Powder compositions for establishing hemostasis

Abstract

In aspects, the present application provides hemostatic powder compositions, and methods of making and using such powder compositions. For example, the present application provides hemostatic powder compositions, comprising a modified chitosan comprising covalently attached hydrophobic grafts and / or benzenediol groups, and optionally oxidized forms of the benzenediol groups, along the chitosan backbone.

Description

[0001] POWDER COMPOSITIONS FOR ESTABLISHING HEMOSTASIS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 668,846, filed July 9, 2024, the entire contents of which are hereby incorporated by reference in their entirety.

[0004] BACKGROUND

[0005] Modified chitosans have been described with more advanced hemostatic properties, including hydrophobically-modified chitosans. See e.g., United States Patent 8,932,560. However, hydrophobically-modified chitosans can lose properties of tissue adhesion and material cohesive properties. That is, while the modified chitosan can provide advanced hemostatic action, the ability of the modified chitosan to adhere to the wounded tissue and maintain cohesiveness especially in the presence of significant blood flow can be compromised.

[0006] Accordingly, there remains a need for hemostatic compositions that rapidly stop bleeding, including for surgical bleeds (e g., organ bleeds) and cavity bleeds. The subject matter of the present application addresses these and other objectives.

[0007] BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGs. 1A-C shows the structure and relative darkness of brown color of (Fig. 1A) a non-functionalized hydrophobically modified chitosan (hmC-INT); (Fig. IB) non-oxidized catechol -hmC (hmC-X); and (Fig. 1C) oxidized catechol -hmC (hmC-O).

[0009] FIG. 2 depicts a pre-loaded bellows apparatus containing the dry powder composition for administration.

[0010] FIG. 3 depicts formation of a granular gel (formed via capillary adhesion) upon contact between dry hemostatic powder and blood. DETAILED DESCRIPTION OF THE INVENTION

[0011] In various aspects and embodiments, the present application contemplates hemostatic powder compositions, as well as methods and uses of the composition for treating bleeds, as well as methods of making the compositions.

[0012] In aspects, the present application contemplates a hemostatic powder composition comprising a modified chitosan comprising covalently attached hydrophobic grafts and / or benzenediol groups along the chitosan backbone. In embodiments, the benzenediol groups comprise a catechol moiety. In embodiments, a portion of the benzenediol groups (e.g., catechol moi eties) are oxidized to the corresponding quinone. In embodiments, the modified chitosan is in the form of granules having a mean diameter of from about 50 microns to about 1,000 microns. In embodiments, the granules have a mean diameter of from about 100 microns to about 500 microns, and optionally of from about 100 microns to about 425 microns.

[0013] In embodiments, the hemostatic powder composition comprises a modified chitosan comprising covalently attached benzenediol groups, for example comprising a catechol moiety, along the chitosan backbone. In embodiments, the benzenediol groups are grafted to the chitosan at a density of from about 1% to about 10% of chitosan monomers (e.g., from about 1% to about 7% of chitosan monomers). In embodiments, the modified chitosan has a degree of acetylation (or alternatively a degree of C1-C4 hydrocarbon groups attached to the chitosan backbone) of from about 10% to about 40% of chitosan monomers. In embodiments, the powder is in the form of granules having a mean diameter (e.g., the longest dimension) of from about 100 to about 500 microns. The modified chitosan granules can be formulated (e.g., granulated) with various pharmaceutically acceptable excipients, including various mono-, di-, oligo-, or poly-saccharides, sugar alcohols, or in other embodiments, gelatin granules.

[0014] Without wishing to be bound by theory, the present application demonstrates that the hemostatic powder composition according to the disclosure clusters into a closed-pack array in the presence of fluid (e.g., water), thus quickly stopping, for example, surgical or organ bleeds by forming a coat or seal. In embodiments, the powder composition of the disclosure demonstrates a surprising ability to tune and balance the properties of tissue adhesion, material cohesion, and hemostatic action of the material.

[0015] Chitosan is the common name of the linear, random copolymer that consists of |3-(1- 4)-linked D-glucosamine and N-acetyl-D-glucosamine. The molecular structure of chitosan consists of a linear backbone linked with glycosidic bonds. Chitosan is the major component of crustacean shells such as crab, shrimp, krill and crawfish shells. Additionally, chitosan is the second most abundant natural biopolymer after cellulose. Commercial chitosan samples are typically prepared by chemical de-N-acetylation of chitin under alkaline conditions. Depending on the source of the natural chitin (extracted from shells) and its production process, chitosan can differ in size (average molecular weight, Mw) and degree of N- acetylation (%DA). While the poor solubility of chitosan in water and in common organic solvents restricts its applications, reactive amino groups in the chitosan backbone make it possible to chemically conjugate chitosan with various molecules to modulate its properties.

