Flowable paste with ellagic acid-nickel complex and its use for the activation of blood clotting

Abstract

The present invention relates to a hemostatic composition comprising a gallate-metal complex and its use for activating the endogenous pathway of blood coagulation. In particular, a hemostatic composition comprising gallate complexed with nickel ions is disclosed, the complex being dispersed or embedded within an absorbable polymer matrix, such as gelatin. A method for preparing the hemostatic composition is also disclosed.

Description

Technical Field

[0001] The present invention relates in particular to hemostatic compositions comprising ellagic acid-nickel complexes and their use in activating coagulation. Background Technology

[0002] Topical hemostatic agents have become essential tools for minimizing bleeding and reducing the associated risk of serious complications during surgery or emergencies. Adjunctive hemostatic agents used by surgical teams can contain a variety of agents, such as gelatin, fibrin sealants, oxidized regenerated cellulose (ORC), or synthetic hydrogels and adhesives. Products based on animal (e.g., swine and bovine) gelatin and plant-derived ORCs are at the forefront of bleeding management because they are applicable to a variety of surgical procedures and because of their low cost.

[0003] Products based on flowable gelatin are local hemostatic agents composed of a matrix that forms a viscous gel capable of penetrating and coating hard-to-reach spaces. To improve the efficacy of these gelatin-based matrices and accelerate in situ coagulation, thrombin (the terminal serine protease of the coagulation cascade) is typically added to the paste.

[0004] Although human plasma-derived thrombin is generally considered safe and approved by the U.S. Food and Drug Administration (FDA) and other regulatory agencies, it is purified from human plasma, thus posing a theoretical risk of disease transmission. Furthermore, the cost of thrombin (even as low as 2000 IU) significantly increases the cost of gelatin hemostatic agents. Finally, thrombin (an active enzyme) is typically supplied in lyophilized powder form, which must be reconstituted before use. If thrombin could be omitted from the formulation, the stability and ease of preparation of the gelatin paste would be improved.

[0005] Ellagic acid (EA) is a natural phenolic antioxidant found in many fruits and vegetables and is available as a dietary supplement. EA can complex with a variety of metal ions and has been used for decades in in vitro coagulation assays (Bock et al., Biochemistry 1981, 20, 25, 7258–7266) to provide a rapid activator of the endogenous pathway, sometimes referred to as the “contact activation pathway.” The primary use of nickel tannate complexes is for activating the aPTT (prothrombin time). For example, the use of EA complexed with metal ions to improve existing assays, such as aPTT, which utilize less sensitive and consistent activators of the endogenous pathway, has been previously disclosed in, for example, EP0525035B1.

[0006] US5709889A discloses novel assays and reagents for determining the coagulation properties of blood or plasma, as well as reagents and methods for hemostasis. The new ellagic acid-based activator consists essentially of an aqueous solution of ellagic acid, phenol, and a suitable metal ion, and provides new limiting values ​​for platelet assays. The coagulation reagent of the invention, comprising propyl gallate or tannin, is also disclosed, along with an aPTT reagent exhibiting significantly superior sensitivity to heparin and factor deficiency compared to previous aPTT reagents.

[0007] US5041558A relates to promoters of hydrolytic enzyme activity, particularly promoters that promote the activation of serine protease precursors (including coagulation factor XII), and promoters that promote the enzyme activity of serine proteases generated from the activation of said precursors. Furthermore, the present invention relates to coagulation promoters using said promoters.

[0008] EP3258974A1 discloses a hemostatic composition comprising water retention, adhesive dust suppression, inorganic and organic hemostatic agents, and a hemostatic device comprising a hemostatic agent composition and a container holding the hemostatic composition.

[0009] EP1011727A1 discloses that the hemostatic product of the present invention is active in all patients, including those treated with heparin. It consists of a viscous, biocompatible, biodegradable composition and / or capable of being biologically eliminated, but not a collagen composition, containing hemostatic extracts of snake venom, such as batroxobin or ancrolein. The viscous composition is specifically formed from hyaluronic acid, optionally esterified.

[0010] US7371722B2 relates to a pharmaceutically active substance for producing a thrombin-producing or thrombin-containing drug and compositions comprising the same. The pharmaceutically active substance comprises (A) prothrombin (coagulation factor II) obtained from plasma or through genetic engineering, (B) coagulation factors V, VIII, IX, X (which may be present at least partially in their activated states) obtained from plasma or through genetic engineering, and coagulation factor XIa obtained from plasma or through genetic engineering, and (C) a prion-safe, procoagulant phospholipid, wherein the phospholipid is optionally contained in liposomes.

[0011] WO2009098029A2 relates to wound pads or wound dressings that can promote or accelerate blood clotting.

[0012] CN107281472A relates to the field of medical materials, and more specifically, to a medical nano-silver antibacterial thermosensitive hydrogel and its preparation method.

[0013] Marcińczyk et al. (Front Pharmacol. 2022 Jan 13; 12:806891) discussed tannins as hemostatic modifiers. Summary of the Invention

[0014] This invention relates in particular to ellagic acid (EA)-nickel complexes and their use in activating coagulation. One object of this invention is to provide compositions capable of rapidly activating the intrinsic coagulation pathway.

[0015] This invention utilizes a combination of gelatin paste and a bioactive polyphenolic compound (e.g., EA complexed with nickel ions (EA-Ni)) to produce a flowable paste capable of rapidly activating the intrinsic coagulation pathway. In vitro assays using whole blood were used to optimize the coagulation properties of the EA-Ni complex alone and the combination of the EA-Ni complex and gelatin, and to demonstrate the mechanism of action. In vivo studies were conducted to compare the performance of this novel EA-Ni gelatin paste with standard gelatin pastes with and without thrombin. In vivo studies showed that comparable hemostatic performance could be achieved using the EA-Ni gelatin paste compared to gelatin pastes with thrombin. The flowability and consistency of the EA-Ni gelatin paste were similar to those of EA-free gelatin formulations. A wide range of EA-Ni activity was exhibited. Furthermore, the amount of nickel required for rapid coagulation was so low that, from a toxicity perspective, it was below the level of concern. SURGIFLO with an ellagic acid-nickel complex... ® / SURGIFOAM ™ The mixture can produce rapid in vitro clotting time and rapid / effective hemostasis in an in vivo spleen biopsy model. It has also been shown that some optimized compositions with specific buffer solutions may exist.

[0016] According to one aspect of the invention, a hemostatic composition is provided comprising a bioactive polyphenolic compound, such as ellagic acid (EA), which is complexed with metal ions and dispersed or embedded within an absorbable polymer matrix.

[0017] In some embodiments, the polymer matrix comprises collagen or a derivative thereof. In some embodiments, the collagen derivative comprises gelatin. In some embodiments, the gelatin is in a lyophilized or dried form.

[0018] In some embodiments, the metal is selected from the group consisting of nickel, cobalt, and copper. In some embodiments, the metal is nickel.

[0019] In some embodiments, the metal ions and EA are present in a molar ratio of metal to EA ranging from 100:1 to 1:100, or optionally in a molar ratio of metal ions to EA ranging from 10:1 to 1:10.

[0020] In some embodiments, the composition or absorbable polymer matrix (e.g., gelatin) is selected from the following forms: a flowable paste at room temperature, a sponge matrix, a powder, and combinations thereof.

[0021] In some embodiments, the composition or absorbable polymer matrix is ​​in the form of a paste.

[0022] In some embodiments, the paste is characterized by a viscosity in the range of 1000 Pa·s to 5000 Pa·s.

[0023] In some implementations, the matrix comprises a combination of gelatin in paste and powder form.

[0024] In some embodiments, the composition is substantially free of one or more coagulation factors selected from factors II, V, VII, VIII, IX, X, XI, XII and fibrinogen in inactive or active forms. In some embodiments, the composition is substantially free of thrombin.

[0025] In some embodiments, the metal ions are present at a preferred concentration of the matrix (e.g., gelatin) to the metal ions in the range of 11,200:1 to 7:1 by weight / weight, optionally 1120:1 to 70:1.

[0026] In some embodiments, the EA complex is present at a concentration of EA-Ni in the range of 56,000:1 to 35:1 weight / weight or 5600:1 to 350:1 weight / weight, optionally at a matrix (e.g., gelatin) to 5600:1 to 350:1 weight / weight ratio.

[0027] According to another aspect of the invention, a method is provided for treating a subject with diseased or damaged tissue that can benefit from a rapid activation of blood clotting pathways, the method comprising administering to the subject a therapeutically effective amount of ellagic acid (EA) complexed with metal ions, said complex being incorporated into a matrix comprising collagen or a derivative thereof.

[0028] In some implementations, application includes bringing the damaged tissue into contact with the matrix.

[0029] In some implementations of this invention, the metal is nickel.

[0030] In some implementations, blood clotting occurs within approximately 400 seconds after contact.

[0031] In some embodiments of this aspect, the collagen derivative includes gelatin selected from the following forms: flowable paste at room temperature, sponge matrix, compressed tablets, powder, and any combination thereof.

[0032] In some embodiments of this aspect, the matrix comprises a combination of gelatin in paste form and gelatin in powder form.

[0033] According to another aspect of the invention, a method for preparing a hemostatic composition comprising ellagic acid (EA) complexed with metal ions added to an absorbable polymer matrix is ​​provided. The method comprises the step of adding the complex to the matrix at a ratio of EA-Ni complex in the range of 56,000:1 to 35:1 or 5600:1 to 350:1 by weight, respectively.

[0034] In some embodiments of this aspect, the polymer matrix comprises collagen or a derivative thereof.

