Patent Application
20220347341
The present invention provides hemostatic products and methods for treating wounds.
Non-compressible hemorrhage is the leading cause of death due to traumatic bleeding, accounting for 90% of all hemorrhage-related deaths. Non-compressible hemorrhage refers to bleeding that is not accessible to direct pressure, and includes intracavitary bleeding from soft tissues within the abdomen, a common result of shrapnel injuries during military combat and car accidents within civilian settings.
Standard and advanced bandages which require compression to function properly are not applicable for non-compressible hemorrhage bleeds. Currently no effective solutions for non-compressible hemorrhage exist outside of surgical intervention. Because it may require several hours to transport an injured patient to the hospital, a clear need exists for products which can effectively stop non-compressible bleeding both initially on site and subsequently throughout the pre-hospital transfer period.
In various aspects and embodiments, the present invention provides hemostatic products and methods for treating wounds, including cavity wounds and non-compressible hemorrhage, and including surgical bleeds. In various embodiments, the invention employs hydrophobically-modified polymer foams, gels, or pastes.
In one aspect, the invention involves a hydrophobically-modified polymer foam as a hemostatic product. For example, the hemostatic product may comprise a device having at least two compartments each containing a releasable, flowable solution. The first compartment comprises a solution of an acidified hydrophobically-modified polymer that creates hemostasis in the presence of an injury. Such hemostasis is created when the hydrophobically-modified polymer gels in the presence of blood or creates an artificial clot or seal in the presence of blood. An exemplary hydrophobically-modified polymer is hydrophobically-modified chitosan. A second compartment comprises a solution of a bicarbonate or carbonate salt at an alkaline pH. Upon release of the contents through a mixing tip, the flowable products produce a stable foam (due to the production of CO2) that will expand in a body cavity and produce an “artificial clot” or seal with blood cells, thereby resulting in hemostasis. The character and density of the hydrophobic grafts, and solution additives, ensure the production of a stable foam. In some embodiments, the hydrophobic modifications comprise or consist essentially of linear hydrocarbon groups having a variable size.
In some embodiments, the foaming action of the product enables expansion of the material inside a body cavity (e.g., “expansion phase”), and during this expansion, the foam adheres and attaches to tissue in the body cavity, resulting in a halting of the bleeding during the expansion phase. In accordance with embodiments of the invention, varying the hydrophobic chain length of the hydrophobically-modified polymer, with select hydrophobic grafting densities, allows for production of a stable foam that does not quickly dissipate. In some embodiments, the product comprises a double-barrel syringe comprising two compartments and a static mixing tip.
In some embodiments, the linear hydrocarbon groups (or “grafts”) are from about C6 to about C18, or are from about C8 to about C18. In some embodiments, the hydrophobically-modified polymer has hydrocarbon grafts of at least two different sizes attached to the polymer backbone. For example, the hydrocarbon grafts can comprise at least a first hydrocarbon chain length of from C8 to C12, which is sufficient to provide hemostatic properties, and at least a second hydrocarbon chain length of C14 or greater, which provides improved blood gelling properties and foam integrity.
In some embodiments, the hydrophobically-modified polymer has hydrocarbon grafts of at least three different sizes attached to the polymer backbone. In some embodiments, the hydrophobic grafts comprise a combination of the following linear, saturated, hydrocarbon grafts: (a) grafts of C6 to C8 in length; (b) grafts of C10 to C14 in length; and (c) grafts of C16 to C18 in length. This combination of hydrocarbon grafts can provide strong blood gelling properties while also producing a stable foam. Further, the viscosity is amenable to dispensing through a double-barrel syringe.
In some embodiments, the modified polymer is amphiphilic. In some embodiments, the polymer is based on a polysaccharide backbone, such as chitosan, alginate, gelatin, cellulosic, pectin, gellan gum, xanthan gum, dextran, and hyaluronic acid, among others.
In some embodiments, the polymer is a synthetic (i.e., non-natural) polymer, such as polyethylene glycol, poly-lactic acid, poly-glycolic acid, poly lactic co-glycolic acid, poly lactic co-glycolic acid, polymethylmethacrylate, poly ε-caprolactone, polyurethane, silicone, among others. In some embodiments, the modified polymer is hydrophobically-modified chitosan. In some embodiments, the polymer is hydrophobically-modified alginate or hydrophobically-modified gelatin.
The first compartment of the container contains an acidic solution of the hydrophobically-modified polymer. In some embodiments, the principal solvent is water with an organic acid. In some embodiments, the organic acid is selected from one or more of acetic acid, lactic acid, ascorbic acid, and citric acid, and having a pH of from about 2 to about 4.5. For example, in the case of acetic acid and hm-chitosan, the acetic acid may be from 0.5M to 2M (e.g., about 1M). The second compartment of the container comprises a solution of a carbonate or bicarbonate salt, such as a bicarbonate salt (e.g., sodium bicarbonate). In some embodiments, the bicarbonate solution is from about 0.5 to about 2M (e.g., about 1M). The bicarbonate or carbonate solution may be at pH 8 to pH 14. In various embodiments, the first or second compartment further comprises one or more foam stabilizing additives. In some embodiments, the second compartment further comprises a polymer selected from gelatin, alginate, dextran, polyethylene oxide, hydroxyethylcellulose, and hydropropylcellulose. In some embodiments, the polymer added to the second compartment is anionic. In further embodiments, the polymer additive may also be hydrophobically modified. For example, the polymer added to the second compartment may be anionic (e.g., alginate or hm-alginate), and the hydrophobically-modified polymer in the first container may be cationic (e.g., chitosan). Upon releasing the flowable contents, the polymers will form a coacervate.
