Flowable sealant hemostatic agent

Abstract

Disclosed herein are two-phase flowable compositions in the form of a paste, comprising: (i) a powder of fibrinogen, fibrin, and thrombin; and (ii) a dispersant comprising glycerol and an aqueous solution. Also disclosed are methods and kits for preparing the compositions. Also disclosed are uses of the compositions for treating hemorrhage in a subject.

Description

Technical Field

[0001] The present invention relates in particular to flowable hemostatic compositions comprising a blend of fibrinogen, thrombin, and a dispersant. Background Technology

[0002] In various situations during surgery, therapeutic agents are spread throughout the body to prevent postoperative bleeding. Existing agents may have some drawbacks, such as the difficulty in placing tablets during minimally invasive surgery (MIS).

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

[0004] To address these issues, materials for controlling excessive bleeding have been developed. Topically absorbable hemostatic agents (TAHs) are widely used in surgical applications. TAHs encompass products based on oxidized cellulose (OC), gelatin, collagen, chitin, deacetylated chitosan, and more. To improve hemostatic performance, scaffolds based on these materials can be combined with biologically derived coagulation factors such as thrombin and fibrinogen.

[0005] However, difficult-to-access bleeding sites, including deep anatomical locations, narrow crevices, or cavities, are typical scenarios for surgeons. There is a strong need to develop hemostatic agents capable of precisely reaching narrow and deep bleeding sites. It is desirable for the hemostatic agent to exhibit fluidity without compromising adhesion; that is, the hemostatic agent must adhere firmly to the selected tissue even under high fluid pressure, wetting, and dynamic conditions, and remain in place until hemostasis is achieved.

[0006] PCT patent application No. PCT / CN2021 / 087167 discloses a two-component flowable composition comprising: (i) a powder having oxidized cellulose, fibrinogen, and thrombin, wherein the weight ratio of oxidized cellulose to fibrinogen in the powder is in the range of about 1:5 to about 2:1; and (ii) a dispersant comprising glycerol and an aqueous solution, wherein the composition is in the form of a paste suspension at room temperature. Use of these compositions for treating hemorrhagic tissue and methods for preparing these compositions are also disclosed. Summary of the Invention

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

[0008] One object of the present invention is to provide a composition for preparing a topical hemostatic agent that can be easily applied to the desired bleeding site, including areas difficult to reach by the body, and can quickly adhere to the bleeding site and then form a sealant therein. A specific object of the present invention is to develop a flowable hemostatic agent that, after being applied to the bleeding site during surgery, has both fluidity and adhesion to tissues and / or platelets. Inaccessible bleeding sites, including deep anatomical sites, narrow fissures, or cavities, while visible, are difficult to access with conventional medical devices and are typical scenarios in surgical procedures. There is a great need to develop hemostatic agents that can precisely reach and flexibly flow into narrow and deep bleeding sites, with fluidity that does not compromise adhesion. Platelet adhesion is crucial for hemostatic agents, which should precisely reach the bleeding site and remain in place until hemostasis is achieved.

[0009] Fibrinogen and thrombin interact in aqueous media. Finding a suitable formulation with appropriate consistency is not easy because commonly used dispersants such as water or hydrophilic dispersants cannot be used without impairing pasteness, adhesion, and / or hemostatic function. Therefore, for stable liquid / flowable compositions containing both fibrinogen and thrombin, a non-aqueous medium (dispersant) is typically used before application to the bleeding site, for example, for sealing and hemostasis.

[0010] Therefore, this invention is based on the surprising discovery that such a paste-like suspension containing powders of fibrinogen / fibrin and thrombin dispersed in a dispersant comprising, for example, glycerol and an aqueous solution, provides excellent hemostatic properties. Thus, this composition can be applied to the desired site to achieve bioactivity, such as controlling bleeding, particularly in hard-to-reach areas of the body. The inventors have developed a flowable hemostatic paste that remains in place after application to the bleeding site. The disclosed paste can absorb some of the exudated blood, which facilitates rapid and tight adhesion of the paste to the bleeding site and deep into deep bleeding sites; and can form a protein sealant at the bleeding site, which helps to further enhance adhesion to tissue.

[0011] Using an aqueous solution in such a suspension is counterintuitive, and it can be assumed, without being bound by any particular theory or mechanism, that after the porous powder of the present invention is mixed in a dispersant, the uniformly dispersed biopharmaceutical forms at least partially cross-linked fibrin in a glycerol / saline system, thereby forming a paste rich in thrombin that remains in the system.

[0012] Therefore, it has been proven that the disclosed composition is an easy-to-use, stable and effective hemostatic agent.

[0013] According to one aspect of the invention, a two-phase flowable composition in the form of a paste at room temperature is provided, the composition comprising: (i) A powder comprising: fibrinogen and fibrin present together in an amount of at least about 70% by weight; and thrombin; and (ii) A dispersant comprising glycerol and an aqueous solution, in: The dispersants are present in a dispersant-to-powder ratio in the range of about 3:1 to about 5:1 (v / w; ml / g); and glycerol is present at a concentration of more than about 30% by volume of the dispersant.

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

[0015] In some embodiments, the composition comprises about 60% to 80% by weight of fibroin.

[0016] In some embodiments, fibrinogen is present at a concentration ranging from about 12 w / w to about 20 w / w. In some embodiments, fibrin is present at a concentration ranging from about 53 w / w to about 82 w / w. In some embodiments, thrombin is present at a concentration ranging from 4 w / w to 25 w / w.

[0017] In some implementations, the fibrin is at least partially cross-linked.

[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 also contains a calcium salt. In some embodiments, the calcium salt is CaCl2, in an amount of up to about 3%, for example, from 0.5% by weight to 3% by weight, for example, about 2% w / w. The term "CaCl2" as used herein is also intended to further include any hydrate thereof, such as, but not limited to, CaCl2. . H2O (hydrate), CaCl2 . 2H2O (dihydrate) or combinations thereof.

[0020] In some implementations, thrombin is present in amounts typically from about 2000 IU to about 4000 IU per gram of powder.

[0021] In some embodiments, at least 80% of the powder is in the form of particles with a size of less than 500 µm. In some embodiments, at least a portion of the particles is agglomerated. In some embodiments, glycerol is present at a concentration ranging from about 30% to about 80% by weight of the dispersant. In some embodiments, the aqueous solution comprises brine.

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

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

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

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

[0026] In some embodiments, a method of using the composition to treat bleeding tissue includes applying the composition to the bleeding tissue.

[0027] In some implementations, the method includes minimally invasive surgery, such as laparoscopic surgery.

[0028] According to another aspect, a method for preparing a hemostatic composition is provided, characterized in that its viscosity is in the range of about 3000 Pa·s to about 5000 Pa·s at about 25°C and / or its swelling ratio is at most 10% at about 25°C, the method comprising the following steps: (i) Stir the fibrinogen solution to obtain foam; (ii) Spray the thrombin solution into the foam, so that the foam is further infused with thrombin; (iii) Stir the foam containing thrombin to convert fibrinogen into fibrin; (iv) Lyophilized fibrin; (v) Grind the freeze-dried fibroin to obtain a uniform powder, and (vi) Under conditions that allow for the preparation of hemostatic compositions, the powder is mixed with a dispersant comprising an aqueous solution, glycerol and thrombin.

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

[0030] In some implementations, the thrombin solution in step (ii) also contains calcium salts.

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

[0032] According to another aspect, a kit is provided, the kit comprising: (a) A container 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; and (b) A container holding a dispersant containing glycerin and optionally an aqueous solution, and optionally (c) an instruction manual.

[0033] The implementation scheme for the powder is described in full in this article and is also incorporated into the kit aspect.

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

[0035] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While methods and materials similar to or equivalent to those described herein may be used for the operation or testing of embodiments of the invention, the methods and materials described below are exemplary. In case of any conflict, the patent specification and its definitions shall prevail. Furthermore, materials, methods, and examples are exemplary only and are not intended to impose necessary limitations. Attached Figure Description

[0036] This document describes some embodiments of the invention by way of example and in conjunction with the accompanying drawings. Referring now specifically to the drawings, the details shown are intended to be illustrative and for the purpose of discussing embodiments of the invention in a demonstrative manner. In this respect, the description taken in conjunction with the drawings will enable those skilled in the art to understand how to practice embodiments of the invention.

[0037] Figure 1The photographic images presented illustrate a process according to an embodiment of the invention, in which a first component containing powders of fibrinogen and thrombin and a second component containing a dispersant comprising an aqueous solution of glycerol are mixed using an applicator made of two syringes, and the resulting paste is then applied by attaching the tip of the applicator to the applicator. "Component 1" is 1 g of fibrin sealant (FS) powder plus 3300 IU potency thrombin powder with CaCl2 (approximately 21 mg). "Component 2" is 4 ml of 50% v / v glycerol solution (in physiological saline).

[0038] Figures 2A to 2B A flowchart with photographic images is presented, illustrating an exemplary embodiment of the process for preparing the powder of the present invention. Figure 2A ), and photographic images of the obtained paste ( Figure 2B ).