[0035] In some implementations of this invention, collagen derivatives include gelatin.

[0036] In some implementations of this invention, the metal is nickel.

[0037] In some embodiments of this aspect, the matrix, such as gelatin, is selected from the following forms: a flowable paste at room temperature, a sponge matrix, compressed tablets, powders, and combinations thereof.

[0038] In some embodiments of this aspect, hemostatic compositions obtainable by this method are provided.

[0039] According to another aspect of this disclosure, a kit is provided that includes:

[0040] A container holding ellagic acid (EA) complexed with metal ions; a polymer matrix; an applicator for applying the composition to tissues; and optionally, instructions for use.

[0041] In some embodiments of this aspect, the polymer matrix comprises collagen or a derivative thereof.

[0042] In some implementations of this invention, collagen derivatives include gelatin.

[0043] In some implementations of this invention, the metal is nickel.

[0044] In some embodiments of this aspect, the matrix, such as gelatin, is selected from the following forms: a flowable paste at room temperature, a sponge matrix, a powder, and combinations thereof.

[0045] In some implementations of this aspect, the EA complexed with metal ions is provided in a buffer solution.

[0046] In some embodiments of this aspect, the buffer solution comprises a solution containing tetramethylammonium hydroxide (TMAH).

[0047] 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

[0048] To better understand the subject matter disclosed herein and to illustrate how it can be implemented in practice, various embodiments are now described by way of non-limiting example only, with reference to the accompanying drawings, wherein:

[0049] Figure 1 The photographic images show recalcified blood after several minutes, using 12 mg of diatomaceous earth particles (clotting blood in a tube; upper part of the image) and bioactive glass (45 mg).

[0050] Figure 2 A graph showing the comparative activated clotting time (ACT) of diatomaceous earth (12 mg) and bioactive glass (45 mg) is presented.

[0051] Figure 3 A graph showing the ACT time process using diatomaceous earth is presented, which shows the time process of testing diatomaceous earth (12 mg) in an ACT analyzer over a period of 6 hours over a day.

[0052] Figure 4 A graph showing ACT data using different diatomaceous earth concentrations is presented.

[0053] Figure 5 A bar chart showing ACT data using various types of polyphosphates and diatomaceous earth is presented.

[0054] Figure 6 The results are presented relative to the ellagic acid (EA)-Ni complex and without the activator. Figure 2 The bar chart shown here compares the ACT data.

[0055] Figures 7A to 7B The figure presents a dose-response study of blood clotting time relative to the EA-Ni complex: the average of triplicate results at each test concentration ( Figure 7A ), and the concentration range of complexes in which clotting time did not show a significant trend ( Figure 7B ).

[0056] Figure 8A bar graph showing the effect of adding phospholipids (phosphatidylcholine) to the EA-Ni complex on the coagulation rate is presented.

[0057] Figure 9 A bar graph showing the effect of adding a corn trypsin inhibitor (CTI) to the EA-Ni complex on coagulation rate is presented. CTI is an inhibitor of factor XIIa.

[0058] Figure 10 A bar graph is presented showing the comparative results of the hemostatic efficacy of various formulations in an in vivo porcine spleen hemorrhage model.

[0059] Figure 11 The presentation shows the addition of SURGIFLO ® Bar graph showing the effect of various buffer solutions on coagulation rate in preparations containing gelatin and EA-Ni complex.

[0060] Figures 12A to 12B Presented the results of soaking in the EA-Ni complex before (left) and after (right) for uncompressed tablets. Figure 12A ) and compressed tablets ( Figure 12B ( ), a photographic image captured of a tablet made of gelatin sponge.

[0061] Figure 13 A bar graph showing the comparison results of the average clotting times of various gelatin tablets as described in Example 3 is presented. Detailed Implementation

[0062] This invention utilizes a combination of gelatin paste and nickel-ion-complexed EA (EA-Ni) to produce a flowable paste capable of rapidly activating the intrinsic coagulation pathway. In vitro assays using whole blood were used to optimize the coagulation properties of the EA-Ni complex alone and the combination of the EA-Ni complex and gelatin, and to demonstrate the mechanism of action. In vivo studies were conducted to compare the performance of this novel EA-Ni gelatin paste with standard gelatin pastes containing and without thrombin. In vivo studies showed that the EA-Ni gelatin paste achieves comparable hemostatic performance to gelatin pastes containing thrombin. The flowability and consistency of the EA-Ni gelatin paste are similar to those of EA-free gelatin formulations.

[0063] It exhibits a wide range of EA-Ni activities. Furthermore, the amount of nickel required for rapid clotting is so low that, from a toxicity perspective, it is below the level of concern. SURGIFLO, with its ellagic acid-nickel complex... ® / SURGIFOAM ™ The mixture can produce rapid in vitro clotting time and rapid / effective hemostasis in an in vivo spleen biopsy model. It has also been shown that some optimized compositions with specific buffer solutions may exist.

[0064] One object of the present invention is to provide a composition that can be readily applied to bleeding sites, particularly in hard-to-reach areas of the body. Another advantage of the compositions of the present invention is that they are bioabsorbable and therefore can remain after surgery without causing any side effects.

[0065] Before detailing at least one embodiment of the present invention, it should be understood that the invention is not necessarily limited in its application to the details set forth in the following description or examples. The invention can have other embodiments, or can be practiced or implemented in various ways.

[0066] Composition

[0067] According to one aspect of some embodiments of the present invention, a hemostatic composition is provided comprising ellagic acid (EA) complexed with metal ions. In some embodiments, the complex is dispersed, incorporated, or embedded within an absorbable polymer matrix for, for example, uniform distribution of the complex therein.

[0068] As used herein, the term "composition" includes a substance or formulation formed from a combination or mixture of various ingredients. In some embodiments, the composition comprises a homogeneous suspension, formulation, or gel. As used herein, "homogeneous" means a composition, texture, or suspension that is uniform in overall consistency, for example, having a density and / or viscosity that varies within a range of up to ±25%. In some embodiments, the composition is homogeneous.

[0069] The term "complex" refers to a compound comprising one or more metal cations and one or more anion- or neutral ligands (e.g., EA), wherein at least one of the ligands is bonded to the metal, for example, using a donor electron.

[0070] As used herein, the term "ellagic acid" (EA) or "ellagic acid compound" is a general term encompassing ellagic acid, ellagic acid salts, and ellagic acid metal chelates. Ellagic acid can be used as an activator of contact factors involved in the intrinsic coagulation pathway.

[0071] Ellagic acid is known to be an activator of contact factors involved in the intrinsic coagulation pathway. Examples of contact factors include, but are not specifically limited to, prokallikrein, high molecular weight kininogen, factors XII and XI, etc. Ellagic acid can be natural or synthetic. Examples of ellagic acid salts include, but are not specifically limited to, metal salts of ellagic acid, etc. Ellagic acid forms metal chelates with metal ions through coordination bonds to strongly activate contact factors involved in the intrinsic coagulation pathway. Therefore, in the reagents provided herein, the ellagic acid compound is preferably a metal chelate of ellagic acid. In an exemplary embodiment, the metal ion is nickel.

[0072] In some implementations, the intrinsic coagulation pathway refers to factor XII (“FXII”) activation (see [link to implementation]). Figure 9 ).

[0073] As used herein, the term "polymer matrix" refers to a molecule composed of several repeating and linked chemical units, which can be used as sites for one or more reagents to link, embed, or embed therein. Generally, a polymer matrix refers to a biocompatible and / or biodegradable polymer.

[0074] As used throughout this document, the term "polymer" describes an organic substance composed of multiple repeating structural units (main chain units) covalently linked to each other. The term also includes copolymers. As used herein, the term "copolymer" refers to a polymer of at least two chemically distinct monomer units.

[0075] As used herein, the term “absorbable polymer matrix” is defined as a natural or synthetic, biocompatible polymer (including copolymers) that is absorbable and degradable and absorbed by, for example, the body when implanted in humans and animals.

[0076] More specifically, as used herein, the term “bioabsorbable” refers to the ability of a tissue-compatible material to degrade in the body after implantation, and the material mentioned is at least partially biocompatible with materials that are excreted, metabolized, or assimilated by the body after implantation (see, for example, Barrows, “Synthetic Bioabsorbable Polymers,” p. 243, High Performance Biomaterials—A comprehensive Guide to Medical and Pharmaceutical Applications, edited by Michael Szycher, Technomic Publishing, Lancaster, PA, 1991).

[0077] Absorbable polymers readily break down into small fragments when exposed to moist body tissues. These fragments are then absorbed or excreted by the body. More specifically, because the biodegradable fragments are absorbed or pass through the body, the body does not retain permanent traces or residual fragments, thus they do not cause permanent chronic foreign body reactions.

[0078] The term “embedded” refers to terms such as “dispersion,” “in combination with,” or “encapsulated,” “enclosed in,” “coated,” “contained,” and otherwise “provided remotely or physically connected,” or can be considered as part of a polymer structure or polymer particles made therefrom.

[0079] In some embodiments, the polymer matrix comprises collagen or a derivative thereof.

[0080] In some embodiments, the polymer matrix comprises gelatin in particulate form. As used herein, the term "gelatin" also includes gelatin derivatives, such as gelatin derived from aromatic sulfonyl chlorides, carboxyl chlorides, carboxylic anhydrides, aryl isocyanates, etc. The term "gelatin" also includes collagen and its derivatives.

[0081] As used herein, the term “derivative” refers to a substance that contains the same basic carbon skeleton and functional groups as the parent compound, but may also contain one or more substituents of the parent compound.

