HK1194968B - A hemostatic composition - Google Patents

Description

Hemostatic compositions

Technical Field

The present invention relates to hemostatic compositions and methods of making these compositions.

Background

Hemostatic compositions in dry storage stable form comprising a biocompatible, biodegradable, dry stable particulate material are known from e.g. WO98/008550a or WO2003/007845 a. These products have been successfully applied in the prior art for hemostasis.Is an example of a highly effective hemostatic agent consisting of a granulated gelatin matrix that swells in a thrombin-containing solution to form a flowable ointment.

Because such products are intended for human use, it is necessary to provide the highest safety standards for the quality, storage stability and sterility of the final product and its components. In addition, manufacturing and handling should be as convenient and efficient as possible.

In this field, a successful product (as described above)Product) used a gelatin matrix in combination with a reconstituted (reconstituted) lyophilized thrombin solution. The gelatin matrix is applied as gelatin and thrombin in the form of flowable granules, wherein the gelatin content is about 11-14.5%. Lower gelatin content results in a soft product with reduced performance due to difficulty in retaining the product at the treatment site, especially at higher blood flow conditions. Higher gelatin particle concentrations can result in products that are difficult to release by common administration means such as syringes or catheters due to higher flow resistance. It has been suggested (EP0927053B1) to include plasticizers such as polyethylene glycol, sorbitol, glycerol, etc. in the composition to reduce extrusion forces, but inclusion of these materials does not necessarily improve performance.

It is an object of the present invention to provide a hemostatic composition based on cross-linked gelatin (i.e. cross-linked gelatin) and a gelatin product according to the prior art, such asIn contrast, it has improved adhesion and hemostatic properties. It is also an object of the present invention to provide a method of making such a hemostatic composition. The composition will also be provided in a convenient and suitable manner for use, in other words as a flowable ointment that can be used in endoscopic and microsurgical applications. The product must have a pressing force of 40N or less, preferably 35N or less, particularly preferably 20N or less. The product will preferably be provided in the form of: it would be convenient to provide a "ready-to-use" hemostatic composition that can be applied directly to the lesion without involving any time-consuming reconstitution step.

Disclosure of Invention

Accordingly, the present invention provides a haemostatic composition comprising cross-linked gelatin in particulate form suitable for use in haemostasis, wherein the composition is in the form of an ointment comprising 15.0-19.5% (w/w) cross-linked gelatin, preferably 16.0-19.5% (w/w), 16.5-19.5% (w/w), 17.0-18.5% (w/w) or 17.5-18.5% (w/w), more preferably 16.5-19.0% (w/w) or 16.8-17.8% (w/w), especially preferably 16.5-17.5% (w/w); and wherein the composition comprises a compression enhancer, in particular albumin.

The invention also relates to the use of such a hemostatic composition for treating a lesion, comprising administering a hemostatic composition and a kit that can manufacture a hemostatic composition for treating such a lesion, the lesion being selected from the group consisting of: a wound, bleeding, damaged tissue, and/or bleeding tissue.

Detailed Description

The present invention provides a haemostatic composition comprising cross-linked gelatin in particulate form suitable for use in haemostasis, wherein the composition is in the form of an ointment comprising 15.0-19.5% (w/w) cross-linked gelatin (dry cross-linked gelatin weight/final composition weight), preferably 16.0-19.5% (w/w), 16.5-19.5% (w/w), 17.0-18.5% (w/w) or 17.5-18.5% (w/w), more preferably 16.5-19.0% (w/w) or 16.8-17.8% (w/w), especially preferably 16.5-17.5% (w/w) cross-linked gelatin; and wherein the composition comprises an extrusion enhancer.

It has been surprisingly found that in the process of the present invention to provide a suitable level of extrusion enhancer such as albumin, higher gelatin concentrations can be used and that the use of higher gelatin concentrations improves the hemostatic properties of such products. This is an effect that has not been suggested in the prior art. Furthermore, it is surprising that higher concentrations of cross-linked gelatin lead to better adhesion properties (contrary to the results known in the prior art (e.g. fig. 4 of WO2008/076407a 2)).

In order to achieve the preferred properties according to the invention due to the higher gelatin concentration in the ointment, it is necessary to provide the crush enhancer in a suitable amount. The content should be high enough to obtain a squeezing effect, i.e. to obtain a flowable ointment with a cross-linked gelatin content of 15-19.5%, so that the hemostatic composition can be used in e.g. microsurgery; on the other hand, the amount should be low enough to prevent negative functional properties of the hemostatic composition, such as adhesion to a wound or hemostatic properties. For example, if the extrusion enhancer is albumin (in particular, it is particularly preferably human serum albumin), the content must be set between 0.5-5.0% (w/w) (-extrusion enhancer weight/final composition weight), preferably 1.0-5.0% (w/w), preferably 2.0-4.5% (w/w), more preferably 1.5-5.0% (w/w), particularly preferably about 1.5% (w/w).

Another preferred class of extrusion enhancers according to the invention are phospholipids, such as phosphatidylcholine and serine, or complex mixtures such as lecithin or soybean oil.

In another preferred embodiment, the present invention provides a haemostatic composition comprising cross-linked gelatin in particulate form suitable for use in haemostasis, wherein the composition is in the form of an ointment comprising 16.0-19.5% (w/w), preferably 16.5-19.5% (w/w), 17.0-18.5% (w/w), or 17.5-18.5% (w/w), more preferably 16.5-19.0% (w/w) or 16.8-17.8% (w/w), especially preferably 16.5-17.5% (w/w); and wherein the composition comprises an extrusion enhancer. Preferably the compression enhancer is human serum albumin.

In another preferred embodiment, the present invention provides a haemostatic composition comprising cross-linked gelatin in particulate form suitable for use in haemostasis, wherein the composition is in the form of an ointment comprising 15.0-19.5% (w/w) cross-linked gelatin, preferably 16.0-19.5% (w/w), 16.5-19.5% (w/w), 17.0-18.5% (w/w) or 17.5-18.5% (w/w), more preferably 16.5-19.0% (w/w) or 16.8-17.8% (w/w), especially preferably 16.5-17.5% (w/w); and wherein the composition comprises an extrusion enhancer in a concentration of more than 0.8% (w/w), preferably about 3.3% (w/w). Preferably the compression enhancer is human serum albumin, for example, at the concentrations described above.

The hemostatic composition according to the present invention, in particular using albumin as a compression enhancer, has particular advantages, in particular an enhanced in vivo efficacy, compared to compositions using lower amounts (13-14.5%) of cross-linked gelatin. Unexpectedly, the process of the present invention shows that formulations with higher gelatin particle concentrations achieve greater hemostatic performance not only in ex vivo (exvivo) testing methods using whole human blood, but also in preclinical animal testing. The product according to the invention allows to reduce the surgical time and to speed the haemostasis time.

The compositions according to the invention have an average pressing force of 40N or less, preferably 35N or less, particularly preferably 20N or less (using the test method described in example 1).

According to a preferred embodiment of the invention, the hemostatic composition comprises glutaraldehyde-crosslinked gelatin or genipin (methyl (1R,2R,6S) -2-hydroxy-9- (hydroxymethyl) -3-oxabicyclo [4.3.0] -nonane-4, 8-diene-5-carboxylate) crosslinked gelatin, preferably type B gelatin, more preferably type B gelatin of hide origin.

Preferably, the cross-linked gelatin is present as a granular material.

