Improved hemostat methods and devices

Abstract

Disclosed herein are hemostatic materials and methods of production and use thereof.

Description

IMPROVED HEMOSTAT METHODS AND DEVICESFIELD

[0001] The present Specification relates to the preparation and use of hemostatic materials.BACKGROUND

[0002] Control of topical bleeding is of critical importance in wound management, particularly for the management of trauma, e.g., as a result of traumatic injury or surgery. Typical methods of controlling bleeding employ the use of “passive” devices including cotton gauze pads. Passive devices, however, do not initiate or accelerate blood clotting.

[0003] In contrast to passive devices, hemostats are “active” materials that promote hemostasis through the use of hemostatic agents, for example, fibrinogen or thrombin, and actively participate in the coagulation cascade to form a fibrin clot. Thrombin is a serine protease that plays important roles in blood clotting (coagulation). As the key coagulation protease, thrombin converts soluble fibrinogen into fibrin networks crosslinked by a transglutaminase (FXIII). In addition, thrombin is the most potent activator of platelets by stimulating protease-activated receptors (PAR). Upon activation by thrombin, platelets physically alter the conformation of GP llb / llla receptors and provide high-affinity binding sites for fibrinogen, providing fibrinogen-crosslinked platelet aggregation.

[0004] However, commercial hemostats formulated with biologically-derived materials, including high thrombin concentrations, can lead to adverse effects including apoptosis of human keratinocytes. For example, current hemostatic powders often contain animal- and / or human-derived components such as gelatin, thrombin, fibrinogen, or collagen which may induce unwanted immunological responses or carry infectious risks.

[0005] Therefore, improved methods and systems are desirable.SUMMARY

[0006] The instant disclosure provides a novel class of hemostatic materials comprising recombinant hemostatic agents, for example, recombinant human thrombin (rhthrombin), and recombinant substrate materials, for example polysaccharides such as starch.

[0007] Further embodiments comprise methods of making disclosed hemostat materials.

[0008] Disclosed embodiments also comprise methods of use. For example, disclosed methods and devices can be used to reduce or stop bleeding, for example bleeding associated with surgical procedures, injuries, wounds, and the like. Embodiments can comprise treatment of various categories of bleeding, including “VIBe SCALE” bleeding categories:0 No bleeding (<1 .0 mL / min)1 Ooze or Intermittent Flow (>1 .0-5.0 mL / min)2 Continuous Flow (>5.0-10.0 mL / min)3 Controllable Spurting and / or Overwhelming Flow (>10.0-50.0 mL / min)4 Unidentified or Inaccessible Spurting or Gush (>50.0 mL / min)

[0009] Further embodiments comprise kits comprising disclosed hemostat materials.

[0010] Further embodiments comprise an internal dry powder delivery system which is capable of directly applying a disclosed dry powder hemostat to internal tissue / organ sites.DETAILED DESCRIPTION

[0011] Disclosed embodiments combine a hemostatic agent, for example thrombin, such as a recombinant (RH) thrombin, and a substrate material, for example polysaccharide granules (PG).

[0012] Definitions:

[0013] “Administration,” or “to administer” means the step of giving ( / .e. administering) a hemostatic device, material or agent to a subject. The materials disclosed herein can be administered via a number of appropriate routes.

[0014] “Cultured cell” means a cell capable of being grown in liquid or solid media over a number of generations. In the case of cells derived from multicellular organisms, a cultured cell is a cell isolated from the organism as a single cell, a tissue, or a portion of a tissue.

[0015] “DNA construct” means a DNA molecule, or a clone of such a molecule, either single or double-stranded, which has been modified through human intervention to contain segments of DNA combined and juxtaposed in a manner that would not otherwise exist in nature. DNA constructs may contain operably linked elements which direct the transcription and translation of DNA sequence encoding polypeptides of interest. Such elements include promoters, enhancers and transcription terminators. By virtue of the elements contained within the DNA constructs, certain constructs are understood to be capable of directing the expression and / or secretion of the encoded polypeptides. If a DNA sequence encoding a polypeptide of interest contains a secretory signal sequence, the DNA construct containing appropriate elements will be considered to be capable of directing the secretion of the polypeptide.

[0016] “Gia domain” means an amino acid sequence, generally containing from about 26 to about 45 amino acids, generally but not always located in the amino terminal region of a protein, that contains between three and twelve glutamyl residues that are post-translationally modified to y-carboxyglutamyl residues (Gia). In some cases, the Gia domain may be defined by exon-intron boundaries of the genomic sequence. The v -carboxyglutamyl residues in Gia domains facilitate the calcium-mediated binding of vitamin K-dependent proteins to membrane phospholipids. However, prothrombin, which has a gla domain that is encoded within Exon II of the genomic sequence, does not require the gla domain for biological activity. In the absence of a functional gla domain, a gla-domainless prothrombin has a Km of activation by the Factor Xa complex that is markedly higher than wild-type prothrombin.

[0017] “Hemostatic agent” means an agent that can initiate and stabilize blood clot growth during bleeding, including biologies such as thrombin, small molecules such as tranexamic acid (TXA), polymers such as feracrylum, peptides such as Thrombin Receptor Activating Peptides (TRAPs), polysulfonic acid polymers, sulfated icodextrin, sulfated carbohydrates, and inorganic materials such as kaolin.

[0018] “Hemostatic material” means a material comprising a hemostatic agent in a form suitable for application to a patient.

