CA2471994A1 - Hemostatic compositions and methods for controlling bleeding - Google Patents

Abstract

The invention provides hemostatic compositions useful to promote hemostasis at active bleeding wound sites. The hemostatic compositions typically include a n article containing cellulose, e.g., cotton gauze, and a polysaccharide covalently linked to the cellulose, or a polysaccharide ionically cross-link ed and in association with the article. Methods of making and using the hemostatic compositions are also provided.

Description

HEMOSTATIC COMPOSITIONS AND METHODS FOR CONTROLLING
BLEEDING
TECHNICAL FIELD
This invention relates to hemostatic compositions and methods employing the same, and more particularly to hemostatic compositions useful for controlling bleeding at active bleeding wotmd sites.
BACKGROUND
Wounds are generally classified as acute or chronic in accordance with their To healing tendencies. Acute wounds, typically those received as a result of surgery or trauma, usually heal uneventfully within an expected time frame. Acute wounds include wounds such as active bleeding wound sites, e.g, wounds that have detectable, unclotted blood. The rapid control of topical bleeding at active bleeding wound sites is of critical importance in wound management, especially fox the management of trauma, e.g., as a ~ 5 result of military exercises or surgery.
A conventional method of controlling bleeding at active bleeding wound sites, such as an external hemorrhage or a surgical wound, advocates the use of cotton gauze pads capable of absorbing 250 ml of blood. Cotton pads are considered passive, however, because of their inability to initiate or accelerate blood clotting. Other formulations have 2o been reported to promote hemostasis and are described in U.S. Pat. Nos.
6,454,787;
6,060,461; 5,196,190; 5,667,501; 4,793,336; 5,679,372; 5,098,417; and 4,405,324. A
hemostatic composition capable of accelerating the coagulation cascade to form a thrombus would be useful.
SUMMARY
25 Accordingly, the invention relates to hemostatic compositions and methods for making and using the same in order to promote hemostasis at active bleeding wound sites.
The present compositions typically include an article which contains cellulose, e.g., cotton gauze, and a polysaccharide covalently linked to the cellulose. In other embodiments, a polysaccharide is sonically cross-linked and in association with an article comprising cellulose. Hemostatic compositions can include additional polysaccharides covalently linced to either or both of the cellulose and the first polysaccharide or physically trapped by a networlc formed by the covalent linking or ionic cross-liucing of the first polysaccharide.
In one aspect of the invention, a method for controlling bleeding at an active bleeding wound site of an animal is provided. The animal can be a mammal. For example, the animal can be a human, horse, bird, dog, cat, sheep, cow, or monkey. The method includes applying a hemostatic composition to the active bleeding wound site.
The hemostatic composition includes an article which contains cellulose and a o polysaccharide, such as dextran, starch, or alginate, covalently linked to the cellulose. If dextran is used, it may be in the form of a bead, e.g., covalently cross-linced dextran beads. The molecular weight of the dextran can range from about 10,000 to about 2,000,000 Daltons, or from about 20,000 to about 100,000 Daltons. When a polysaccharide is linlced to the cellulose, it can have a molecular weight exclusion limit of 15 greater than about 30,000 Daltons.
Articles which contain cellulose can be barriers, structures, or devices useful in surgery, diagnostic procedures, or wound treatment. For example, an article containing cellulose can be a bandage, suture, dressing, gauze, gel, foam, web, film, tape, or patch.
An article containing cellulose can include a cotton material, e.g., cotton gauze. The 2o article can also optionally include adhesives or polymeric laminating materials.
Hemostatic compositions of the present invention are useful for accelerating blood clotting at an active bleeding wound site. Prior to the application of a hemostatic composition, an active bleeding wound site may be characterized in that it bleeds at a rate of from about 0.5 ml/min to about 1000 ml/min. After application of a hemostatic 2s composition, the active bleeding wound site may bleed at a rate of less than 0.03 ml/min.
For example, the rate of less than 0.03 ml/min. may be achieved in from about 2 to about 20 minutes, and in certain embodiments in less than about 5 minutes.
A hemostatic composition can comprise a second polysaccharide covalently linked to the cellulose and, optionally, to the first polysaccharide. The second so polysaccharide may have a different molecular weight than the first polysaccharide. For example, the second polysaccharide may be dextran having a molecular weight from about 800,000 to about 2M. Alternatively, the second polysaccharide may be physically trapped by the covalent linlcing of the first polysaccharide to the cellulose.
In other embodiments, hemostatic compositions of the present invention can include an auticle comprising cellulose in association with a polysaccharide ionically linked to itself (cross-linked). For example, the article comprising cellulose may be coated with, immersed in, or soaked in the polysaccharide, which is subsequently ionically cross-liuced. The polysaccharide may be further covalently linlced to the cellulose of the article. In addition, in certain embodiments, the polysaccharide may be physically trapped in fibers of the article comprising cellulose.
o One example of a polysaccharide that can be ionically cross-linked is alginate.
Alginate can be ionically cross-linlced to itself with metal cations, including Mg2+; Ni2+;
Ca2+; Sr2+; Ba2+; Zn2+; Cd2+; Cu2+; Pb2+; Fe3+; and A13+. In some embodiments, the cation is Ca2+. A second polysaccharide, such as dextran, can also be physically trapped, e.g., by the network formed by the ionic cross-lincing of the first polysaccharide.
~ 5 Dextran can be in the form of cross-linked beads, e.g., dextran that has been previously cross-linked to itself. Dextran can be covalently linked to the bandage, e.g.
by linlcing dextran to the cellulose with epichlorohydrin.
In another aspect, a hemostatic composition can include dextran-alginate spheres, such as ionically linked dextran-alginate spheres, or covalently linked dextran-alginate 2o spheres, or both ionically and covalently linked dextran-alginate spheres.
In another aspect of the invention, hemostatic compositions are provided that include additional agents, such as analgesics, steroids, antihistamines, anesthetics, bactericides, disinfectants, fungicides, vasoconstrictors, hemostatics, chemotherapeutic drugs, antibiotics, lceratolytics, cauterizing agents, antiviral drugs, epidermal growth 25 factor, fibroblast growth factors, transforming growth factors, glycoproteins, collagen, fibrinogen, fibrin, humectants, preservatives, lymphokines, cytolcines, odor controlling materials, vitamins, and clotting factors.
The invention also provides methods for malting hemostatic compositions.
Hemostatic compositions of the present invention can be made by incubating a linking so agent with a polysaccharide and an auticle comprising cellulose to form a hemostatic composition having the polysaccharide covalently linked to the cellulose.

The linlcing agent may be any liucing agent useful for linking available hydroxyl groups on cellulose with available hydroxyl groups on a polysaccharide.
Examples include epichlorohydrin, dichlorohydrin, diepoxyburan, disepoxypropyl ether, or ethylene-glyco-bis-epoxypropylether. The incubation step may occur in an aqueous alkaline solution. The temperature of the incubation step can range from about 40 °C to about 70°C. In certain embodiments, the temperature is about 50 °C. The incubation step can occur for about 1 to about 24 hours. In addition, the incubation step can be in the presence of a stabilizing solution, e.g., a solution designed to prevent or limit evaporation of water. The stabilizing solution can include cellulose acetate butyrate. The covalently liuced polysaccharide may have a molecular weight exclusion limit of greater than 30,000 Daltons.
In certain embodiments of the method, the polysaccharide is dextran. The dextran can be in the form of covalently cross-linked beads. The molecular weight of the dextran can range from about 10,000 to about 2M, or from about 20,000 to about 100,000 ~ 5 Daltons. The incubation step may be occur in an aqueous alkaline solution having about 12% to about 75% dextral.
In another aspect, the invention provides a method of malting a composition including incubating a polysaccharide and a cation with an article containing cellulose in order to form a hemostatic composition having the article containing cellulose in 2o association with an ionically cross-linked polysaccharide. The polysaccharide may be further covalently linked to the cellulose. The cation may be, for example, Ca2+. The Ca2+ may be in the form of, or derived from, Ca2+-loaded cross-linked dextran beads.
The polysaccharide may be sodium alginate or a derivative of alginic acid, including salts of alginic acid.
25 In certain embodiments, the incubation step includes a second polysaccharide; the second polysaccharide may become physically trapped in the three dimensional network formed by the ionic cross-linlcing of the first polysaccharide. The second polysaccharide may be dextran, e.g., dextran in the form of cross-linked beads. The second polysaccharide may be further covalently linked to the cellulose, e.g., through a linlcing 3o agent such as epichlorohydrin.

