Fluid hemostatic paste

Abstract

The present invention provides a fluid composition for preparing a spreadable, paste-like hemostatic agent having medicinal or pharmacological effects, which can be easily applied to necessary areas of the body, including hard-to-reach areas, and a method for preparing the composition. [Solution] A two-component fluid composition is provided, comprising (i) powders of oxidized cellulose, fibrinogen, and thrombin, each having a weight ratio of oxidized cellulose to fibrinogen in the range of about 1:5 to about 2:1, and (ii) a dispersion medium composed of glycerol and an aqueous solution, wherein the composition is in the form of a paste-like suspension at room temperature. Use of the composition for treating bleeding tissue and a method for preparing the composition are also provided.

Description

Technical Field

[0001] The present invention particularly relates to a fluid hemostatic composition comprising a blend of fibrinogen, thrombin, and a dispersant.

Background Art

[0002] In various situations during surgery, therapeutic agents are spread into the human body to prevent postoperative bleeding. Existing drugs have drawbacks; for example, it is difficult to place a sheet in minimally invasive surgery (MIS).

[0003] The selection of an appropriate method or product for controlling bleeding depends on many factors including, but not limited to, the severity of bleeding, the anatomical location of the bleeding source and the proximity of adjacent important structures, whether the bleeding is from a discrete bleeding source or from a larger surface area, the visibility and accurate identification of the bleeding source, and access to the bleeding source.

[0004] In efforts to address the above problems, substances for controlling excessive bleeding have been developed. Topical absorbent hemostats (TAHs) are widely used in surgical applications. TAHs include products based on oxidized cellulose (OC), gelatin, collagen, chitin, chitosan, etc. To improve hemostatic performance, scaffolds based on the above substances can be combined with biologically induced coagulation factors such as thrombin and fibrinogen.

[0005] Due to their biodegradability, as well as their bactericidal and hemostatic properties, oxidized cellulose (OC) materials, such as oxidized regenerated cellulose (ORC), have long been used as topical hemostatic agents. OC and ORC materials are also used as adhesion barriers. ORC products are used in a variety of surgical procedures, including neurosurgery, abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck surgery, pelvic surgery, and skin and subcutaneous tissue procedures. Several methods are known for forming various types of hemostatic agents based on OC materials, whether in powder, woven, nonwoven, knitted, or other forms. Hemostatic agents currently in use include powders or fabrics containing ORC.

[0006] However, since bleeding control is essential and crucial in surgical procedures to minimize blood loss, reduce postoperative complications, and shorten operating time in the operating room, improved forms and materials are needed to facilitate ease of application, especially at bleeding sites and in hard-to-reach areas.

[0007] U.S. Patent Application No. 2020 / 0,139,021 relates to a composition comprising oxidized cellulose (OC) and glycerol, wherein the ratio of glycerol to OC is at least about 0.5:1 w / w glycerol:OC, and / or the viscosity of the composition is at least 10% higher than the viscosity of glycerol and about 2.6 × 10⁻⁶ 9 A composition is disclosed having a cP of less than 8 w / w% and a total water content of less than approximately 8 w / w%.

[0008] U.S. Patent No. 9,265,858 discloses a dry composition that, upon addition of an aqueous medium, forms a substantially homogeneous paste suitable for use in hemostatic procedures. [Overview of the Initiative] [Means for solving the problem]

[0009] The present invention relates, in particular, to a fluid hemostatic composition comprising a blend of fibrinogen, thrombin, and a dispersant.

[0010] The object of the present invention is to provide a composition for preparing a paste-like, spreadable hemostatic agent having medicinal or pharmacological effects, which can be easily applied to necessary areas, including areas of the body that are difficult to reach.

[0011] Fibrinogen interacts with thrombin in aqueous media. Since commonly used dispersions such as water or hydrophilic dispersions cannot be used without impairing pasteurity, adhesion, and / or hemostatic function, finding a suitable formulation with appropriate viscosity is not easy. Therefore, for stable liquid / flowable compositions containing both fibrinogen and thrombin, a non-aqueous media (dispersant) is generally used before application to the bleeding site, for example, for sealing and hemostasis. Since commonly used solvates or non-solvates, such as water or oil, cannot be used without impairing the active function of OC, finding a suitable formulation to give OC the appropriate consistency is not easy.

[0012] The inventors have developed a composition, for example, one containing oxidized cellulose (OC), for preparing a spreadable paste or powder for use as a hemostatic agent, which can be easily applied to the required site.

[0013] Therefore, the present invention is based on the remarkable finding that such a paste-like suspension containing powders of OC, fibrinogen, and thrombin dispersed in a dispersion medium, for example, glycerol and an aqueous solution, provides excellent hemostatic properties. Thus, such a composition can be applied to the required site to obtain biological activity, such as controlling bleeding, particularly in hard-to-reach areas of the body.

[0014] While using an aqueous solution with such a suspension is counterintuitive, without being bound by any specific theory or mechanism, it can be assumed that after mixing the powder in a dispersion medium, the uniformly dispersed biopharmaceutical forms fibrin in a glycerol / saline system, which, together with OC, forms a porous paste, leaving abundant thrombin in the system. Subsequently, when applied to the bleeding site, the porous, fluid ORC-fibrin matrix (which is insoluble in water) allows blood to easily penetrate through its spaces and be exposed to a high concentration of thrombin, thereby accelerating the formation of an endogenous fibrin clot upon contact with blood. Thus, without being bound by theory, it can be assumed that such a porous matrix of OC-fibrin also provides an environment for red blood cells and platelets to adhere and aggregate, and that the fluid matrix can flow into deep fissures and irregular areas, allowing it to adhere closely to the bleeding site after gentle pressure.

[0015] Thus, the disclosed compositions have been demonstrated to be easy to use, stable, and effective hemostatic agents.

[0016] According to aspects of the present disclosure, a two-component fluid composition is provided, comprising (i) a powder comprising oxidized cellulose, fibrinogen, and thrombin, wherein the weight ratio of oxidized cellulose to fibrinogen is in the range of 1:5 to 2:1, and (ii) a dispersion medium comprising glycerol and an aqueous solution, and being in the form of a paste-like suspension at room temperature.

[0017] In some embodiments, the weight ratio of OC to fibrinogen is in the range of 1:2 to 2:1.

[0018] Referring to Figure 2, it is shown that using 3 mL of dispersion medium (an aqueous solution containing 50 vol% glycerol) per 1.2 g of powder results in a composition that is too concentrated, while using 5 mL results in a composition that is too dilute. Therefore, in some embodiments, the dispersion medium exists in a dispersion medium-to-powder ratio ranging from approximately 2.5:1 to approximately 5:1 (v / w, in mL / g units) (including any value and range between these), for example, 2.5:1, 3:1, 3.5:1, 4:1, or 5:1.

[0019] In some embodiments, OC includes oxidized regenerated cellulose (ORC).

[0020] In some embodiments, at least a portion of the powder is in an aggregated form. In some embodiments, the OC is in a pulverized form.

[0021] In some embodiments, the composition further comprises fibrin. In some embodiments, the fibrin is at least partially crosslinked.

[0022] In some embodiments, thrombin is present in an amount of about 2000 to about 4000 IU per gram of powder.

[0023] In some embodiments, the composition includes a calcium salt.

[0024] In some embodiments, the composition includes a buffer, for example, in the form of a powder. In some embodiments, the buffer is selected from Tris, Lysine, or a combination thereof.

[0025] In some embodiments, the particle size of at least 90% of the powder is in the range of 10 to 2,000 μm. In some embodiments, the particle size is in the range of about 200 to about 900 μm.

[0026] In some embodiments, glycerol is present at a concentration greater than about 15% by volume of the dispersion medium. In some embodiments, glycerol is present at a concentration greater than about 40% by volume of the dispersion medium. In some embodiments, glycerol is present at a concentration of about 70% by volume or less of the dispersion medium.

[0027] In some embodiments, the composition is stable for at least about 20 hours at a pH of 4.5 - 6.5 at about room temperature.

[0028] In some embodiments, the aqueous solution includes physiological saline.

[0029] In some embodiments, the powder is spray dried and / or high shear mixed.

[0030] In some embodiments, fibrinogen is present in a concentration range of 60% to 95% by weight or 70% to 90% by weight of the powder. In some embodiments, the composition is for use as a hemostatic agent at the bleeding site of tissue.

[0031] In one aspect, there is provided the use of the composition in a method of treating bleeding tissue, the method comprising applying the composition of any of its embodiments onto the bleeding tissue. In some embodiments, the method includes minimally invasive surgery (MIS) such as laparoscopic surgery.

[0032] In another aspect, there is provided a method of making the composition of any of its embodiments, the method comprising combining OC (e.g., ORC) fibers / powder with fibrinogen powder and thrombin powder under conditions that allow for the formation of an aggregated form of OC fibers / powder, fibrinogen, and thrombin, and combining the aggregates with a dispersion medium comprising glycerol and an aqueous solution.

[0033] In some embodiments, the conditions allow for at least partial conversion of fibrinogen to fibrin. In some embodiments, the conditions allow for at least partial crosslinking of fibrin. In some embodiments, the conditions are selected from one or more of the following: (i) to (iii): (i) a temperature in the range of 0 to 30°C, (ii) a pH in the range of 4.5 to 6.5, and (iii) a dispersion medium having a dispersion medium-to-powder ratio in the range of 3:1 to 4:1 (v / w mL / g), respectively.

[0034] In another embodiment, a kit is provided which includes a container for containing the composition of any embodiment, an applicator for applying the composition onto tissue, and optionally, instructions for use.

[0035] In another embodiment, a kit is provided comprising a container for containing a powder comprising oxidized cellulose, fibrinogen, and thrombin, each having a weight ratio of oxidized cellulose to fibrinogen in the range of about 1:5 to about 2:1; a container for containing a dispersion medium comprising glycerol and an aqueous solution; and optionally, instructions for use.