[0019] “Patient” means a human or non-human subject receiving medical or veterinary care.

[0020] “Therapeutically effective amount” means the level, amount or concentration of an agent, material, or composition needed to achieve a treatment goal.

[0021] “Treat,” “treating,” or “treatment” means an alleviation or a reduction (which includes some reduction, a significant reduction, a near total reduction, and a total reduction), resolution or prevention (temporarily or permanently) of a symptom, disease, disorder or condition, so as to achieve a desired therapeutic, such as by healing of injured or damaged tissue.

[0022] Hemostat Materials

[0023] The instant disclosure provides a novel class of hemostatic materials comprising a substrate material, for example a polysaccharide, such as starch, and a hemostatic agent, for example, thrombin, such as recombinant thrombin.

[0024] In embodiments, the hemostatic materials can comprise, for example, particles, granules, such as polysaccharide granules (PG), polysaccharide microspheres, powders, sponges, foams, gels, and films.

[0025] Disclosed embodiments provide distinct advantages relative to current hemostats. For example, in embodiments, both the hemostatic agent and the substrate material are non-animal-derived, thereby mitigating the risk of viral transmission. In contrast, other commercially available thrombin materials are isolated from human or animal plasma, and typical hemostat substrate materials (often gelatin or collagen) are derived from animal tissues.

[0026] Further advantages provided by the instant disclosure include rapid degradation; the recombinant thrombin is inactivated in vitro within hours by proteases, and PG are degraded within hours, in contrast to collagen and gelatin granules that can take months to fully degrade.

[0027] In addition, the use of PG provides additional mass to a hemostatic powder, resisting blood flow and aiding in localizing the hemostat. Further, PG can adsorb fluids and concentrate clotting factors, accelerating the clotting process.

[0028] Hemostatic Agents

[0029] Disclosed hemostatic materials comprise hemostatic agents. For example, in embodiments, the hemostatic agent can comprise, for example, thrombin, such as recombinant thrombin.

[0030] Disclosed herein are methods for producing rhthrombin using recombinant methods and host cells, for example eukaryotic host cells. A feature of the present disclosure is the use of an expression vector comprising a DNA sequence encoding a gla-domainless prothrombin. An additional feature of the present disclosure is the use of expression vectors within host cells to produce thrombin precursors that are activated either in vivo or in vitro to thrombin.

[0031] In an embodiment, the rhthrombin is produced by a method comprising: a. introducing into a host cell a DNA construct capable of directing the expression of a thrombin precursor and comprising the following operably linked elements: a transcriptional promoter, a DNA sequence encoding a gla-domainless prothrombin and a transcriptional terminator; b. growing the host cell under suitable conditions to allow the expression of the thrombin precursor encoded by the DNA sequence of step a; and c. isolating the thrombin precursor from the host cell. In one embodiment of the disclosure, the thrombin precursors are secreted from the host cell. In another aspect of the disclosure, the thrombin precursors are activated in vivo or may be activated in vitro.

[0032] In an embodiment, the gla-domainless prothrombin comprises kringle 1 , kringle 2, the A chain, an activation site, and the serine protease domain of prothrombin. In another embodiment, the gla-domainless prothrombin comprises kringle 2, the A chain, an activation site, and the serine protease domain of prothrombin. In yet another embodiment, the gla-domainless prothrombin comprises the A chain, an activation site, and the serine protease domain of prothrombin.

[0033] Host cells for use in practicing disclosed embodiments comprise mammalian, avian, plant, insect, fungal and bacterial cells. Fungal cells, including species of yeast (e.g., Saccharomyces spp., Schizosaccharomyces spp.) or filamentous fungi (e.g.,Aspergillus spp., Neurospora spp.) may be used as host cells within the present disclosure. Strains of the yeast Saccharomyces cerevisiae are particularly preferred.

[0034] Thrombin precursors produced according to the present disclosure can be purified by conventional chromatography, for example by using cibacron blue F3GA dye that has been adhered to a solid matrix such as AFFI-GEL® blue affinity gel (Bio-Rad, Richmond, Calif.). Thrombin precursors can be eluted from the column by exposure to high salt of between 0.6 and 1 M NaCI, preferably 1 M NaCI. The peak fractions, as determined by measuring the absorbance at 280 nm, are pooled.

[0035] In embodiments, the pool is diluted in 25 mM Tris, pH 7.4, and the thrombin precursors are activated by the addition of between 1 :1 ,000 and 1 :2,000 (w / w) snake venom activator based on an estimated protein concentration. The activated material is purified by chromatography preferably using para-aminobenzamidine coupled to a solid matrix such as Benzamidine Sepharose 4B (Pharmacia LKB Biotechnology Inc., Piscataway, NJ). Thrombin may be eluted from the column by exposure to 15 mM benzamidine. The fractions are assayed for chromogenic activity by diluting aliquots of the fractions and adding a thrombin chromogenic substrate. The pooled fractions can be desalted by applying the material to a G-25 column (Bio-Rad).