[0016] The modified chitosan included in the hemostatic powder composition of the present application in some embodiments is prepared using fatty acid anhydride chemistry, resulting in amide bonds with the chitosan polymer and the hydrocarbon chains. See U.S. Patent No. 11,274,194, which is hereby incorporated by reference in its entirety. Amide bonds can generally be susceptible to hydrolysis in solution. Hydrolysis of amide bonds is generally known to occur in the presence of acids, which act as catalysts for the reaction between the amide and water. Amides are also susceptible to alkaline hydrolysis. However, the amide bonds formed between chitosan and fatty acid anhydrides are stable in the presence of dilute acids that are required to maintain solubility of the modified chitosan. Accordingly, the modified chitosans may be prepared using a one-pot synthesis, without the need for harsh reagents, including reducing agents.

[0017] In embodiments, the modified chitosan is derived from a deacteylated chitin, which may be derived from one or more of crab, shrimp, krill, and crawfish. The charge density of chitosan is an important parameter for its tissue adherent properties. The amount of acetylation can be tuned by adding C1-C4 hydrocarbon groups (e g., acetyl groups) back to the chitosan (e.g., reacetylation), according to this disclosure. Thus, according to embodiments of this disclosure, the modified chitosan will have a free amine at about 40% of its monomers or more, or at about 50% of its monomers or more, or at about 60% of its monomers or more, or at about 70% of its monomers or more. In various embodiments, the modified chitosan according to this disclosure comprises a free amine on about 30% to about 80% of it monomers, or on about 40% to about 80% of its monomers, or on about 50% to about 80% of its monomers, or on about 55% to about 80% of its monomers. The structure of chitosan (shown deacetylated) is depicted in Formula 1 :

[0018] In embodiments, the chitosan is a small molecular weight chitosan, a medium molecular weight chitosan, a large molecular weight chitosan, or a combination thereof. In embodiments, the average molecular weight of the chitosan ranges from about 25,000 to about 1,500,000 grams per mole. In embodiments, the chitosan is substantially or predominantly a low molecular weight chitosan In embodiments, the chitosan is substantially or predominantly a low molecular weight chitosan having an average molecular weight of less than about 150,000 Daltons, or in embodiments less than about 125,000 Daltons, or less than about 100,000 Daltons. In other embodiments, the chitosan is substantially or predominantly a medium molecular weight chitosan having an average molecular of about 150,000 to about 350,000 grams per mole. In yet other embodiments, the chitosan comprises a high molecular weight chitosan having an average molecular weight of more than about 350,000 grams per mole. In embodiments, the average molecular weight of the chitosan ranges from about 25,000 to about 150,000 grams per mole, or from about 50,000 to about 150,000 grams per mole, or from about 100,000 to about 150,000 grams per mole. In embodiments, the average molecular weight of the chitosan ranges from about 200,000 to about 1,500,000 grams per mole, or from about 250,000 to about 1,500,000 grams per mole, or from about 300,000 to about 1,500,000 grams per mole, or from about 350,000 to about 1,500,000 grams per mole, or from about 400,000 to about 1,500,000 grams per mole, or from about 450,000 to about 1,500,000 grams per mole, or from about 500,000 to about 1,500,000 grams per mole.

[0019] As used herein, the term “molecular weight” means average molecular weight. Methods for determining average molecular weight of bio-polymers include low angle laser light scattering (LLS) and Size Exclusion Chromatography (SEC). In performing low angle LLS, a dilute solution of a polysaccharide, typically 2% or less, is placed in the path of a monochromatic laser. Light scattered from the sample hits the detector, which is positioned at a low angle relative to the laser source. Fluctuation in scattered light over time is correlated with the average molecular weight of the polysaccharide in solution. In performing SEC measurements, again a dilute solution of biopolymer, typically 2% or less, is injected into a packed column. The chitosan is separated based on the size of the dissolved polymer molecules and compared with a series of standards to derive the molecular weight.