[0082] In some embodiments, the matrix, such as gelatin, is selected from the following forms: a flowable paste at room temperature, a sponge-like structure (e.g., a matrix or scaffold), a powder, a compressed tablet, and combinations thereof.

[0083] In some embodiments, the matrix comprises both gelatin powder and gelatin gel. In an exemplary embodiment of the invention, a hemostatic composition is provided comprising ellagic acid (EA) complexed with nickel ions, wherein the complex is dispersed, incorporated, or embedded in gelatin powder distributed within a gelatin gel.

[0084] In an additional exemplary embodiment of the invention, a hemostatic composition is provided comprising ellagic acid (EA) complexed with nickel ions, wherein the complex is dispersed, incorporated, or embedded within a gelatin gel, and a suitable buffer, optionally without gelatin powder. Non-limiting exemplary buffers are selected from CHES and bicarbonate buffers (see [link to relevant documentation]). Figure 11 ).

[0085] As used herein, the term "sponge-like structure" refers to a flexible three-dimensional structure, such as a porous, elastic, fibrous, and / or resilient reversible compressible foam.

[0086] The term "powder" refers to a substance containing finely ground, pulverized, or otherwise dispersed solid particles. The term "solid" characterizes 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. Unlike "liquid" compounds and compositions, solids do not flow under their own weight.

[0087] As used herein, the term "compressed tablet" refers to a tablet prepared by a compression step, which results in a size-stable tablet.

[0088] In an additional exemplary embodiment of the invention, a hemostatic composition is provided comprising ellagic acid (EA) complexed with nickel ions, wherein the complex is incorporated into or embedded within a compressed gelatin tablet. See also Figure 13 It shows that tablets soaked in EA-Ni and then freeze-dried and compressed have shorter clotting times than similar uncompressed tablets.

[0089] In some embodiments, the composition is in the form of a paste. As used herein, the term "paste" refers to the consistency of the composition at at least one temperature of about room temperature and may refer to a fluid mixture of solid particles. The term "paste" according to this disclosure may also include a slurry. A slurry may be functionally considered as a thin, aqueous paste. Pastes according to this disclosure may also include pores formed by expandable gases such as air. Thus, the composition is a paste, or has a paste (or greasy) consistency at about room temperature.

[0090] The consistency of the composition can also be classified into more fluid pastes (referred to herein as "flowable") and more doughy pastes (referred to herein as "non-flowable" or "non-flowing").

[0091] Therefore, in some embodiments, the composition is flowable at at least one temperature near room temperature, such as at one or more of the following temperature values: 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, and 40°C. This composition is referred to below as a "flowable" composition.

[0092] Therefore, in some embodiments, the composition is non-flowable at at least one temperature near room temperature, such as at one or more of the following temperature values: 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, and 40°C. This composition is hereinafter referred to as a "non-flowable" or "non-flowing" composition.

[0093] The term "non-flowable" (or "non-freezing") paste is also referred to herein as "putty." As used herein, in the context of pastes, the term "flowable" refers to a greater fluid consistency at approximately room temperature, which remains flowable after the composition has been applied to / on the bleeding site. The term "non-flowable" or "putty" refers to a more doughy consistency at 37°C, which requires a longer time to settle and has better shape retention compared to "flowable" pastes.

[0094] While the consistency of a composition as a "paste" has been defined above, alternatively or otherwise, the composition may also be defined according to its resistance under certain conditions, as detailed below. Generally, viscosity is a measure of the resistance of a liquid to flow.

[0095] In some embodiments, the composition has a viscosity of about 1000 cP to 5000 cP at one or more of the following temperature values: 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, and 40°C. In some embodiments, the composition has a viscosity of about 2500 cP to 3500 cP at one or more of the following temperature values: 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, and 40°C. In some embodiments, the composition has a viscosity of about 1000 cP, about 1500 cP, about 2000 cP, about 2500 cP, about 3000 cP, about 3500 cP, about 4000 cP, about 4500 cP, or about 5000 cP at one or more of the following temperature values: 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, and 40°C, including any values ​​and ranges therebetween.

[0096] In some embodiments, the composition is sterile. As used herein, the term "sterile" means having a low bioload and being effectively sterile, such as being free of microorganisms (e.g., bacteria and viruses). Sterilization is the process of reducing the bioload to an effectively sterile level. Sterile liquids are generally defined as liquids that have undergone sterile filtration. Therefore, in some embodiments, the sterile composition has been sterilized by sterile filtration, for example, through a filter of 0.22 μm or smaller.

[0097] In some embodiments, the metal ion is a divalent ion. In some embodiments, the metal ion is selected from zinc, iron, and sodium.

[0098] In some embodiments, the metal ion is a divalent ion. In some embodiments, the metal ion is selected from nickel, cobalt, and copper. In some embodiments, the metal ion is nickel.

[0099] In some embodiments, metal (e.g., nickel) ions and EA are present in a molar ratio ranging from 1:100 to 100:1, or in some embodiments, a metal (e.g., nickel) ion to EA ratio of 1:10 to 10:1. In some embodiments, metal (e.g., nickel) ions and EA are present in a metal to EA molar ratio ranging from about 1:5 to 5:1. In some embodiments, metal (e.g., nickel) ions and EA are present in a metal to EA molar ratio of about 1:4 to 4:1. In some embodiments, metal (e.g., nickel) ions and EA are present in a metal to EA molar ratio of 4:1, 5:1, 6:1, 7:1, or 8:1, including any values ​​and ranges therein. Typically, but not exclusively, metal (e.g., nickel) ions are in excess, and the complex remains in suspension rather than forming a single large network of EA and metal (e.g., nickel) ions. In an exemplary embodiment, metal (e.g., nickel) ions and EA are present in a molar ratio of about 1:1 (e.g., 0.89:1).

[0100] In some embodiments, the metal ions are present at a gelatin-to-metal ratio concentration (e.g., nickel) ranging from 11,200:1 to 7:1 weight / weight. In some embodiments, the metal ions are present at a gelatin-to-metal ratio concentration (e.g., nickel) ranging from 1120:1 to 70:1 weight / weight. In some embodiments, the metal ions are present at a gelatin-to-metal ratio concentration (e.g., nickel) ranging from 1120:1 to 7:1 weight / weight. In some embodiments, the metal ions are present at a gelatin-to-metal ratio concentration (e.g., nickel) ranging from 11,200:1 to 70:1 weight / weight.

[0101] In an exemplary implementation, based on dose-response studies, EA-Ni can be diluted 1:40 and still accelerate clotting (i.e., less than 200 seconds) (see [link to relevant documentation]). Figures 7A to 7B The preferred range for calculating the gelatin to EA-Ni ratio is 56,000:1 to 35:1. Furthermore, based on dose-response studies, EA-Ni can be diluted 1:4 to rapidly accelerate clotting (i.e., approximately 100 seconds). Therefore, the preferred range for calculating the gelatin to EA-Ni ratio is 5600:1 to 350:1.

[0102] Therefore, in some embodiments, the EA complex is present at a gelatin-to-metal ion (e.g., nickel) concentration in the range of 56,000:1 to 35:1 weight / weight. In some embodiments, the EA complex is present at a gelatin-to-metal ion (e.g., nickel) concentration in the range of 5600:1 to 35:1 weight / weight. In some embodiments, the EA complex is present at a gelatin-to-metal ion (e.g., nickel) concentration in the range of 5600:1 to 350:1 weight / weight. In some embodiments, the EA complex is present at a gelatin-to-metal ion (e.g., nickel) concentration in the range of 56,000:1 to 350:1 weight / weight. In an exemplary embodiment, the weight ratio of gelatin to EA-Ni is from about 1350:1 to about 1450:1, for example 1409:1, weight / weight.

[0103] In some embodiments, the gelatin is in lyophilized form. As used herein, the term "lyophilization" refers to the preparation of a dried composition formed by rapid freezing and thawing (sometimes referred to as "sublimation") in a frozen state. This process can be carried out under vacuum at reduced air pressure, due to drying at temperatures below, for example, atmospheric pressure.

[0104] In some implementations, the matrix comprises a combination of gelatin in paste and powder form.

[0105] In some embodiments, the composition is substantially free of enzymes (thrombins) that can activate mammalian clotting factors.

[0106] The terms "coagulation factors" or "thrombins" refer to plasma proteins, such as enzymes that interact with platelets in a complex cascade of enzyme-catalyzed reactions, leading to fibrin formation, which is used to initiate blood clots during the blood clotting process. The term refers to components in the intrinsic, extrinsic, and common coagulation pathways. Coagulation factors are typically serine proteases when activated, but also include glycoproteins (factors VIII and V) or other types of enzymes, such as transglutaminase (factor XIII).

[0107] As used herein, the term "plasma protein" refers to any protein, especially any industrially or therapeutically important protein contained in plasma.

[0108] The prothrombins, proteins, and peptides may be naturally occurring, recombinant, or synthetic, and may also be selected from: prothrombin, fibrin, fibronectin, heparinase, factor X / Xa, factor VII / VIIa, factor IX / IXa, factor XI / XIa, factor XII / XIIa, tissue factor, batroxobin, ancrolein, venom, von Willebrand factor, platelet surface glycoproteins, vasopressin and vasopressin analogs, adrenaline, selectin, procoagulant venom, plasminogen activator inhibitors, platelet activators, synthetic peptides with hemostatic activity, derivatives of the above, and any combination thereof.

[0109] In some embodiments, the composition is substantially free of one or more coagulation factors in inactive or active forms selected from factors II, V, VII, VIII, IX, X, XI, XII and fibrinogen.