[0036] Disclosed embodiments further comprise preparation of a thrombin composition for later use in producing disclosed hemostatic materials. In embodiments, the composition can comprise 0.1 mg / mL to 5.0 mg / mL thrombin, 2% to 4% (w / v) sucrose, 3.5% to 5% (w / v) mannitol, 50 mM to 300 mM NaCI, 0-5 mM CaCl2, 0.03% to 1 % (w / v) of a surfactant or high-molecular-weight polyethylene glycol, and a physiologically acceptable buffer, in aqueous solution at pH 5.7-7.4, wherein concentration of the buffer is selected to provide approximately physiological pH upon application of the composition in a surgical setting and wherein the ratio of mannitoksucrose is greater than 1 :1 but not greater than 2.5:1 (w / w).

[0037] In other embodiments the ratio of mannitol to sucrose is 1.33:1 (w / w). Within other embodiments of the disclosure the molar ratio of sucrose:thrombin is at least 700:1 or at least 2000:1. In other embodiments, the thrombin concentration is 0.5-3.0 mg / ml or the thrombin concentration is 1 mg / ml. Within other embodiments of the disclosure, the sucrose concentration is 3% (w / v) and / or the mannitol concentration is4% (w / v). Within a further embodiment the surfactant or high-molecular-weight polyethylene glycol is PEG 3350. Within a related embodiment the surfactant or high- molecular-weight polyethylene glycol is PEG 3350 at a concentration of 0.1% (w / v). Within additional embodiments, the pH of the composition is 6.0. Within further embodiments the buffer is selected from the group consisting of histidine, citrate, phosphate, Tris, and succinate buffers. Within related embodiments the buffer is histidine buffer, such as 2.0-10 mM histidine buffer or 5 mM histidine buffer. A disclosed embodiment comprises 0.1 mg / mL to 5.0 mg / mL thrombin, 2% to 4% (w / v) sucrose, 3.5% to 5% (w / v) mannitol, 100 mM to 200 mM NaCI, 1 mM to 5 mM CaCl2, 0.03% to 1 % (w / v) PEG3350, and 2 mM to 10 mM histidine in aqueous solution at pH 5.5-6.5, wherein the ratio of mannitoksucrose is greater than 1 :1 but not greater than 2.5:1 (w / w). A second such composition of the disclosure consists essentially of 1 mg / ml thrombin, 3% (w / v) sucrose, 4% (w / v) mannitol, 4 mM CaCl2, 0.1 % (w / v) PEG3350, 150 mM NaCI, and 5 mM histidine in aqueous solution at pH 6.0.

[0038] Substrates

[0039] In embodiments the substrate material comprises a biodegradable polymer. The polymer is usually selected to breakdown into subunits which are biocompatible with the surrounding tissue. The persistence of a biodegradable polymer in vivo depends on its molecular weight and degree of cross-linking.

[0040] In various embodiments, the substrate material comprises a recombinant polymer. In particular, the recombinant polymer can be a polysaccharide, for example, cellulose, alkyl cellulose, methylcellulose, alkylhydroxyalkyl cellulose, hydroxyalkyl cellulose, cellulose sulfate, salts of carboxymethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, chitin, carboxymethyl chitin, hyaluronic acid, salts of hyaluronic acid, alginate, alginic acid, propylene glycol alginate, glycogen, dextran, dextran sulfate, curdlan, pectin, pullulan, xanthan, chondroitin, chondroitin sulfates, carboxymethyl dextran, carboxymethyl chitosan, chitosan, heparan, heparan sulfate, dermatan sulfate, keratan sulfate, carrageenans, chitosan, starch, amylose, amylopectin, poly-N- glucosamine, polymannuronic acid, polyglucuronic acid, polyguluronic acid, derivatives of said polysaccharides, or combinations thereof.

[0041] The present biocompatible substrate material can also be based on a synthetic polymer. The synthetic absorbable polymer can be an aliphatic polyester polymer, an aliphatic polyester copolymer, or combinations thereof.

[0042] In embodiments, the polymer is capable of being cross-linked and hydrated to form a hydrogel. Exemplary polymers include a polysaccharide, such as a glycosaminoglycan (e.g., hyaluronic acid or chondroitin sulfate), a starch derivative, a cellulose derivative, a hemicellulose derivative, xylan, agarose, alginate, chitosan, and combinations thereof. As a further alternative, the polymer may comprise a non-biologic hydrogel-forming polymer, such as polyacrylates, polymethacrylates, polyacrylamides, polyvinyl polymers, polylactide-glycolides, polycaprolactones, polyoxyethylenes, and derivatives and combinations thereof.

[0043] Disclosed substrate materials can comprise, for example, polysaccharides such as polysaccharide granules (PG). For example, disclosed polysaccharide granules can comprise starch, for example a modified starch.

[0044] Starch is a glucosan. At room temperature, raw starch is generally not soluble in water, nor does it readily absorb water. Raw starch normally absorbs water at temperatures above 60°C and swells to an adhesive, translucent and colloidal solution. Disclosed modified starch is raw starch processed through physical and chemical modifications, resulting in physical and chemical changes which make its characteristics and properties suitable for applications in various industries. Starch is generally classified by its origin, such as potato starch or corn starch, etc. There are two types of glucose chains in starch, including a simple chain called amylose and a complex branched form called amylopectin. The diameter range of starch grains is normally between 1 to about 100 pm and the average diameter is from 15 to 30 pm.

[0045] Natural raw starch has minimal hemostatic characteristics because starch grains are small and light and the hydrophilic properties are unsatisfactory at room temperature.