[0036] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. Similar or equivalent methods and materials may be used to perform or test embodiments of the present invention, but exemplary methods and / or materials are described below. In case of any inconsistency, this specification shall prevail, including definitions. The materials, methods, and examples are illustrative and not necessarily intended to be limiting. [Brief explanation of the drawing]

[0037] Several embodiments of the present invention will be described herein with reference to the accompanying drawings for illustrative purposes only. It should be emphasized that the details shown herein are illustrative and intended to illustrate embodiments of the present invention. In this regard, the description with reference to the drawings will make it clear to those skilled in the art how embodiments of the present invention may be carried out. [Figure 1] As described in Example 2, comparative photographic images showing the fluidity of the selected sample (1.2 g of powder in 4 mL of 50 vol% glycerol in physiological saline) are presented, from left to right: (ii) powder with an ORC to fibrinogen weight ratio of 2:1 (containing thrombin, i.e., a dose of 3300 IU per 1.2 g), (ii) powder with an ORC to fibrinogen weight ratio of 2:1 but without thrombin, (iii) powder containing ORC and BSA (bovine serum albumin), with an ORC to BSA weight ratio of 2:1 and containing thrombin, (iv) ORC, (v) powder containing ORC and thrombin (without fibrinogen). [Figure 2] Comparative photographic images showing the fluidity of the selected samples are presented according to Table 1 in the following Examples section (each panel is labeled with the corresponding sample number from left to right). [Figure 3A] Comparative photographic images are presented showing the fluidity and coagulation time of tested samples with various ORC to fibrinogen weight ratios ("ratios") ranging from 2:1 to 1:5, specifically 70% or 80% glycerol in physiological saline with ratios of 2:1, 1:1, 1:2, and 1:5 (Figure 3A, from left to right), 70% glycerol with ratios of 1:2 and 1:5 (Figure 3B, from left to right), blood coagulation (80% glycerol with a ratio of 1:5, after more than 180 seconds, Figure 3C), and blood coagulation (70% glycerol with ratios of 1:2 and 1:5, after 70 seconds, Figure 3D, from left to right). [Figure 3B]Comparative photographic images are presented showing the fluidity and coagulation time of tested samples with various ORC to fibrinogen weight ratios ("ratios") ranging from 2:1 to 1:5, specifically 70% or 80% glycerol in physiological saline with ratios of 2:1, 1:1, 1:2, and 1:5 (Figure 3A, from left to right), 70% glycerol with ratios of 1:2 and 1:5 (Figure 3B, from left to right), blood coagulation (80% glycerol with a ratio of 1:5, after more than 180 seconds, Figure 3C), and blood coagulation (70% glycerol with ratios of 1:2 and 1:5, after 70 seconds, Figure 3D, from left to right). [Figure 3C] Comparative photographic images are presented showing the fluidity and coagulation time of tested samples with various ORC to fibrinogen weight ratios ("ratios") ranging from 2:1 to 1:5, specifically 70% or 80% glycerol in physiological saline with ratios of 2:1, 1:1, 1:2, and 1:5 (Figure 3A, from left to right), 70% glycerol with ratios of 1:2 and 1:5 (Figure 3B, from left to right), blood coagulation (80% glycerol with a ratio of 1:5, after more than 180 seconds, Figure 3C), and blood coagulation (70% glycerol with ratios of 1:2 and 1:5, after 70 seconds, Figure 3D, from left to right). [Figure 3D] Comparative photographic images are presented showing the fluidity and coagulation time of tested samples with various ORC to fibrinogen weight ratios ("ratios") ranging from 2:1 to 1:5, specifically 70% or 80% glycerol in physiological saline with ratios of 2:1, 1:1, 1:2, and 1:5 (Figure 3A, from left to right), 70% glycerol with ratios of 1:2 and 1:5 (Figure 3B, from left to right), blood coagulation (80% glycerol with a ratio of 1:5, after more than 180 seconds, Figure 3C), and blood coagulation (70% glycerol with ratios of 1:2 and 1:5, after 70 seconds, Figure 3D, from left to right). [Figure 4] This document presents graphs showing the time-dependent viscosity curves of various samples with ORC to fibrinogen weight ratios of 2:1 ("1"), 1:1 ("2"), 1:2 ("3"), and 1:5 ("4") dispersed in an 80 vol% glycerol solution in physiological saline. [Figure 5] This graph shows the results of amplitude sweep tests performed on various samples of ORC to fibrinogen weight ratios of 2:1 ("1", "1'"), 1:1 ("2", "2'"), 1:2 ("3", "3'"), and 1:5 ("4", "4'") dispersed in a 50 vol% glycerol solution in physiological saline. In the plot, square symbols indicate G' and triangular symbols indicate G''. [Figure 6] As described in Example 4, a graph showing the porcine blood coagulation time (ex vivo test) using samples with different formulations 2 hours and 20 hours after reconstitution (physiological saline and glycerol of various concentrations) is presented (see "Effects of pH and glycerol concentration on stability"; circles indicate solid-liquid phase separation after 20 hours for sample 6). [Figure 7] As described in Example 4, graphs showing the clotting times of porcine blood at various samples and pH levels 0.5 hours, 2 hours, and 20 hours after reconstitution (physiological saline and glycerol of various concentrations) are presented (see Table 3). [Figure 8] As described in Example 5, graphs showing the effect of glycerol concentration on the time-dependent thrombin stability of ORC vs. fibrinogen preparations in 2:1 and 1:1 ratios at various pH values ​​are presented (see Table 4). [Figure 9] As described in Example 5, graphs showing the effect of pH on the time-dependent thrombin stability of 2:1 and 1:1 ORC to fibrinogen ratio formulations at various pH values ​​(in physiological saline + 50% volume glycerol) are presented (see Table 5). [Figure 10] As described in Example 6, a graph showing the thickness in millimeters over time for the selected ORC to fibrinogen ratio sample (1.2 g in 4 mL of 50% vol. glycerol + physiological saline) is presented (see Table 6). [Figure 11]An image illustrating the grades of bleeding levels used in a spleen biopsy punch model is presented. White circles represent punches, gray circles represent the background, and black circles represent blood flowing from the biopsy punch site (from left to right panel: no bleeding "0", exudation "1", very mild "2", mild "3", moderate "4", severe "5"). [Figure 12] A bar graph summarizing the hemostatic success rate for various formulations, as determined in in vivo punch model tests using a 6 mm biopuncher with a spleen biopunch model under heparinization conditions, is presented (N=5, see Example 7). [Figure 13] A bar graph summarizing the hemostatic success rate for various formulations, as determined in in vivo dog punch model tests using a 6mm biological puncher with a spleen biological punch model under heparinization conditions, is presented (N=5, see Example 7). [Modes for carrying out the invention]

[0038] The creation of easily prepared, fluid hemostatic fibrin complex matrix media for intraoperative application to hard-to-reach bleeding sites is particularly challenging, especially during minimally invasive surgery (MIS), such as endoscopic surgery.

[0039] The object of the present invention is to provide a composition that can be easily applied to the necessary bleeding site, particularly in areas of the body that are difficult to reach. A further advantage of the compositions of the present invention is that they are bioabsorbable and therefore can be left in place postoperatively without causing side effects.

[0040] "Applied to the necessary bleeding site" means, for example, applying the composition locally to the surgical site where tissue is bleeding or to its vicinity.

[0041] According to aspects of the present disclosure, a fluid composition is provided comprising (i) a powder comprising oxidized cellulose, fibrinogen, and thrombin, and (ii) a dispersion medium comprising a polyol compound and an aqueous solution. In some embodiments, the composition is in the form of a suspension at about room temperature. In some embodiments, the suspension is a paste-like suspension. In some embodiments, the polyol compound comprises a sugar alcohol, such as glycerol. In some embodiments, the weight ratio of oxidized cellulose to fibrinogen is about 1:5 to about 2:1, respectively. In some embodiments, the composition is a two-component composition.

[0042] In some embodiments, the composition is bioabsorbable. The term "bioabsorbable" refers to the ability of a tissue-compatible material to be broken down in the body into non-toxic products that are eventually removed from the body or metabolized after implantation.

[0043] Where used herein, unless otherwise specified, the terms “weight ratio,” “by weight,” “w / w,” “weight percent,” or “weight %” are used interchangeably herein to describe the concentration of a particular substance in the total weight of the corresponding mixture, solution, suspension, formulation, or composition. Where used herein, unless otherwise specified, the terms “volume ratio,” “volume,” “v / v,” “volume percent,” or “volume %” are used interchangeably herein to describe the concentration of a particular substance in the total volume of the corresponding mixture, dispersion medium, solution, suspension, formulation, or composition.

[0044] A two-component composition can be prepared by mixing a first component and a second component to create a suspension. As used herein, the term "two-component composition" refers to a composition comprising at least two components. Typically, but not limited to, the two components are stored in separate containers and mixed to create a mixture or suspension, which is then applied to a substrate. Specifically, in embodiments of the two-component composition used in the present invention, the first component and the second component are mixed to create a dispersed mixture within two hours, or possibly within 0.5 hours, before applying the mixture to a substrate, for example, a bleeding site.

[0045] As used herein, the term “mixture” means any combination of components in any physical form, such as a blend, solution, suspension, or dispersion.

[0046] The term “suspended substance” or any grammatical derivative thereof, as used herein, refers to a heterogeneous mixture of solids in the form of fine solute-like particulate matter dispersed in a liquid or solvent-like phase. Typically, suspensions tend to settle; that is, particulate matter of solid substance may tend to settle after a period of more than 20 hours in some embodiments. This period may depend on many factors, including the substances of the particulate matter and liquid, temperature, pH, and other physical parameters such as stirring and shaking, as well as the presence of other substances such as dispersants, emulsifiers, surfactants, and thickeners.

[0047] As used herein, the term “bleeding” refers to the spillage of blood from any component of the circulatory system. Thus, “bleeding” encompasses undesirable, uncontrolled, and often excessive bleeding associated with surgery, trauma, or other forms of tissue injury, as well as undesirable bleeding in patients with hemorrhagic disorders. The term “blood” or any grammatical variation thereof also includes blood fractions such as plasma.

[0048] The term "trauma" is defined as an injury caused by physical force, and non-exclusive examples include the consequences of vehicle accidents, gunshot wounds, and burns.

[0049] The term "powder" refers to a solid particulate material, typically in the form of dispersed dry solid particles, such as a plurality of solid particles, each characterized by a small size of at least one dimension, typically ranging from 0.1 to 1000 micrometers, and sometimes less than 100 micrometers.

[0050] The term “solid” refers to the state of a compound or composition at room temperature (e.g., 25°C) and atmospheric pressure (760 mmHg), i.e., a highly viscous compound or composition that retains its form during storage. In this application, this term also refers to non-fluid particles or dissolved substances. In contrast to “liquid” compounds and compositions, solids do not flow under their own weight. “Powdered” and “fine particles” are interchangeable herein.

[0051] As used herein, the term “paste” defines a fluid mixture of solid particles with respect to the consistency of the composition at at least one temperature near room temperature. Typically, a paste does not have a fixed shape and is neither solid nor gaseous. The term “paste” as used herein may also include slurries, ointments, and creams. A slurry can be functionally considered a thin, oily paste. Pastes as used herein may also contain pores containing an expandable gas such as air. Thus, in some embodiments, the composition is a paste or has a paste-like viscosity at at least one temperature near room temperature.

[0052] The term "fluidity" includes viscous solutions. The term "viscous solution" refers to a solution that has high flow resistance but is still flowable. In some embodiments, the weight ratio of oxidized cellulose to fibrinogen is in the range of 1:5 to 2:1, and the composition has a viscosity of over approximately 400 and less than approximately 3000 Pa·s. See Figure 4 for details.

[0053] In some embodiments, the paste is characterized by a viscosity of 400 to 2950, ​​for example, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 2990 Pa·s (including any values ​​and ranges between these).

[0054] The term "viscosity" refers to a property of fluids and slurries that exhibits flow resistance, defined as the ratio of shear stress to shear rate. Viscosity typically has a shear rate that is described or understood. Throughout this specification, unless otherwise stated, the shear rate with respect to viscosity is 0.5 seconds. -1 Viscosity is measured under ambient pressure and temperature conditions (referred to as "ambient conditions," i.e., around room temperature and atmospheric pressure). The term "shear rate" refers to the velocity gradient measured over the diameter of a fluid flow path, if it is a pipe, ring, or other shape. Shear rate is the rate of change in the velocity at which one layer of fluid passes over an adjacent layer.

[0055] In some embodiments, the composition is in an injectable form. As used herein, the term “injectable form” means a fluid composition (for example, in the form of particles small enough to be injected into a human subject, e.g., via a syringe and needle), and more preferably of high purity and non-toxic when injected into a subject. The composition must be homogeneous and have rheological properties to pass smoothly through needles of various inner diameters (e.g., 14–32 gauge). Thus, in some embodiments, the composition can pass through an applicator.