[0110] In some embodiments, the composition is substantially free of thrombin.

[0111] 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.

[0112] Human thrombin is a 295-amino acid protein composed of two polypeptide chains linked by disulfide bonds. Both human and non-human thrombin (e.g., bovine, porcine, and salmon thrombin) can be used within the scope of this disclosure.

[0113] As used herein, the term “substantially absent” with respect to a component of a composition means a component that is present in or absent in the composition at a concentration of less than 0.1 wt% of the total composition.

[0114] In some embodiments, the composition is free of thrombin. In some embodiments, the composition is free of one or more coagulation factors selected from factors II, V, VII, VIII, IX, X, XI, XII and fibrinogen in inactive or active forms.

[0115] In some embodiments, the composition is platelet-free. In some embodiments, the composition is free of added phospholipids (see [link to original text]). Figure 8 This showed that adding phospholipids to fresh whole blood did not improve clotting time.

[0116] 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.

[0117] In some implementations, the pharmaceutical active agent is selected from therapeutic active agents and labeling agents.

[0118] In some embodiments, the composition further comprises an additional pharmaceutically active agent contained within or on the surface of the composition. In some embodiments, the pharmaceutically active agent is selected from therapeutic agents and labeling agents.

[0119] In some embodiments, the therapeutic active agent is selected from: stem cells, growth factors, bone morphogenetic proteins, cells, cytokines, hormones, drugs, minerals, plasmids with therapeutic potential, and combinations thereof. In some embodiments, the therapeutic active agent may be an antifibrinolytic agent, such as aprotinin, tranexamic acid (Cyklokapron), ε-aminocaproic acid (Amicarn), nafamostat mesylate, lysine, lysine derivatives, and combinations thereof.

[0120] In some embodiments, the composition is stable. When referring to the disclosed compositions, the terms "stable" and "reliable" mean, for example, that the active component in thrombin retains at least 70% of its activity after a specific temperature and for a specific duration, i.e., the ability to form fibrin clots. In some embodiments of any aspect of the compositions disclosed herein, the composition may comprise one or more additives as described below, such as calcium salts and / or one or more excipients selected, for example, from, but not limited to, one or more amino acids, albumin, sugars, and / or sugar derivatives.

[0121] The term “additive” is intended to be understood as any substance that can be added to a composition or is derived from a source material, and may also include active additives such as calcium salts as described below. As used herein, the term “excipient” means an inactive or non-therapeutic agent added to a pharmaceutical composition to, for example, provide a desired consistency or stabilizing effect.

[0122] Calcium is an important element in the coagulation cascade and may be required for the activation of factor XIII to factor XIIIa, which crosslinks and stabilizes fibrin to form insoluble clots. Calcium is also important for the production of various enzymes in the coagulation cascade, including factors IXa, Xa, and IIa, thus making it a key component of the intrinsic coagulation pathway.

[0123] Therefore, in some embodiments of any aspect of the disclosed compositions, the compositions also contain additives, such as, but not limited to, calcium. Calcium used with the present invention may be in the form of a salt (e.g., calcium chloride). Alternatively, other salts, such as calcium acetate and / or calcium citrate, may be used.

[0124] Treatment

[0125] In another aspect of the invention, a method is provided for treating a subject with diseased or damaged tissue having an endogenous pathway that can benefit from rapid activation of blood clotting, the method comprising administering to the subject a therapeutically effective amount of ellagic acid (EA) complexed with a metal ion, the complex being incorporated into a buffer or matrix comprising, for example, collagen or a derivative thereof.

[0126] Not bound by any specific mechanism, ellagic acid needs to complex with metal ions to form aggregates in order to activate, for example, factor XII. This provides a surface for protein binding and activation.

[0127] The term "treatment of disease or damaged tissue" refers to reducing blood loss at the site of bleeding in tissue (within the body), such as in a patient undergoing surgery. The term "treatment" includes eliminating, substantially inhibiting, slowing, or reversing the progression of a condition, substantially improving the clinical or aesthetic symptoms of a condition, or substantially preventing the occurrence of the clinical or aesthetic symptoms of a condition.

[0128] 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 to the aqueous solution to promote rapid clotting time. Therefore, in some such embodiments, the composition is used as a hemostatic agent at the bleeding site in any embodiment of the tissue.

[0129] 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. In one embodiment, the subject for whom the subject is needed is a patient.

[0130] The term "application" means providing a subject with a specific strain of the invention or a specific composition containing that strain as an active ingredient by any suitable method. In some embodiments, the complex is applied to the subject via a topical application route selected from topical, subcutaneous, or intradermal application.

[0131] In some implementations, application is directed to the site of injury or bleeding in an organ. The term “application to (the subject’s) site” means application to: (a) a location on a body surface corresponding to or near the “site”; and / or (b) a location on a body surface that provides an accessible path to the “site”.

[0132] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined using standard pharmaceutical procedures in vitro, in cell cultures, or in laboratory animals. Data obtained from these in vitro cell culture assays and animal studies can be used to formulate dosage ranges for human use. This dosage may vary depending on the dosage form used and the route of administration. The exact formulation, route of administration, and dosage can be selected by an individual physician based on the patient's condition (see, for example, Fingl E. et al., (1975), "The Pharmacological Basis of Therapeutics", Chapter 1, page 1).

[0133] Dosage and administration intervals can be individually adjusted to provide sufficient plasma levels of the active ingredient to induce or inhibit biological effects (i.e., coagulation activity). Coagulation activity will vary for each formulation but can be estimated from in vitro data. The dose required to achieve coagulation activity will depend on individual characteristics and route of administration. Bioassays can be used to determine plasma concentrations.

[0134] Examples of dosage forms for topical application include, but are not limited to, transdermal patches, creams, foams, gels, lotions, ointments, pastes, powders, shaken lotions, solids, sponges, tapes, tinctures, vapors, injections, drops, rinses, sprays, and solutions. A “unit dose” or “dose unit” may be configured to provide a full unit dose or a fraction thereof (e.g., or 1 / 4 of the dose).

[0135] In some implementations, as used herein, the term “dosage” means, for a site, the amount of drug or active ingredient taken by an individual subject per administration, specifically the total amount of drug or active ingredient taken by an individual subject per administration.

[0136] In some implementations, as used herein, the term "dosage form" refers to the unit of administration of the active agent. Examples of dosage forms include tablets, capsules, injections, suspensions, liquids, emulsions, creams, ointments, suppositories, inhalable forms, etc.

[0137] The predetermined amount in each unit dose may depend on factors including, but not limited to, the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the art of producing and administering such unit doses. For example, a unit dose may be a transdermal patch, a spray (i.e., a single spray in spray application), a droplet in drip application, a tape of a specific length, an ointment the size of a rice grain or a bean, or a spoonful or scoop of ointment.

[0138] A unit measuring device or kit, such as a cup, spoon, syringe, dropper, spittoon, or colonic flushing device, can contain dosage forms such as creams, foams, gels, lotions, ointments, pastes, powders, shaken lotions, and solids, wherein the amount of composition measured is equal to the total unit dose or a fraction thereof (e.g., 1 / 2, 1 / 3, or 1 / 4 of the dose). In a single dose administration, a single unit dose or multiple unit doses may be present. The kit may include instructions regarding the size of the unit dose or its fraction.

[0139] In some embodiments, the EA complexed with metal ions is in a pharmaceutically acceptable form. In some embodiments, the EA complexed with metal ions is in a dosage form.

[0140] In some implementations, the term "unit dose" or "dose unit" refers to a dosage form of a composition or component thereof configured to deliver a specified amount or dose.

[0141] Depending on the severity and responsiveness of the condition being treated, administration may be a single or multiple doses (e.g., weekly or bi-weekly), and the treatment may last from a few minutes to several hours, or until a cure (i.e., clotting) or a reduction in the disease state is achieved.

[0142] The term “pharmaceutically acceptable” is defined herein as those compounds, materials, compositions, and / or dosage forms that are suitable for contact with a patient’s tissues without excessive toxicity, irritation, allergic reactions, or other problems and complications commensurate with a reasonable benefit / risk ratio, within the bounds of reasonable medical judgment.

[0143] 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 chemical means. The term “injured tissue” also encompasses contused tissue, as well as tissue that has been cut, punctured, or torn, 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.

[0144] The term "tissue" can refer to tissues selected from soft tissue and bone tissue. 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 tissues include, but are not limited to, connective tissues (such as tendons, ligaments, fascia, skin, fibrous tissue, fat, and synovium), muscles, and internal organs. Generally, soft tissue is intended to exclude bone tissue.

[0145] Therefore, in some embodiments, the disclosed composition may be used or administered, for example, during minimally invasive surgery (MIS) (such as, for example, endoscopy) at hard-to-reach bleeding sites. One common form of endoscopy is laparoscopy, which is a minimally invasive examination and surgery inside the abdominal cavity.

[0146] 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. The sealant forms within a relatively short clotting period, after which the disclosed composition is applied to the damaged tissue in any aspect and / or embodiment.

[0147] As shown in the following embodiments, in some embodiments, blood clotting occurs in less than about 400 seconds after application or contact of the damaged tissue with the matrix. In some embodiments, blood clotting occurs in less than about 300 seconds upon application or contact of the damaged tissue with the matrix. In some embodiments, blood clotting occurs in less than about 250 seconds upon application or contact of the damaged tissue with the matrix. Typically, after tamponade for 1 to 3 minutes, a localized clot can form that stops bleeding, but a longer time may be required for the coagulation reaction to reach the paste surface and form a complete clot.