[0020] In various embodiments, the modified chitosan contains hydrophobic grafts (i.e., hydrophobic substituents) that are hydrocarbon chains of greater than 6 carbon atoms, such as from 8 to 18 carbon atoms. In various embodiments, the hydrocarbon chains are linear (i.e., not branched) hydrocarbon chains. In some embodiments, the hydrocarbon groups comprise an arylalkyl group. As used herein, the term “arylalkyl group” means a group containing both aromatic and aliphatic structures. In embodiments, the hydrophobic grafts can be a uniform size or a combination of sizes. In some embodiments, the hydrophobic grafts are attached to the chitosan backbone on about 0.01% to about 15% or about 0.01% to about 10% of the chitosan monomers. In some embodiments, the modified chitosan has from about 0.5% to about 5%, or from about 0.5% to about 3% (e.g., about 1%) of chitosan monomers modified with a hydrocarbon chain greater than C6, such as independently selected from the range of C8 to Cl 8. In embodiments, the hydrocarbon group comprises one or more of linear C8, CIO, C12, C14, C16, and C18 hydrocarbon chains. In some embodiments the hydrocarbon groups (e.g., C8 to C18 groups) are alkyl groups. In some embodiments, the modified chitosan comprises hydrophobic grafts selected from one or a combination of C8, CIO, Cl 2, Cl 4, Cl 6, and Cl 8. In embodiments, the hydrophobic grafts comprise one or more selected from C8, CIO, and Cl 2. In embodiments, the hydrophobic grafts comprise C12. In some embodiments, the modified chitosan comprises C12 hydrocarbon chains present on about 0.5% to about 3% of chitosan monomers. In some embodiments, the modified chitosan comprises C12 hydrocarbon chains present on about 1% of chitosan monomers.

[0021] In embodiments, the modified chitosan does not have any hydrocarbon grafts greater than C6, or in embodiments, the modified chitosan does not have any hydrocarbon grafts greater than C4 or C3.

[0022] In various embodiments, the modified chitosan comprises smaller hydrocarbon substituents (such as Cl to C4, including C2, i.e., acetyl substituents as in chitin) to tune the density of positive charges. In some embodiments, the smaller hydrocarbon substituents are selected from the range of a Cl to C4 hydrocarbon. In various embodiments, the Cl to C4 hydrocarbon substituents (e.g., C2) along the chitosan backbone are present in the range of about 5% to about 50% of chitosan monomers, or in the range of about 10% to about 50% of chitosan monomers, or in the range of about 10% to about 45% of chitosan monomers, or in the range of about 10% to about 40%, or about 10% to about 35%, of chitosan monomers. In embodiments, the Cl to C4 hydrocarbon substituents (e.g., C2) are present on about 20% to about 40%, or about 25% to about 40%, or about 30% to about 40% of chitosan monomers. In embodiments, the hydrophobic groups comprise Cl to C4 hydrocarbon groups (e.g., C2) present on at least 10% of chitosan monomers. In embodiments, C2 hydrocarbon groups are present on at least 10% of chitosan monomers. In embodiments, C2 hydrocarbon groups are present in the range of about 10% to about 35% of chitosan monomers. In embodiments, the desired level of small hydrocarbon substituents is added to the chitosan backbone using anhydride reagents as described elsewhere herein (e g., reacetylation).

[0023] In embodiments, the modified chitosan comprises benzenediol groups substituted along the biopolymer backbone, and optionally where a portion of the benzenediol groups are oxidized. In embodiments, the benzenediol groups comprise a catechol moiety and optionally oxidized forms thereof. In embodiments, the catechol moiety is grafted to the modified chitosan using dihydrocaffeic acid or L-DOPA reagents. In embodiments, the modified chitosan has catechol moieties attached to the chitosan backbone, and which are substantially (e.g., more than 90%) or entirely unoxidized. The addition of benzenediol groups to the modified chitosan compositions increases the tissue adhesive properties (e.g., mucoadhesive properties) of the modified chitosan compositions. Further, such benzenediol groups added to the modified chitosan help the chitosan to remain in solution and form materials that are easy to use. The following is the chemical structure of native chitosan to which the benzenediol groups have been added by conjugation to available amines:

[0024] The following figure illustrates each monomer unit separated by brackets (m monomer comprises catechol moiety; n monomer comprises a free amine; and p monomer comprises acetyl:

[0025] The following formula shows a modified chitosan molecule in which a benzenediol substituent has been fully oxidized to its corresponding quinone:

[0026] The following formula illustrates a modified chitosan of the disclosure, having benzenediol and C8 hydrocarbon substituents, and where a portion of the benzenediol groups are oxidized:

[0027] According to this formula, hydrophobic grafts (C8 illustrated above) are present with benzenediol and its oxidized form in various ratios and densities, as described herein. The modified chitosan may also comprise C1-C4 groups (e.g., acetyl) as described elsewhere herein. In various embodiments, the modified chitosan can be described according to the following formula, in which an amount of chitosan monomers having a substituent comprising catechol are represented by an integer m; an amount of chitosan monomers having a substituent having a free amine are represented by an integer n; and an amount of chitosan monomers having a hydrophobic substituent (i.e., greater than C6) are represented by an integer q:

[0028] In various embodiments, the benzenediol groups and oxidized forms thereof (m and p) are grafted to the modified chitosan at a density of from 0.1% to about 15% of chitosan monomers. For example, the benzenediol groups and oxidized forms thereof (m and p above) are grafted to the modified chitosan at a density of from about 0.1% to about 10% of chitosan monomers, or from about 0.1% to about 7% of chitosan monomers, or from about 0.1% to about 5% of chitosan monomers, or from about 0.1% to about 2.5% of chitosan monomers. In embodiments, the benzenediol groups and oxidized forms thereof (m and p above) are grafted to the modified chitosan at a density of from about 1% to about 5% of chitosan monomers. In embodiments, about 10% to about 100% of the benzenediol groups are oxidized (monomer p in the formula above), or from about 25% to about 75% of the benzenediol groups are oxidized, or from about 30% to about 60% of the benzenediol groups are oxidized to the corresponding quinone. In embodiments, the ratio of unoxidized benzenediol (e.g., catechol) to oxidized benzenediol (e.g. quinones) is about 1:2, or about 1:1; or in other embodiments, the ratio may be about 2: 1; or about 5: 1. As the level of oxidation increases, cohesiveness of the material increase. Similarly, the greater the level of unoxidized molecules the greater adhesiveness of the modified chitosan. In embodiments, at least 90% of the benzenediol or oxidized moieties (m and p) are unoxidized (m). In embodiments, essentially all (e.g., at least 95%) of the benzenediol groups are unoxidized. The modified chitosan in the foregoing formula may further comprise monomers having Cl- 4 hydrocarbon substituents (e.g., acetyl groups) as already described. The partial oxidation of the benzenediol groups allows for a degree of tunability of both the adhesive and cohesive properties of the modified chitosan, e.g., in dry powder format that forms a coat upon mixing with liquid. There are two measures of functionality of hemostatic powders: cohesiveness and adhesiveness. The cohesive nature of a hemostatic powder is exemplified by the ability of the coat formed from powder to maintain integrity while under pressure. For example, in burst pressure tests in which a stream of water is directed at a coat formed from powder, the higher cohesiveness of the coat formed from powder results in the higher pressure necessary for the water to break through the coat. On the other hand, the adhesive nature of the coat formed from powder can be measured in a similar way. The higher the level of adhesiveness, the more pressure that is required to dislodge the coat from a tissue or surface to which the coat is adhered. The hemostatic coats formed from powders of the present disclosure have both high cohesive properties and high adhesive properties.

[0029] While hydrocarbon chains as hydrophobic grafts also provide a framework to tune or balance adhesive and cohesive properties of resulting modified chitosan (as already described), the optional partial oxidation of benzenediol groups along the backbone provide an additional layer of tunability, independent of the hydrophobic interactions conferred by the hydrophobic groups. For example, non-oxidized benzenediol groups amplify the adhesive properties of the chitosan (e.g., mucoadhesive properties), whereas oxidized benzenediol groups amplify the cohesive properties of the chitosan. In aggregate, the combination of hydrophobic modification, along with the addition of both non-oxidized and oxidized benzenediol groups onto the chitosan backbone, results in a highly tunable system for optimized adhesive and cohesive properties, as well as hemostatic action, so as to effectively treat bleeding from injured tissues (including moderate to severe surgical bleeds) and manage wound exudate. Cohesive properties of a material can be measured based on the elastic modulus. For example, according to various embodiments, the elastic modulus of a material according to the disclosure ranges from about 50 to about 5,000 pascals. In some embodiments, the elastic modulus is less than about 4000 pascals, or less than about 2000 pascals, or less than about 1000 pascals, or less than about 500 pascals. An exemplary process for adding benzenediol groups to modified chitosan involves the following steps. Modified chitosan is dissolved and / or suspended in distilled water. An acid, such as HCL, is added to the modified chitosan to bring the solution to a desired pH and further solubilize the modified chitosan. A catalyst such as l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) hydrochloride and ethanol are added to the mixture. The source of the benzenediol, such as 3,4 dihydroxy hydrocinnamic acid (hydrocaffeic acid) (HCA) is added to the mixture, which is allowed to react for about one hour. While the reaction is taking place, an oxidizing agent such as NaOH is added to bring the pH to from about 7.0 to about 12.5 for several minutes. For example, the reaction can be allowed to proceed for up to 10 minutes, at which time the reaction is shocked with ethanol to stop oxidation. The higher the pH that the solution is allowed to achieve, the higher the level of oxidation. After the reaction is stopped, the resulting solution can be collected and processed, for example, dried and / or ground to a powder.