The hemostatic composition according to the present invention preferably comprises a gelatin polymer, especially a type B gelatin polymer. Type B gelatin has proven to be particularly advantageous for use as a hemostatic agent because the matrix treatment is highly effective in producing gelatin with appropriate properties, as well as in mitigating viral and infectious animal infections. Particularly preferred gelatin products can be prepared by the following process: the calf dermis was processed with 2n naoh at room temperature for approximately 1 hour, neutralized to pH7-8, and heated to 70 ℃. The dermis is then fully solubilized to gelatin with 3-10% (w/w), preferably 7-10% (w/w) gelatin in solution. The solution can be cast, dried and ground to provide a type B gelatin powder.

Preferably, the gelatin has a bloom strength (bloomtrength) of 200 to 400, particularly preferably the type B gelatin has a bloom strength of 200 to 400. Bloom is a test value for measuring the strength of gelatin. This test value determines the weight (in grams) required to tilt the surface of the gel by 4mm without breaking by means of a probe (typically 0.5 inches in diameter). The result is expressed as bloom (grade). For the bloom test (Bloomtest) on gelatin, a 6.67% gelatin solution was held at 10 ℃ for 17-18 hours prior to testing.

The hemostatic composition according to the invention preferably contains cross-linked gelatin in the form of particles, especially a granular material. The particulate material is capable of swelling rapidly when exposed to a liquid (i.e. a diluent) and in this swollen form enables the flowable ointment to be applied to the bleeding site to be assisted. According to a preferred embodiment, the crosslinked gelatin is provided by drying the crosslinked gelatin. The dry cross-linked gelatin powder can be rapidly prepared as a re-hydrate if contacted with a pharmaceutically acceptable diluent. Gelatin particles, in particular in the form of gelatin powder, preferably comprise relatively large particles, also referred to as chips or subunits, as described in WO98/08550a and WO2003/007845 a. Preferred (median) particle sizes will be in the range of 10 to 1.000 μm, preferably 200 to 800 μm, although particle sizes outside this preferred range may find use in many environments.

Typically, the gelatin particles have an average particle size ("average particle size" is the average particle size measured by laser diffraction method; "median particle size" (or mass median particle diameter) is the particle diameter dividing the frequency distribution in half; fifty percent of the particles of a given product have a larger diameter and fifty percent of the particles have a smaller diameter) of 10-1000 μm, preferably 50-700 μm, 200-700 μm, 300-550 μm, and particularly preferably 350-550 μm. Although the terms "powder" and "granular (or particulate)" are sometimes used to distinguish between different types of materials, powder is defined herein as a particular subclass of granular material. In particular, powder refers to a granular material having a finer particle size and therefore a greater tendency to form agglomerates when flowing. The particles comprise coarse aggregate material that does not tend to form agglomerates except when wet.

The crosslinked gelatin in particulate form suitable for use in hemostasis of the present invention may comprise a dimensionally isotropic or non-isotropic form. For example, the cross-linked gelatin in the kit according to the present invention may be a particle or a fiber; and may be present in a discontinuous structure, for example in powder form.

The dried gelatin composition is liquid-absorbent. For example, crosslinked gelatin, upon contact with a liquid such as an aqueous solution or suspension (especially a buffer or blood), absorbs the liquid and will exhibit some degree of swelling depending on the degree of hydration. The material preferably can absorb at least 400 wt%, preferably from about 500 wt% to about 2000 wt%, especially preferably from about 500 wt% to about 1300 wt% of water or aqueous buffer solution, with a nominal increase in diameter or width of the individual particles corresponding to the subunits ranging, for example, from about 50% to about 500%, typically from about 50% to about 250%. For example, if the (dry) particles have a particle size range of preferably 0.01mm to 1.5mm, particularly preferably 0.05mm to 1mm, the fully hydrated composition (e.g. after administration to a wound or after contact with an aqueous buffer solution) may have a particle size range of 0.05mm to 3mm, particularly preferably 0.25mm to 1.5 mm.

The dried composition will also exhibit significant "equilibrium swelling" when exposed to an aqueous re-hydrate medium (a pharmaceutically acceptable diluent, also referred to as a reconstitution medium). Preferably, depending on the intended use, the degree of swelling will be in the range 400% to 1300%, preferably 400% to 1000%, more preferably 500% to 1100%, especially preferably 500% to 900%. This equilibrium swelling can be controlled, for example (for crosslinked polymers), by varying the degree of crosslinking, which in turn is achieved by varying the crosslinking conditions such as exposure time of the crosslinking agent, concentration of the crosslinking agent, crosslinking temperature, and the like. Materials with different equilibrium swell values behave differently in different applications. The ability to control crosslinking and balance swelling allows the compositions of the present invention to be optimized for a variety of uses. In addition to equilibrium swelling, it is also important to control hydration of the material immediately prior to release to the target site. Hydration and equilibrium swelling are of course closely related. The 0% hydrated material was not swellable. A 100% hydrated material will be that it is at equilibrium moisture content. Hydration between 0% and 100% will correspond to swelling between the minimum and maximum levels. The "equilibrium swelling" can be determined by subtracting the dry weight of the gelatin hydrogel powder from the weight when the gelatin hydrogel powder is fully hydrated and thus fully swollen. This difference is then divided by the dry weight and multiplied by 100 to give the swell measurement. The dry weight should be measured after exposing the material to an elevated temperature for a time sufficient to remove substantially all residual moisture, e.g., 120 ℃, two hours. Equilibrium hydration of the material can be achieved by immersing the dried material in a pharmaceutically acceptable diluent such as saline for a period of time sufficient for the water content to become constant, typically 18 to 24 hours at room temperature.

The cross-linked gelatin may be provided as a film which can then be milled to form a granular material. The majority of the particles (e.g. more than 90% w/w) comprised in the particulate material preferably have a particle size of 10-1.000. mu.m, preferably 50-700. mu.m, 200-700. mu.m, 300-550. mu.m, particularly preferably 350-550. mu.m.

Preferably, the flowable form of the haemostatic composition contains more than 50% (w/w) particles of size 100-1000 μm, preferably more than 80% (w/w) particles of size 100 to 1000 μm.

Examples of suitable gelatin materials for cross-linking are described in examples 1 and 2 of EP1803417B1, and in examples 14 and US6,063,061A of US6,066,325A. Gelatin may also be used with processing aids such as PVP, PEG and/or dextran as rehydration aids.

In a particular aspect of the invention, the composition comprises a cross-linked gelatin powder having a water content of 20% (w/w) or less, wherein the powder is cross-linked in the presence of a rehydration aid such that the rehydration rate of the powder is at least 5% higher than the rehydration rate of a similar powder prepared without the rehydration aid. "rehydration rate" is defined according to EP1803417B1 and means the amount of aqueous solution, typically 0.9% (w/w) saline, taken up by 1 gram of powder (dry weight composition) in 30 seconds, expressed as g/g. The rehydration rate was measured by the following method: the crosslinked gelatin was mixed with the saline solution for 30 seconds and the wet gelatin was deposited on a filter membrane under vacuum to remove the free aqueous solution. The weight of wet gelatin remaining on the filter is then recorded, dried (e.g., at 120 ℃ for 2hr), and the dry weight of gelatin is then recorded, calculating the weight of solution absorbed per gram of dry gelatin.