[0056] In some embodiments, the composition is homogeneous. As used herein, “homogeneous” means that the composition and texture are uniform throughout, i.e., the powder is substantially uniformly dispersed throughout the dispersion medium. In this specification, “substantially uniformly dispersed” means that the concentration of the powder in the dispersion medium varies within ±30% by weight, ±20% by weight, ±10% by weight, or ±5% by weight throughout the dispersion medium, i.e., there is no significant solid-liquid phase separation. Typically, the term “homogeneous” refers to a product (e.g., a paste) that appears uniform and withstands liquid / solid phase separation at 8–40°C, 10–30°C, or 20–30°C (e.g., less than 4% by weight).

[0057] The terms “liquid medium,” “medium,” or “dispersant” may be used interchangeably throughout this Spec. Herein, the term “dispersant” means a liquid medium, optionally, a liquid medium having a viscosity that provides or is capable of providing a paste-like viscosity. Therefore, the term “dispersant” may be used in a general sense and may be used, rather than the term “solvent,” only to clarify, for example, that a disclosed blend or powder comprising fibrinogen, thrombin, and OC fibers or powder is not substantially soluble in such a liquid medium, but rather forms a solid-liquid system.

[0058] "Around room temperature" means at least one temperature value within the range of 10°C to 40°C or 15°C to 37°C (including any value in between), for example, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C.

[0059] In some embodiments, the weight ratio of OC to fibrinogen is in the range of 1:5 to 2:1. In some embodiments, the weight ratio of OC to fibrinogen is in the range of 1:4 to 2:1. In some embodiments, the weight ratio of OC to fibrinogen is in the range of 1:3 to 2:1. In some embodiments, the weight ratio of OC to fibrinogen is in the range of 1:2 to 2:1. Referring to the Examples chapter, for example, Figure 10, it is shown that when the ORC to fibrinogen ratio is less than 2:1 (1:1, 1:2, and 1:5), the reconstituted paste becomes harder and thicker, and at a ratio of about 1:5 it becomes too hard.

[0060] The term "oxidized cellulose" (or "OC") refers to a cellulose derivative in which at least a portion of the primary alcohol group, for example, the carbon at position 6 of the anhydrous glucose unit, is oxidized to a carboxylic acid and optionally functionalized.

[0061] Oxidized cellulose (OC) can be produced by applying an oxidizing agent to cellulose. The oxidizing agent can be selected from, but is not limited to, chlorine, hydrogen peroxide, peracetic acid, chlorine dioxide, nitrogen dioxide, persulfates, permanganates, dichromates-sulfuric acids, hypochlorous acid, hypohalous acid, periodates, or any combination thereof, and / or various metal catalysts. Depending on the properties of the oxidizing agent and the reaction conditions, the oxidized cellulose may contain carboxylic acids, aldehydes, and / or ketone groups in place of, or in addition to, the original hydroxyl groups of the starting material cellulose.

[0062] The OC used in the compositions of this disclosure is typically in the form of fibers or powder (also called granular OC, ground / crushed OC, or ground / crushed OC in aggregate form), but is not limited thereto. Ground / crushed OC can be prepared by a variety of methods, including from existing products, some non-limiting examples of such products are described below. Since some of the existing products are in the form of fabric, OC powder may be prepared by grinding or crushing the fabric to obtain a powder.

[0063] In some embodiments, OC includes oxidized regenerated cellulose (ORC).

[0064] Woven or nonwoven ORC material, shredded or ball-milled ORC material, can be further roller-compressed to achieve a desired low aspect ratio and high density of ORC particles.

[0065] Examples of OC-based products that are in aggregate form or can be crushed or ground and thus used to prepare particles of a composition include, but are not limited to, INTERCEED® absorbent adhesion barrier, SURGICEL® Original absorbent hemostatic agent, SURGICEL® NU-KNIT® absorbent hemostatic agent, SURGICEL® FIBRILLAR® absorbent hemostatic agent, SURGICEL® SNoW® absorbent hemostatic agent, and SURGICEL® powder absorbent hemostatic agent, and GelitaCel® absorbent cellulose bandage (manufactured by Gelita Medical BV (Amsterdam, The Netherlands)).

[0066] While the usual source of organic compounds (OCs) is plant material, it is understood that OCs may also be derived from bacterial sources. In some embodiments, the OCs are derived from plant sources.

[0067] As described herein, in one embodiment, the fibers or powder for producing the disclosed composition are prepared by grinding a cellulosic material. Prior to the grinding step, a step may be performed in which the cellulosic raw material is cut into elongated pieces to form material fragments.

[0068] In some embodiments, the disclosed compositions have OC having a carboxyl content of about 12% to about 24%.

[0069] When used herein with reference to OC, the terms “oxidation level,” “degree of oxidation,” “carboxyl content,” and “carboxylation level” are interchangeable and may be determined in accordance with United States Pharmacopeia (USP) 23-NF18.

[0070] Therefore, in some embodiments, the carboxyl content of OC is approximately 12% to 24% (w / w). In some embodiments, the carboxyl content of OC is 12% to 23% (w / w). In some embodiments, the carboxyl content of OC is 12% to 22% (w / w). In some embodiments, the carboxyl content of OC is approximately 12% to approximately 21% (w / w).

[0071] Non-limiting exemplary powders include solid particles containing ORC fibers and / or granules (also known as “granular ORC”).

[0072] The term "glycerol" refers to 1,2,3-propanetriol, also known as "glycerin," and can also include its derivatives. The term "derivative" refers to a compound that contains essential elements of the parent substance.

[0073] As used herein, "thrombin" refers to the activating enzyme resulting from the proteolytic cleavage of prothrombin (factor II). Thrombin may be produced by various methods known in the art, including but not limited to recombinant thrombin and plasma-derived thrombin.

[0074] Human thrombin is a 295-amino acid protein composed of two polypeptide chains joined by a disulfide bond. Both human thrombin and non-human (e.g., bovine) thrombin can be used within the scope of this disclosure.

[0075] The origin of the thrombin used in this disclosure may be derived from one or more sources, including but not limited to plasma (e.g., porcine plasma), recombinant bacteria and / or cells (e.g., Vu et al., 2016, J. Viet. Env. 8(1):21-25), whole blood (which may or may not be pooled from several transfusions), and / or blood fractions (which may be pooled from multiple transfusions, e.g., plasma). Thrombin is available from manufacturers such as Johnson and Johnson, Baxter, and CSL Behring as a standalone product, e.g., EVITHROM®, or as a component of a product, e.g., EVICEL®, TISEEL®, Beriplast®, etc.

[0076] In some embodiments, thrombin is present in an amount of about 2000 to about 4000 IU per gram of powder. In some embodiments, thrombin is present in an amount of about 3000 IU per gram of powder. In some embodiments, thrombin is present in an amount of about 2000, 3000, or 4000 IU (including any value and range between these) per gram of powder.

[0077] In this specification, "U" represents a coagulation factor unit, i.e., the unit of physiological measurement of coagulation factors in 1 mL of normal human plasma.

[0078] "IU" refers to the international units of a coagulation factor, which is a physiological measurement of a given sample when compared to an appropriate international standard, for example, a coagulation assay against an internal reference standard for potency concentration measurement calibrated against the World Health Organization (WHO) Second International Standard for Thrombin, 01 / 580.

[0079] The term "fibrinogen" is intended to include any type of fibrinogen, even if not specified further. "Fibrinogen" refers to monomeric and dimeric fibrinogen molecules, hybrid molecules, and their variants, whether naturally occurring, modified, or synthetic, possessing the monomeric structure (AαBβγ). The term "fibrinogen" may refer to human-derived fibrinogen, but may also include fibrinogen from any species, particularly mammalian species, such as fibrinogen produced from porcine plasma.

[0080] The term "aqueous solution" refers to a solution containing water as a solvent. This term may include water, saline solution, or a bleeding site. In some embodiments, the aqueous medium is selected from water, saline solution, calcium chloride solution, or a buffered aqueous medium.

[0081] In exemplary embodiments, the aqueous solution includes physiological saline. As used herein, the term “physiological saline” means water containing a salt concentration greater than about 0.5% by weight and less than about 20% by weight, for example, a solution of about 0.9 w / w% sodium chloride in desalinated water.

[0082] In some embodiments, at least a portion of the powder is in the form of aggregates or granules. The term “aggregates” describes particles formed from the combined components. Aggregates may optionally be produced by one of the following steps: humidifying the powder composition; forming aggregates by compressing the powder, for example, with rollers and / or striking the powder; dehumidifying; grinding; sieving the aggregates; or optionally introducing the resulting aggregates into a storage container or delivery device. The terms “granules” or “granular material” may particularly mean aggregates of particles typically less than 900 micrometers, less than 800 micrometers, less than 700 micrometers, or even less than 500 micrometers. That is, at least a portion of the particles are not in the form of discrete solids. In some embodiments, high-shear mixing is used to construct aggregates from particles of fibrinogen, thrombin, and optionally CaCl2, and ORC.

[0083] In another embodiment, the composition is in the form of a mixture. In an exemplary embodiment, the mixture may contain a first spray-dried fine particle containing fibrinogen combined with a second spray-dried fine particle containing thrombin, such as human thrombin, which together form particles of, for example, 10 micrometers to about 250 micrometers or larger.

[0084] Additionally or alternatively, the powder compositions according to this disclosure, including fibers and compounds, are optionally compressed into aggregate form using, for example, drying, grinding / grinding, and sieving steps as described in U.S. Patent No. 10,034,957. The sieves used define the particle size of the powder.

[0085] In some embodiments of any aspect of this disclosure, the plurality of particles are characterized by a median particle size of up to about 900 μm.

[0086] In some embodiments, the particle size of at least 90% of the powder is in the range of 10 to 2,000 μm. In some embodiments, the particle size of at least 90% of the powder is in the range of 10 to 1,000 μm. In some embodiments, the particle size of at least 90% of the powder is in the range of 100 to 1,000 μm. In some embodiments, the particle size of at least 90% of the powder is in the range of 200 to 1,000 μm. In some embodiments, the particle size of at least 90% of the powder is in the range of 100 to 900 μm. In some embodiments, the particle size of at least 90% of the powder is in the range of 200 to 900 μm.

[0087] In some embodiments of any aspect of this disclosure, the plurality of particles are characterized by a median particle size of about 100 to about 900 μm. In some embodiments of any aspect of this disclosure, the plurality of particles are characterized by a median particle size of about 200 to about 900 μm.

[0088] The term "median," also known as "D50," refers to the particle size at which 50% of a group is either larger or smaller than the median.

[0089] The term "at least a portion of the powder is in aggregate form" refers to a situation where a portion of the particles in the powder, for example, at least 10% to 100%, form aggregates.

[0090] In some embodiments, the composition further comprises fibrin.

[0091] The term “fibrin” refers not only to fully solidified fibrinogen, but also to any mixture of fibrin and fibrin that may result during the formation of fibrin from fibrinogen using thrombin, and therefore includes any conceivable ratio of fibrinogen / fibrin and any degree of gelation and / or solidification, as long as it does not adversely affect the final paste-like texture of the composition. Furthermore, the term “fibrin” further includes any partially or fully crosslinked, gelled, or solidified form.