[0039] Figures 3A to 3C The present invention presents a flowable paste ( Figure 3A (The bar chart in the middle) and oxidized regenerated cellulose (ORC) paste ( Figure 3B (The bar chart in the image shows the comparative viscosity after standing at room temperature for a specified time of up to 24 hours; to test the homogeneity of the sample, the sample is removed from various parts of the syringe: such as...) Figure 3C The diagram shows F (front), M (middle), and E (rear).

[0040] Figures 4A to 4B The swelling ratio of the flowable paste of the present invention was presented compared to that of the gelatin matrix: the samples were first incubated in phosphate-buffered saline (PBS) solution for 24 hours to allow them to swell ( Figure 4A Then, the weight increase of each sample was measured. Five parts of the test sample were tested.

[0041] Figures 5A to 5B Comparative photographic images are presented, demonstrating the blood absorption capacity of the flowable paste of the present invention. Figure 5A ) and the blood absorption capacity of ORC paste ( Figure 5B The results showed that, compared with ORC paste, the flowable paste of the present invention is easily and uniformly absorbed into the blood, thereby accelerating the coagulation cascade reaction to form endogenous fibrin clots.

[0042] Figures 6A to 6B Efficacy testing in an in vivo spleen perforation model is presented; photographic images of the steps from left to right ( Figure 6A: A wound was created through a biopsy puncture (6 mm in diameter, 3 mm in depth); the central core (tissue plug) was removed; moderate bleeding was in progress; a test sample was applied to cover the wound site; and the hemostasis result (success or failure) was evaluated after applying pressure with moist gauze for 2 minutes; paste tests were performed on five samples, and the success rate is shown in a bar chart. Figure 6B From left to right: negative control - 0%, fibrin sealant of the embodiment of the present invention - 80%; gelatin matrix - 20% and ORC paste - 40%.

[0043] Figures 7A to 7B Scanning electron microscope (SEM) images of fibrin sealant powder prepared according to some embodiments of the present invention are presented. Figure 7A (Scale bar is 100µm), indicating a loose, porous powder, compared to a flowable powder prepared with ORC (ORC powder), indicating a non-porous structure. Figure 7B (Scale bar is 50 μm). Detailed Implementation

[0044] The production of flowable hemostatic agents with fibrin composite matrices that are easy to prepare and apply to surgical procedures involving difficult-to-access bleeding sites is challenging, especially during minimally invasive procedures (MIS), such as endoscopy.

[0045] Based on the characteristics of the "bleeding site," including inaccessible areas such as deep anatomical sites, narrow fissures, or cavities, although visible, are difficult to access with traditional medical devices, which is a typical scenario in surgical procedures.

[0046] Therefore, one object of the present invention is to provide an improved composition that can be readily applied to bleeding sites where necessary, particularly in hard-to-reach areas of the body. Another advantage of the compositions of the present invention is that they are bioabsorbable and thus can remain after surgery without causing any side effects.

[0047] The term "applied to the bleeding site where needed" means, for example, applying the composition topically to the bleeding surgical site of the tissue or on / near it.

[0048] According to one aspect of this disclosure, a two-phase flowable composition in paste form at about room temperature is provided, the composition comprising: (a) a powder comprising: (i) at least about 70% by weight of fibrinogen; (ii) thrombin; (iii) a calcium salt; and (b) a dispersant comprising glycerol and an aqueous solution. In some embodiments, the dispersant is present in a dispersant-to-powder ratio in the range of about 3:1 to about 5:1 (v / w; ml / g); in some embodiments, glycerol is present at a concentration greater than about 30% by volume of the dispersant.

[0049] The terms “liquid medium,” “medium,” or “dispersant” are used interchangeably throughout this document. Here, the term “dispersant” means a liquid medium, optionally a liquid medium having a viscosity that is either pasty or capable of providing a pasty consistency. Therefore, the term “dispersant” is used in its general sense and is distinct from the word “solvent,” which may only be used to clarify that the disclosed blends or powders, composed of, for example, fibrinogen or thrombin, are substantially insoluble in such liquid media and instead form a solid-liquid system.

[0050] In some embodiments, the dispersant is present as a dispersant-to-powder ratio in the range of about 2.5:1 to about 6:1 (v / w, in ml / g), such as 2.5:1, 3:1, 3.5:1, 4:1 or 5:1 (inclusive of any values ​​and ranges therein).

[0051] The term "approximately room temperature" means at least one temperature value in the range of 10°C to 40°C or 15°C to 37°C, such as 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C or 40°C, including any value in between.

[0052] The term "glycerol" refers to 1,2,3-propanetriol and is also known as "glycerin," and may also be intended to encompass its derivatives. The term "derivative" refers to a compound containing the basic element of its parent substance.

[0053] As used herein, “thrombin” refers to the activated enzyme produced by the proteolytic cleavage of prothrombin (factor II). Thrombin can be produced by a variety of methods known in the art, including but not limited to recombinant thrombin and plasma-derived thrombin.

[0054] Human thrombin is a 295-amino acid protein composed of two polypeptide chains linked by disulfide bonds. Human and non-human (e.g., bovine) thrombins may be used within the scope of this disclosure.

[0055] The thrombin used in this disclosure may originate from one or more sources, including but not limited to: plasma (e.g., porcine plasma), recombinant bacteria and / or cells (see, for example, Vu et al., 2016, J. Viet. Env., 8(1):21-25), whole blood (collected from or not from several blood donations) and / or blood fractions (collected from several blood donations, such as plasma). Thrombin may be manufactured by manufacturers such as Johnson & Johnson, Baxter, and CSL Behring as standalone products (e.g., EVITHROM). ® ) or in the form of product components (e.g., EVICEL) ®TISEEL ® Beriplast ® Provided by (etc.).

[0056] In exemplary embodiments, the powder comprises fibrinogen at a concentration of 12% w / w to 20% w / w, fibrin at a concentration of 53% w / w to 82% w / w, thrombin at a concentration of about 2% w / w to 25% w / w, and calcium. In some embodiments, calcium is present at an amount of about 2% w / w. In some embodiments, calcium is present at an amount of about 2% w / w to about 4.5% w / w. In some embodiments, calcium is present at an amount of about 2% w / w to about 3% w / w. In some embodiments, the powder is in the form of porous microparticles. As used herein, the term "porous" refers to a material having voids throughout its internal structure.

[0057] In some embodiments, thrombin is present in amounts of about 2000 IU to about 4000 IU per gram of powder. In some embodiments, thrombin is present in amounts of about 3000 IU per gram of powder. In some embodiments, thrombin is present in amounts of about 2000 IU, 3000 IU, or 4000 IU per gram of powder, including any values ​​and ranges in between.

[0058] In this article, "U" represents the coagulation factor unit, which is the physiological measurement unit of coagulation factors in 1 mL of normal human plasma.

[0059] “IU” refers to the International Unit of a coagulation factor, which is a physiological measurement in a given sample compared to an appropriate intentional standard, such as that determined by a coagulation assay performed against an internal reference standard for measuring potency concentration, which has been calibrated against, for example, the World Health Organization (WHO) Second International Standard 01 / 580 for thrombin.

[0060] The term "fibrinogen" (without further ado) is intended to include any type of fibrinogen. "Fibrinogen" refers to monomeric and dimeric fibrinogen molecules, hybrid molecules, and their variants having a monomeric structure (AαBβγ), whether naturally occurring, modified, or synthetic. The term "fibrinogen" may refer to fibrinogen derived from humans, but may include fibrinogen from any species, particularly mammalian species, such as fibrinogen produced from porcine plasma.

[0061] The term "powder" refers to solid particulate material, typically in the form of dispersed, dry solid particles, for example, in the form of multiple solid particles typically ranging from 0.1 micrometer to 1000 micrometers (µm), sometimes less than 500 µm, and sometimes less than 100 µm, characterized by a smaller size in at least one dimension.

[0062] 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 compound or composition that retains its form with high viscosity during storage. In this application, the 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. "Powder" and "particles" are used interchangeably herein.

[0063] As used herein, the term "paste" refers to the consistency of a composition at at least one temperature of about room temperature and defines a fluid mixture of solid particles. Typically, a paste does not have a fixed shape and is therefore neither a solid nor a gas. The term "paste" according to this disclosure may also include slurries, ointments, and creams. A slurry can be functionally considered as a thin, oily paste. Pastes according to this disclosure may also include pores containing expandable gases, such as air. Thus, in some embodiments, the composition is a paste, or has a paste-like consistency at at least one temperature of about room temperature.

[0064] The term "aqueous solution" refers to a solution containing water as a solvent. This term can include water, saline solution, or the 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.

[0065] In an exemplary embodiment, the aqueous solution comprises brine. As used herein, the term "brine" refers to water containing a salt concentration greater than about 0.5% by weight and less than about 20% by weight, such as water containing about 0.9 w / w% sodium chloride solution, typically softened water.