In another aspect, the invention provides a method for manufacturing a composition, where the method includes the step of mixing all aqueous phase alkaline polysaccharide solution with an organic phase stabilizing agent solution to form a mixture having polysaccharide spheres; incubating a cross-linking agent with the mixture to cross-liuc the polysaccharide spheres; isolating the cross-linked polysaccharide spheres; and coating an article comprising a sodium alginate solution with the cross-linced polysaccharide spheres. The method can include removing the organic phase stabilizing agent from the mixture, e.g., prior to isolating the cross-linlced polysaccharide spheres.
The method can also include exposing the cross-linked polysaccharide spheres to a o solution comprising Ca2+ ions, e.g., washing the cross-linked polysaccharide spheres in a Ca2+ solution. The polysaccharide may be dextran, and the organic phase stabilizing agent solution may include cellulose acetate butyrate.
In certain embodiments of the method,~cross-linlced polysaccharide spheres are between about 30 to about 500 ~,m in size. The mixing and incubating steps may occur at ~5 a temperature of from about 40 °C to about 70 °C. The coating step can include spraying the article with the cross-linked polysaccharide spheres. The invention also relates to hemostatic compositions manufactured according to the above method.
In a further aspect, another method for manufacturing a composition is provided.
The method includes the steps of providing an aqueous phase alkaline solution having 2o dextran and sodium alginate therein; preparing dextran-alginate spheres from the aqueous phase alkaline solution; and incubating the dextran-alginate spheres with a linking agent to link said dextran-alginate spheres. Dextran-alginate spheres can be prepared by any method conventional in the art, including the use of a mechanical droplet generator. A
linlcing agent may covalently or ionically linlc or cross-linlc the dextran-alginate spheres.
25 Accordingly, a linking agent may be epichlorohydrin or a Ca2+-containing salt such as calcium chloride. The dextran-alginate spheres may be linlced with a Ca2+
linlcing agent, axed then linked with an epichlorohydrin linking agent, or the lincing can be performed in the reverse order, or simultaneously. The method can fizrther including coating, e.g., spraying, an article, such as an article comprising sodium alginate, with the linked 3o dextran-alginate spheres. The invention also includes hemostatic compositions manufactured according to the method.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as corrunonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are s described below. All publications, patents, patent applications, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not meant to be limiting.
The details of one or more embodiments of the invention are set forth in the o accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF DRAWINGS
Lilce reference symbols in the various drawings indicate lilce elements.

As used herein, the terms "linking" or "linlced" are meant to indicate either a covalent or ionic link, either direct or mediated by a chemical moiety or an ion, between two chemically distinct entities, e.g., dextran liu~ed to cellulose. The teen "cross-link" is meant to indicate a covalent or ionic linlc, either direct or mediated by a chemical moiety 20 or ion, between two chemically similar moieties, e.g., dextran cross-linked to itself;
alginate cross-linlced to itself. The chemically similar moieties do not have to be identical. For example, dextran having a particular average molecular weight range includes dextran molecules of a variety of molecular weights, and thus the dextran molecules are not identical but chemically similar. When dextran molecules having ail 25 average molecular weight range are linked, e.g., covalently linked with epichlorohydrin, they are said to be "cross-liuced."
The terms "spheres," "particles," or "beads," when used in the context of the present invention, are not meant to imply different sizes, but are meant to be interchangeable terms describing an embodiment of a composition.