[0046] Disclosed modified starch acquires certain chemical and physical characteristics by cutting, rearranging, or adding other chemical groups that change the structure of the raw starch molecular chain. The modified starch can be categorized primarily intophysically modified starch, chemically modified starch, enzymatically modified starch, and naturally modified starch, according to the performed modification process.

[0047] An advantage of the modified starch hemostatic materials according to the present disclosure is their rapid dissolution in water, facilitating the easy removal of excess modified starch particles from the wound by simple saline irrigation. The residual modified starch not actively involved in hemostasis can be rinsed away by irrigation. In the treatment of battle wounds, self rescue, or first aid, the hemostatic material remaining in small amounts will be absorbed by the body and the irritation wound debridement or gauze removal is avoided.

[0048] In embodiments, the modified hemostatic starch material has properties of stability, extended shelf life, resistance to high and low pressure, resistance to high (up to 60° C) and low (down to -40° C) temperatures, storage convenience, and physical stability. Therefore, it can be employed as a hemostatic material for military, emergency, and first-aid uses. Particularly, it can be adapted in extreme environmental conditions such as desert areas, polar regions, alpine areas, outer space, and underwater probes. The modified starch sponge has the physical properties of pliability, flexibility, moldability, and curling. It can be adapted for wound surfaces with various shapes, sizes, and features, such as deep and irregular anatomical wounds and organs, both inside and outside the lacuna surfaces, and may be applied via endoscope, laparoscope or open surgery.

[0049] In embodiments comprising a starch substrate, the starch can be modified, for example by carboxylation or hydroxylation of a glucose unit contained in the starch, wherein the biocompatible modified starch exhibits, for example: a. water absorbency capacity of at least 1 times its own weight; and b. a viscosity of a 6.67% suspension not lower than 30 mPas at 37°C; wherein the hemostatic product has an adhesiveness sufficient to form a starch-blood coagulation matrix upon contacting a wound tissue; and wherein said hemostatic product is degradable by amylase.

[0050] In embodiments, the modified starch comprises one or more groups of pregelatinized starch, acid modified starch, dextrin, oxidized starch, esterified starch, etherified starch, and cross-linked starch. In embodiments, the starch can be pre-gelatinized by the following physical modifying processes: a) dry-process, such as an extrusion process and a roller process; b) wet-process, such as a spray drying process.

[0051] In embodiments, the chemical modifying according to the present disclosure comprises acidolysis, oxidation, esterification, etherification, cross-linking, chemical agent grafting, or multiple modifying processes including at least two of the above processes, or one of the above modifying processes performed at least twice.

[0052] By adding the functional group on the raw starch glucose units with chemical agents, e.g. by carboxylation modification, or hydroxylation modification, the starch captures hydrophilic groups in its molecular structure and obtains hydrophilic properties. By using bifunctional or polyfunctional chemical agents to cross-link the raw starch macromolecules or grafting external macromolecular hydrophilic groups to the raw starch, the starch acquires enhanced hydrophilic properties and viscosity / adhesiveness in a water solution. The viscosity of modified starch relates to the raw starch origin and the degree of substitution of external and cross-linked or grafted functional groups, etc. When contacting blood, the hydrophilic and adhesive properties of the modified starch of the present disclosure produce a "starch-blood coagulation matrix" with strong adhesive characteristics which can seal wounded tissue and stop bleeding. In addition, the interaction between the formed blood coagulation matrix and the functional groups of tissue proteins causes the "starch-blood coagulation matrix" to adhere to and seal the wounded tissue, resulting in hemostasis.

[0053] In embodiments, the disclosed substrates comprise two or more modified starches to satisfy the physical and chemical properties of a hemostatic agent, where the weight ratio of the two modified starch groups can be 99:1 .about.1 :99.

[0054] Specifically, in embodiments the weight ratio of the two modified starch groups can be: 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, or 50:50.

[0055] The main physical parameters of an embodiment of the modified starch are provided below: a. A modified starch of the present disclosure has a molecular weight over 15,000 daltons (for instance, 15,000 to about 2,000,000 daltons). b. a water absorbency capacity not lower than one time its weight (i.e. 1 gram of described modified starch can absorb 1 gram or more of water);whereas it can be 1 to about 500 times generally and 2 to about 100 times preferably.

[0056] In embodiments a modified starch composition of the present disclosure comprises at least one carboxymethyl starch, hydroxyethyl starch, and cationic starch. For example, the carboxymethyl starch is a polymer of linear structure as expressed in the following formula:

[0057] Modified starches such as carboxymethyl starch (CMS) and hydroxyethyl starch are known clinically as plasma substitutes. These modified starches exhibit biocompatibility and safety with no toxic side effects when employed in the human circulatory system. The hemostatic composition, according to the present disclosure, can further include other plasma substitutes by means of well-known pharmacokinetics approaches and specified physical / chemical properties to produce safe and reliable hemostatic agents.

[0058] When cationic starch of the modified starches is selected as a hemostatic material, the surface positive charge of the cationic starch attracts and interacts with electronegative blood erythrocytes, accelerating the blood coagulation process. Furthermore, when contacting blood, the positively charged modified starch adheres tightly to tissue, seals the wound, and rapidly stops bleeding. The cationic starch can be used independently as a hemostatic material or mixed with other modified starches as a composite hemostatic material.

