites. In surgery, controlling bleeding is crucial for minimizing blood loss, reducing postoperative complications, and shortening the duration of surgical procedures in the operating room. The selection of appropriate methods or products for controlling bleeding depends on many factors, including but not limited to the severity of the bleeding, the anatomical location of the source and its proximity to adjacent critical structures, whether the bleeding has a discrete source or originates from a wider surface area, the visibility and precise identification of the source, and the accessibility of the source.

[0005] Conventional methods for achieving hemostasis include the use of surgical techniques, sutures, ligatures or clips, and energy-based coagulation or cauterization. When these conventional measures are ineffective or impractical, adjunctive hemostatic techniques and products are typically used.

[0006] To address the aforementioned issues, materials have been developed for controlling excessive bleeding or as adjunctive hemostasis. Topically absorbable hemostatic agents (TAHs) are widely used in surgical applications. TAHs encompass a variety of products, such as those based on woven or nonwoven fabrics or sponges, and are typically made at least partially of absorbable materials ranging from natural polymers to synthetic polymers and combinations thereof, including lactide-glycolic acid copolymers (such as polylactic acid 910), oxidized cellulose, oxidized regenerated cellulose (ORC), gelatin, collagen, chitin, chitosan, starch, etc. Gelatin is used in various forms with or without a local thrombin solution. Additionally, bioactive local hemostatic products (local thrombin solutions, fibrin sealants, etc.) and various synthetic local sealants are widely used.

[0007] To improve hemostatic performance, scaffolds based on the aforementioned TAH material can bind to biologically derived coagulation factors such as thrombin and fibrinogen.

[0008] Due to its biodegradability and its bactericidal and hemostatic properties, oxidized cellulose and oxidized regenerated cellulose have long been used as topical hemostatic wound dressings in various surgical procedures, including neurosurgery, abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck surgery, pelvic surgery, and skin and subcutaneous tissue surgery. Numerous methods for forming various types of hemostatic agents based on oxidized cellulose materials are known, regardless of whether the hemostatic agent is made into powder, woven, nonwoven, knitted, or other forms. Currently utilized hemostatic wound dressings include knitted or nonwoven fabrics containing oxidized regenerated cellulose (ORC), which is oxidized cellulose with increased homogeneity of cellulose fibers.

[0009] Introduced in the 1960s, ORC provided safe and effective hemostasis for many surgical procedures. The mechanism of action of ORC hemostatic agents is believed to begin with the material absorbing water at the bleeding site and then slightly swelling to provide a packing. ORC fibers initially trap fluid, blood proteins, platelets, and cells, forming a gel-like "pseudoclot" that acts as a barrier to blood flow, and subsequently serves as a matrix for the formation of a solid fibrin clot. ORC fabric has a loosely knitted structure in its matrix and adapts to its surrounding environment, making it easier to manage than other absorbable preparations because it does not adhere to surgical instruments and its size can be easily trimmed. This allows surgeons to hold the cellulose firmly in place until bleeding has completely stopped.

[0010] One of the most commonly used topical hemostatic agents is The original absorbable hemostatic agent is made from oxidized regenerated cellulose (ORC). It is used when ligation or other conventional control methods are impractical or ineffective. Absorbable hemostatic agents are used to help control bleeding from capillaries, veins, and small arteries during surgery. The absorbable hemostatic agents portfolio consists of four main product groups, all of which are available from Johnson & Johnson Wound Management Worldwide, a division of Johnson & Johnson in Somerville, New Jersey. The original hemostatic agent is a white fabric with a pale yellow hue and a faint caramel-like aroma. It is strong and can be sewn or cut without wear.

[0011] Absorbable hemostatic agents are similar, but with a higher weave density, resulting in greater tensile strength. They are particularly recommended for use in trauma and transplant surgeries because they can be used to wrap or suture in situ to control bleeding.

[0012] FIBRILLAR TM Products in absorbable hemostatic form have a layered structure, which allows surgeons to use clamps to peel and grasp any amount needed to achieve hemostasis at a specific bleeding site. FIBRILLAR TM Hemostatic agents. For bleeding sites that are difficult to reach or have irregular shapes. FIBRILLAR TM The hemostatic form may be more convenient than the braided form. It is particularly recommended for orthopedic / spinal and neurosurgical procedures.

[0013] This product SNoW TM The absorbable hemostatic agent is in the form of a structured nonwoven fabric. Due to the structured nonwoven fabric, SNoW TM Hemostatic agents may be more effective than other forms of hemostatic agents. It is better suited for endoscopic use. It is highly adaptable and recommended for both open and minimally invasive surgeries.

[0014] Other examples of commercially available absorbable hemostatic agents containing oxidized cellulose include those from Gelita Medical BV in Amsterdam, Netherlands. Absorbable cellulose surgical dressings. The aforementioned commercially available oxidized cellulose hemostatic agents are knitted or nonwoven fabrics with a porous structure used to provide hemostasis.

[0015] Fibrinogen and thrombin are key proteins involved in achieving hemostasis after vascular injury and are crucial for clot formation. Fibrinogen and thrombin can be in powder form or bound in non-aqueous suspensions without initiating a typical coagulation reaction, thus preventing fibrin clot formation until these proteins are hydrated in an aqueous medium or other liquid environment in which proteins are soluble. Incorporations of these protein powders have a variety of potential biomedical applications, including local hemostasis, tissue repair, drug delivery, etc. Additionally, these protein incorporations can be loaded in powder form onto carriers or substrates or other medical devices to form products that can be used, for example, as hemostatic devices.

[0016] Fibrin sealants, also known as fibrin glue, have been used clinically for decades. Typically, fibrin sealants consist of two liquid components: a component containing fibrinogen and a component containing thrombin, which are cryopreserved due to their inherent instability. Sometimes, fibrin sealant products consist of two lyophilized components, requiring reconstitution just before use and delivery via a connected syringe or other dual-barrel delivery device. Lyophilized formulations are generally stable, but the fibrinogen component is difficult to reconstitute. Many hemostatic agents currently available on the market or under development utilize lyophilized fibrinogen, often combined with lyophilized thrombin, and are applied as dry powders, semi-liquid pastes, or liquid formulations, or optionally placed on support structures such as absorbable fabric stents.

[0017] To enhance the hemostatic, tissue-sealing, and adhesive properties of dressings, therapeutic agents, including but not limited to thrombin, fibrin, and fibrinogen, have been combined with dressing carriers or substrates (including gelatin-based carriers, polysaccharide-based carriers, glycolic acid or lactic acid-based carriers, and collagen matrices). Examples of such dressings are disclosed in the following patents: U.S. Patent 6,762,336 “Hemostatic sandwich bandage”, U.S. Patent 6,733,774 “Carrier with solid fibrinogen and solid thrombin”, PCT Publication WO2004 / 064878 “Hemostatic Materials”, and European Patent EP1809343B1 “A reinforced absorbable multilayered hemostatic wound dressing and method of making”.

[0018] European patent EP1493451B1, “Haemostatic devices and compositions comprising oxidized cellulose particles and a polysaccharide binder,” discloses that the use of carboxylic acid oxidized cellulose as a carrier for acid-sensitive substances (such as thrombin and fibrinogen) and other acid-sensitive biological agents and pharmaceuticals is problematic. It also discloses a hemostatic composition comprising: biocompatible oxidized cellulose particles having an average specified nominal particle size of 0.035 mm to 4.35 mm; a biocompatible porous water-soluble polysaccharide binder component other than chitosan; and optionally, a hemostatic agent selected from thrombin, fibrinogen, or fibrin, wherein the weight ratio of the water-soluble polysaccharide to the oxidized cellulose particles is 3:97 to 15:85, and wherein the composition is a porous foam sponge, which can be obtained by a method comprising the steps of: providing a polymer solution having the polysaccharide binder component dissolved in a suitable solvent; providing the biocompatible oxidized cellulose particles; contacting the polymer solution with the oxidized cellulose particles under conditions that effectively disperse the oxidized cellulose particles substantially uniformly throughout the polymer solution to form a substantially uniform dispersion; subjecting the polymer solution having the particles dispersed therein to conditions that effectively solidify the substantially uniform dispersion; and removing the solvent from the solidified dispersion, thereby forming the hemostatic composition.

