KR20190099476A - Hemostatic composition comprising an anion exchanger and calcium salt - Google Patents

Abstract

A composition comprising an anion exchanger and a calcium salt for use as a medicament for inducing hemostasis at the bleeding site; And a method for producing the same.

Description

Hemostatic composition comprising an anion exchanger and calcium salt

The present invention relates to the field of hemostatic compositions. More specifically, the present invention relates to hemostatic compositions comprising anion exchangers and calcium and methods of use thereof.

Bleeding is a term commonly used to describe the leakage of blood from the circulatory system of vertebrates. Bleeding may occur inside the body (internal bleeding) or outside the body (external bleeding). The bleeding site can be almost any area of the body. Typically, internal bleeding occurs when blood leaks through damage to blood vessels or organs. External bleeding occurs when blood exits through torn portions of the skin, or when blood exits through the body's natural openings, such as the mouth, nose, ears, vagina, or rectum.

Bleeding is a traumatic injury (including abrasions, grazes, lacerations, incisions, cuts from objects such as needles or knives, crushing injuries and gunshot wounds) or certain medical conditions, eg For example, it can be caused by a wide variety of events or conditions, including those associated with coagulation that endanger subjects with clotting factor deficiency. In addition, bleeding may be associated with some nonsteroidal anti-inflammatory drugs (NSAIDs) or anti-coagulant drugs such as warfarin, low molecular weight heparin, Apixaban (ELIQUIS®), Dabigatran (PRADAXA®), Edoxaban (SAVAYSA®) and Rivaroxaban (XARELTO) Trademarks)).

Continued, untreated bleeding can lead to exsanguination leading to death, ie excessive reduction in blood volume (hypoglycemia).

Stopping or controlling bleeding is called hemostasis, which involves blood coagulation, and the promotion, acceleration or improvement of this mechanism is an important part of both first aid and surgeries. Agents and compositions that enhance, promote or accelerate hemostasis are referred to as "hemostatic agents".

Hemostatic agents can include hemostats, sealants or adhesives. Typically, hemostatic agents are subdivided into mechanical hemostatic agents (gelatin, collagen, oxidized regenerated cellulose, etc.), active hemostatic agents (eg thrombin), flow hemostatic agents (eg gelatin matrix with thrombin), and fibrin sealant.

Some known hemostatic agents require long preparation before use, which involves a number of steps that waste valuable time in an emergency.

Biological hemostatic agents are very effective but carry a potential safety risk and are expensive to produce.

Many known hemostatic agents require refrigeration, which may be unavailable or very expensive in various situations (including battlefields, isolated areas, emergencies) as well as in developing countries or during electrical failures. Moreover, refrigeration requirements increase the cost of production, transportation and storage.

Many known hemostatic agents are not effective for patients using blood-thinning drugs such as heparin, aspirin or coumadin.

Many known hemostatic agents include procoagulants, which carry a potential risk of contamination, have high production costs, short expiration times, and usually require refrigeration.

Thus, there is a need for a safe and effective hemostatic agent that is free of at least some of the disadvantages of the prior art.

Background art is described in US Pat. Nos. 5,502,042 and 8,741,335; US Patent Application Publication No. 2009/0062849; And International Patent Publications WO 2013/071235 and 1993/05822.

The present invention provides a hemostatic composition comprising an anion exchanger, calcium, and a pharmaceutically acceptable carrier; And methods of use thereof for inducing hemostasis.

According to one aspect of some embodiments of the invention, there is provided a method of inducing hemostasis at a bleeding site of a subject in need of said hemostasis, the method comprising: Applying to.

According to some embodiments, the anion exchanger comprises one or more positively charged groups bonded to the matrix.

According to some embodiments, the positively charged group (also referred to as polycationic) is provided by a base selected from the group consisting of strong bases, weak bases, and combinations thereof.

According to some embodiments, the strong base comprises a quaternary amino group.

According to some embodiments, the weak base comprises an amino group selected from the group consisting of primary amino groups, secondary amino groups, tertiary amino groups, and combinations thereof.

According to some embodiments, the weak base consists of a diethylaminoethyl (DEAE) group.

According to some embodiments, the matrix is selected from the group consisting of aliphatic polyesters, polysaccharides, polypeptides, polystyrene-divinylbenzenes, proteins (eg, collagen, gelatin or albumin), silica and combinations thereof.

According to some embodiments, the matrix is crosslinked and optionally covalently cross-linked.

According to some embodiments, the composition is substantially free of any protein of the blood clotting cascade.

According to some embodiments, the composition is in a form selected from the group consisting of slurries, powders, fibers, films, patches, and liquids.

According to some embodiments, the composition in slurry or liquid form further comprises a pharmaceutically acceptable carrier.

According to some embodiments, the applying step is performed by selectively applying pressure to the composition towards the bleeding site.

According to one aspect of some embodiments of the invention, an anion exchanger; Calcium salts; And optionally, a pharmaceutically acceptable carrier.

According to some embodiments, the anion exchanger comprises one or more positively charged groups bonded to the matrix.

According to some embodiments, the anion exchanger is linked to a solid phase.

According to some embodiments, the positively charged group consists of a base selected from the group consisting of strong bases, weak bases, and combinations thereof.

According to some embodiments, the strong base comprises a quaternary amino group.

According to some embodiments, the weak base comprises an amino group selected from the group consisting of primary amino groups, secondary amino groups, tertiary amino groups, and combinations thereof.

According to some embodiments, the weak base consists of a diethylaminoethyl (DEAE) group.

According to some embodiments, the matrix is selected from the group consisting of aliphatic polyesters, polysaccharides, polypeptides, polystyrene-divinylbenzenes, silicas, and combinations thereof.

According to some embodiments, the matrix is crosslinked and optionally covalently crosslinked.

According to some embodiments, the polysaccharide is selected from the group consisting of cellulose, dextran, agarose, and combinations thereof.

According to some embodiments, the protein is a structural protein such as collagen or gelatin, or a protein having a high abundance in plasma, such as albumin.

According to some embodiments, the composition is substantially free of any protein of the blood coagulation cascade.

According to some embodiments, the composition is in a form selected from the group consisting of slurry, powder, film, patch and liquid.

According to some embodiments, the composition in slurry or liquid form further comprises a pharmaceutically acceptable carrier.

According to one aspect of some embodiments of the invention, diethylaminoethyl (DEAE) bound to a matrix; And a calcium salt is provided.

