# Patent

## Title
Process for making dry and stable hemostatic compositions

## Abstract

Described is a process for making a dry and stable hemostatic composition, said process comprising

    
    
        a) providing a first component comprising a dry preparation of a coagulation inducing agent,
        b) providing a second component comprising a dry preparation of a biocompatible polymer suitable for use in hemostasis,
        c) mixing said first component and said second component under conditions effective to form a wet paste while essentially preventing degradation of the second component by said first component in a final container or transferring said wet paste into a final container,
        d) freezing and lyophilizing said paste in said container thereby obtaining a dry and stable hemostatic composition comprising said first and said second component in lyophilized form, and
        e) finishing said dry and stable hemostatic composition in said final container to a storable pharmaceutical device containing said first component and said second component in a combined form as a dry and stable hemostatic composition.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a nonprovisional of, and claims the benefit of priority to, U.S. Provisional Application No. 61/350,214, filed Jun. 1, 2010, the entire contents of which is incorporated herein by reference for all purposes.


FIELD OF THE INVENTION
The present invention relates to processes for making hemostatic compositions in storage-stable form.
BACKGROUND OF THE INVENTION
Hemostatic compositions in dry storage-stable form that comprise biocompatible, biodegradable, dry stable granular materials are known e.g. from WO 98/008550 A or WO 2003/007845 A. These products have been successfully applied on the art for hemostasis. Floseal® is an example for a powerful and versatile haemostatic agent consisting of a granular gelatin matrix swollen in a thrombin-containing solution to form a flow-able paste.
Since such products have to be applied to humans, it is necessary to provide highest safety standards for quality, storage-stability and sterility of the final products and the components thereof. On the other hand, manufacturing and handling should be made as convenient and efficient as possible. If the Hemostatic compositions require a thrombin component for use, provision of this thrombin component in the final product is challenging. Since thrombin and the matrix material usually have different properties concerning manufacture requirements, they have to be manufactured and provided separately. For example, sterilization requirements may differ significantly between relatively stable granular (often also crosslinked) matrix material and proteinaceous components, such as thrombin. Whereas such matrix materials can usually be sterilized by powerful sterilization methods (such as autoclaving, gamma-irradiation, etc.), thrombin (as an enzyme) has to be treated with more care. Those powerful sterilization methods are usually not possible for thrombin, because of loss of enzymatic activity caused by such harsh treatments. For stability reasons, such products (as well as the products according to the present invention) are usually provided in a dry form and brought into the “read-to-use” form (which is usually in the form of a (hydro-)gel, suspension or solution) immediately before use, necessitating the addition of wetting or salvation (suspension) agents and the mixing of the matrix material component with the thrombin component. Thrombin reconstitution or the mixing step of a thrombin solution with the granular matrix material are steps which usually require some time and handling and can cause problems especially in intensive health care.
It is an object of the present invention to overcome such problems and provide suitable methods for making dry and storage-stable hemostatic composition with are conveniently providable and usable. These methods should provide product formats enabling a convenient provision of “ready-to-use” hemostatic compositions, especially in intensive care medicine wherein the number of handling steps should be kept as low as possible.
BRIEF SUMMARY OF THE INVENTION
Therefore, the present invention provides a process for making a dry and stable hemostatic composition, said process comprising:

    
    
        a) providing a first component comprising a dry preparation of a coagulation inducing agent, such as a dry thrombin preparation,
        b) providing a second component comprising a dry preparation of a biocompatible polymer suitable for use in hemostasis,
        c) mixing said first component and said second component under conditions effective to form a wet paste while preventing degradation of the second component by said first component in a final container or transferring said wet paste into a final container.
        d) freezing and lyophilizing said paste in said container thereby obtaining a dry and stable hemostatic composition comprising said first and said second component in lyophilized form, and
        e) finishing said dry and stable hemostatic composition in said final container to a storable pharmaceutical device containing said first component and said second component in a combined form as a dry and stable hemostatic composition.