[0019] Russian patent publication RU2235539C1, "Method for preparing powder-like material for cessation bleeding," discloses a method for preparing a powdery material that produces a hemostatic effect. This method involves mixing partially oxidized cellulose as a base with thrombin and fibrinogen in an aqueous medium. Additionally, gelatin, ε-aminocaproic acid, and lysozyme are added to a specified substance, and dialdehyde cellulose, used as a fabric, is used as the partially oxidized cellulose, with the aldehyde content at 4% to 6% in the following proportions: dialdehyde cellulose, 1g; fibrinogen, 18mg-22mg; gelatin, 27mg-33mg; ε-aminocaproic acid, 45mg-55mg; lysozyme, 9.5mg-10.5mg; thrombin, 350U; water, 6.5ml. The method involves preparing a solution containing fibrinogen, ε-aminocaproic acid, and half the total amount of gelatin in half the total amount of water, and separately preparing a solution of thrombin, lysozyme, and the remaining amount of gelatin in the remaining water. In the prepared solutions, half the amount of dialdehyde cellulose is kept for 3-4 hours, the semi-finished product is extruded, air-dried, and then combined and ground.

[0020] US Patent Publication 20060159733A1, "Method of providing hemostasis to a wound," discloses that the acidity of carboxylic acid oxidized cellulose substrates can rapidly denature upon contact and inactivate acid-sensitive proteins, including thrombin or fibrinogen. A significant portion of the enzymatic activity of thrombin and factor XIII may be lost during the reaction. This makes it difficult to use carboxylic acid oxidized cellulose as a carrier for thrombin, fibrinogen, fibrin, or other acid-sensitive biological agents and pharmaceuticals. It also discloses that hemostatic wound dressings containing neutral carboxylic acid oxidized cellulose and protein-based hemostatic agents (such as thrombin, fibrinogen, and fibrin) are known. Neutral carboxylic acid oxidized cellulose materials are prepared by treating acidic carboxylic acid oxidized cellulose with an aqueous or alcoholic solution of a weak organic acid-base salt prior to the addition of thrombin, thereby raising the pH of the cellulose material to 5 to 8 to make it compatible with thrombin by neutralizing the acid groups on the cellulose. A thrombin-based hemostatic patch is disclosed, wherein thrombin is added to an acidic carboxylic acid oxidized regenerated cellulose or other substance in the presence of an acid neutralizer, ε-aminocaproic acid (EACA), to raise the pH of the substance to a range where thrombin can function as a hemostatic agent. Although this neutral carboxylic acid oxidized cellulose may be thrombin-compatible, it is no longer bactericidal because the antimicrobial activity of the oxidized cellulose is due to its acidity.

[0021] US Patent 7094428B2, "Hemostatic compositions, devices and methods," discloses a hemostatic composition comprising at least one procoagulant metal ion such as silver (I) or mercury (II), and at least one procoagulant biopolymer such as collagen, thrombin, prothrombin, fibrin, fibrinogen, heparinase, factor VIIa, factor VIII, factor IXa, factor Xa, factor XII, von Willebrand factor, selectin, procoagulant toxin, plasminogen activator inhibitor, glycoprotein IIb-IIIa, protease, or plasma. The composition is available in paste, dough, adhesive, liquid, lyophilized powder, or foam form for application to wounds. A hemostatic composition comprising at least one procoagulant biopolymer and a procoagulant metal ion, wherein, in the absence of the procoagulant biopolymer, the level of the procoagulant metal ion present in the composition is below its effective hemostatic concentration, wherein the hemostatic composition is selected from the group consisting of: silver (I) and collagen, silver (I) and thrombin, silver (I) and prothrombin, silver (I) and fibrin, silver (I) and fibrinogen, silver (I) and heparinase, silver (I) and factor VIIa, silver (I) and factor VIII, silver (I) and factor IXa, silver (I) and factor Xa, silver (I) and factor XII, silver (I) and von Willebrand factor, silver (I) and selectin, silver (I) and procoagulant toxin, silver ( Mercury (II) and plasminogen activator inhibitors, silver (I) and glycoproteins IIb-IIIa, silver (I) and protease, silver (I) and plasma, mercury (II) and collagen, mercury (II) and thrombin, mercury (II) and prothrombin, mercury (II) and fibrin, mercury (II) and fibrinogen, mercury (II) and heparinase, mercury (II) and factor VIIa, mercury (II) and factor VIII, mercury (II) and factor IXa, mercury (II) and factor XII, mercury (II) and von Willebrand factor, mercury (II) and selectin, mercury (II) and procoagulant toxin, mercury (II) and plasminogen activator inhibitors, mercury (II) and glycoproteins IIb-IIIa, mercury (II) and protease, mercury (II) and plasma. The hemostatic compositions of the present invention may also include carriers, such as, but not limited to, polyethylene glycol, hyaluronic acid, cellulose, oxidized cellulose, methylcellulose, or albumin. These can be used to provide a matrix, suitable viscosity, delivery capability, adhesion, or other properties desired to impart to the compositions herein that facilitate application to wounds. Many other carriers that impart these characteristics are included herein.

[0022] US Patent 6,162,241A, “Hemostatic tissue sealants,” discloses a hemostatic tissue sealant comprising: a biocompatible, biodegradable hydrogel tissue sealant including a crosslinkable group in which an effective amount of hemostatic agent is bound to prevent blood from flowing out of the tissue for a medically acceptable period of time.

[0023] US Patent 6,177,126B1, “Process for the production of a material forsealing and healing wounds,” discloses a method for producing a material for sealing and / or healing wounds, the method comprising: i) filling a liquid composition into a container having two or more plates, at least two of said plates being perforated to have one or more flow holes, and at least one of said perforated plates being movable relative to the other of said perforated plates; ii) transporting a carrier under the container in a transport direction; and iii) continuously moving the perforated plates relative to each other so that the liquid composition drips onto the carrier transported under the container, thereby applying the liquid composition substantially uniformly onto the carrier.

[0024] PCT publication WO2014135689A2 "Powder formulation" discloses a sterile powder composition comprising thrombin and fibrinogen suitable for medical use, wherein the thrombin powder is prepared from a liquid raw material comprising a solution or suspension (preferably a solution) of thrombin, wherein the powder is prepared by removing liquid using a method selected from sterile spray drying or sterile fluidized bed drying, and wherein the powder produced by removing liquid from the raw material exhibits at least 80% of the thrombin potency or activity of the liquid raw material, and wherein the fibrinogen powder is prepared by removing liquid from the raw material using sterile spray drying or sterile fluidized bed drying, wherein the raw material comprises a solution or suspension (preferably a solution) of fibrinogen, and wherein the composition is packaged into a sterile final pharmaceutical product for medical use.

[0025] US Patent Publication: 2010 / 0119563 "SOLID FIBRINOGEN PREPARATION" discloses a solid fibrinogen formulation comprising fibrinogen, which further comprises: (a) albumin; (b) a nonionic surfactant; (c) a basic amino acid or a salt thereof; and (d) at least two amino acids selected from the group consisting of: acidic amino acids, acidic amino acid salts, neutral amino acids and their salts.