Preferred compositions of the invention have a rehydration rate of at least 3g/g, preferably at least 3.5g/g, typically above 3.75 g/g. Similar powders prepared without the use of rehydration aids typically have a rehydration rate below 3 and a percentage increase in rehydration rate of generally at least 5%, preferably at least 10%, more preferably at least 25% or more.

Crosslinking can be carried out using any suitable crosslinking agent, such as glutaraldehyde as described in WO98/08550A and WO 2003/007845A. Crosslinking can also be carried out using non-toxic crosslinking agents such as genipin and the like.

The genipin cross-linked gelatin product according to the present invention has a lower production cost than the glutaraldehyde cross-linked product because of the lower reagent, energy, and time costs. The genipin cross-linked gelatin reaction can be performed in water at neutral pH at room temperature for <16 hours. The product can be purified by ethanol and/or water washing, which is not only cheaper but, more importantly, safe for the operator.

The process preferably uses gelatin which is present in dry form prior to the crosslinking step.

A preferred genipin type of cross-linking agent according to the invention is of course genipin (methyl (1R,2R,6S) -2-hydroxy-9- (hydroxymethyl) -3-oxabicyclo [4.3.0] nonane-4, 8-diene-5-carboxylate). However, other crosslinkers of the iridoid type or secoiridoid glycoside (secoiridoid) type, such as oleuropein, may also be used. Preferred concentrations of genipin for crosslinking are in the range of 0.5-20mM, preferably 1-15mM, especially 2-10 mM.

According to a preferred embodiment of the invention, the genipin cross-linked gelatin is subjected to a quenching/oxidation step with an oxidizing agent such as bleach, tBu hydroperoxide or the like, preferably sodium percarbonate, sodium hypochlorite, chlorine water or hydrogen peroxide (H)₂O₂) Treatment, particularly preferably with sodium percarbonate or H₂O₂The treatment is carried out, and percarbonate is optimally used for treatment.

Preferably H₂O₂The concentration is 0.5-20% (w/w), particularly preferably 1-15% (w/w), more preferably about 5% (w/w). In a particularly preferred embodiment, the genipin concentration is between 5 and 10mM, the reaction time of gelatin with genipin is between 3 and 10 hours, particularly preferably 6 hours, H₂O₂Concentration between 3 and 5% (w/w), genipin cross-linked gelatin with H₂O₂The reaction time of (a) is about 20 hours.

Preferably the percarbonate concentration is between 1 and 10% (w/w), especially preferably 1 to 5% (w/w), more preferably 1 to 4% (w/w). In a particularly preferred embodiment, the genipin concentration is between 5-10mM (particularly preferably about 8mM), the reaction time of gelatin with genipin is between 3 to 10 hours (particularly about 5 hours), the percarbonate concentration is between 1-10% (w/w), particularly preferably between 1 to 4% w/w, and the reaction time of genipin cross-linked gelatin with percarbonate is between 1 to 20 hours, preferably between 1 to 5 hours (e.g. 1, 2 or 3 hours).

Quenching may also be performed in the presence of an antioxidant such as sodium ascorbate, or by controlling the oxidation potential of the reaction environment, such as quenching in an inert environment such as nitrogen or argon and/or genipin reaction.

Preferred crosslinking reaction conditions include in aqueous solution, preferably in Phosphate Buffered Saline (PBS)/ethanol buffer, especially pH4-12, preferably 5.0-10.0, especially 6-8 buffer, or in deionized water or other buffered aqueous solutions which may contain 0-50% water miscible organic solventsIs carried out in liquid. PBS buffer contains physiological amounts of NaCl and KCl in phosphate buffer at physiological pH. Examples of PBS buffer contain 137mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄·2H₂O、1.76mMKH₂PO₄(pH 7.4). Another example of PBS buffer is composed of 137mM NaCl, 2.7mM KCl, 4.3mM Na₂HPO₄And 1.4mMKH₂PO₄(pH 7.5).

The reaction may also be carried out in aqueous buffered solutions containing up to 50% water-miscible organic solvents and/or processing aids such as PEG, polyvinylpyrrolidone (PVP), mannitol, sodium percarbonate, sodium lactate, sodium citrate, sodium ascorbate, and the like.

Preferably, the crosslinking step is carried out at a temperature of 4 ℃ to 45 ℃, preferably 15 ℃ to 45 ℃, especially 20 ℃ to 40 ℃.

The crosslinking step may be followed by a quenching step, in particular with an amino group containing quencher, preferably an amino acid, especially glycine. By using a quencher, the genipin-type cross-linking agent that has not reacted is deactivated (e.g., by reacting with excess quencher) in order to inhibit further cross-linking. Quenching can also be performed by raising the pH of the solution to between 8 and 14, or by using nucleophilic compounds containing amino, thiol, or hydroxyl groups, and combinations of raising the pH and using nucleophilic compounds. Performing the quenching step after the genipin-gelatin crosslinking reaction according to the present invention can positively impart desired physical properties, such as swelling and TEG, which are important determinants of hemostatic activity, superior to the common genipin crosslinking alone.

The crosslinked gelatin is preferably washed after the crosslinking step, preferably with methanol, ethanol or water, particularly preferably with deionized water. Another preferred washing step employs an aqueous buffered solution containing up to 50% (v/v) of a water-miscible organic solvent and/or one or more processing aids.

According to a preferred embodiment, the crosslinked gelatin is dried. In such a dry state, the hemostatic composition is stable for long periods of time even at elevated temperatures (e.g., above 20 ℃, above 30 ℃, or even above 40 ℃). Preferred drying conditions include crosslinked biocompatible polymers which are dried to have a water content of less than 15% (w/w), preferably less than 10%, more preferably less than 5%, especially preferably less than 1%. In another preferred embodiment, the product may be provided in a hydrated or wet state in which the hydrolysis solution may be a biocompatible buffer or solution.

Glutamic acid-gel products have a tendency to be camouflaged by surrounding tissue, since they are yellowish, mixed with surrounding tissue. This makes the visual assessment of the intended application uncertain. The genipin cross-linked gelatin product according to the present invention exhibits variable color from light yellow to deep blue or green based on the extent of the cross-linking reaction conditions, and subsequent processing and finishing steps. This color adjustment capability and the ability to achieve a desired color in the final product has the added advantage of providing a visual indication to the physician when applying the appropriate product to the wound site, since the color can distinguish it from the surrounding tissue, rather than possibly being masked by the surrounding tissue. This is another novel feature of the present invention. On the other hand, the color can be removed to give a colorless product, depending on the requirements of the final product.

In a preferred embodiment, a biocompatible polymer, such as gelatin crosslinked with a genipin-type crosslinking agent, such as genipin, is a homogeneously crosslinked polymer that can be shown, for example, by fluorescence measurements, as described in example 3 herein. In a particularly preferred embodiment, the biocompatible polymer, such as gelatin, is present as a biocompatible polymer, such as gelatin, in particulate form that is homogeneously cross-linked by genipin.

The hemostatic composition according to the present invention preferably contains an excipient, such as a lubricant, e.g. hyaluronic acid, therein.

In another embodiment of the invention, no excipients, such as lubricants, e.g. hyaluronic acid, are present.

The pharmaceutically acceptable diluent is preferably an aqueous solution, and may contain a substance selected from the group consisting of: NaCl, CaCl₂And sodium acetate.