[0030] In embodiments, the modified chitosan further comprises one or more dihydrocaffeic acid groups (e.g., prepared by conjugation of dihydrocaffeic acid to free amines of the chitosan polymer, forming an amide linkage), and optionally oxidized forms thereof, to tune adhesive and cohesive properties of the composition. In embodiments, the one or more dihydrocaffeic acid groups comprise a catechol moiety, and optionally oxidized forms thereof. In embodiments, at least some of the dihydrocaffeic acid groups are oxidized to the corresponding quinone. In embodiments, the dihydrocaffeic acid groups, and optionally oxidized forms thereof, are grafted to the modified chitosan at a density from about 0.1 mol% to about 15 mol% of chitosan monomers, or from about 1 mol% to about 15 mol% of chitosan monomers, or from about 0.1 mol% to about 12 mol% of chitosan monomers, or from about 0.1 mol% to about 10 mol% of chitosan monomers, or from about 0.1 mol% to about 8 mol% of chitosan monomers, or from about 1 mol% to about 7 mol% or from about 1 mol% to about 5 mol% of chitosan monomers. In various embodiments, from about 1% to about 70% of the dihydrocaffeic acid groups are oxidized to the corresponding quinone, or from about 1% to about 10% of the dihydrocaffeic acid groups are oxidized to the corresponding quinone, or from about 5% to about 50% of the dihydrocaffeic acid groups are oxidized to the corresponding quinone, or from about 15% to about 50% of the dihydrocaffeic acid groups are oxidized to the corresponding quinone, or from about 20% to about 50% of the dihydrocaffeic acid groups are oxidized to the corresponding quinone, or from about 25% to about 50% of the dihydrocaffeic acid groups are oxidized to the corresponding quinone, or from about 30% to about 50% of the dihydrocaffeic acid groups are oxidized to the corresponding quinone, or from about 40% to about 50% of the dihydrocaffeic acid groups are oxidized to the corresponding quinone. In embodiments, less than about 90% of the dihydrocaffeic acid groups are oxidized to the corresponding quinone, or less than about 95% of the dihydrocaffeic acid groups are oxidized to the corresponding quinone.

[0031] In embodiments, the hemostatic powder comprises one or more pharmaceutically- acceptable excipients. In embodiments, the modified chitosan is granulated with one or more such excipients. In embodiments, the excipients comprise one or more pharmaceutically acceptable monosaccharides, disaccharides, oligosaccharides, or polysaccharides (including sugar alcohols). Exemplary saccharides and sugar alcohols include trehalose, maltose, sucrose, lactose, dextrose, glucose, mannitol, sorbitol, xylitol, and erythritol, among others. Other excipients that may be used include gelatin, alginate, carrageenan, cellulose, modified cellulose (e.g., methylcellulose or hydroxypropyl methylcellulose (HPMC)), pectin, hyaluronic acid, oligoethylene glycol (OEG), or polyethylene glycol (PEG). In embodiments, the hemostatic powder comprises gelatin granules. In embodiments, the hemostatic powder is granulated with trehalose.

[0032] In embodiments, the hemostatic powder composition comprises from about 10% to about 75% by weight of pharmaceutically acceptable excipient, or in embodiments from about 25% to about 75% by weight excipient, or from about 40% to about 60% by weight excipient, such as about 50% by weight excipient.

[0033] In embodiments, the hemostatic powder composition comprises trehalose or gelatin. In embodiments, the powder comprises from about 10% to about 75% by weight of trehalose or gelatin, or in embodiments from about 25% to about 75% by weight trehalose or gelatin, or from about 40% to about 60% by weight trehalose or gelatin, such as about 50% by weight trehalose or gelatin. In embodiments, the hemostatic powder composition of the present application is produced by a process comprising the steps of: (a) preparing a modified chitosan with catechol groups, and optionally oxidizing the catechol groups (as described); (b) grinding the modified chitosan to a powder (optionally with one or more excipients as described); and (c) sieving the powder to a granule size having a mean diameter of from about 100 to about 1,000 microns (or otherwise as described). In embodiments, the preparation of the modified chitosan comprises preparing a modified chitosan solution by combining modified chitosan with hydrochloric acid.

[0034] In embodiments, about 0.25 M to about 2.0 M hydrochloric acid is used to prepare the modified chitosan. In embodiments, the preparation further comprises adding a 1-Ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC)-hydrocaffeic acid solution to the chitosan solution.