[0026] Other patent documents related to the formulation of hemostatic agents include: US9717821B2“Formulations for wound therapy” US7052713B2“Carrier with solid fibrinogen and solidthrombin” US9724213B2“Nanocrystalline cellulose materials andmethods for theirpreparation”US8840877B2“Polysaccharide-protein conjugates reversibly coupledvia imine bonds”US6200587B1“Tissue sealant containing fibrinogen,thrombin andcarboxymethyl cellulose or salt thereof” US8846105B2“Drypowder fibrin sealant” US20160015792A1“POWDER FORMULATION COMPRISING THROMBIN ANDFIBRINOGEN” US6596318B2“Fibrin tissue adhesive formulation andprocess for itspreparation” KR1624625B1“Improved absorbable hemostatic material andmethodforpreparing the same”KR1588633B1“Composition and kit for forming gel forhemostasis and adhesion inhibition”| KR804434B1“Fibrin-based glue granulate and corresponding productionmethod|Fibrin-based glue granulate and amethod ofmanufacturing the same” US9795773B2 "Medicament unit dose cartridge and delivery device" US7351422B2 "Hemostatic soluble cellulose containing fibers coagulating protein for treating wound and process for producing the same" CN101716383A "Preparation method of bleeding stopping and adherencepreventing material | Preparation method for stanching and anti-blocking material" US5484913A "Calcium-modified oxidized cellulose hemostat" EP918548B1 "USE OF OXIDIZED CELLULOSE AND COMPLEXES THEREOF FORCHRONIC WOUND HEALING".

[0027] There is a need for improved forms and materials of hemostasis that are easy to apply and provide rapid hemostasis. Summary of the Invention

[0028] This invention relates to hemostatic materials comprising aggregates, said aggregates comprising fibrinogen, thrombin, and oxidized regenerated cellulose fibers. In some aspects, the hemostatic material further includes additives such as calcium chloride and Tris. In another aspect, this invention relates to a method for preparing the aforementioned hemostatic material, wherein the method involves suspending a mixture of fibrinogen, thrombin, and ORC powder in a non-aqueous solvent, spraying the suspension through a nozzle onto a substrate, removing the hemostatic material from the substrate, and sieving it.

[0029] On the other hand, the present invention relates to a method for treating a wound by applying the above-mentioned hemostatic material to and / or therein a patient's wound.

[0030] In one embodiment, the present invention relates to a method for forming a powdered hemostatic composition by: forming a suspension of a mixture comprising particles of fibrinogen, thrombin, and ORC fibrils in a non-aqueous low-boiling-point solvent; spraying the suspension onto a substrate through a nozzle to evaporate the non-aqueous solvent; separating the composition from the substrate and sieving the composition; thereby forming a powdered hemostatic composition. The non-aqueous low-boiling-point solvent may be hydrofluoroether C4F9OCH3, such as, but not limited to, HFE7100. The suspension may also include Tris and / or calcium chloride. The liquid suspension may contain fibrin sealing agent powder comprising about 90% by weight of fibrinogen, about 8% by weight of thrombin, and about 2.5% by weight of calcium chloride. The weight ratio of fibrin sealing agent powder to ORC in the suspended powdered hemostatic composition may be from about 1:1 to about 10:1. The suspended powdered hemostatic composition may be in powder form having a measured particle size primarily in the range of about 250 micrometers to about 850 micrometers, more preferably from about 355 micrometers to about 850 micrometers. The resulting powdered hemostatic composition comprises at least partially integrated aggregated ORC fibers, fibrinogen, and thrombin, and may also include Tris and / or calcium chloride.

[0031] The present invention also relates to a method of treating a wound by applying the hemostatic composition obtained above to and / or therein. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the manufacturing process.

[0033] Figure 2 The images show test bottles used to evaluate the gelation of the compositions of the present invention and comparative compositions in water.

[0034] Figure 3 The image shows a test bottle used to evaluate the coagulation of blood upon contact with the composition of the present invention and a comparative composition.

[0035] Figure 4 The image shows a test bottle used to evaluate the coagulation of blood upon contact with the composition of the present invention and a comparative composition.

[0036] Figure 5 This is a composite image showing the results of a solubilization test on the comparative compositions.

[0037] Figure 6 This is a composite image showing the results of a solubilization test of the composition of the present invention.

[0038] Figure 7 This is a composite image showing the results of a comparative solubilization test of the composition.

[0039] Figure 8This is a composite image showing the solubilization test results of the compositions of the present invention at different particle sizes.

[0040] Figure 9 A SEM image of one of the compositions of the present invention is shown.

[0041] Figure 10 This is a schematic diagram of the HSM manufacturing process.

[0042] Figure 11 This is a schematic diagram of the HSM manufacturing reactor.

[0043] Figure 12 The hemostatic powder of the present invention prepared by the HSM method of Example 9 is shown.

[0044] Figure 13 A graph showing gel strength expressed in kPa is presented.

[0045] Figure 14 The results of experiments on hemostatic powders prepared using different solvents / binders in animal models are shown.

[0046] Figure 15 A schematic block diagram of an HSM manufacturing method for preparing hemostatic powder using acetone and polymer RG502 is shown.

[0047] Figure 16 A schematic block diagram of a method for manufacturing hemostatic powder using acetone and water is shown.

[0048] Figure 17 A schematic block diagram of a method for manufacturing hemostatic powder using 96% ethanol is shown.

[0049] Figure 18 A schematic block diagram of a method for manufacturing hemostatic powder using HFE / water is shown.

[0050] Figure 19 The density characteristics of hemostatic powders obtained with various solvents / binders are shown.

[0051] Figure 20 The gel strength properties of hemostatic powders obtained with various solvents / binders are shown.

[0052] Figure 21 A lung leakage model with a 1.2 cm long incision is shown.

[0053] Figure 22 The hemostatic powder of the present invention forms a gel on a constricted lung (timed 2 minutes).

[0054] Figure 23The hemostatic powder of the present invention forms a gel that seals pulmonary leakage at 30 cm H2O.

[0055] Figure 24 A lung leakage model with a 1 cm long incision is shown.

[0056] Figure 25 The hemostatic powder of the present invention forms a gel that seals pulmonary leakage at 30 cm H2O.

[0057] Figure 26 The hemostatic powder of the present invention forms a partially layered gel under a pressure of 40 cm H2O (arrows indicate the layered areas). Detailed Implementation

[0058] The inventors have discovered a hemostatic material and a method for preparing the same, which possesses surprising and highly beneficial properties for hemostasis.

[0059] The hemostatic material according to the invention is made of oxidized cellulose-based fibrous material, more preferably of oxidized regenerated cellulose powder, fibrinogen powder, and thrombin powder. The hemostatic material according to the invention represents at least partially integrated ORC fibers, fibrinogen, and thrombin in powder form.

[0060] See Figure 1 A schematic block diagram illustrating the process of preparing the hemostatic material according to the present invention is shown, which includes the following steps: • Preparation of dry powders containing fibrinogen, thrombin, and ORC • A mixture of fibrinogen, thrombin, and ORC powder is suspended in a non-aqueous solvent that can evaporate rapidly under ambient conditions. • The suspension is sprayed onto the substrate through a nozzle. • Hemostatic material obtained by evaporating and drying a non-aqueous solvent • The hemostatic material is removed / separated from the substrate and sieved to form at least partially integrated ORC fibers, fibrinogen, and thrombin in powder form. In one embodiment, powdered Tris or tris(hydroxymethyl)aminomethane buffer is added to the mixture of fibrinogen, thrombin, and ORC to adjust the pH. The mixed composition is then added to HFE to form a suspension. In one embodiment, during the jetting process, the cooling / quenching effect of the material stream due to HFE evaporation causes some ambient moisture to be absorbed onto or into the resulting hemostatic material. Any excess moisture thus absorbed is removed by drying in a vacuum oven in the final drying step.

[0061] According to one aspect of the invention, the ratio of fibrinogen / thrombin mixture to ORC powder in the hemostatic material of the invention is from about 1:1 to about 10:1 by weight.

