Fluid sealant hemostatic agent

Abstract

To provide a fluid hemostatic composition. [Solution] Disclosed herein is a two-phase fluid composition in the form of a paste comprising (i) powders of fibrinogen, fibrin, and thrombin, and (ii) a dispersion medium of glycerol and an aqueous solution. Processes and kits for preparing the composition are also disclosed. Use of the composition for treating bleeding in a subject is also disclosed.

Description

Technical Field

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

Background Art

[0002] In various situations during surgery, therapeutic agents are spread within the human body to prevent postoperative bleeding. Existing medicaments may have drawbacks, for example, it is difficult to place sheets in minimal invasive surgeries (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 the bleeding, the anatomical location of the bleeding source and the proximity of adjacent critical structures, whether the bleeding originates from a discrete bleeding source or a broader surface area, the visibility and accurate identification of the bleeding source, and the access to the bleeding source.

[0004] In efforts to address the above-mentioned problems, substances for controlling excessive bleeding have been developed. Topical Absorbable Hemostat (TAH) is widely used in surgical applications. TAH includes products based on oxidized cellulose (OC), gelatin, collagen, chitin, chitosan and the like. To improve hemostatic performance, scaffolds based on the aforementioned substances can be combined with biologically derived coagulation factors such as thrombin and fibrinogen.

[0005] However, difficult-to-access bleeding sites, including deep anatomical areas, narrow clefts, or voids, are important cases in surgical procedures. There is a great need to develop hemostatic agents that can accurately reach narrow and deep bleeding sites. The hemostatic agent should ideally exhibit fluidity without compromising adhesion. That is, it must adhere strongly to selected tissue even under high fluid pressure, wet, and dynamic conditions, and remain in place until hemostasis is achieved.

[0006] PCT Patent Application No. PCT / CN2021 / 087167 discloses a two-component fluid composition 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 comprising glycerol and an aqueous solution, wherein the composition is in the form of a paste-like suspension at room temperature. The use of the composition for treating bleeding tissue and a method for preparing the composition are also disclosed. [Overview of the Initiative] [Means for solving the problem]

[0007] The present invention relates, in particular, to a fluid hemostatic composition comprising a blend of fibrinogen, thrombin, and a dispersant.

[0008] The object of the present invention is to provide a composition for preparing a spreadable hemostatic agent that can be easily applied to the required bleeding site, including hard-to-reach areas of the body, adheres rapidly to the bleeding site, and then forms a sealant therein. A particular object of the present invention is to develop a fluid hemostatic agent that, after being applied to the bleeding site during surgery, possesses both fluidity and adhesion to tissues and / or platelets. Inaccessible bleeding sites, including deep anatomical areas, narrow clefts, or voids, are visible but difficult to access with conventional medical devices and are important cases in surgeries. There is a great need to develop a hemostatic agent that can accurately reach and flexibly flow into narrow, deep bleeding sites while possessing fluidity without compromising adhesion. Platelet adhesion is important for hemostatic agents that must accurately reach the bleeding site and remain in place until hemostasis is achieved.

[0009] 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.

[0010] Therefore, the present invention is based on the remarkable finding that such a paste-like suspension containing fibrinogen / fibrin and thrombin powder 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. The inventors have developed a fluid hemostatic paste that can remain in place after being applied to a bleeding site. The disclosed paste can absorb some of the overflowing blood, thereby helping the paste adhere quickly and closely to deep bleeding sites, and the paste can form a protein sealant at the bleeding site, thereby further enhancing its adhesion to tissue.

[0011] While it is counterintuitive to use an aqueous solution in such a suspension, without being bound by any particular theory or mechanism, it can be assumed that after mixing porous powders in a dispersion medium, the uniformly dispersed biological product forms fibrin, which is at least partially crosslinked in a glycerol / saline system, forming a paste in which thrombin remains abundant in the system.

[0012] Thus, the disclosed compositions have been demonstrated to be easy to use, stable, and effective hemostatic agents.

[0013] According to one aspect of the present invention, a two-phase fluid composition is provided that is in the form of a paste at room temperature. (i) A powder containing fibrinogen and fibrin, as well as thrombin, present together in an amount of at least about 70% by weight, (ii) A dispersion medium comprising glycerol and an aqueous solution, The dispersion medium is present in a ratio of dispersion medium to powder in the range of approximately 3:1 to 5:1 (v / w, mL per g), and glycerol is present at a concentration exceeding approximately 30% by volume of the dispersion medium.

[0014] In some embodiments, the powder is in the form of porous particles. In some embodiments, at least 50% of the particles are characterized by having a pore size in the range of about 10 to about 30 μm.

[0015] In some embodiments, the composition contains fibrin in an amount of about 60% to 80% by weight.

[0016] In some embodiments, fibrinogen is present in a concentration range of about 12 to about 20 w / w%. In some embodiments, fibrin is present in a concentration range of about 53 to about 82 w / w%. In some embodiments, thrombin is present in a concentration range of 4 to 25 w / w%.

[0017] In some embodiments, the fibrin is at least partially crosslinked.

[0018] In some embodiments, fibrinogen and / or fibrin are present in the powder in an amount of about 80% by weight.

[0019] In some embodiments, the powder further comprises a calcium salt. In some embodiments, the calcium salt is CaCl2, in an amount of up to about 3%, for example, 0.5 to 3% by weight, for example, about 2 w / w%. In this specification, “CaCl2” also means further comprising any of their hydrates, for example, but not limited to CaCl2·H2O(hydrate), CaCl2·2H2O(dihydrate), or combinations thereof.

[0020] In some embodiments, thrombin is typically present in an amount of about 2000 to about 4000 IU per gram of powder.

[0021] In some embodiments, at least 80% of the powder is in the form of particles smaller than 500 μm. In some embodiments, at least a portion of the particles are aggregated. In some embodiments, glycerol is present in the dispersion medium at a concentration ranging from about 30% to about 80% by weight. In some embodiments, the aqueous solution contains physiological saline.

[0022] In some embodiments, the composition (paste) is characterized by a uniform viscosity. In some embodiments, the paste is characterized by a viscosity in the range of about 3000 to about 5000 Pa·s at about 25°C. In some embodiments, the viscosity varies within a range of less than about 30% after storage at 25°C for 24 hours. In some embodiments, the composition is characterized by a swelling rate of up to 10% at about 25°C upon contact with blood.

[0023] In some embodiments, the composition does not comprise oxidized cellulose.

[0024] In some embodiments, the composition does not comprise a buffer agent.

[0025] In some embodiments, the composition is a flowable hemostatic composition.

[0026] In some embodiments, the composition is for use in a method for treating bleeding tissue, which comprises applying the composition to the bleeding tissue.

[0027] In some embodiments, the method comprises minimally invasive surgery, such as laparoscopic surgery.

[0028] According to another aspect, provided is a method of making a hemostatic composition characterized by a viscosity in the range of about 3000 to about 5000 Pa·s at about 25°C and / or a swelling rate of up to 10% at about 25°C, the method comprising the following steps performed subsequently: (i) aerating a fibrinogen solution to obtain a foam; and (ii) spraying a thrombin solution into the foam so that the foam further incorporates thrombin; and (iii) agitating the thrombin-containing foam to partially convert fibrinogen to fibrin; and (iv) lyophilizing the fibrin; and (v) pulverizing the lyophilized fibrin, thereby obtaining a uniform powder. (vi) The process includes the step of mixing the powder with a dispersion medium containing an aqueous solution, glycerol, and thrombin, under conditions that enable the preparation of a hemostatic composition.