[0035] In various embodiments, the present application contemplates methods for using the hemostatic powder composition for achieving hemostasis in a subject in need thereof. For example, the present application contemplates that the hemostatic powder composition can be used to achieve hemostasis at a bleeding site (e.g., including but not limited to a site of diffuse bleeding).

[0036] In embodiments, the hemostatic powder composition of the present application is used to treat a bleed in a method comprising applying the powder composition to the bleed. In embodiments, the bleed is a surgical bleed or a trauma bleed. In embodiments, the bleed is a cavity bleed or organ bleed. For example, the bleed can be an arterial or venous bleed, or, in embodiments, is an organ bleed. In embodiments, the bleed is a liver bleed, or large or small bowel bleed. In embodiments, the cavity bleed or organ bleed presents as diffuse bleeding.

[0037] In embodiments, the hemostatic powder composition forms a granular gel on a tissue surface upon contact with liquid. In embodiments, the liquid is a bodily fluid, for example blood. In embodiments, the granular gel that is formed on a tissue surface comprises a thickness of from about 0.1 to about 10 mm, and optionally of from about 0.5 to about 5 mm, or about 0.5 to about 3 mm. In embodiments, the hemostatic powder composition of the present application achieves hemostasis of a bleed that is a scale 1 to 3 bleed (out of scale of 5). In embodiments, the hemostatic powder composition of the present application achieves hemostasis of a bleed that is at least a scale 3 bleed. In embodiments, the hemostatic powder composition of the present application achieves hemostasis of a bleed that is a scale 4 bleed. Generally, the bleeding scale can be defined as: 0 (no bleeding, hemostasis); 1 (minimal bleeding); 2 (mild bleeding); 3 (moderate bleeding); 4 (severe bleeding), and 5 (extreme bleeding). Due to the cohesive and adhesive properties of the material, and its hemostatic action, even moderate to severe bleeds can be controlled using the compositions of the disclosure. In this context, bleeding scales (e.g., for skin lacerations) can be modeled as described in U.S. Patent No. 10,283,015, which is hereby incorporated by reference. Devices can be used to simulate bleeds of different scales ex vivo by pumping natural or synthetic blood through a porous surface (e.g., determined by an interchangeable plate) and under different flow rates.

[0038] In embodiments, the present application contemplates that the use of the hemostatic powder composition described herein in a subject can reach hemostasis at a bleed site in less than about 15 minutes, or less than about 10 minutes, or less than about 5 minutes.

[0039] In embodiments, the present application contemplates a method of treating a bleed in a subject in need thereof in which the dry hemostatic powder composition described herein is administered to the bleed site with a bellows. The contemplated bellows comprises preloaded dry hemostatic powder. In embodiments, the bellows comprises a handle, a tip applicator, and a cap on said tip applicator. Without wishing to be bound by theory, the use of the preloaded bellows allows for ease of application of the dry hemostatic powder composition described herein. In embodiments, the bellows apparatus further allows for ease of storage (e.g., without the need for special storage).

[0040] In embodiments, the hemostatic powder composition of the present application is administered as a unit of weight of at least about 0.05 g, or at least about 0.1 g, or at least about 1g, or at least about 2 g. In embodiments, the composition is administered in the range of from about 0.05 g to about 10 g, and optionally in the range of 0.1 g to about 5 g, and optionally in the range of 0.1 g to about 3 g, or optionally in the range of about 0.5 g to about 2 g-

[0041] As used herein, the term “about” means ± 10% of a reference value, unless the context requires otherwise.

[0042] Other aspects and embodiments of this disclosure will be apparent from the following working examples and claims.

[0043] EXAMPLES

[0044] Example 1: Assessment of Hemostatic Properties in Hydrophohicall -Modified Chitosan with Catechol Groups and Oxidized Forms Thereo f

[0045] The following Example was conducted to investigate functionalized hydrophobically modified chitosans with catechol groups and oxidized forms thereof, and in particular in the form of a dry powder and for its physical properties when mixed with water.

[0046] Figs. 1A-C depict modified chitosan materials for preparing dry hemostatic powders. Modified chitosans include (Fig. 1 A) a hydrophobically modified chitosan (hmC-INT); (Fig. IB) a non-oxidized catechol -hmC (hmC-X); and (Fig. 1C) an oxidized catechol -hmC (hmC- O). These compositions, when formulated as hemostatic powders, can be administered using pre-loaded bellows, as illustrated in Fig. 2.