According to one aspect of some embodiments of the invention, preparing an anion exchanger by covalently bonding at least one positively charged group to a crosslinked matrix; And adding a calcium salt to the anion exchanger is provided.

According to one aspect of some embodiments of the present invention, there is provided a hemostatic composition obtainable by the method disclosed herein.

As used herein, the term “inducing hemostasis” refers to causing, causing and / or promoting, and / or accelerating and / or enhancing hemostasis.

As used herein, the term “bleeding site” refers to a site that actively bleeds and that may be vulnerable or susceptible to bleeding complications, such as, for example, surgical sites, anastomosis sites, and / or suture sites.

As used herein, the term "pharmaceutically acceptable carrier" refers to any inert diluent or vehicle that has no biological activity and is suitable for use in humans or other animals. Carriers include phosphate buffered saline (PBS), saline, sodium chloride solution, calcium chloride solution, lactate ringer (LR), 5% dextrose in physiological saline, various sugars, sugar alcohols (e.g. mannitol, sorbitol) and It may be selected from any of the carriers known in the art, such as but not limited to water for injection.

As used herein, the term “slurry” refers to a thick, soft, moist material. Typically, the slurry is produced by mixing the dry ingredients (eg powder or solid hydrophilic particles) with the liquid. The dry component concentration can be 0.5% to 99% w / w of the total slurry composition. Typically, the slurry is a material that is moldable in the temperature range of 15-40 ° C.

As used herein, the term "without" in reference to the components of a composition refers to the components present in the composition at a concentration of less than 0.1% w / w of the total composition.

As used herein, the terms “comprising”, “comprising”, “having” and their grammatical variants are considered to specify the features, integers, steps, or components mentioned, but one or more of them may be added. It does not exclude the addition of features, integers, steps, components or groups. These terms include the terms "consisting of" and "consisting essentially of".

As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more" unless the context clearly dictates otherwise.

As used herein, the term "about" refers to ± 10%.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, the descriptions, materials, methods, and examples are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

Some embodiments of the invention are described in the present invention with reference to the accompanying drawings. The detailed description with drawings will make apparent to those skilled in the art how some embodiments of the invention may be practiced. The drawings are for the purpose of illustrative discussion and are not intended to represent the structural details of the embodiments in more detail than necessary for a basic understanding of the invention. For clarity, some articles shown in the figures are not drawn to scale.
In the drawing:
FIG. 1 shows a compression time of 30 seconds (for DEAE Sephadex ™ A-50) or 60 seconds (for commercial gelatin hemostatic agent) with DEAE Sephadex ™ A-50 (10% w / v) and commercial gelatin hemostatic agents After application, the reduction in bleeding is shown in an in-vivo heparinized porcine spleen circular punch model (4 mm diameter / 2 mm depth) in vivo .
2A shows the device used in the Porcine Spleen Problematic Bleeding Model to create a "bullet-like" wound. 2B shows the wound produced by the device of FIG. 2A in a pig spleen. As can be seen in FIG. 2B, the wound results in severe bleeding from the spleen. DEAE Sephadex ™ A-50 was prepared as a slurry of 10% w / v in 20 mM CaCl ₂ and applied to the wound. After application of the slurry and a compression time of 4 minutes, post-application bleeding intensity was evaluated as described for FIG. 1. 2C shows gunshot-like wounds after application and tamponade of DEAE Sephadex ™ A-50, achieving complete hemostasis of severely bleeding wounds.

The present invention provides a hemostatic composition comprising an anion exchanger, a calcium salt, and optionally, a pharmaceutically acceptable carrier; And methods of use thereof in achieving hemostasis.

The principles, use and practice of the teachings of the invention may be better understood with reference to the attached detailed description. Upon reading the detailed description, those skilled in the art can practice the invention without undue effort or experiment.

Before describing at least one embodiment in detail, it will be appreciated that the invention is not necessarily limited in its application to the details and arrangements of the components and / or methods described in the following description. The invention may be capable of other embodiments or of being practiced or carried out in various ways.

The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

According to one aspect of some embodiments of the invention, there is provided a method of inducing hemostasis at a bleeding site of a subject in need thereof, wherein the method applies an effective amount of a hemostatic composition comprising an anion exchanger and a calcium salt to the bleeding site It includes a step.

According to one aspect of some embodiments of the invention, a hemostatic composition is provided comprising an anion exchanger and a calcium salt for use in inducing hemostasis at a bleeding site.

According to one aspect of some embodiments of the invention, there is provided the use of a hemostatic composition comprising an anion exchanger and a calcium salt in the manufacture of a medicament for inducing hemostasis.

According to one aspect of some embodiments of the invention, there is provided the use of a pharmaceutical hemostatic composition comprising an anion exchanger and a calcium salt in the manufacture of a medicament for inducing hemostasis.

According to some embodiments of the methods, compositions for use, uses, or methods of manufacture disclosed herein, the anion exchanger is a group of polyvalent ions (at a polycation) in one or more amounts (at a pH of 2 to 10) bound to the matrix. Referred to). In some such embodiments, the hemostatic composition is free of polyanions.

According to some embodiments of the methods, pharmaceutical compositions for use, uses, or methods of preparation disclosed herein, the anion exchanger is a group (polycation) that is charged in an amount of one or more (at a pH of 2 to 10) bound to the matrix (Also referred to as). In some such embodiments, the hemostatic composition is free of polyanions (eg, polyanionic polymers).

Polyanions are molecules or chemical complexes with more than one negative charge. Polycationics are molecules or chemical complexes with more than one positive charge.

In one embodiment, the matrix may comprise anionic residues; The total net charge of the anion exchanger will be positive.

In some embodiments, the positively charged group is present in the hemostatic composition at a total ionic capacity of at least 2 mmol / g, such as from 2 to 5, 3 to 4 mmol / g.

As used herein, the term “total ionic capacity” refers to the total amount of charged sites in the composition that are available for exchange. Total ion capacity is expressed on a dry weight, wet weight or wet volume basis.

According to some embodiments, the anion exchanger is at a concentration of 0.5-99% w / v of the total hemostatic composition, optionally 5-15% w / v of the total hemostatic composition, for example 5%, 6%, 7 Present in concentrations of%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%.

According to some embodiments in which the anion exchanger is present at a concentration of at least 10% w / v of the total hemostatic composition, the ratio between the anion exchanger and calcium in the hemostatic composition ranges from 1: 5 to 1:70. In some embodiments, the ratio is about 1:34.