[0059] Composite modified starch can comprise at least pre-gelatinized hydroxypropyl distarch phosphate. Specifically, the hydroxypropyl distarch phosphate is produced by cross-linking and etherifying the starch with propylene oxide and phosphoric acid, followed by pre-gelatinization modification through a spray drying process. The hydroxypropyl distarch phosphate of the present disclosure has high adhesiveness, strong water absorbency and robust hemostatic effects. It is stable in acidic or alkali environments and can be used as a biocompatible, hemostatic material, a surgical sealant, a tissue healing composition, an anti-adhesion agent, and a tissue repair material.

[0060] In embodiments, a cross-linked starch of the present disclosure comprises at least one of epichlorohydrin cross-linked starch and cross-linked carboxymethyl starch. Cross-linking of the polysaccharide may be achieved in any conventional manner. For example, cross-linking may be achieved using a suitable cross-linking agent, such as an aldehyde, sodium periodate, epoxy compounds, and the like. Alternatively, cross-linking may be induced by exposure to radiation, such as y-radiation or electron beam radiation. Polysaccharides and non-biologic polymers may also be cross-linked using suitable cross-linking agents and radiation.

[0061] In embodiments, the grafted starches of the present disclosure comprise at least a propylene ester-carboxymethyl starch grafted copolymer and an acid-carboxymethyl starch grafted copolymer. Grafted starch has both enhanced water absorption capability and high viscosity / adhesiveness. Therefore, it has a profound effect on hemostasis when applied to wound surfaces, especially combat wounds, traumatic wounds, and profuse bleeding from large arteries and large veins due to aneurysms or large phlebangioma ruptures.

[0062] In embodiments, modified starch granules or particles of the present disclosure can have a diameter range of 10 to about 1000 pm, or 30 to about 500 pm. Starch granules with diameters of 30 to about 500 pm represent no less than, for example, 95% of the total starch granules in an embodiment. In embodiments, the measured, optical diameter of the starch granules can be between 50 and 25 pm.

[0063] Specifically, because pre-agglomerated modified starch granules are small and lightweight, they readily form a colloid on the particle surface with the moisture in blood.In this case, it affects the hemostatic outcome by preventing water molecules from further dispersing to other starch particles. Disclosed embodiments can be produced using agglomeration processing technologies in the food and pharmaceutical industries to accumulate microscopic modified starch granules in the general 5 to about 50 pm diameter range, creating clinically applicable particles with a diameter range of 30 to about 500 pm. Modified starch granules produced by the process disclosed herein exhibit rapid water absorption, strong hydrophilic properties and rapid dispersion in blood to achieve improved hemostatic outcomes, while not readily forming a colloidal protecting layer which may disrupt the hemostatic effect.

[0064] The modified starch material according to the present disclosure comprises starch modified physically, chemically, naturally, or enzymatically, and starch modified repeatedly with at least one of the above methods or a combination of two or more of the above methods.

[0065] The physical modifying process according to the present disclosure comprises irradiation, mechanical, and steam treatment.

[0066] Physically modified starch, for example, a pre-gelatinized starch treated solely with spray drying or irradiation process, is remarkably safe as a bio-absorbable, hemostatic material since it is not treated with any chemical agents.

[0067] In embodiments, methods of making the modified starch can comprise dissolving or swelling in water and the subsequent formation of adhesive glue or adhesive gel.

[0068] In embodiments, chemically modified starch is produced by processing the raw starch with chemical agents and changing the molecular structure to achieve the desired modified starch properties. Specifically, the chemically modified starch is categorized primarily as acid modified starch, oxidized starch, baking dextrin, esterified starch, etherified starch, grafting starch, etc.

[0069] Further embodiments comprise a modified starch acquisition of hydrophilic groups in its molecular chains through the modification process.

[0070] Granular substrates of the present disclosure can be made by an agglomeration process and pellet fabrication. Normally, modified starch grain dimensions are relatively small and light and may need to agglomerate into larger sizes and heavier weights which can readily disperse into the excess blood and generate coagulation close to thebroken vessels to achieve an optimal hemostatic outcome. The agglomeration process may not be necessary for large sized modified starch particles such as grafted starch or cross-linked starch.

[0071] Production of Hemostatic Materials

[0072] Hemostatic materials as disclosed herein can be produced via a number of technologies known in the art. Example 3 provides a suitable method of producing the materials.

[0073] Commercial Products / Kits

[0074] The present hemostatic materials can be finished as a commercial product by the usual steps performed in the present field, for example by appropriate sterilization and packaging steps. The material according to the present disclosure can be finally sterile- wrapped so as to retain sterility until use and packaged (e.g. by the addition of specific product information leaflets) into suitable containers (boxes, etc.).

[0075] According to further embodiments, the hemostatic material can be provided in kit form combined with other components necessary for administration of the material to the patient. For example, if the substrate material is provided in flowable dry form (e.g. as granules or as a powder) or as a flowable paste, it is preferred to provide such material with a liquid component comprising the hemostatic agent which can be added shortly before administration to the patient. Buffer components such as phosphate, carbonate, TRIS, etc., divalent metal ions, preferably Ca2+ions, or other functional components (if not already present on or in the substrate), such as anti-bacterial agents, immunosuppressive agents, anti-inflammatory agents, anti-fibrinolytic agents, such as aprotinin or ECEA, growth factors, vitamins, cells, etc. The kit may further contain means for administering or preparing administering the hemostatic material, such as syringes, tubes, catheters, forceps, scissors, sterilizing pads or lotions, etc.