[0047] Specifically, the materials were made by chemically modifying the chitosan by hydrophobic modification (hmC-INT), and then functionalizing the modified chitosan with catechol groups (hmC-X), followed by partial oxidation in some embodiments (hmC-0). These materials can be dried and ground to a powder, followed by sieving to specific granule sizes. Preparation of hydrophobically-modified chitosan is described in US Patent No. 8,932,560; US Patent No. 10,179,145; and US Patent No. 11,274,194, which are hereby incorporated by reference in their entireties. Preparation of hmC-X and hmC-0 are described in International Publication WO 2024 / 073359A1, which is hereby incorporated by reference in its entirety. In one embodiment, the resulting powder comprises hmC-0 having from 1% to 4% catechol modification (e.g., about 2.5% catechol modification, and which is substantially oxidized), about 1% C12 modification, and from about 30% to about 40% C2 modification. The material is sieved to desired particle size.

[0048] For the purpose of demonstration, in an in vivo liver injury, it was shown that the granules (having diameters of from about 50 to about 500 microns) transform into a granular gel coat or seal upon contact with blood (formed by capillary adhesion), effectively stopping the bleeding (Fig. 3). Each pair of granules is believed to be linked by a liquid bridge.

[0049] Thus, it was demonstrated that the catechol-hmC powder composition described herein, when it comes into contact with blood, is able to exhibit robust mechanical properties by gel formation via capillary adhesion, thereby forming a stable barrier (e.g., a seal or coat around the wound) to stop the bleed.

[0050] Example 2: Assessment of Hemostatic Properties in Chitosan Modified with Catechol Groups

[0051] The following Example was conducted to investigate chitosan modified with catechol groups.

[0052] Specifically, modified chitosan was synthesized by conjugating catechol to native low molecular weight chitosan, where the catechol was 1 mol% to 5 mol%, in a single-step process. In particular, samples were prepared of low molecular weight chitosan having 5 mol% catechol (“PWDR-100,” “PWDR-101,” and “PWDR-102”), 2.5 mol% catechol (“PWDR-103”), or 1 mol% catechol (“PWDR-104”). The PWDR-100, PWDR-101, and PWDR-102 samples were E-Beam sterilized, while PWDR-103 and PWDR-104 were nonsterile. Each of the Samples contained powder particles having a range of 106-425 microns.

[0053] These samples were subjected to an animal efficacy test in which powder was applied to a wound lesion and characterized by the Surface Bleeding Severity Scale (SBSS), as well as observed for other properties, such as appearance, wetting, and ease of removal. SBSS scores characterize the severity of a bleeding wound: 0 = none, visually dry; 1 = minimal, visually oozing; 2 = mild, visually pooling; 3 = moderate, visually flowing; 4 = severe (not immediately life-threatening), streaming; and 5 = extreme (immediately life-threatening), gushing. Timepoints 1, 2, and 3 occurred within 10 minutes of the Baseline measurement. In particular, Timepoint 1 occurred at either 1 minute or 2 minutes after the Baseline measurement. Timepoint 2 occurred in the range of 2.5 to 5 minutes after the Baseline measurement. Timepoint 3 occurred 6 or 7 minutes after the Baseline measurement.

[0054] The results of these studies are presented in the Table below:

[0055] In sum, each of the sample powders exhibited comparable satisfactory efficacy in the animal bleed study, and the material extrinsic to the blood clot was easily removed for all samples following endpoint.

Claims

CLAIMSWhat is claimed is:

1. A hemostatic powder composition, comprising: a modified chitosan comprising covalently attached hydrophobic grafts and / or benzenediol groups, and optionally oxidized forms of the benzenediol groups, along the chitosan backbone; and wherein the modified chitosan is in the form of granules having a mean diameter of from about 50 microns to about 1,000 microns.

2. The powder composition of claim 1, wherein the granules have a mean diameter of from about 100 microns to about 500 microns, and optionally of from about 100 microns to about 425 microns.

3. The powder composition of claim 1 or 2, wherein the chitosan is a low molecular weight chitosan.

4. The powder composition of any one of claims 1 to 3, wherein the benzenediol groups comprise a catechol moiety and optionally oxidized forms thereof.

5. The powder composition of claim 4, wherein the catechol moiety is grafted to the modified chitosan using hydrocaffeic acid or L-DOPA reagents.

6. The powder composition of any one of claims 1 to 5, wherein the benzenediol groups, and optionally oxidized forms thereof, are grafted to the chitosan at a density of from 0.1% to about 15% of chitosan monomers.