[0092] Therefore, it is understood that fibrin itself exists as a high-molecular-weight, insoluble matrix containing fibrils, and each fibril contains many fibrin molecules.

[0093] In some embodiments, glycerol is present at a concentration exceeding approximately 15% by volume of the dispersion medium. In some embodiments, glycerol is present at a concentration exceeding approximately 40% by volume of the dispersion medium. In some embodiments, glycerol is present at a concentration of approximately 80% by volume or less of the dispersion medium. In some embodiments, glycerol is present at a concentration of approximately 70% by volume or less of the dispersion medium. In some embodiments, glycerol is present at a concentration of approximately 60% by volume or less of the dispersion medium.

[0094] In some embodiments, glycerol is present in a concentration of more than about 15% to about 80% by volume of the dispersion medium. In some embodiments, glycerol is present in a concentration of more than about 15% to about 80% by volume of the dispersion medium. In some embodiments, glycerol is present in a concentration of more than about 15% to about 60% by volume of the dispersion medium. In some embodiments, glycerol is present in a concentration of more than about 40% to about 80% by volume of the dispersion medium. In some embodiments, glycerol is present in a concentration of more than about 40% to about 60% by volume of the dispersion medium.

[0095] In some embodiments, glycerol is present in the dispersion medium at concentrations of about 15 vol%, about 20 vol%, about 25 vol%, about 30 vol%, about 35 vol%, about 40 vol%, about 45 vol%, about 50 vol%, about 55 vol%, about 60 vol%, about 65 vol%, about 70 vol%, about 75 vol%, or about 80 vol% (including any values ​​and ranges in between).

[0096] Referring to Figures 3A to 3D, the 70% glycerol sample provided a paste-like composition when dispersed in the aforementioned dispersion medium, while the 80% glycerol sample showed higher fluidity, mainly for samples with an ORC to fibrinogen weight ratio ("ratio") of 2:1 to 1:2. As a result, the composition had a lower ability to remain at the wound site, and the coagulation rate was slower (exceeding 180 seconds for the 1:5 sample, while the blood coagulation time for the 70% glycerol sample (1:2 and 1:5 ratios) was 70 seconds). Further results, as shown in Figure 6, indicated that (i) for ORC to fibrinogen weight ratios of 2:1 to 1:1, the preferred concentration of glycerol in the dispersion medium (which does not impair enzyme activity, as described below) was between 15% and 50% by volume, providing a coagulation time of less than 60 seconds at each time point, and (ii) liquid-solid phase separation occurred in the 15% glycerol sample after 20 hours.

[0097] In some embodiments, the powder is spray-dried and / or high-shear mixed.

[0098] The term “spray drying” is used herein in a broad sense and includes, but is not limited to, processes for converting solids dissolved or suspended in liquids into powders. Typically, spray drying is a drying method used to produce powders from solutions, suspensions, or emulsions by spraying them through a spray nozzle into a stream of hot air and drying them immediately. A spray drying process for producing thrombin and fibrinogen powders that retain their activity can be controlled by several process parameters, including, but is not limited to, column airflow and temperature, nozzle size and spray air velocity, and material flow rate. Non-limiting exemplary parameters used to establish a robust process for producing a blend of thrombin and fibrinogen (e.g., in powder form) that retains their activity are provided in the following Examples chapter.

[0099] The term "high shear mixing" generally refers to turbulent mixing. In some embodiments, high shear mixing is characterized by mixing blade speeds and / or cutting blade speeds in the range of 150 to 500 rpm.

[0100] Additionally or alternatively, other methods may be used to obtain the dried composition, such as freeze-drying and then micronizing the fibrinogen solution and / or thrombin solution, for example. Typically, after freeze-drying the solution, a porous, spongy solid material called a "cake" (which may also be referred to herein as a "solid composition") is obtained.

[0101] The term "micronization" is used, for example, when the diameter of particles produced from a solid composition is only a few micrometers. Micronization can be achieved by processes including, but not limited to, jet milling, pearl ball milling, high-pressure homogenization, RESS (rapid expansion of supercritical solution) process, SAS (supercritical poor solvent) method, or PGSS (particles from gas-saturated solution) method. Typically, as a result of micronization, protein powders are reduced in size by 30 to 400 times from their original size, but this is not limited to this.

[0102] In some embodiments, the composition is stable.

[0103] The terms “stable” and “stability,” when referring to the disclosed paste, mean, for example, that the active components in thrombin remain at least 70% active after a specific period of time at a specific temperature, i.e., that they can form fibrin clumps.

[0104] In some embodiments, the composition is a storage-stable composition. The term “storage-stable” is typically understood to mean a formulation or composition present in a container, e.g., a vial or syringe, that is stable under pre-selected storage conditions (e.g., pH), including a pre-selected storage temperature and a pre-selected physical state of the formulation. Stability can be determined by methods known in the art, for example, by testing whether there are any visible aggregates, remnants, and / or fibrin clumps in the formulation under pre-selected storage conditions when the formulation is in liquid or paste form. Furthermore, according to this disclosure, when referring to a stable sealant composition or formulation, it is understood that it has an effective solidification time at use, regardless of the storage conditions of some formulations, for example, a sealant formulation solidifies in essentially the same time whether it is stored at room temperature (e.g., 8–40°C) or at a lower temperature (e.g., below 8°C).

[0105] In some embodiments, the composition has a pH of about 4 to about 6.5 at about room temperature. For this, please refer to the following Examples chapter, for example, Figures 7-9. In some embodiments, the composition has a pH of about 4, 4.5, 5, 5.5, 6, or about 6.5 (including any value and range between these) at about room temperature.

[0106] In some embodiments, the composition in any of those embodiments remains stable at a pH of 4–6.5 at approximately room temperature for at least about 20 hours.

[0107] In some embodiments, less than 25% by weight, less than 24% by weight, less than 23% by weight, less than 23% by weight, less than 22% by weight, less than 21% by weight, or less than 20% by weight of remnants were detected at approximately room temperature and a pH of 4–6.5 after at least 20 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days.

[0108] In some embodiments, the composition further comprises additional pharmaceutically active agents contained within or on the surface of the composition, in any embodiment thereof.

[0109] In some embodiments, the pharmaceutically active agent is selected from the group consisting of therapeutic agents and labeling agents.

[0110] In some embodiments, the therapeutic activator is selected from the group consisting of stem cells, growth factors, bone morphogenetic proteins, cells, cytokines, hormones, pharmaceuticals, minerals, therapeutic plasmids, and combinations thereof. As described herein, in some embodiments, the composition may further contain at least one biological activator. Non-limiting biological activators that may be included in the composition include calcium, as well as therapeutic agents such as antibiotics, anti-inflammatory agents, growth factors, or coagulation factors. Specifically, the disclosed composition may also contain components that promote mass formation, such as but not limited to calcium salts, factor VIII, factor XIII, fibronectin, vitronectin, von Willebrand factor (vWF), and these components may be provided as separate components or combined in a blend or paste.

[0111] Optionally, a pharmaceutically active agent is conjugated to a protein (i.e., fibrinogen or fibrin) matrix (e.g., covalently to the protein). This agent may be conjugated to the matrix after the matrix has been prepared, and / or to a component (e.g., a protein and / or reducing sugar), which is then used to prepare a protein matrix, such as cross-linked fibrin.

[0112] Alternatively or additionally, the agent is absorbed by a crosslinked protein matrix. Absorption can be achieved, for example, by contacting a crosslinked fibrinogen or fibrin matrix or a non-crosslinked fibrin matrix with a solution containing the agent (e.g., by immersion, soaking, or washing), or by adding the agent before crosslinking (e.g., by including the agent in a crosslinking solution, or by including the agent in a solution of fibrinogen and thrombin).

[0113] The compositions described herein, and the contents of the kits described above, may, if desired, be provided in packs or dispenser devices that can contain one or more unit dosage forms containing the compositions or components (e.g., fibrinogen) and / or reagents (e.g., dispersion media) for preparing the compositions. Packs such as blister packs may contain, for example, metal or plastic foil. The packs or dispenser devices may be accompanied by instructions for administration. The packs or dispenser devices may be accompanied by notices in the form prescribed by government agencies that regulate the manufacture, use, or sale of pharmaceuticals, which reflect the approval of such government agencies of the form of the composition for administration to humans or animals. Such notices may include, for example, the labeling approved by the U.S. Food and Drug Administration (FDA) for prescription drugs, or approved product inserts. Compositions containing the preparations of the present invention, formulated in a pharmaceutically acceptable carrier, may also be prepared, placed in appropriate containers, and labeled for use for specified uses and / or for the treatment of specified conditions, as further detailed herein. The term “preparation” means one that is physiologically suitable for therapeutic use.

[0114] It is understood that the compositions of the embodiments of this disclosure may be attached to or included in a medical device, for example, to promote wound healing.

[0115] In another aspect of this disclosure, any composition disclosed in any aspect or embodiment thereof is intended for use in a method of preparing a fibrin sealant in / on damaged tissue of a subject by applying the composition disclosed in any aspect and / or embodiment thereof to the surface of the tissue.

[0116] Another aspect of the present disclosure provides a method for preparing a fibrin sealant in / on the damaged tissue of a subject by, for example, applying the composition disclosed in any aspect and / or embodiment thereof to the surface of the tissue as a hemostatic agent.

[0117] Another aspect of the present disclosure provides a method for preparing a composition according to that embodiment, comprising: combining OC or ORC fibers with fibrinogen powder and thrombin powder under conditions that enable the formation of aggregated forms of ORC fibers, fibrinogen, and thrombin; and combining the aggregates with a dispersion medium comprising glycerol and an aqueous solution.

[0118] In some embodiments, such conditions allow for at least partial conversion of fibrinogen to fibrin. In some embodiments, these conditions allow for at least partial polymerization of fibrin, for example, by adding a specific amount of aqueous solution to a dispersion medium, as detailed throughout this specification.

[0119] Conditions that enable the formation of powder / aggregate forms of ORC fibers, fibrinogen, and thrombin include, for example, pulverization of the powder and OC fibers by a high-shear process, a dispersion medium pH of, for example, 4-6, and a temperature range such as, but not limited to, 10-50°C, 45-50°C, or 20-30°C.

[0120] In the context of the present invention, the term "sealant," also called "fibrin sealant" and "biological glue," should be understood as an adhesive, glue, or hemostatic agent having components that, upon contact with or proximity to tissue and / or blood, react to subsequently form a mass and further act as a tissue adhesive, thereby preventing, reducing, or stopping bleeding, joining structures, and / or sealing physiological leakage of, for example, cerebrospinal fluid (CSF), lymph, bile, gastrointestinal (GI) contents, air leakage from the lungs, etc., such as originating from or derived from the disclosed compositions. In some embodiments, the sealant formulation also includes one or more therapeutic agents, as a result, the therapeutic agents are released when the mass formed in the body spontaneously decomposes. The therapeutic agents may be, but are not limited to, drugs such as antibiotics, analgesics, anti-inflammatory drugs, and anticancer drugs, or cells, for example, any type of stem cell of human or other origin, such as germinal stem (ES) cells, adult stem cells, induced pluripotent stem cells (iPSCs), etc.

[0121] The term "hemostasis" refers to the ability to prevent, reduce, or stop blood loss from surgical wounds or other wounds, for example, by promoting blood clot formation.