[0148] In another aspect of the invention, a method of treating a wound is provided, comprising the step of applying (e.g., contacting) the disclosed composition in any aspect and / or embodiment thereto to a wound and / or in a subject in need of it. In some embodiments, the treatment is performed in vivo or locally (i.e., at the site of bleeding). The term "applied to a site of bleeding in need" means, for example, applying the composition locally to the site of bleeding in tissue at or near the surgical site.

[0149] The compositions and treatment methods disclosed herein can be used for any therapeutic purpose. The term "any therapeutic purpose" means any curative or preventative treatment of a subject. Exemplary therapeutic purposes include, but are not limited to, sealing drill holes formed in tissues or organs (e.g., bone); anastomosis at blood vessels; joining tissue portions (e.g., soft tissue portions); treating or preventing dura mater defects, such as tearing and leakage, cracks, or fissures after injection; treating or preventing bleeding; treating or preventing air leakage, such as after lung resection; treating or preventing defects after intestinal perforation; treating or preventing defects after anastomosis in any tissue such as the uterus, esophagus, stomach, pancreas, pancreatic duct, gallbladder, bile duct, intestine (including small and large intestine), and rectum; and treating or preventing defects after anastomosis in any tissue such as the uterus, esophagus, stomach, pancreas, pancreatic duct, and gallbladder. Leakage in the bile duct, intestine (including the small and large intestine), and rectum; for example, preventing or reducing postoperative leakage at a suture or suture site by applying the composition according to the invention alone or in combination with an additional matrix (e.g., a patch or pad) to at least a portion of the defect (such as a suture staple / suture); for securely fixing prostheses, such as during hernia surgery; for suture staple / suture reinforcement; for preventing or reducing air leakage, such as alveolar air leakage; for treating or preventing kidney defects; for treating or preventing fistulas; for treating or preventing heart defects, such as penetrating heart wounds; for reinforcing vascular graft prostheses; and for treating or preventing cerebrospinal fluid leakage.

[0150] In some embodiments, the composition, complex, or matrix of any of the embodiments disclosed herein is used to provide hemostasis, leakage sealing, and / or connection structures. In some embodiments, a method is provided to provide hemostasis, leakage sealing, and / or connection structures in a subject in need of such a subject, the method comprising using a composition, complex, or matrix of any of the embodiments disclosed herein.

[0151] 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 help stop bleeding. Hemostasis (or "haemostasis") refers to the first stage of wound healing. It is the process of stopping bleeding. The term "auxiliary hemostasis" means helping to reduce or stop bleeding. The term "application to the bleeding site" means, for example, applying a composition topically to the bleeding site (e.g., at a surgical site) to control bleeding.

[0152] Bleeding control is required in a variety of situations, including wound management. As used herein, the terms “control,” “prevent,” or “reduce” (which may be used interchangeably in the context of bleeding herein, including any grammatical variations thereof) indicate that the rate of blood exudation is substantially eliminated or reduced, for example, by at least 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 has not come into contact with the bleeding site. Methods for determining the level of bleeding occurrence are known in the art.

[0153] In addition, in some implementations, the terms “control,” “prevent,” or “reduce” in the context of bleeding also mean covering at least partial closure of the blood vessel.

[0154] 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, and severe postnatal bleeding, including bleeding associated with cesarean section. The term “blood” or any grammatical variations thereof also includes blood fractions such as plasma or red blood cells.

[0155] As used herein, a wound is any damage to any tissue of a patient that results in the loss of fluids from the circulatory system and / or from the patient's body. The injury can be caused by any agent or source, including traumatic injury, infection, or surgical intervention. A wound can be located in soft tissue such as an organ, or in hard tissue such as bone. The tissue can be internal, such as an organ or blood vessel, or external, such as skin. Blood loss can be internal, such as from a ruptured organ, or external, such as from a laceration.

[0156] In some implementations, the matrix comprises a combination of gelatin in paste and powder form.

[0157] This document also incorporates other embodiments described above under “Compositions”, such as embodiments of polymer matrices, tablets, powders, and gelatin.

[0158] Method for preparing hemostatic compositions

[0159] According to another aspect of the present invention, a method for preparing a hemostatic composition comprising ellagic acid (EA) complexed with metal ions added to an absorbable polymer matrix is ​​provided, the method comprising the step of adding the complex to the matrix.

[0160] The term "addition" refers to contact with a reactant, reagent, solvent, or buffer solution in which the reagent or reactant (e.g., EA) is present. Reactants, reagents, solvents, catalysts, etc., can be added individually, simultaneously, or separately, and can be added in any order.

[0161] In an exemplary embodiment, an EA-metal ion (e.g., Ni) complex is first combined with gelatin powder, and then combined with a gelatin paste.

[0162] As described in Example 2, it was first compared with SURGIFOAM at the same concentration of EA-Ni complex. ™ Conversely, combining the EA-Ni complex with SURGIFLO alone... ® The paste (34µg EA-Ni / ml paste) combination resulted in a fourfold decrease in coagulation potency (approximately 400 seconds in a 4ml whole blood gelation test).

[0163] In some embodiments, the matrix may be added to the complex in any ratio described above under “Composition”, for example, the polymer matrix complex to the EA-metal ion (e.g., nickel) complex in the range of 56,000:1 to 35:1 (weight / weight) by weight, or for example, the matrix to the EA-metal ion (e.g., nickel) complex in the range of 5,600:1 to 350:1 by weight.

[0164] This article incorporates other embodiments described above under “Compositions”, such as other embodiments of polymer matrices, tablets, powders and gelatin.

[0165] In some embodiments, the EA-metal ion (e.g., nickel) complex is suspended together with a matrix (e.g., gelatin) powder and then combined with a paste matrix such as gelatin (see Example 2 below).

[0166] In some embodiments, a hemostatic composition is provided that is obtainable through any embodiment of the method for preparing the hemostatic composition disclosed herein. As used herein, the term "available" also includes the term "obtained". In one embodiment, the term "available" means "obtained".

[0167] Kits and additional aspects and implementation schemes

[0168] In some aspects, in any of its embodiments, the disclosed compositions are provided as kits. According to one aspect of the invention, a kit is provided comprising: a container holding ellagic acid (EA) complexed with a metal (e.g., nickel) ion; a polymer matrix; and an applicator for applying the composition to tissue. According to another aspect of this disclosure, a kit is provided comprising: a container holding EA; a container holding a metal (e.g., nickel) ion; a polymer matrix; and an applicator for applying the composition to tissue.

[0169] In some implementations of this aspect, the EA complexed with metal ions is provided in a buffer solution.

[0170] In some embodiments of this aspect, the buffer solution comprises a solution containing tetramethylammonium hydroxide (TMAH).

[0171] If desired, the compositions and kit contents described herein may be contained in a packaging or dispenser device that contains one or more unit dosage forms containing the composition or fractions and / or reagents (e.g., buffer solutions or aqueous solutions) for preparing the composition. 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 a suitable container, 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 something physiologically suitable for therapeutic use.

[0172] In some respects, methods are provided for activating endogenous pathways of blood clotting, comprising administering to a subject a therapeutically effective amount of ellagic acid (EA) complexed with a metal (e.g., nickel) ion, optionally incorporating the complex into a matrix containing collagen or a derivative thereof or in a suitable buffer solution as described herein.

[0173] 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.

[0174] In another aspect of this disclosure, the disclosed composition is used in any aspect or embodiment of a method for preparing a fibrin sealant in or near a subject’s damaged tissue by applying the disclosed composition in any aspect or embodiment of the present invention to the surface or vicinity of a tissue.

[0175] 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.

[0176] 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, joining structures, and / or sealing physiological leaks, such as leaks of cerebrospinal fluid (CSF), lymph, bile, gastrointestinal (GI) contents, air leaks from the lungs, etc.

[0177] Therefore, it should be understood that fibrin itself exists as an insoluble matrix containing polymers of fibrils, and each fibril contains many fibrin molecules.

[0178] 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.

[0179] According to another embodiment of the invention, a kit is provided for producing the composition described herein. It should be understood that the consistency of the composition allows it to be applied, for example, by spreading or by directly adhering the composition to damaged tissue and / or bleeding sites. 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 damaged tissue and / or bleeding sites. However, suitable applicators, such as syringes, can be used to apply, spread, or adhere the composition to bleeding sites for easy access and treatment.

[0180] In additional aspects and / or embodiments, the invention provides a kit comprising: a) a container holding the composition of the invention as described above; b) an applicator for applying the composition to tissue; and c) optionally, instructions for use. In another embodiment, the invention also provides a hemostatic kit comprising a container holding the composition disclosed herein in one embodiment thereof.

[0181] Additionally or alternatively, the hemostatic 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, such as dissolved EA, and powders, such as metal (e.g., nickel) salts, 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.

[0182] 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. In some embodiments, a syringe is provided in addition to the container of the kit. The term "container" can refer to any generic structure, such as a container or vial.

[0183] 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 with an injection needle that optionally retracts automatically from the patient's body organ 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.

[0184] The hemostatic kit of the present invention can be a kit for reducing, preventing, or stopping blood flow, such as in an open wound, and can be used during, before, or after surgical procedures such as laparoscopic surgery, neurosurgery, abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck surgery, pelvic surgery, and skin and subcutaneous tissue surgery. The kit can be used to reduce or prevent blood flow from the skin or internal organs.

[0185] 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.

[0186] Any aspect and embodiment of the compositions disclosed herein may be incorporated into aspects and embodiments of the methods and kits with necessary modifications.