7. The powder composition of claim 6, wherein the benzenediol groups, and optionally oxidized forms thereof, are grafted to the chitosan at a density of from about 1% to about8. The powder composition of claim 7, wherein the benzenediol groups, and optionally oxidized forms thereof, are grafted to the chitosan at a density of from about 1% to about 7% of chitosan monomers.

9. The powder composition of any one of claims 1 to 8, wherein none of the benzenediol groups are oxidized.

10. The powder composition of any one of claims 1 to 8, wherein at least some of the benzenediol groups are oxidized to a corresponding quinone.

11. The powder composition of claim 10, wherein from about 10% to about 100% of the benzenediol groups are oxidized, or from about 25% to about 75% of the benzenediol groups are oxidized, or from about 30% to about 60% of the benzenediol groups are oxidized to the corresponding quinone.

12. The powder composition of any one of claims 1 to 11, wherein the chitosan comprises C1-C4 hydrocarbon groups on about 10% to about 40% of the chitosan monomers.

13. The powder composition of any one of claims 1 to 11 , wherein the hydrophobic grafts comprise linear hydrocarbons of from 6 to 18 carbon atoms.

14. The powder composition of claim 13, wherein the hydrophobic grafts comprise one or more selected from C8, CIO, C12, C14, and C16.

15. The powder composition of claim 13, wherein the hydrophobic grafts comprise one or more selected from C8, CIO, and C12.

16. The powder composition of claim 13, wherein the hydrophobic grafts comprise C12.

17. The powder composition of any one of claims 1 to 16, wherein the modified chitosan has from about 0.01% to about 10% of chitosan monomers modified with a hydrophobic graft.

18. The powder composition of claim 17, wherein the modified chitosan has from about 0.1% to about 5%, or from about 0.5% to about 3%, or about 1% of chitosan monomers modified with a hydrophobic graft.

19. The powder composition of claim 17 or 18, wherein from 5% to about 50% of the chitosan monomers comprise an acetyl group; and optionally from about 10% to about 40% of the modified chitosan monomers comprise an acetyl group.

20. The powder composition of claim 19, wherein the modified chitosan comprises a free amine on about 40% to about 75% of it monomers.

21. The powder composition of any one of claims 1 to 20, wherein the chitosan granules are formulated with one or more pharmaceutically acceptable excipients selected from mono-, di-, oligo-, or poly-saccharides, sugar alcohols, and gelatin granules.

22. The powder composition of claim 21, wherein the powder comprises from about 25% to 75% by weight pharmaceutically acceptable excipient, optionally about 50% by weight.

23. The powder composition of claim 21 or 22, wherein the excipient is trehalose.

24. The powder composition of claim 21 or 22, wherein the excipient is gelatin granules.

25. The powder composition of any one of claims 1 to 24, wherein the powder does not significantly expand upon contact with liquid.

26. A method of making the powder composition of any one of claims claim 1 to 25, comprising:(a) preparing a modified chitosan with catechol groups, and optionally oxidizing the catechol groups;(b) grinding the modified chitosan to a powder optionally with one or more excipients; and(c) sieving the powder to a granule size having a mean diameter of from about 100 to about 1,000 microns.

27. The method of claim 26, wherein the preparation of the functionalized modified chitosan comprises preparing a modified chitosan solution by combining modified chitosan with hydrochloric acid, optionally about 0.25 M to about 2.0 M hydrochloric acid.

28. The method of claim 27, wherein the preparation further comprises adding a 1-Ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC)-hydrocaffeic acid solution to the modified chitosan solution.

29. A method for treating a bleed, comprising applying the powder composition of any one of claims 1 to 25 to said bleed.

30. The method of claim 29, wherein the bleed is a surgical bleed or a trauma bleed.

31. The method of claim 30, wherein the bleed is a cavity bleed or organ bleed, and optionally presents as diffuse bleeding.

32. The method of claim 30, wherein the bleed is at a site at risk of pressure or compression related injury.

33. The method of any one of claims 29 to 32, wherein the bleed is a scale 1 to 3 bleed.

34. The method of any one of claims 29 to 32, wherein the bleed is at least a scale 3 bleed.

35. The method of any one of claims 29 to 32, wherein the bleed is a scale 4 bleed.

36. The method of any one of claims 29 to 35, wherein the powder composition is administered with a bellows.

37. The method of any one of claims 29 to 36, wherein the powder composition is administered as a unit of weight in the range of from about 0.05 g to about 10 g.

38. The method of any one of claims 29 to 37, wherein the powder composition forms a granular gel upon contact with liquid.

39. The method of claim 38, wherein the liquid is a bodily fluid, and optionally blood.

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