The term "active bleeding wound site" means, at a minimum, that unclotted blood is present in the wotmd, e.g., extravascular blood, particularly where the surface of a tissue has been broken or an artery, vein, or capillary system has been compromised. The rate of blood flow from an active bleeding wound site can vary, depending upon the nature of the wound. In some cases, an active bleeding wound site will exhibit blood flow at a rate from about at a rate of from about 0.5 ml/min to about 1000 ml/min. Some active bleeding wound sites may exhibit higher rates of blood flow, e.g., punctures of major arteries such as the aorta. After application of the hemostatic composition, the active bleeding wound site may bleed at a rate of less than 0.03 ml/min. For example, the o rate of less than 0.03 ml/min. may be achieved in from about 2 to about 20 minutes, and in certain embodiments in less than about 5 minutes.
Hemostc~tic Cor~cpositioras The invention relates to hemostatic compositions used to promote hemostasis at ~5 active bleeding wound sites. While not being bound by any theory, it is believed that the hemostatic compositions of the present invention control bleeding by initiating and accelerating blood clotting. The hemostatic compositions of the present invention activate platelets and concentrate high molecular weight components of the coagulation cascade (e.g., clotting factors) by excluding high molecular weight components of the 2o cascade, while absorbing the lower molecular weight components in blood.
Accordingly, coagulation cascade components having a molecular weight higher than about 30,000 Daltons are excluded, including fibrinogen (MW 340,000); prothrombin (MW
70,000);
thrombin (MW 34,000); Factor V (MW 330,000); Factor VII (MW 50,000); Factor VIII
(MW 320,000); von Willebrand factor (MW >850,000); Factor IX (MW 57,000);
Factor 25 X (MW 59,000); Factor XI (MW 143,000); Factor XII (MW 76,000); Factor XIII
(MW
320,000); high MW lcininogen (Fitzgerald Factor) (MW 120,000 - 200,000), and prelcallilcrein (Fletcher Factor) (MW 85,000 -100,000). In addition, laboratory experiments indicate that platelets aggregate around the hemostatic compositions of the present invention when exposed to blood. The net result is that concentrated clotting 3o factors (coagulation cascade components) and activated platelets activate the conversion of prothrombin to thrombin in the presence of Ca2+, which subsequently catalyzes the conversion of fibrinogen to insoluble fibrin multimers, e.g, a fibrin clot.
Additional information on the clotting cascade and hemostatic compositions containing fibrin can be found in U.S. Pat. No. 5,773,033.
Hemostatic compositions typically include an article comprising cellulose, e.g., s cotton gauze, and a polysaccharide covalently linced to the cellulose. In other embodiments, hemostatic compositions include an article comprising cellulose in association with a polysaccharide that is ionically cross-linked. The polysaccharide can be fuuther covalently linlced to the cellulose. Hemostatic compositions can include additional polysaccharides covalently linked to either or both of the cellulose and the first polysaccharide. Other embodiments of hemostatic compositions include linlced and cross-linked polysaccharide spheres, optionally loaded with a ration, e.g., Ca2+.
It should be noted that certain hemostatic compositions comprise both a macroscopic structure (e.g., an article) and a microscopic structure (e.g., networlcs of polysaccharide cross-linkages or networks of polysaccharide covalent linkages to ~s cellulose). Some hemostatic compositions therefore form three dimensional networks of a polysaccharide, either as ionically linked chains or covalently bound to the cellulose of the article. Accordingly, in some embodiments, a second polysaccharide may be physically trapped by the network formed by the first polysaccharide.
Accordingly, in one aspect, a hemostatic composition includes an article 2o containing cellulose and a polysaccharide, such as dextran, starch, or alginate, covalently linked to the cellulose. The article may include natural or synthetic celluloses (e.g., cellulose acetate, cellulose butyrate, cellulose propionate). The polysaccharide chosen should be safe for in vivo use, e.g., non-allergenic, non-toxic, and preferably non-metabolized. Polysaccharides for clinical use are lcnown in the art and available from a 25 variety of sources. See, e.g., U.S. Patent No. 6,303,585.
As used herein, covalent linkages encompass bonds from any of the available chemical moieties of the polysaccharide to any of the available chemical moieties of the cellulose. For example, if the polysaccharide dextran is used, hydroxyl moieties on dextran can be covalently linked to hydroxyl moieties on cellulose through the linlcing 0o agent epichlorohydrin. In that case, a glyceryl bridge linking dextran to cellulose is formed. For additional information, see Flodin, P., and Ingelman, B., "Process for the Manufacture of Hydrophilic High Molecular Weight Substances," British Patent No. 854, 715; and Flodin, P. "Chapter 2: The Preparation of Dextran Gels," Dextran Gels and Their Applications in Gel Filtration, Pharmacia, Uppsala Sweden, 1962, pages 14-26.
The average molecular weight range of the polysaccharide can vary, but typically ranges from about 10,000 to about 2M Daltons. The molecular weight range chosen will affect the molecular weight exclusion limit of the covalently linced polysaccharide, and thus its ability to exclude the coagulation components and concentrate them.
Dextran is a high molecular weight polysaccharide that is water-soluble. It is not metabolized by humans, and is non-toxic and tolerated well by most animals, including humans. The average molecular weight of dextran used in the present invention can range from about 10,000 to about 2,000,000 Daltons, or from about 20,000 to about 100,000 Daltons.
Dextran can be in the form of beads, e.g., covalently cross-linced beads, before it is liuced covalently to the cellulose. Dextran beads can exhibit a range of sizes, e.g., ~5 from about 30 to about 500 Vim. Dextran beads are commercially available, e.g., as SephadexTM (Pharmacia); see, for example UI~ 974,054. Alternatively, dextran beads or particles may be formed during the preparation of the hemostatic composition, e.g., from the covalent cross-linking of previously uncross-linlced dextran molecules.
In other embodiments, dextran may be in solution form, e.g., uncross-linlced, 2o before it is covalently linked to the cellulose. Dextran may be covalently linked to the cellulose and covalently cross-linked to itself, e.g., when exposed to a liucing agent such as epichlorohydrin. When dextran is in solution form (e.g., uncross-linlced), the dextran molecules may coat all or a component of the article, such as fibers of a cotton bandage, so that it subsequently forms a three-dimensional microscopic linked network or mesh 25 when it is covalently linked to the cellulose and covalently cross-linked to itself. Dextran beads linked to cellulose or a cellulose-dextran mesh as described previously contribute to the ability of a hemostatic composition to exclude high molecular weight components of the coagulation cascade.
The average molecular weight of the polysaccharide, the degree of linlcing of the 3o polysaccharide to cellulose, and any cross-linking of the polysaccharide (e.g., to itself) are factors in the molecular weight exclusion limit of the polysaccharide in a hemostatic compositon and the water regain of a hemostatic composition. Water regain is defined as the weight of water taken up by 1 g of dry hemostatic composition and can be determined by methods known in the art. For example, it is lcnown that small changes in dextran concentration or liucing agent concentration (e.g., epichlorohydrin) can result in dramatic changes in water regain. Typically, at lower molecular weights of dextran, a higher water regain results. See Flodin, P., "Chapter 2: The Preparation of Dextran Gels,"
Dextran Gels and Their Applications in Gel Filtration, Pharmacia, Uppsala Sweden, 1962, pages 14-26.
Similarly, the degree of hydration of the polysaccharide also affects the molecular 1 o weight exclusion limit. As the degree of hydration increases, the molecular weight exclusion Iimit of the polysaccharide usually increases. Typically, when dextran is linced to cellulose, the dextran will have a molecular weight exclusion limit of greater than about 30,000 Daltons, thus effectively excluding the components of the coagulation cascade and concentrating them on the microscopic surface of the hemostatic 15 composition.
Articles which contain cellulose can be any barriers, structures, or devices useful in surgery, diagnostic procedures, or wound treatment. For example, an article containing cellulose can be a bandage, suture, dressing, gauze, gel, foam, web, film, tape, or patch.
An article containing cellulose can include a cotton material, e.g., cotton gauze. The 2o article should allow the polysaccharide linked to the cellulose to interact with the wound site.
A hemostatic composition can comprise a second polysaccharide covalently linked to cellulose. The second polysaccharide may have a different molecular weight than the first polysaccharide. For example, the second polysaccharide may be dextran 25 having a molecular weight from about 800,000 to about 2M. The second polysaccharide may be covalently linked to the cellulose at a time after the first polysaccharide, at the same time as the first polysaccharide, or at a time before the first polysaccharide.
In other embodiments, hemostatic compositions of the present invention can include an article containing cellulose in association with an ionically cross-linked so polysaccharide. The polysaccharide may be further covalently linked to the cellulose. In this context, ionic linkages include ion-mediated bonds between available chemical to moieties on the polysaccharide. Typical chemical moieties that can be mediated with an ion (e.g., a cation) include hydroxyl moieties. For example, sodium alginate or alginic acid salts can be sonically liuced with metal cations, including Mg2+, Ni2+, Ca2+, Sr2+, Ba2+, Zn2+, Cd2+, Cu2+, Pb2+, Fe3+, and Al3+. Typically, Ca2+ may be used. The alginate can be of any type, including type G (L-guluronic acid) or type M (D-mammronic -acid), or mixed M and G For more information on alginate, see U.S.
Pat.
No. 5,144,016.
In certain embodiments, a second polysaccharide, such as dextran, can be physically trapped in the networlc formed by the ionic cross-linking of the first polysaccharide. Dextran can be in the form of covalently cross-Iinlced beads, e.g., dextran that has been previously cross-linked to itself with epichlorohydrin, or SephadexTM beads.
Alternatively, the dextran can be in solution form (e.g., mlcross-linked), as described above. In addition, dextran can be covalently linlced to the cellulose, e.g.
by linking dextran to the cellulose with epichlorohydrin. Accordingly, dextran may become cross-15 Iinced to itself.
Other embodiments of hemostatic compositions include dextran-alginate spheres, such as sonically linked dextran-alginate spheres, or covalently linlced dextran-alginate spheres, or both sonically and covalently linced dextran-alginate spheres. In addition, cross-linlced dextran spheres loaded with Ca2+ ions are also included as hemostatic 2o compositions of the present invention.
Hemostatic compositions can include additional agents, such as analgesics, steroids, antihistamines, anesthetics, bactericides, disinfectants, fungicides, vasoconstrictors, hemostatics, chemotherapeutic drugs, antibiotics, Iceratolytics, cauterizing agents, antiviral drugs, epidermal growth factor, fibroblast growth factors, 25 transforming growth factors, glycoproteins, collagen, fibrinogen, fibrin, humectants, preservatives, lympholcines, cytolcines, odor controlling materials, vitamins, and clotting factors. For further information on these additional agents for incorporation, refer to WO
00/27327.
Hemostatic compositions may be used in combination with polymeric laminating so materials and adhesives to provide both mechanical support and flexibility to an article and to facilitate adhesion to the wound. Additional information on such polymeric laminating materials and adhesives for use in the present invention can be found in, e.g., WO 00/27327.
Pha~maceutieal Compositions The present invention also contemplates pharmaceutical compositions comprising certain hemostatic compositions of the present invention, e.g., dextran-alginate linlced spheres or calcium-loaded cross-linced dextran spheres. Pharmaceutical compositions may be formulated in conventional mamlers using one or more physiologically acceptable carriers containing excipients and auxiliaries. Proper formulation is dependent upon the route of administration chosen.
A "pharmaceutically acceptable carrier" (also referred to herein as an "excipient") is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more hemostatic compositions to a subject. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected 15 With the planned manner of administration in mind so as to provide for the desired bulls, consistency, and other pertinent transport and chemical properties, when combined with a hemostatic composition. Other components may be present in a pharmaceutical composition, if desired.
Pharmaceutical compositions of the present invention can be administered by a 2o number of methods depending upon the area to be treated. Administration can be, for example, topical or parenteral. Administration can be rapid (e.g., by injection) or can occur over a period of time. For treating tissues in the central nervous system, pharmaceutical compositions can be administered by injection or infusion into the cerebrospinal fluid, preferably with one or more agents capable of promoting penetration 25 of the pharmaceutical composition across the blood-brain barrier.
Formulations for topical administration include, for example, sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents and other suitable additives. Pharmaceutical compositions and formulations for topical so administration can include patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thiclceners and the like may be necessary or desirable.
Compositions and formulations for parenteral administration can include sterile aqueous solutions, which also can contain buffers, diluents and other suitable additives (e.g., penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers).
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, aqueous suspensions, and liposome-containing formulations.
These compositions can be generated from a variety of components that include, for o example, preformed liquids, self emulsifying solids and self emulsifying semisolids.
Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other; in general, emulsions are either of the water-in-oiI (w/o) or oil-in-water (o/w) variety. Emulsion formulations have been widely used for oral delivery of therapeutics due to their ease of formulation and efficacy of ~ 5 solubilization, absorption, and bioavailability.
Pharmaceutical compositions of the invention further encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. The term 20 "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the hemostatic compositions of the invention (i.e., salts that retain the desired biological activity without imparting undesired toxicological effects). Examples of pharmaceutically acceptable salts include, but are not limited to, salts formed with cations (e.g., sodium, potassium, calcium, or polyamines such as spermine);
acid addition 2s salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or nitric acid); salts formed with organic acids (e.g., acetic acid, citric acid, oxalic acid, pahnitic acid, or fumaric acid); and salts formed from elemental anions (e.g., chlorine, bromine, and iodine).