According to some embodiments, the positively charged group is selected from the group consisting of strong bases (eg, including quaternary amino groups), weak bases (eg, primary amino groups, secondary amino groups, tertiary amino groups Comprising an amino group) and a combination thereof.

According to some embodiments, the weak base consists of a diethylaminoethyl (DEAE) group.

According to some embodiments, the positively charged group is bound to the matrix, for example the matrix support, via a linking group present between the matrix support and the positively charged group.

According to some embodiments, the matrix is an aliphatic polyester, polysaccharide, polypeptide (eg gelatin, bovine serum albumin (BSA) or collagen, or a combination thereof), polyacrylamide, acrylate-copolymer, polystyrene-die Vinylbenzene, silica, and combinations thereof.

According to some embodiments, the matrix is crosslinked and optionally covalently crosslinked. In some such embodiments, the matrix is free of ionic crosslinks.

According to some embodiments, the polysaccharide is selected from the group consisting of cellulose, dextran, agarose, and combinations thereof.

According to some embodiments, the matrix comprises Sephadex ™ (dextran), Sephacel ™ (cellulose) or TOYOPEARL ™ (hydroxylated methacryl polymer) or combinations thereof. According to some embodiments, the composition is in a form selected from the group consisting of slurry, powder, film, patch and liquid. According to some such embodiments, the composition in slurry or liquid form further comprises a pharmaceutically acceptable carrier.

According to some embodiments of the methods disclosed herein, applying the hemostatic composition to the bleeding site is performed by applying pressure to the composition (eg, using gauze) towards the bleeding site.

According to one aspect of some embodiments of the invention, an anion exchanger; Calcium salts; And optionally, a pharmaceutically acceptable carrier.

According to some embodiments, the anion exchanger comprises one or more positively charged groups (also referred to as polycationics) bound to the matrix. In some such embodiments, the hemostatic composition is substantially free of polyanions.

In some embodiments, the positively charged group is present in the hemostatic composition at a total ionic dose of at least 2 mmol / g, such as from 2 to 5, 3 to 4 mmol / g.

According to some embodiments in which the anion exchanger is present at a concentration of at least 10% w / v of the total hemostatic composition, the ratio between the anion exchanger and calcium in the hemostatic composition ranges from 1: 5 to 1:70. In some embodiments, the ratio is about 1:34.

According to some embodiments, the anion exchanger is at a concentration of 1 to 99% w / v of the total hemostatic composition, optionally 5 to 15% w / v of the total hemostatic composition, eg, 5%, 6%, 7 Present in concentrations of%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%.

According to some embodiments, a positively charged group is a strong base (eg, comprising a quaternary amino group), a weak base (eg, primary amino group, secondary amino group, tertiary amino group, and combinations thereof Comprising an amino group selected from the group consisting of: and combinations thereof.

According to some embodiments, the weak base comprises a diethylaminoethyl (DEAE) group.

According to some embodiments, the positively charged group is bonded to the matrix via a linking group present between the matrix support and the positively charged group.

According to some embodiments, the matrix is a polysaccharide, polypeptide (eg, gelatin, bovine serum albumin (BSA) or collagen, or a combination thereof), polyacrylamide, acrylate-copolymer, polystyrene-divinylbenzene, silica And combinations thereof.

According to some embodiments, the matrix is crosslinked and optionally covalently crosslinked. In some such embodiments, the matrix is free of ionic crosslinks.

According to some embodiments, the polysaccharide is selected from the group consisting of cellulose, dextran, agarose, and combinations thereof.

According to some embodiments, the matrix comprises Sephadex ™ (dextran), Sephacel ™ (cellulose) or Toyopearl ™ (hydroxylated methacryl polymer) or a combination thereof.

According to some embodiments, the composition is in a form selected from the group consisting of slurry, powder, film, patch and liquid. According to some such embodiments, the composition in slurry or liquid form further comprises a pharmaceutically acceptable carrier.

According to some embodiments, the salt used in the present invention is a positive divalent cation.

According to some embodiments of the methods, compositions for use, uses or hemostatic compositions disclosed herein, the calcium is calcium chloride, calcium acetate, calcium lactate, calcium oxalate, calcium carbonate, calcium gluconate, calcium phosphate, calcium glycophosphate or It is present in hemostatic compositions as calcium salts such as combinations thereof. In some embodiments, the calcium salt is calcium chloride, optionally as a solution, further optionally present at a concentration of 1-100 mM. In some such embodiments, the calcium present in the hemostatic composition is calcium chloride.

According to some embodiments of the methods, compositions for use, uses or hemostatic compositions disclosed herein, the hemostatic composition is substantially free of all proteins of the blood coagulation cascade.

According to some embodiments, the matrix is free of the following polyanionic polymers: alginate and / or hyaluronate.

According to some embodiments, the matrix includes polystyrene sulfonates (eg, sodium polystyrene sulfonate), polyacrylates (eg sodium polyacrylate), polymethacrylates (eg sodium polymethacrylate). ), Polyvinyl sulfate (e.g. sodium polyvinyl sulfate), polyphosphate (e.g. sodium polyphosphate), iota carrageenan, kappa carrageenan, gellan gum, carboxy methyl cellulose, Carboxyl methyl agarose, carboxyl methyl dextran, carboxyl methyl chitin, carboxyl methyl chitosan, polymers modified with carboxyl methyl groups, alginates (e.g. sodium alginate), polymers containing a plurality of carboxylate groups At least one crosslinkable polyanionic polymer selected from the group consisting of xanthan gum, and combinations thereof The.

According to some embodiments, the polymer of the matrix is not modified by the addition of carboxymethyl (CM) groups.

According to some embodiments, the biocompatible polymer is modified with diethylaminoethyl (DEAE) groups to obtain a cationic functional group to become a polycationic polymer.

According to some embodiments, the polycationic polymer comprises chitosan (eg, chitosan chloride), chitin, diethylaminoethyl-dextran, diethylaminoethyl-cellulose, diethylaminoethyl-agarose, diethylaminoethyl -Alginate, a polymer modified with diethylaminoethyl group, a polymer containing a plurality of protonated amino groups, and a polypeptide having an average residue isoelectric point greater than 7, and combinations thereof. Preferably, the polycationic polymer is diethylaminoethyl-dextran (DEAE-dextran).