[0029] In some embodiments, the conditions are selected from one or more of the following: (a) the dispersion medium contains thrombin in an amount of about 200 to about 800 IU per 1 mL; (b) the dispersion medium is added to the powder in a dispersion medium-to-powder ratio ranging from 3:1 to 5:1 (v / w, mL / g); and (c) at least about 70% of the foamy substance obtained in step (ii) contains fibrin.

[0030] In some embodiments, the thrombin solution of step (ii) further comprises a calcium salt.

[0031] In some embodiments of this model, the calcium salt is CaCl2, with a concentration of 0.5 to about 3 w / v%.

[0032] According to another embodiment, (a) A container for containing a powder comprising fibrinogen, thrombin and optionally fibrin, wherein fibrinogen + fibrin is present in a total amount of at least about 70% by weight, (b) A kit is provided comprising a container for containing a dispersion medium, which optionally contains glycerol and an aqueous solution.

[0033] Embodiments of the powder are described throughout this specification and are similarly incorporated into the kit.

[0034] In some embodiments, the kit includes an applicator device.

[0035] 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]

[0036] Several embodiments of the present invention will be described herein with reference to the accompanying drawings for illustrative purposes only. With further detailed reference to the drawings, the details shown are illustrative and intended to provide an illustrative description of 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] The image shows a photograph illustrating a process according to one embodiment of the present invention, in which a first component containing fibrinogen and thrombin powder is mixed with a second component containing a dispersion medium containing glycerol and an aqueous solution using an applicator consisting of two syringes, and the resulting paste is applied by connecting an applicator tip to the applicator. "Component 1" is thrombin powder with a potency of 3300 IU, containing 1 g of fibrin sealant (FS) powder + CaCl2 (approximately 21 mg). "Component 2" is 4 mL of 50 v / v% glycerin solution (in physiological saline). [Figure 2A] Figure 2A shows a flowchart with photographic images illustrating an exemplary embodiment of the process for producing the powder of the present invention, and Figure 2B shows a photographic image of the obtained paste. [Figure 2B] Figure 2A shows a flowchart with photographic images illustrating an exemplary embodiment of the process for producing the powder of the present invention, and Figure 2B shows a photographic image of the obtained paste. [Figure 3A]The bar graph in Figure 3A shows the comparative viscosity of the fluid paste of the present invention versus the oxidized regenerated cellulose (ORC) paste (bar graph in Figure 3B) after standing at room temperature for a maximum of 24 hours as indicated. As shown in Figure 3C, samples were taken from various parts of a syringe: F (front), M (middle), and E (end) to test the homogeneity of the samples. [Figure 3B] The bar graph in Figure 3A shows the comparative viscosity of the fluid paste of the present invention versus the oxidized regenerated cellulose (ORC) paste (bar graph in Figure 3B) after standing at room temperature for a maximum of 24 hours as indicated. As shown in Figure 3C, samples were taken from various parts of a syringe: F (front), M (middle), and E (end) to test the homogeneity of the samples. [Figure 3C] The bar graph in Figure 3A shows the comparative viscosity of the fluid paste of the present invention versus the oxidized regenerated cellulose (ORC) paste (bar graph in Figure 3B) after standing at room temperature for a maximum of 24 hours as indicated. As shown in Figure 3C, samples were taken from various parts of a syringe: F (front), M (middle), and E (end) to test the homogeneity of the samples. [Figure 4A] The swelling rate of the fluid paste of the present invention is shown in comparison to the swelling rate of the gelatin matrix. Samples were first incubated in phosphate-buffered saline (PBS) solution for 24 hours to allow them to swell (Figure 4A), and then the weight increase of each sample was measured. Five portions of the sample were tested. [Figure 4B] The swelling rate of the fluid paste of the present invention is shown in comparison to the swelling rate of the gelatin matrix. Samples were first incubated in phosphate-buffered saline (PBS) solution for 24 hours to allow them to swell (Figure 4A), and then the weight increase of each sample was measured. Five portions of the sample were tested. [Figure 5A]Figure 5A shows comparative photographic images demonstrating the blood absorption capacity of the fluid paste of the present invention and the blood absorption capacity of ORC paste (Figure 5B). Compared to ORC paste, the fluid paste of the present invention absorbs blood easily and uniformly, thereby promoting the coagulation cascade and forming an endogenous fibrin clot. [Figure 5B] Figure 5A shows comparative photographic images demonstrating the blood absorption capacity of the fluid paste of the present invention and the blood absorption capacity of ORC paste (Figure 5B). Compared to ORC paste, the fluid paste of the present invention absorbs blood easily and uniformly, thereby promoting the coagulation cascade and forming an endogenous fibrin clot. [Figure 6A] Figure 6A shows photographic images of the process from left to right, illustrating the efficacy test in an in vivo spleen punch model: a wound was created by biopsy punching (6 mm in diameter and 3 mm deep), the central core (tissue fragment) was removed, moderate bleeding persisted, the test sample was applied to cover the wound site, and pressed in with moist gauze for 2 minutes. The hemostasis result was then evaluated (success or failure), and five samples of the paste were tested. The success rates are shown in a bar graph (Figure 6B, from left to right: negative control - 0%, fibrin sealant of one embodiment of the present invention - 80%, gelatin matrix - 20%, and ORC paste - 40%). [Figure 6B] Figure 6A shows photographic images of the process from left to right, illustrating the efficacy test in an in vivo spleen punch model: a wound was created by biopsy punching (6 mm in diameter and 3 mm deep), the central core (tissue fragment) was removed, moderate bleeding persisted, the test sample was applied to cover the wound site, and pressed in with moist gauze for 2 minutes. The hemostasis result was then evaluated (success or failure), and five samples of the paste were tested. The success rates are shown in a bar graph (Figure 6B, from left to right: negative control - 0%, fibrin sealant of one embodiment of the present invention - 80%, gelatin matrix - 20%, and ORC paste - 40%). [Figure 7A]The images show scanning electron microscope (SEM) images of fibrin sealant powder (Figure 7A, bar 100 μm) prepared according to some embodiments of the present invention, which exhibit a coarse porous powder compared to a fluid powder prepared from ORC (ORC powder) which exhibits a non-porous structure (Figure 7B, bar 50 μm). [Figure 7B] The images show scanning electron microscope (SEM) images of fibrin sealant powder (Figure 7A, bar 100 μm) prepared according to some embodiments of the present invention, which exhibit a coarse porous powder compared to a fluid powder prepared from ORC (ORC powder) which exhibits a non-porous structure (Figure 7B, bar 50 μm). [Modes for carrying out the invention]

[0037] The creation of easily prepared, fluid hemostatic fibrin complex matrices for intraoperative application to hard-to-access bleeding sites is particularly difficult, especially during minimally invasive surgery (MIS), such as endoscopic surgery.

[0038] Based on the characteristics of the "bleeding site," inaccessible areas including deep anatomical sites, narrow clefts, or voids, while visible, are difficult to access with conventional medical equipment and represent important cases in surgical procedures.

[0039] The object of the present invention is therefore to provide an improved composition that can be easily applied to the required bleeding site, particularly in hard-to-reach areas of the body. A further advantage of the compositions of the present invention is that they are bioabsorbable and can therefore 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 or its vicinity where tissue is bleeding.

[0041] According to one aspect of the present disclosure, a two-phase fluid composition is provided that is in the form of a paste at room temperature, comprising (a) (i) a powder containing at least about 70 wt% of fibrinogen, (ii) thrombin, and (iii) a calcium salt, and (b) a dispersion medium containing glycerol and an aqueous solution. In some embodiments, the dispersion medium is present in a dispersion medium-to-powder ratio ranging from about 3:1 to about 5:1 (v / w, mL per g), and in some embodiments, the glycerol is present at a concentration of more than about 30 volume percent of the dispersion medium.