Methods of Corrt~°olling Bleeding hi one aspect of the invention, a method for controlling bleeding at an active bleeding wound site of an animal is provided. The method includes applying a hemostatic composition to the active bleeding wound site. Application of the hemostatic composition typically includes contacting the hemostatic composition with the wound or bleeding site surface. The hemostatic composition is maintained in contact with the wound or bleeding site for a period of time sufficient to control the bleeding, e.g., to clot the blood, slow the rate of bleeding, or stop the bleeding. The application may include the use of pressure, e.g., by using an elastic bandage to maintain contact with the bleeding 1 o site. Alternatively, an internal wound may be paclced with a hemostatic composition until hemostasis is achieved. In other embodiments, a hemostatic composition is delivered to the wound site. For example, a catheter or needle may be used to deliver a hemostatic composition to an intravascular puncture site or to a biopsy site. The catheter or the needle may be optionally coated with a hemostatic composition of the present invention.
Usually a hemostatic composition can control bleeding, for example, to a rate of less than 0.03 mlhnin, in a period of from about 2 to about 20 minutes. In certain embodiments, bleeding stops immediately, or in less than about 5 minutes.
Typically a hemostatic compositions of the present invention will be used to inhibit or completely stop bleeding of a parenchymal organ, such as the liver, kidney, 2o spleen, pancreas, or lungs; or to control bleeding during surgery (e.g., abdominal, vascular, gynecological, dental, tissue transplantation surgery, etc.). For example, percutaneous needle biopsies are common interventional medical procedures.
Possible complications of needle biopsies, however, include bleeding at the biopsy site. The amount of bleeding is related to the needle size, tissue sample size, location of the biopsy, and vascularization of the tissue. Hemostatic compositions of the present invention can be used to promote hemostasis at needle biopsy sites. Biopsy needles may either be coated with hemostatic compositions of the present invention, or may be used to deliver a hemostatic composition to the biopsy site. For more information on biopsy tracts, see U.S. 6,447,534.
so Similarly, catheterization and interventional procedures, such as angioplasty and stenting, generally are performed by inserting a hollow needle through a patient's skin and muscle tissue into the vascular system. A guide wire is then typically passed through the needle lumen into a blood vessel. The needle is removed and an introduces sheath is advanced over the guide wire into the vessel, and a catheter is typically passed through the lumen of the introduces sheath and advanced over the guide wire for p051tlolllllg.
s Upon completion of the medical procedure, the catheter and introduces sheath are removed, often leaving a puncture site in the vessel, with associated bleeding.
Hemostatic compositions of the present invention may be used to coat the exterior of catheters, stems, introduces sheath, and guide wires, etc., or may be delivered, e.g., via a catheter, to the puncture site in order to promote hemostasis. For additional information, o see U.S. Pat. No. 6,391,048.
The amount of hemostatic composition to be used will vary with the patient, the wound, and the composition employed. For example, hemostatic compositions with varying water regains can be assembled (e.g., stacked in descending order) for use in major bleeding to attain hemostasis.
Methods fog Making HenZOStatic Conzpositaohs In another aspect, the invention provides methods for malting hemostatic compositions. The hemostatic compositions of the present invention can be made by incubating a linking agent with a polysaccharide and an article containing cellulose to 2o form a hemostatic composition having the polysaccharide covalently lil~lced to the cellulose.
Any biologically compatible bifunctional or heterobifunctional reagent may be used as the linking agent, including reagents with halogens, epoxides, hydroxy succinimide esters, aldehydes, activated thiols, or other moieties for reacting free amines, 25 hydroxides, hydroxyls, or sulfhydryls on the bandage or on the polysaccharide. The bandage may be modified, e.g., derivatized, to incorporate reactive moieties such as amines or sulfhydryls for reacting with a particular linking agent. The polysaccharide may also be modified, e.g., derivatized, in a similar manner, provided that the polysaccharide so derivatized remains pharmaceutically suitable for animal, e.g., human 3o use. The linlcing agent may be epichlorohydrin, dichlorohydrin, diepoxyburan, disepoxypropyl ether, or ethylene-glyco-bis-epoxypropylether. For additional information, see Flodin, P., and Ingelman, B., "Process for the Manufacture of Hydrophilic High Molecular Weight Substances," British Patent No. 854, 715;
and Flodin, P., "Chapter 2: The Preparation of Dextran Gels," Dextran Gels and Their Applications in Gel Filtration, Phannacia, Uppsala Sweden, 1962, pages 14-26.
The incubation step may occur in an aqueous alkaline solution. Typically, the polysaccharide is from about 10% to about 80% wt/vol of the aqueous alkaline solution.
The concentration of the liucing agent in the incubation step can range from about 2% to about 20% wt/wt of the polysaccharide.
The temperature of the incubation step can range from about 40 °C to about 70°C.
In certain embodiments, the temperature is about 50 °C. The incubation step can occur for about 1 to about 24 hours. The incubating step may also include agitation of the reagents. In addition, the incubation step can be in the presence of a stabilizing solution, e.g., a solution designed to prevent or limit evaporation of water. The stabilizing solution can include cellulose acetate butyrate. The method can also include neutralizing the ~5 aqueous allcaline solution, e.g., with an acid such as HCl at a concentration of from 1 to SM.
The hemostatic composition can be washed with an aqueous solution, e.g., distilled water, or an aqueous alcoholic wash, e.g., 50/50 vol/vol EtOH/water.
The hemostatic composition can be serially washed in increasing amounts of an alcoholic 2o wash, such as 25%, 50%, 75%, and 100% EtOH. The alcohol wash solution can contain a humectant, e.g., glycerin, at a concentration of about 0.1 to about 2.0%.
The hemostatic composition can be dried, e.g., at about 50°C to about 80 °C.
For example, the hemostatic composition can be dried at 70°C After drying, the covalently linked polysaccharide may have a molecular weight exclusion limit of greater than 30,000 25 Daltons.
In certain embodiments of the method, the polysaccharide is dextran. The dextran can be in the form of covalently cross-linked beads. The molecular weight of the dextran can range from about 10,000 to about 2M, or from about 20,000 to about 100,000 Daltons. Typically, dextran of MW 40,000 is used. The incubation step may be occur in 3o an aqueous allcaline solution having about 12 to about 75% dextran wt/vol.