[0076] Disclosed kits, such as for use in surgery and / or in the treatment of injuries and / or wounds, can comprise a disclosed hemostatic material and at least one administration device, for example a buffer, a syringe, a tube, a catheter, forceps, scissors, gauze, a sterilizing pad or lotion.

[0077] In embodiments, the buffer solution further comprises an anti-bacterial agent, immunosuppressive agent, anti-inflammatory agent, anti-fibrinolytic agent, especiallyaprotinin or ECEA, growth factor, vitamin, cell, or mixtures thereof. Alternatively, the kit can also further comprise an anti-bacterial agent, immunosuppressive agent, antiinflammatory agent, anti-fibrinolytic agent, especially aprotinin or ECEA, growth factor, vitamin, cell, or mixtures thereof.

[0078] Exposure to radiation, such as y-radiation, can also be carried out in order to sterilize the compositions before or after packaging. In embodiments, x-ray, e-beam, and beta sterilization can be used.

[0079] When the compositions are composed of radiation-sensitive materials, it will be necessary to protect the compositions from the undesirable effects of sterilizing radiation. For example, in some cases, it will be desirable to add a stabilizer, such as ascorbic acid, in order to inhibit degradation and / or further excessive cross-linking of the materials by free radical mechanisms.

[0080] The kits are designed in various forms based on the specific deficiencies they are designed to treat. For example, additional components, for example cannula or desiccating materials can be added.

[0081] Methods of Use

[0082] In embodiments, the modified starch of the present disclosure has a bacteriostatic and anti-inflammatory effect on bleeding wound surfaces. Disclosed modified starch material has a hemostatic effect which controls bleeding, reduces blood and tissue fluid exudation, and maintains a moist wound surface. As a result, it suppresses the growth of bacteria and reduces the inflammatory response, diminishing local irritation and relieving pain. Furthermore, to strengthen the anti-inflammatory response, known antibiotic or other anti-pyrotic agents can be added to the modified starch during the manufacturing process to produce hemostatic powders, sponges and foams, hemostatic glues and gels, etc., all of which are suitable for topical and internal clinical applications.

[0083] The present disclosure provides a modified starch material with the biocompatible properties of hemostasis, adhesion prevention, tissue healing, absorbable tissue sealing and tissue bonding. More specifically, the modified starch material, which is rapidly degraded by humans and animals when applied directly to the wound surface of humans and mammals, including a bleeding or exudating woundsurface, is able to stop bleeding, prevent adhesions, promote tissue healing, seal wounded tissue, prevent bleeding and fluid exudation from tissue, bond and repair tissue or organs injured during trauma or surgery, and avoid or minimize surgical sutures.

[0084] In embodiments, the topical application of modified starch can be used as a hemostatic agent to manage and control bleeding wound surfaces in humans, mammals, birds, or reptiles, and the internal application for hemostasis of bleeding wound surfaces within human bodies, tissues and organs following surgical operations and trauma treatments, under open surgery or nasoscope, laryngoscope, endoscope and laparoscope.

[0085] The modified starch hemostatic composition, according to the present disclosure, can be applied for hemostasis on bleeding bone tissue caused by surgery or trauma, particularly for hemostasis in spongy bone tissue. In thoracic or neurological operations involving some patients, such as children, elderly people, and patients with osteoporosis, sternal bleeding and skull bleeding is difficult to control. It is common to apply bonewax to the sternum or skull, however, bonewax is slow to absorb and may cause complications such as non-union or infection. The modified starch composition, according to the present disclosure, is a biocompatible substitute for bonewax to control bone bleeding and mechanically seal the wound caused by surgery or trauma with its robust hydrophilic properties, strong adhesiveness and ease of molding. The modified starch hemostatic material degrades rapidly after surgery and avoids the complicating issues of non-union and infection associated with bonewax. When the modified starch is applied as a hemostatic material, other biological benefits of the described modified starch are worthy of attention. It is essential to evaluate the described modified starch composition as having further positive effects on wound inflammation, tissue adhesion and tissue healing while acting as a hemostat.

[0086] Embodiments of the present disclosure have further application as an absorbable, postoperative tissue adhesion barrier. The adhesion barrier of the modified starch, according to the present disclosure, prevents wounded tissue or organs from adhering to other tissue or organs in the vicinity, thereby reducing local bleeding andexudation, by mechanically isolating the wound or wound surface from adjacent tissue and surrounding organs, such as the peritoneum.

[0087] Disclosed embodiments can promote tissue healing, including skin, subcutaneous soft tissue, musculature, bone tissue, neurological tissue, nerve tissue, and wounded tissue of the liver, kidney, spleen, etc., through proper dosage and application. The modified starch can be a "scaffold" for skin tissue cells to promote healing and the growth of skin tissue from large wound surfaces due to burns, a "scaffold" for osteocyte growth and propagation in bone defects from trauma, bone tumor resection, etc., and a "scaffold" for neurological tissue cell growth and propagation when applied to injured neurological tissue caused by brain trauma, brain tumor resection, etc. The modified starch, according to the present disclosure, has further applications as a biocompatible surgical sealant, capable of forming a protective layer of colloid or film on the wound surface to seal and prevent drainage of blood, tissue fluids, lymph fluid, cerebrospinal fluid, bile, gastric fluid, and other intestinal fluids resulting from surgery and trauma treatment. This sealing effect will prevent lymph fistula leakage, bile flaccidity, pleural flaccidity, intestinal flaccidity, cerebrospinal flaccidity, and vascular flaccidity.