According to some embodiments of the methods, compositions for use, uses or hemostatic compositions disclosed herein, the matrix is free of alginic acid and pectic acid.

According to one aspect of some embodiments of the invention, diethylaminoethyl (DEAE) bound to a matrix; And a calcium salt is provided.

According to one aspect of some embodiments of the invention, preparing an anion exchanger by covalently bonding at least one positively charged group to a crosslinked matrix; And adding a calcium salt to the anion exchanger is provided.

According to a further aspect of such an embodiment of the present invention, there is provided a hemostatic composition obtainable by the method disclosed herein.

According to some embodiments of the methods, compositions for use, uses or hemostatic compositions disclosed herein, the hemostatic composition is substantially free of all biological hemostatic agents, ie all protein components of the blood coagulation cascade, namely fibrinogen, fibrin, factor V Factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XIa, factor XI, factor XII, factor XIIa, tissue factor (TF), and thrombin, and Substantially free of prothrombinase complex, prothrombin, and vWF, tenase complex, high molecular weight kininogen (HMWK), prekallikrein, kallikrein, thromboplastin.

According to some embodiments of the methods, compositions for use, uses or hemostatic compositions disclosed herein, the hemostatic composition is substantially free of all proteins (eg, thrombin, prothrombin and fibrinogen) in the blood coagulation cascade.

In some embodiments, the anion exchanger may be a matrix ("support", "backing", "background"), which may be solid or semi-solid, optionally in the form of beads with one or more positively charged groups bound thereto. ), Also referred to as "base bead" or "resin."

Advantageously, the matrix can support the pressure as typically applied during surgery when using the auxiliary material to stop bleeding without disintegration.

In some embodiments, the matrix comprises a crosslinked polymer. In some embodiments, the solid or semisolid matrix material does not dissolve or disintegrate (at least one minute from application of the hemostatic composition) until hemostasis is achieved.

According to some embodiments, the polymer forming the matrix is insoluble in water and is preferably porous with an exclusion limit of at least 20K Da.

According to some embodiments, the matrix does not disintegrate when under manual physical stress.

As used herein, the term “positively charged group” refers to a molecule comprising a chemical group having a positive charge in the pH range of 2.0-10, for example ammonium, alkyl ammonium, dialkylammonium, trialkyl ammonium, 4 Primary ammonium, diethylaminoethyl (DEAE), dimethylaminoethyl (DMAE), triethylaminoethyl, trimethylaminoethyl, alkyl group, amino functional group (e.g., NR ₂ H ⁺ ), diethyl- (2 -Hydroxypropyl) aminoethyl, trimethylamino-hydroxypropyl, and combinations thereof.

In one embodiment, the ion exchanger has a plurality of pKa values in the range of 6-14. In further embodiments, the ion exchanger has a single pKa value of greater than nine.

According to some embodiments, the anion exchanger comprises DEAE bound to any matrix known to have hemostatic properties to increase the hemostatic efficacy of the matrix. Examples of suitable matrices include, without limitation, gelatin, cellulose, collagen, and starch.

According to some embodiments, the hemostatic composition comprises at least a blend of anion exchanger and calcium. The term “blend” is intended to refer to any form of mixture, at least homogeneous or heterogeneous, of at least an anion exchanger and calcium. The blend may optionally further comprise other ingredients.

According to some embodiments, the blend is substantially free or free of any protein component of the blood coagulation cascade (ie, such component is present in the composition at a concentration of less than 0.1% w / w of the total composition), for example a blend May be substantially free or free of thrombin and fibrinogen.

According to some embodiments, the blend is a slurry, powder or liquid blend.

According to some embodiments, the blend is provided in a frozen state such that before use, the product is thawed and brought to room temperature (ie, in the range of 15-40 ° C.), where the blend is in its usable state.

According to some embodiments, the compositions disclosed herein stop bleeding of the wound within 1 minute.

As used herein, the term "hemostatic agent" or "hemostatic composition" refers to a material or composition that functions by coagulating blood, that is, induces hemostasis. Typically, hemostatic agents increase blood clotting.

In one embodiment, the term “induces hemostasis” in the context of a composition refers to a composition that coagulates blood by activation of a clotting factor, such as prothrombin, causing bleeding to stop or a decrease in bleeding intensity. .

As used herein, the term “stop bleeding” or “stop bleeding” in connection with a composition, when applied to a wound site, is, for example, 0 (such as described in the 'Materials and Methods' section below). No bleeding) to a scale of 5 to refer to a composition that does not cause bleeding.

As used herein, the term “reduction of bleeding intensity” (also referred to herein as “hemostatic efficacy”) refers to the difference between initial bleeding intensity and post-application bleeding intensity.

As used herein, the term "initial bleeding intensity", as assessed immediately after wound formation and prior to application of the composition, for example on a scale of 0 to 5 as described in the 'Materials and Methods' section below. Refers to the intensity of bleeding.

As used herein, for the specified compression time, the term “post-applied bleeding intensity”, after application of the composition and after the compression time, is for example 0 to 5 as described in the 'Materials and Methods' section below. Refers to the intensity of bleeding as assessed by the scale.

The hemostatic efficacy of the composition can be assessed in terms of compression time when applied to bleeding wounds.

As used herein, the term "compression time" refers to the time that manual compression is applied to the bleeding wound after application of the composition. Typically, this force is equal to the strength normally applied by the surgeon when using the assistive product to achieve hemostasis. In some embodiments where no compression is applied, the compression time is referred to as 0 seconds.

In some embodiments, the compression time is about 8-12 minutes. In some embodiments, in problematic bleeding, the compression time is about 5 minutes. In some embodiments, in the bleeding encountered in a general surgical procedure, the compression time is about 1 to 2 minutes. "Problem bleeding" is defined as class III bleeding or more according to the WHO classification. Typically, class III bleeding involves a loss of 30-40% of the circulating blood volume. Typical symptoms include a drop in the patient's blood pressure, an increase in heart rate, and a decrease in peripheral perfusion (shock).

Without wishing to be bound by any one theory, the inventors assume that thrombin is produced in situ when the composition as disclosed herein is applied to a bleeding wound or site. Surprisingly, this in situ thrombin production has been found to occur to a sufficient degree and at a rate sufficient to achieve hemostasis.

Advantageously, the presence of the physical matrix allows the hemostatic composition to be easily applied to the bleeding site, optionally using compression. Moreover, the matrix itself can contribute to coagulation by capturing platelets similarly to oxidized regenerated cellulose (ORC).