[0187] General

[0188] The terms “comprising,” “including,” “having,” and variations thereof mean “including, but not limited to.” The term “consisting of” means “including and limited to.” The term “substantially consisting 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.

[0189] 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.

[0190] 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.

[0191] As used herein, unless the context clearly indicates otherwise, the singular forms “an,” “a,” and “the” include plural references. For example, the term “a compound” or “at least one compound” may include multiple compounds (including mixtures thereof). In those cases where conventions such as “at least one of A, B, and C” are used, such structures are generally intended to have the meaning of the convention that will be understood by those skilled in the art (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 will 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”.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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 features of the invention described in the context of a single embodiment or several embodiments 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.

[0197] As used herein, the terms “about” or “approximately” are intended to include values ​​that are 10% higher or lower than the indicated value. Unless otherwise specified, all figures such as those representing, for example, ratios, weights, moles per mole, quantities, viscosity, temperatures, etc., should 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 ​​that can vary by up to ±10% according to the desired properties sought to be obtained according to the invention.

[0198] The various embodiments and aspects of the invention described above and claimed in the claims section below are experimentally supported in the following examples.

[0199] Example

[0200] The following embodiments, together with the foregoing description, illustrate some implementations of the invention in a non-limiting manner.

[0201] Example 1 - Determination of clotting time using various activators

[0202] Exemplary procedures were performed to evaluate various hemostatic agents that function via the natural physiological pathway of coagulation. The aim was to assess the rate at which the activator coagulates fresh whole blood. Two test methods were developed to evaluate the activator: (i) the Actalyke Activated Coagulation Time (ACT) Analyzer manufactured by Helena Laboratories and (ii) a whole blood (WB) gelation test.

[0203] These clotting time assays assess the material's ability to activate endogenous pathways to form fibrin clots in whole blood following recalcification of citric blood. Fresh citric blood is used to preserve clotting factors. These tests are well-suited for particle activators such as diatomaceous earth, kaolin, and bioglass.

[0204] ACT Test: Once the analyzer is turned on and equilibrated to 37°C, a biological control plasma is evaluated using diatomaceous earth to confirm the analyzer's operation. The general procedure involves combining the activator with citric acid-treated bovine blood (2 mL) into an empty ACT tube (containing only the magnet) and mixing immediately. After mixing the blood and activator, 1.0 mL of calcium salt solution (nominal saline solution of 25 mM CaCl2, 0.9% sodium chloride solution) is added to the tube and inserted into the analyzer. Clotting time is detected based on the displacement of the magnet. It is important to note that because ACT is designed for small-particle activators (such as diatomaceous earth and kaolin), larger biological materials (such as gelatin particles) cannot be adequately mixed. This is why the Western blot gelation test is primarily used to test gelatin formulations.

[0205] Western Blot (WB) gelation assay: Compared to small-volume ACT tubes, the WB gelation assay uses a larger volume of blood to accommodate biological materials such as gelatin and collagen, allowing for the assessment of interactions between activators and biological materials. The Actalyke ACT analyzer is designed to test small-particle activators such as diatomaceous earth and kaolin (for clinical applications). However, when the amount of activator or biological material (e.g., gelatin) is too large, the analyzer introduces errors. To address this limitation, the WB gelation assay was developed, featuring a heated housing and a larger tube to accommodate a larger volume of blood and a greater amount of activator. The setup includes an acrylic housing with a heating unit for testing at 37°C. A temperature sensor within the housing maintains the temperature at 37°C ± 1°C. The housing contains an orbital rotator to gently mix the blood with other tested materials while keeping the blood warm. Calcium saline and a tube rocker are also located within the housing. The housing has a front hinge to allow access from the front of the unit.

[0206] The stability of Western blot (WB) was assessed using ACT. 12 mg of diatomaceous earth was added to an empty tube (containing only the magnet) and immediately mixed with citric acid-treated bovine blood (2 mL). After mixing the blood and activator, 1.0 mL of calcium salt solution was added to the tube and the tube was inserted into the analyzer. The rate of clot formation was determined based on the displacement of the magnet. The goal of this test was to demonstrate that WB was stable throughout the day, thus showing that the blood did not change, for example, due to degradation of clotting factors, and that measurements taken throughout the day were comparable.

[0207] In the exemplary procedure, citric acidified whole blood (4 ml) is added to a 15 ml conical tube containing the activator / biomaterial and mixed. Then, a calcium salt solution (2 ml) is added to the tube and a timer is started. A heated shell is used to approximate in vivo or in vitro studies. The tube is placed on a rocker and observed until more than 90% clot formation is observed. Five parallel assays are performed for each formulation.

[0208] In the exemplary procedure, the bioactive glass particles were tested with fresh whole blood. Diatomaceous earth was used as a positive control (diatomaceous earth was purchased from EP Minerals, Reno, USA, product code EP-300, diatomaceous earth filter media), and the bioactive glass was purchased from MO-SCI, Rolla, USA, 45S5 (bioglass material, also known as "bioglass 45S5" or "calcium sodium phosphate silicate", is specifically a bioactive glass composed of 45% by weight SiO2).

[0209] In the ACT test, 45 mg and 65 mg of bioglass were tested in 2 mL of blood. These samples either did not clot at all or required a prolonged time to clot (i.e., the blood flowed freely in the tube after more than 4 minutes). Figure 1The image shows the use of 12 mg of diatomaceous earth particles (clotting in a tube; upper part of the image). Figure 2 Comparative ACT data for diatomaceous earth (12 mg) and bioactive glass (45 mg) are shown. Diatomaceous earth particles rapidly coagulated blood (below 200 seconds), however, bioactive glass particles showed longer clotting times, clotting within the range of 700-840 seconds. Testing of bioactive glass within a certain concentration range (12 mg-65 mg) did not improve clotting time; data are not shown.

[0210] Figure 3 The ACT time process using diatomaceous earth is presented, showing the time process of testing diatomaceous earth (12 mg) in an ACT analyzer over a 6-hour period over a day. These data show that WB is stable over the 6-hour test duration, which is the limit for whole blood use.

[0211] Figure 4 ACT data using different diatomaceous earth concentrations are presented. The dose-response of diatomaceous earth was assessed by combining different amounts of diatomaceous earth (0–50 mg) with 2 mL of citric acid-treated bovine whole blood into empty tubes (containing only the magnet). After mixing the blood and activator, 1.0 mL of calcium salt solution was added to the tube and inserted into the ACT analyzer. The rate of clot formation was determined. These data show that WB clotting time decreased with 0–12 mg of diatomaceous earth and then plateaued, resulting in no further acceleration of clotting time observed after 12 mg of activator.

[0212] In an additional exemplary procedure, medium-chain and long-chain polyphosphates (MCPPs) were compared with diatomaceous earth for their ability to clot whole blood. Two amounts (12 mg and 20 mg) of MCPPs (purchased from Kerafast, Boston, USA, P100 and P700 polyphosphates) were evaluated by combining them with 2 mL of citric acid-treated bovine whole blood in empty tubes (containing only the magnet). After mixing the blood and activator, 1.0 mL of calcium salt solution was added to the tube and inserted into the ACT analyzer. The rate of clot formation was determined. In the same study, 12 mg of raw diatomaceous earth (from EP Minerals, Reno, USA, product code EP-300, diatomaceous earth filter media) and 14 mg of Helena diatomaceous earth (from Helena Laboratories) were tested. The mean clotting time for the medium-chain and long-chain polyphosphate groups ranged from 288 to 600 seconds, while the mean clotting time for the diatomaceous earth group was 197 seconds for reserve diatomaceous earth and 218 seconds for Helena diatomaceous earth.

[0213] Figure 5The ACT results for various types of polyphosphates and diatomaceous earth are presented. These data show that the WB clotting time of the diatomaceous earth activator is significantly faster than that of medium-chain and long-chain polyphosphates (12 mg and 20 mg), thus demonstrating that diatomaceous earth is an excellent activator in this WB clotting assay.

[0214] Ellagic acid

[0215] Preparation of the EA-Ni complex: 1.8 mL of 10% (w / v) tetramethylammonium hydroxide (TMAH; purchased from Sigma-Aldrich) was dissolved in 250 mL of water. Then, 0.034 g of ellagic acid (ellagic acid hydrate; purchased from Sigma-Aldrich; product number 372749; EA molecular weight 302.2 g / mol, giving 0.0001125 mol EA) was dissolved in the TMAH solution. Next, 1.5 g of phenol was dissolved in the TMAH-ellagic acid solution, followed by the addition of 1.0 mL of 0.1 M nickel chloride (purchased from Sigma-Aldrich; i.e., 0.0001 mol nickel). The resulting solution was mixed for 10 minutes on a stirring plate using a Teflon-coated stir bar. Then, 1.2 g of TRIS-HCl was dissolved in the solution. The EA-Ni complex was tested in a whole blood gelation assay.

[0216] The EA-Ni complex, tested at a concentration of 0.085 mg / mL in the Western blot gelation test, produced impressive results. The whole blood clotting time was significantly faster (mean 84 seconds) compared to 24 mg of diatomaceous earth (mean 173 seconds). Ellagic acid, without nickel (or any other metal), was tested, but no clotting was observed within 1200 minutes. Bioactive glass (as described above) produced a clotting time of 763 seconds. The comparative results are shown in the box plot. Figure 6 It is worth noting that the EA-copper complex was also tested, but the clotting time was significantly longer by 4 times (data not shown), although it was still faster than that of bioactive glass.