[0042] The terms “liquid medium,” “medium,” or “dispersion medium” may be used interchangeably throughout this Spec. Herein, the terms “dispersion medium,” “liquid medium,” and “liquid medium” mean a liquid medium having a viscosity that optionally provides a paste-like viscosity. Therefore, the term “dispersion medium” may be used in a general sense and may be used rather than “solvent” only to clarify that a disclosed blend or powder, for example, composed of fibrinogen or thrombin, is not substantially soluble in such a liquid medium, but rather forms a solid-liquid system.

[0043] In some embodiments, the dispersion medium exists in a ratio of approximately 2.5:1 to approximately 6:1 (v / w, mL / g units) (including any value and range between these), for example, a dispersion medium to powder ratio of 2.5:1, 3:1, 3.5:1, 4:1, or 5:1.

[0044] "Around room temperature" means a temperature range of 10°C to 40°C, or 15°C to 37°C, for example, at least one temperature value among 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C (including any value between these values).

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The origin of 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 & 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.

[0049] In exemplary embodiments, the powder contains fibrinogen at a concentration of 12–20 w / w%, fibrin at a concentration of 53–82 w / w%, thrombin at a concentration of about 2–25 w / w%, and calcium. In some embodiments, calcium is present in an amount of about 2 w / w%. In some embodiments, calcium is present in an amount of about 2 w / w% to about 4.5 w / w%. In some embodiments, calcium is present in an amount of about 2 w / w% to about 3 w / w%. In some embodiments, the powder is in the form of porous particles. As used herein, the term “porous” refers to a material having voids throughout its internal structure.

[0050] 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.

[0051] 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.

[0052] "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.

[0053] 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.

[0054] 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, typically ranging in size from 0.1 to 1000 micrometers (μm), sometimes less than 500 μm, and sometimes less than 100 μm, characterized by a small size of at least one dimension.

[0055] 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.

[0056] 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.

[0057] 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, in some embodiments, a buffered aqueous medium.

[0058] In exemplary embodiments, the aqueous solution includes physiological saline. As used herein, the term “physiological saline” refers to water, in water, typically desalinated water, for example, a sodium chloride solution of about 0.9 w / w%, containing a salt concentration greater than about 0.5 wt% and less than about 20 wt%.

[0059] In some embodiments, at least 90% of the particles in the powder are in the range of 10 to 800 μm. In some embodiments, at least 90% of the particles in the powder are in the range of 10 to 700 μm. In some embodiments, at least 90% of the particles in the powder are in the range of 100 to 800 μm. In some embodiments, at least 90% of the particles in the powder are in the range of 200 to 700 μm. In some embodiments, at least 90% of the particles in the powder are in the range of 100 to 600 μm. In some embodiments, at least 90% of the particles in the powder are in the range of 200 to 800 μm.

[0060] 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 600 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 500 μm.

[0061] 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 size, effectively dividing the group in half.

[0062] In some embodiments of any aspect of this disclosure, the plurality of particles are characterized by a median particle size of less than about 500 μm.

[0063] In some embodiments, at least a portion of the powder is in the form of one or more aggregates, one or more aggregates, or one or more granules. The term “aggregate” describes particles formed from aggregated components. Aggregates may optionally be produced by one of the following steps: humidifying the powder composition; compressing the powder, for example, with a roller and / or pounding the powder to form aggregate structures; dehumidifying; grinding; sieving the aggregates; or optionally introducing the resulting aggregates into a storage container or delivery device. The term “granules” or “granular material” may particularly mean aggregates of particles typically less than 900 μm, less than 800 μm, less than 700 μm, or even less than 500 μm. 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 aggregate particles of fibrinogen, thrombin, and optionally CaCl2 into aggregates.

[0064] In some embodiments, at least a portion of the powder or solid blend is in the form of aggregates or clumps. The term “clump” refers to a weak association of primary particles or aggregate particles that are typically held together by charge or polarity.

[0065] The term "at least a portion of the powder" refers to a portion of the particle population in the powder, for example, at least 10% to 100% of the powder, or sometimes, for example, at least 25% of the powder. Therefore, the term "at least a portion of the powder is in aggregate form" refers to a portion of the particle population in the powder, for example, at least 10% to 100%, forming aggregates.

[0066] The term "fluidity" also encompasses viscous solutions. A "viscous solution" refers to a solution that has high flow resistance but is still flowable. In some embodiments, the composition has a viscosity greater than about 400 Pa·s and less than about 8000 Pa·s.

[0067] The term "viscosity" refers to a fluid and slurry property that exhibits flow resistance, defined as the ratio of shear stress to shear rate. Viscosity typically has a described or understood shear rate. Throughout this specification, unless otherwise stated, with respect to viscosity, the shear rate is 0.5 sec⁻¹, and 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 of one layer of fluid as it passes over an adjacent layer.

[0068] In some embodiments, the composition has a viscosity greater than about 400 Pa·s and less than about 8000 Pa·s. In some embodiments, the composition has a viscosity in the range of about 1000 to about 7000 Pa·s. In some embodiments, the composition has a viscosity in the range of about 2000 to about 6000 Pa·s. In some embodiments, the composition has a viscosity in the range of about 3000 to about 5000 Pa·s. In some embodiments, the composition has a viscosity of about 2000 Pa·s, about 2500 Pa·s, about 3000 Pa·s, about 3500 Pa·s, about 4000 Pa·s, about 4500 Pa·s, and about 5000 Pa·s (including any values ​​and ranges between these).

[0069] In some embodiments, the composition has a uniform viscosity. The term “uniform viscosity” or homogeneous viscosity refers to a viscosity of at least about 3000 Pa·s, and varies by Pa·s value within the range of less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%.

[0070] In some embodiments, the composition has a stable viscosity. The term “stable viscosity” refers to a viscosity that changes within the range of less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% over at least 24 hours at temperatures near room temperature.

[0071] In some embodiments, the paste is characterized by a viscosity of approximately 2500 to approximately 4500 Pa·s, or 3000 to 4500 Pa·s, for example, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, or 4500 Pa·s (including any values ​​and ranges between these).

[0072] 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 typically may have homogeneous rheological properties for smooth passage while being injected through needles of various bore diameters (e.g., 14–32 gauge). Thus, in some embodiments, the composition can pass through an applicator. In some embodiments, the composition is characterized by a homogeneous viscosity, i.e., the viscosity of the composition changes within the range of less than |±40%|, less than |±30%|, less than |±20%|, less than |±10%|, or less than |±5%| within 4 hours after the dispersion medium is added to the powder (see Figure 3A).

[0073] As used herein, “homogeneous” or “uniform” means that the composition, viscosity, and texture are uniform throughout, and typically the powder is substantially uniformly dispersed throughout the dispersion medium. “Substantially uniformly dispersed” means that the concentration of the powder in the dispersion medium of the composition varies throughout the dispersion medium within the range of less than |±30% by weight|, less than |±20% by weight|, less than |±10% by weight|, or less than |±5% by weight|, and typically there is no significant solid-liquid phase separation. In addition, the term “homogeneous” typically 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).

[0074] In some embodiments, the composition is characterized by a low swelling rate. As used herein, the term “swelling rate” refers to the change in the percentage change in the volume of the paste when it is immersed in a liquid, such as blood, compared to the volume of the paste before immersion in the liquid; that is, the degree of expansion of the paste between the state before adsorbing the liquid (e.g., blood) and the state after adsorbing the maximum possible amount of liquid. In this specification, “low swelling rate” means a swelling rate of less than about ±5%.