In another aspect, the invention provides a method of malting a hemostatic composition including incubating a polysaccharide slid a cation with an article containing cellulose in order to form a hemostatic composition having the article containing cellulose in association with an ionically cross-liuced polysaccharide. The polysaccharide may be further covalently liuced to the cellulose. The cation may be as described previously, including, for example, Ca2+. The Ca2+ may be in the form of, or derived from, Ca2+-loaded cross-liuced, dextran beads. The polysaccharide may be sodium alginate or derivatives of alginic acid, including salts of alginic acid. Aqueous and alcoholic washes of the hemostatic composition can be performed, as described previously.
o In certain embodiments, the incubation step includes a second polysaccharide.
The second polysaccharide may be dextran, e.g., dextran in the form of covalently cross-linlced beads. The second polysaccharide may be physically trapped, e.g., in the three-dimensional network formed by the ionic cross-lii~lcing of the first polysaccharide. The second polysaccharide may be further covalently linlced to the bandage, e.g., through a 15 linking agent such as epichlorohydrin.
In one embodiment, an article such as cellulose gauze is immersed in a solution of a first polysaccharide (e.g., about 1 to 5% sodium alginate) and a second polysaccharide (e.g., 20% dextran, avg. molecular weight 40,000). The first polysaccharide is ionically cross-linlced with a canon solution, e.g., Ca2+ from a solution having about 0.5 to about 20 10% aqueous calcium chloride. The Ca2+ concentration can be reduced with serial washes, e.g., to reduce the Ca2+ concentration to about 0.5% Ca2+. The first or second polysaccharide, or both, may then be covalently linlced to the cellulose and/or cross-linl~ed using an aqueous alkaline solution (e.g., 20% NaOH) of a linking agent, e.g., epichlorohydrin (e.g., at about 3 - 6% of the weight of the second polysaccharide). The 25 resulting hemostatic composition may be dried as described previously.
In another embodiment of the present invention, cross-linked polysaccharide spheres can be mixed with a Ca2+-alcoholic wash solution (e.g., 1% calcium chloride in neat alcohol). The cross-linced polysaccharide spheres can be purchased, e.g., as SephadexTM, or can be prepared from an aqueous allcaline polysaccharide solution and a so cross-linking agent (e.g., dextran cross-linked with epichlorohydrin), as discussed previously. After washing in the Ca2+-alcohol solution, the cross-linked polysaccharide spheres have Ca2+ in their pores, e.g., are Ca2+-loaded spheres, and can be used to coat an article, e.g., sprayed onto a bandage, that has been previously soaked or innnersed in a polysaccharide solution, e.g., sodium alginate, at a concentration of about 0.5 - 5%
polysaccharide. In certain embodiments, Ca2+ from the cross-liuced spheres exchanges with the sodium from the sodium alginate solution, resulting in ionically cross-linced calcium alginate, which physically traps cross-linlced polysaccharide spheres in the three-dimensional network of ionic bonding. The hemostatic composition so formed may be dried as discussed previously.
In another aspect, the invention provides a method for manufacturing a o composition, where the method includes the step of mixing an aqueous phase allcaline polysaccharide solution with an organic phase stabilizing agent solution to form a mixture having polysaccharide spheres; incubating a cross-linl~ing agent with the mixture to cross-linlc the polysaccharide spheres; isolating the cross-linl~ed polysaccharide spheres; and coating an article comprising a sodium alginate solution with the cross-linked polysaccharide spheres. The method can include removing the orgaxlic phase stabilizing agent from the mixture, e.g., prior to isolating the cross-linked polysaccharide spheres.
The method can also include exposing the cross-liuced polysaccharide spheres to a solution comprising Ca2+ ions, e.g., washing the cross-linked polysaccharide spheres in a Ca2+ solution. The polysaccharide may be dextran, and the organic phase stabilizing 2o agent solution may include cellulose acetate butyrate.
In certain embodiments of the method, the cross-linlced polysaccharide spheres are between about 30 to about 500 ~m in size. The mixing and incubating steps may occur at a temperature of from about 40 °C to about 70 °C. The coating step can include spraying the article with the cross-linlced polysaccharide spheres. The invention also relates to hemostatic compositions manufactured according to the above method.
In a further aspect, another method for manufacturing a composition is provided.
The method includes the steps of providing an aqueous phase allcaline solution having dextran and sodium alginate therein; preparing dextran-alginate spheres from the aqueous phase allcaline solution; and incubating the dextran-alginate spheres with a linlcing agent so to form linlced dextran-alginate spheres. The dextran-alginate spheres can be prepared by any method conventional in the art, including the use of a mechanical droplet generator or an air lazife.
The linking agent may covalently or ionically linlc and/or cross-liuc the dextran alginate spheres. Accordingly, the linking agent may be epichlorohydrin or a cation as described previously, e.g., a Ca2+-containing salt. The dextran-alginate spheres may be first linked with a cation liucing agent, and then linked with an epichlorohydrin linlcing agent, or vice versa. The dextran-alginate spheres may be liuced simultaneously with a canon linking agent and an epichlorohydrin lincing agent. The method can further including coating, e.g., spraying, an article with the linked dextran-alginate spheres. The o invention also is directed to hemostatic compositions manufactured according to the method.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
EXAMPLES
Example 1: Cotton-Dextran Compositions Pharmaceutically acceptable cotton-based compositions were incubated with 2o dextran (40,000 MW) in an allcaline epichlorohydrin solution (20% dextran in NaOH
wt/vol; epichlorohydrin at about 3 to 6% wt/wt dextran). The solutions were allowed to react for about 1 to about 16 hours at a temperature range from about ambient room temperature to about 60°C. The resulting cross-linlcing reactions were subsequently neutralized using a 1 to 5 Molar HCl solution.
The cross-linked and linlced dextran-cotton hemostatic compositions were washed about 4 times with distilled water. The products were further washed twice with a 50%
distilled water/alcohol solution, then with a 75% alcohol solution, and lastly with a 100%
alcohol solution, to remove excess epichlorohydrin. A final alcohol wash solution contained about 0.1 to about 2% glycerin to keep the composition from becoming brittle.
so The hemostatic composition was dried at about 70°C overnight.