In another aspect, the invention provides a gel or paste based on the hydrophobically-modified polymer. In accordance with this aspect of the invention, hydrogel particles or granules, such as quaternized dimethylaminoethyl methacrylate (QDM) or dextran particles, are incorporated into the hydrophobically-modified polymer solution. The resulting material will have paste-like characteristics that is easy to handle and apply, e.g., to treat or manage surgical bleeding. Further the material will create flow-resistance against blood that would enter the polymer matrix upon application to a bleeding site. In these embodiments, the hemostatic product is useful for treating minor soft tissue bleeds.
A product of the present disclosure may be used in methods related to treating a wound, including but not limited to non-compressible hemorrhage, cavity bleeds including soft tissue bleeds and internal bleeding during surgery. In various embodiments, the wound is treated by releasing the contents of the product onto the site of bleeding. For example, the contents of the container may be released into one or more body cavities to halt a cavity bleed or non-compressible hemorrhage. The contents of the container may also be released into one or more body cavities during surgery. In some embodiments, the flowable product expands by foaming action in the cavity. In some embodiments, the wound has high exudate or blood flow. In some embodiments, the gel or paste employing hydrophobically-modified polymer is applied to a bleeding wound, such as a soft tissue bleed during surgery.
Other aspects and embodiments of the invention will be apparent from the following detailed disclosure.
One barrel contains HM-CS in acidic solution, and the second barrel contains sodium bicarbonate. Upon dispensing the solutions through a mixing catheter, the reaction between sodium bicarbonate and the acidic solution produces a foam due to the generation of CO2. The character of the HM-CS hydrophobic grafts and formulation additives can stabilize the HM-CS foam to prevent immediate or quick dissipation, and ensure sufficient foam integrity.
In various aspects and embodiments, the present invention provides hemostatic products and methods for treating wounds, including surgical bleeds, cavity wounds and non-compressible hemorrhage. In various embodiments, the invention employs hydrophobically-modified polymer foams, gels, or pastes.
In one aspect, the invention involves a hydrophobically-modified polymer foam as a hemostatic product. For example, the hemostatic product may comprise a container having at least two compartments each containing a releasable, flowable solution. The first compartment comprises a solution of an acidified hydrophobically-modified polymer that creates hemostasis, for example, by forming a gel, artificial clot, or seal, in the presence of blood or bleeding injury. The second compartment comprises a solution of a bicarbonate or carbonate salt at an alkaline pH. Upon release of the contents through a mixing tip, the flowable products produce a stable foam (due to the production of CO2) that will expand in a body cavity and produce a gel or “artificial clot” or seal with blood cells. The character and density of the hydrophobic grafts, and solution additives (including polymer additives), ensure the production of a stable foam. In some embodiments, the hydrophobic modifications comprise or consist essentially of linear hydrocarbon groups having a variable size.
In some embodiments, the foaming action of the product enables expansion of the material inside a body cavity (e.g., “expansion phase”), and during this expansion, the foam adheres and attaches to tissue in the body cavity, resulting in a halting of the bleeding during the expansion phase. In accordance with embodiments of the invention, varying the hydrophobic chain length of the hydrophobically-modified polymer, with select hydrophobic grafting densities, allows for production of a stable foam that does not quickly dissipate.
In some embodiments, the container is any type or shape that allows for controlled release and mixing of the contents, such as a double barrel syringe. The ratio of volume released in various embodiments can be from about 1:0.5 to 0.5:1 of the first and second containers. In some embodiments, the ratio of the solution volume in the first compartment and in the second compartment is about 1:1. In some embodiments, each compartment has a solution volume of from about 1 mL to about 100 mL, or from about 1 mL to about 50 mL. In some embodiments, each compartment has a solution volume of from about 10 mL to about 50 mL, or from about 20 mL to about 50 mL. In some embodiments, the product comprises a double-barrel syringe comprising two compartments and a static mixing tip.
In some embodiments, the linear hydrocarbon groups (or “grafts”) are from about C6 to about C18, or are from about C8 to about C18. In some embodiments, the hydrophobically-modified polymer has hydrocarbon grafts of at least two different sizes attached to the polymer backbone. For example, the hydrocarbon grafts can comprise at least a first hydrocarbon chain length of from C8 to C12, which is sufficient to provide hemostatic properties, and at least a second hydrocarbon chain length of from C14 or greater, which provides improved blood gelling properties and foam integrity. In some embodiments, the second chain length is C16 or C18.
In some embodiments, the hydrophobically-modified polymer has hydrocarbon grafts of at least three different sizes attached to the polymer backbone. In some embodiments, the hydrophobic grafts comprise a combination of the following linear, saturated, hydrocarbon grafts: (a) grafts of C6 to C8 in length; (b) grafts of C10 to C14 in length; and (c) grafts of C16 to C18 in length. This combination of hydrocarbon grafts can provide strong blood gelling properties while also producing a stable foam. Further, the viscosity is amenable to dispensing through a double-barrel syringe. In various embodiments, the hydrophobically-modified polymer has a grafting density from about 1% to about 50% of the available functional groups on the polymer backbone. In some embodiments, particularly with regard to hm-chitosan, the hydrocarbon groups of (a) have a grafting density of about 3% to about 10% substitution of available amines (e.g., about 5 to 7% substitution of available amines). In some embodiments, the hydrocarbon groups of (b) have a grafting density of about 1% to about 5% substitution of available amines (e.g., from about 1% to 3% substitution of available amines), and the hydrocarbon groups of (c) have a grafting density of about 0.5 to about 3% substitution of available amines (e.g., about 1% substitution of available amines). In some embodiments, the chitosan is a medium molecular weight chitosan, having an average molecular weight (e.g., about 250 kDa), and level of de-acetylation as described below. In some embodiments, the hydrophobic grafts comprise C8 hydrocarbon groups, C12 hydrocarbon groups, and C16 hydrocarbon groups. In some embodiments, the linear hydrocarbon groups consist essentially of or consist of C8, C12, and C16 hydrocarbon groups. Generally, the ratios are selected to provide strong blood gelling properties (e.g., the gel can hold its weight for at least one hour), while enhancing foam integrity, and having a solution viscosity that can be easily dispensed from a double-barrel syringe. When employing alternative hydrophobically-modified polymers, equivalent grafting densities can be determined based on the availability of functional groups on the polymer backbone.