For example, the pharmaceutically acceptable diluent comprises water for injection, and independently of each other, 50-200mM NaCl (preferably 150mM), 10-80mM CaCl₂Preferably 40mM, and 1-50mM sodium acetate, preferably 20 mM. In another embodiment, the pharmaceutically acceptable diluent contains less than 35g/L mannitol, preferably less than 25g/L mannitol, more preferably less than 10g/L mannitol, and it is especially preferred that the pharmaceutically acceptable diluent is substantially free of mannitol.

According to a preferred embodiment, the pharmaceutically acceptable diluent comprises thrombin, preferably 10-1000I.U. thrombin/mL, particularly preferably 250-700I.U. thrombin/mL. Preferably, the hemostatic composition is in a ready-to-use form comprising 10-100,000 International units (I.U.) of thrombin, more preferably 100-10,000IU, especially preferably 500-5,000 I.U.S. of thrombin. Thrombin (or any other coagulation inducing agent such as snake venom, platelet activators, thrombin receptor activating peptides and fibrinogen precipitating agents) can be derived from any (i.e. pharmaceutically acceptable) thrombin preparation suitable for use in humans. Suitable thrombin sources include human and bovine blood, plasma or serum (other animal sources of thrombin can be used if an adverse immune response is not anticipated), recombinant sources of thrombin (e.g., human recombinant thrombin) and autologous human thrombin can be preferred for some applications.

The pharmaceutically acceptable diluent is used in an amount to achieve the desired final concentration in the ready-to-use composition. The thrombin preparation may contain other useful components such as ions, buffers, excipients, stabilizers, etc. Preferably, the thrombin preparation contains human albumin as a compression enhancer. Preferred salts are NaCl and/or CaCl₂Both are applied to thrombin in conventional amounts and concentrations (e.g., 0.5-1.5% NaCl (e.g., 0.9%) and/or 20-80mM CaCl₂(e.g., 40 mM)).

In another embodiment, the diluent may also comprise a buffer or buffer system to buffer the pH of the reconstituted dry composition, preferably at pH3.0-10.0, more preferably at pH6.4-7.5, especially preferably at pH 6.9-7.1.

Suitable amounts of cross-linked gelatin, diluent and extrusion enhancer can be established in a kit according to the above prerequisites: for example, a) vials with 0.736-0,995g of dry cross-linked gelatin (corresponding to 15.0-19.5% (w/w) in the final product) and b) 4mL of diluent with 60-240mg albumin and optionally thrombin and/or 40 mcaacl at a concentration of 500i.u./mL can be provided₂The second vial of (a). Alternatively, albumin may be added to the dry gelatin component of a) of the kit in a freeze-dried form. For example, there may be provided: a) vials with 0.573-0.775g dry cross-linked gelatin (corresponding to 15.0-19.5% (w/w) in the final product) containing 48-192mg albumin; and b) thrombin and/or 40mM CaCl with 3.2mL of diluent and optionally at a concentration of 500I.U./mL₂The second vial of (a).

The cross-linked gelatin component of the kit according to the invention is preferably provided as a dry composition, wherein the cross-linked gelatin is present in dry form.

The residual moisture content of the substantially dry crosslinked gelatin compositions according to the present invention may be about equivalent to that of comparable commercially available products such as(Floseal for example has a moisture content of about 8-12% of the dry product).

In the kit according to the invention, the dry cross-linked gelatin in particulate form suitable for use in haemostasis is preferably gelatin in powder form, particularly preferably the median particle size of the powder particles is from 10 to 1000. mu.m, preferably from 50 to 700. mu.m, 200-550. mu.m, 300-550. mu.m, particularly preferably 350-550. mu.m. The "dried crosslinked gelatin particulate products" according to the invention are generally known and are described, for example, in WO 98/08550A. Preferably, the crosslinked gelatin is a biocompatible, biodegradable, dry stable particulate material.

According to another aspect, the present invention relates to a hemostatic composition according to the present invention for use in the treatment of a lesion selected from the group consisting of: a wound, bleeding, damaged tissue, bleeding tissue, and/or bone injury.

Another aspect of the invention is a method of treating a lesion selected from the group consisting of: a wound, a hemorrhage, damaged tissue and/or bleeding tissue, the treatment comprising administering a hemostatic composition according to the invention to the site of injury.

According to another aspect, the present invention also provides a method for releasing a hemostatic composition according to the present invention to a target site in the body of a patient, the method comprising releasing a hemostatic composition manufactured according to the method of the present invention to the target site. Although the dry composition can also be administered directly to the target site (and optionally contacted with a diluent at the target site if necessary), it is preferred to contact the dry hemostatic composition with a pharmaceutically acceptable diluent prior to administration to the target site, so as to obtain a hemostatic composition according to the invention in the form of an ointment.

In such a method, a kit for making a flowable cross-linked gelatin ointment may be used for treating a lesion selected from the group consisting of: a wound, bleeding, damaged tissue, and/or bleeding tissue, the kit comprising:

a) a dry haemostatic composition to be reconstituted into a flowable ointment comprising cross-linked gelatin in particulate form, the flowable ointment comprising 15.0-19.5% (w/w) cross-linked gelatin (═ dry gelatin weight/final composition weight), preferably 16.0-19.5% (w/w), 16.5-19.5% (w/w), 17.0-18.5% (w/w) or 17.5-18.5% (w/w), more preferably 16.5-19.0% (w/w) or 16.8-17.8% (w/w), especially preferably 16.5-17.5% (w/w) cross-linked gelatin; and

b) a pharmaceutically acceptable diluent for reconstituting the hemostatic composition,

wherein the composition or diluent comprises an appropriate amount of a compression enhancer, especially albumin, for example in an amount (for albumin) such that the albumin concentration in the reconstituted ointment is between 0.5-5.0% (w/w) (-weight of compression enhancer/weight of final composition), preferably 1.0-5.0% (w/w), preferably 2.0-4.5% (w/w), more preferably 1.5-5.0% (w/w), especially preferably about 1.5% (w/w).

Preferred further components of such a kit, in particular if the hemostatic composition is contained in dry form, are diluents (i.e. rehydration media) for reconstituting the hemostatic composition. Other components of the kit may be administration tools such as syringes, catheters, brushes, etc., if the composition is not provided in the administration tool, or other components necessary for use in medical practice (surgery) such as replacement needles or catheters, additional vials or other wound covering means. Preferably, the kit according to the invention comprises a syringe containing the dried and stabilized hemostatic composition, and a syringe containing the diluent (or provided for removing the diluent from another diluent container).

In a preferred embodiment, the pharmaceutically acceptable diluent is provided in a separate container. It can preferably be a syringe. The diluent in the syringe can then be easily applied to the final container for reconstitution of the dried hemostatic composition according to the present invention. If the final container is also a syringe, both syringes can be completed together in one package. It is therefore preferred to provide the dry hemostatic composition according to the present invention in a syringe, which is completed with a diluent syringe having a pharmaceutically acceptable diluent, for reconstitution of the dry and stable hemostatic composition.

According to a preferred embodiment, the final container further contains an effective amount of a stabilizer to inhibit alteration of the polymer upon exposure to sterilizing radiation, the stabilizer preferably being ascorbic acid, sodium ascorbate, other salts of ascorbic acid, or antioxidants.