Example 2: Cotton-Alginate-Dextran Compositions A stabilizing agent, such as cellulose acetate butyrate was dissolved in ethylene dichloride at 3% wt/vol and heated to about 50 °C while stirring at about 200 RPM in a 1-2 liter cylindrical reaction vessel. Dextran (MW 40,000) was dissolved in water at 15%
wt/vol with SN NaOH at 2% of the dextran weight. The dextran solution was gradually added to the stabilizing mixture with continued heating and stirring. When droplets of the desired size were formed (30-500 ~.m), a cross-linlcing agent such as epichlorohydrin was added to the vessel at 20% of the dextran weight. The reaction formed cross-linlced gel spheres in 1 - 3 hours, but was allowed to proceed up to 16 hours before termination.
o Acetone was added and decanted twice to remove the stabilizer (cellulose acetate butyrate). The spheres were then treated with NaOH (equal parts of 2N NaOH and 95%
ethyl alcohol) for about 15 rains., and neutralized with dilute acid (1N HCl) before filtration and washing with water. The swollen spheres were shrunc with alcohol treatment (25, 50, 75, 100% serial alcohol washes).
~5 Dry calcium chloride was mixed with a second 100% alcohol wash solution (1%
calcium chloride in alcohol), which was used to wash the cross-linked dextran particles.
The alcohol was evaporated off, trapping the calcium in the pores of the dextran particles.
The final products Were dry cross-liuced dextran-calcium ion compositions.
Pharmaceutically acceptable cotton-based compositions were irrnnersed (dipped) 2o into a sodium alginate liquid solution (0.5 - 4%). After removal from the solution, the wet sodium alginate coated cotton materials were sprayed or dusted with the cross-linked dextran calcium compositions. Calcium exchanged with sodium, resulting in cross-linced calcium alginate. The cotton-dextran-alginate hemostatic compositions were then dried at 70 °C overnight.
Example 3: Alternative Method to Prepare Cotton-Dextran-Alginate Compositions Cotton gauzes were immersed in solutions of 1 to 5% sodium alginate and 20%
dextran (40,000 MW). The mixtures were cross-linked and linked with about 0.5%
to 3o about 10% aqueous calcium chloride solution. The compositions were washed to reduce Ca2+ concentration to about 0.5% Ca2+.

Dextran-alginate cross-linking solutions were prepared using an aqueous alkaline epichlorohydrin solution, where the concentration of the epichlorohydrin was fiom about 3 to about 6% by weight of the dextran. The solutions included about 20% NaOH.
The resulting hemostatic compositions were allowed to dry at ambient to about 60 °C
overnight.
Example 4: Cotton-Dextran Compositions Dextran (MW 40,000) was dissolved in 1N NaOH at a range of 18-69% wt/vol of dextran in the alkaline solution. Epichlorohydrin was added to a concentration of 20% of o the dextran by weight at room temperature. Pharmaceutically acceptable cotton (cellulose) gauzes were added to the allcaline epichlorohydrin solutions. The solutions were allowed to react with agitation at 25 °C to 70°C. The mixtures were heated until a cross-liuced dextran-cellulose gel formed on the gauze fibers, from 1-6 hours, up to 24 hours. After neutralization with dilute HCl (1N), successive washes removed excess 5 reaction products and impurities: four times in distilled water, then with increasing concentrations of alcohol (25, 50, 75, 100%). A final alcohol wash of 100%
EtOH
contained from 0.1 to 2.0% glycerin to keep the compositions pliable. The resulting hemostatic compositions were dried at about 70 °C overnight.
The water regain for the hemostatic compositions ranged from 2.5 ml/g to 35 2o ml/g. For information on water regain, see Flodin, P., Dextran Gels and Their A~~lications in Gel Filtration, Pharmacia, Uppsala Sweden, 1962, pages 31-32.
Example 5: Cellulose-Dextran Composition 150 g of dextran (MW 40,000) was dissolved in 300 ml 1N NaOH. 30 g 25 epichlorohydrin was quiclcly mixed with the dextran solution at room temperature. A
pharmaceutically acceptable cellulose gauze was dipped in the alkaline epichlorohydrin solution to thoroughly coat the fibers with the reaction solution, then placed in a flat-bottomed dish. The gauze was heated to 50 °C in a humidified atmosphere, with gentle roclcing after 1 hour, until a dextran-cellulose gel formed on the gauze fibers, typically in 30 1-2 hours. Heating was continued until the desired end-point, up to 24 hours. The gauze was neutralized, washed, treated with glycerin, and dried as described previously. The water regain was 7.5 ml/g.
Example 6: Cellulose-Dextran Com osition Dextran (MW 40,000) was dissolved in 1N NaOH (34% dextran wt/vol of the alkaline solution). Epichlorohydrin was added to a concentration of 20% of the dextran by weight at room temperature. A pharmaceutically acceptable cellulose fiber based composition, 16-ply 4 x 4 gauze, was dipped in the alkaline epichlorohydrin solution to thoroughly coat the fibers with the reaction solution, then placed in a flat bottomed dish.
~o To prevent concentrating the reaction solution by evaporation, a stabilizing solution of cellulose acetate butyrate in ethylene dichloride (3% wt/vol), which is immiscible in water, was used to cover the gauze. The gauze was heated at 50 °C, with gentle rocking after 1 hr., until a cross-linlced and liuced dextran-cellulose gel formed on the gauze fibers, typically in about 2 to 3 hours. Heating was continued until the desired endpoint, ~5 up to 24 hours. Acetone was added and decanted twice to remove the stabilizer. The gauze was neutralized with dilute HCl and washed as described previously in aqueous alcohol and alcohol solutions and dried at about 70 °C overnight. The water regain was ml/g.
2o Example 7 Cellulose Dextran Composition Dextran (MW 40,000) was dissolved in 1N NaOH and reacted with gauze in the presence of epichlorohydrin, followed by neutralization, washing, and drying as described above. Cellulose-dextran compositions were made by varying the volume of dextran in the solvent from 12 to 75 wt/vol to produce hemostatic composition with water regains ranging from 5 ml/g to 35 ml/g. The compositions can be assembled for use in major bleeding by stacking them in descending order (e.g., 35 ml/g to 5 ml/g) to attain hemostasis.