[0066] In some embodiments, at least 90% of the powder has a particle size in the range of 10 µm to 800 µm. In some embodiments, at least 90% of the powder has a particle size in the range of 10 µm to 700 µm. In some embodiments, at least 90% of the powder has a particle size in the range of 100 µm to 800 µm. In some embodiments, at least 90% of the powder has a particle size in the range of 200 µm to 700 µm. In some embodiments, at least 90% of the powder has a particle size in the range of 100 µm to 600 µm. In some embodiments, at least 90% of the powder has a particle size in the range of 200 µm to 800 µm.

[0067] In some embodiments of any aspect of this disclosure, the plurality of particles are characterized by a median particle size of about 100 µm 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 µm to about 500 µm.

[0068] In the context of granularity, the term "median" (also known as "D50") refers to the granularity at which 50% of the population is larger or smaller than the population size when dividing the population into two halves.

[0069] 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.

[0070] In some embodiments, at least a portion of the powder is in the form of aggregates, agglomerates, or particles. The term "agglomerate" describes microparticles formed by assembled components. Agglomerates may optionally be prepared by the following steps: humidifying the powder composition; compacting, for example, by rolling and / or heavy pressing of the powder to form an aggregate structure; dehumidifying; grinding; sieving the aggregates; and optionally quantitatively adding the resulting aggregates to a storage container or delivery device. The term "particles" or "particulate material" may specifically refer to aggregates of microparticles typically below 900 µm, below 800 µm, below 700 µm, or even below 500 µm. That is, at least a portion of the microparticles is not in the form of discrete solids. In some embodiments, microparticles of fibrinogen, thrombin, and optionally CaCl2 are assembled into aggregates using high-shear mixing.

[0071] In some embodiments, at least a portion of the powder or solid blend is in the form of aggregates or clusters. The term "agglomerate" refers to a weak association of primary particles or aggregated particles that are typically held together by charge or polarity.

[0072] The term "at least a portion of the powder" refers to a portion of the microparticle population in the powder, such as 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 microparticle population in the powder, such as at least 10% to 100% forming aggregates.

[0073] The term "flowable" also covers viscous solutions. The term "viscous solution" refers to a solution that has increased flow resistance but is still able to flow. In some embodiments, the composition has a viscosity greater than about 400 Pa·s and less than about 8000 Pa·s.

[0074] The term "viscosity" refers to the property of fluids and slurries that indicates their flow resistance and is defined as the ratio of shear stress to shear rate. Viscosity typically has a specified or known shear rate. In this document, unless otherwise stated, the shear rate is 0.5 s⁻¹ in the case of viscosity, and viscosity is measured under ambient pressure and temperature conditions (referred to as "ambient conditions," i.e., approximately room temperature and atmospheric pressure). The term "shear rate" refers to the velocity gradient measured along the diameter of a fluid flow channel, which can be a pipe, ring, or other shape. Shear rate is the rate of change of velocity of a fluid layer passing through an adjacent layer.

[0075] 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 Pa·s to about 7000 Pa·s. In some embodiments, the composition has a viscosity in the range of about 2000 Pa·s to about 6000 Pa·s. In some embodiments, the composition has a viscosity in the range of about 3000 Pa·s to about 5000 Pa·s. In some embodiments, the composition has viscosities 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 therebetween.

[0076] 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 it varies within a range of less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the Pa·s value.

[0077] In some embodiments, the composition has a stable viscosity. The term "stable viscosity" refers to a viscosity that changes by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of its Pa·s value over at least 24 hours at a temperature near room temperature.

[0078] In some embodiments, the paste is characterized by a viscosity of about 2500 Pa·s to about 4500 Pa·s, or 3000 Pa·s to 4500 Pa·s, such as 2500 Pa·s, 2600 Pa·s, 2700 Pa·s, 2800 Pa·s, 2900 Pa·s, 3000 Pa·s, 3100 Pa·s, 3200 Pa·s, 3300 Pa·s, 3400 Pa·s, 3500 Pa·s, 3600 Pa·s, 3700 Pa·s, 3800 Pa·s, 3900 Pa·s, 4000 Pa·s, 4100 Pa·s, 4200 Pa·s, 4300 Pa·s, 4400 Pa·s, or 4500 Pa·s, including any value and range therebetween.

[0079] In one embodiment, the composition is in an injectable form. As used herein, the phrase "injectable form" means a flowable composition (e.g., comprising cross-linked fibrinogen, polymerized fibrin, and / or cross-linked fibrin in particulate form, such particles being small enough to allow injection into a human subject, e.g., via a syringe and needle), and having suitable purity and non-toxicity for injection into a subject. The composition should be homogeneous and generally possess homogeneous rheological properties that allow for smooth passage through needles of various inner diameters (e.g., 14-32 gauge). Thus, in some embodiments, the composition is permeable to an applicator. In some embodiments, the composition is characterized by a homogeneous viscosity, i.e., the viscosity of the composition varies within less than ±40%, less than ±30%, less than ±20%, less than ±10%, or less than ±5% within 4 hours after the dispersant is added to the powder (see [link to relevant documentation]). Figure 3A ).

[0080] As used herein, "homogeneous" or "uniform" means a uniform composition, viscosity, and texture throughout the dispersion, typically indicating that the powder is substantially uniformly dispersed throughout the dispersion. In this context, "substantially uniformly dispersed" means that the concentration of the powder within the dispersion of the composition varies within the range of less than ±30% by weight, less than ±20% by weight, less than ±10% by weight, and less than ±5% by weight, typically without significant solid-liquid phase separation. Furthermore, the term "homogeneous" generally refers to a product (e.g., a paste) that exhibits uniformity in resisting liquid-phase / solid-phase separation (e.g., less than 4% by weight) at temperatures ranging from 8°C to 40°C, 10°C to 30°C, or 20°C to 30°C.

[0081] In some embodiments, the composition is characterized by a low swelling ratio. As used herein, the term "swelling ratio" refers to the percentage change in volume of the paste when immersed in a liquid (e.g., blood), compared to the volume of the paste before immersion in the liquid; that is, the degree of swelling of the paste formed between its state before adsorbing the liquid (e.g., blood) and its state after adsorbing the maximum possible amount of liquid. In this document, "low swelling ratio" means a swelling ratio less than about |±5%|.

[0082] Not bound by any specific mechanism, it is speculated that a low swelling ratio allows for improved hemostasis at small defects without an occupation effect, meaning the paste can remain at the bleeding site for an extended period. See [reference needed] in this regard. Figure 5A .

[0083] In some embodiments, the composition is bioabsorbable. The term "bioabsorbable" refers to the ability of a tissue-compatible material to degrade in the body after implantation into a non-toxic product, which is eventually eliminated or metabolized from the body.

[0084] As used herein, and unless otherwise indicated, the terms “weight ratio,” “by weight,” “w / w,” “weight percentage,” or “weight %” are used interchangeably herein, describing the concentration of a particular substance in the total weight of the respective mixture, solution, suspension, formulation, or composition. As used herein, and unless otherwise indicated, the terms “volume ratio,” “by volume,” “v / v,” “volume percentage,” or “volume %” are used interchangeably herein, describing the concentration of a particular substance in the total volume of the respective mixture, dispersant, solution, suspension, formulation, or composition.

[0085] Phase component compositions can be prepared by mixing a first component and a second component to prepare a suspension. As used herein, the term "phase component composition" refers to a composition comprising at least two phases. Typically, but not exclusively, the two phases can be stored in separate containers and mixed prior to use to produce a mixture or a suspension thereof, for example, when the mixture or suspension is applied to a subject's site (e.g., as shown in the image). Figure 1 (As shown).

[0086] As used herein, the term “mixture” refers to, but is not limited to, a combination of components in any physical form (e.g., blends, solutions, suspensions, dispersions, etc.).

[0087] As used herein, the term "suspension" or any grammatical variation thereof refers to a heterogeneous mixture of solids in fine solute particles dispersed in a liquid or solvent phase. Typically, suspensions will have a tendency to settle, meaning that the fine particles of solid matter may have a tendency to settle after a period of time, which in some embodiments is longer than 24 hours. The time period can depend on many factors, such as, but not limited to, the composition of the particles and liquid, temperature, pH, and other physical parameters (such as stirring and agitation) and the presence of other substances (such as dispersants, emulsifiers, surfactants, thickeners, etc.).

[0088] In some embodiments, the composition further comprises fibrinogen. The term "fibrin" refers not only to fully coagulated fibrinogen, but also includes any mixture of fibrin and fibrinogen that may occur during the formation of fibrin from fibrinogen using thrombin, and therefore includes any ratio of fibrinogen / fibrin and any degree of conceivable gelation and / or coagulation, provided it does not negatively affect the final paste-like texture of the composition. Furthermore, the term "fibrin" also includes any partially or fully cross-linked, gelled, or coagulated form.

[0089] Therefore, it should be understood that fibrin itself exists as an insoluble matrix containing polymers of fibrils, and each fibril contains many fibrin molecules.

[0090] In some embodiments, glycerol is present at a concentration greater than about 30% by volume of the dispersant. In some embodiments, glycerol is present at a concentration greater than about 40% by volume of the dispersant. In some embodiments, glycerol is present at a concentration of up to about 80% by volume of the dispersant. In some embodiments, glycerol is present at a concentration of up to about 70% by volume of the dispersant. In some embodiments, glycerol is present at a concentration of up to about 60% by volume of the dispersant.