In some embodiments, hydrophobic grafts of C6 to C8 make up at least 50% of the hydrophobic grafts, and/or grafts of C16 to C18 make up no more than 20% or 15% of the hydrophobic grafts. In some embodiments, the hydrocarbon groups have a grafting density selected from samples X, Y, and Z from Table 1. As described, these samples exhibited strong gelation with blood, and the gel held its weight for a significant period of time (at least 3 hours) even with vial tapping. These materials suggest a unique property for treating non-compressible hemorrhage.
In some embodiments, the modified polymer is amphiphilic. In some embodiments, the polymer is based on a polysaccharide backbone, such as chitosan, alginate, gelatin, cellulosic, pectin, gellan gum, xanthan gum, dextran, and hyaluronic acid, among others. In some embodiments, the polymer is a synthetic (i.e., non-natural) polymer, such as polyethylene glycol, poly-lactic acid, poly-glycolic acid, poly lactic co-glycolic acid, poly lactic co-glycolic acid, polymethylmethacrylate, poly ε-caprolactone, polyurethane, silicone, among others. In some embodiments, the polymer is hydrophobically-modified chitosan. In some embodiments, the polymer is hydrophobically-modified alginate. In some embodiments, the polymer is hydrophobically modified gelatin. In some embodiments, one or more compartments may comprise an unmodified polymer, including but not limited to chitosan, alginate, gelatin, pectin, dextran, polyethylene oxide, carboxymethylcellulose, hydroxyethylcellulose, hydropropylcellulose, and xanthum gum, among others.
The first compartment of the container contains an acidic solution of the hydrophobically-modified polymer, which is optionally a cationic polymer such as chitosan as described. In some embodiments, the principal solvent is water with an organic acid. The modified polymer (e.g., hm-chitosan) may be present in the first container at a concentration of from about 0.1 to about 5% by weight, or from about 1% to about 3% by weight, or in some embodiments about 2% by weight. The modified polymer may be formulated at a pH of from about 2.0 to 4.5, and in some embodiments is in the range of pH 3 to pH 4.5. In some embodiments, the organic acid is selected from one or more of acetic acid, lactic acid, ascorbic acid, and citric acid, and having a pH of from about 2 to about 4.5. For example, in the case of acetic acid and hm-chitosan, the acetic acid may be from 0.5M to 2M (e.g., about 1M).
In various embodiments, the hydrophobically-modified polymer solution has a viscosity (Pa·s) of about 0.5 to about 10, or a viscosity (Pa·s) of about 0.5 to about 5. In some embodiments, the viscosity (Pa·s) is from about 1 to about 5. The viscosity is low enough to allow the material to easily pass through the syringe.
The second compartment of the container comprises a solution of a carbonate or bicarbonate salt, such as a bicarbonate salt (e.g., sodium bicarbonate). In some embodiments, the bicarbonate solution is from about 0.5 to about 2M, or from about 0.7M to about 1.5M (e.g., about 1M). The bicarbonate or carbonate solution may be in the range of pH 8 to pH 14, or from about pH 8 to about pH 12. In some embodiments, the second compartment further comprises a polymer that acts to stabilize the foam, such as a polymer selected from gelatin, alginate, dextran, polyethylene oxide, hydroxyethylcellulose, and hydropropylcellulose, among others. In some embodiments, the second compartment comprises an anionic polymer (e.g. alginate or hm-alginate) and the first compartment comprises a cationic polymer (e.g., hm-chitosan as already described). Upon releasing the contents of the double barrel syringe, the contents will foam and form a coacervate. The coacervate contributes to the formation of a stable foam.
In exemplary embodiments, the first compartment comprises hydrophobically modified chitosan (as described), and the second compartment comprises hydrophobically modified alginate. Because the hm-chitosan will be cationic and the hm-alginate will be anionic, when combined upon release, the modified polymers will form a foam stabilizing coacervate. The hm alginate can be modified with hydrophobic grafts generally comprising hydrocarbon groups (e.g., alkyl groups), which can be independently selected from the range of C1 to C18 in various embodiments. In some embodiments, the hydrocarbon groups are independently selected from the range of C4 to C14, or independently selected from the range of C4 to C12. The hydrocarbon groups may be linear or branched, saturated or unsaturated, and may be variable in size. Exemplary hydrocarbon groups are linear, saturated, C6, C8, C10, and C12 hydrocarbon chains. The hydrocarbon groups or chains can be conjugated to carboxyl groups of the alginate backbone. For example, in various embodiments, from about 5% to about 50% of the carboxyl groups in the alginate backbone include a hydrophobic graft. In some embodiments, from about 10% to about 40% of the carboxyl groups in the alginate backbone have a hydrophobic graft. In some embodiments, the hydrophobically-modified alginate has C6, C8, and/or C10 hydrocarbon chains conjugated to from 10% to about 40% (e.g., about 25%) of the backbone carboxyl groups. In some embodiments, larger hydrophobic grafts are employed (i.e., larger than C10), and are conjugated to the polymer backbone at a density of less than about 15%, or less than about 10%, or less than about 5% (but at least about 1%) of the backbone carboxyl groups. Alternatively, the grafts may be smaller than C6, and conjugated to the polymer at a density of more than about 30%, or about 40%, or about 50% of the backbone carboxyl groups. In various embodiments, the hm-alginate (e.g., with C6 to C10 grafts as described) is present in the solution at 0.5 wt % to about 5wt %, or at 1 wt % to about 3wt % (e.g., about 2 wt %) of the solution.