[0075] While not constrained by any particular mechanism, a low swelling rate may allow for improved hemostatic effect in small defect sites in the absence of spatial occupancy effects; that is, the paste is assumed to remain at the bleeding site for an extended period. See Figure 5A for details.

[0076] 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.

[0077] 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.

[0078] Phase component compositions can be prepared by mixing a first component and a second component to create a suspension. As used herein, the term “phase component composition” refers to a composition comprising at least two phases. Typically, but not limited to, the two phases may be stored in separate containers and mixed before use to create the mixture or suspension, such as by applying the mixture or suspension to the site of application (for example, as shown in Figure 1).

[0079] As used herein, the term “mixture” means any combination of components in any physical form, such as a blend, solution, suspension, dispersion, etc.

[0080] The term “suspended substance” or any grammatical variation 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 24 hours in some embodiments. This period may depend on many factors, but are not limited to, 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.

[0081] In some embodiments, the composition further comprises fibrin. The term “fibrin” refers not only to fully solidified fibrinogen but also further comprises any mixture of fibrin and fibrinogen 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 gelling and / or solidification, as long as it does not adversely affect the final paste-like texture of the composition. Furthermore, the term “fibrin” further comprises any partially or fully crosslinked, gelled, or solidified form.

[0082] Therefore, it is understood that fibrin itself exists as a high-molecular-weight, insoluble matrix containing fibrils, and that each fibril contains many fibrin molecules.

[0083] In some embodiments, glycerol is present at a concentration greater than approximately 30% by volume of the dispersion medium. In some embodiments, glycerol is present at a concentration greater than 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.

[0084] In some embodiments, glycerol is present at a concentration of approximately 25% to over 80% by volume of the dispersion medium. In some embodiments, glycerol is present at a concentration of approximately 35% to over 80% by volume of the dispersion medium. In some embodiments, glycerol is present at a concentration of approximately 15% to over 60% by volume of the dispersion medium. In some embodiments, glycerol is present at a concentration of approximately 30% to over 80% by volume of the dispersion medium. In some embodiments, glycerol is present at a concentration of approximately 40% to over 60% by volume of the dispersion medium.

[0085] In some embodiments, glycerol is present in the dispersion medium at a concentration of 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 value and range between these).

[0086] In some embodiments, the dispersion medium has a pH in the range of about 4.5 to about 7.

[0087] Samples with approximately 50% glycerol provided a paste-like composition that offered both optimal viscosity and adherence to the target area during dispersion in the aforementioned dispersion medium, while retaining thrombin activity. On the other hand, samples with more than 80% glycerol exhibited high fluidity, resulting in a state where the composition could not adhere to the target area, such as a wound site. Further results showed that liquid-solid phase separation occurred in samples with less than 30% glycerol after 20 hours.

[0088] In some embodiments, the composition is stable. The terms “stable” and “stability,” when referring to the disclosed paste, mean, for example, that at least 70% of the activity of the active ingredient in thrombin remains after a certain period of time at a certain temperature, i.e., that it can form a fibrin mass.

[0089] In some embodiments, the composition is stable. 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 or a pre-selected physical state of the composition. Stability can be determined by methods known in the art, for example, by testing whether there are any visible aggregates, remnants, and / or undesirable clumps in the formulation under pre-selected storage conditions when the composition 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, that a sealant solidifies in essentially the same period of time whether it has been stored at room temperature (e.g., 8–40°C) or at a lower temperature (e.g., below 8°C).

[0090] In some embodiments, less than 25% by weight, less than 24% 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 a pH of 4–6.5 near room temperature 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.

[0091] In some embodiments, the composition further comprises additional pharmaceutically active agents contained within or on the surface of the composition, in any embodiment thereof.

[0092] In some embodiments, the pharmaceutically active agent is selected from the group consisting of therapeutic agents and labeling agents.

[0093] 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.

[0094] 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.

[0095] Alternatively or additionally, the drug is absorbed by a cross-linked protein matrix. Absorption can be achieved, for example, by contacting a cross-linked fibrinogen or fibrin matrix or a non-cross-linked fibrin matrix with a solution containing the drug (e.g., by immersion, soaking, or washing), or by adding the drug before cross-linking (e.g., by including the drug in a cross-linking solution, or by including the drug in a solution of fibrinogen and thrombin).

[0096] 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 for the form of the compositions 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 for use for specified uses and / or for the treatment of specified conditions, placed in appropriate containers, and labeled, as further detailed herein. The term "preparation" refers to a composition that is physiologically suitable for therapeutic use.

[0097] 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.

[0098] In another aspect of this disclosure, any of the compositions disclosed in those aspects or embodiments are intended for use in a method for preparing a fibrin sealant in a damaged tissue of interest by applying the composition to the surface of the tissue.

[0099] 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.

[0100] In some embodiments, the composition is substantially free of oxidized cellulose. The term “oxidized cellulose” (or “OC”) refers to a cellulose derivative in which at least a portion of a primary alcohol group, for example, the carbon at position 6 of an anhydrous glucose unit, is oxidized to a carboxylic acid and optionally functionalized. In some embodiments, the composition is substantially free of oxidized regenerated cellulose (ORC).

[0101] In some embodiments, the composition is substantially free of buffering agents. In some such embodiments, the buffering agent refers to Tris or Tris(hydroxymethyl)aminomethane buffer, solubilizer. In some such embodiments, the buffering agent refers to Tris or Tris(hydroxymethyl)aminomethane buffer, solubilizer in powder form.

[0102] The term “substantially absent” is defined herein as meaning almost absent or completely absent; that is, less than 1(w / w)%, less than 0.5(w / w)%, less than 0.3(w / w)%, less than 0.1(w / w)%, or 0% of the total weight of the composition.

[0103] Another aspect of the present disclosure provides a method for preparing a composition according to one embodiment thereof, for example, a hemostatic composition characterized by a viscosity in the range of about 3000 to about 5000 Pa·s at about 25°C and / or a swelling rate of up to 10% at about 25°C, the method comprising the following steps: (i) A step of foaming the fibrinogen solution to obtain a foamy substance, (ii) A step of spraying thrombin solution into a foamy substance so that the foamy substance further incorporates thrombin, (iii) A step of stirring a foamy substance containing thrombin so as to partially convert fibrinogen to fibrin, (iv) A step of freeze-drying the fibrin, (v) A step of grinding freeze-dried fibrin to obtain a uniform powder, (vi) A step of mixing the powder with a dispersion medium containing an aqueous solution, glycerol, and optionally thrombin, under conditions that enable the preparation of a hemostatic composition.

[0104] In some embodiments, the fibrinogen in the solution is at least 20 mg / mL, for example, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL (including any values ​​and ranges between these).

[0105] As used herein, the term “whisking” refers to the action of using a mixer to rapidly incorporate air and create a foamy substance by moving it back and forth, whisking, or beating the sample.

[0106] As used herein, the term “foaming” refers to a substance formed by trapping gas pockets in a liquid or solid. The term “spray” should be understood in a broad sense and may also include terms such as watering, atomizing, and misting. The term “grinding” refers to the process of reducing particles by collision with a rapidly moving surface in a mill. In some embodiments, the mill is a ball mill, bead mill, jet mill, or disc mill. The term “mixing” refers to blending or stirring together components (e.g., powder and dispersion medium).

[0107] In some embodiments, about 60 to about 95% by weight of fibrinogen, or in some embodiments, about 70 to about 85% by weight of fibrinogen, coagulates (i.e., is converted to fibrinogen).