[0091] In some embodiments, glycerol is present at a concentration of more than about 25% to about 80% by volume of the dispersant. In some embodiments, glycerol is present at a concentration of more than about 35% to about 80% by volume of the dispersant. In some embodiments, glycerol is present at a concentration of more than about 15% to about 60% by volume of the dispersant. In some embodiments, glycerol is present at a concentration of more than about 30% to about 80% by volume of the dispersant. In some embodiments, glycerol is present at a concentration of more than about 40% to about 60% by volume of the dispersant.

[0092] In some embodiments, glycerol is present at concentrations of about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% by volume of the dispersant, including any values ​​and ranges between them.

[0093] In some implementations, the pH of the dispersant is in the range of about 4.5 to about 7.

[0094] The results showed that samples containing approximately 50% glycerol, when dispersed in the aforementioned dispersant, provided a paste-like composition while offering optimal viscosity and retention in place, as well as thrombin potency retention. Samples containing more than approximately 80% glycerol exhibited high fluidity, resulting in the composition being less able to retain in place, for example, at wound sites. Further results showed that for samples containing less than 30% glycerol, liquid-solid phase separation occurred after 20 hours.

[0095] In some embodiments, the composition is stable. When referring to the disclosed paste, the terms "stable" and "stable" mean, for example, that the active component in thrombin retains at least 70% of its activity after a certain temperature and duration, i.e., it is capable of forming fibrin clots.

[0096] In some embodiments, the composition is stable. The term "storage stable" should be understood to mean a formulation or composition that is normally present in a container (e.g., vial or syringe) and stable under pre-selected storage conditions (e.g., pH), including pre-selected storage temperature or pre-selected physical state of the composition. Stability can be determined by methods known in the art, such as by testing for the absence of visible aggregates, residues, and / or undesirable clots in the formulation under pre-selected storage conditions (e.g., when the composition is in liquid or paste form). Furthermore, according to this disclosure, when referring to a stable sealant composition or formulation, it should be understood as a formulation that has an effective clotting time at use regardless of the storage conditions of some formulations, for example, the sealant coagulates in substantially the same time period whether stored at room temperature (e.g., 8°C to 40°C) or even at lower temperatures (e.g., below 8°C).

[0097] In some implementations, 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 were detected 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 at approximately room temperature and at a pH of 4 to 6.5.

[0098] In some embodiments, the composition further comprises, in any embodiment thereof, an additional pharmaceutically active agent contained within or on the surface of the composition.

[0099] In some implementations, the pharmaceutical active agent is selected from the group consisting of a therapeutic active agent and a labeling agent.

[0100] In some embodiments, the therapeutic active agent is selected from the group consisting of stem cells, growth factors, bone morphogenetic proteins, cells, cytokines, hormones, drugs, minerals, plasmids with therapeutic potential, and combinations thereof. As described herein, in some embodiments, the composition may also contain at least one bioactive agent. Non-limiting bioactive agents that may be included in the composition include calcium, as well as therapeutic agents such as antibiotics, anti-inflammatory agents, growth factors, or clotting factors. Specifically, the disclosed compositions may also contain components that promote clot formation, such as, but not limited to, calcium salts, factor VIII, factor XIII, fibronectin, vitrin, and von Willebrand factor (vWF), which may be provided as individual components or formulated with blends or pastes.

[0101] The pharmaceutical active agent optionally attaches to a protein matrix (i.e., fibrinogen or fibrin) (e.g., covalently linked to the protein). This agent may attach to the matrix and / or to components (e.g., proteins and / or reducing sugars) after matrix preparation, which are then used to prepare the protein matrix, such as cross-linked fibrin.

[0102] Alternatively or additionally, the agent is absorbed by the cross-linked protein matrix. Absorption can be achieved, for example, by contacting the cross-linked fibrinogen or fibrin matrix or the non-cross-linked fibrin matrix with a solution containing the agent (e.g., by impregnation, soaking, washing), or by adding the agent prior to cross-linking (e.g., including the agent in a cross-linked solution, or including the agent in a solution of fibrinogen and thrombin).

[0103] If desired, the compositions described herein and the contents of the aforementioned kits may be contained in a packaging or dispenser device containing one or more unit dosage forms containing the composition or ingredient (e.g., fibrinogen) and / or reagents for preparing the composition (e.g., dispersants). The packaging may include, for example, metal or plastic foil, such as blister packs. The packaging or dispenser device may be accompanied by instructions for use. The packaging or dispenser device may also be accompanied by notices in the form prescribed by a government agency regulating the manufacture, use, or sale of the drug, reflecting the form in which the agency approves the composition for human or animal administration. Such notices may, for example, include labels or approved product inserts for prescription drugs approved by the U.S. Food and Drug Administration (FDA). Compositions of the preparations of the present invention, formulated in a pharmaceutically acceptable carrier, placed in suitable containers, and labeled for the specified application and / or for the treatment of a specified condition, as further detailed herein, may also be prepared. The term "preparation" refers to a composition that is physiologically suitable for therapeutic use.

[0104] It should be understood that the compositions of the embodiments of this disclosure may be attached to or included in a medical device, such as for promoting wound healing.

[0105] In another aspect of this disclosure, the disclosed composition, according to any aspect or embodiment thereof, is used in a method of preparing a fibrin sealant in / on damaged tissue of a subject by applying the composition to the surface of a tissue.

[0106] In another aspect of this disclosure, a method is provided for preparing a fibrin sealant in / on damaged tissue of a subject by applying the disclosed composition as a hemostatic agent to the surface of tissue in any aspect or embodiment thereof.

[0107] 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 some primary alcohol groups on the C6 of, for example, glucan units are oxidized to carboxylic acids and optionally functionalized. In some embodiments, the composition is substantially free of oxidized regenerated cellulose (ORC).

[0108] In some embodiments, the composition is substantially free of buffers. In some such embodiments, the buffer refers to Tris or tris(hydroxymethyl)aminomethane buffer or lysin. In some such embodiments, the buffer refers to Tris or tris(hydroxymethyl)aminomethane buffer or lysin in powder form.

[0109] The term “substantially non-existent” is defined herein as meaning almost non-existent or completely non-existent. That is, it means less than 1%, less than 0.5%, less than 0.3%, less than 0.1% (w / w) of the total weight of the composition, or 0% of the total weight of the composition.

[0110] In another aspect of this disclosure, a method for preparing a composition according to embodiments thereof, such as a hemostatic composition, is provided, characterized in that the viscosity is in the range of about 3000 Pa·s to about 5000 Pa·s at about 25°C and / or the swelling ratio is at most 10% at about 25°C, the method comprising the following steps performed sequentially: (i) Stir the fibrinogen solution to obtain foam; (ii) Spray the thrombin solution into the foam, so that the foam is further infused with thrombin; (iii) Stir the foam containing thrombin to convert fibrinogen into fibrin; (iv) Lyophilized fibrin; (v) Grind the freeze-dried fibroin to obtain a uniform powder, and (vi) Under conditions that allow for the preparation of hemostatic compositions, the powder is mixed with a dispersant comprising an aqueous solution, glycerol, and optionally thrombin.

[0111] In some embodiments, the concentration of fibrinogen in the solution is at least 20 mg / ml, such as 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, or any value and range therein.

[0112] As used in this article, the term "whipping" refers to the action of using a forward and backward movement of a whisk or mixer to beat a sample in order to quickly incorporate air and create foam.

[0113] As used in this specification, the term "foam" refers to a substance formed by encapsulating a gaseous sac within a liquid or solid. The term "spray" should be understood broadly and may also include terms such as spraying, atomizing, and misting. The term "grinding" refers to the process of reducing particle size by collision with a rapidly moving surface in a grinding mill. In some embodiments, the mill is a ball mill, sand mill, jet mill, or disc mill. The term "mixing" refers to blending or stirring components (e.g., powders and dispersants) together.

[0114] In some embodiments, about 60% to about 95% by weight, or in some embodiments, about 70% to about 85% by weight, of fibrinogen coagulation (i.e. conversion to fibrinogen).

[0115] The foam obtained in step (iii) can be freeze-dried (lyophilized) and stored for later use, or it can be freeze-dried and then immediately ground or milled into powder. As used herein, the term "lyophilized" or any grammatical variation thereof refers to a product obtained by freezing a solution and then reducing the concentration of water to a level that does not support biological or chemical reactions, for example by sublimation, to form a "cake". Such a freeze-dried "cake" with low protein density can have a high solubility. To dissolve the "cake" in an aqueous solvent, a solid dispersion can optionally be prepared by mechanically crushing and / or grinding the "cake".

[0116] Typically, after the solution is freeze-dried, a porous and sponge-like solid material is obtained, which is referred to as a "cake" (or "solid composition" in this document).