In various embodiments, the first or second compartment further comprises one or more alternative or additional foam stabilizing additives. In some embodiments, the one or more foam stabilizing additives create flow resistance against blood that would otherwise enter the flowable product upon application to a bleeding site. In some embodiments, the additives comprise at least one of the following: hydrogel particles or granules (e.g., quaternized dimethylaminoethyl methacrylate (QDM)), dextran particles, polymers such as polyethylene oxide, and non-ionic surfactants. In some embodiments, the additive is polyethylene oxide. In some embodiments, the additive is a non-ionic surfactant, such as Pluronic 123. In some embodiments, the additive is a polymer, such as one or more selected from alginate, gelatin, pectin, dextran, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, and xanthum gum. The additives can be added to either the first or second compartment, depending on their solubility at acidic or basic pH.
Blood gelation of the compositions disclosed herein can be evaluated using dynamic rheology, by perform a time-to-gelation evaluation. For example, dynamic time sweeps after foams are mixed with blood at equal volume. For treating severe bleeds, it is important to understand how quickly a hemostatic material can halt blood flow. Hm-chitosan solution and blood can be added to the rheometer stage sequentially. An oscillatory shear can then be actuated as quickly as possible to achieve the best representation of time-to-gelation. G′ and G″ are then recorded as a function of time, i.e., a “time sweep.” Any time at which G′ becomes higher than G″ indicates a time-to-gelation point at which the material began acting more like an elastic solid, rather than a viscous liquid.
Tissue adhesion plays an important role for hemostatic materials which attempt to seal damaged tissue against a pressurized blood flow. The hydrophobic grafts on hm-chitosan are expected to attach and anchor onto fatty tissue and thereby improve adhesion to tissue relative to unmodified chitosan. This can be evaluated, for example, by examining the adhesion of solid foams to a bovine tissue using an automated strain-gauge. Based on rheological results, the number of grafted hydrophobes per chitosan chain are believed to be correlated with tissue adhesion.
As an exemplary means of testing tissue-material interaction, an adhesion test can be designed and developed from a standard tissue adhesion test.4 For example, foams can be tested with an Instron machine, which involves placing a dry foam sample between a cover plate and loading platform. The foam can be mixed with 0.25 ml of bovine blood to coincide with in vivo large animal models. A 200 mm2 PVC adhesion surface area can be brought into contact with the blood-foam mixture and loaded at a rate of 10 N/s to 10 N or 50 kPa and held for 3 minutes. The adhesion-testing surface is then pulled away from the bandage at a rate of 1 mm/s, and the adhesion strength (kPa) can be determined by the maximum force divided by the contact surface area.
Upon mixing of the first compartment and the second compartment using the mixing tip, an expanding foam is generated due to stabilization of bubbles formed from the generation of CO2 gas. In general, a smaller bubble size yields a longer drainage time, and therefore, longer stability. Foam stability (i.e., ‘foamability’) can be adjusted based on hydrophobic modification degrees and types, and also on the hydrophobically modified polymer's molecular weight. In addition, the static mixer foams of the present disclosure are able to form in situ, such that foam may be generated within the blood.
In another aspect, the invention provides a gel or paste based on a hydrophobically-modified polymer (as described above). The gel or paste can be based on single or variable-sized hydrophobic grafts as described above. In some embodiments, the gel or paste employs hydrophobically modified chitosan as described. In accordance with this aspect of the invention, hydrogel particles or granules, such as quaternized dimethylaminoethyl methacrylate (QDM) or dextran particles, are incorporated into the hydrophobically-modified polymer solution. In some embodiments, QDM is added at around 0.02 g/mL to about 0.1 g/mL, or from about 0.04 g/mL to about 0.075 g/ mL (e.g., about 0.05 g/mL). In some embodiments, an amount of dextran particles is employed such that the resulting formulation comprises dextran particles at from about 0.1% by weight to about 2% by weight. In some embodiments, the formulation comprises dextran particles from about 0.1% to about 1.5% by weight, or from about 0.1% to about 1.0% by weight. In some embodiments, the formulation comprises dextran particles from about 0.5% to about 1.5% by weight, or from about 0.5% to about 1.0% by weight. For example, the formulation may comprise dextran particles at about 0.1% by weight; or about 0.2% by weight; or about 0.3% by weight; or about 0.4% by weight; or about 0.5% by weight; or about 0.6% by weight; or about 0.7% by weight; or about 0.8% by weight; or about 0.9% by weight; or about 1.0% by weight. The result will be a material with paste-like characteristics that is easy to handle and apply (e.g., during surgery). Further the material will create flow-resistance against blood that would enter the polymer matrix upon application to a bleeding site. In these embodiments, the hemostatic product is useful for treating minor soft tissue bleeds.
As disclosed herein, a product of the present disclosure may be used in methods related to treating a wound, including but not limited to non-compressible hemorrhage, cavity bleeds including soft tissue bleeds internal bleeding during surgery. In various embodiments, the wound is treated by releasing the contents of the product onto the site of bleeding. For example, the contents of the container may be released into one or more body cavities to halt a non-compressible hemorrhage. The contents of the container may also be released into one or more body cavities during surgery. In some embodiments, the flowable product expands by foaming action in the cavity. In some embodiments, the wound has high exudate or blood flow.