[0062] According to one aspect of the invention, the hemostatic material of the invention comprises particles with a size of 250 micrometers to 850 micrometers.

[0063] According to one aspect of the invention, the hemostatic material of the invention comprises at least partially integrated ORC fibers, fibrinogen, and thrombin in powder form that are substantially uniformly distributed.

[0064] According to one aspect of the present invention, the hemostatic material of the present invention has high uniformity, integration, rapid gelation / solidification, and strong adhesion.

[0065] According to one aspect of the invention, the collection surface or substrate to which the suspension is sprayed comprises an inert nonwoven felt or a mesh or a steel plate.

[0066] According to another embodiment of the invention, the present invention relates to a hemostatic material comprising aggregates containing fibrinogen, thrombin, and oxidized regenerated cellulose fibers. In some aspects, the hemostatic material further includes additives such as calcium chloride and buffer solutions (such as Tris and / or lysine).

[0067] On the other hand, the present invention relates to a method for preparing the aforementioned hemostatic material by suspending a mixture of fibrinogen, thrombin, and ORC powder in a non-aqueous solvent in a high-shear mixing (HSM) apparatus, followed by high-shear mixing and conventional mixing and / or stirring, while allowing the volatile non-aqueous solvent to evaporate through one or more suitable ports (such as through a gas-sealed structure of the mixing blades / impeller and / or through a vacuum tube). Optionally, a small amount of water or an aqueous solution is introduced into the reactor during mixing to aid particle binding / aggregation / flocculation. The non-aqueous low-boiling-point solvent may be hydrofluoroether C4F9OCH3, such as, but not limited to, HFE7100.

[0068] According to one embodiment of the present invention, the hemostatic aggregate powder comprises ORC fine fibers or powder, thrombin, fibrinogen, lysine (as a buffer / pH adjuster), and calcium salt.

[0069] According to one embodiment of the present invention, a method for preparing such a powdered hemostatic product includes the following steps: mixing the various components as powders with HFE (volatile non-aqueous solvent), and generating a suspension in HFE (volatile non-aqueous solvent) in a high-shear mixing / shear reactor having low-speed mixing blades and high-speed shearing blades. During mixing, the HFE can evaporate through the gas-sealed structure of the mixing blades and / or a vacuum tube. A small amount of water is introduced into the reactor to aid in particle binding / aggregation / clustering.

[0070] According to one embodiment of the present invention, a method for preparing a hemostatic powder includes the following steps: forming a suspension of ORC powder, thrombin, fibrinogen, lysine, and calcium salt in HFE (volatile non-aqueous solvent); stirring the suspension with a high-speed shear blade (and optionally simultaneously and continuously mixing these components with a low-speed mixer); introducing a small amount of water into a reactor to help the particles bind / aggregate / cluster; allowing the HFE to completely evaporate from the reactor; thereby forming a hemostatic powder.

[0071] Example 1: Preparation of hemostatic composition The components of the hemostatic composition of the present invention are prepared as follows.

[0072] Fibrinogen can be prepared using any method for preparing fibrinogen powder, including lyophilization, freeze-drying, etc. In this embodiment, the fibrinogen powder was prepared by spray drying (spray dryer manufacturer: ProCepT, model: 4M8-TriX). The fibrinogen solution formulation was available from Beixiu Biotechnology Co., Ltd., located in Guangzhou, China, and contained fibrinogen, albumin, and other reagents required for WFI. The fibrinogen solution was first atomized through a nozzle in a hot gas stream and then dried immediately. The spray drying parameters are shown in Table 1.

[0073] Table 1 Feed rate 130ml / h Drying column 3 (Mode II) Columnar airflow <![CDATA[0.6m 3 / min]]> Inlet air temperature 150℃ Nozzle diameter 0.8mm atomized airflow 12L / min Swirling gas <![CDATA[0.15m 3 / min]]> Thrombin: Thrombin powder can be prepared using any method, including lyophilization, freeze-drying, etc. In this embodiment, the thrombin powder is prepared by spray drying with a thrombin preparation solution. The thrombin solution preparation is available from Beixiu Biotechnology Co., Ltd., located in Guangzhou, China, and contains thrombin, albumin, and other reagents required in WFI. The spray drying parameters are shown in Table 2.

[0074] Table 2 Feed rate 258±20ml / h Drying column 2 Columnar airflow <![CDATA[0.3m 3 / min]]> Cooling airflow <![CDATA[0.3m 3 / min]]> Inlet air temperature 160℃ Nozzle diameter 0.4mm atomized airflow 7L / min Swirling gas <![CDATA[0.1m 3 / min]]> The thrombin and fibrinogen powders were then mixed together to prepare a complex with the following weight ratio: fibrinogen 89.7%, thrombin 7.8%, and calcium chloride 2.5%, thus forming a fibrin sealing agent powder.

[0075] Fibrinogen and thrombin are derived from porcine plasma, which is fractionated to obtain fibrinogen and thrombin, and is supplied by Bioseal Biotech CO.LTD, located in Guangzhou, China.

[0076] ORC powder can be obtained by treating the original Surgicel fabric. Reference is made to U.S. Provisional Patent Application 62 / 251773, filed November 6, 2015, entitled "Compacted Hemostatic Cellulosic Aggregates," the entire contents of which are incorporated herein by reference for all purposes.

[0077] In short, ORC powder is obtained by treating the original Surgicel fabric through the following process: 1) Divide the fabric and cut it into pieces approximately 2″ x 8″. 2) The fabric is ground into powder using a known grinding method, with a particle size D50 of less than 94 micrometers. One method used in the preparation is ball milling – approximately 100 grams of fabric is placed in a 500 ml zirconia wide-mouth bottle, then 12 to 13 20 mm zirconia balls (agate) are placed in the same bottle, the bottle is sealed, and the bottle is fixed on a Retsch planetary ball mill (model PM100). The fabric is ground at 450 rpm for 20 minutes. The ground powder is then transferred to a sieve with an 8” diameter and 300 micrometer openings. The agate and powder are separated by gentle shaking, and the powder is collected.

[0078] The hemostatic composition of the present invention is prepared by means of a co-spraying method as follows: 1 part by weight of ORC fiber is combined with 1 part, 2 parts, 5 parts, or 10 parts of fibrin sealing agent powder. Thus, as an example, 10g of ORC powder is combined with 10g, 20g, 50g, or 100g of fibrin sealing agent mixed powder to produce 20g, 30g, 50g, or 100g of mixture.

[0079] For each ORC:fibrin sealant ratio considered, a small amount of Tris was added to adjust the pH to 7.0. The pH was adjusted by placing the powder on a wetted surface and measuring the resulting pH, then assessing the amount of Tris required to achieve neutral pH 7. The sample was then discarded. The appropriate amount of dry Tris powder was then added to the powder mixture prior to co-spraying.

[0080] Since Tris is added in dry form, ORC itself is not neutralized. ORC is neutralized only during application when the entire powder formulation is wetted and Tris dissolves. A suspension of FS and ORC powders is prepared using a low-boiling-point non-aqueous solvent. Hydrofluoroether C4F9OCH3, obtained as HFE 7100, is used, for example, Novec 7100 engineered fluid with a boiling point of 61°C supplied by 3M. The HFE 7100 solvent is added to the powder composition and filtered through a 150 μm sieve. A uniformly distributed suspension is formed by continuous stirring at 90 rpm / min in a reservoir at 20 ± 5°C. These suspended components are sprayed through a 0.7 mm diameter nozzle. The nozzle type used is B1 / 8VAU-316SS+SUV67-316SS manufactured by Spraying Systems. The flow rate is 130 ml / min through the nozzle and onto a nonwoven fabric or stainless steel substrate at 20 ± 5°C.