[0108] The foam obtained in step (iii) may be freeze-dried and stored for later use, or freeze-dried and then immediately ground or pulverized into a powder. As used herein, the term “freeze-dried” or any grammatical variation thereof refers to a product obtained by a process of freezing a solution and then reducing the concentration of water by sublimation to a level that does not sustain, for example, biological or chemical reactions, thereby forming a “cake.” Such freeze-dried “cakes” having a low protein density may have a higher level of solubility. A solid dispersion system may optionally be prepared by mechanically crushing and / or grinding the “cake” to dissolve the “cake” in an aqueous solvent.

[0109] 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.

[0110] Typically, the grinding process may also be referred to as micronization. The term "micronization" is used, for example, when the diameter of the particles produced from a solid composition is at most a few hundred micrometers. Micronization can be achieved by processes including, but not limited to, jet mill grinding, pearl ball mill grinding, 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 powder is reduced in size by 30 to 400 times from its original size, but this is not limited to this.

[0111] In some embodiments, thrombin is present in the thrombin solution (step ii) at a concentration of about 1500 to about IU / mL. In some embodiments, thrombin is present in the thrombin solution at a concentration of about 1550 to about 2000 IU / mL.

[0112] In some embodiments, the thrombin solution further comprises, but is not limited to, one or more additives such as calcium salts such as calcium chloride, and / or one or more excipients selected from, for example, one or more amino acids, albumin, sugars, and / or sugar derivatives.

[0113] 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 herein.

[0114] 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.

[0115] In some embodiments, the method further comprises adding a calcium salt to the powder obtained in step (v). In some embodiments, the calcium salt is CaCl2 and is in an amount of up to approximately 3% by weight of the powder.

[0116] In some embodiments, a calcium salt, such as chloride, is dissolved in the thrombin solution. In some embodiments, the amount of calcium chloride dissolved in the thrombin solution is in the range of about 0.60 to about 3 w / v%, or in some embodiments, it is in the range of about 0.65 to about 2.6 w / v%.

[0117] As used herein, the term “excipient” means, for example, an inactive or non-therapeutic substance added to a pharmaceutical composition to provide a desired viscosity or stabilizing effect.

[0118] In some embodiments, a foamy substance, such as a freeze-dried sample, is ground into a powder having a median particle size of less than 1000 μm. In some embodiments, a foamy substance, such as a freeze-dried sample, is ground into a powder having a median particle size of less than 500 μm.

[0119] In some embodiments, the dispersion medium contains about 30 to about 80 v / v% glycerol in physiological saline. In some embodiments, the dispersion medium contains about 40 to about 80 v / v% glycerol in physiological saline. In some embodiments, the dispersion medium contains about 40 to about 70 v / v% glycerol in physiological saline. In an exemplary embodiment, the dispersion medium contains about 50% glycerol in physiological saline.

[0120] In some embodiments, the dispersion medium contains thrombin. In some embodiments, thrombin is added to the dispersion medium to catalyze blood clot formation when the paste is used at the bleeding site. In some embodiments, the amount of thrombin in the dispersion medium is in the range of about 100 to about 900 IU / mL. In some embodiments, the amount of thrombin in the dispersion medium is in the range of about 200 to about 800 IU / mL. In some embodiments, the amount of thrombin in the dispersion medium is in the range of about 300 to about 600 IU / mL. In an exemplary embodiment, the amount of thrombin in the dispersion medium is about 400 IU / mL.

[0121] In some embodiments, the method is (a) to (e): (a) thrombin is present in an amount of about 2000 to about 4000 IU per gram, (b) the dispersion medium is present in a dispersion medium to powder ratio in the range of 3:1 to 5:1 (v / w, mL / g), (c) at least about 70% of the foamy substance obtained in step (ii) contains fibrinogen, (d) the method is carried out at a temperature in the range of 0 to 30°C, and (e) The pH of the dispersion medium is in the range of 4.5 to 7. The procedure is carried out under conditions selected from one or more of these. In some embodiments, the conditions are selected from one or more of (a) to (c).

[0122] In some embodiments, such conditions allow for at least partial conversion of fibrinogen to fibrin.

[0123] In another aspect, the compositions disclosed in all of those embodiments are for use in methods for treating bleeding tissue, which include applying the compositions to bleeding tissue by obtaining a sealant at the site of application. Thus, a method for controlling bleeding is provided, which includes applying the compositions disclosed herein to a bleeding site.

[0124] 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., 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.

[0125] The term "hemostasis" refers to the ability to prevent, reduce, or stop bleeding from a wound, such as a surgical wound or trauma, or to assist in hemostasis, for example, by promoting blood clot formation. "Hemostasis (or Haemostasis)" refers to the first stage of wound healing. This is the process of stopping bleeding. "To assist in hemostasis" 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. Bleeding control is required in a variety of situations, including wound treatment.

[0126] 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.

[0127] 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.

[0128] As used herein, the terms “control,” “prevent,” or “reduce” may be used interchangeably herein with respect to bleeding, meaning that the rate of hemorrhage is essentially nullified or reduced by, for example, 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 not in contact with the bleeding site. Methods for determining the level of bleeding occurrence are known in the art.

[0129] Furthermore, with respect to bleeding, the terms “control,” “prevent,” or “reduce” may include, for example, at least partially sealing blood vessels at the site of bleeding in soft tissue.

[0130] 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.

[0131] 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 any chemical means. The term “injured tissue” also includes bruised, punctured, or lacerated tissue, 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.

[0132] The term "tissue" may refer to tissue selected from soft tissue and bone tissue.

[0133] As used herein, the term "soft tissue" refers to body tissue that is not solidified or calcified. The term also refers to soft tissue that is angiogenic and therefore a potential 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 viscera. Generally, soft tissue excludes bone tissue.

[0134] Therefore, in some embodiments, the disclosed compositions may be used by being applied to hard-to-reach bleeding sites during MIS, such as endoscopic surgery, in any of its embodiments. One common form of endoscopic surgery is laparoscopic surgery, which is minimally invasive examination and surgery performed inside the abdominal cavity.

[0135] 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. In 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. Sealant formation occurs within a relatively short coagulation time after application of the disclosed composition to the injured tissue, according to any aspect and / or embodiment thereof. Typically, when used after 1-3 minutes of tampon filling, an incomplete mass may form, which can stop bleeding, although more time may be required for the coagulation reaction to reach the paste surface and form a complete mass.

[0136] 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.

[0137] "Healing wounds" means, in particular, reducing blood loss at the site of tissue bleeding (in vivo) in patients undergoing surgery. Referring to Figure 6B, the high success rate of hemostasis (over 75%) of the tested samples disclosed herein is shown.

[0138] 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 physiological 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 for use as a hemostatic agent at a site of tissue bleeding.

[0139] 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 (see Figure 1).

[0140] 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.

[0141] In another embodiment, the present invention further provides a hemostatic kit comprising a container for containing the composition disclosed herein in that embodiment. In another embodiment, the present invention provides a hemostatic kit comprising: (b) a container for containing a powder comprising fibrinogen or fibrin and thrombin, wherein the fibrinogen or fibrin is present in an amount of at least about 70% by weight; (b) a container for containing a dispersion medium comprising glycerol and optionally an aqueous solution; and optionally (c) instructions for use.

[0142] In some embodiments, the kit further includes an applicator device.

[0143] In some embodiments, a powder containing fibrin and thrombin is obtained by the method described herein in one embodiment.

[0144] In some embodiments, the dispersion medium further comprises thrombin powder. Alternatively, the kit contains thrombin powder or thrombin solution in a separate container, such as a vial. The amount of thrombin powder may be in the range of 200 to 600 IU per 1 mg or 1 mL, for example, about 400 IU.