As shown in the Examples section below, the matrix was found to be a prerequisite for the hemostatic capacity of a positively charged functional group, for example DEAE. Typically, the matrix does not dissolve upon initial contact with the liquid and maintains its integrity until hemostasis is achieved, e.g., for at least 1 minute, and causes aggregation of blood proteins to be concentrated locally at the wound site It must have properties that allow the initiation of the cascade. Advantageously, the matrix is stable under the pressure normally applied by the surgeon during normal surgery to achieve hemostasis. Advantageously, the property of the matrix is to be dispensed onto and / or in the wound after application, optionally using compression.

According to the present invention, various Sephadex types and commercial gelatin hemostatic agents have been found to fail to stop bleeding (in a hepatic bleeding model), ie no decrease in bleeding intensity has been observed.

The various Sephadex types surprisingly reduced bleeding when the same base polymer included crosslinked dextran provided as a powder from which the slurry was prepared.

Moreover, after application of DEAE Sephadex, the spleen was manually manipulated by folding the trachea from both sides and no rebleeding occurred.

Commercial gelatin hemostatics failed to stop bleeding after 60 seconds of compression time, but DEAE Sephadex ™ A-50 10% (w / v) was found to successfully stop bleeding after a short compression time of 30 seconds. It has also been found that a composition comprising an anion exchanger, for example DEAE bound to a matrix, with a calcium salt results in complete hemostasis.

These compositions cause substantially complete hemostasis regardless of the particular matrix used.

In addition, compositions comprising, with calcium salts, DEAEs bound to matrices such as anion exchangers such as Sephadex ™, Sephacel ™, and Toyopearl ™ (dextran, cellulose and hydroxylated methacryl polymers, respectively) It has been found to cause complete hemostasis.

More specifically, it has been found that DEAE Sephadex in CaCl ₂ can stop bleeding after 60 and 30 seconds of compression.

DEAE Sephadex ™ A-50 (8% w / v) application has been found to be used without compression to reduce bleeding intensity.

The hemostatic capacity of a composition comprising DEAE Sephadex (eg, DEAE Sephadex ™ A-50) and calcium salt is, for example, when using the same compression time (eg, 30 seconds or 10 seconds of compression), It has been shown to show similar efficacy as commercial gelatin hemostatics with thrombin. However, the hemostatic ability of anion exchangers comprising DEAEs bound to the matrix, and hemostatic agents based on calcium salts, was significantly superior to the hemostatic capacity of commercial gelatin hemostatic agents in the absence of biologically active ingredients such as thrombin.

According to the present invention it has been found that compositions comprising DEAE Sephadex prepared using NaCl but lacking calcium salts do not reduce the bleeding intensity.

The results show that the use of a composition comprising DEAE groups bound to the matrix in the presence of calcium salts effectively achieved hemostasis.

The results also showed that a compression time of 30 seconds and 60 seconds after DEAE Sephadex application in the presence of calcium salts resulted in complete hemostasis.

Hemostatic time (TTH) of normal plasma in the presence of calcium (as measured by coagulation analysis) is about 200 seconds. In contrast, according to the invention, the TTH of normal plasma in the presence of calcium and an anion exchanger ranges from about 10 to 180 seconds, for example from about 10 to 60 seconds, from about 10 to 30 seconds, from about 15 to 60 seconds. Range, about 15 to 30 seconds, and about 30 to 60 seconds. In one embodiment, the TTH is about 30 seconds.

According to the present invention, anion exchangers such as DEAE bound to the matrix have been shown to provide complete hemostasis with calcium salts. These results were obtained regardless of the matrix used. The results are comparable to those obtained when using commercially available hemostatic agents such as gelatin with thrombin.

QAE Sephadex ™ has been shown to reduce bleeding with calcium salts.

Compositions without calcium salt and / or matrix did not affect bleeding intensity.

These results by DEAE and QAE suggest that compositions containing anion exchangers bound to the matrix and comprising calcium salts are effective as hemostatic agents.

The hemostatic capacity of DEAE bound to the crosslinked polymer in the presence of calcium salts is determined by Holcomb JB, Pusateri AE, Harris RA, which is incorporated by reference as if fully set forth herein. More of problem bleeding, modified from the disclosure of Effect of dry fibrin sealant dressings versus gauze packing on blood loss in grade V liver injuries in resuscitated swine. J Trauma. 1999; 46: 49-58). Further confirmation from the challenging model.

DEAE bound to the crosslinked matrix according to the present invention was successful in achieving complete hemostasis in vivo in the heparinized pig spleen round punch model, whereas DEAE not bound to the matrix failed to reduce bleeding intensity. .

The density of positively charged groups bound to the matrix as disclosed herein (ie, Presence) is shown to be important for achieving hemostasis. The charge on the matrix may advantageously be present at a density sufficient to achieve hemostasis according to the present invention.

It has been found in accordance with the present invention that not all positive charges evaluated provide the same level of hemostatic efficacy. Thus, to assess charge density and charge type, an analysis can be performed to ensure that an optimal density range exists.

For example, the synthesized matrix can be monomerized, for example by acid hydrolysis, and is capable of separating monomers based on different charges of the monomers (eg, high pressure liquid chromatography, Gas chromatography). Analysis can be performed in this manner to assess the charge density on a given molecule.

Example

Materials and methods

TABLE 1

In vivo circular punch model.

This model is based on the model previously described in WO 2012087774 A1, with some modifications. This model evaluates the efficacy of the tested compositions in reducing bleeding in vivo (hemostatic efficacy).

Initially, the organ to be studied for hemostasis was exposed and then a single biopsy punch was applied (4 mm diameter, 2 mm depth in Examples 1 and 5; 4 mm diameter, 2 mm in Examples 2, 2). Depth or 8 mm diameter, 3 mm depth). The tissue in the punch was removed. Initial bleeding intensity was rated on a scale of 0 to 5 (“initial bleeding”), where 0 is “no bleeding”; 1 is "oozing"; 2 is "very mild bleeding"; 3 is "light bleeding"; 4 is "moderate bleeding"; 5 is "severe bleeding".

To assess the hemostatic efficacy of each tested composition, approximately 0.5 ml of slurry or 100 mg of powder (for compositions applied as a powder) was applied to the bleeding punch wound. The composition applied as a slurry was applied to the wound using a syringe; The composition applied as a powder was applied directly onto the wound.