In some embodiments, the gel or paste employing hydrophobically-modified polymer is applied to a bleeding wound, such as a soft tissue bleed during surgery.
An exemplary hm-polymer material for use in accordance with embodiments of the invention is chitosan. Chitosan is the common name of the linear, random copolymer that consists of β-(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 and to modulate its properties for use as a hemostatic product.
In various embodiments, the degree of deacetylation of chitin may range from about 40-100%, or in some embodiments, from 60 to 100%, or from 40 to about 90%, or from 50 to about 100%, which determines the charge density. The structure of chitosan (deacetylated) is depicted in Formula 1:
These repeating monomeric units include a free amino group, which makes molecules or compounds containing chitosan or its derivatives readily reactive. The hydrophobic modification of the chitosan backbone is through the association of an amphiphilic compound with the amino group, such that the hydrophobic tail of the amphiphilic compound is bound with the hydrophilic backbone structure. For determination of grafting density, the degree of acetylation is assumed to be about 85%.
The polymer that forms the backbone is chitosan, or similar polymer of synthetic or natural origin, including for example, water-soluble polysaccharides and water-soluble polypeptides. In some embodiments, the polymer is one or more hm-polysaccharides, including but not limited to cellulosics, chitosans, alginates, and gelatin, all of which are abundant, natural biopolymers. In some embodiments, the hm-biopolymer contains cationic groups.
The natural origin of these polysaccharides varies, cellulosics are found in plants, whereas chitosans and alginates are found in the exoskeleton or outer membrane of a variety of living organisms. Many of these naturally occurring polymers, in addition to being able to form long stable chains for forming the polymer backbone, have properties that are beneficial for a hemostat application, including anti-microbial properties.
In some embodiments, the hm-chitosan is derived from a deacteylated chitin, which may be derived from one or more of crab, shrimp, krill, and crawfish.
The form of the natural polymers used may vary to include standard states, derivatives and other various formulations. For example, the hm-cellulosics may be formed from, without limitation, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, and/or hydroethyl methyl cellulose. Hm-chitosans may be prepared from, without limitation, the following chitosan salts: chitosan lactate, chitosan salicylate, chitosan pyrrolidone carboxylate, chitosan itaconate, chitosan niacinate, chitosan formate, chitosan acetate, chitosan gallate, chitosan glutamate, chitosan maleate, chitosan aspartate, chitosan glycolate and quaternary amine substituted chitosan and salts thereof. Hm-alginates may be prepared from, without limitation, sodium alginate, potassium alginate, magnesium alginate, calcium alginate, and/or aluminum alginate. It is to be understood that various other forms of any of these natural polysaccharides that provide the proper functional capabilities may be employed without departing from the scope and spirit of the present invention.
In some embodiments, the polymeric component is a mixture of polysaccharides. For instance, the mixture may be of various different sub-classes of a single polymer class. Alternatively, the mixture may include two or more different classes of polymer, for instance a chitosan and an alginate.
In various embodiments, the biopolymer is an hm-chitosan, which may be prepared according to International PCT Application No. PCT/US18/25742, which is hereby incorporated by reference in its entirety.
In some embodiments, the molecular weight of the polysaccharides used as the biopolymer range from about 25,000 to about 1,500,000 grams per mole. In various embodiments, the molecular weight of the biopolymer ranges from about 40,000 to about 500,000 grams per mole, or from about 50,000 to about 250,000 grams per mole, or from about 50,000 to about 100,000 grams per mole. In some embodiments, the chitosan has an average molecular weight of from 200,000 to 300,000 grams per mole (e.g., about 250,000 grams per mole). 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 the 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 polysaccharide is separated based on the size of the dissolved polymer molecules and compared with a series of standards to derive the molecular weight.
The biopolymer (such as chitosan) backbone includes a hydrophilically reactive functional group (e.g., amino groups) that binds with the hydrophilically reactive head groups (e.g., carbonyl functional group) of an amphiphilic compound (e.g., aldehyde), to form the hm-chitosan or other hm-polymer. The head group is further associated with a hydrophobic tail group. In the current embodiment, the hydrophobic tail may be, for example, a hydrocarbon. Thus, a hydrophobic tail is associated with the biopolymer backbone providing the hydrophobic modification to the molecule that extends from the backbone and may interact with a surrounding environment in numerous ways, such as through hydrophobic interaction with materials. Examples of procedures for modifying polymers are as follows.
Alginates can be hydrophobically modified by exchanging their positively charged counterions (e.g. Na+) with tertiary-butyl ammonium (TBA) ions using a sulfonated ion exchange resin. The resulting TBA-alginate is dissolved in dimethylsulfoxide (DMSO) where reaction occurs between alkyl (or aryl) bromides and the carboxylate groups along the alginate backbone. Alginate can also be modified by fatty amine groups (e.g. dodecyl amine), followed by addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, via EDC coupling.
Cellulosics can be hydrophobically modified by first treating the cellulosic material with a large excess highly basic aqueous solution (e.g. 20 wt % sodium hydroxide in water). The alkali cellulose is then removed from solution and vigorously mixed with an emulsifying solution (for example, oleic acid) containing the reactant, which is an alkyl (or aryl) halide (e.g. dodecyl bromide).
Chitosans can be hydrophobically modified by reaction of alkyl (or aryl) aldehydes with primary amine groups along the chitosan backbone in a 50/50 (v/v) % of aqueous 0.2 M acetic acid and ethanol. After reaction, the resulting Schiff bases, or imine groups, are reduced to stable secondary amines by dropwise addition of the reducing agent sodium cyanoborohydride.