[0088] When the hydrophilic and enhanced adhesive modified starch according to the present disclosure is applied to bleeding wound sites, it rapidly absorbs water in the blood and concentrates blood components. Meanwhile, this interaction creates an adhesive matrix formed with the blood and plasma which adheres to the bleeding wound, mechanically seals the broken blood vessels and stops bleeding.

[0089] Another object of the present disclosure is to provide a modified starch composition as an agent for anti-adhesion therapy, for promoting tissue healing, for sealing wounds and bleeding vessels, for adhesive sealing and tissue bonding, and for promoting bacteriostatic and anti-inflammatory effects on bleeding wound sites. In addition to its hemostatic performance, the application will support anti-adhesions, tissue healing, surgical sealing and tissue bonding and reinforcement when the bleeding wound is on the skin surface or in the internal organs and whether the application is in an open surgical operation, trauma treatment, or delivered under laryngoscope, endoscope, and laparoscope.

[0090] In addition, the modified starch in the present disclosure can be used as a biocompatible, an anti-adhesion material, a tissue healing agent, a surgical sealant, and a tissue bonding / reinforcement substance for tissue repair.

[0091] Disclosed methods of use comprise application of disclosed embodiments to a site where bleeding is desired to be reduced, such as a site of injury or surgical procedure. These methods are further described in the following Examples.

[0092] Delivery Systems

[0093] Further embodiments provide an internal dry powder delivery system which is capable of directly applying dry powder to internal tissue / organ site.

[0094] Further embodiments provide an internal dry powder delivery system via working channel of endoscopic cannula to overcome the difficulties when directly applying powdered medication to the tissue / organ site of human gastro-intestine through the narrow working channel of an endoscope.

[0095] Further embodiments provide a new dry powder-format drug delivery method and system thereof via working channel of endoscopic cannula, and its clinical applications, which overcome the most common problems associated with delivering dry powder medication through the working channel of an endoscopic cannula, including that dry powder particles often occlude because of resistance in the long and narrow cannula through the working channel of endoscope and excess liquid and pressurized air mixed with dry particles could cause occlusion at the tip of the delivery catheter, in which these common problems minimize physicians' ability to deliver powdered medications directly and precisely to the tissue / organ site of the human gastro-intestine via endoscope.

[0096] Further embodiments provide physicians an innovative powder medication delivery system and method thereof, a new diagnosis and treatment alternatives when performing a minimally invasive procedure, such as via endoscope.

[0097] Further embodiments provide physicians an innovative powder medication delivery system and method thereof, a new diagnosis and treatment alternatives when performing a minimally invasive procedure, such as via endoscope.

[0098] Further embodiments provide an innovative internal powder delivery system and method thereof which can directly reach the internal tissues in the internal cavities and deliver internal powder thereto.

[0099] Further embodiments provide a new powder medication delivery system through the working channel of the endoscope, to directly reach the internal tissues in the gastrointestinal tract, abdominal, chest and pelvic cavity and deliver biomaterial for the purpose of tissue healing, sealing, anti-adhesion, fistula closure hemostasis etc.

[0100] Further embodiments provide a new powder form medication delivery method for physicians via endoscope. This method could be used with various purposes such as hemostatic, anti-inflammation, tissue repair, mucosal protection, ulcer repair and antineoplastic treatment, etc.EXAMPLES

[0101] The following non-limiting Examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments. This example should not be construed to limit any of the embodiments described in the present specification.Example 1Production of Substrate

[0102] Carboxymethyl starch material (66#) is added into an agglomerator at 40 - 50°C. Distilled water is added. The processes include particles coagulation (agglomeration) and pellet making. Molecule weight of the carboxymethyl starch (66#) is 15,000 to about 2,000,000 dalton. Diameter of the particles is 10 to about 1000 pm. Particle diameter is between 30 and 500 pm in no less than 95% of the particles. Viscosity of a 6.67% suspension at 37° C is 557.9 mPas. Work of adhesion under room temperature is 68.1 gsec when the modified starch is saturated with water.Example 2 Production of Substrate

[0103] Hydroxyethyl starch (88#) material and distilled water are added into an agglomerator 40 - 50°C. The processes include particles coagulation (agglomeration) and pellet making Molecule weight of the hydroxyethyl starch (88#) is 15,000 to about 1 ,000,000 dalton. Diameter of hydroxyethyl starch (88#) particles is 10.about.1000 pm. Particle diameter is between 50 and 500 pm in no less than 95% of the particles. Workof adhesion under room temperature is 420.9 gsec when the modified starch is saturated with water.Example 3Production of Hemostatic Material

[0104] There are several possible methods to prepare the RH thrombin / PG material, including but not limited to the following. The vialed lyophilized product can be opened, decanted, and further comminuted as described supra. Alternatively, the bulk drug substance can be lyophilized in bulk and the resulting cake can be further comminuted by grinding or pulverizing. The bulk drug substance can also be suitably formulated to enable it to be spray-dried into a powder with appropriate potency and handling characteristics. The final mixture of the RH thrombin I polysaccharide can be prepared by any number of standard powder blending techniques, including but not limited to tumbling, rotary mixing, and suspending in a fluidized bed. Alternatively, the RH thrombin can be dissolved in an appropriate diluent and the resulting thrombin solution can be mixed with the polysaccharide powder, with the combined RH thrombin / polysaccharide mixture subsequently dried or lyophilized. This results in a powder product containing individual granules that are each a mixture of RH thrombin and polysaccharide.