    
    


The process provides the dry and stable composition according to the invention in a convenient manner allowing the composition to be easily reconstituted for medical use. The invention further relates to a method for delivering a hemostatic composition to a target site in a patient's body, said method comprising delivering a hemostatic composition produced by the process of the present invention to the target site. According to another aspect, the present invention relates to a finished final container obtained by the process according of the present invention. The invention also relates to a method for providing a read-to-use hemostatic composition comprising contacting a hemostatic composition produced by the process of the present invention with a pharmaceutically acceptable diluent as well as to a kit comprising the finished final container and other means for applying the composition (e.g., a diluent container). The compositions according to the present invention are particularly useful for providing hemostasis at bleeding sites, including surgical bleeding sites, traumatic bleeding sites and the like. An exemplary use of the compositions may be in sealing the tissue tract above a blood vessel penetration created for vascular catheterization.


DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an improvement for the delivery and handling of hemostatic compositions, mainly by providing a two-component product in a convenient single-composition format. The hemostatic compositions according to the invention contain a first component comprising a dry preparation of a coagulation inducing agent, such as a dry thrombin preparation (the “coagulation inducing agent component” or “thrombin component”) and a second component comprising a dry preparation of a biocompatible polymer suitable for use in hemostasis (the “hemostatic biocompatible polymer component”). Further components may be present. Products of this kind are known in principle in the art, yet in a different format: Usually, the components are provided as separate entities in dry form. Before mixing the components for administration to a patient, the dry components are usually contacted separately with suitable diluents. Mixing of the components is then performed by mixing the separately reconstituted components. For example, a dry preparation of a coagulation inducing agent such as e.g., a thrombin component may be provided which is reconstituted by a pharmaceutically acceptable (aqueous) diluent. The solution of a coagulation inducing agent, such as a thrombin solution obtained after reconstitution is then used for wetting or solubilizing the polymer, usually under formation of a hydrogel which is then applied to the patient. Since this is at least a two-step process before the product is “ready-to-use”, it would be more convenient if a product would necessitate only one step before it is ready to use. However, as stated above, the nature of the two components prevents a simple admixture of the components in the course of the production method, mainly due to stability and activity losses.
With the present invention, production processes are provided which enable that the two components are provided already in a combined dry form ready to be reconstituted together. The processes according to the present invention are not only feasible for scientific bench experiments but are suitable for industrial pharmaceutical mass production. With the present invention it was possible to provide this already admixed hemostatic composition without the risk of unwanted degradation or loss of enzyme activity. The resulting compositions have a storage-stability comparable to the previously known products, but are more convenient in handling because separate reconstitution and admixture before medical administration is not necessary with the products obtainable with the present invention. Providing a ready-to-use hydrogel, suspension or solution of the hemostatic composition is possible in a one step process, simply by adding a suitable pharmaceutically acceptable diluent to the composition in the final container. The final container is preferably a syringe designed to directly administer the reconstituted hemostatic composition after contact with the diluent.
The coagulation inducing agent is a substance selected from the group consisting of thrombin, a snake venom, a platelet activator, a thrombin receptor activating peptide and a fibrinogen precipitating agent, preferably it is thrombin.
The “thrombin solution” can be made from any thrombin preparation which is suitable for use in humans (i.e. pharmaceutically acceptable). Suitable sources of thrombin include human or bovine blood, plasma or serum (thrombin of other animal sources can be applied if no adverse immune reactions are expected) and thrombin of recombinant origin (e.g., human recombinant thrombin); autologous human thrombin can be preferred for some applications. The concentration of the thrombin solution provided in the first component is usually adjusted to the planned thrombin concentration in the reconstituted hemostatic composition. Preferably, the hemostatic composition contains 10 to 100.000 International Units (I.U.) of thrombin, more preferred 100 to 10.000 I.U., especially 500 to 5.000 I.U. The thrombin concentration in the “read-to-use” composition is preferably in the range of 10 to 10.000 I.U., more preferred of 50 to 5.000 I.U., especially of 100 to 1.000 I.U. The diluent is used in an amount to achieve the desired end-concentration in the “ready-to-use” composition.