[0081] During the spray suspension process, most of the HFE 7100 solvent evaporates. Any HFE 7100 solvent residue and ambient moisture absorbed by the powder (if present) can be evaporated in a vacuum drying oven at 20±5°C for 24h±5h.

[0082] The resulting hemostatic composition is scraped, peeled, or otherwise separated from the substrate and then passed sequentially through sieves with openings of 850 μm, 355 μm, and 250 μm.

[0083] Table 3 shows the parameters used for the co-spray composition.

[0084] Table 3: Parameters of the co-spraying composition - Fan pressure 0.3 bar atomization pressure 3 bars Flow rate 130ml / min Liquid pressure 16 kPa Nozzle diameter 0.7mm Stirring speed 90 rpm / min As a comparative example, a pure fibroin sealant mixture composition without ORC and without Tris was also prepared using the same spraying method HFE7100.

[0085] Example 2: Comparison of gelation of hemostatic compositions obtained by mechanical mixing and jet mixing Rapid gelation and formation of strong gels are important for hemostatic materials.

[0086] The hemostatic composition of the present invention was prepared by co-spraying method as described above.

[0087] A contrasting mechanically mixed composition was prepared by manually shaking the dry powders (i.e., the FS and ORC powders obtained as described above) to achieve a homogeneous mixture in a container without co-spraying. The composition comprises all the same components (including Tris), differing only in the mixing method.

[0088] The tested ratios of fibrin sealant powder to ORC were 1:1, 2:1, 5:1, and 10:1 (by weight). Therefore, for a 1:1 FS / ORC ratio, 1 part fibrin sealant powder (containing fibrinogen, thrombin, and calcium chloride) was combined with 1 part ORC powder (by weight).

[0089] The mechanically mixed composition and the spray-mixed composition of the present invention were then added to 20 ml of water in a 50 ml bottle, with 200 mg of the mixture positioned at the top of the water surface. After gelation for 2 minutes, the bottle was inverted and observed to see if a gel layer of the composition had formed. In this case, it was observed that the water was sealed by the gel layer and held at the bottom of the bottle by the formed gel layer.

[0090] See now Figure 2 It shows an image of the test bottle inverted at the end of the experiment, in which • Test vial 1 shows a mechanically mixed composition with an FS / ORC ratio of 1:1. • Test vial 2 shows a mechanically mixed composition with an FS / ORC ratio of 2:1. • Test vial 3 shows a mechanically mixed composition with an FS / ORC ratio of 5:1. • Test vial 4 shows a mechanically mixed composition with an FS / ORC ratio of 10:1. • Test vial 5 shows a mechanically mixed composition containing only fibrin sealant powder and no ORC powder. • Test vial 6 shows the composition of the present invention with an FS / ORC ratio of 1:1. right Figure 2 Analysis of the results shows that in test bottles 1-5, gelation was insufficient to retain the fluid, and the fluid was visible at the bottom of the bottle. In test bottle 6, the fluid was visible at the top of the bottle, meaning the water was held in place by the gel layer and prevented from moving to the bottom of the bottle under gravity. Therefore, gelation was insufficient for all proportions of the mechanically mixed compositions and the ORC-free fibrin sealing compositions, while the hemostatic composition of the present invention prepared in a 1:1 ratio exhibited surprisingly strong gelation.

[0091] Example 3: In vitro blood coagulation test The following tests were conducted on the in vitro coagulation of several compositions of the present invention and comparative compositions.

[0092] Add 20 ml of citrate-treated whole blood (pig) to a 50 ml bottle. Add 200 mg of the hemostatic composition to be tested to the top of the blood surface. After 2 minutes of coagulation, invert the bottle and observe the blood coagulation. In the case of complete coagulation, the coagulated blood remains at the top of the inverted bottle. In the case of incomplete coagulation, the blood remains fluid and flows to the bottom of the bottle under gravity.

[0093] The comparative excipients added to the fibrin sealant powder instead of ORC were trehalose, PEG4000, mannitol, and alpha-cellulose (α-cellulose). All excipients were purchased from Aladdin Industries, Inc. The fibrin sealant powder mixture containing trehalose, PEG4000, mannitol, or α-cellulose was prepared by spraying as described above at a ratio of 10:1, i.e., 10 parts fibrin sealant (FS) powder to 1 part of the corresponding excipient. The total amount of the composition of the present invention and the comparative composition added to 20 ml of blood was 200 mg.

[0094] See now Figure 3 It shows an image of the test bottle inverted at the end of the experiment, in which • Test vial 8 shows the hemostatic composition of the present invention with a 2:1 FS / ORC ratio. • Test vial 9 shows the hemostatic composition of the present invention with a 5:1 FS / ORC ratio. • Test vial 10 shows the hemostatic composition of the present invention with a 1:1 FS / ORC ratio. • Test bottle 11 shows a comparative composition prepared by co-spraying, consisting only of fibrin sealant powder and containing no ORC. • Test bottle 12 shows a comparative composition including fibrin sealant powder and α-cellulose with added α-cellulose at a FS / α-cellulose weight ratio of 10:1. • Test bottle 13 shows a comparative composition comprising fibrin sealant powder and trehalose added at a FS / trehalose weight ratio of 10:1. • Test bottle 14 shows a comparative composition comprising fibrin sealant powder and PEG4000 added at a weight ratio of 10:1 (FS / PEG4000). • Test bottle 15 shows a comparative composition comprising fibrin sealant powder and mannitol added at a FS / mannitol weight ratio of 10:1. right Figure 3Analysis of the presented results shows that in test bottles 8-10 containing the hemostatic composition of the present invention, the blood coagulated, with the blood clot visible in the upper part of the inverted bottle, preventing the coagulated blood from moving to the lower part of the bottle under gravity. Therefore, the hemostatic compositions of the present invention prepared at FS / ORC ratios of 2:1, 5:1, and 1:1 exhibit surprisingly strong blood coagulation. Additionally, the comparative sample in bottle 12 containing α-cellulose showed blood coagulation. Comparative examples in bottles 11 (containing only fibrin sealant powder without ORC), 13 (containing fibrin sealant powder and added trehalose), 14 (containing fibrin sealant powder and added PEG4000), and 15 (containing fibrin sealant powder and added mannitol) showed no coagulation or insufficient coagulation, thus insufficient to retain fluid, and the fluid was visible in the lower part of the bottle, i.e., the blood remained fluid and flowed towards the lower part of the bottle under gravity. The hemostatic composition of the present invention exhibits a surprisingly strong coagulation effect.

[0095] Using the same test methods, additional in vitro coagulation tests were conducted on the hemostatic composition of the present invention, a comparative mechanically mixed composition prepared by manually shaking dry powder in a container, and a composition containing only fibrin sealing agent powder without ORC. See now Figure 4 It shows an image of the test bottle inverted at the end of the experiment, in which • Test vial 1 shows a mechanically mixed composition with an FS / ORC ratio of 1:1. • Test vial 2 shows a mechanically mixed composition with an FS / ORC ratio of 2:1. • Test vial 3 shows a mechanically mixed composition with an FS / ORC ratio of 5:1. • Test vial 4 shows a mechanically mixed composition with an FS / ORC ratio of 10:1. • Test vial 5 shows a comparative composition (200 mg) comprising compacted ORC powder aggregates prepared as described in U.S. Provisional Patent Application 62 / 251773 entitled “Compacted Hemostatic Cellulosic Aggregates”, filed November 6, 2015 by Yi-Lan Wang. • Test bottle 6 shows a comparative composition prepared by co-spraying, consisting only of fibrin sealant powder and containing no ORC. • Test vial 7 shows the hemostatic composition of the present invention with a 1:1 FS / ORC ratio. • Test vial 8 shows the hemostatic composition of the present invention with a 2:1 FS / ORC ratio. • Test vial 9 shows the hemostatic composition of the present invention with a 5:1 FS / ORC ratio. • Test vial 10 shows the hemostatic composition of the present invention having a 10:1 FS / ORC ratio. right Figure 4 Analysis of the presented results indicates that the comparative examples in test bottles 1-6 (comprising all proportions of mechanically mixed compositions, compositions containing only ORC powder, and compositions containing fibrin sealant powder but without ORC) showed no coagulation or insufficient coagulation, thus insufficient coagulation to retain fluid, and the fluid was visible at the bottom of the bottle, i.e., the blood remained fluid and flowed downwards under gravity. Conversely, and compared to... Figure 3 The results were similar; in test vials 7-10 containing the hemostatic composition of the present invention, the blood had coagulated, with the blood clot visible in the upper part of the inverted vial, preventing the coagulated blood from moving to the lower part of the vial under gravity. Therefore, the hemostatic compositions of the present invention prepared at FS / ORC ratios of 1:1-10:1 exhibit a surprisingly strong blood-coagulating effect.