[0145] According to some embodiments, a kit is provided for preparing a composition comprising (i) a powder containing fibrinogen, fibrin, and optionally thrombin, and optionally (ii) a dispersion medium containing glycerol and optionally an aqueous solution. During preparation, the composition is in the form of a paste-like suspension at room temperature. (i) and (ii) may each be present in separate containers.

[0146] In some embodiments, the powder contains at least 60% by weight of fibrin, or in some embodiments, at least 70% by weight of fibrin.

[0147] Any aspect and embodiment of the compositions disclosed throughout this specification may be incorporated into the aspects and embodiments of the kit, as well as the embodiments of the dispersion medium and / or powder.

[0148] Optionally, fibrinogen or fibrin and thrombin are packaged separately in the kit (e.g., in dry form or in solution). Thus, each of the two components is packaged in separate packaging materials in addition to the overall kit packaging material. In some embodiments, fibrinogen, fibrin, and thrombin are packaged together in the kit in the same packaging material (e.g., as dry powder), and optionally, the dispersion medium is provided in an additional container.

[0149] In some embodiments, the kit further includes 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.

[0150] Optionally, the kit further includes a dispersant. The dispersion medium, such as glycerol, may be in pure form or in solution with another liquid (e.g., water, saline solution, or aqueous buffer). Optionally, the dispersant is packaged separately from the powder. Alternatively, the dispersant is packaged together with the powder, for example, as 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.

[0151] 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 combine liquids and powders that were initially separate, and then produce a substantially homogeneous paste mixture by moving the blended contents back and forth between two connected syringes through 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.

[0152] In some embodiments, at least one of the containers in the kit is a pre-filled syringe. In some embodiments, a syringe is provided in addition to the containers in the kit. In some embodiments, the container is of a specific type, such as a vial or an applicator such as a syringe.

[0153] 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” can mean any general structure, such as a container or vial, that can contain a powder, a dispersion medium disclosed herein, or any component thereof, or a paste.

[0154] 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 and a tip or needle configured to connect to the applicator. In one embodiment, the applicator has a syringe equipped with an injection 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.

[0155] 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 a surgical procedure, including before or after a surgical procedure 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.

[0156] 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.

[0157] 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.

[0158] In some embodiments of the kit or composition, the composition contains a calcium salt, such as calcium chloride (CaCl2), 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 of the powder. In some embodiments, the calcium salt, such as calcium chloride, is present in the blend at concentrations 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). In some embodiments, the calcium salt is present in the thrombin solution.

[0159] In some embodiments, fibrinogen is present in a concentration range of 65% to 80% by weight, 65% to 95% by weight, or 70% to 90% by weight of the powder. In some embodiments, fibrinogen is present in a concentration of 65% to 70% by weight, 75% to 80% by weight, 85% by weight, or 90% by weight of the powder (including any value and range between these). In some embodiments, fibrinogen is present in a concentration of about 80% by weight of the powder. In some embodiments, fibrin is present in a concentration range of 65% to 80% by weight, 65% to 95% by weight, or 70% to 90% by weight of the powder. In some embodiments, fibrin is present in a concentration of 65% to 70% by weight, 75% to 80% by weight, or 85% by weight, or 90% by weight of the powder (including any value and range between these). In some embodiments, fibrin is present in a concentration of about 80% by weight of the powder.

[0160] In exemplary embodiments, the blend or powder comprises approximately 70–80% by weight of fibrinogen or fibrin powder and 2000–4000 IU of thrombin / dose powder relative to the total weight of the powder. In this specification, “dose” means approximately 1.2 g.

[0161] 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 paste is supported on a pad to facilitate application or tamponade.

[0162] 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.

[0163] 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. Sterile liquids, powders, or pastes are generally defined as liquids, powders, or pastes that have undergone sterile filtration.

[0164] general The terms “comprises,” “comprising,” “includes,” “including,” and “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 structure may include additional components, processes, and / or parts, provided that these additional components, processes, and / or parts do not substantially alter the basic and novel properties of the claimed composition, method, or structure.

[0165] 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.

[0166] 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.

[0167] As used herein, unless otherwise explicitly indicated in the context, the singular forms "a," "an," and "the" refer to multiple objects. For example, the terms "compound" or "at least one compound" may refer to multiple compounds, including mixtures thereof.

[0168] 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.

[0169] 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.

[0170] As used herein, the term “method” means methods, means, techniques and procedures for accomplishing a given task, and includes, but is not limited to, methods, means, techniques and procedures that are known or readily developed as well-known methods, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical fields.

[0171] As used in the present invention, the term "treating" includes inhibiting, 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.

[0172] 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 possibilities of "A" or "B" or "A and B".

[0173] 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.

[0174] 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, including, but not limited to, 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.

[0175] 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]

[0176] Herein, in conjunction with the above description, the following examples illustrate some embodiments of the present invention in a non-limiting manner.

[0177] Example 1: Preparation of a fluid composition The objective of this research is to develop a fluid hemostatic agent that possesses both fluidity and tissue adhesion after being applied to the bleeding site during surgery.

[0178] Materials and methods

[0179] [Table 1] * For instructions on preparing "ORC Paste," please refer to the following.

[0180] The source of fibrinogen and thrombin was porcine plasma supplied by Bioseal Biotech Co. Ltd., located in Guangzhou, China, which was fractionated to obtain fibrinogen and thrombin.

[0181] Preparation of fibrinogen sealant powder Fibrinogen / fibrin sealant powder (FSP) was obtained by freeze-drying and pulverizing fibrin foam. This foam was prepared by pre-mixing porcine plasma-extracted fibrinogen and thrombin as follows: First, a fibrinogen solution (at least 20 mg / mL) was foamed until completely foamy (the foam should not slide off when scooped up and inverted). Foaming was performed to help create a porous structure containing proteins, which in turn allows for crosslinking reactions to occur in larger spaces, resulting in uniform and efficient crosslinking.

[0182] Next, a small amount of CaCl2 (0.65–2.6 w / v%) was completely dissolved in thrombin solution (1555–2000 IU / mL) and thoroughly mixed. Then, using a spray nozzle attached to a syringe applicator, the thrombin solution (containing CaCl2) was sprayed onto the foamy material and subsequently stirred to crosslink. It is estimated that approximately 70–85% of the fibrinogen coagulated during this process.

[0183] Next, a freeze-drying process was carried out. After freeze-drying, the water inside the porous structure could be easily evaporated, and the remaining porous structure ("cake") was easily and uniformly ground to prepare a homogeneous fluid paste. The freeze-dried sample was ground into a powder with a particle size of less than 500 μm. Although not constrained by the mechanism, it is noteworthy that freeze-drying helps maintain the activity of unreacted fibrinogen (approximately 20-30% of fibrinogen remains unreacted), which can be used to form an in-situ sealant when applied to bleeding sites. The freeze-drying parameters were pre-freezing at -45°C for 2 hours, primary drying at 15°C for 96 hours, and secondary drying at 37°C for 24 hours (using a SCIENTZ dry vacuum pump freeze-dryer, SCIENTZ-50F / B model, Ningbo Scientz Freeze-Drying Equipment Co.,Ltd).

[0184] The weight ranges of each component in the obtained powder were 12-20 w / w% for fibrinogen, 53-82 w / w% for fibrin, 4-25 w / w% for thrombin, approximately 2 w / w% for CaCl2, or 2.3 w / w% for CaCl2·H2O, and 2.6 w / w% for CaCl2·2H2O.