[0117] The milling process is also commonly referred to as micronization. The term "micronization" is used when, for example, microparticles are produced from a solid composition and the diameter of these microparticles ranges from a few micrometers to at most a few hundred micrometers. Micronization can be achieved through processes including, but not limited to, jet milling, ball milling, high-pressure homogenization, RESS (rapid expansion of supercritical solution) processes, SAS (supercritical antisolvent) methods, or PGSS (particles in gas-saturated solution) methods. Typically, but not exclusively, micronization results in a reduction in protein powder size by 30 to 400 times from its original size.

[0118] In some embodiments, thrombin is present in the thrombin solution at a concentration of about 1500 IU / ml to about 1 U / ml (in step ii). In some embodiments, thrombin is present in the thrombin solution at a concentration of about 1550 IU / ml to about 2000 IU / ml.

[0119] In some embodiments, the thrombin solution also contains one or more additives, such as, but not limited to, calcium salts such as calcium chloride, and / or one or more excipients, such as, but not limited to, selected from one or more amino acids, albumin, sugars and / or sugar derivatives.

[0120] The term “additive” is intended to be understood as any substance that may be added to a composition, and may also include active additives such as calcium salts as described herein.

[0121] Calcium is an important element in the coagulation cascade, and calcium is required for the activation of factor XIII to factor XIIIa, which crosslinks and stabilizes fibrin to form insoluble clots.

[0122] In some embodiments, the method further includes adding a calcium salt to the powder obtained in step (v). In some embodiments, the calcium salt is CaCl2, and its amount is up to about 3% by weight of the powder.

[0123] In some embodiments, a certain amount of a calcium salt, such as calcium chloride, is dissolved in a thrombin solution. In some embodiments, the amount of calcium chloride dissolved in the thrombin solution is in the range of about 0.60% w / v to about 3% w / v, or in some embodiments, in the range of about 0.65% w / v to about 2.6% w / v.

[0124] As used herein, the term "excipient" refers to an inactive or non-therapeutic agent that is added to a pharmaceutical composition to, for example, provide a desired consistency or stabilizing effect.

[0125] In some embodiments, the foam (e.g., in lyophilized form) is ground into a powder with a median particle size of less than 1000 μm. In some embodiments, the foam (e.g., lyophilized sample) is ground into a powder with a median particle size of less than 500 μm.

[0126] In some embodiments, the dispersant comprises an aqueous saline solution of about 30% v / v to about 80% v / v glycerol. In some embodiments, the dispersant comprises an aqueous saline solution of about 40% v / v to about 80% v / v glycerol. In some embodiments, the dispersant comprises an aqueous saline solution of about 40% v / v to about 70% v / v glycerol. In an exemplary embodiment, the dispersant comprises an aqueous saline solution of about 50% glycerol.

[0127] In some embodiments, the dispersant contains thrombin. In some embodiments, thrombin is added to the dispersant to catalyze a blood clot when the paste is applied to the bleeding site. In some embodiments, the amount of thrombin in the dispersant is in the range of about 100 IU / ml to about 900 IU / ml. In some embodiments, the amount of thrombin in the dispersant is in the range of about 200 IU / ml to about 800 IU / ml. In some embodiments, the amount of thrombin in the dispersant is in the range of about 300 IU / ml to about 600 IU / ml. In an exemplary embodiment, the amount of thrombin in the dispersant is about 400 IU / ml.

[0128] In some embodiments, the method is carried out under conditions selected from one or more of (a) to (e): (a) thrombin is present in an amount of about 2000 IU to about 4000 IU per gram; (b) a dispersant is present in a dispersant-to-powder ratio in the range of 3:1 to 5:1 (v / w; ml / g); (c) at least about 70% of the foam obtained in step (ii) contains fibrinogen; (d) the method is carried out at a temperature in the range of 0°C to 30°C; and (e) the pH of the dispersant is in the range of 4.5 to 7. In some embodiments, the conditions are selected from one or more of (a) to (c).

[0129] In some implementations, such conditions allow fibrinogen to be converted at least partially into fibrin.

[0130] According to another aspect, in a method of treating bleeding tissue with the disclosed composition according to any embodiment thereof, the method includes applying the composition to the bleeding tissue by obtaining a sealant at the site upon application. Therefore, a method for controlling bleeding is provided, comprising applying the disclosed composition herein to the bleeding site.

[0131] In the context of this invention, the term "sealant," also known as "fibrin sealant" and "bioglue," should be understood as an adhesive, glue, or hemostatic agent, such as that derived from or derived from the disclosed compositions, having components that react upon contact with or proximity to tissue and / or blood to subsequently form a clot, and further acting as a tissue adhesive and thereby preventing, reducing, or stopping bleeding, binding structures, and / or sealing physiological leaks, such as leaks of cerebrospinal fluid (CSF), lymph, bile, gastrointestinal (GI) contents, air leaks from the lungs, etc. In some embodiments, the sealant formulation also comprises one or more therapeutic agents that are released when the clot formed in the body naturally degrades. The therapeutic agents may be (but are not limited to) drugs (such as antibiotics, analgesics, anti-inflammatory drugs, anticancer drugs, etc.) or cells (including, for example, any type of stem cell, such as embryonic stem (ES) cells from humans or other sources, adult stem cells, induced pluripotent stem cells (iPSCs), etc.).

[0132] The term "hemostasis" refers to, for example, preventing, reducing, or stopping blood loss from a wound, such as a surgical or traumatic one, by promoting the formation of a blood clot, or the ability to assist in hemostasis. Hemostasis ("hemostasis" or "haemostasis") refers to the first stage of wound healing. It is the process of stopping bleeding. The term "assist in hemostasis" means helping to reduce or stop bleeding. The term "applied to the bleeding site" means, for example, applying a composition topically to the bleeding site (e.g., in a surgical site) to control bleeding. Bleed control is required in many situations, including wound management.

[0133] As used herein, the term “bleeding” refers to the extravasation of blood from any component of the circulatory system. Therefore, “bleeding” encompasses unintended, uncontrolled, and often excessive bleeding associated with surgery, trauma, or other forms of tissue damage, as well as unintended bleeding in patients with hemorrhagic disorders. The term “blood” or any grammatical variations thereof also includes blood fractions, such as plasma.

[0134] The term “trauma” is defined as injury caused by physical force; non-limiting examples include the consequences of vehicle accidents, shootings, and burns.

[0135] As used herein, the terms “control,” “prevent,” or “reduce” (which may be used interchangeably in the context of bleeding herein) indicate that the rate of blood exudation is substantially eliminated or reduced, for example, by 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of the initial bleeding rate, compared to a situation where the disclosed composition does not come into contact with the bleeding site. Methods for determining the level of bleeding occurrence are known in the art.

[0136] In addition, the terms “control,” “prevent,” or “reduce” in the context of bleeding may also cover at least partially sealing the blood vessels at the site of bleeding (e.g., in soft tissue).

[0137] As used herein, the term "subject" shall mean any animal, including but not limited to humans, mice, rats, rabbits, non-human primates, or any other mammal. In some embodiments, the subject is a human, such as a human patient. The subject may be male or female.

[0138] As used herein, the term “damaged tissue” (or “injured tissue”) refers to a disruption of the normal continuity of a structure caused by physical (e.g., mechanical) forces, such as incisions caused by surgical, biological (e.g., thermal or photochemical) or any chemical means. The term “damaged tissue” also encompasses contused tissue, punctured or torn tissue, and openings, punctures, and injuries caused, for example, by tearing, scratching, squeezing, and biting. The term “damaged tissue” also encompasses wounds and bleeding sites as described throughout this document.

[0139] The term "tissue" can refer to tissues selected from soft tissues and bone tissues.

[0140] As used herein, the term "soft tissue" refers to body tissue that is not hardened or calcified. This term particularly refers to vascularized soft tissue, which can therefore be a source of bleeding. Examples of such tissue include, but are not limited to, connective tissue (such as tendons, ligaments, fascia, skin, fibrous tissue, fat, and synovium), muscles, and internal organs. Generally, the term "soft tissue" is intended to exclude bone tissue.

[0141] Therefore, in some embodiments, the disclosed composition may be applied, for example, during MIS (e.g., endoscopy) to hard-to-reach bleeding sites. One common form of endoscopy is laparoscopy, which is a minimally invasive examination and surgery inside the abdominal cavity.

[0142] The term "surgery" also encompasses the time during or after a surgical or diagnostic procedure. Other non-limiting examples of surgery include neurosurgery, abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck surgery, pelvic surgery, and skin and subcutaneous tissue surgery. For at least one of these situations, the compositions of the present invention can be used as suitable sealants that allow adhesion to the bleeding wound and thus achieve hemostasis regardless of the condition of the injured tissue, such as the severity of the bleeding. Sealant formation occurs within a relatively short clotting period, followed by application of the disclosed composition to the damaged tissue according to any aspect and / or embodiment. Typically, after tamponade for 1 to 3 minutes, an incomplete clot may form that stops the bleeding, but a longer time may be required for the coagulation reaction to reach the paste surface, thereby forming a complete clot.