After application of the composition, gauze was used to selectively apply manual compression for the specified time (also referred to herein as "compression time"). After compression, the gauze was removed and post-applied bleeding intensity was assessed qualitatively (with or without) or quantitatively (using a scale of 0 to 5 as described above) immediately or after an additional minute.

Tested compositions that reduce the bleeding intensity (starting at 3 or more) to 1 (flow) or 0 (no bleeding) were considered effective. For qualitative determination, the presence or absence of bleeding was visually inspected, as well as a piece of gauze pressed on the edge of the treated area.

The heparinized biopsy punch model is considered to be a suitable model for assessing strong hemostasis.

Hemostatic time (TTH) was assessed after application of the composition. Hemostatic time (TTH) was assessed after application of the composition. TTH is defined as the time interval from application of the composition until complete hemostasis (score 0) is observed.

Example 1: Hemostatic properties of a composition comprising an anion exchanger and a calcium salt in an in vivo spleen model.

In an in vivo heparinized pig spleen round punch model as described above, using DEAE covalently bound to Sephadex (DEAE- Sephadex ™) as an anion exchanger, the composition of the composition comprising an anion exchanger and calcium salt Initial evaluation of hemostatic properties was performed. In this experiment, the punch size was 4 mm diameter, 2 mm deep. A compression time of 30 or 60 seconds was used after application. DEAE Sephadex ™ A-50 was tested at two concentrations. In this experiment, post-application bleeding intensity was qualitatively assessed.

The following compositions (see above in Table 1 above) were evaluated for hemostatic efficacy:

1. DEAE Sephadex ™ A-50, prepared as a 10% w / v slurry in 20 mM CaCl ₂ solution (0.5 ml contains 50 mg of DEAE Sephadex ™ A-50) (30 sec compression time);

2. DEAE Sephadex ™ A-50, prepared as a 6.6% w / v slurry in 20 mM CaCl ₂ solution (0.5 ml contains 33 mg of DEAE Sephadex ™ A-50) (60 sec compression time);

3. Sephadex ™ G-75 superfine, prepared as a 10% w / v slurry in 20 mM CaCl ₂ solution (0.5 ml contains 50 mg of Sephadex ™ G-75 superfine) (60 sec compression time );

4. Sephadex ™ G-50 Medium (0.5 ml contains 50 mg of Sephadex ™ G-50 Medium) prepared as 10% w / v slurry in 20 mM CaCl ₂ solution (60 sec compression time);

5. Commercial gelatin hemostatic agent (0.5 ml contains 55 mg of gelatin) prepared as a slurry (60 sec compression time).

All four Sephadex ™ samples comprise the same base polymer, crosslinked dextran. Compositions 1-4 were provided as powders and slurries therefrom were prepared as described in Table 1 above. Commercial gelatin flow hemostatics were used as controls.

Sephadex ™ G-50 Medium, Sephadex ™ G-75 Superpine and commercial gelatin hemostatic agents have been shown to fail to stop bleeding, ie no decrease in bleeding intensity has been observed (results not shown).

Surprisingly, DEAE Sephadex ™ A-50 reduced bleeding at all tested compression times. After application of DEAE Sephadex ™ A-50, the spleen was manually manipulated by folding the trachea from both sides. No rebleeding occurred at any of the concentrations tested and after two different compression times (results not shown). Because hemostasis occurred only in the matrix supplemented with DEAE phase, it was concluded that the hemostatic effect was due to the presence of DEAE phase.

1 shows exemplary results obtained using DEAE Sephadex ™ A-50 10% (w / v) and commercial gelatin. As shown in the figure, commercial gelatin hemostatic agents failed to stop bleeding after a 60 second compression time, but DEAE Sephadex ™ A-50 10% (w / v) successfully bleeded after a shorter compression time of 30 seconds. Stopped.

Example 2 Effect of Compositions Comprising Anion Exchanger and Calcium on Hemostasis in an In Vivo Porcine Liver Model

In the examples below, using the in vivo heparinized pig liver round punch model, as described above, the effects on the hemostasis of the individual components of the composition comprising an anion exchanger and calcium salt, individually and in combination Evaluated. This experiment identifies which of the components of the composition are needed to achieve hemostasis.

The preparation of each composition is described in Table 1 above. Compression times are listed in Table 2 below. In this experiment, initial bleeding intensity and post-application bleeding intensity were evaluated according to a scale of 0-5.

The following compositions were evaluated:

1. DEAE Sephadex ™ A-50 (0.5 ml contains 40 mg of DEAE Sephadex ™ A-50) prepared as an 8% w / v slurry in 20 mM CaCl ₂ solution;

2. Commercial gelatin hemostatic agent prepared as a slurry (0.5 ml contains 55 mg of gelatin);

3. Commercial gelatin hemostatic agent with thrombin, prepared as a slurry (0.5 ml contains 55 mg of gelatin);

4. Sephadex ™ G-50 Medium (0.5 ml contains 70 mg of Sephadex ™ G-50 Medium) prepared as a 14% w / v slurry in 20 mM CaCl ₂ solution;

5. DEAE Sephadex ™ A-50 (0.5 ml contains 40 mg of DEAE Sephadex ™ A-50) prepared as an 8% w / v slurry in 20 mM NaCl solution;

6. SP Sephadex ™ C-50 (0.5 ml contains 40 mg of SP Sephadex ™ C-50) prepared as an 8% w / v slurry in 20 mM CaCl ₂ solution;

7. QAE Sephadex ™ (0.5 ml contains 40 mg of QAE Sephadex ™) prepared as an 8% w / v slurry in 20 mM CaCl ₂ solution;

8. DEAE Sephacel ™ prepared as a slurry (100 mg); And

9. Toyopearl DEAE-650M ™ prepared in powder form (100 mg).

Compression time and bleeding intensity results after application of each tested composition are shown in Table 2. Bleeding intensity reduction was calculated by subtracting post-application bleeding intensity from initial bleeding intensity.

TABLE 2

TABLE 3

In general, it can be seen that a composition comprising an anion exchanger, such as DEAE, bound to a matrix together with a calcium salt results in complete hemostasis (DEAE Sephadex ™ A-50, DEAE Sephacel ™ and Toyopearl DEAE-650M See Table 2 for ™, all of which contain calcium salts). These compositions cause substantially complete hemostasis regardless of the particular matrix used. For example, matrices such as Sephadex ™, Sephacel ™ and Toyopearl ™ (dextran, cellulose and hydroxylated methacrylic polymers, respectively) had a similar effect in reducing bleeding intensity.