[0122] "Hemostasis" (or "haemostasis") refers to the first stage of wound healing. This is the process of stopping bleeding. "To help stop bleeding" means to help reduce or stop bleeding. "To apply to bleeding tissue" means to apply a composition topically to the bleeding site, such as a surgical site, in order to control bleeding. Controlling bleeding is necessary in a variety of situations, including wound treatment.

[0123] As used herein, the terms “control,” “prevent,” or “reduce” may be used interchangeably herein with respect to bleeding (including any grammatical variations thereof), meaning to reduce the rate of hemorrhage by 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of the initial bleeding rate compared to a situation in which the disclosed composition is essentially nullified or not in contact with the bleeding site. Methods for determining the level of bleeding occurrence are known in the art.

[0124] Furthermore, in some embodiments, with respect to bleeding, the terms “control,” “prevent,” or “reduce” also mean, for example, at least partially sealing the blood vessels at the site of bleeding in soft tissue.

[0125] As used herein, the term “subject” means any animal, but is not limited to humans, mice, rats, rabbits, non-human primates, or any other mammal. In some embodiments, the subject is a human (e.g., a human patient). The subject may be male or female.

[0126] As used herein, the term “injured tissue” (or “damaged tissue”) means the disruption of the normal continuity of a structure caused by physical (e.g., mechanical) force, such as surgical incision, biological means (e.g., thermal or chemical radiation), or chemical means. The term “injured tissue” also includes bruised tissue, and tissue that has been cut, punctured, or lacerated, as well as injuries caused by opening, puncture, and, for example, tearing, scratching, pressure, and biting. The term “injured tissue” also includes wounds and bleeding sites as described throughout this specification.

[0127] The term "tissue" may refer to tissue selected from soft tissue and bone tissue.

[0128] The term "soft tissue," as used in this specification, refers to body tissue that is not solidified or calcified. This term refers to soft tissue that is angiogenic and therefore a source of bleeding. Examples of such tissues include, but are not limited to, connective tissue (tendons, ligaments, fascia, skin, fibrous tissue, fat, and synovial membrane), muscle, and internal organs. Generally, soft tissue excludes bone tissue.

[0129] Therefore, in some embodiments, the disclosed compositions may be used, in any embodiment, by being applied to hard-to-reach bleeding sites during minimally invasive surgery (MIS), such as endoscopic surgery. One common form of endoscopic surgery is laparoscopic surgery, which is minimally invasive examination and surgery performed inside the abdominal cavity.

[0130] The term “surgery” also encompasses the time during or after a surgical or diagnostic procedure. Further non-limiting examples of procedures include neurological surgery, abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck surgery, pelvic surgery, and skin and subcutaneous tissue procedures. For at least one of these situations, the compositions of the present invention may function as suitable sealants that can adhere to bleeding wounds and thus address hemostasis regardless of the condition of the wound tissue, e.g., the severity of the bleeding. The formation of the sealant occurs within a relatively short coagulation time after applying the composition disclosed in any aspect and / or its embodiments to the injured tissue. Typically, when used after 1-3 minutes of tampon filling, an incomplete mass may form, which can stop the bleeding, although more time may be required for the coagulation reaction to reach the paste surface and form a complete mass.

[0131] Another aspect of the present invention provides a method for treating a wound, comprising the step of applying (e.g., contacting) a composition disclosed in any aspect and / or its embodiments onto and / or into the wound of a subject requiring treatment.

[0132] "To treat a wound" further means, for example, reducing blood loss at the site of tissue bleeding (in vivo) in a patient undergoing surgery. Referring to Figures 12 and 13, the high success rate of hemostasis (over 60%) of the tested samples disclosed herein is shown.

[0133] Accordingly, in some embodiments, the method is for reducing blood loss at a site of tissue bleeding and / or forming a sealant layer in / on such tissue, for example, in a patient undergoing surgery, and includes bringing the composition disclosed in that embodiment into contact with the bleeding site and / or its vicinity. Optionally, the method includes first applying an aqueous solution, such as saline, to such tissue to promote a rapid coagulation time, and then applying the disclosed composition onto the aqueous solution. Accordingly, in some such embodiments, the composition is intended to be used as a hemostatic agent at a site of tissue bleeding in any embodiment.

[0134] According to another embodiment of the present invention, a kit for producing the compositions described herein is provided. The consistency of the composition is understood to be such that it can be applied, for example, by spreading or adhering the composition directly onto the bleeding site. Therefore, the composition does not need to be further spread or applied onto a solid surface, object, or other solid medium such as a strip or film in order to be in a form suitable for application to the bleeding site. Nevertheless, a suitable applicator, such as a syringe, may be used for easy access and handling in order to apply, spread, or adhere the composition onto the bleeding site.

[0135] In further embodiments and / or other embodiments, the present invention provides a kit comprising: a) a container for containing the composition of the present invention described above; b) an applicator for applying the composition to tissue; and c) optionally instructions for use.

[0136] In another embodiment, the present invention further provides a hemostatic kit comprising a container for containing the composition disclosed herein.

[0137] According to several embodiments, a kit is provided for preparing a composition in the form of a paste-like suspension at room temperature, comprising (i) a powder comprising oxidized cellulose, fibrinogen, and thrombin, wherein the weight ratio of oxidized cellulose to fibrinogen is in the range of 1:5 to 2:1, respectively, and optionally (ii) a dispersion medium comprising glycerol and optionally an aqueous solution. (i) and (ii) may each be present in separate containers.

[0138] Any aspect and embodiment of the compositions disclosed throughout this specification may be incorporated into aspects and embodiments of kits, including embodiments of the compositions, dispersions, and / or powders.

[0139] Optionally, fibrinogen and thrombin are packaged separately in the kit (e.g., in dry form or solution). Therefore, each of the two components is packaged in separate packaging materials in addition to the overall kit packaging materials.

[0140] Alternatively, fibrinogen, thrombin, and oxidized cellulose may be packaged together in the kit (e.g., as dry powders) in the same packaging material, and optionally, a dispersion medium may be provided in an additional container. In some embodiments, the kit may further include instructions on how to combine the components of the kit and / or how to combine the components of the kit with additional components (e.g., a dispersion medium) to produce a desired composition.

[0141] Optionally, the kit further includes a dispersion medium. The dispersion medium, such as glycerol, may be in pure form or in a solution with another liquid (e.g., water, saline solution, or aqueous buffer). Optionally, the dispersion medium is packaged separately from the powder. Alternatively, the dispersion medium is packaged together with the powder as, for example, a ready-to-use composition as described herein. In such embodiments, the kit may further include measuring means for measuring the volume of the dispersion medium or its components, such as a measuring cylinder.

[0142] Additionally or alternatively, the hemostatic kit may include a syringe containing a blend, mixture, or powder, and another syringe containing a dispersion medium. For example, a dual-syringe mixer can initially mix separate liquids and powders, and then produce a substantially homogeneous paste mixture by moving the blended contents back and forth between two connected syringes via interconnected outlets. Therefore, a low pressure force for dispensing the paste from the syringe may be preferable to facilitate mixing and, ultimately, to develop the resulting paste. The desired pressure force may be less than 1.51 lbf.

[0143] In some embodiments, at least one of the containers in the kit is a pre-filled syringe. In some embodiments, syringes are provided in addition to the container(s) of the kit. In some embodiments, the container(s) are of a specific type, such as a vial or an applicator such as a syringe.

[0144] In some embodiments, at least one of the containers in the kit is a pre-filled syringe. In some embodiments, syringes are provided in addition to the containers in the kit. The term “container” may refer to any general structure, such as a container or vial, that can hold a paste.

[0145] The kit may be applied using an applicator device that can be used to administer the composition injectable solution several times in succession. In one embodiment, the applicator device allows for multiple injections of a fixed dose of the mixed component onto a 2D surface of tissue while moving the device. In one embodiment, the applicator has a syringe equipped with a needle, which optionally automatically retracts from the patient's skin after the injection is complete, without requiring the administerer to lift the device above the injection surface. In one embodiment, the kit may be used to administer a sealant.

[0146] The hemostatic kit of the present invention may be, for example, a kit for use in reducing, blocking, or stopping blood flow in an open wound, and it can be used to reduce, block, or stop blood flow during, before, or after surgical procedures such as laparoscopic surgery, neurosurgery, abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck surgery, pelvic surgery, and skin and subcutaneous tissue surgery. The kit can be used to reduce or block blood flow from the skin or internal organs.

[0147] In one embodiment, the kit can be stored at room temperature, for example, in the range of 8 to 40°C, or at a lower temperature.

[0148] In some embodiments of any form of the kit or composition disclosed herein, the powder or blend may contain an additive, such as a calcium salt, and / or one or more excipients selected from, for example, one or more amino acids, albumin, sugars, and / or sugar derivatives.

[0149] The term "additive" is understood to mean any substance that may be added to a composition, and may include active additives such as calcium salts as described below.

[0150] As used herein, the term “excipient” means, for example, an inactive or non-therapeutic agent added to a pharmaceutical composition to provide a desired viscosity or stabilizing effect.

[0151] Calcium is an important element in the coagulation cascade and may be necessary to activate factor XIII, which cross-links and stabilizes fibrin to form factor XIIIa, generating an insoluble mass.

[0152] Accordingly, in some embodiments of any part of the disclosed kit and / or composition, the blend, mixture, or powder further comprises an additive such as calcium, for example, but is not limited to calcium. The calcium used in the present invention may be in the form of a salt, such as a calcium chloride salt. Alternatively, additional salts such as calcium acetate and / or calcium citrate may be used. In the kit, the calcium salt may be provided in a composition containing a powder. Alternatively, the excipient and / or calcium salt may be provided in a separate container in the kit, or the excipient and / or calcium salt may be provided in the kit in the same container as the blend / mixture / powder component.

[0153] In some embodiments of the kit or composition, a calcium salt, such as calcium chloride (CaCl2), is present in the powder at a concentration of 0.5% to about 4% by weight, or 2% to 4% by weight, or 2.5% to 3.5% by weight. In some embodiments, a calcium salt, such as calcium chloride, is present in the blend at a concentration of 0.5% by weight, 1% by weight, 1.5% by weight, 2% by weight, 2.5% by weight, 3% by weight, 3.5% by weight, or 4% by weight (including any values ​​and ranges between these).

[0154] Since oxidized cellulose is known to be acidic, and this acidity immediately denatures plasma proteins such as thrombin and fibrinogen, simultaneous administration of oxidized cellulose with thrombin and / or fibrinogen may negate the effects of plasma proteins.

[0155] In some embodiments, buffers are added to the composition to adjust the pH.

[0156] In some such embodiments, a buffer, e.g., Tris, or Tris(hydroxymethyl)aminomethane buffer, or lysine, is added in powder form to the fibrinogen, thrombin, and ORC blend / mixture / powder to adjust the pH (for example, to about pH 7). In some embodiments, the buffer, e.g., Tris or lysine, is present in concentrations of 0–25% by weight, 4%–8% by weight, or 5%–7% by weight of the blend / mixture / powder. In some embodiments, the buffer, e.g., Tris or lysine, is present in concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25% by weight (including any values ​​and ranges between these).

[0157] In some embodiments of any part of the kit and / or composition, the powder further comprises an excipient selected from amino acids, such as lysine. In some embodiments of any part of the kit and / or composition, the blend or powder comprises OC and lysine.