With such a pharmaceutically acceptable diluent, a ready-to-use hemostatic composition of the present invention may be provided, which can then be directly applied to a patient. Thus, there is also provided a method for providing a ready-to-use hemostatic composition according to the invention, wherein the hemostatic composition is provided in a first syringe, a diluent for reconstitution is provided in a second syringe, the first and second syringes are in communication with each other, and liquid is brought into the first syringe to produce a flowable form of the hemostatic composition; and optionally returning the flowable form of the hemostatic composition to the second syringe at least once. Preferably, the ready-to-use article is present as, or provided as, a hydrogel. Such products are generally known in the art in different forms. Thus, a preferred embodiment of the present invention also provides a method for providing a ready-to-use hemostatic composition according to the present invention, wherein the hemostatic composition is provided in a first syringe, a diluent for reconstitution is provided in a second syringe, the first and second syringes are in communication with each other, and the diluent is brought into the first syringe to produce a flowable form of the hemostatic composition; and optionally returning the flowable form of the hemostatic composition to the second syringe at least once. This method, also known as "swirling", provides a suitable ready-to-use form of the composition according to the invention, which can be manufactured easily and efficiently in a short time, e.g. in an emergency during surgery. The flowable form of the hemostatic composition provided by such a method is particularly suitable for treating a lesion selected from the group consisting of: a wound, bleeding, damaged tissue, bleeding tissue, and/or bone injury.

For stability reasons, these products (and the products according to the invention) are usually provided in the final container in dry form and immediately before use form a ready-to-use form, usually in the form of a (aqueous) gel, suspension or solution, necessitating the addition of a pharmaceutically acceptable diluent (the rehydration medium).

According to another aspect, the present invention relates to a method for providing a ready-to-use hemostatic composition according to the present invention, wherein the hemostatic composition is provided in a first syringe, a diluent for reconstitution is provided in a second syringe, the first and second syringes are in communication with each other, and a liquid is brought into the first syringe to produce a flowable form of the hemostatic composition; and optionally returning the flowable form of the hemostatic composition to the second syringe at least once.

Preferably, the haemostatic composition according to the invention in flowable form contains more than 50% (w/w) particles with a size of 100-.

Once the biocompatible hemostatic cross-linked polymer according to the present invention is applied to a wound, an effective matrix capable of forming a blood flow barrier is formed. In particular, the swelling properties of the hemostatic polymer can make it an effective mechanical barrier to inhibit bleeding and repeated bleeding procedures.

The compositions of the present invention may additionally contain a hydrophilic polymeric component (also referred to as a "reactive hydrophilic component" or a "hydrophilic (polymeric) crosslinker") which may further enhance the adhesive properties of the compositions of the present invention. The hydrophilic polymeric component of the hemostatic composition according to the present invention functions as a hydrophilic cross-linking agent that is capable of reacting with its reactive groups upon application of the hemostatic composition to a patient (e.g., to a wound of the patient or to another site on the patient where hemostatic activity is desired). Thus, it is important for the present invention that the reactive groups in the hydrophilic polymeric component are reactive when administered to a patient. Thus, in manufacturing the hemostatic composition according to the present invention, it is necessary that the reactive groups in the polymeric component are retained during the manufacturing process, which should react once the hemostatic composition is applied to a wound.

Since the reactive groups of the hydrophilic polymeric crosslinker are hydrolyzable, the hemostatic composition must be protected from premature contact with water or aqueous solutions prior to administration of the hemostatic composition to a patient, particularly during manufacture. However, during manufacture, processing of the hydrophilic polymeric component may also be carried out in an aqueous medium under conditions (e.g., at low pH) in which reaction of the reactive groups is inhibited. If the hydrophilic polymeric component is capable of being melted, the melted hydrophilic polymeric component can be sprayed, or printed, onto the matrix of cross-linked gelatin. It is also possible to mix the hydrophilic polymeric component in dry form (e.g. as a powder) with the crosslinked gelatin in dry form. If necessary, a temperature increase can be applied to melt the hydrophilic polymeric component sprinkled onto the cross-linked gelatin, so as to obtain a durable coating of the hemostatic composition. Alternatively, these hydrophilic polymeric components can be added to an inert organic solvent (inert compared to the reactive groups of the hydrophilic polymeric components) and added to the matrix of cross-linked gelatin. Examples of such organic solvents are dry ethanol, dry acetone or dry dichloromethane (which are inert towards hydrophilic polymeric components, such as PEGs substituted with NHS-esters, for example). Optionally, nucleophilic groups (e.g., PEG-SH) may also be added.

In a preferred embodiment, the hydrophilic polymer component is a single hydrophilic polymer component and is a polyalkylene oxide polymer, preferably PEG comprising a polymer. The reactive group in the reactive polymer is preferably an electrophilic group.

The reactive hydrophilic component may be a poly-electrophilic polyalkylene oxide polymer, such as poly-electrophilic PEG. The reactive hydrophilic component can comprise more than two electrophilic groups, preferably the PEG comprises more than two groups selected from succinimidyl ester (-CON (COCH)₂)₂) Aldehyde (-CHO), and isocyanate (-N ═ C ═ O), such as the components disclosed in WO2008/016983a (which is incorporated herein by reference in its entirety).

Preferred electrophilic groups in the hydrophilic polymeric crosslinkers according to the present invention are groups that are reactive with amino, carboxyl, thiol and hydroxyl groups in proteins, or mixtures thereof.

Preferred reactive groups specific for amino groups in the presence of carbodiimide isocyanate, or THPP (β - [ tris (hydroxymethyl) phosphino ] propionic acid), are NHS-ester, imino-ester, aldehyde, carboxyl groups, and especially preferred are pentaerythritol poly (ethylene glycol) ether tetrasuccinimide glutarate (═ pentaerythritol tetrakis [1-1 '-oxy-5' -succinimidyl valerate-2-polyoxyethyleneglycol ] ether (═ NHS-PEG, MW10,000).

Preferred reactive groups specific for carboxyl groups are amino groups in the presence of carbodiimides.

Preferred reactive groups specific for thiol groups are maleimides or haloacetyl groups.

Preferred reactive groups specific for hydroxyl groups are isocyanate groups.

The reactive groups on the hydrophilic crosslinker may be the same (functional homotype) or different (functional heterotype). The hydrophilic polymeric component may have two reactive groups (homobifunctional or heterobifunctional) or more reactive groups (homotrifunctional/heterobifunctional).

In a particular embodiment, the material is a synthetic polymer, preferably comprising PEG. The polymer may be a derivative of PEG that contains reactive side groups suitable for crosslinking and adhering to tissue.

By virtue of these reactive groups, the hydrophilic reactive polymer has the ability to crosslink blood proteins, as well as tissue surface proteins. Crosslinking with crosslinked gelatin is also possible.

The poly-electrophilic polyalkylene oxide can include two or more succinimide groups. The poly-electrophilic polyalkylene oxide may include more than two maleimide groups.

Preferably, the multi-electrophilic polyalkylene oxide may be polyethylene glycol or a derivative thereof.

In a most preferred embodiment, the hydrophilic polymeric component is pentaerythritol poly (ethylene glycol) ether tetrasuccinimidyl glutarate (═ COH102, i.e., pentaerythritol tetrakis [1-1 '-oxo-5' -succinimidyl valerate-2-polyoxyethyleneglycol ] ether).