More specifically, DEAE Sephadex ™ A-50 8% w / v in CaCl ₂ could stop bleeding after 60 and 30 seconds of compression. The results also show that DEAE Sephadex ™ A-50 (8% w / v) application can be used without compression to reduce bleeding intensity (Table 2).

The hemostatic ability of a composition comprising DEAE Sephadex ™ A-50 and calcium salt has similar efficacy as with commercial gelatin hemostatics with thrombin when using the same compression time (30 seconds) and even using only 10 seconds of compression. Indicated. However, the hemostatic ability of anion exchangers comprising DEAEs bound to the matrix, and hemostatic agents based on calcium salts, was significantly superior to the hemostatic capacity of commercial gelatin hemostatic agents in the absence of biologically active ingredients such as thrombin.

As shown in Table 3, a composition comprising DEAE Sephadex ™ prepared using NaCl and lacking a calcium salt does not reduce the bleeding intensity, thus it can be concluded that the sample is not effective at stopping bleeding.

In evaluating the effect of ion exchange groups on hemostatic capacity, SP Sephadex ™ and calcium salts containing sulfopropyl (SP), an anionic group, did not appear to reduce bleeding intensity. In other words, materials having negative (SP) groups instead of positive (DEAE) groups were not effective as hemostatic agents.

In addition, quaternary aminoethyl, QAE Sephadex ™ and calcium salts have been shown to reduce bleeding intensity.

As in Example 1, the matrix alone (Separdex ™ G-50 with calcium salt but without DEAE group) also showed no hemostatic efficacy in the absence of functional groups.

The results show that the use of a composition comprising DEAE groups bound to the matrix in the presence of calcium salts effectively achieved hemostasis.

The results also showed that compression times of 30 seconds and 60 seconds after application of DEAE Sephadex ™ A-50 (8% w / v) resulted in complete hemostasis, thus TTH was defined as 30 seconds.

Thus, anion exchangers such as DEAE bound to the matrix have been shown to provide complete hemostasis with calcium salts. These results were obtained regardless of the matrix used. The results are comparable to those obtained when using commercially available hemostatic agents such as gelatin with thrombin.

QAE Sephadex ™ has been shown to reduce bleeding with calcium salts.

Compositions without calcium salt and / or matrix did not affect bleeding intensity.

These results suggest that a composition comprising an anion exchanger and a calcium salt is effective as a hemostatic agent.

Example 3: Effect of a composition comprising an anion exchanger and a calcium salt on hemostasis in a swine spleen model in vivo.

Previous experiments have shown that a composition comprising an anion exchanger consisting of DEAE bound to a crosslinked polymer, and a calcium salt, is effective in reducing bleeding intensity in an in vivo splenic round punch model.

In this experiment, the hemostatic activity of the composition was confirmed in another model, an in vivo heparinized pig spleen round punch model (performed as described above). This model was more severe than the previous model in terms of bleeding intensity. Compression time for all samples was 30 seconds. For Samples 1-6, the punch size was 4 mm diameter, 2 mm deep. For Samples 7-9, the punch size was 8 mm diameter, 3 mm deep. Typically, the severity of bleeding increased with increasing punch size and / or addition of heparin.

The following samples were tested for hemostatic efficacy:

1. DEAE Sephadex ™ A-50 (0.5 ml contains 40 mg of DEAE Sephadex ™ A-50) prepared as an 8% w / v slurry in 20 mM CaCl ₂ solution;

2. DEAE Sephadex ™ A-50 (0.5 ml containing 50 mg of DEAE Sephadex ™ A-50) prepared as 10% w / v slurry in 20 mM CaCl ₂ solution, repeated twice;

3. Commercial gelatin hemostatic agent prepared as a slurry (0.5 ml contains 55 mg of gelatin);

4. Commercial gelatin hemostatic agent with thrombin, prepared as a slurry (0.5 ml contains 55 mg of gelatin);

5. Sephadex ™ G-50 Medium (0.5 ml contains 70 mg of Sephadex ™ G-50 Medium) prepared as a 14% w / v slurry in 20 mM CaCl ₂ solution;

6. DEAE Sephadex ™ A-50 (0.5 ml contains 40 mg of DEAE Sephadex ™ A-50) prepared as an 8% w / v slurry in 20 mM NaCl solution;

7. Pure DEAE;

8. CaCl ₂ (20 mM); And

9. DEAE with CaCl ₂ .

In the presence of CaCl ₂ DEAE Sephadex ™ A-50 8% and 10% w / v were observed to be able to reduce bleeding intensity (a decrease of about 2-3 points was observed), with higher percentages being more Provided excellent results. In this model, the hemostatic capacity of DEAE Sephadex ™ A-50 was superior to that of commercial gelatin-based hemostatic agents with or without thrombin.

As shown for liver experiments, both Sephadex ™ G-50 alone and DEAE Sephadex ™ with sodium salt failed to reduce bleeding intensity.

Pure DEAE failed to reduce bleeding intensity. Thus, if DEAE did not bind to the crosslinked polymer, it was concluded that it did not function as an effective hemostatic agent (ie no significant bleeding reduction occurred). The addition of calcium salt did not improve the hemostatic capacity of DEAE in the absence of the matrix, as can be seen for compositions comprising DEAE with the calcium salt composition (zero reduction). Calcium salts alone have also been demonstrated to have no hemostatic capacity.

This experiment further supported previous experiments and showed that a combination of calcium salt, DEAE groups and matrix is required for proven hemostatic efficacy.

Example 4: Hemostatic properties of a solution comprising an anion exchanger and calcium salt in an in vivo swine spleen problem bleeding model.

In this example, the hemostatic ability of DEAE bound to a crosslinked polymer in the presence of calcium salts is disclosed by Holcombe J.B., Fustheta A., Harris R. et al., Incorporated herein by reference as if fully set forth herein. Further testing in a more challenging model of problem bleeding, modified from the Effect of dry fibrin sealant dressings versus gauze packing on blood loss in grade V liver injuries in resuscitated swine.J Trauma. 1999; 46: 49-58. It was.