[0158] In some embodiments, fibrinogen is present in the powder at concentrations ranging from 20% to 80% by weight, 60% to 95% by weight, or 70% to 90% by weight. In some embodiments, fibrinogen is present at concentrations of 20% to 30% to 40% to 50% to 60% to 70% to 80% to 90% by weight (including any values ​​and ranges in between) of the powder.

[0159] In exemplary embodiments, the blend or powder comprises 20–80% by weight of fibrinogen powder, 2000–4000 IU of thrombin / dose powder, 10–50% of ORC powder, 2–4% of CaCl2 powder, and 0–25% of lysine powder, relative to the total weight of the powder. In this specification, “dose” means approximately 1.2 g.

[0160] In some embodiments of any part of the kit and / or composition, the disclosed composition may also be used in conjunction with a backing material, pad, bandage, gauze, sponge, scaffold, or matrix to provide mechanical strength for covering the surface of a wound. In this case, the matrix is ​​supported on a pad to facilitate application or tampon filling.

[0161] In some embodiments of any part of the kit and / or composition, the composition is sterile. Sterility, specifically viral inactivation, is a critical issue, especially when handling blood products. Generally, viral inactivation can be carried out by any number of methods, including solvent detergents, thermal inactivation, irradiation, and nanofiltration. Typically, standards for viral inactivation require the use of two different methods. Furthermore, FDA standards for sterility require filtration.

[0162] As used herein, the term “sterile” means having a low degree of biological contamination, effectively free from pathogens, for example, essentially or absolutely free from microorganisms, such as bacteria and viruses. Sterilization is the process of reducing the degree of biological contamination to a level that is effectively free from pathogens. A sterile liquid or paste is generally defined as a liquid or paste that has undergone sterile filtration.

[0163] The terms “comprises,” “comprising,” “includes,” “including,” “having,” and combinations thereof mean “including but not limited to.” The term “consisting of” means “including and limited to.” The term “consisting essentially of” means that a composition, method, or configuration may include additional components, processes, and / or parts, provided that the additional components, processes, and / or parts do not substantially alter the basic and novel properties of the claimed composition, method, or configuration.

[0164] The term “exemplary” in this specification means “serving as an example, case, or illustration.” Any embodiment described as “exemplary” is not necessarily construed to be preferable or advantageous to other embodiments, and / or does not necessarily exclude the incorporation of features from other embodiments.

[0165] In this specification, the term “optionally” means “provided in some embodiments but not in other embodiments.” Any particular embodiment of the present invention may include several “optional” features, provided that such features do not conflict.

[0166] In the present invention, unless otherwise explicitly indicated, the singular forms "a," "an," and "the" refer to multiple references. For example, the terms "compound" or "at least one compound" may refer to multiple compounds, including mixtures thereof.

[0167] Throughout this application, various embodiments of the invention may be presented in range form. It should be understood that the range form description is for convenience and brevity only and should not be interpreted as a firm limitation on the scope of the invention. Accordingly, the range description should be considered to specifically disclose all possible subranges and the individual numbers within those ranges. For example, a range description such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numbers within those ranges, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0168] Whenever a numerical range is shown herein, it means that any cited number (fraction or integer) within the indicated range is included. The terms “ranging / ranges” between the first and second indicated numbers and “ranging / ranges from” the first indicated number to the second indicated number are used interchangeably herein, meaning that the first and second indicated numbers and all fractions and integers between them are included.

[0169] As used in the present invention, the term “method” means a method, means, technique and procedure for accomplishing a given task, and includes, but is not limited to, methods, means, techniques and procedures known or readily developed by practitioners of the chemical, pharmacological, biological, biochemical and medical fields.

[0170] As used in the present invention, the term "treating" includes suppressing, substantially inhibiting, slowing, or reversing the progression of a disease, substantially improving the clinical or aesthetic symptoms of a condition, or substantially preventing the appearance of the clinical or aesthetic symptoms of a condition.

[0171] Where expressions similar to "at least one of A, B, and C" are used, such expressions are generally intended to be understood in a way that a person skilled in the art would understand (for example, "a composition having at least one of A, B, and C" is not limiting, but includes compositions having only A, only B, only C, both A and B, both A and C, both B and C and / or all of A, B and C). A person skilled in the art will further understand that virtually all separators and / or phrases presenting two or more alternative terms in the specification, claims, or drawings should be understood to take into account the possibility of including one of the terms, either of the terms, or both. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B".

[0172] Certain features of the present invention are described in the context of separate embodiments for clarity, but it is understood that these may also be presented in combination in a single embodiment. Conversely, various features of the present invention are described in the context of a single embodiment for brevity, but these may also be provided separately, in any preferred partial combination, or in any other described embodiment of the present invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment would be unable to operate without those elements.

[0173] As used herein, the term “about” means that values ​​that are 10% higher or lower than the specified value are intended to be included. Unless otherwise specified, all numbers, such as those representing ratios, weights, moles / moles, quantities, viscosity, temperature, etc., should be understood in all cases as being modified by the term “about.” Therefore, unless otherwise specified, the numerical parameters described herein and in the appended claims are approximations that may vary by up to ±10% depending on the desired properties that the invention seeks to achieve.

[0174] Various embodiments and aspects of the present invention, as detailed above in this specification and claimed in the following claims, will be experimentally supported in the following examples. [Examples]

[0175] Herein, in conjunction with the above description, the following examples illustrate some embodiments of the present invention in a non-limiting manner.

[0176] Example 1: Process for producing ORC, thrombin, and fibrinogen powders Materials and methods: The source of fibrinogen and thrombin is porcine plasma supplied by Bioseal Biotech CO.LTD, located in Guangzhou, China, which is fractionated to obtain fibrinogen and thrombin. Bovine serum albumin (BSA) - SIGMA VETEC, V900933, L-lysine-ALADDIN, L103479-500g Oxidized regenerated cellulose (ORC): Characteristics of ORC: Particle size <70 μm, fibrous form. ORC powder was obtained by processing SURGICEL® original fabric. In short, ORC powder is obtained by processing SURGICEL® original fabric in the following steps: 1) Divide the fabric into approximately 2-inch x 8-inch pieces and cut them; 2) Using a known grinding method, grind the fabric to a powder particle size (typically less than 94 micrometers, D50, but not limited to these); place approximately 100 grams of fabric into a 500 mL zirconia jar, then place 12-13 20 mm zirconia balls (marbles) into the same jar, close the jar, and fix it to a Retsch planetary ball mill (model PM100). Grind the fabric at 450 rpm for 20 minutes. Transfer the ground powder onto an 8-inch diameter, 300-micrometer mesh sieve, shake gently to separate the marbles from the powder, and finally collect the powder.

[0177] In exemplary procedures, particularly by spray drying, the hemostatic compositions of the present invention were prepared as follows: 1 part by weight of ORC fiber relative to the weight of fibrinogen was combined with 1 part by weight, 2 parts by weight, 5 parts by weight, or 0.5 parts by weight of fibrinogen or fibrin sealant (a blend of fibrinogen and thrombin) powder. For example, 10 g of ORC powder was combined with 5 g of fibrinogen in a fibrin sealant mixed powder.

[0178] Spray drying is a drying method used to produce powders from solutions, suspensions, or emulsions by spraying them into a high-temperature airflow through a spray nozzle and drying them immediately. The spray drying process is controlled by several process parameters, including column airflow and temperature, nozzle size and spray air velocity, and material flow rate.

[0179] In the exemplary procedure, thrombin and fibrinogen powders were produced (one at a time) using spray drying (using a 4M8-TriX spray dryer (with ProCepT NV, Zelzate, Belgium)) as follows.

[0180] The prepared thrombin and fibrinogen porcine fibrinogen solution was drawn into a syringe and supplied in varying amounts to the syringe pump of the spray dryer. The feed valve was closed and the syringe pump was set to the desired flow rate. While the feed valve of the syringe pump was closed, the spray dryer was started and the desired spray gas flow rate, drying gas flow rate, drying gas temperature, cooling gas flow rate, and cyclone gas flow rate were set.

[0181] The cooling gas flow rate and temperature were selected so as not to disturb the laminar flow in the drying tower, but to lower the gas flow temperature below the glass transition temperature of the composition in order to prevent the powder from adhering to the glass portion.

[0182] The spray dryer was run until the measured parameter values ​​reached the set levels and remained constant, achieving a steady state. The feed valve was then opened, and the thrombin / fibrinogen solution was flowed through the feed inlet to the spray nozzle, atomized into small droplets by the spray gas flow, and then dried in the drying tower. The spray-dried powder was then collected at the powder outlet of the spray dryer's cyclone.

[0183] The spray-dried powder recovered from the cyclone was weighed into a dehumidifier with a relative humidity of less than 30% and divided into 100-200 mg samples. Each sample was individually sealed in a stoppered test tube and sealed with Parafilm® (Bemis, Oshkosh, Wisconsin, USA) until evaluation.

[0184] Spray parameters: Fan pressure 0.3 bar, spray pressure 3 bar, flow rate 130 mL / min, liquid pressure 16 kPa, nozzle diameter 0.7 mm, stirring speed 90 rpm / min.

[0185] In an exemplary procedure, the powder was further granulated using a high-shear process.

[0186] Equipment details: Balance: SS1000, High-shear mixer: Mini-CG 1L, Syringe pump: LSP01-1C, Vacuum dryer: Vacucell 111, Vibrating screen: Fritsch analyzette 3.

[0187] Environment: Temperature: 22-26°C, Relative humidity: 50-70%.

[0188] In the exemplary procedure, granulated powder was prepared in a high-shear mixing / shear reactor having mixing blades with speeds of 150-500 rpm and cutting blades with speeds of 150-1000 rpm. Specifically, in the pre-mixing step, the material was weighed to a total weight of 100 g. The material was placed in a 1 L bowl of the high-shear mixer. The chopper speed was set to 100 rpm and the impeller speed to 200 rpm, and the mixture was mixed for 5 minutes. Next, in the granulation step, the chopper speed was set to 300 rpm, the impeller speed to 1000 rpm, and the sealing pressure was set to 0.05 MPa. Using a syringe pump, 8-16 mL of glycerol solution was sprayed into the bowl at a flow rate of 4 mL / min and a nozzle size of 0.4 mm to bind the particles. The spray pressure was 0.05 MPa. Granulation time: 2-4 minutes.

[0189] Next, in the post-granulation process, the chopper speed was adjusted to 300 rpm and the impeller speed to 1000 rpm. Granulation was then continued for 40-120 seconds. Note that no liquid was visible because glycerol-water was used as a binder during the process.

[0190] Next, the first vacuum drying was applied: the obtained composition was transferred to a tray and placed in a vacuum drying box. The vacuum pressure was set to approximately 0 Pa over 0.5 to 1 hour. Next, the powder was sieved: the powder was transferred to a vibrating sieve tray, with sieve sizes of 500 / 355 micrometers in appropriate order. The amplitude was set to 1.5 mm, and the sieving time was 10 minutes. Next, the second vacuum drying was applied: the powder was transferred to a vacuum drying box and maintained at a vacuum pressure of approximately 0 Pa for 3 hours to allow it to dry. Finally, the product was collected.