Example 8 Dextran-Calcimn Spheres A non-ionic polymer substance was dissolved in a suitable solvent with an alkaline solution added as a cross-liucing catalyst. A stabilizer was dissolved in a solvent that was immiscible with the polymer solvent and placed in a cylindrical vessel. The stabilizer solution formed the continuous phase and was heated with regular stirring.
When the polymer solution was added to the stabilizer solution, a biphasic system formed in which the polymer droplets became the dispersed phase. A bifunctional cross-linking agent was added to the system which caused co-polymerization (cross-linking) of the polymer to form gel spheres.
o After purification and drying, the water regain of the spheres was determined in order to classify the molecular sieving capability of the cross-linced polymer. Water regain can be determined by methods well known in the art, and generally involves hydrating 1 gram of dry composition, and determining the amount of water absorbed by thel g of the dry composition. Generally, greater swelling capacity relates to larger pores ~5 (e.g., less cross-linking) and a higher molecular weight exclusion.
More specifically, a stabilizing agent, such as cellulose acetate butyrate was dissolved in ethylene dichloride at 3% wt/vol and heated to about 50 °C
while stirring at about 200 RPM in a 1-2 liter cylindrical reaction vessel. Dextran (MW 40,000) was dissolved in water at 15% wt/vol with SN NaOH at 2% of the dextran weight. The 2o dextran solution was gradually added to the stabilizing mixture with continued heating and stirring. When droplets of the desired size were formed (30-500 ~,m), a cross-linking agent such as epichlorohydrin was added to the vessel at 20% of the dextran weight. The reaction formed cross-linced gel spheres in 1 - 3 hours, but was allowed to proceed up to 16 hours before termination. Acetone was added and decanted twice to remove the 25 stabilizer (cellulose acetate butyrate). The spheres were then treated with NaOH (equal parts of 2N NaOH and 95% ethyl alcohol) for about 15 rains., and neutralized with dilute acid (1N HCl) before filtration and washing with water. The swollen spheres were shrunk with alcohol treatment (25, 50, 75, 100% serial alcohol washes) as described previously. A final alcohol wash contained calcium chloride (0.04-1%). The alcohol was 3o evaporated by drying at about 70 °C, thus trapping the calcium in the pores and on the surface of the dextran spheres. The final product was a dry cross-linced dextran-calcitun ion composition. The water regain was 20 ml/g.
Example 9: Cotton-Alginate-Dextran Compositions A pharmaceutically acceptable cotton (cellulose) gauze was immersed in or sprayed with a sodium alginate solution (0.5 - 4%). The wet sodium alginate coated gauzes were sprayed or dusted with dextran-calcium spheres, prepared as described previously. Calcium exchanged with sodium in the alginate, resulting in cross-linced calcium alginate. Ionic bonds formed between cellulose, alginate, and dextran, thereby o chemically incorporating the spheres into the gauze to form the hemostatic compositions.
Example 10 Dextran-Alginate Spheres 43 g dextran (MVV 40,000) was dissolved in 50 ml 2N NaOH and mixed with a 50 ml solution of 2% sodium alginate (43% dextran and 1% alginate in 100 ml of 1 N
NaOH). 50-200 ~m dextran-alginate droplets were linked and cross-linked in a solution of 1.7% calciwn chloride. The spheres were further linked and cross-linked by adding 7.2 ml epichlorohydrin to 100 ml of the calcium chloride solution at 45 °C with agitation.
The reaction continued for up to 16 hours until the desired amount of linking and cross-linlcing occurred. The spheres were neutralized with dilute HCI, washed in increasing 2o concentrations of alcohol (25, 50, 75, 100%) and dried at about 70 °C overnight. The water regain was 10 ml/g.
Example 11 Cotton-Dextran-Alginate there Composition Cross-linked and linced dextran-alginate spheres, prepared as described previously, were washed in a final calcium chloride alcohol wash (0.04-1%
calcium chloride). The alcohol was evaporated to trap the calcium in the pores of the spheres.
The spheres were then chemically incorporated into gauze containing sodium alginate, as described previously.

Examule 12: Comuositions Having Dextrans of Different Molecular Weights 100 g of dextran (MW 2,000,000) dissolved in 2000 ml O.SN NaOH was mixed with 30 g of dextran spheres, prepared as described previously. The cross-linked spheres and dextran solutions were mixed, and 100 g epichlorohydrin added. A
pharmaceutically acceptable cellulose gauze was placed in a flat bottomed dish and the allcaline epichlorohydrin/dextran sphere/dextran solution was slowly poured over the gauze to thoroughly coat the fibers with the dextran spheres and dextran solution The coated gauze was heated at 50 °C with gentle rocking, until a gel formed on the gauze fibers, typically in 1 - 2 hours. The coated gauze was removed from the solution and placed in a o flat bottom dish and heating was continued until the desired end-point, approximately 8 12 hours. The gauze was neutralized, washed, and dried, as described previously.
Example 13: Tests of Cotton-Alginate-Dextran Compositions and Calcium-Dextran S_pher es ~ 5 Two different alginates (G, M) were applied in 0.5% and/or 1 %
concentrations to 12-ply 2 x 2 cotton gauze squares, Type VII, in either 2 or 4 mI; amounts. 0.5 g of calcium-dextran spheres (containing 3, 6, 12, or 24 mM calcium) were ionically bonded to the sodium alginate on the gauze. Freshly drawn human blood was added to each test sample, with 0.5 mls added to each calcium-dextran-alginate gauze and 0.1 ml added to 2o the calcium-dextran spheres alone. Gauze irmnersed only in sodium alginate was used as a control.
The blood added to the calcium-dextran spheres clotted upon contact. All of the calcium-dextran-alginate gauzes clotted the blood in less than 5 minutes, whereas alginate only gauzes produced wealcly clotted blood in > Smins. See Table 1 below.

Table 1: Clotting Times for Hemostatic Compositions gauze + gauze + gauze + gauze +

4 4 ml 2 ml 4 ml ml .5 % 1 % alginate1 % alginate1 % alginate alginate G G M
G

0 mM Ca++

> 5 min. > 5 min. > 5 min. > 5 min.

control 3 mM Ca++

dextran instantly not tested< 5 min. not testednot tested spheres 6 mM Ca++ instantly < 5 min. < 5 min. < 5 min. < 5 min.

dextran spheres 12 mM

instantly < 5 min. < 5 min. < 5 min. < 5 min.
Ca++

dextran spheres 24 mM < 5 min. < 5 min. < 5 min. < 5 min.
instantly Ca++

dextran spheres OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims.
Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (87)

WHAT IS CLAIMED IS:

1. A method for controlling bleeding at an active bleeding wound site of an animal, said method comprising applying a hemostatic composition to said active bleeding wound site, said hemostatic composition comprising an article comprising cellulose and a polysaccharide covalently linked to said cellulose.

2. The method of claim 1, wherein said polysaccharide is selected from the group consisting of dextran, starch, and alginate.

3. The method of claim 2, wherein said polysaccharide is dextran.

4. The method of claim 3, wherein said dextran is in the form covalently cross-linked beads.

5. The method of claim 3, wherein the molecular weight of said dextran is from about 10,000 to about 2,000,000.

6. The method of claim 5, wherein the molecular weight of said dextran ranges from about 20,000 to about 100,000.

7. The method of claim 1, wherein said covalently linked polysaccharide has a molecular weight exclusion limit greater than about 30,000 Daltons.

8. The method of claim 1, wherein said article comprising cellulose is selected from the group consisting of a bandage, suture, dressing, gauze, gel, foam, web, film, tape, or patch.

9. The method of claim 1, wherein said article comprising cellulose comprises cotton.

10. The method of claim 9, wherein said article comprising cellulose is cotton gauze.

11. The method of claim 1, wherein prior to said application of said hemostatic composition, said active bleeding wound site bleeds at a rate from about 0.5 ml/min to about 1000 ml/min.

12. The method of claim 11, wherein after application of said hemostatic composition, said active bleeding wound site bleeds at a rate of less than 0.03 ml/min.

13. The method of claim 12, wherein said rate of less than 0.03 ml/min is achieved from about 2 to about 20 minutes after application of said bandage.

14. The method of claim 7, wherein said hemostatic composition further comprises a second polysaccharide covalently linked to said cellulose.

15. The method of claim 7, wherein said hemostatic composition further comprises dextran having a molecular weight from about 800,000 to about 2M
covalently linked to said cellulose.

16. A method for controlling bleeding at an active bleeding wound site of an animal, said method comprising applying a hemostatic composition to said active bleeding wound site, said hemostatic composition comprising an article comprising cellulose in association with an ionically cross-linked polysaccharide.

17. The method according to claim 16, wherein said ionically cross-linked polysaccharide is alginate.

18. The method according to claim 17, wherein said alginate is ionically cross-linked with a cation selected from the group consisting of: Mg2+; Ni2+; Ca2+;
Sr2+;
Ba2+; Zn2+; Cd2+; Cu2+; Pb2+; Fe3+; and Al3+.

19. The method according to claim 18, wherein said cation is Ca2+.

20. The method according to claim 17, further comprising a second polysaccharide physically trapped in a network formed by said ionically-cross-linked polysaccharide.