[0096] Example 4: Solubilizing effect of the composition When in contact with bodily fluids, the rapid solubilization or solubility of powdered hemostatic compositions can contribute to establishing rapid hemostasis and indicate rapid interaction with fluids. A visual test was conducted as follows: 1 gram of the tested hemostatic powder composition was uniformly applied to an area of ​​a wetted substrate placed in a tray containing pure water. The substrate consisted of a nonwoven fabric topped with a sponge material. Visual observation of the composition's solubility was performed after application to the wetted substrate surface, and results were recorded at zero time (immediately after application), 1 minute after application, and 2 minutes after application.

[0097] See now Figure 5 Composite images show the test results of comparative mechanically mixed compositions prepared by manually shaking dry powder in a container. Images were taken at 0, 1, and 2 minutes for FS / ORC ratios of 1:1, 2:1, 5:1, 10:1, and FS powder without ORC. The results show that for the comparative examples, solubilization was poor even at the 2-minute time point.

[0098] See now Figure 6Composite images showing test results of the hemostatic compositions of the present invention prepared by spraying are presented. Images were taken at 0, 1, and 2 minutes for FS / ORC ratios of 1:1, 2:1, 5:1, 10:1, and FS powder without ORC. The results show good solubilization even at the 1-minute time point and very good solubilization at the 2-minute time point, with rapid and complete solubilization observed at 1 minute for the 1:1 and 2:1 ratios and good solubilization observed at 2 minutes for all ratios. For the comparative examples, pure FS showed poor solubilization even at the 2-minute time point.

[0099] See now Figure 7 Composite images showing test results for a comparative composition comprising FS powder in which excipients are added to fibrin sealant powder in place of ORC, and FS powder without ORC. The excipients are trehalose, PEG 4000, and mannitol. A mixture of fibrin sealant powder containing trehalose, PEG 4000, and mannitol was prepared by spraying as described above at a 10:1 FS / excipient ratio. Images taken at 0, 1, and 2 minutes are shown. The results indicate that, for the comparative example, solubilization was poor even at the 2-minute time point.

[0100] The solubility of the hemostatic composition of the present invention prepared by spraying is affected by the concentration of the ORC component. Even at low concentrations of ORC, the solubility of the composition is improved.

[0101] Example 5: Effect of Particle Size The effect of particle size on the performance of the hemostatic composition of the present invention was evaluated. Particle size was controlled by sequentially passing the composition through sieves with pore sizes of 850 μm, 355 μm, and 250 μm. The powder of the composition of the present invention was divided into size groups primarily larger than 850 μm, primarily 355 μm-850 μm, primarily 250 μm-355 μm, and primarily smaller than 250 μm. Solubility tests were performed on the hemostatic composition of the present invention prepared with a 5:1 FS / ORC ratio.

[0102] See now Figure 8Composite images showing test results of the hemostatic compositions of the present invention prepared by spray method are presented. Images were taken at 0, 1, and 2 minutes for different powder size ranges. The results show that at the 2-minute time point, compositions with a predominantly 355 μm-850 μm particle size exhibit particularly excellent solubilizing activity, compositions with a predominantly 250 μm-355 μm particle size exhibit good solubilizing activity, and compositions with a predominantly larger than 850 μm particle size and a predominantly smaller than 250 μm particle size exhibit inefficient solubilizing activity. Therefore, compositions with a predominantly 250 μm-850 μm particle size range show good solubilizing activity and are the preferred particle size range, with particles predominantly in the 355-850 μm range being particularly preferred. The resulting powder is an aggregate of fibrinogen, thrombin, and ORC, and many of the particles are larger than the particle size of the raw materials.

[0103] Example 6: Effect of adding Tris Peeling tests were conducted on the compositions of the present invention with and without Tris. The addition of Tris was titrated to achieve pH = 7. The Tris powder was milled and passed through a 150 μm sieve. Powder smaller than 150 μm was collected and added in a predetermined amount to the dried mixture prior to co-spraying to adjust the pH of the composition.

[0104] The peel test was performed as follows. 0.5 g of the composition of the present invention was applied to dermal tissue, covered with a composite bilayer matrix, pressed into the powder for 3 minutes, and the separation force between the sample and the tissue was measured using an Instron tensile testing machine and recorded as force / unit width (N / m). The composite bilayer matrix comprises a synthetic absorbable poly(glycolic acid-lactide) (PGL, 90 / 10 mol / mol) nonwoven layer needle-punched into a woven oxidized regenerated cellulose carboxylate (ORC) fabric, as described in U.S. Patent 7,666,803 entitled “Reinforced absorbable multilayered fabric for use in medical devices” by D. Shetty et al., which is incorporated herein by reference.

[0105] Referring now to Table NN, the adhesive strength of the formulations of this invention is shown as a function of the amount of ORC added. Adhesive strength is lower at higher ORC contents, even though a 1:1 FS powder:ORC fiber formulation exhibits considerable peel strength.

[0106] Referring now to Table 4, the adhesive forces of the formulations of the present invention, with and without Tris, are shown for different FS / ORC ratios and corresponding pH values. Tris was added in the listed weight percentages to adjust the pH to 7.0. Although all compositions exhibited high peel strength, the presence of Tris clearly resulted in higher peel strength for the same FS / ORC ratio, with some showing 2-4 times higher peel strength.

[0107] Table 4: Adhesion strength of the formulations of this invention containing and without Tris Data analysis shows that the addition of Tris remarkably improves the adhesive or peel strength of the compositions of the present invention with a neutral pH, although the strength is somewhat small.

[0108] Example 7: Characterization of Particles See now Figure 9 The image shows a magnified SEM image of the FS / ORC composition of the present invention at a ratio of 5:1. It is clear that the components of the composition are at least partially integrated, i.e., attached to or encapsulating each other, rather than simply mechanically mixed.

[0109] Examination of the powdered composition of the present invention showed that the components were well mixed and that the biopharmaceutical adhered tightly to the ORC fibers.

[0110] Example 8: Hemostasis Test The following in vivo tests were conducted to assess the hemostatic efficacy of the hemostatic composition of the present invention in a liver abrasion model. A liver abrasion model was created by establishing a 3cm × 3cm exudate area on the surface of a pig liver. 0.5g of the hemostatic composition of the present invention, with a FS / ORC ratio of 5:1, was applied to cover the exudate area without any packing material. Hemostasis was achieved within 2 minutes.

[0111] The following is an in vivo test of the hemostatic efficacy of the hemostatic composition of the present invention in a liver resection model. The liver resection model was created using a portal vein occlusion technique (a surgical procedure used in some abdominal surgeries), which requires the application of a large amount of non-invasive hemostatic agent via clamps. First, portal vein occlusion was applied to control bleeding, then a 5 cm long and 5 cm wide incision was made along the edge of the liver to expose the bile ducts. Immediately afterwards, the hemostatic composition powder of the present invention was applied to cover the cross-section, while saline was sprayed until bleeding stopped. The Pringle clamps were then released to examine the results. Hemostasis was observed, and bile leakage was prevented after the Pringle clamps were released. Hemostasis was achieved within 2 minutes.