Alternatively, fatty acid anhydride chemistry may be used for hydrophobic modification of chitosan, resulting in amide bonds with the chitosan polymer and the hydrocarbon chains. While hydrolysis of amide bonds is generally known to occur in the presence of dilute acids, which acts as a catalyst for the reaction between the amide and water, the amide bonds formed between chitosan and alkyl anhydrides are shelf stable, even in the presence of dilute acids that are required to maintain solubility of the hydrophobically-modified chitosan. Accordingly, the modified polymers may be prepared using a one-pot synthesis, without the need for harsh reagents, including reducing agents. The materials can be precipitated following the reaction and dried for processing and incorporation into products, including solutions, gels, and foams, among others.
The degree of substitution of the hydrophobic substituents on the polymer is up to 50% of available functional groups, for example, amines in the case of chitosan. For example, in some embodiments, the hydrophobic substituents can be added to from 1 to 50% of available amines, or from 10 to 50% of available amines, or from 20 to 50% of available amines. In some embodiments, the grafting density is no more than about 15% of available amines.
In some embodiments, the hydrophobic substituent is derived from an amphiphilic compound, meaning it is composed of a hydrophilic Head group and a hydrophobic Tail group. The Head group binds with the polymer and positions the Tail group to extend from the backbone of the polymer scaffold. This makes the hydrophobic Tail group available for hydrophobic interactions. The Tail group is a hydrocarbon of various forms.
The hydrophobic Tail group of the amphiphilic compound bound to the polymer backbone of the current invention is capable of branching and/or allowing the inclusion of side chains onto its carbon backbone. It may be understood that the strength of the hydrophobic interaction is based upon the available amount of “hydrophobes” that may interact amongst themselves or one another. Thus, it may further promote the hydrophobic effect by increasing the amount of and/or hydrophobic nature of the hydrophobic Tail group that is interacting. For instance, a hydrophobic Tail group, which in its original form may include a hydrocarbon chain, may promote an increase in its hydrophobicity (ability to hydrophobically bond and strength of hydrophobic interaction) by having a hydrophobic side chain attach to one of the carbons of its carbon backbone.
In some embodiments, the current invention contemplates the use of various molecules and/or compounds that may increase hemorrhage control, durability, water repellent properties, and/or flexibility of the hemostat product. The side chains may be linear chains, aromatic, aliphatic, cyclic, polycyclic, or any various other types of hydrophobic side chains as contemplated by those skilled in the art.
As used herein, the term “about” means ±10% of the associated numerical value.
Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.
Other aspects and embodiments of the invention will be apparent to the skilled artisan from this disclosure.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Disclosed herein are compositions and methods related to variable-length hydrophobically-modified polymers comprising hydrophobes that can effectively treat a non-compressible hemorrhage in a patient. The compositions and methods of the present disclosure are capable of promoting hemostasis and/or a hemostatic response through the interaction of the hydrophobes with cells and/or tissue.
One key feature of the material composition is the attachment of hydrophobes (e.g., nano-scale sized “stickers”) along the backbone of chitosan chains. The top of
Blood can be gelled upon mixing with hm-chitosan.2 Further studies on the effect of level of hydrophobic grafting density or hydrophobic chain length (i.e. the hydrophobic design) have been limited with regard to blood gelling ability. Changing variables for an amphiphilic polymer (i.e., a polymer containing both hydrophilic and hydrophobic segments) can significantly alter the phase behavior of the polymer. As such, these variables were viewed as optimization levers for the development of a foam material that functions via self-assembly in biological fluid. The experiments of this example sought to understand the effects of varying the components of hm-chitosan as they relate to blood gelation and tissue adherence.
The following variants of hm-Chitosan (Chitosan Source: Primex, Medium MW) were synthesized in 2 g batches. Hm-chitosan was synthesized by attaching acyl groups to the chitosan backbone via reaction with a range of fatty anhydrides (from C8 to C18 chain length). Briefly, 1 wt % chitosan solution was dissolved in 0.2 M Acetic Acid in water and 50% (v/v) ethanol. Under stirring, the fatty anhydride was added to the reaction mixture, forming an N-acylated attachment at the —NH2 position of the chitosan backbone. Grafting density was determined by the percentage of available amines along the chitosan backbone on a molar basis. The reaction was left to stir overnight, and the chitosan was then precipitated from solution by adding 0.1 M NaOH dropwise. The precipitates (e.g., fatty acids of the corresponding chain length of the utilized fatty anhydride group) were washed 5×with ethanol and 5× with water to ensure sample purification from excess reactants. In the above reaction synthesis of hm-Chitosan, for example, palmitoyl anhydride yields palmitic acid hydrophobic grafts.
Table 1 below shows a selected list of synthesized hm-Chitosan batches. Sample A in Table 1 is unmodified, or can be referred to as native chitosan. All other lettered samples in Table 1 (left column) have at least some degree of hydrophobic modification. Hence, Sample A acts an important control in the experiments of this disclosure.
In total, 120 HMC variants were synthesized. The above 25 HMC samples listed in Table 1 were studied due to a pre-screening of blood gelation scoring (see Table 2 below). Only samples with a score of 3 or higher were selected for follow up characterization. In Table 2 below, visual blood gelation scores of 0 to 5 were given to each sample according to the following scale after mixing an HMC solution (1.5 wt % in 0.1 M acetic acid) with blood (citrated bovine blood (Lampire)) at 2:1 polymer:blood ratio using a vortex mixer for about 1 second. Samples X, Y, and Z, which have a range of hydrophobic graft lengths between C8 and C16, and a theoretical grafting density of between 7-9% mol (i.e., substitution of 7-9% of available amines), displayed very strong blood gelation.