The “dry preparation of a biocompatible polymer” according to the present invention is known e.g. from WO 98/08550 A. Preferably, the polymer is a biocompatible, biodegradable dry stable granular material. The “dry” polymer according to the present invention is usually provided with particle sizes of 0.1 to 5.000 μm. Usually, the polymer particles have a mean particle diameter (“mean particle diameter” is the median size as measured by laser diffractometry; “median size” (or mass median particle diameter) is the particle diameter that divides the frequency distribution in half; fifty percent of the particles of a given preparation have a larger diameter, and fifty percent of the particles have a smaller diameter) from 10 to 1000 μm, especially 50 to 500 μm (median size). Applying larger particles is mainly dependent on the medical necessities; particles with smaller mean particle diameters are often more difficult to handle in the production process. The dry polymer is therefore provided in granular form. Although the terms powder and granular (or granulates) are sometimes used to distinguish separate classes of material, powders are defined herein as a special sub-class of granular materials. In particular, powders refer to those granular materials that have the finer grain sizes, and that therefore have a greater tendency to form clumps when flowing. Granulars include coarser granular materials that do not tend to form clumps except when wet.
A “dry” hemostatic composition according to the present invention has only a residual content of moisture which may approximately correspond t the moisture content of comparable available products, such as Floseal® (Floseal, for example, has about 12% moisture as a dry product). Usually, the dry composition according to the present invention has a residual moisture content below these products, preferably below 10% moisture, more preferred below 5% moisture, especially below 1% moisture. The hemostatic composition according to the present invention can also have lower moisture content, e.g. 0.1% or even below. Preferred moisture contents of the dry hemostatic composition according to the present invention are 0.1 to 10%, especially 0.5 to 5%.
According to the present invention, the hemostatic composition is provided in dry form in the final container. In the dry form, degradation or inactivation processes for the components are significantly and appropriately reduced to enable storage stability. Suitable storage stability can be determined based on the thrombin activity. Accordingly, a dry hemostatic composition of the present kind is storage stable, if no less than 400 I.U./ml (for a 500 I.U./ml product) after reconstitution after 24 months storage in dry form at room temperature (25° C.) are still present (i.e. 90% thrombin activity or more remaining compared to the initial activity before lyophilization). Preferably, the composition according to the present invention has higher storage stability, i.e. at least 90% thrombin activity remaining, especially at least 95% thrombin activity remaining after this 24 months storage.
However, providing a wet paste made by mixing a solution of a coagulation inducing agent, such as e.g. a thrombin solution and a biocompatible polymer to a wet paste and lyophilization thereof is not trivial, because mixture and lyophilization have to be performed in a way so that relevant degradation of the polymer and/or the coagulation inducing agent, e.g. thrombin is prevented. This is safeguarded by the present invention by carrying out the mixing step under conditions preventing degradation and freezing of the wet paste resulting from the mixing process in the final container. Then the composition can be properly lyophilized without the risk of further degradation processes. It is important for the present invention that even the contact time (between the solution of a coagulation inducing agent, e.g. the thrombin solution and the polymer) in wet state during mixing and before lyophilization in the paste is held as short as possible. The maximum contact times during wet state according to the present invention are dependent on various parameters based on which appropriate contact times for the present method can easily be adjusted by a person skilled in the art based on the info disclosed herein. The most important parameters for the defining an appropriate contact time are temperature, water content and thrombin concentration. For example, for a thrombin solution with 500 I.E./ml in a paste with 4 ml thrombin solution and 0.8 g polymer (e.g. gelatin granules associated with water and corresponding to 0.704 g of dry gelatin powder), the maximum contact times with the polymer in wet state (i.e. in the paste form) are—for about 4° C., 15° C. and room temperature (25° C.)—25 h, 6 h and 2 h. Temperatures especially used in the present invention are from about 2° C. to about 25° C., preferably form about 2° C. to 15° C., especially preferred about 4° C. Higher concentrations of the coagulation inducing agent, such as thrombin, or higher water contents amount to shorter maximum contact times during wet state. Accordingly, preferred contact times in wet state are in the range of 5 min to 6 h, even more preferred 5 min to 2 h, especially 5-30 min. Preferably, the mixing step is carried out at low temperatures, e.g. between 1 and 10° C., especially between 2 and 6° C.