[0143] In another aspect of the invention, a method of treating a wound is provided, the method comprising the step of applying (e.g., contacting) the disclosed composition in any aspect and / or embodiment thereof to a wound and / or in a subject to whom the wound is desired.

[0144] The term "wound management" specifically refers to reducing blood loss at sites of internal bleeding, such as in patients undergoing surgery. (See reference) Figure 6B This demonstrates a high hemostasis success rate (above 75%) for the test samples disclosed in this paper.

[0145] Therefore, in some embodiments, the method is used to reduce blood loss at bleeding sites in tissues, such as those of patients undergoing surgery, and / or to form a sealant layer in / on such tissues. The method includes, in one embodiment, contacting the disclosed composition with the bleeding site and / or its vicinity. Optionally, the method includes first applying an aqueous solution (such as saline) to such tissues, and then applying the disclosed composition over the aqueous solution to promote rapid coagulation time. Therefore, in some such embodiments, the composition serves as a hemostatic agent at the bleeding site in the tissue.

[0146] According to another embodiment of the invention, a kit for generating the compositions described herein is provided. It should be understood that the consistency of the composition allows it to be applied, for example, by spreading or by adhering the composition directly to the bleeding site. Therefore, the composition does not require further spreading to or application to a solid surface, object, or other solid medium, such as a strip or film, to achieve a suitable form for application to the bleeding site. However, suitable applicators (such as syringes) can be used to apply, spread, or adhere the composition to the bleeding site for easy access and handling; see [link to relevant documentation]. Figure 1 .

[0147] In another aspect and / or embodiment, the present invention provides a kit comprising: a) a container containing the composition of the present invention as described above, b) an applicator for applying the composition to tissues, and c) optionally, instructions for use.

[0148] In another embodiment, the invention also provides a hemostasis kit comprising a container holding a composition disclosed herein in one embodiment. In another embodiment, the invention provides a hemostasis kit comprising: (b) a container holding 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; and (c) a container holding a dispersant comprising glycerol and optionally an aqueous solution; and optionally (d) instructions for use.

[0149] In some implementations, the kit also includes an applicator device.

[0150] In some embodiments, the powder containing fibrin and thrombin is obtained by the method described herein in its embodiments.

[0151] In some embodiments, the dispersant comprises thrombin powder. Alternatively, the kit includes thrombin powder or thrombin solution in a separate container (e.g., in a vial). The amount of thrombin powder can range from 200 IU / mg or IU / ml to 600 IU / mg or IU / ml, for example, about 400 IU / mg or IU / ml.

[0152] 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 dispersant containing glycerol and optionally an aqueous solution. During preparation, the composition is in the form of a paste suspension at room temperature. (i) and (ii) may each be present in separate containers.

[0153] In some embodiments, the powder contains at least 60% by weight of fibrin, or in some embodiments, it contains at least 70% by weight of fibrin.

[0154] Any aspect and embodiment of the compositions disclosed herein may be incorporated into aspects and embodiments of the kit, as well as embodiments of the dispersant and / or powder.

[0155] Optionally, fibrinogen or fibrin and thrombin are each individually packaged (e.g., in dry form or in solution) in the kit. Thus, each of the two components is packaged in a separate packaging material, in addition to the overall packaging material of the kit. In some embodiments, fibrinogen or fibrin and thrombin are packaged together (e.g., as a dry powder) in the same packaging material in the kit, and optionally a dispersant is provided in a separate container.

[0156] In some embodiments, the kit also includes instructions on how to combine the kit components and / or how to combine the kit components with other components (e.g., dispersants) to produce a desired composition.

[0157] Optionally, the kit also contains a dispersant. The dispersant (such as glycerol) may be in pure form or in the form of a solution with another liquid (e.g., water, saline, or aqueous buffer). Optionally, the dispersant is packaged separately from the powder. Alternatively, the dispersant is packaged in combination with the powder, for example as a ready-to-use composition described herein. In such embodiments, the kit may additionally contain a measuring device, such as a graduated cylinder, for measuring the volume of the dispersant or its components.

[0158] Additionally or alternatively, the hemostasis kit may include a syringe containing a blend, mixture, or powder and another syringe containing a dispersant. For example, a dual-syringe mixing device can produce a substantially homogeneous paste mixture by combining initially separated liquids and powders and then passing the blended contents back and forth between two connected syringes via interconnected outlets. Therefore, a low pressure output for dispensing the paste from the syringe may be preferred to facilitate mixing and ultimately deployment of the resulting paste. The desired pressure output may be less than 1.51 lbf.

[0159] In some embodiments, at least one container in the kit is a pre-filled syringe. In some embodiments, a syringe is provided in addition to the container of the kit. In some embodiments, the container is of a specific type, such as a vial or applicator, such as a syringe.

[0160] In some embodiments, at least one container in the kit is a pre-filled syringe. In some embodiments, a syringe is provided in addition to the container of the kit. The term "container" can refer to any general structure, such as a vessel or vial, that may contain powder, a dispersant as disclosed throughout the document, or any component or paste thereof.

[0161] An applicator device can be used to administer the kit, which can be used for multiple and continuous injections of the composition. In one embodiment, the applicator device enables multiple injections of a fixed dose of the mixed components onto a 2D surface of tissue while the device is moved. In one embodiment, the applicator has a syringe and a tip or needle configured to be attached to the applicator. In one embodiment, the applicator has a syringe equipped with an injection needle that optionally retracts automatically from the patient's skin after the injection is completed without requiring the applicator to lift the device up from the injection surface. In one embodiment, the kit can be used to administer a sealant.

[0162] The hemostatic kit of the present invention can be a kit for reducing, preventing, or stopping blood flow, such as in open wounds, and can be used during surgery, including before or after surgical procedures such as laparoscopic surgery, neurosurgery, abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck surgery, pelvic surgery, and skin and subcutaneous tissue surgery. This kit can be used to reduce or prevent blood flow from the skin or internal organs.

[0163] In one implementation, the kit can be stored at room temperature, such as in the range of 8°C to 40°C, or at a lower temperature.

[0164] In some embodiments of any aspect of the kits or compositions disclosed herein, the powder or blend may contain additives, such as calcium salts and / or one or more excipients, such as those selected from, but not limited to, one or more amino acids, albumins, sugars and / or sugar derivatives.

[0165] In some embodiments of the kit or composition, the composition comprises, by weight of the powder, a calcium salt, such as calcium chloride (CaCl2), at a concentration of, for example, from 0.5% to about 4%, or 2% to 4%, or 2.5% to 3.5%. In some embodiments, the calcium salt (e.g., calcium chloride) is present at a concentration of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% (inclusive of any values ​​and ranges therein) by weight of the blend. In some embodiments, the calcium salt is present in a thrombin solution.

[0166] In some embodiments, fibrinogen is present at a concentration ranging from 65% to 80%, 65% to 95%, or 70% to 90% by weight of the powder. In some embodiments, fibrinogen is present at a concentration of 65%, 70%, 75%, 80%, 85%, or 90% (inclusive of any values ​​and ranges between them) by weight of the powder. In some embodiments, fibrinogen is present at a concentration of about 80% by weight of the powder. In some embodiments, fibrin is present at a concentration of 65% to 80%, 65% to 95%, or 70% to 90% by weight of the powder. In some embodiments, fibrin is present at a concentration of 65%, 70%, 75%, 80%, 85%, or 90% (inclusive of any values ​​and ranges between them) by weight of the powder. In some embodiments, fibrin is present at a concentration of about 80% by weight of the powder.

[0167] In an exemplary embodiment, the blend or powder contains about 70% to 80% by weight of fibrinogen or fibrin powder; 2000 IU to 4000 IU of thrombin / dose powder; and "dose" in this document means about 1.2 g.

[0168] In some embodiments of the kit and / or composition, the disclosed composition may be used in conjunction with a backing, pad, bandage, gauze, sponge, scaffold, or matrix to provide mechanical strength to cover the wound surface. In this case, the example paste is supported on a pad to facilitate application or packing.

[0169] In some embodiments of the kit and / or composition, the composition is sterile. Sterility is critical, especially when handling blood products, particularly regarding virus inactivation. Generally, virus inactivation can be performed by any method, including solvent washing, heat inactivation, irradiation, and nanofiltration. Typically, virus inactivation standards require the use of two different methods. Additionally, FDA sterility standards require filtration.

[0170] As used herein, the term "sterile" means having a low bioload and being effectively sterile, such as being substantially or even absolutely free of microorganisms (e.g., bacteria and viruses). Sterilization is the process of reducing the bioload to an effectively sterile level. Sterile liquids, powders, or pastes are generally defined as liquids or pastes that have undergone sterile filtration.

[0171] The present invention also provides the following technical solutions.

[0172] 1. A two-phase flowable composition in paste form at room temperature, said composition comprising: (iii) A powder comprising: fibrinogen and fibrin, present together in an amount of at least about 70% by weight; thrombin; and (iv) A dispersant comprising: glycerol and an aqueous solution, in: The dispersants are present in a dispersant-to-powder ratio in the range of about 3:1 to about 5:1 (v / w; ml / g); and wherein: The glycerol is present at a concentration of more than about 30% by volume of the dispersant.