[0191] Materials in the formulation: 20-80% by weight of fibrinogen powder, 2000-4000 IU / dose of thrombin powder, 10-50% by weight of ORC powder, 2-4% by weight of CaCl2 powder, and 0-25% by weight of lysine powder.

[0192] In an exemplary preliminary procedure, 1.2–1.5 g of the powder sample to be tested was combined with 4–5 mL of a dispersion medium containing physiological saline and glycerol at specific ratios (v / v): 15% glycerol, 30% glycerol, 40% glycerol, 50% glycerol, and 70% glycerol. As comparative samples, the following samples were further tested: 1:1 ORC vs. fibrinogen, thrombin-free, and 2:1 ORC vs. BSA, thrombin-free (ratios are by weight). Details of the tested samples are provided in Example 2 below.

[0193] Example 2: Characterization of the fluidity properties of selected samples - Preliminary test In the exemplary procedure, experiments were conducted to test various samples in terms of their paste-like viscosity.

[0194] Preliminary test: In an exemplary procedure, 1.2 g of the powder sample to be tested was dispersed in 4 mL of a dispersion medium containing physiological saline and 50% glycerol (v / v). The samples tested were: (i) the aforementioned powder having a weight ratio of ORC to fibrinogen of 2:1 (containing thrombin, 3300 IU per 1.2 g); (ii) the aforementioned powder having a weight ratio of ORC to fibrinogen of 2:1 but without thrombin; (iii) a powder containing ORC and BSA, with an ORC to BSA weight ratio of 2:1 and containing thrombin; (iv) ORC; and (v) a powder containing ORC and thrombin (without fibrinogen).

[0195] The results are shown in Figure 1 (the photograph was taken immediately after the blend was incorporated into the dispersion medium). The results show that only sample (i) provided a paste-like composition when dispersed in the above dispersion medium. The other samples showed higher fluidity, and as a result, the composition could not remain at the wound site, resulting in a slower gelation rate.

[0196] Determination of dispersion amount: In the exemplary procedure, a dispersion medium containing physiological saline and 50% glycerol (v / v), and powders having ORC to fibrinogen weight ratios of 1:1 and 2:1 (with or without thrombin, 3300 IU / dose, i.e., 1.2 g) were selected. The samples further contained calcium and lysine as detailed in Example 1 above. The samples were evaluated for their fluidity with the aim of providing a fluid composition that was neither too thick (making it difficult to extrude) nor too thin. The tested samples are summarized in Table 1 below.

[0197] [Table 1]

[0198] It can be seen that the composition provided by 3 mL of dispersion medium is too concentrated, and the composition becomes very dilute with 5 mL. The results are shown in Figure 2. Taking these results into consideration, the sample tested in the following examples contains 1.2 g of powder dispersed in 4 mL of dispersion medium.

[0199] Additional exemplary tests were conducted on the fluidity and solidification time of tested samples containing 70%-80% glycerol in a ratio of 1:2-1:5 for ORC to fibrinogen weight ratios of 2:1, 1:1, 1:2, and 1:5. In the exemplary procedure, 1.2 g of the test sample was dispersed in 4 mL of a dispersion medium containing physiological saline and 70% or 80% glycerol (v / v). The sample further contained thrombin, calcium, and lysine, as detailed in Example 1 above.

[0200] Figure 3 shows the results for porcine blood coagulation time in vitro (the photograph was taken immediately after incorporating the blend into the dispersion medium). The results showed that the 70% glycerol sample in the dispersion medium provided a paste-like composition when dispersed in the aforementioned dispersion medium, while the 80% glycerol sample (mainly for the 2:1 to 1:2 samples) exhibited higher fluidity, resulting in a lower ability of the composition to remain at the wound site and a slower coagulation rate (blood coagulation time was 70 seconds for the 70% glycerol sample (1:2 and 1:5 ratios), exceeding 180 seconds for the 1:5 sample). It can be concluded that approximately 70% glycerol is the upper limit of the concentration of the compositions disclosed herein.

[0201] Example 3: Rheological properties In the exemplary procedure, the sample was tested for its viscosity.

[0202] A 1.2 g portion of the powder sample to be tested was dispersed in 4 mL of a dispersion medium containing physiological saline and 50% glycerol (v / v) with an ORC to fibrinogen weight ratio of 1:5 to 2:1. The sample further contained thrombin (3300 IU per 1.2 g, calcium, and lysine, as detailed in Example 1 above).

[0203] Viscosity: The mixed sample was sandwiched between two 25mm parallel plates and a Peltier plate surface. For all measurements, the gap between the two plates was set to 1.00mm. A rotation mode test with a shear rate of 0.5 1 / s and a temperature of 25°C was used for all measurements.

[0204] Viscosity was determined by the ratio of shear stress to shear rate. The results are shown in Figure 4, which indicates that when the weight ratio of ORC to fibrinogen is in the range of 1:5 to 2:1, the viscosity is above approximately 400 and below approximately 3000 Pa·s.

[0205] Amplitude Sweep Tests: In an exemplary series of experiments, the shear loss (G') and shear storage (G'') moduli of the specimens were evaluated via a shear rheometer. Measurements were performed using an Anton Paar Modular Compact Rheometer MCR102. All tests were conducted at a temperature of 25°C. In strain sweep tests, the test frequency was fixed and the amplitude was gradually increased. Strain sweep tests were performed to determine the linear viscoelastic regime of the specimens. Next, sweep tests were performed in the shear strain (vibration) range of 0.01–100% and angular frequencies up to 10 rad / s. The amplitude is the maximum value of the vibrational motion. For analysis, the storage modulus G' and loss modulus G'' are plotted against deformation. If G' > G'', the specimen exhibits a gel-like or solid structure and can be called a viscoelastic material. The limits of the linear viscoelastic region are also determined first. In the linear viscoelastic region, the functions of G' and G'' show a constant plateau value, which indicates the range in which tests can be performed without destroying the structure of the specimen. The intersection of G' and G'' is the yield point (τ). τ = F / A (where τ is the shear stress, F is the shear force in Newtons, and A is the shear area in m²). 2 ) is ).

[0206] The results presented in Figure 5 show a linear viscoelastic region in which the storage modulus (G') and loss modulus (G") remain unchanged, meaning that samples with ORC to fibrinogen weight ratios of 1:5 to 2:1 exhibit strong stability. The results also show that the 2:1 sample has the highest yield point and a longer linear viscoelastic region, while the 1:5 sample has the shortest linear viscoelastic region, meaning that a 1:5 ORC to fibrinogen ratio may not be very favorable.

[0207] Example 4: Coagulation and Stability Test Ex vivo coagulation of porcine blood: Blood coagulation with several compositions of the present invention and comparative compositions ex vivo was tested as follows. A 1.2 g portion of each sample was dissolved in 4 mL of physiological saline + glycerol (v / v) for 20-40 minutes to form a paste. 2 mL of blood was taken into a penicillin bottle, 1 mL of the sample (approximately 0.24 g of powder or 0.5 g of SURGIFLO® gelatin matrix) was added, and the bottle was then gently shaken three times using a vortex meter (IKA VORTEX 3). Coagulation time was observed and recorded along with the state of each sample at different time points. The time of coagulation formation was evaluated by tilting the vial over time. Samples were tested at 0.5 hours, 2 hours, and 20 hours after sample preparation (reconstitution, then mixing the powder with the dispersion medium several times back and forth to form a paste). Coagulation time was determined by visual observation of blood flow through an inverted sample vial. At the end of the coagulation time, the blood clot did not detach from the vial. The results are summarized in Table 2 below.

[0208] [Table 2]

[0209] The results show that, at ORC to fibrinogen weight ratios of 2:1 to 1:1, the preferred concentration of glycerol in the dispersion medium (which does not impair enzyme activity, as described below) is greater than 15% by volume and less than 50% by volume, providing a coagulation time of less than 60 seconds at each time point.

[0210] Effect of pH and glycerol concentration on stability: Additional experiments were conducted to test the stability of various samples after reconstitution at different pH values ​​using L-lysine to adjust the pH. Samples tested: (1) ORC vs fibrinogen 2:1, physiological saline + 50% glycerol, pH 4.48 ("2:1, 50%, 4.48"), and accordingly: (2) 2:1, 50%, 5.38, (3) 2:1, 50%, 6.53, (4) 2:1, 40%, 4.44, (5) 2:1, 30%, 4.44, (6) 2:1, 15%, 4.33, (7) 1:1, 50%, 5.03, (8) 1:1, 50%, 5.46, (9) 1:1, 40%, 5.05, (10) 1:1, 30%, 4.81, (11) 1:1, 15%, 4.82, (12) SURGIFLO® + thrombin. The sample further contains thrombin, calcium, and lysine, as detailed in Example 1 above. The results shown in Figure 6 demonstrate liquid-phase separation for the 15% glycerol sample after 20 hours.

[0211] Effect of pH on clotting time: In the exemplary procedure, the effect of pH on clotting time was further investigated using 2:1 and 1:1 formulations (50% glycerol (v / v), physiological saline solution). The protocol for testing thrombin activity is detailed above and was performed at room temperature. The results are presented in Table 3 and Figure 7 below.

[0212] [Table 3]

[0213] These results indicate a preferred pH range of approximately 4.5–6.5, within which the blood clotting time is further confirmed to be 50 seconds or less in at least two tests.

[0214] Example 5: Activity test over time Tested Samples with 2:1 to 1:1 Ratios: Thrombin activity in the tested samples is expressed as specific activity reported as IU / mg. To limit thrombin activity and minimize fibrin formation, the tested samples were hydrated with alkaline carbonate. A small amount of sample dissolved in the carbonate solution was neutralized with a large amount of buffered fibrinogen solution to enable the coagulation reaction. In an exemplary procedure, 1.2 g of the tested sample was dispersed in 4 mL of dispersion medium with varying concentrations of glycerol (%v / v) and pH values ​​in physiological saline. Table 4 shows the results for thrombin (enzyme) activity (IU / 1.2 g) at 0.5 hours, 1.5 hours, 2.5 hours, 4.5 hours, and 20 hours for each tested sample with specific ORC to fibrinogen weight ratios of 2:1 or 1:1 and glycerol concentration (%v / v), according to the following protocol.

[0215] The results are further shown in Figure 8.

[0216] [Table 4]

[0217] Effect of pH on thrombin stability over time: In an exemplary procedure, the effect of pH on thrombin stability over time was tested using 2:1 and 1:1 formulations (50% glycerol (v / v), physiological saline solution). pH was adjusted using L-lysine powder, and pH was measured using a surface electrode pH meter (METTLER TOLEDO). The protocol for testing thrombin activity is detailed above and was performed at room temperature. The results are presented in Table 5 and Figure 9 below.

[0218] [Table 5]

[0219] In summary, the results indicate that thrombin activity did not significantly decrease within 20 hours under different formulations and pH conditions, thus demonstrating good stability within 20 hours. Nevertheless, the preferred pH range is approximately 4.5–6.5, because within this range, thrombin activity did not decrease to a noticeable degree within 20 hours and remained stable.

[0220] Example 6: Effect on thickness In the exemplary procedure, specific amounts of various samples were taken and stacked on the test platform of a thickness tester (CDK). The sample thickness was allowed to decrease naturally while compressing it. The sample thickness was then recorded, and the change in sample thickness over time was observed. Various samples with specific ORC to fibrinogen weight ratios of 2:1 or 1:1 and glycerol concentrations (%v / v, 4 mL / 1.2 g) were tested, and the results are presented in Table 6. The samples further contained thrombin, calcium, and lysine, as detailed in Example 1 above.