It is also important that the mixing step according to the present invention results in a wet paste which does not contain a considerable liquid phase, i.e. the past according to the present invention is almost free of free liquid. The wet paste according to the present invention is flowable (has a degree of fluidity), yet it is also sufficiently viscous to be handled as a paste (e.g. preferred viscosities of about 10 to 100 Pa·s and above). This is important to prevent degradation processes during mixing before lyophilization. Mixing the components in the soluble (suspended) form (and not in the past form according to the present invention) and then beginning the drying process results in intolerable degradation of material. For example, even if thrombin and gelatin are kept at 4° C., a clear degradation is visible after 24 h.
Another important parameter in this connection is the amount of the coagulation inducing agent, e.g. thrombin component in the mixing step. It has to be high enough to create a wet paste but not as high to create a significant liquid phase. Accordingly, addition of more than 85% w/w of coagulation inducing agent, e.g. thrombin solution can result in more than insignificant excess liquid coagulation inducing agent, e.g. thrombin in the wet paste so that more than 85% w/w should preferably be avoided. Preferred mixing ratios start at about 80% w/w downwards (i.e. 80 ml thrombin solution/20 g dry polymer. Lower content of a coagulation inducing agent e.g. thrombin content can easily be adjusted for specific polymers by testing the handing performance of the resulting wet paste. Usually, a paste which is close to a 60 to 80% solution of a coagulation inducing agent, e.g. thrombin solution content is easier to handle; for extrusion mixtures also lower contents of coagulation inducing agent, e.g. thrombin contents do not create major obstacles in handling. Wet pastes formed with less than a 50% solution of a coagulation inducing agent, e.g. thrombin solution could become problematic due to a possible loss of fluidity which could make them problematic in handling (e.g. in syringes).
Accordingly, the present invention uses in principle two embodiments for arriving at this aim. The first principle includes mixing the two components in the final container, then lyophilizing the mixture; alternatively, the components can be mixed outside the container to form the wet paste and then transferred into a final container, e.g. via extrusion. Mixing can be achieved e.g. by “swooshing” between two connected containers (e.g. syringes) or by brining the two components into an extruder and extruding the extrusion product into the final container. Preferably, the mixture obtained (i.e. the wet slurry) is frozen and lyophilized.
Preferably, the process according to the present invention is carried out in an aseptic environment, especially the mixing step should be performed aseptically. It is also preferred to start the process by components which have already been appropriately sterilized and then to perform all further steps aseptically.
The final step of the method is the finishing step. During this step, the final container is appropriately sealed and made ready for storage and/or sale. The finishing step may comprise labeling of the final container, packaging and performing (further) sterilization processes (performed e.g. on the final container or on the packaged product or kit comprising the final container).
Preferably, the finishing step comprises an EO (ethylene oxide) sterilization step. EO sterilization is common in the present filed of technology. Ethylene oxide gas kills bacteria (and their endospores), mold, and fungi. EO sterilization is used to sterilize substances that would be damaged by high temperature techniques such as pasteurization or autoclaving.
Other preferred embodiments for sterilization are application of ionizing irradiation such as β or γ-irradiation or use of vaporized hydrogen peroxide.
According to a preferred embodiment, the final container further contains an amount of a stabilizer effective to inhibit modification of the polymer when exposed to the sterilizing radiation, preferably ascorbic acid, sodium ascorbate, other salts of ascorbic acid, or an antioxidant.
The final container can be any container suitable for housing (and storing) pharmaceutically administrable compounds. Syringes, vials, tubes, etc. can be used; however, providing the hemostatic compositions according to the present invention in a syringe is specifically preferred. Syringes have been a preferred administration means for hemostatic compositions as disclosed in the prior art also because of the handling advantages of syringes in medical practice. The compositions may then preferably be applied (after reconstitution) via specific needles of the syringe or via suitable catheters. The reconstituted hemostatic compositions (which are preferably reconstituted to form a hydrogel) may also be applied by various other means e.g. by a spatula, a brush, a spray, manually by pressure, or by any other conventional technique. Usually, the reconstituted hemostatic compositions according to the present invention will be applied using a syringe or similar applicator capable of extruding the reconstituted composition through an orifice, aperture, needle, tube, or other passage to form a bead, layer, or similar portion of material. Mechanical disruption of the compositions can be performed by extrusion through an orifice in the syringe or other applicator, typically having a size in the range from 0.01 mm to 5.0 mm, preferably 0.5 mm to 2.5 mm. Preferably, however, the hemostatic composition will be initially prepared from a dry form having a desired particle size (which upon reconstitution, especially by hydration, yields subunits of the requisite size (e.g. hydrogel subunits)) or will be partially or entirely mechanically disrupted to the requisite size prior to a final extrusion or other application step. It is, of course evident, that these mechanical components have to be provided in sterile form (inside and outside) in order to fulfill safety requirements for human use.