[0173] 2. The composition according to claim 1, wherein the composition comprises about 60% to 80% by weight of fibroin.

[0174] 3. The composition according to claim 1 or 2, wherein the fibrin is at least partially cross-linked.

[0175] 4. The composition according to any one of claims 1 to 3, wherein the fibrinogen and / or fibrin are present in the powder in an amount of about 80% by weight.

[0176] 5. The composition according to any one of claims 1 to 4, wherein the powder further comprises a calcium salt.

[0177] 6. The composition according to claim 5, wherein the calcium salt is CaCl2 or its hydrate, and the amount of the calcium salt is at most about 3 by weight.

[0178] 7. The composition according to any one of claims 1 to 6, wherein the thrombin is present in an amount of about 2000 IU to about 4000 IU per gram of powder.

[0179] 8. The composition according to any one of claims 1 to 7, wherein at least 80% of the powder is in the form of particles with a size of less than 500 µm.

[0180] 9. The composition according to any one of claims 1 to 8, wherein at least a portion of said microparticles is porous.

[0181] 10. The composition according to any one of claims 1 to 9, wherein the glycerol is present at a concentration ranging from about 30% to about 80% by weight of the dispersant.

[0182] 11. The composition according to any one of claims 1 to 10, wherein the aqueous solution comprises brine.

[0183] 12. The composition according to any one of items 1 to 11, characterized by a uniform viscosity.

[0184] 13. The composition according to any one of claims 1 to 12, characterized in that it has a viscosity of about 3000 Pa·s to about 5000 Pa·s in the range of about 25°C.

[0185] 14. The composition according to item 13, wherein after storage at about 25°C for 24 hours, the viscosity changes by less than about 30%.

[0186] 15. The composition according to any one of claims 1 to 14, characterized in that it has a swelling ratio of up to 10% when in contact with blood at about 25°C.

[0187] 16. The composition according to any one of items 1 to 15, wherein the composition is free of oxidized cellulose.

[0188] 17. The composition according to any one of items 1 to 16, wherein the composition does not contain a buffer.

[0189] 18. The composition according to any one of items 1 to 17, wherein the composition is a hemostatic composition.

[0190] 19. The composition according to any one of claims 1 to 18, in a method of treating hemorrhagic tissue, the method comprising applying the composition to the hemorrhagic tissue.

[0191] 20. The composition according to any one of claims 1 to 19, wherein the method comprises minimally invasive surgery, such as laparoscopic surgery.

[0192] 21. A method for preparing a hemostatic composition, characterized in that its viscosity is in the range of about 3000 Pa·s to about 5000 Pa·s at about 25°C and / or its swelling ratio is at most 10% at about 25°C, the method comprising the following steps: (vii) Stir the fibrinogen solution to obtain foam; (viii) Spray the thrombin solution into the foam, so that the foam is further incorporated with the thrombin; (ix) Stir the foam containing the thrombin to at least partially convert fibrinogen into fibrin; (x) Freeze-dry the fibrin; (xi) Grind the freeze-dried fibroin to obtain a uniform powder, and (xii) The powder is mixed with a dispersant comprising an aqueous solution, glycerol and thrombin, wherein the method is carried out under conditions that allow for the preparation of the hemostatic composition.

[0193] 22. The method according to item 21, wherein the condition is selected from one or more of (a) to (c): (a) The dispersant contains thrombin in an amount of about 200 IU to about 800 IU per 1 ml; (b) The dispersant is added to the powder at a dispersant-to-powder ratio in the range of 3:1 to 5:1 (v / w; ml / g); and (c) At least about 70% of the foam obtained in step (ii) contains fibroin.

[0194] 23. The method according to any one of items 21 or 22, wherein the thrombin solution in step (ii) further comprises a calcium salt.

[0195] 24. The method according to item 23, wherein the calcium salt is CaCl2 and has a concentration of 0.5% w / v to about 3% w / v.

[0196] 25. A kit comprising: (a) A container for containing powder comprising fibrinogen, fibrin, and thrombin, wherein the fibrinogen and the fibrin are present in a total amount of at least about 70% by weight; and (b) A container for containing a dispersant comprising glycerol and optionally an aqueous solution, and optionally... (c) Instruction manual.

[0197] 26. The kit according to claim 25, further comprising an applicator device.

[0198] General The terms “comprising,” “including,” “containing,” “having,” and their variations mean “including but not limited to.” The term “composed of” means “including and limited to.” The term “essentially composed of” means that a composition, method, or structure may contain additional ingredients, steps, and / or components, provided that such additional ingredients, steps, and / or components do not substantially alter the essential and novel characteristics of the composition, method, or structure protected by the claims.

[0199] The term “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any implementation described as “exemplary” is not necessarily to be construed as preferred or advantageous over other implementations and / or excludes features of other implementations.

[0200] The term "optionally" is used herein to mean "provided in some embodiments but not in others." Any specific embodiment of the invention may include several "optional" features unless such features conflict.

[0201] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references. For example, the terms “a compound” or “at least one compound” can include multiple compounds (including mixtures thereof).

[0202] Throughout this application, various embodiments of the invention may be presented in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as a fixed limitation on the scope of the invention. Therefore, a scope description should be considered to have all clearly disclosed possible sub-scopes and the respective numerical values ​​within those scopes. For example, a scope such as 1 to 6 should be considered to have clearly disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the respective numerical values ​​within those scopes, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the scope.

[0203] Whenever this document indicates a range of numbers, it means that any of the enumerated numbers (fractions or integers) within the indicated range are included. The phrases “range is” between the first and second indicated numbers and “range is from” the first indicated number to the second indicated number are used interchangeably in this document and refer to all fractions and integers including the first and second indicated numbers and those in between.

[0204] As used herein, the term “method” refers to the manner, means, techniques and procedures used to accomplish a given task, including but not limited to those manner, means, techniques and procedures known to or readily developed from known manner, means, techniques and procedures by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine.

[0205] As used herein, the term “treatment” includes eliminating, substantially inhibiting, slowing or reversing the progression of a disease, substantially improving the clinical or aesthetic symptoms of a disease, or substantially preventing the occurrence of the clinical or aesthetic symptoms of a disease.

[0206] In cases where conventions such as "at least one of A, B, and C" are used, such structures are generally intended to have a meaning that those skilled in the art will understand (e.g., "a composition having at least one of A, B, and C" will include, but is not limited to, compositions having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art should also understand that, in practice, whether in the detailed description, claims, or drawings, transitional words and / or phrases presenting two or more alternative terms should be understood to cover the possibility of including one of these terms, any one of these terms, or both of these terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B".

[0207] It should be understood that, for clarity, certain features of the invention described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various structures of the invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination, or as appropriately provided in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiments would not function without those elements.

[0208] As used herein, the term "about" means and is intended to include values ​​that are 10% higher or lower than the indicated value. Unless otherwise specified, all figures such as, but not limited to, those representing, for example, ratios, weights, moles per mole, quantities, viscosity, temperatures, etc., shall in all cases be understood to be modified by the term "about". Therefore, unless stated to the contrary, the numerical parameters listed in this specification and the appended claims are approximate values, which may vary by up to ±10% according to the desired properties sought to be obtained according to the invention.

[0209] The various embodiments and aspects of the invention described above and claimed in the claims section below are experimentally supported in the following examples. Example

[0210] The following embodiments, together with the foregoing description, illustrate some implementations of the invention in a non-limiting manner.

[0211] Example 1: Preparation of a flowable composition The aim of this study is to develop a flowable hemostatic agent that, when applied to the bleeding site during surgery, exhibits both fluidity and tissue adhesion.

[0212] Materials and methods The source of fibrinogen and thrombin is porcine plasma, which is fractionated to obtain fibrinogen and thrombin, and is supplied by Bioseal Biotech Co. Ltd., located in Guangzhou, China.

[0213] Preparation of fibrinogen sealant powder Fibrinogen / fibrin sealant powder (FSP) was obtained by freeze-drying and grinding fibrin foam. The foam was generated by pre-mixing fibrinogen and thrombin extracted from porcine plasma as follows: First, the fibrinogen solution (at least 20 mg / ml) was stirred until fully foamed (the foam should not slip when scooped and turned). Foaming helps the protein form a porous structure, allowing the cross-linking reaction to occur in a larger space, resulting in uniform and effective cross-linking.

[0214] Next, a small amount of CaCl2 (0.65% w / v - 2.6% w / v) was completely dissolved in the thrombin solution (1555 IU / ml - 2000 IU / ml) and thoroughly mixed. Then, the thrombin solution (containing CaCl2) was sprayed onto the foam using a nozzle attached to the syringe applicator, and then stirred to allow cross-linking. It is estimated that approximately 70%-85% of the fibrinogen coagulated during this process.