The hydrophilic polymeric component is a hydrophilic cross-linking agent. According to a preferred embodiment, the cross-linking agent has more than two reactive groups ("arms") for cross-linking, for example 3, 4, 5, 6, 7, 8, or more arms with reactive groups for cross-linking. For example, NHS-PEG-NHS is an effective hydrophilic crosslinker according to the invention. However, for some embodiments, a 4-arm polymer (e.g., 4-arm-p-NP-PEG) may be more preferred; based on the same principle, for those embodiments in which multiple reactive crosslinks are advantageous, an 8-arm polymer (e.g., 8-arm-NHS-PEG) may be even more preferred. In addition, hydrophilic crosslinkers are polymers, i.e., macromolecules (macromers) composed of repeating structural units, which are typically linked by covalent chemical bonds. The hydrophilic polymer component should have a molecular weight of at least 1000Da (in order to suitably function as a cross-linking agent in the hemostatic composition according to the invention); the crosslinked polymers according to the invention preferably have a molecular weight of at least 5000Da, particularly preferably at least 8000 Da.

For some hydrophilic crosslinkers, the presence of alkaline reaction conditions (e.g., at the site of administration) is preferred or necessary for functional properties (e.g., for faster crosslinking reactions at the site of administration). For example, carbonate or bicarbonate ions (e.g. as a buffer at a ph of 7.6 or more, preferably 8.0 or more, especially 8.3 or more) may additionally be provided at the site of administration (e.g. as a buffer solution, or as a fabric or pad impregnated with such a buffer), allowing to improve the performance of the hemostatic composition according to the invention, or allowing to be effectively used as a hemostatic material and/or a wound adhesive material.

The reactivity of the hydrophilic polymeric component (which, as noted above, acts as a cross-linking agent) in the composition according to the present invention is retained in the composition. This means that the reactive groups of the cross-linking agent have not reacted with the hemostatic composition and have not been hydrolyzed by water (or at least not in significant amounts, without having a negative consequence on the hemostatic function of the composition of the present invention). This can be achieved by combining the cross-linked gelatin with a hydrophilic cross-linking agent in a manner that does not result in the reactive groups in the cross-linking agent reacting with the hemostatic polymer, or with water. Typically, this includes omitting wetting in an aqueous environment (or wetting), especially non-acidic conditions (provided the crosslinker is non-reactive under acidic conditions). This allows to obtain a reactive hemostatic material.

Preferably, in the hemostatic composition according to the invention, the ratio of crosslinked gelatin to hydrophilic polymeric component is 0.1-50% (w/w), preferably 5-40% (w/w).

Other components may also be present in the hemostatic composition according to the present invention. According to a preferred embodiment, the hemostatic composition according to the invention may further comprise a substance selected from the group consisting of: anti-fibrinolytic agents, coagulants, platelet activators, antibiotics, vasoconstrictors, pigments, growth factors, bone morphogenic proteins, and analgesics.

The invention also relates to a self-contained final container containing a hemostatic composition according to the invention. The final container contains the hemostatic composition according to the present invention in a sterile, storage-stable and marketable form. The final container can be any container suitable for containing (and storing) a pharmaceutically administrable compound. Syringes, vials, test tubes, etc. may be used; however, it is particularly preferred to provide the hemostatic composition according to the present invention in a syringe. Syringes have been the preferred means of administration for hemostatic compositions, as disclosed in the art, and also because of the handling advantages of syringes in medical practice. The composition may then be applied, preferably through a special syringe needle or through a suitable catheter (after reconstitution). The reconstituted hemostatic composition, which is preferably reconstituted to form a hydrogel, may also be applied by a variety of other means, such as by spatula, brush, spray, manual pressure, or by any other conventional technique. It is particularly preferred that the reconstituted hemostatic composition is administered to the patient by endoscopic (laparoscopic surgery) means. Typically, the reconstituted hemostatic composition according to the present invention is applied by using a syringe or similar applicator that is capable of extruding the reconstituted composition through a port, opening, needle, tube, or other means to form a bead, layer, or similar partial material. Mechanical dissociation of the composition can be effected by squeezing an orifice in the syringe or other applicator, typically having a size in the range 0.01mm to 5.0mm, preferably 0.5mm to 2.5 mm. However, it is preferred that the hemostatic composition is prepared initially from a dry form having the desired particle size (prepared upon reconstitution, especially by hydration, to produce subunits of the requisite size (e.g., hydrogel subunits)), or is partially or fully mechanically dissociated to the requisite size prior to the final extrusion or application step. Obviously, the components of these machines need to be provided in sterile form (inside and outside) in order to meet safety requirements for human use.

The hemostatic composition according to the invention is preferably applied to a patient in the form of a paste from a container as described in example 1, said container having a squeezing pressure below 40N, such as below 30N, or below 20N, preferably in the range of 15-30N.

Another aspect of the invention relates to a method for providing a ready-to-use hemostatic composition comprising contacting a hemostatic composition according to the invention.

The invention will be further described in connection with the following examples and the accompanying drawings to which the invention is not limited.

FIG. 1 shows the average squeeze force (the squeeze force required to push the product out of the syringe at a compression rate of 250 mm/min, measured at a distance of 35 mm; all products were incubated at room temperature for 30 minutes, and rapidly "re-vortexed" shortly before the squeeze force measurement, the ointment containing 17.5% (w/w) of cross-linked gelatin with varying concentrations of human serum albumin in the thrombin component.

Figure 2 shows the consistency of a cross-linked gelatin ointment containing 17.5% (w/w) (depending on the human serum albumin concentration) cross-linked gelatin.

Figure 3 shows the average squeeze force for genipin cross-linked gelatin ointments containing 17.5% (w/w) gelatin with different concentrations of human serum albumin in the thrombin component. The X-axis shows the concentration of human serum albumin in the thrombin component [ g/L ] and the Y-axis shows the mean extrusion force [ N ].

Figure 4 shows an assessment and approximation of the bleeding severity after application of the test article.

Fig. 5 to 8 show the hemostatic efficacy of different preparations in a porcine liver perforation-biopsy model. The X-axis shows the time [ sec ] after application and the Y-axis shows the percentage of hemostatic performance (defined as "no bleeding" in fig. 5 and "no bleeding" or "oozing" in fig. 6).

In fig. 5 and 6, the symbols refer to:

_____ glutaraldehyde cross-linked gelatin with 50g/L human serum albumin in thrombin solution (n ═ 8)

Glutaraldehyde cross-linked gelatin with 75g/L human serum albumin in thrombin solution (n ═ 8)

In fig. 7 and 8, the symbols refer to:

_____ ≈ 17.5% (w/w) glutaraldehyde crosslinked gelatin

14.5% (w/w) glutaraldehyde cross-linked gelatin

… …. approximant to 17.5% (w/w) glutaraldehyde cross-linked gelatin + 2.5% PEG10,000 in thrombin solution

Examples

Example 1: measurement of Extrusion Force (EF)

The extrusion force required to extrude the product from the syringe was measured using an Instron (tensile tester) mechanical tester model 5544 equipped with a 100N load cell, operating at a longeron speed (cross-beam) of 250 mm/min. The required squeeze force was measured during the full ram displacement (34mm offset), which corresponds to the distance the syringe plunger was moved to squeeze almost all of the product out of the syringe. From these pressures, the average extrusion force was calculated according to the following formula:

the samples used for this test were prepared as follows: a 5mL standard syringe (with a cylinder with an internal diameter of 12.2 mm) with a male lock interface (maleluer lock) system (in which the internal orifice lumen to which the adapter is attached measures 2.54mm in diameter) is filled with a solid sample of 0.704g dry matter (drawing up about 12% residual moisture, calculated to be about 0.8 g). And the diluent, 3.2mL of thrombin solution containing 500i.u./mL thrombin in 40mM calcium chloride and 0, 5, 15, 25, 50 or 75mg/mL human serum albumin was used. The diluent and solid components are mixed by communicating a syringe holding the diluent (a standard 5ml syringe with a female lock interface (femaleluerlock) system) with the syringe holding the dry components and pushing the contents back and forth at least 10 times (this mixing technique is referred to as "vortexing"). Thereafter the samples were incubated at room temperature for 30 minutes before measurement. After incubation, each sample was "vortexed" twice and the syringe holding the product (syringe previously holding the dry component as described above) was communicated to the applicator (female locking connector system, inner tube diameter 2.29mm, holding double line, total length 141.5 mm). The syringe was assembled to the applicator and placed in the set Instron and the test started.