[0112] Example 9: Manufacturing method in a high-shear reactor According to one embodiment of the present invention, a method for preparing a powdered hemostatic product includes the following steps: mixing the various components as powders with HFE (volatile non-aqueous solvent), and generating a suspension in HFE (volatile non-aqueous solvent) in a high-shear mixing / shear reactor having low-speed mixing blades and high-speed shearing blades. During mixing, the HFE can evaporate through the gas-sealed structure of the mixing blades and / or a vacuum tube. A small amount of water is introduced into the reactor to aid particle binding / aggregation / clustering. The amount of water is sufficient for a small portion of the biological agent to react and form particles, but insufficient for all the biological agents, i.e., thrombin and fibrinogen cannot react completely to convert all fibrinogen into fibrin. The fibrinogen portion converted into fibrin is about 0.1% to about 50%, more preferably 1% to 25%, and even more preferably 2% to 10%. The hemostatic powder prepared according to the present invention always contains a certain amount of coagulating fibrinogen, which can react when the powder is applied to or in connection with a wound.

[0113] According to one embodiment of the present invention, a method for preparing a hemostatic powder includes the following steps: forming a suspension of ORC powder, thrombin, fibrinogen, lysine, and calcium salt in HFE (volatile non-aqueous solvent); stirring the suspension with a high-speed shear blade (and optionally simultaneously and continuously mixing these components with a low-speed mixer); introducing a small amount of water into a reactor to help the particles bind / aggregate / cluster; allowing the HFE to completely evaporate from the reactor; thereby forming a hemostatic powder.

[0114] See Figure 10 A schematic block diagram of a method for preparing a hemostatic material according to the present invention is shown, and includes the following steps: • Prepare a dry powder mixture of fibrinogen, thrombin, ORC, optionally calcium chloride, and optionally buffer compounds (such as Tris and / or lysine); • The dry powder mixture is suspended in a non-aqueous solvent that can evaporate rapidly under ambient conditions in a high-shear mixer reactor. • The suspension is stirred with high-speed shear blades, and optionally, the components are continuously mixed simultaneously with low-speed mixer blades. • Introduce a small amount of water into the reactor to help particles bind / aggregate / flocculate. • Evaporate the non-aqueous solvent from the reactor and dry the resulting hemostatic material. • The resulting hemostatic material is removed from the reactor and optionally sieved to form at least partially integrated ORC fibers, fibrinogen, and thrombin in aggregated powder form.

[0115] In one embodiment, lysine or Tris in powder form is added to the mixture of fibrinogen, thrombin, and ORC to adjust the pH.

[0116] According to one aspect of the invention, the hemostatic material of the invention comprises at least partially integrated ORC fibers, fibrinogen, and thrombin in powder form that are substantially uniformly distributed.

[0117] According to one aspect of the present invention, the hemostatic material of the present invention has high uniformity, integration, rapid gelation / solidification, and strong adhesion.

[0118] See now Figure 11 A schematic diagram of a high-shear mixing (HSM) reactor is shown, wherein a container or bowl 5 and an impeller 1 are used for mixing and suspending the feedstock (low-speed mixer); high-speed shear blades or shredder blades 2; a spray nozzle 3 is used to add water (binder); a vent 4 is used to remove HFE volatile fluids; and a channel 6 is used to optionally introduce air or gas through the mixing and / or shear blade sealing structure to remove HFE volatile fluids. Alternatively, a dedicated air inlet (not shown) may be provided. Alternatively, vent 4 may be connected to a vacuum.

[0119] HSM reactors are commercially available. The reactor used in this embodiment is an HSM-type Mini-CG purchased from Chuangzhi Electromechanical Co., Ltd. (China).

[0120] Example 10: Comparison of hemostatic powders prepared by the spraying method of Example 1 and the HSM method of Example 9 test The inventors compared the particles prepared by the HSM method (Example 9) with those prepared by spraying an HFE suspension through a nozzle (Example 1). The experimental results showed that the hemostatic powder prepared by the HSM method possessed comparable physicochemical properties and functional properties, such as tensile strength and adhesive strength, and exhibited comparable hemostatic efficacy to the powder prepared by the spraying method of Example 1 in animal studies.

[0121] A comparison of the powder of Example 1 prepared with Tris buffer and the powder prepared with lysine as a buffer compound in Example 9 (HSM method) showed no significant difference in the performance of the hemostatic powder.

[0122] Advantageously, the HSM method for preparing hemostatic powder particles offers the advantage of providing an HSM device, which is a substantially sealed system, allowing the powder to be granulated in a sealed environment with no material loss during the process. Bioburden can be minimized compared to the spray method of Example 1.

[0123] See Figure 12 This illustrates a hemostatic powder prepared by the HSM method of Example 9.

[0124] Example 11: Preparation of hemostatic compositions using the HSM method: Comparison of various solvents / binders The inventors used the HSM method (Example 9) to manufacture hemostatic powders using several different solvents / binders. See also Figure 13The graph shows gel strength expressed in kPa, where the gel is formed from the hemostatic powder of the present invention, thereby preparing the powder using one of four different solvents and / or binders. The gel strength at several different test points is shown. The first section of the graph shows the gel strength with acetone as the volatile suspension solvent and polymer RG502 (D,L-lactide-co-glycolic acid) as the binder. The second section shows the gel strength with an ethanol / water combination as both the volatile solvent and binder. The third section shows the gel strength with an acetone / water combination as both the volatile solvent and binder. The fourth section shows the gel strength with an HFE / water combination as both the volatile solvent and binder. Figure 13 As can be seen from the chart, the HFE / water combination exhibits the highest gel strength.

[0125] See now Figure 14 This study presents the experimental results of hemostatic powders prepared using different solvents / binders in animal models (hemostasis test of a cortical liver surface model). From Figure 14 It can be seen that the combinations of acetone / RG502, ethanol / water, and acetone / water all showed bleeding 3 minutes after application. In contrast, the HFE / water combination of the present invention showed that the bleeding stopped after only 1 minute.

[0126] Example 12: Preparation of hemostatic composition using HSM method and polymer RG502 / acetone 200g of raw material powder comprising fibrinogen powder / thrombin powder / ORC powder / calcium chloride powder and Tris was prepared according to the formulation shown in Table 5. The binder / suspension was prepared from 74.7g of acetone and 8.3g of polymer RG502 (D,L-lactide-co-glycolic acid), wherein the polymer was completely dissolved and thoroughly mixed.

[0127] Table 5: Formulation of Example 12 The principle block diagram of the manufacturing method is as follows: Figure 16 As shown. All dried materials were transferred to a bowl in an HSM and premixed for 5 minutes using a mixing impeller speed of 75 rpm and a high-shear blade shredder speed of 1000 rpm. The binder / suspension (RG502 / acetone) was then sprayed onto the dried materials via a peristaltic pump and nozzle at a feed flow rate of 60 g / min. The polymer / acetone solution began to bind the raw material particles to form a powder.

[0128] Then increase the impeller speed to 150 rpm and the shredder speed to 3000 rpm, and continue the post-granulation process for 5 minutes.

[0129] After granulation, the material was sieved using a 1.7 mm sieve, and the resulting powder was collected below the sieve. The powder was transferred to a vacuum drying oven and dried for 1 hour. The product was then sieved using two sieves (106 μm and 425 μm sieves). The final powder fraction was collected between the two sieves.