21. The method according to claim 20, wherein said second polysaccharide is dextran.

22. The method according to claim 21, wherein said dextran is in the form of covalently cross-linked beads.

23. The method according to claim 20, wherein said polysaccharide is further covalently linked to said cellulose.

24. The method according to claim 17, wherein said alginate is further covalently linked to dextran.

25. The method according to claim 24, wherein said dextran is in the form of covalently cross-linked beads.

26. The method according to claim 1 or claim 16, wherein said hemostatic composition further comprises an agent selected from the group consisting of analgesics, steroids, antihistamines, anesthetics, bactericides, disinfectants, fungicides, vasoconstrictors, hemostatics, chemotherapeutic drugs, antibiotics, keratolytics, cauterizing agents, antiviral drugs, epidermal growth factor, fibroblast growth factors, transforming growth factors, glycoproteins, collagen, fibrinogen, fibrin, humectants, preservatives, lymphokines, cytokines, odor controlling materials, vitamins, and clotting factors.

27. A method for manufacturing a composition, said method comprising incubating a linking agent, a polysaccharide, and an article comprising cellulose to form a hemostatic composition comprising said polysaccharide covalently linked to said cellulose.

28. The method according to claim 27, wherein said linking agent is epichlorohydrin.

29. The method according to claim 27, wherein said incubating occurs in an aqueous alkaline solution.

30. The method according to claim 27, wherein said incubating occurs at a temperature of from about 40 °C to about 70 °C.

31. The method according to claim 30, wherein said incubating occurs at a temperature of about 50 °C.

32. The method according to claim 27, wherein said incubating is allowed to occur for from about 1 to about 24 hours.

33. The method according to claim 29, further comprising performing said incubating step in the presence of a stabilizing solution.

34. The method according to claim 33, wherein said stabilizing solution comprises cellulose acetate butyrate.

35. The method according to claim 27, wherein said polysaccharide is dextran.

36. The method of claim 35, wherein said dextran is in the form of covalently cross-linked beads.

37. The method of claim 35, wherein the molecular weight of said dextran is from about 10,000 to about 2,000,000.

38. The method of claim 37, wherein the molecular weight of said dextran is from about 20,000 to about 100,000.

39. The method of claim 29, wherein said incubating is carried out in an aqueous alkaline solution having about 12% to about 75% dextran.

40. The method of claim 27, wherein said covalently linked polysaccharide has a molecular weight exclusion limit greater than about 30,000 Daltons.

41. The method of claim 27, wherein said article comprising cellulose comprises cotton.

42. The method of claim 41, wherein said article comprising cellulose is cotton gauze.

43. A method for manufacturing a composition, said method comprising incubating an article comprising cellulose in a solution of a polysaccharide and a cation to form a hemostatic composition wherein said polysaccharide is ionically cross-linked and associated with said article.

44. The method according to claim 43, wherein said polysaccharide is alginate.

45. The method according to claim 43, wherein said cation is Ca2+ ion.

46. The method according to claim 45, wherein said Ca2+ is derived from Ca2+-ion-loaded porous, cross-linked, dextran beads.

47. The method according to claim 43, wherein said solution further comprises a second polysaccharide.

48. The method according to claim 47, wherein said second polysaccharide is dextran.

49. The method according to claim 48, wherein said dextran is in the form of covalently cross-linked beads.

50. The method according to claim 47, wherein said second polysaccharide is physically trapped by a network formed by said ionically cross-linked polysaccharide.

51. The method according to claim 43, further comprising incubating a second polysaccharide and a linking agent with said article comprising cellulose to form a hemostatic composition comprising said second polysaccharide covalently linked to said cellulose.

52. The method according to claim 51, wherein said linking agent is epichlorohydrin.

53. The method according to claim 51, wherein said second polysaccharide is dextran.

54. A method for manufacturing a hemostatic composition, said method comprising:
(a) mixing an aqueous phase alkaline polysaccharide solution with an organic phase stabilizing agent solution to form a mixture comprising polysaccharide spheres;
(b) incubating a cross-linking agent with said mixture to cross-link said polysaccharide spheres;
(c) isolating said cross-linked polysaccharide spheres; and (d) coating an article comprising a sodium alginate solution with said cross-linked polysaccharide spheres.

55. The method of claim 54, wherein said isolating step comprises removing the organic phase stabilizing agent from said mixture.

56. The method of claim 54, further comprising exposing said cross-linked polysaccharide spheres to a solution comprising Ca2+ ions.

57. The method of claim 54, wherein said polysaccharide comprises dextran.

58. The method of claim 54, wherein said organic phase stabilizing agent solution comprises cellulose acetate butyrate.

59. The method of claim 54, wherein said polysaccharide spheres are between about 30 to about 500 µm in size.

60. The method of claim 54, wherein said mixing and said incubating steps occur at a temperature of from about 40 °C to about 70 °C.

61. The method of claim 54, wherein said coating step occurs by spraying said article with said cross-linked polysaccharide spheres.

62. A hemostatic composition manufactured according to the method of claim 54.

63. A method for manufacturing a hemostatic composition, said method comprising:
(a) providing an aqueous phase alkaline solution comprising dextran and sodium alginate;

(b) preparing dextran-alginate spheres from said aqueous phase alkaline solution; and (c) incubating said dextran-alginate spheres with a linking agent to link said dextran-alginate spheres.

64. The method of claim 63, wherein said dextran-alginate spheres are prepared with a mechanical droplet generator.

65. The method of claim 63, wherein said linking agent is epichlorohydrin.

66. The method of claim 63, wherein said linking agent is Ca2+ linking agent.

67. The method of claim 63, wherein said dextran-alginate spheres are incubated with a Ca2+ linking agent and an epichlorohydrin linking agent.

68. The method of claim 63, further comprising coating an article with said linked dextran-alginate spheres.

69. The method of claim 68, wherein said coating is performed by spraying said linked dextran-alginate spheres on said article.

70. The method of claim 68, wherein said article comprises a sodium alginate solution.

71. A hemostatic composition manufactured according to the method of claim 63.

72. A hemostatic composition comprising an article comprising cellulose having a polysaccharide covalently linked to said cellulose.

73. The hemostatic composition of claim 72, wherein said polysaccharide is selected from the group consisting of dextran, starch, and alginate.

74. The hemostatic composition of claim 73, wherein said polysaccharide is dextran.

75. The hemostatic composition of claim 74, wherein said dextran linked to said cellulose is in the form of covalently cross-linked beads.

76. The hemostatic composition of claim 73, wherein the molecular weight of said dextran ranges from about 10,000 to about 2,000,000.

77. The hemostatic composition of claim 76, wherein the molecular weight of said dextran ranges from about 20,000 to about 100,000.

78. The hemostatic composition of claim 72, wherein said covalently linked polysaccharide has a molecular weight exclusion limit greater than about 30,000 Daltons.

79. The hemostatic composition of claim 73, wherein said article comprising cellulose comprises cotton.

80. The hemostatic composition of claim 79, wherein said article comprising cellulose is cotton gauze.

81. A hemostatic composition comprising an article comprising cellulose in association with cross-linked alginate and dextran.

82. The hemostatic composition of claim 81, wherein said alginate is covalently linked to said cellulose.

83. The hemostatic composition of claim 81, wherein said dextran is in the form of covalently cross-linked beads.

84. The hemostatic composition of claim 83, wherein said covalently cross-linked dextran beads comprise Ca2+.

85. A hemostatic composition comprising ionically linked dextran-alginate spheres.

86. The hemostatic composition of claim 85, wherein said ionically linked dextran-alginate spheres are linked ionically with Ca2+.

87. The hemostatic composition of claim 86, wherein said ionically linked dextran-alginate spheres are further cross-linked covalently.
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