[0185] Paste preparation A paste was obtained by mixing the powder (1 g) with a dispersion medium (3-5 mL, approximately 50 v / v% glycerol in physiological saline) containing thrombin powder (3300 IU potency from approximately 21 mg of CaCl2). The dispersion medium had to have a viscosity high enough to remain at the bleeding site for a certain period of time. It was found that using a low concentration of glycerol (less than 30 vol%) in the dispersion medium resulted in phase separation of glycerol and water. On the other hand, if the concentration was too high (more than 70%), a paste with excessive viscosity was produced. The dispersion medium had a pH of approximately 4.5-7.

[0186] Thrombin (400 IU / mL) was further added along with the dispersion medium to ensure that a sufficient amount of thrombin was present to catalyze blood clot formation when the paste was applied at the bleeding site.

[0187] Figures 2A and 2B show a flowchart with photographic images illustrating an exemplary embodiment of the process for producing the powder of the present invention (Figure 2A), and a photographic image of the resulting paste (Figure 2B).

[0188] Comparative formulation Comparative fibrinogen and thrombin preparations ("ORC paste") were also prepared from porcine fibrinogen and thrombin solutions. These solutions were processed into powders by spraying and combined with ORC, lysine, and calcium salts. ORC powder was prepared by grinding or pulverizing the corresponding textile fibers, without a foaming process. The weight ratio of ORC to fibrinogen was 1:1 to 1:2. ORC paste contained buffers, for example, in the form of powders (Tris, lysine, or a combination thereof). ORC paste components: ORC: 0.6 g, fibrinogen: 0.3 g, thrombin: 3300 IU (approximately 5 mL) per syringe paste, lysine: 0.2 g, CaCl2: 0.02 g, dispersion medium: 50% by volume glycerol in physiological saline. ORC paste was prepared without foaming or freeze-drying processes. Lysine was added to reduce the acidity of ORC. Such acidity can denature plasma proteins (e.g., thrombin).

[0189] Example 2: Characterization of fibrin sealant paste viscosity The FS paste viscosity test was performed using a 25 mm diameter parallel plate vibrating rheometer (Anton Paar Rheometer) with a 1.25 mm gap. The results are shown in Figures 3A and 3B. The reconstituted fibrin paste was confirmed to have a stable and uniform viscosity at room temperature for at least 24 hours. The FS paste was found to be more stable and have a higher viscosity compared to the paste prepared using ORC, which also allows the FS paste to remain on the wound site for a longer period of time.

[0190] Swelling rate In an exemplary procedure, the swelling rate of the FS paste was tested by adding fibrinogen (70 mg / mL, 2 mL) to a paste (1 g of FS powder + 4.5 mL of dispersion medium), then incubating it at 37°C for 30 minutes to obtain a solidified paste. The solidified paste was cut into approximately 1.0 g sections. After weighing, the sections were incubated in phosphate-buffered saline (PBS) for 24 hours to swell (see Figure 4A), and the swelling rate due to weight increase was then calculated. The results are shown in Figure 4B. A comparative test was conducted using a gelatin matrix with SURGIFLO® hemostatic matrix containing thrombin as the paste to which fibrinogen was added. The results show that the FS paste of the present invention has a lower swelling rate compared to the gelatin sample. Due to the lower swelling rate, the FS paste does not have a "space-occupying effect" and can remain at the bleeding site for a longer time, providing an improved hemostatic effect.

[0191] In vitro coagulation time In the exemplary procedure, 0.5 g of FS paste was placed in a glass vial, followed by 4 mL of porcine blood anticoagulant (sodium citrate, 3.2–3.8 w / v%). The vial was gently shaken or vortexed for several seconds to bring the blood and paste into contact and mix them. The vial was then tilted to observe blood coagulation. If complete coagulation occurred, the coagulated blood remained at the top of the vial when tilted or inverted. If coagulation was incomplete, the blood remained fluid and flowed to the bottom of the vial by gravity. The time at which the blood stopped flowing was recorded. The results showed coagulation times of 14–16 seconds, i.e., less than 20 seconds, which demonstrated good efficacy of in vitro blood coagulation.

[0192] Test samples taken from vials show that, compared to ORC paste (Figure 5B), the fluid paste of the present invention absorbs blood more easily and uniformly (Figure 5A), thereby improving its ability to promote the coagulation cascade and form endogenous fibrin clots in vivo.

[0193] Animal studies - Efficacy studies in an in vivo spleen punch model In the exemplary procedure, a modified 6mm biopsy punch (with a rubber band placed around the punch as a 3mm deep fastener) was used to create a wound defect approximately 6mm in diameter and 3mm deep. The central core (tissue fragment) was removed using surgical forceps and scissors. A new site was created for each test substance or control substance, and defects of similar size were created for each treatment group. The sample test was added to the wound site (with moderate bleeding), applied to cover the wound site, and then pressed into the wound with moist gauze for 2 minutes. The gauze was then gently removed after 2 minutes, and the bleeding from the wound was observed. See Figure 6A. If no active bleeding was observed, hemostasis was successful; otherwise, it was considered unsuccessful. The negative controls were moist gauze pressing and ORC fluid paste. The success rate of hemostasis is the number of successful hemostasis cases divided by the total number of sample tests. The results are shown in Figure 6B, which represents the success rate of hemostasis for FS paste. As shown in Figure 6B, FS paste demonstrated an 80% success rate in hemostasis compared to 40% success rate for ORC paste.

[0194] In summary, compared to ORC-fluidity, the present invention enhances hemostatic effect by improving the formulation with extra FSP (fibrin sealant powder) that helps increase blood absorption. In addition, although not constrained by a specific mechanism, the porous powder in the fluid paste allows for easy absorption of blood and exposure to high concentrations of thrombin, thereby promoting the coagulation cascade and forming an endogenous fibrin clot. Furthermore, although not constrained by a specific mechanism, this porous structure remains in the powder at least partially (20-30% of the pores) after grinding the porous cake, resulting in more voids and contact with the bleeding site, thereby improving the hemostatic effect. This fluid hemostatic composition can be applied to bleeding sites that are difficult to access and hemostasis can be achieved. Figure 7A shows the porous structure of the powder of the present invention compared to the structure of the powder in ORC paste (Figure 7B). The estimated pore size of the powder of the present invention is in the range of approximately 10-30 μm.

[0195] 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.

[0196] [Implementation Method] (1) A two-phase fluid composition that is in the form of a paste at room temperature, (iii) A powder containing fibrinogen and fibrin present together in an amount of at least about 70% by weight, and thrombin, (iv) A dispersion medium comprising glycerol and an aqueous solution, The aforementioned dispersion medium is present in a ratio of dispersion medium to powder in the range of approximately 3:1 to approximately 5:1 (v / w, mL per g), A two-phase fluid composition in which the glycerol is present at a concentration exceeding approximately 30% by volume of the dispersion medium. (2) The composition according to Embodiment 1, comprising fibrin in an amount of approximately 60% to 80% by weight. (3) The composition according to Embodiment 1 or 2, wherein the fibrin is at least partially crosslinked. (4) The composition according to any one of Embodiments 1 to 3, wherein the fibrinogen and / or fibrin are present in the powder in an amount of about 80% by weight. (5) The composition according to any one of embodiments 1 to 4, wherein the powder further comprises a calcium salt.