0=freely flowing, no gelation at all;
1=still freely flowing, slight increase in viscosity;
2=still freely flowing, significant increase in viscosity;
3=mild gelation and then initial gelation, quickly followed by flowing; does not hold weight upon tapping the vial;
4=moderate gelation, and then the gel holds weight for significant period of time, even with tapping the vial; flows again within 1 hour of gelation;
5=strong gelation, and then the gel holds weight for significant period of time, even with tapping the vial; holds weight for greater than 3 hours after initial gelation.
The experiments of this example sought to further understand the result of the synthesis on an analytical chemical level. Hydrophobic modification of chitosan is best characterized in a quantitative chemical sense with 1H-NMR as the hydrogen molecules along the grafted hydrocarbon backbone will have a distinctly different resonance than those normally attached to the chitosan backbone.
Synthesized hm-chitosans were dissolved in 0.2 M CD3COOD in D2O and submitted to the NMR facility at the University of Maryland for 1H-NMR characterization. Calculation of the actual degrees of graft substituted depended upon peak integrals for the H-2 hydrogen (present on every monomer unit, chemical shift δ≈3.1 ppm), and the graft hydrogens (chemical shift δ≈1.25 ppm).
Table 3 shown below displays key 1H-NMR data and analysis on all synthesized samples. The reactions for the C-12 grafts generated relatively low deviations from theoretically expected results relative to the C-18 grafts. The large deviations from expected degree of substitution values for the C-18 hm-chitosans may be attributed to low solubility during reaction. Aside from C-18 grafts, the modifications to the chitosan backbone were fairly close to theoretical values. This gives assurance that hydrophobic modification did occur, and the experiments of this example provide a mechanism of comparing real versus theoretical values of hydrophobic modification.
1H-NMR Characterization Summary Of Synthesized hm-Chitosan Batches
The experiments of this example were based on dynamic and steady state rheology profiling on foams created from a double-barrel delivery device.
Rheology is a useful method to understand how viscoelastic materials behave under shear stress. This a particularly fitting characterization method for hydrophobically modified polymers, as addition of hydrophobes to the backbone of the polymer increases the viscosity of the resulting solution. Hence, rheology is a relatively efficient way of determining trends in the solution properties of amphiphilic polymers.
Steady and dynamic rheological experiments were performed on a Rheometrics AR2000 stress-controlled rheometer. A cone-and-plate geometry of 40 mm diameter and a 4° cone angle was used and samples (1 wt % in solution, 0.2M acetic acid) were run at the physiological temperature of 37° C. Dynamic frequency spectra were obtained in the linear viscoelastic regime of the samples, as determined from dynamic strain sweep experiments. Frequency sweeps of all samples displayed fluid-like mechanical properties with G″ (viscous modulus)>G′ (elastic modulus) for a practical range of frequencies, with both being strong functions of frequency. This is an important gating criterion for the experiments of this example as the samples must be able to flow through the double barrel syringe in order to be delivered into a bleeding cavity. A comparison of apparent viscosities (η (Pa·s)) of all samples is shown in Table 4 below.
The apparent viscosities are amenable to the double barrel syringe foam concept.
Chitosan samples were loaded into double barrel syringes at 2 wt % in 1M Acetic Acid (with 1M Sodium Bicarbonate in the adjacent barrel), and were mixed with heparinized bovine blood at a ratio of 50:50 v/v, resulting in final mixture concentrations of 0.5 wt % polymer and 50% blood. All samples studied in this rheology series had a polymer concentration of 0.5 wt % and a blood concentration of 50% by volume. Thus, as an example, “Sample C” in this results section describes a mixture of 0.5 wt % of Sample C and 50% blood by volume.
As previously described above, a Rheometrics AR2000 stress-controlled rheometer was used to test the mixtures under the same testing parameters as described above. Steady shear rheology testing results from this set of samples is shown in Table 5 below. It is important to note the viscosity values recorded for Sample A (unmodified chitosan+blood) over the studied range stress values. Sample A displays an “apparent viscosity” (i.e., viscosity as shear stress approaches 0) of 10−2Pa·s, which is about 10 times the viscosity of water. In contrast, Sample G and Sample Z show apparent viscosity values on the order of 103 and 104 Pa·s, respectively. For Sample Z, this represents a million-fold increase in apparent viscosity relative to Sample A, and for Sample G, this represents a hundred-thousand-fold increase in apparent viscosity. Visually, both Samples G and Z held their own weight upon vial inversion, whereas Sample A remained a freely-flowing viscous liquid. Samples B, C, D, E, J, K, N, O and T each displayed similar apparent viscosities on the order of 102, which represent a ten thousand-fold increase in viscosity relative to Sample A. Visually, samples B, C, D, E, J, K, N, O and T showed a large increase in viscosity, but soon after gelation, the sample would begin to flow again. This result suggests that level of hydrophobicity of the chitosan backbone, assuming the same hydrophobe type, positively correlates with its ability to gel blood. However, by varying the hydrophobe length, even stronger gelation was achieved even with a lower grafting density.
A portion of this set of samples mixed with blood underwent dynamic rheology testing in order to understand their behavior over a range of timescales.