The design of the final container can preferably be adapted to the lyophilization process in the final container.
The dry hemostatic compositions according to the present invention are usually reconstituted (re-hydrated) before use by contacting the dry composition with a suitable diluent. The diluent according to the present invention may be any suitable reconstitution medium for the dry hemostatic composition which allows suitable wetting of the dry composition. Preferably, the dry hemostatic composition is reconstituted into a hydrogel as a “ready-to-use” format.
Suitable diluents are pharmaceutically acceptable aqueous fluids, e.g. pharmaceutical grade de-ionized water (if all ionic or buffer components are already provided in the dry composition; “water-for-injection”) or pharmaceutical grade aqueous solutions containing specific ions and/or buffers. These aqueous solutions may further contain other ingredients, such as excipients. An “excipient” is an inert substance which is added to the solution, e.g. to ensure that e.g. thrombin retains its chemical stability and biological activity upon storage (or sterilization (e.g. by irradiation)), or for aesthetic reasons e.g. color. Preferred excipients include human albumin, mannitol and sodium acetate. Preferred concentrations of human albumin in the reconstituted product are from 0.1 to 100 mg/ml, preferably from 1 to 10 mg/m. Preferred mannitol concentrations can be in the concentration range of from 0.5 to 500 mg/ml, especially from 10 to 50 mg/ml. preferred sodium acetate concentrations are in the range of from 1 to 10 mg/ml, especially 2 to 5 mg/ml.
For example, a suitable diluent comprises water for injection; and—independently of each other—NaCl (preferably 50 to 150 mM, especially 110 mM), CaCl2 (preferably 10 to 80 mM, especially 40 mM), human albumin (preferably up to 2% w/w, especially 0.5% w/w), sodium acetate (preferably 0 to 50 mM, especially 20 mM) and mannitol (preferably up to 10% w/w, especially 2% w/w). Preferably, the diluent can also include a buffer or buffer system so as to buffer the pH of the reconstituted dry composition, preferably at a pH of 6.4 to 7.5, especially at pH of 6.9 to 7.1.
In a preferred embodiment, the diluent is provided in a separate container. This can preferably be a syringe. The diluent in the syringe can then easily be applied to the final container for reconstitution of the dry hemostatic compositions according to the present invention. If the final container is also a syringe, both syringes can be finished together in a pack. It is therefore preferred to provide the dry hemostatic compositions according to the present invention in a syringe which is finished with a diluent syringe with a pharmaceutically acceptable diluent for reconstituting said dry and stable hemostatic composition.
The dry preparation of a biocompatible polymer suitable for use in hemostasis (the “dry hemostatic polymers”) of the present invention may be formed from biologic and non-biologic polymers. Suitable biologic polymers include proteins, such as gelatin, soluble collagen, albumin, hemoglobin, casein, fibrinogen, fibrin, fibronectin, elastin, keratin, and laminin; or derivatives or combinations thereof. Particularly preferred is the use of gelatin or soluble non-fibrillar collagen, more preferably gelatin, an exemplary gelatin formulations are set forth below. Other suitable biologic polymers include polysaccharides, such as glycosaminogylcans, starch derivatives, xylan, cellulose derivatives, hemicellulose derivatives, agarose, alginate, and chitosan; or derivatives or combinations thereof. Suitable non-biologic polymers will be selected to be degradable by either of two mechanisms, i.e. (1) break down of the polymeric backbone or (2) degradation of side chains which result in aqueous solubility. Exemplary nonbiologic hydrogel-forming polymers include synthetics, such as polyacrylates, polymethacrylates, polyacrylamides, polyvinyl resins, polyactideglyocolides, polycarprolactones, and polyoxyethylenes; or derivatives or combinations thereof. Also combinations of different kinds of polymers are possible (e.g. proteins with polysaccharides, proteins with non biologic hydrogel-forming polymers, etc.)