[0215] Next, a freeze-drying step is performed. After freeze-drying, the water inside the porous structure can easily evaporate, and the remaining porous structure (“cake”) can be easily and uniformly ground to prepare a homogeneous, flowable paste. The freeze-dried sample is ground into a powder with a particle size <500 μm. Notably, regardless of the mechanism, freeze-drying helps to retain the activity of unreacted fibrinogen (approximately 20% to 30% of fibrinogen remains unreacted), which can be used to form an in-situ sealant when applied to the bleeding site. The freeze-drying parameters are: pre-freezing at -45°C for 2 hours; primary drying at 15°C for 96 hours; secondary drying at 37°C for 24 hours (using a SCIENTZ-50F / B vacuum pump freeze dryer manufactured by Ningbo Scientz Freeze-Drying Equipment Co., Ltd.). The weight ranges of the components in the obtained powder are as follows: fibrinogen 12% w / w-20% w / w, fibrin 53% w / w-82% w / w, thrombin 4% w / w-25% w / w, and CaCl2 approximately 2% w / w, or in CaCl2... . The percentage is 2.3% w / w for H2O and for CaCl2. . 2H2O has a w / w content of 2.6%.

[0216] Paste preparation A paste is obtained by mixing the powder (1 g) with a dispersant (3 ml to 5 ml, approximately 50% v / v saline solution of glycerol) and thrombin powder (3300 IU potency and approximately 21 mg CaCl2). The dispersant should have a sufficiently high viscosity to remain at the bleeding site for a certain period of time. It was found that using low concentrations of glycerol (below 30% v / v) in the dispersant resulted in phase separation of glycerol and water. On the other hand, excessively high concentrations (above 70%) resulted in an overly viscous paste. The pH of the dispersant was approximately 4.5 to 7.

[0217] Thrombin (400 IU / ml) is also added along with the dispersant to ensure that there is a sufficient amount of thrombin to catalyze the blood clot when the paste is used at the bleeding site.

[0218] Figures 2A to 2B A flowchart with photographic images is presented, illustrating an exemplary embodiment of the process for preparing the powder of the present invention. Figure 2A ), and photographic images of the obtained paste ( Figure 2B ).

[0219] Comparative formulation The contrast fibrinogen and thrombin preparation (“ORC paste”) is also made from porcine fibrinogen and thrombin solution. The solution is processed into a powder by spraying and combined with ORC, lysine, and calcium salts. ORC powder is prepared by milling or grinding the corresponding fabric fibers to obtain the powder without a foaming step. The weight ratio of ORC to fibrinogen is in the range of 1:1 to 1:2. The ORC paste contains a buffer, such as a buffer in powder form (Tris, lysine, or a combination thereof). ORC paste component data: ORC: 0.6 g; fibrinogen: 0.3 g; thrombin: 3300 IU, in one syringe paste (approximately 5 ml); lysine: 0.2 g; CaCl2: 0.02 g; dispersant: 50 vol% glycerol saline solution. The ORC paste is prepared without foaming and lyophilization steps. Lysine is added to reduce the acidity of the ORC. This acidity may denature plasma proteins such as thrombin.

[0220] Example 2: Characterization of Fibrin Sealant Paste Viscosity The viscosity of the FS paste was tested using an oscillating rheometer (Anton Paar Rheometer) with a parallel plate geometry of 25 mm diameter and 1.25 mm gap. The results show... Figures 3A to 3B The results show that the reconstituted fibrin paste maintains a stable and uniform viscosity at room temperature for at least 24 hours. It also shows that the FS paste is more stable and has a higher viscosity compared to the paste prepared with ORC, thus allowing the FS paste to remain at the wound site for a longer period.

[0221] swelling ratio In an exemplary procedure, the swelling ratio of the FS paste was tested by adding fibrinogen (70 mg / mL, 2 ml) to a paste (1 g FS powder + 4.5 ml dispersant) and then incubating it at 37°C for 30 minutes to obtain a cured paste. The cured paste was cut into slices of approximately 1.0 g each. After weighing, the slices were incubated in phosphate-buffered saline (PBS) for 24 hours to allow them to swell. See [link to relevant documentation]. Figure 4A The swelling ratio was then calculated by increasing the weight. The results are shown in... Figure 4B In a comparative test using a gelatin matrix, SURGIFLO containing thrombin was used. ™ The hemostatic matrix is ​​a paste fortified with fibrinogen. Results showed that the FS paste of the present invention has a low swelling ratio compared to gelatin samples. Due to the low swelling ratio, the FS paste does not exhibit a "occupancy effect" and can remain at the bleeding site for a longer period, providing improved hemostasis.

[0222] In vitro clotting time In the exemplary procedure, 0.5 g of FS paste was placed in a glass vial, followed by the addition of 4 mL of porcine blood anticoagulant (sodium citrate, 3.2% w / v–3.8% w / v). The vial was gently shaken or swirled for a few seconds to allow the blood and paste to come into contact and mix. Next, the vial was tilted to observe the clotting. In cases of complete clotting, the coagulated blood remained at the top of the vial once tilted or inverted. In cases of incomplete clotting, the blood remained fluid and flowed to the bottom of the vial under gravity. The time it took for the blood to stop flowing was recorded. The results showed a clotting time of 14 to 16 seconds, less than 20 seconds, indicating good efficacy for in vitro blood clotting.

[0223] Test samples taken from the vials showed that, compared with ORC paste ( Figure 5B Compared to other methods, the flowable paste of this invention is easily and uniformly absorbed into the blood. Figure 5A This gives it the potential to improve the accelerated coagulation cascade and the formation of endogenous fibrin clots in vivo.

[0224] Animal studies—Efficacy testing in an in vivo model of spleen perforation In the exemplary procedure, a wound defect approximately 6 mm in diameter and 3 mm deep is created using a modified 6 mm biopsy puncturer (with an elastic band placed around the circumference of the puncturer as a 3 mm depth stop). The central core (tissue plug) is removed using surgical forceps and scissors. A new site is prepared for each test or control article, and a defect of similar size is prepared for each treatment group. The test sample is added to the wound site (with moderate bleeding) and applied to cover the wound, then the sample is pressed into the wound with moist gauze for 2 minutes. After 2 minutes, the gauze is gently removed, and the wound is observed for continued bleeding. See also Figure 6A If no active bleeding is found, hemostasis is considered successful; otherwise, hemostasis fails. Negative controls are moist gauze application and ORC flowable paste. The hemostasis success rate is the percentage of successful hemostasis divided by the total number of sample tests. Results are shown in... Figure 6B The hemostatic success rate of FS paste is presented in the table. For example... Figure 6B As shown, compared with the 40% hemostasis success rate of ORC paste, FS paste showed an 80% hemostasis success rate.

[0225] In summary, compared to ORC-flowable pastes, this invention enhances hemostatic efficacy by modifying the formulation with additional FSP (fibrin sealant powder), which contributes to enhanced blood absorption. Furthermore, and without being bound by a specific mechanism, the porous powder in the flowable paste allows for easy absorption of blood and exposure to high concentrations of thrombin, thereby accelerating the coagulation cascade to form an endogenous fibrin clot. Moreover, without being bound by a specific mechanism, after grinding the porous cake, the porous structure is at least partially (20% to 30% of the pores) retained in the powder, providing more pores and contact with the bleeding site, thus improving hemostasis. This flowable hemostatic composition can be applied to hard-to-reach bleeding sites and achieve hemostasis. Figure 7A The structure of the powder in the ORC paste was presented. Figure 7B Compared to the porous structure of the powder of the present invention, the estimated pore size of the powder of the present invention ranges from about 10 μm to 30 μm.

[0226] Although the invention has been described in conjunction with its specific embodiments, many alternatives, modifications, and variations will obviously be apparent to those skilled in the art. Therefore, this document is intended to cover all such alternatives, modifications, and variations that fall within the substantive and broad scope of the appended claims.

Claims

1. A two-phase flowable composition in paste form at room temperature, said composition comprising: (iii) A powder comprising: fibrinogen and fibrin, present together in an amount of at least about 70% by weight; thrombin; and (iv) A dispersant comprising: glycerol and an aqueous solution, in: The dispersants are present in a dispersant-to-powder ratio in the range of about 3:1 to about 5:1 (v / w; ml / g); and wherein: The glycerol is present at a concentration of more than about 30% by volume of the dispersant.

2. The composition according to claim 1, wherein the composition comprises about 60% to 80% by weight of fibroin.

3. The composition according to claim 1 or 2, wherein the fibroin is at least partially cross-linked.

4. The composition according to any one of claims 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 claims 1 to 4, wherein the powder further comprises a calcium salt.

6. The composition according to claim 5, wherein the calcium salt is CaCl2 or its hydrate, and the amount of the calcium salt is at most about 3 by weight.

7. The composition according to any one of claims 1 to 6, wherein the thrombin is present in an amount of about 2000 IU to about 4000 IU per gram of powder.

8. The composition according to any one of claims 1 to 7, wherein at least 80% of the powder is in the form of particles with a size of less than 500 µm.

9. The composition according to any one of claims 1 to 8, wherein at least a portion of the microparticles is porous.

10. The composition according to any one of claims 1 to 9, wherein the glycerol is present at a concentration ranging from about 30% to about 80% by weight of the dispersant.

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