In the experiments of this example (
Particles were added into hm-chitosan gels to create flow resistance against blood that would enter the polymer matrix upon application to a bleeding site in vivo. These experiments were performed to assess how well quaternized dimethylaminoethyl methacrylate (QDM) particles and hm-chitosan gels would perform in an in vivo model of bleeding. To carry out these experiments, the “mashed potato” like substance (i.e., QDM particles and hm-chitosan gels) was taken to an animal lab and tested on 15 mm×15 mm liver squares in swine (
The experiments of this example show further increased stability of foams by introduction of amphiphilic polymer at the gas/hm-chitosan polymer interface. (see
In the experiments of this example, pluronics (i.e., non-ionic surfactants) were added in the hm-chitosan foams. The addition of pluronics in the hm-chitosan foams increased the stability of the foams by introducing an amphiphilic polymer at the gas/hm-chitosan polymer interface. To carry out the experiments, a double barrel syringe was used, where one barrel was filled with 2 wt % hm-chitosan and the second barrel was filled with 5 wt % Pluronic 123, and foams were generated. The experiments demonstrated that the foams generated with pluronics were more stable than hm-chitosan foams without the addition of pluronics. The pluronic-hm-chitosan foams did not get dissipated on tapping with spatula, and retained their structure even after multiple tapping. Therefore, hm-chitosan-pluronics foams have good mechanical integration and elasticity that would be useful for achieving hemostasis without getting dissipated easily by gauze compression.
The purpose of the experiments detailed in this example was to show that foams based on mixtures of two hemostatic biopolymers, hmC (cationic) and hmA (anionic), have enhanced rheological properties as compared to foams based on hmC alone. For example, it was shown that the carbon dioxide (CO2) gas bubbles in this foam were stabilized by hmC and hmA chains that collectively form a coacervate (i.e., a distinct phase formed by liquid-liquid phase separation in mixtures of oppositely charged polymers or surfactants) that surrounds the bubbles, and that this coacervate stabilization has significant effects on the properties of the foam.
To synthesize hmC, 1 wt % chitosan was first dissolved in 0.2 M acetic acid. An equal volume of ethanol was then added, and the solution was heated to 65° C. Palmitic anhydride was dissolved in a separate beaker with ethanol and heated to 65° C. The anhydride solution was then added to the chitosan solution such that the stoichiometry corresponded to 1.5 mol % of the amines on the chitosan. The mixture was allowed to react overnight, whereupon the hmC was formed. To precipitate the hmC from this solution, the pH was raised by adding NaOH. The precipitate was then washed with ethanol several times and left to dry, then ground into a powder. The resulting hmC had C16 hydrophobes with a degree of hydrophobe modification of 1.5%.
To synthesize hmA, 2 wt % alginate was dissolved in water using HCl to make the solution acidic (with a pH of approximately 3.4). An aqueous solution of EDC (0.66 grams of EDC for every gram of alginate) was then added to this solution, followed by the addition of n-octylamine (0.91 grams of octylamine for every gram of alginate) dissolved in 50/50 water/ethanol. The reaction was allowed to run for 24 hours to form the hmA. The hmA was precipitated out by adding acetone into the reaction mixture. This precipitate was then washed several times with acetone and allowed to dry, then ground into a powder. The resulting hmA had Cg hydrophobes with a degree of hydrophobe modification of 25%.
The phenomenon of coacervation in chitosan-alginate solutions was shown.
Similar results were observed in hmC-hmA solutions. The hmC has hexadecyl (C16) tails attached to 1.5% of the amines along the chitosan backbone. The hydrophobes allow hmC chains to associate and thereby thicken the solution. This is why a 2% hmC solution is extremely viscous (see,
To generate the foams in the study, a double-barrelled syringe (DBS) was used, as schematized in
CH3COOH+NaHCO3→CH3COONa+CO2 (g)+H2O (1)
The net result is the formation of carbon dioxide (CO2) gas in the form of bubbles. The experiment investigates surfactant-free foams due to their intended use in biomedical applications. Therefore, an amphiphilic polymer (hmC or hmA or both) must be included for foam formation.
The key contrast in this experiment is between a foam made with hmC or hmA alone and one with both hmC and hmA. In the case of the hmC foam (
First, visual observations and optical microscopy were used to contrast an hmC-hmA foam and one with hmC. The foams with hmC or hmA alone are similar in most respects, and so the contrast is made with the hmC one.
Next, the mechanical (rheological) properties of the foams in their stable, expanded state were studied. The different rheology exhibited by the foams is reflected in the photos in
Then, the hmC and hmC-hmA foams were characterized by optical microscopy. Representative images are provided for hmC foams (
The rheological properties of hmC and hmC-hmA foams were then measured. First, foams with 4% hmC and 2% hmC+2% hmA were compared using dynamic rheology (oscillatory shear).
Additionally, the variation of the foam modulus G′ with polymer concentration was examined for both the hmC and the hmC-hmA foams. The latter had equal concentrations of the two polymers—e.g., a 2% foam corresponded to 1% hmC+1% hmA. Dynamic rheology was performed on each sample and data for G′ at a frequency of 10 rad/s are plotted in
The use of hmC-hmA foams as hemostatic materials, i.e., to stop bleeding were examined. To examine this ability, experiments were conducted in vitro with the foams in conjunction with blood. A simple tube-inversion test served as a preliminary indicator in this regard. In brief, 10 mL of heparinized bovine blood was first added to a 50 mL centrifuge tube. The foam (volume of 2 mL in each barrel of the DBS=4 mL total) was injected into the tube from the nozzle of the DBS. The foam then expanded and filled the headspace in the tube. The tube was then inverted to see whether the foam could hold back the blood (
To probe the interactions between the foams and blood, optical microscopy was also used. For these experiments, heparinized blood was diluted 10×in saline to ensure visibility of blood cells. The foams were mixed with this blood and then studied under bright-field microscopy. The images in
The hemostatic efficacy of hmC-hmA foams was tested in pig liver-injury models. Initially a 2% hmC foam (out of a canister) and a 4% hmC+4% hmA foam (out of a DBS) were compared over an actively bleeding pig injury (
A test with both foams was conducted and the results are depicted in
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This application claims the benefit of U.S. Provisional Application No. 62/910,080, filed on Oct. 3, 2019, the entire contents of which are hereby incorporated in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/54268 | 10/5/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62910080 | Oct 2019 | US |