A non-cross-linked polymer together with a suitable re-hydration aid may be cross-linked in any manner suitable to reconstitute, e.g. to form a suitable hydrogel bas. For example, polymeric molecules may be cross-linked using bi- or poly-functional cross-linking agents which covalently attach to two or more polymer molecules changes. Exemplary bifunctional cross-linking agents include aldehydes, epoxides, succinimides, carbodiimides, maleimides, azides, carbonates, isocyanates, divinyl sulfone, alcohols, amines, imidates, anhydrides, halides, silanes, diazoacetate, aziridines, and the like. Alternatively, cross-linking may be achieved by using oxidizers and other agents, such as periodates, which activate side-chains or moieties on the polymer so that they may react with other side-chains or moieties to form the cross-linking bonds. An additional method of cross-linking comprises exposing the polymers to radiation, such as gamma radiation, to activate the polymer chains to permit cross-linking reactions. Dehydrothermal cross-linking methods may also be suitable. Preferred methods for cross-linking gelatin molecules are described below.
According to a preferred embodiment, the biocompatible polymer suitable for use in hemostasis therefore contains a crosslinked polysaccharide, a crosslinked protein, or a crosslinked non-biologic polymer; or mixtures thereof.
Preferably, the biocompatible polymer suitable for use in hemostasis is a granular material. This granular material can rapidly swell when exposed to a fluid (i.e. the diluent) and in swollen form is capable of contributing to a flowable paste that can be applied to a bleeding site. The biocompatible polymer, e.g. gelatin, may be provided as a film which can then be milled to form a granular material. Most of the particles contained in this granular material have preferably particle sizes of 100 to 1.000 μm, especially 300 to 500 μm.
According to a preferred embodiment, the biocompatible polymer suitable for use in hemostasis is a cross-linked gelatin. Dry cross-linked gelatin powder can be prepared to re-hydrate rapidly if contacted with a suitable diluent. The gelatin powder preferably comprises relatively large particles, also referred to as fragments or sub-units, as described in WO 98/08550 A and WO 2003/007845 A. A preferred (median) particle size will be the range from 20 to 1.000 μm, preferably from 100 to 750 μm, especially from 150 to 500 μm, but particle sizes outside of this preferred range may find use in many circumstances. The dry compositions will also display a significant “equilibrium swell” when exposed to an aqueous re-hydrating medium (=diluents). Preferably, the swell be in the range from 400% to 1000%. “Equilibrium swell” may be determined by subtracting the dry weight of the gelatin hydrogel powder from its weight when fully hydrated and thus fully swelled. The difference is then divided by the dry weight and multiplied by 100 to give the measure of swelling. The dry weight should be measured after exposure of the material to an elevated temperature for a time sufficient to remove substantially all residual moisture, e.g., two hours at 120° C. The equilibrium hydration of the material can be achieved by immersing the dry material in a suitable diluent, such as aqueous saline, for a time period sufficient for the water content to become constant, typically for from 18 to 24 hours at room temperature.
A non-cross-linked gelatin together with the re-hydration aid may be cross-linked in any manner suitable to form a suitable hydrogel base. Dry cross-linked gelatin powders according to this preferred embodiment are preferably obtained by preparing the powders in the presence of certain re-hydration aids. Such re-hydration aids will be present during the preparation of the powders, but will usually be removed from the final products. For example, re-hydration aids which are present at about 20% of the total solids content will typically be reduced to below 1% in the final product, often below 0.5% by weight. Exemplary re-hydration aids include polyethylene glycol (PEG), preferably having a molecular weight of about 1000; polyvinylpyrrolidone (PVP) preferably having an average molecular weight of about 50,000; and dextran, typically having an average molecular weight oaf about 40,000. it is preferred to employ at least two of these re-hydration aids when preparing the compositions of the present invention, and more particularly preferred to employ all three.