[0105] The powder product can be delivered to the treatment site by several means including a bellows, syringe, sprinkling, or using a spatula. In embodiments, the powder products can be packaged separately and co-administered.

[0106] In embodiments, RH thrombin can be added in an amount sufficient to provide an “excess” to account for loss of activity, for example during sterilization.

[0107] In embodiments, the PG can be replaced with polysaccharide microspheres.

[0108] PG can be generated via granulation, spray drying, solvent precipitation, fluidized bed drying, or crystallization.

[0109] In embodiments, rather than PG, a sheet, paste, spray, or solution can be used.

[0110] In a specific method, 5000 III of recombinant thrombin, RECOTHROM® Thrombin Topical [Recombinant] commercially available from Baxter HealthcareCorporation was opened and the lyophilized powder was pulverized to a fine particulate. Next, a 5g PERCLOT® Polysaccharide Hemostatic Powder, commercially available from Baxter Healthcare Corporation, was obtained and the application top was removed to allow access to the bellows. All of the RH thrombin powder was added to the bellows making a nominal dose of 1000 IU RH thrombin per gram polysaccharide granules. The PERCLOT® applicator was re-sealed and shaken by hand for 1 minute to evenly disperse the powders.Example 4Use of Hemostatic Material

[0111] A VIBe grade 1 bleed was created using a standard hepatic abrasion model. The produced RH thrombin I polysaccharide material was applied on one of a pair of lesions with the other treated with HEMOBLAST®. The two lesions were covered with a wet gauze that was held approximate to the wound for 1 minute. Both lesions were then evaluated for treatment. The produced RH thrombin / PG material reduced the bleeding to VIBe grade zero with similar efficacy to the HEMOBLAST®.Example 5Use of Hemostatic Material

[0112] A VIBe grade zero bleed was created using a laparoscopic porcine abdominal wall abrasion model. The produced RH thrombin / PG material was applied to the ventral surface and found to disperse through the trocar and adhere to the bleeding site.Example 6Treatment of Injury

[0113] An automobile accident victim sustains traumatic injuries to the abdomen. To stop blood loss, a disclosed hemostatic material is applied to the injury site using a disclosed hemostatic material delivery device. Blood loss is reduced within minutes.Example 7Treatment of Surgical Incision

[0114] To stop blood loss, a disclosed hemostatic material is applied to the site of a surgical incision using a disclosed hemostatic material delivery device. Blood loss is reduced within minutes, the hemostatic material also provides an antimicrobial effect.

[0115] In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and / or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. Accordingly, embodiments of the present disclosure are not limited to those precisely as shown and described.

[0116] Certain embodiments are described herein, comprising the best mode known to the inventor for carrying out the methods and devices described herein. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. Accordingly, this disclosure comprises all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0117] Groupings of alternative embodiments, elements, or steps of the present disclosure are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be comprised in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groups used in the appended claims.

[0118] Unless otherwise indicated, all numbers expressing a characteristic, item, quantity, parameter, property, term, and so forth used in the present specification and claims are to be understood as being modified in all instances by the term “about.” As used herein, the term “about” means that the characteristic, item, quantity, parameter, property, or term so qualified encompasses a range of plus or minus ten percent above and below the value of the stated characteristic, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the disclosure are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.

[0119] The terms “a,” “an,” “the” and similar referents used in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope otherwise claimed. No language in the present specificationshould be construed as indicating any non-claimed element essential to the practice of embodiments disclosed herein.

[0120] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the present disclosure so claimed are inherently or expressly described and enabled herein.

Claims

CLAIMS1 . A hemostatic material comprising; a recombinant hemostatic agent and a substrate material.

2. The hemostatic material of claim 1 , wherein said recombinant hemostatic agent comprises thrombin.

3. The hemostatic material of claim 2, wherein said recombinant thrombin comprises recombinant human thrombin (rhthrombin).

4. The hemostatic material of claim 1 , wherein said substrate material comprises a polymer.

5. The hemostatic material of claim 4, wherein said polymer comprises a polysaccharide.

6. The hemostatic material of claim 5, wherein said polysaccharide comprises starch.

7. The hemostatic material of claim 6, wherein said starch comprises a recombinant starch.

8. The hemostatic material of claim 3, wherein said substrate material comprises a modified starch.

9. The hemostatic material of claim 8, wherein said hemostatic agent comprises rhthrombin.

10. A kit for use in establishing local hemostasis, said kit comprising a hemostatic material comprising; a hemostatic agent and a substrate material.11 . The kit of claim 10, wherein said hemostatic agent comprises thrombin.

12. The kit of claim 11 , wherein said thrombin comprises rhthrombin.

13. The kit of claim 10, wherein said substrate material comprises a polymer.

14. The kit of claim 13, wherein said polymer comprises a polysaccharide.

15. The kit of claim 14, wherein said polysaccharide comprises starch.

16. The kit of claim 15, wherein said starch comprises a recombinant starch.

17. The kit of claim 15, wherein said hemostatic agent comprises rhthrombin.

18. A method of establishing hemostasis in a patient in need thereof, comprising administering a hemostatic composition comprising rhthrombin and a modified starch substrate to a bleed in the patient.

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