[0130] Example 13: Preparation of a hemostatic composition using the HSM method and water / acetone Prepare 200g of raw material powder comprising fibrinogen powder / thrombin powder / ORC powder / calcium chloride powder and Tris according to the formulation shown in Table 6. The binder / suspension is prepared by thoroughly mixing 68g of acetone and 17g of purified water.

[0131] Table 6: Formulation of Example 13 The principle block diagram of the manufacturing method is as follows: Figure 18 As shown. All dried materials are transferred to a bowl in an HSM and premixed for 5 minutes using a mixing impeller speed of 75 rpm and a high-shear shredder speed of 1000 rpm. The binder / suspension (water / acetone) is then sprayed onto the dried materials via a peristaltic pump and nozzle at a feed flow rate of 60 g / min. The solution begins to bind the raw material particles to form a powder.

[0132] Then the impeller speed was increased to 120 rpm and the shredder speed was increased to 3000 rpm, and the post-granulation process continued for 3 minutes.

[0133] After granulation, the material was sieved using a sieve (1.7 mm aperture) and the resulting powder was collected below the sieve. The powder was transferred to a vacuum drying oven and dried under vacuum for 1 hour. The product was then sieved using two sieves (106 μm and 425 μm aperture). The final powder fraction was collected between the two sieves.

[0134] Example 14: Preparation of a hemostatic composition using the HSM method and 96% ethanol Prepare 150g of raw material powder comprising fibrinogen powder / thrombin powder / ORC powder / calcium chloride powder and Tris according to the formulation shown in Table 7. The binder / suspension is prepared from 60g of 96% ethanol.

[0135] Table 7: Formulation of Example 14 The principle block diagram of the manufacturing method is as follows: Figure 20As shown. All dried materials were transferred to a bowl in an HSM and premixed for 3 minutes using a mixing blade impeller speed of 75 rpm and a high-shear blade shredder speed of 1000 rpm. The binder / suspension (96% ethanol) was then sprayed onto the dried materials through a peristaltic pump and nozzle at a feed flow rate of 4 g / min. The solution began to bind the raw material particles to form a powder.

[0136] Then increase the impeller speed to 120 rpm and the shredder speed to 1500 rpm, and continue the post-granulation process for 3 minutes.

[0137] After granulation, the material was sieved using a sieve (pore size 710 μm) and the resulting powder was collected below the sieve. The powder was then transferred to a tray oven for drying and dried at 45°C for 0.5 hours.

[0138] The product was then sieved using two sieves (100 μm and 315 μm aperture sizes). The final powder fraction was collected between the two sieves. Example 15: Preparation of hemostatic composition using HSM method and HFE / water Prepare 100g of raw material powder comprising fibrinogen powder / thrombin powder / ORC powder / calcium chloride powder and Tris according to the formula shown in Table 8. Utilize 500g of HFE7100.

[0139] Table 8: Formulation of Example 15 The principle block diagram of the manufacturing method is as follows: Figure 22 As shown. Transfer all dried material to a bowl in an HSM container and add 500g of HFE7100. Premix the composition for 3 minutes using a mixing blade impeller speed of 200rpm to form a suspension. Then, inject 9g of water into the suspension through a peristaltic pump and nozzle, with a feed flow rate of 4.5g / min. The solution begins to bind the raw material particles to form a powder.

[0140] Then adjust the impeller speed to 100-300 rpm and the shredder speed to 150-1000 rpm, and continue the granulation process for 10 minutes. Adjust the sealing pressure of the impeller and shredder to 0.02 MPa to blow out the HFE and dry the composition.

[0141] After granulation, the material is sieved using a sieve (pore size 710 μm) and the resulting powder is collected below the sieve. The powder is then transferred to a vacuum chamber for drying under a vacuum of 0-10 Pa for 12-24 hours.

[0142] The product was then sieved using two sieves (pore sizes 106 μm and 355 μm). The final powder fraction was collected between the two sieves.

[0143] Example 16: Comparison of hemostatic powder formulations obtained in Examples 12, 13, 14, and 15The properties of the hemostatic powders obtained as described in Examples 12, 13, 14, and 15 were compared. Water content was determined using the Karl Fischer method; thrombin potency was tested based on coagulation time; coagulating proteins were tested by quantitative fibrinogen testing; particle size was tested using a laser particle size analyzer; density was measured as bulk density; and gel strength was measured by tensile strength testing. The results are shown in Table 9 and... Figure 19 , Figure 20 middle.

[0144] Table 9: Properties of hemostatic powders obtained with various solvents / binders Advantageously, the volatility of HFE potentially increases the porosity of the final hemostatic powder product, which will lead to a lower density of the final product and accelerate solubility or redispersion time. Compared with other methods, the powder of Example 15 has the lowest density, while the resulting gel has the highest strength. Figure 14 As shown, it also has better hemostatic properties.

[0145] Example 17: Experiment of the hemostatic powder of the present invention in a lung closure model Two studies were conducted to test the sealing function of the hemostatic powder of the present invention in a lung leakage model. Both studies showed that at a pressure of 30 cm H2O, the gel formed by the powder of the present invention adhered tightly to the lung tissue and effectively sealed the leakage. When the pressure was 40 cm H2O, the gel began to delaminate, but it could still seal the leakage.

[0146] Figure 21 A lung leakage model with a 1.2 cm long incision is shown; Figure 22 The hemostatic powder of the present invention forms a gel on a constricted lung (timed for 2 minutes); Figure 23 This invention demonstrates the formation of a gel by the hemostatic powder of the present invention to seal lung leakage at 30 cm H2O. Figure 24 A lung leakage model with a 1cm long incision is shown; Figure 25 This invention demonstrates the formation of a gel by the hemostatic powder of the present invention to seal lung leakage at 30 cm H2O. Figure 26 The hemostatic powder of the present invention forms a partially layered gel under a pressure of 40 cm H2O (arrows indicate the layered areas).

[0147] Advantageously, it has been demonstrated that the hemostatic powder of the present invention can seal lung leakage.

Claims

1. A method for forming a powdered hemostatic composition, comprising the following steps: a) Forming a suspension comprising a mixture of particles in a non-aqueous low-boiling solvent, said particles comprising fibrinogen, thrombin and oxidized regenerated cellulose (ORC) fibers, said non-aqueous low-boiling solvent being hydrofluoroether; b) Stirring and shearing the suspension in a high-shear mixing reactor; c) Add at least 9g of water per 100g of the particles, causing at least 0.1% of fibrinogen to be converted into fibrin at least 25% to form aggregated particles containing said fibrinogen, thrombin and ORC fibrin, while preventing the fibrinogen from completely coagulating. d) Evaporate the non-aqueous solvent; e) Dry and sieve the composition; and This forms the powdered hemostatic composition.

2. The method according to claim 1, wherein the non-aqueous low-boiling solvent comprises hydrofluoroether C4F9OCH3.

3. The method according to claim 2, wherein the non-aqueous low-boiling solvent comprises HFE7100.

4. The method according to claim 1, wherein the suspension further comprises lysine or Tris.

5. The method according to claim 1, wherein the suspension further comprises calcium chloride.

6. Use of the hemostatic composition formed according to claim 1 in the preparation of a medicament for treating a wound by applying the hemostatic composition to and / or into the wound.

7. Use of the hemostatic composition formed according to claim 1 in the preparation of a medicament for treating air leakage of the lung by applying the hemostatic composition to and / or into the lung.

8. A hemostatic composition formed by the method of any one of claims 1-5.

9. A hemostatic material comprising aggregates, said aggregates comprising fibrinogen, thrombin, and oxidized regenerated cellulose fibers.

10. A method for preparing a hemostatic material, the method comprising the following steps: A mixture of fibrinogen, thrombin, and ORC powder is suspended in a non-aqueous solvent. The suspension is sprayed onto a substrate through a nozzle, and the hemostatic material is removed from the substrate and sieved.

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