The syringe and applicator are part of a commercially available Floseal hemostatic matrix product from Baxter.

The test results for glutaraldehyde cross-linked gelatin in Floseal are depicted in fig. 1 and the test results for genipin cross-linked gelatin are depicted in fig. 3 below, and these results are also shown in corresponding tables 1 and 2.

The consistency of the cross-linked gelatin ointment containing 17.5% (w/w) cross-linked gelatin depending on the albumin concentration is shown in figure 2 (providing 0, 25, 50 and 75g/L human serum albumin in the thrombin component).

Table 1:

albumin concentration in the Thrombin fraction [ g/L]	Extrusion force [ N ]]	Standard deviation of
			0	40	2,4
5	38	1,5
			15	30	2,6
25	25	2,2
			50	19	1,5
60	19	1,0

Table 2:

albumin concentration in the Thrombin fraction [ g/L]	Extrusion force [ N ]]	Standard deviation of
			0	54	1.9
15	29	3.6
			50	17	2.2

Preparation of genipin cross-linked gelatin:

bovine derived collagen is processed by alkaline treatment followed by rinsing with deionized process water (DIW) to remove residual salts. Extracting gelatin by heat treatment, and drying to obtain sheet. The sheet was ground into powder and treated with genipin as a cross-linking agent.

1kg of gelatin granules was added to 20L of 10mM genipin DIW solution. The reaction was carried out at neutral pH (7.2) in a jacket temperature-controlled tank at 23 ℃. Mixing was carried out for 6 hours, the solution was drained off, the solid remained in the grid, washed with DIW and the residual genipin was washed off. Resuspend the material in 5% H₂O₂The solution was left for 20 hours. The material was washed with DIW to remove H₂O₂. The solid was pre-dried on filter paper under vacuum and then oven dried for 2.5 days. Grinding the dried matrix to powder, and exposing to light_γPrior to irradiation, each plastic syringe was filled.

Example 2: determination of hemostatic efficacy

Materials and methods:

animal model

For this model, a midline laparotomy was performed, followed by electrocautery to terminate bleeding from the surgical incision. The liver was exposed and the lobes separated. Biopsy with 10mm diameter punch produced 2 sequential incomplete thickness lesions approximately 5mm deep, with nuclear tissue removed. A pretreatment test was performed on the scars, including collecting blood flow from each scar for 10 seconds using pre-weighed gauze.

The test article was randomized and provided to a surgeon unaware of the sample treatment. Approximately 1.0mL of the indicated test article was applied topically to the lesion. Saline-wetted gauze was used to help evaluate the test article at the designated scar, starting the timer. The saline-wetted estimate gauze was removed after 30 seconds.

The extent of bleeding was evaluated 30, 60, 90, 120, 300, and 600 seconds after the test articles were applied to their designated lesions as described in fig. 3 (bleeding fraction: 0: no bleeding (product saturated with blood), 1: bleeding (blood out of product but no blood drop), 2: very slight (blood drop on product), 3: slight (blood drop down), 4: moderate (small blood flow), 5: severe (large blood flow)).

Products saturated with blood, but without active bleeding, are scored as "0" (zero). After the 300 second test, the excess test article was rinsed with saline away from the scar. This procedure was repeated in multiple lobes. A surgeon generates, processes, and performs the observational tests.

Test article synthesis

Test articles for in vivo evaluation in a pig liver model were prepared by preparing cross-linked gelatin ointments (concentration of 14.5% and 17.5% with 25 or 50g/L human serum albumin in thrombin solution (with or without additional 2.5% PEG)).

The results are depicted in fig. 5 to 8, showing improved performance with 17.5% gelatin, but little efficacy in the presence of Plasticizer (PEG).

Example 3

Gelatin samples were prepared according to the Floseal packaging instructions, except for the coupling key. First, gelatin was formulated using sodium chloride instead of calcium chloride and 125% solids instead of 100%. The gelatin/thrombin formulation was allowed to stand for 25 minutes, and then 1mL of the preparation was discarded. Another 1ml of material was applied to the Topical Hemostatic System (THS). The THS device is pre-filled with platelet-deficient plasma.

THS is a device designed to simulate bleeding wounds. The artificial wound is a cylindrical cavity in a silicone substrate. The surface of the silicone cylinder is coated with a layer of fibrinogen. The syringe pump discharges the clotting liquid (whole blood, plasma, etc.), in this case platelet-poor plasma, while the back pressure is recorded. In this experiment, plasma was flowed at a fixed rate of 0.25 ml/min through a small hole in the center of the bottom of a cylindrical wound. Immediately prior to application of the hemostatic matrix, excess plasma was aspirated with gauze. As the plasma continued to flow, 1ml of hemostatic matrix was applied to the cylindrical wound. The wound was immediately covered with moist gauze and a fixed pressure was applied. After 30 seconds, the weight was removed and the plasma continued to flow for 8-10 minutes, at which point the flow was terminated and the clot was set aside in the humidity test vessel for more than 2 hours. At the end of two hours, the clot was mounted on a vibrating microtome at 8 ℃ and sections of approximately 500 μm thickness were cut from the clot. These sections were immersed in PBS buffer. When not in use, the sections were stored in a 5 ℃ refrigerator. Sections were placed on cover slips and photographed with a NikonA1R confocal microscope running NIS-elementsadvancedesreacchv3.22.00build 710 software. To collect the micrographs, a planar fluorescent 10x objective was used, the laser excited 488nm light, and the emission collection window was 500-550 nm. While transmitted light images are collected using a transmitted light detector. With these image parameters, automated stitching by software is used to generate a macroscopic sample atlas. The smaller area of the sample was also characterized by collecting images of the 3 Dz-stack with an optical slice thickness of 5.125 μm. A composite confocal map was used to identify gelatin particles that were located on the surface and sectioned. This is important for the localization of elasticity measurements in atomic pressure microscopy (AFM). Clot slices were mounted in VeecoMultimodeAFM. The multimode (multimode) was equipped with a Nanoscope V controller and a JV piezoelectric scanner. Pressure measurements were performed using a NovascanAFM cantilever loaded with 4.5 μm polystyrene foam spheres. The pressure constant of the cantilever was determined to be 0.779N/m by the thermal coordination method (thermaltunethod). A cantilever was placed over the center of the gelatin particle and a 16 x 16 array of pressure measurements was taken. Each pressure curve involved moving the gelatin particles up into contact with the styrofoam ball and continued to move the particles until the cantilever deflection reached the preset trigger value of 2 volts, at which point the gelatin was retracted a distance of 1.00 microns from the trigger position.