DEAE Sephadex ™ A-50 was prepared as a slurry of 10% w / v in 25 mM CaCl ₂ . Punching was performed using a dedicated device (see FIG. 2A) to create an X-shaped wound with each arm 5.5 cm long, with a central hole 1.5 cm deep, bullet shaped and 9.5 mm in diameter (FIG. 2B). The wound showed problematic bleeding (eg, a gunshot wound).

About 20 ml of the tested composition were applied to the wound. Manual compression was applied to the wound for 4 minutes. The tested composition stopped bleeding.

The experiment was repeated three times (two more times) and similar results were obtained.

If there was not enough material to achieve complete hemostasis on the first attempt, additional material was applied and pressed for an additional 3 minutes. Subsequently complete hemostasis was achieved.

Example  5: Evaluation of Matrix Requirements

In the examples below, an in vivo heparinized pig spleen round punch model as described above was used to further evaluate the requirements of the matrix to which DEAE is bound. The preparation of the composition is described in Table 1 above. Compression time was 60 seconds. In this experiment, initial bleeding intensity and post-application bleeding intensity were evaluated according to a scale of 0-5.

DEAE Dextran 500 is a polycationic derivative of dextran, prepared from dextran having an average molecular weight of 500 kD, in which the dextran chains are not crosslinked.

The following compositions were evaluated:

1. DEAE Sephadex ™ A-50 (0.5 ml contains 40 mg of DEAE Sephadex ™ A-50) prepared as a 10% w / v slurry in 20 mM CaCl ₂ solution;

2. DEAE Dextran 500 prepared using 10% (w / w) CaCl ₂ powder (100 mg of powder contains 90 mg of DEAE dextran and 10 mg of CaCl ₂ ).

The bleeding intensity results of the different tested compositions are shown in Table 4.

TABLE 4

DEAE bound to the crosslinked matrix was successful in achieving complete hemostasis, while DEAE not bound to the matrix failed to reduce bleeding intensity.

Example  6: positive for hemostasis Charged  Effect

The density of positively charged groups bound to the matrix as disclosed herein (ie, Presence) is shown to be important for achieving hemostasis. The charge on the matrix should advantageously be present at a density sufficient to achieve hemostasis as described above.

It was shown in the previous examples that not all positive charges evaluated provided the same level of hemostatic efficacy. Thus, in order to assess charge density and charge type, an analysis is performed to ensure that an optimal density range exists.

To this end, an analytical instrument (eg, high pressure liquid chromatography, gas chromatography) capable of monomerizing the synthesized matrix, for example by acid hydrolysis, and separating monomers based on the different charges the monomer has Applies to). Analysis is performed in this manner to assess the charge density on a given molecule.

It is appreciated that certain features of the invention described in connection with the individual embodiments for clarity may also be provided in combination with a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or suitably from any other described embodiment of the invention. Certain features described in connection with the various embodiments are not essential features of these embodiments, so long as the embodiments can be used without these members.

Although the present invention has been described in connection with specific embodiments thereof, it is apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the scope of the appended claims.

The mention or recognition of any reference in this application should not be understood as allowing such reference to be used as prior art to the present invention.

Claims (29)

A composition comprising an anion exchanger and a calcium salt for use as a medicament for inducing hemostasis at a bleeding site. The composition of claim 1, wherein the anion exchanger comprises at least one positively charged group bonded to the matrix. The composition of claim 2, wherein the positively charged group is provided by a base selected from the group consisting of strong bases, weak bases, and combinations thereof. The composition of claim 3, wherein the strong base comprises a quaternary amino group. The composition of claim 3, wherein the weak base comprises an amino group selected from the group consisting of primary amino groups, secondary amino groups, tertiary amino groups, and combinations thereof. The composition of claim 3, wherein the weak base consists of a diethylaminoethyl (DEAE) group. The composition of claim 2, wherein the matrix is selected from the group consisting of aliphatic polyesters, polysaccharides, polypeptides, polystyrene-divinylbenzene, silica, and combinations thereof. 8. The composition of claim 2, wherein the matrix is crosslinked. 9. The composition of claim 8, wherein the matrix is covalently cross-linked. The composition of claim 1, wherein the composition is substantially free of any protein of a blood clotting cascade. The composition of claim 1, wherein the composition is in a form selected from the group consisting of slurry, powder, film, patch and liquid. The composition of claim 11, wherein the composition in slurry or liquid form further comprises a pharmaceutically acceptable carrier. The composition of claim 1, wherein the use involves applying pressure to the composition, optionally towards the bleeding site. Anion exchanger;
Calcium salts; And
Optionally, a hemostatic composition comprising a pharmaceutically acceptable carrier. The hemostatic composition of claim 14, wherein the anion exchanger comprises one or more positively charged groups bonded to the matrix. The hemostatic composition according to claim 15, wherein said positively charged group is provided by a base selected from the group consisting of strong bases, weak bases, and combinations thereof. The hemostatic composition of claim 16, wherein the strong base comprises a quaternary amino group. The hemostatic composition of claim 16, wherein the weak base comprises an amino group selected from the group consisting of primary amino groups, secondary amino groups, tertiary amino groups, and combinations thereof. The hemostatic composition according to claim 16, wherein the weak base consists of a diethylaminoethyl (DEAE) group. 20. The hemostatic composition according to any one of claims 14 to 19, wherein the matrix is selected from the group consisting of aliphatic polyesters, polysaccharides, polypeptides, polystyrene-divinylbenzene, silica and combinations thereof. 21. The hemostatic composition according to any one of claims 15 to 20, wherein the matrix is crosslinked. The hemostatic composition of claim 21 wherein the crosslinked polymer is covalently crosslinked. The hemostatic composition according to any one of claims 20 to 22, wherein the polysaccharide is selected from the group consisting of cellulose, dextran, agarose, and combinations thereof. The hemostatic composition according to any one of claims 15 to 23, wherein the composition is substantially free of any protein of the blood coagulation cascade. The hemostatic composition according to any one of claims 15 to 24, wherein the composition is in a form selected from the group consisting of slurry, powder, film, patch and liquid. The hemostatic composition of claim 25, wherein the composition in slurry or liquid form further comprises a pharmaceutically acceptable carrier. Diethylaminoethyl (DEAE) bound to the matrix; And
Hemostatic composition comprising a calcium salt. Preparing an anion exchanger by covalently bonding at least one positively charged group to the crosslinked matrix; And
Adding a calcium salt to the anion exchanger, a method for producing a hemostatic composition. A hemostatic composition obtained by the method of claim 28.

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