[0197] (6) The composition according to Embodiment 5, wherein the calcium salt is CaCl2 or its hydrate, and is in an amount of up to about 3% by weight. (7) The composition according to any one of Embodiments 1 to 6, wherein the thrombin is present in an amount of about 2000 to about 4000 IU per gram of powder. (8) The composition according to any one of embodiments 1 to 7, wherein at least 80% of the powder is in the form of particles with a diameter of less than 500 μm. (9) The composition according to any one of embodiments 1 to 8, wherein at least a portion of the particles is porous. (10) The composition according to any one of Embodiments 1 to 9, wherein the glycerol is present in the dispersion medium at a concentration in the range of about 30% to about 80% by weight.

[0198] (11) The composition according to any one of embodiments 1 to 10, wherein the aqueous solution contains physiological saline. (12) A composition according to any one of embodiments 1 to 11, characterized by uniform viscosity. (13) A composition according to any one of Embodiments 1 to 12, characterized by a viscosity in the range of about 3000 to about 5000 Pa·s at about 25°C. (14) The composition according to Embodiment 13, wherein the viscosity changes within a range of less than about 30% after being stored at about 25°C for 24 hours. (15) The composition according to any one of Embodiments 1 to 14, characterized by a maximum swelling rate of 10% at approximately 25°C when in contact with blood.

[0199] (16) A composition according to any one of Embodiments 1 to 15, which does not contain oxidized cellulose. (17) A composition according to any one of embodiments 1 to 16, which does not contain a buffering agent. (18) A hemostatic composition, as described in any of Embodiments 1 to 17. (19) A composition according to any one of Embodiments 1 to 18 for use in a method for treating hemorrhagic tissue, comprising applying the composition to hemorrhagic tissue. (20) The composition according to any one of embodiments 1 to 19, wherein the method includes minimally invasive surgery, such as laparoscopic surgery.

[0200] (21) A method for producing a hemostatic composition characterized by a viscosity in the range of approximately 3000 to approximately 5000 Pa·s at approximately 25°C, and / or a swelling rate of up to 10% at approximately 25°C, the method comprising the following steps: (vii) A step of foaming the fibrinogen solution to obtain a foamy substance, (viii) A step of spraying a thrombin solution into the foamy substance so that the foamy substance further incorporates the thrombin, (ix) A step of stirring the foamy substance containing thrombin so as to convert fibrinogen to fibrin, (x) A step of freeze-drying the fibrin, (xi) A step of grinding the freeze-dried fibrin to obtain a uniform powder, (xii) A method comprising the step of mixing the powder with a dispersion medium containing an aqueous solution, glycerol, and thrombin, wherein the method is carried out under conditions that enable the preparation of the hemostatic composition. (22) The above conditions are (a) The dispersion medium contains thrombin in an amount of about 200 to about 800 IU per 1 mL. (b) The dispersion medium is added to the powder in a ratio of dispersion medium to powder in the range of 3:1 to 5:1 (v / w, mL / g), and The method according to Embodiment 21, selected from one or more of the following: (c) the foamy substance obtained in step (ii) contains fibrin in at least about 70% of it. (23) The method according to either embodiment 21 or 22, wherein the thrombin solution in step (ii) further comprises a calcium salt. (24) The method according to Embodiment 23, wherein the calcium salt is CaCl2 and has a concentration of 0.5 to about 3 w / v%. (25) It is a kit, (a) A container for containing a powder comprising fibrinogen, fibrin and thrombin, wherein the fibrinogen and fibrin are present in a total amount of at least about 70% by weight, (b) A container for containing a dispersion medium containing glycerol and optionally an aqueous solution, and optionally (c) Kit including instruction manual.

[0201] (26) The kit according to embodiment 25, further comprising an applicator device.

Claims

1. A two-phase fluid composition that is in the form of a paste at room temperature, (iii) A powder containing fibrinogen and fibrin present together in an amount of at least about 70% by weight, and thrombin, (iv) A dispersion medium comprising glycerol and an aqueous solution, The dispersion medium is present in a ratio of dispersion medium to powder in the range of approximately 3:1 to approximately 5:1 (v / w, mL per g), A two-phase fluid composition in which the glycerol is present at a concentration exceeding approximately 30% by volume of the dispersion medium.

2. The composition according to claim 1, comprising fibrin in an amount of approximately 60% to 80% by weight.

3. The composition according to claim 1, wherein the fibrin is at least partially crosslinked.

4. The composition according to claim 1, wherein the fibrinogen and / or fibrin are present in the powder in an amount of about 80% by weight.

5. The composition according to claim 1, wherein the powder further comprises a calcium salt.

6. The calcium salt is CaCl ２ The composition according to claim 5, or its hydrate, in an amount of up to about 3% by weight.

7. The composition according to claim 1, wherein the thrombin is present in an amount of about 2000 to about 4000 IU per gram of powder.

8. The composition according to claim 1, wherein at least 80% of the powder is in the form of particles having a diameter of less than 500 μm.

9. The composition according to claim 1, wherein at least a portion of the particles is porous.

10. The composition according to claim 1, wherein the glycerol is present in a concentration in the range of about 30% to about 80% by weight of the dispersion medium.

11. The composition according to claim 1, wherein the aqueous solution contains physiological saline.

12. The composition according to claim 1, characterized by uniform viscosity.

13. The composition according to claim 1, characterized by a viscosity in the range of approximately 3000 to approximately 5000 Pa·s at approximately 25°C.

14. The composition according to claim 13, wherein the viscosity changes within a range of less than about 30% after being stored at about 25°C for 24 hours.

15. The composition according to claim 1, characterized by a maximum swelling rate of 10% at approximately 25°C upon contact with blood.

16. The composition according to claim 1, which does not contain oxidized cellulose.

17. The composition according to claim 1, which does not contain a buffering agent.

18. The composition according to claim 1, which is a hemostatic composition.

19. The composition according to claim 1, for use in a method for treating hemorrhagic tissue, comprising applying the composition to the hemorrhagic tissue.

20. The composition according to any one of claims 1 to 19, wherein the method includes minimally invasive surgery, such as laparoscopic surgery.

21. A method for producing a hemostatic composition characterized by a viscosity in the range of approximately 3000 to approximately 5000 Pa·s at approximately 25°C, and / or a swelling rate of up to 10% at approximately 25°C, wherein the method comprises the following steps: (vii) A step of foaming the fibrinogen solution so that a foamy substance is obtained, (viiii) A step of spraying a thrombin solution into the foamy substance so that the foamy substance further incorporates the thrombin, (ix) A step of stirring the foamy substance containing thrombin so as to convert fibrinogen to fibrin, (x) A step of freeze-drying the fibrin, (xi) A step of grinding the freeze-dried fibrin to obtain a uniform powder, (xi) A method comprising the step of mixing the powder with a dispersion medium containing an aqueous solution, glycerol and thrombin, wherein the method is carried out under conditions that enable the preparation of the hemostatic composition.

22. The aforementioned conditions (a) The dispersion medium contains thrombin in an amount of about 200 to about 800 IU per 1 mL. (b) The dispersion medium is added to the powder in a ratio of dispersion medium to powder in the range of 3:1 to 5:1 (v / w, mL / g), and The method according to claim 21, wherein one or more of the following are selected: (c) the foamy substance obtained in step (ii) comprises at least about 70% fibrin.

23. The method according to any one of claim 21 or 22, wherein the thrombin solution in step (ii) further comprises a calcium salt.

24. The calcium salt is CaCl ２ The method according to claim 23, wherein the concentration is 0.5 to about 3 w / v%.

25. It's a kit, (a) A container for containing a powder comprising fibrinogen, fibrin and thrombin, wherein the fibrinogen and fibrin are present in a total amount of at least about 70% by weight, (b) A container for containing a dispersion medium which contains glycerol and optionally an aqueous solution, and optionally (c) A kit including the instruction manual.

26. The kit according to claim 25, further comprising an applicator device.

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