Discussion of the related Art

Fluorescence data show that glutaraldehyde cross-linked gelatin is heterogeneously cross-linked. Instead, the crosslink density near the edges of the particle appears to be higher, with the central portion of the particle crosslinking significantly less than the edges. In contrast, the genipin cross-linked gelatin appeared to be uniformly (homogeneously) cross-linked throughout the particle. There is no substantial edge effect on the fluorescence intensity. The fluorescence intensities of the genipin-crosslinked material and the glutaraldehyde-crosslinked material cannot be directly compared because of possible fluorescence differences due to the crosslinking agent itself. However, the elastic modulus measurements of the AFM measurements show that genipin crosslinked gelatin is harder than glutaraldehyde crosslinked gelatin, which is now softer (more flexible).

Claims (45)

1. A haemostatic composition comprising cross-linked gelatin in particulate form suitable for use in haemostasis, wherein the composition is in the form of an ointment comprising 15.0-19.5% (w/w) cross-linked gelatin; and wherein the composition comprises an extrusion enhancer which is albumin in an amount of 0.5-5.0% (w/w).

2. The hemostatic composition of claim 1, wherein the composition comprises 16.0-19.5% (w/w) cross-linked gelatin.

3. The hemostatic composition of claim 1, wherein the composition comprises 16.5-19.5% (w/w) cross-linked gelatin.

4. The hemostatic composition of claim 1, wherein the composition comprises 17.0-18.5% (w/w) cross-linked gelatin.

5. The hemostatic composition of claim 1, wherein the composition comprises 17.5-18.5% (w/w) cross-linked gelatin.

6. The hemostatic composition of claim 1, wherein the composition comprises 16.5-19.0% (w/w) cross-linked gelatin.

7. The hemostatic composition according to claim 1, wherein the composition comprises 16.8-17.8% (w/w) cross-linked gelatin.

8. The hemostatic composition of claim 1, wherein the composition comprises 16.5-17.5% (w/w) cross-linked gelatin.

9. The hemostatic composition of claim 1, wherein the compression enhancer is albumin in an amount of 1.0-5.0% (w/w).

10. Hemostatic composition according to claim 1, wherein the albumin is present in an amount of 2.0-4.5% (w/w).

11. Hemostatic composition according to claim 1, wherein the albumin is present in an amount of 1.5-5.0% (w/w).

12. Hemostatic composition according to claim 1, wherein the albumin is present in an amount of 1.5% (w/w).

13. Hemostatic composition according to claim 1 or 9, wherein the cross-linked gelatin is glutaraldehyde cross-linked gelatin or genipin cross-linked gelatin.

14. Hemostatic composition according to claim 1 or 9, wherein the cross-linked gelatin is type B gelatin.

15. Hemostatic composition according to claim 1 or 9, wherein the cross-linked gelatin is present as a granular material.

16. Hemostatic composition according to claim 1 or 9, wherein the particle size of the cross-linked gelatin is 100-1000 μm.

17. Hemostatic composition according to claim 1 or 9, wherein the particle size of the cross-linked gelatin is 200-800 μm.

18. Hemostatic composition according to claim 1 or 9, wherein the particle size of the cross-linked gelatin is 350-550 μm.

19. Hemostatic composition according to claim 1 or 9, wherein the composition comprises thrombin.

20. Hemostatic composition according to claim 1 or 9, wherein the composition comprises thrombin in a concentration of 10-1000i.u. thrombin/mL.

21. The hemostatic composition according to claim 1 or 9, wherein the composition comprises thrombin at a concentration of 250 and 700i.u. thrombin/mL.

22. A haemostatic composition according to claim 1 or 9, for use in treating a lesion selected from the group consisting of: wounds, damaged tissue, bleeding tissue, and/or bone damage.

23. Use of a hemostatic composition according to any one of claims 1 to 21 in the preparation of a medicament for treating an injury comprising administering a hemostatic composition according to any one of claims 1 to 21 to a site of injury selected from the group consisting of: wounds, damaged tissue, and/or bleeding tissue.

24. A kit for manufacturing a flowable cross-linked gelatin ointment for treating a lesion selected from the group consisting of: a wound, damaged tissue, and/or bleeding tissue, the kit comprising:

a) a dry hemostatic composition comprising crosslinked gelatin in particulate form to be reconstituted into a flowable ointment comprising 15.0-19.5% (w/w) crosslinked gelatin; and

b) a pharmaceutically acceptable diluent for reconstituting the hemostatic composition,

wherein the composition or the diluent comprises albumin in an amount such that the concentration of albumin in the reconstituted ointment is between 0.5-5.0% (w/w).

25. The kit of claim 24, wherein the composition comprises 16.0-19.5% (w/w) cross-linked gelatin.

26. The kit of claim 24, wherein the composition comprises 16.5-19.5% (w/w) cross-linked gelatin.

27. The kit of claim 24, wherein the composition comprises 17.0-18.5% (w/w) cross-linked gelatin.

28. The kit of claim 24, wherein the composition comprises 17.5-18.5% (w/w) cross-linked gelatin.

29. The kit of claim 24, wherein the composition comprises 16.5-19.0% (w/w) cross-linked gelatin.

30. The kit of claim 24, wherein the composition comprises 16.8-17.8% (w/w) cross-linked gelatin.

31. The kit of claim 24, wherein the composition comprises 16.5-17.5% (w/w) cross-linked gelatin.

32. The kit of claim 24, wherein the amount of albumin is such that the concentration of albumin in the reconstituted ointment is 1.0-5.0% (w/w).

33. The kit of claim 24, wherein the amount of albumin is such that the concentration of albumin in the reconstituted ointment is 2.0-4.5% (w/w).

34. The kit of claim 24, wherein the amount of albumin is such that the concentration of albumin in the reconstituted ointment is 1.5-5.0% (w/w).

35. The kit of claim 24, wherein the amount of albumin is such that the concentration of albumin in the reconstituted ointment is 1.5% (w/w).

36. The kit of claim 24, wherein the pharmaceutically acceptable diluent comprises a buffer or a buffer system.

37. The kit of claim 24, wherein the pharmaceutically acceptable diluent comprises a buffer or buffer system having a pH of 3.0 to 10.0.

38. The kit of claim 24 or 36, wherein the pharmaceutically acceptable diluent comprises thrombin.

39. The kit of claim 24 or 36, wherein the pharmaceutically acceptable diluent comprises thrombin at a concentration of 10-1000i.u. thrombin/mL.

40. The kit of claim 24 or 36, wherein the pharmaceutically acceptable diluent comprises thrombin at a concentration of 250 and 700i.u. thrombin/mL.

41. The kit of claim 24 or 36, wherein the pharmaceutically acceptable diluent comprises a substance selected from the group consisting of: NaCl, CaCl₂And sodium acetate.

42. The kit of claim 38, wherein the pharmaceutically acceptable diluent comprises a substance selected from the group consisting of: NaCl, CaCl₂And sodium acetate.

43. A method for providing a ready-to-use form of the hemostatic composition of any one of claims 1 to 21, wherein the hemostatic composition is provided in a first syringe, a diluent for reconstitution is provided in a second syringe, the first and second syringes are in communication with one another, and liquid is brought into the first syringe to produce a flowable form of the hemostatic composition; and optionally returning the flowable form of the hemostatic composition to the second syringe at least once.

44. The method of claim 43, wherein the flowable form of the hemostatic composition comprises greater than 50% (w/w) particles having a size of 100-1000 μm.

45. The method of claim 43, wherein the flowable form of the hemostatic composition comprises greater than 80% (w/w) particles having a size of 100 to 1000 μm.