[0221] [Table 6]

[0222] When the ORC-to-fibrinogen ratio is less than 2:1 (1:1, 1:2, and 1:5), the reconstituted paste becomes harder and thicker, and at a ratio of 1:5, it becomes too hard. Figure 10, which shows the results of representative tested samples, further illustrates this characteristic.

[0223] Example 7: In vivo evaluation in porcine liver and spleen models In the exemplary procedure, selected samples were tested to evaluate the hemostatic efficacy of the fluid hemostatic composition using a pig punch bleeding model (spleen and liver biopsy punch bleeding model) under heparinized conditions.

[0224] In the exemplary procedure, the samples shown in Table 8 were tested (in 50% v / v glycerol / saline).

[0225] In the exemplary procedure, a 6mm puncher was used to create a 5mm deep punch hole on the surface of the spleen or liver. Before testing the application of the article, excess blood was absorbed / removed from the target bleeding site by using gauze or suction so that the hemostatic paste could be applied directly to the bleeding site with a bleeding surface. A sufficient amount of paste was applied to the site, covering the entire bleeding area in multiple layers.

[0226] First, the tampon was inflated and maintained for 3 minutes, then the gauze was lifted from the site and the site was observed for up to 60 seconds. If free-flowing blood was observed at any point during the observation period, the tampon was reapplied to the gauze and held for another minute. Hemostasis success or failure was determined from the pressed samples at 3, 5, and 10 minutes after application. The test was interrupted and the results were recorded as raw data for over 10 minutes. Once the bleeding stopped, excess sample was washed away with 10 mL of saline solution.

[0227] Acceptance Criteria: Hemostasis was determined by the cessation of free-flowing bleeding at 1, 2, 3, and 4 minutes after application, using a 0-5 rating scale (Table 7 and Figure 11) to measure bleeding.

[0228] Hemostasis was considered successful if "no bleeding - 0" or "exudation - 1" was achieved after application of the substance.

[0229] [Table 7]

[0230] The results are summarized in Table 8.

[0231] [Table 8]

[0232] Further results are shown in Figure 12.

[0233] In a separate in vivo study, a dog punch model (n=5) using different formulations was conducted using a protocol similar to that used for the liver. Dogs received an initial loading intravenous bolus of 100–300 IU / kg of heparin sodium. Activated clotting time (ACT) was measured approximately 10 minutes after injection. Animals were considered heparinized if the ACT was >×3 baseline reading. The results are summarized in Table 13.

[0234] In summary, the results above show a high success rate of hemostasis (over 60%) for the tested samples, with the 1:1 ratio being slightly superior.

[0235] While the present invention has been described in conjunction with its specific embodiments, it is evident that many alternative, modified, and variant forms will be obvious to those skilled in the art. Therefore, this is intended to encompass all such alternative, modified, and variant forms that fall within the spirit and broad scope of the appended claims.

[0236] [Implementation Method] (1) A two-component fluid composition comprising (i) a powder containing oxidized cellulose, fibrinogen, and thrombin, wherein the weight ratio of oxidized cellulose to fibrinogen is in the range of about 1:5 to about 2:1, and (ii) a dispersion medium containing glycerol and an aqueous solution, which is in the form of a paste-like suspension at room temperature. (2) The composition according to Embodiment 1, wherein the weight ratio of OC to fibrinogen is in the range of 1:2 to 2:1. (3) The composition according to Embodiment 1 or 2, wherein the OC comprises oxidized regenerated cellulose (ORC). (4) The composition according to any one of Embodiments 1 to 3, wherein at least a portion of the powder is in an aggregated form. (5) The composition according to any one of Embodiments 1 to 4, wherein the glycerol is present in a concentration exceeding approximately 15% by volume of the dispersion medium.

[0237] (6) The composition according to any one of Embodiments 1 to 5, wherein the glycerol is present in a concentration exceeding approximately 40% by volume of the dispersion medium. (7) The composition according to any one of Embodiments 1 to 6, wherein the glycerol is present in the dispersion medium at a concentration of about 70% by volume or less. (8) The composition according to any one of Embodiments 1 to 7, wherein the dispersion medium is present in a dispersion medium-to-powder ratio in the range of 3:1 to 4:1 (v / w; mL / g). (9) The composition according to any one of embodiments 1 to 8, wherein the OC is in a pulverized form. (10) The composition according to any one of embodiments 1 to 9, further comprising fibrin.

[0238] (11) The composition according to Embodiment 10, wherein the fibrin is at least partially crosslinked. (12) The composition according to any one of Embodiments 1 to 11, wherein the thrombin is present in an amount of about 2000 to about 4000 IU per gram of powder. (13) The composition according to any one of embodiments 1 to 12, further comprising a calcium salt. (14) The composition according to any one of embodiments 1 to 13, further comprising a buffering agent. (15) The composition according to Embodiment 14, wherein the buffering agent is in the form of a powder.

[0239] (16) The composition according to Embodiment 14 or 15, wherein the buffer is selected from Tris, Lysine, or a combination thereof. (17) The composition according to any one of embodiments 1 to 16, wherein at least 90% of the powder has a particle size in the range of 10 to 2,000 μm. (18) The composition according to any one of Embodiments 1 to 17, wherein the particle size is in the range of about 200 to about 900 μm. (19) A composition according to any one of Embodiments 1 to 18, which is stable at pH 4 to 6.5 and at room temperature for at least about 20 hours. (20) The composition according to any one of embodiments 1 to 19, wherein the aqueous solution contains physiological saline.

[0240] (21) The composition according to any one of embodiments 1 to 20, wherein the powder is spray-dried and / or high-shear mixed. (22) The composition according to any one of Embodiments 1 to 21, wherein the fibrinogen is present in a concentration range of 60% to 95% by weight or 70% to 90% by weight of the powder. (23) A composition according to any one of Embodiments 1 to 22 for use as a hemostatic agent at a bleeding site in tissue. (24) Use of a composition according to any one of embodiments 1 to 23 in a method for treating hemorrhagic tissue, wherein the method includes applying the composition to the hemorrhagic tissue. (25) The use of the method according to Embodiment 24, wherein the method includes minimally invasive surgery, such as laparoscopic surgery.

[0241] (26) A method for preparing the composition according to any of Embodiments 1 to 23, comprising: combining the OC fibers with fibrinogen powder and thrombin powder under conditions that enable the formation of aggregated forms of OC fibers, fibrinogen and thrombin; and combining the aggregates with a dispersion medium comprising glycerol and an aqueous solution. (27) The method according to embodiment 26, wherein the conditions enable at least partial conversion of fibrinogen to fibrin. (28) The method of Embodiment 27, wherein the conditions enable at least partial crosslinking of the fibrin. (29) The method according to either Embodiment 26 or Embodiment 28, wherein the above conditions are selected from one or more of the following: (i) to (iii): (i) a temperature in the range of 0 to 30°C, (ii) a pH of the dispersion medium in the range of 4.5 to 6.5, and (iii) a dispersion medium to powder ratio in the range of 3:1 to 4:1 (v / w; mL / g), respectively. (30) Kit, (a) A container for containing powders having oxidized cellulose to fibrinogen and thrombin, each having a weight ratio of oxidized cellulose to fibrinogen in the range of approximately 1:5 to approximately 2:1, (b) A container for containing a dispersion medium containing glycerol and optionally an aqueous solution, and optionally (c) Kit including instruction manual.

Claims

1. (i) a powder comprising oxidized regenerated cellulose, fibrinogen, and thrombin, wherein the weight ratio of the oxidized regenerated cellulose to the fibrinogen is in the range of about 2:1 to about 1:1, and (ii) a dispersion medium comprising glycerol and an aqueous solution, which is in the form of a paste-like suspension at room temperature. The glycerol is present in the dispersion medium at a concentration of approximately 50% by volume. The thrombin is present in an amount of approximately 2000 to 4000 IU per gram of the powder. A two-component fluid composition in which the dispersion medium is present in a dispersion medium-to-powder ratio in the range of 3:1 to 4:1 (v / w; mL / g).

2. The two-component fluid composition according to claim 1, wherein at least a portion of the powder is in an aggregated form.

3. The two-component fluid composition according to claim 1 or 2, wherein the oxidized regenerated cellulose is in a pulverized form.

4. A two-component fluid composition according to any one of claims 1 to 3, further comprising fibrin.

5. The two-component fluid composition according to claim 4, wherein the fibrin is at least partially crosslinked.

6. A two-component fluid composition according to any one of claims 1 to 5, further comprising a calcium salt.

7. A two-component fluid composition according to any one of claims 1 to 6, further comprising a buffering agent.

8. The two-component fluid composition according to claim 7, wherein the buffering agent is in the form of a powder.

9. The two-component fluid composition according to claim 7 or 8, wherein the buffer is selected from Tris, lysine, or a combination thereof.

10. The two-component fluid composition according to any one of claims 1 to 9, wherein at least 90% of the particle size of the powder is in the range of 10 to 2,000 μm.

11. The two-component fluid composition according to claim 10, wherein the particle size is in the range of about 200 to about 900 μm.

12. A two-component fluid composition according to any one of claims 1 to 11, which is stable at a pH of 4 to 6.5 and at room temperature for at least about 20 hours.

13. The two-component fluid composition according to any one of claims 1 to 12, wherein the aqueous solution contains physiological saline.

14. The two-component fluid composition according to any one of claims 1 to 13, wherein the powder is spray-dried and / or high-shear mixed.

15. A two-component fluid composition according to any one of claims 1 to 14, for use as a hemostatic agent at a bleeding site in tissue.

16. A method for preparing a two-component fluid composition according to any one of claims 1 to 15, comprising: combining oxidized regenerated cellulose fibers with fibrinogen powder and thrombin powder under conditions that enable the formation of aggregate forms of oxidized regenerated cellulose fibers, fibrinogen and thrombin; and combining the aggregates of oxidized regenerated cellulose fibers, fibrinogen and thrombin with a dispersion medium containing glycerol and an aqueous solution.

17. The method according to claim 16, wherein the conditions enable at least partial conversion of the fibrinogen to fibrin.

18. The method according to claim 17, wherein the conditions enable at least partial crosslinking of the fibrin.

19. The method according to any one of claims 16 to 18, wherein the above conditions are selected from one or more of the following: (i) to (iii): (i) a temperature in the range of 0 to 30°C, (ii) a pH of the dispersion medium in the range of 4.5 to 6.5, and (iii) a dispersion medium to powder ratio in the range of 3:1 to 4:1 (v / w; mL / g), respectively.

20. It's a kit, (a) A container for containing a powder comprising oxidized regenerated cellulose, fibrinogen, and thrombin, wherein the weight ratio of the oxidized regenerated cellulose to the fibrinogen is in the range of approximately 2:1 to approximately 1:1, (b) A container for containing a dispersion medium which contains glycerol and optionally an aqueous solution, and optionally (c) Instruction manual and, The glycerol is present in the dispersion medium at a concentration of approximately 50% by volume. The thrombin is present in an amount of approximately 2000 to 4000 IU per gram of the powder. The aforementioned dispersion medium is present in a dispersion medium-to-powder ratio in the range of 3:1 to 4:1 (v / w; mL / g) in each kit.

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