[0217] Ellagic acid dose-response

[0218] In an exemplary procedure, dose-response studies were conducted using the EA-Ni complex, such as... Figure 7AThe details are presented in the figure, showing the average of triplicate results at each test concentration. Different amounts of the EA-Ni complex were tested in a Western blot gelation assay. The results showed a wide range of EA-Ni complex concentrations capable of rapidly clotting blood. Therefore, the EA-Ni complex proved to be a very effective activator, with even levels as low as approximately 2 μg / mL in blood activating rapidly (approximately 100 seconds). Even at levels as low as 2 μg / mL of EA-Ni complex in blood, it coagulated whole blood at a rate similar to that of 24 mg of diatomaceous earth. Without the activator, the blood did not coagulate within 900 seconds. Ellagic acid, which does not contain nickel (or any other metal), was tested, but no coagulation was observed within 1200 minutes. For blood EA-Ni complex concentrations ranging from 0.002125 mg / mL to 0.034 mg / mL (2 μg / mL to 34 μg / mL, which can serve as the preferred range for EA-Ni), there was no statistically significant trend in mean clotting time (p=0.944), thus demonstrating a wide range of EA-Ni complex concentrations capable of rapidly activating clotting. See [link to relevant documentation]. Figure 7B It showed no significant trend in clotting time for blood EA-Ni complex concentrations ranging from 0.002125 mg / mL to 0.034 mg / mL.

[0219] Nickel ellagic acid complex with phospholipid supplementation

[0220] Another set of exemplary experiments was conducted to determine whether the addition of phospholipids could improve clotting rates, i.e., platelet / phospholipid dependence. The goal was to determine the criticality of platelets and whether the addition of phospholipids could further improve clotting rates. EA-Ni was tested using whole blood and added phospholipids.

[0221] Using the aforementioned WB gelation test procedure, the EA-Ni complex was tested with whole blood and blood supplemented with phosphatidylcholine. Phosphatidylcholine is a rabbit brain-derived coagulation reagent containing a mixture of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), suitable for coagulation assays (phosphatidylcholine is manufactured by BioData Corporation and is a reagent used for inactive aPTT. In this test system, phosphatidylcholine is used in its undiluted form, at 1 and 5 times the standard amount).

[0222] The result is Figure 8The results showed that when a small volume of phospholipids (0.1 mL) was added to 4 mL of blood, the average clotting time using the EA-Ni complex was 89 seconds, compared to 87 seconds without any added phospholipids. When a larger volume of phospholipids (0.5 mL) was added to 4 mL of blood, the average clotting time remained unchanged at 87 seconds; that is, no improvement in clotting time was observed when phospholipids were added to fresh whole blood.

[0223] Nickel ellagic acid complex inhibited by corn trypsin inhibitor (CTI)

[0224] The goal of this test was to confirm that the mechanism of action of the EA-Ni complex is activation via FXII through the use of corn trypsin inhibitor (CT) (an inhibitor of FXIIa). CT was purchased from Prolytix (formerly Haematologic Technologies).

[0225] Using the previously described Western blotting test procedure, adding 0.1 mL of 2.5 mg / mL CTI to 4 mL of blood resulted in a mean clotting time of 141 seconds with the EA-Ni complex, compared to a mean clotting time of 87 seconds without any added CTI. To further confirm the inhibitory effect, a larger amount of CTI (2.5 mg / mL, 0.5 mL) was added to the blood, and the resulting clotting time was significantly longer (mean 407 seconds).

[0226] The results are presented in Figure 9 The study showed that at low concentrations of CTI, clotting time was slightly prolonged, while at high concentrations, the effect on clotting time was significant. This confirms that the ellagic acid-nickel complex acts through the inherent coagulation pathway.

[0227] Example 2 - Test of the nickel ellagic acid complex with flowable gelatin

[0228] In vitro model

[0229] SURGIFLO ® / SURGIFOAM ™ EA-Ni flowable paste formulation: Add 15 ml of the above nickel ellagicate complex solution to 1 g SURGIFOAM ™ In, and in accordance with SURGIFOAM ™ IFU was reconstructed. A solution containing 8 mL of SURGIFOAM was prepared. ™ / Instrument containing nickel ellagicate paste and 8 mL of SURGIFLO suspended in saline. ® The preparation of gelatin involves mixing with a syringe. 8 mL of SURGIFOAM is then transferred via syringe exchange. ™ / Nickel ellagicate paste and 8mL SURGIFLO ® Gelatin is thoroughly mixed (e.g., a 6-to-mix process, see SURGIFLO). ® (Simple preparation of hemostatic matrix kit). The method involves rapidly passing the paste formulation between syringes to produce a uniform paste.

[0230] SURGIFLO, which has an ellagic acid-nickel complex, was discovered. ® / SURGIFOAM ™ The mixture produced rapid in vitro clotting time and rapid / effective hemostasis in an in vivo porcine spleen biopsy hemorrhage model. This was observed in comparison with SURGIFLO. ® Prior to combination, the EA-Ni complex was combined with SURGIFOAM ™ Powder suspension is important for maintaining the good efficacy of the EA-Ni complex. More specifically, this is achieved by first suspending it with SURGIFOAM at the same concentration of EA-Ni complex. ™ The combination (approximately 100 seconds clotting time) is the opposite of the combination of EA-Ni complex and SURGIFLO alone. ® The paste (34 µg EA-Ni / ml paste) combination resulted in a fourfold decrease in coagulation potency (approximately 400 seconds in a 4 ml whole blood gelation test). It was also found that when prepared as described herein, 1 ml of SURGIFLO... ® / SURGIFOAM ™ The EA-Ni complex (85 μg EA-Ni / ml paste) formulation also coagulated in approximately 100 seconds for a 4 mL whole blood gelation test. For reference, in the absence of any gelatin paste, a 1 mL EA-Ni complex solution produced a whole blood gelation test time of approximately 75-90 seconds. 1 mL of SURGIFLO... ® Or 1 ml of reconstituted SURGIFOAM ™ (Using saline) was used as a negative control in a whole blood gelation test that did not cause blood clotting after 1200 seconds. It is not yet known why it appears to inactivate the EA-Ni complex in in vitro models, however, with SURGIFOAM. ™ The preparation of complexes does not deactivate the compound.

[0231] Based on the above, it can be concluded that EA-Ni complexes can be used as effective activators of the intrinsic coagulation pathway, even at low concentrations. EA-Ni can interact with SURGIFOAM. ™ Powder combination, then with SURGIFLO ® This combination produces a gelatin paste with excellent in vitro and in vivo coagulation properties. The flow and consistency properties of the EA-Ni gelatin paste are similar to those of conventional gelatin pastes, such as SURGIFLO. ®.

[0232] The presence of nickel may be a concern; however, its amount will be below the 20 μg / day limit set by the ICH Q3D (Elemental Impurities Guidelines). It should also be noted that TMAH (tetramethylammonium hydroxide) is the solvent used during manufacturing and is safely available in diluted form.

[0233] in vivo model

[0234] Preliminary studies show that the combination of SURGIFOAM ™ Powder and SURGIFLO ® (Gelatin formulations) can be combined with EA-Ni complexes to produce formulations that can rapidly achieve hemostasis in animal models. To prepare an EA-Ni complex paste for in vivo testing, a 4X nickel ellagicate complex solution was prepared according to Example 2 of European Patent 0525035 B1, except that 250 mL of deionized water was used instead of 1 L to prepare the 4X solution. First, the SURGIFOAM™ / nickel ellagicate complex was prepared by combining 15 mL of the 4X nickel ellagicate complex solution with 1 g of SURGIFOAM™ powder and reconstructing each IFU by vigorous shaking. Next, 8 mL of SURGIFOAM… ™ Transfer the ellagic acid paste to a 20 mL syringe, and transfer 8 mL of SURGIFLO prepared according to IFU with saline into another syringe. ® Paste. Mix the contents of the two syringes by exchanging syringes until visibly homogeneous. In total, the total volume (16 mL) of SURGIFOAM... ™ / SURGIFLO ® The total weight of gelatin in the paste is 1.533 g. The total weight of EA-Ni is 1.088 mg. Therefore, the weight ratio of gelatin to EA-Ni is 1409:1, weight / weight. It is noteworthy that, based on dose-response studies, EA-Ni can be diluted 1:40 and still accelerates clotting (i.e., less than 200 seconds). Therefore, a preferred range for calculating the weight ratio of gelatin to EA-Ni is 56,000:1 to 35:1. Furthermore, based on dose-response studies, EA-Ni can be diluted 1:4 to rapidly accelerate clotting (i.e., approximately 100 seconds). Another preferred weight range for calculating the ratio of gelatin to EA-Ni would be 5600:1 to 350:1.

[0235] A hemorrhagic lesion (6 mm diameter × 3 mm depth) was induced in the spleen using a biopsy perforation model. Flowable gelatin with an EA-Ni complex performed exceptionally well, achieving hemostasis time comparable to that of SURGIFLO with thrombin administration as described below. ® Quite. For SURGIFLO ®+SURGIFOAM ™ + EA-Ni and SURGIFLO ® There was no statistically significant difference in the average hemostasis time between thrombin and hemostasis, which were 173.0 seconds and 173.4 seconds, respectively.

[0236] In the exemplary procedure, after the target bleeding site is dried as much as possible, sufficient product (approximately 2 mL) is applied to the site to cover the entire bleeding area. A moistened gauze / pad is then immediately applied to keep the test product in contact with the bleeding area. The initial packing pressure is then maintained for 2 minutes.

[0237] Then gently lift the gauze / pad to examine the area. Perform a hemostasis confirmation assessment and continue for up to another minute. If no free-flowing bleeding is observed during this one-minute observation period, record the time of hemostasis as the time when the packing was stopped.

[0238] However, if free-flowing bleeding is observed after tamponade, an additional test product may be administered by the surgeon at their discretion, and tamponade pressure shall be immediately reapplied and maintained for an additional 30-second period. The observation and treatment period continues in this manner until hemostasis is achieved or until the test period reaches five minutes. If bleeding continues at five minutes, the test is terminated as a failure and recorded as ">5:00" (greater than 5 minutes).

[0239] The result is Figure 10 Presented in the image. This shows the effect of the above-mentioned formulation (such as SURGIFLO as described for in vitro testing) in a porcine spleen biopsy perforation model. ® / SURGIFOAM ™ 85µg EA-Ni / ml paste) resulted in a reaction with standard SURGIFLO containing thrombin. ® Equivalent time to hemostasis (TTH) (170-173 seconds). Negative control SURGIFLO ® / SURGIFOAM ™ The ("SF / SFm") paste resulted in a TTH of approximately 231 seconds. The mean hemostasis time of SF / SFm + EA-Ni was statistically significantly different from that of SF / SFm (t-test, p < 0.001). SURGIFLO is preferred. ® / SURGIFOAM ™ Another reason for using formulations in in vivo studies is to provide optimal paste consistency when tested in animal models.

[0240] Flowable paste with buffer In the absence of gelatin powder, a flowable gelatin paste formulation (SURGIFLO) is prepared. ®The solution was combined with the EA-Ni complex. The EA-Ni complex formulation was the same as that described in the previous in vitro tests. To evaluate the effects of the various buffers, 1 mL of a solution containing 1 M phosphate, bicarbonate, ergot, or CHES buffer was combined with 8 mL of gelatin paste containing 1.5 mL of the EA-Ni complex by rapidly exchanging the contents between two syringes. All buffers were reagent grade and purchased from Sigma Aldrich. Triple tests were performed using the Western blot gelation assay as described above, with gelatin paste for each buffer.

[0241] Significant differences in clotting times were observed between the buffer solutions. The mean clotting times for phosphate, bicarbonate, ergot, or CHES buffers were 627.3 seconds, 109.3 seconds, 345.3 seconds, and 117.7 seconds, respectively. See [link to relevant documentation]. Figure 11 For reference, in the WB gelation test, the previously described SURGIFLO was used. ® / SURGIFOAM ™ The average clotting time achieved by the / EA-Ni complex formulation is approximately 100 seconds.

[0242] Example 3: Lyophilized tablets containing nickel tannate complex

[0243] In addition to incorporating nickel ellagicate complexes into pastes, EA-Ni complexes can also be absorbed or coated onto a sponge-like tablet matrix serving as a carrier. Dengen Dental Dengofoam gelatin sponge (1cm × 1cm × 1cm) was used as the carrier.

[0244] An exemplary procedure was performed as follows: For some samples, sponge tablets were pressed in one dimension to a size of 0.2 cm × 1 cm × 1 cm using a Dulytek DW8000 press. Unpressed and pressed gelatin sponge tablets were soaked in 1 mL of 0.136 mg / mL EA-Ni complex (representing a concentration 4 times (4x) higher than that described in Example 2 (page 11) of EP Patent 0525035 B1). Some of the soaked sponge tablets were lyophilized using a 4-day cycle to remove water from the EA-Ni sponge. Photographs of the unpressed tablets were captured before and after soaking in EA-Ni, see [link to illustrative image]. Figure 12A And photographs of the compressed tablets were captured before and after soaking in EA-Ni, see [link to photograph]. Figure 12B .

[0245] Whole blood gelation test of sponge tabletsUsing the aforementioned Western blot gelatinization test procedure, tablets containing and without the EA-Ni complex were tested by adding them to 4 mL of recalcified blood tubes. The clotting time of the compressed gelatin tablets without EA-Ni was 583 seconds. When the compressed tablets were immersed in the EA-Ni complex, the average clotting time was significantly reduced to 109 seconds. The tablets can also be lyophilized for preservation and long-term storage. The average clotting times of the compressed and uncompressed lyophilized gelatin tablets were 129 seconds and 161 seconds, respectively. Results in... Figure 13 The results show that soaking gelatin tablets in EA-Ni significantly reduces whole blood clotting time. Tablets lyophilized after soaking in EA-Ni also exhibit significantly reduced clotting time. Therefore, EA-Ni retains its activity after lyophilization. Interestingly, lyophilized and compressed tablets soaked in EA-Ni have shorter clotting times than similar uncompressed tablets. On the other hand, if EA-Ni lyophilized tablets are first soaked in saline for 30 seconds before being added to whole blood, the clotting time is even longer.

[0246] 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 hemostatic composition comprising ellagic acid (EA) complexed with metal ions, the complex being dispersed or embedded within an absorbable polymer matrix.

2. The composition of claim 1, wherein the polymer matrix comprises collagen or a derivative thereof.

3. The composition according to claim 2, wherein the collagen derivative comprises gelatin.

4. The composition according to any one of claims 1 to 3, wherein the metal is selected from the group consisting of nickel, cobalt and copper.

5. The composition according to any one of claims 1 to 4, wherein the metal is nickel.

6. The composition according to any one of claims 1 to 5, wherein the metal ion and the EA are present in a molar ratio of metal to EA in the range of 100:1 to 1:100, or optionally in a molar ratio of metal ion to EA in the range of 10:1 to 1:

10.

7. The composition according to any one of claims 1 to 6, wherein the absorbable polymer matrix, such as gelatin, is selected from the following forms: a flowable paste at room temperature, a sponge matrix, a powder, and combinations thereof.

8. The composition according to claim 7, wherein the composition is in the form of a paste.

9. The composition according to claim 7 or 8, wherein the paste is characterized in that its viscosity is in the range of 1000 Pa·s to 5000 Pa·s.

10. The composition according to claim 3, wherein the gelatin is in a freeze-dried or dried form.

11. The composition according to any one of claims 2 to 10, wherein the matrix comprises a combination of gelatin in paste form and gelatin in powder form.

12. The composition according to any one of claims 1 to 11, wherein the composition is substantially free of thrombin.

13. The composition according to any one of claims 1 to 11, wherein the composition is substantially free of one or more coagulation factors selected from factors II, V, VII, VIII, IX, X, XI, XII and fibrinogen in inactive or active forms.

14. The composition according to any one of claims 1 to 13, wherein the metal ions are present in a matrix, such as gelatin, at a preferred concentration in the range of 11,200:1 to 7:1 by weight / weight, optionally 1120:1 to 70:

1.

15. The composition according to any one of claims 1 to 14, wherein the EA complex is present at a concentration of EA-Ni in the range of 56,000:1 to 35:1 weight / weight or 5600:1 to 350:1 weight / weight, optionally in the range of 5600:1 to 350:1 weight / weight matrix, such as gelatin.

16. A method of treating a subject with diseased or damaged tissue that can benefit from an endogenous pathway that rapidly activates blood clotting, the method comprising administering to the subject a therapeutically effective amount of ellagic acid (EA) complexed with a metal ion, the complex being incorporated into a matrix comprising collagen or a derivative thereof.

17. The method of claim 16, wherein the application comprises bringing the damaged tissue into contact with the matrix.

18. The method according to claim 16 or 17, wherein the metal is nickel.

19. The method according to claims 16 to 18, wherein blood coagulation occurs in less than about 400 seconds upon contact.

20. The method according to any one of claims 16 to 19, wherein the collagen derivative comprises gelatin in the form of: a flowable paste at room temperature, a sponge matrix, a compressed tablet, a powder, and any combination thereof.

21. The method according to claims 16 to 20, wherein the matrix comprises a combination of gelatin in the form of the paste and gelatin in the form of the powder.

22. A method for preparing a hemostatic composition, said hemostatic composition comprising ellagic acid (EA) complexed with metal ions added to an absorbable polymer matrix, said method comprising the following steps: The complex is added to the matrix at a ratio of 56,000:1 to 35:1 or 5,600:1 to 350:1 by weight of the polymer matrix to the EA-Ni complex.

23. The method of claim 22, wherein the polymer matrix comprises collagen or a derivative thereof.

24. The method of claim 23, wherein the collagen derivative comprises gelatin.

25. The method according to any one of claims 22 to 24, wherein the metal is nickel.

26. The method according to any one of claims 22 to 25, wherein the matrix, such as gelatin, is selected from the following forms: a flowable paste at room temperature, a sponge matrix, a compressed tablet, a powder, and combinations thereof.

27. A hemostatic composition, said hemostatic composition being obtainable by the method according to any one of claims 22 to 26.

28. A kit comprising: (a) A container containing ellagic acid (EA) complexed with metal ions. (b) Polymer matrix; (c) An applicator for applying the composition to tissue; and optional (d) Instruction manual.

29. The kit of claim 28, wherein the polymer matrix comprises collagen or a derivative thereof.

30. The kit of claim 28 or 29, wherein the collagen derivative comprises gelatin.

31. The kit according to any one of claims 28 to 30, wherein the metal is nickel.

32. The kit according to any one of claims 28 to 31, wherein the matrix, such as gelatin, is selected from the following forms: a flowable paste at room temperature, a sponge matrix, a powder, and combinations thereof.

33. The kit according to any one of claims 28 to 32, wherein the EA complexed with the metal ions is provided in a buffer solution.

34. The kit of claim 33, wherein the buffer comprises a solution containing tetramethylammonium hydroxide (TMAH).

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