Exemplary methods for producing cross-linked gelatins are as follows. Gelatin is obtained and suspended in an aqueous solution to form a non-cross-linked hydrogel, typically having a solids content from 1% to 70% by weight, usually from 3% to 10% by weight. The gelatin is cross-linked, typically by exposure to either glutaraldehyde (e.g., 0.01% to 0.05% w/w, overnight at 0° C. to 15° C. in aqueous buffer), sodium periodate (e.g., 0.05 M, held at 0° C. to 15° C. for 48 hours) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (“EDC”) (e.g., 0.5% to 1.5% w/w overnight at room temperature), or by exposure to about 0.3 to 3 megarads of gamma or electron beam radiation. Alternatively, gelatin particles can be suspended in alcohol, preferably methyl alcohol or ethyl alcohol, at a solids content of 1% to 70% by weight, usually 3% to 10% by weight, and cross-linked by exposure to a cross-linking agent, typically glutaraldehyde (e.g., 0.01% to 0.1% w/w, overnight at room temperature). In the case of aldehydes, the pH should be held from about 6 to 11, preferably from 7 to 10. when cross-linking with glutaraldehyde, the cross-links are formed via Schiff bases which may be stabilized by subsequent reduction, e.g., by treatment with sodium borohydride. After cross-linking, the resulting granules may be washed in water and optionally rinsed in an alcohol, and dried. The resulting dry powders may then be provided in the final container as described herein.
After cross-linking, at least 50% (w/w) of the re-hydration aid will be removed from the resulting hydrogel. Usually, the re-hydration aid is removed by filtration of the hydrogel followed by washing of the resulting filter cake. Such filtration/washing steps can be repeated one or more additional times in order to clean the product to a desired level and to remove at least 50% of the re-hydration aid, preferably removing at least 90% (w/w) of the re-hydration aid originally present. After filtration, the gelatin is dried, typically by drying the final filter cake which was produced the dried filter cake may then be broken up or ground to produce the cross-linked powder having a particle size in the desired ranges set forth above.
According to another aspect, the present invention also provides a method for delivering a hemostatic composition to a target site in a patient's body, said method comprising delivering a hemostatic composition produced by the process according to the present invention to the target site. Although in certain embodiments, also the dry composition can be directly applied to the target site (and, optionally be contacted with the diluent a the target site, if necessary), it is preferred to contact the dry hemostatic composition with a pharmaceutically acceptable diluent before administration to the target site, so as to obtain a hemostatic composition in a wetted form, especially a hydrogel form.
The present invention also refers to a finished final container obtained by the process according to the present invention. This finished container contains the combined components in a sterile, storage-stable and marketable form.
Another aspect of the invention concerns a method for providing a ready-to-use hemostatic composition comprising contacting a hemostatic composition produced by the process according to the present invention with a pharmaceutically acceptable diluent.
The present invention also concerns a kit comprising the dry and stable hemostatic composition according to the present invention in finished form and a container with a suitable diluent. Further components of the kit may be instructions for use, administration means, such as syringes, catheters, brushes, etc. (if the compositions are not already provided in the administration means) or other components necessary for use in medical (surgical) practice, such as substitute needles or catheters, extra vials or further wound cover means. Preferably, the kit according to the present invention comprises a syringe housing the dry and stable hemostatic composition and a syringe containing the diluent (or provided to take up the diluent from another diluent container). Preferably, these two syringes are provided in a form adapted to each other so that the diluent can be delivered to the dry hemostatic composition by another entry than the outlet for administering the reconstituted composition.
The invention is further described in the examples below, yet without being restricted thereto.
EXAMPLES
1. Preparation of the Dry Hemostatic Composition According To the Invention
Materials And Methods
All variants use the same scheme of presenting a kit with one syringe containing both the Floseal gelatin matrix and thrombin in a stable form, and one syringe containing a suitable liquid reconstitution medium (e.g. 0.9% NaCl, or 40 mM CaCl2). Both syringes are sterile inside and outside, so the entire reconstitution can take place on the scrub nurse side of the operation theater. Reconstitution is achieved by coupling the two syringes in the familiar fashion and mixing the contents of the two syringes by “swooshing” (i.e. repeated transfer of the contents back and forth between the two syringes).
“In Syringe Lyo”
The composition according to the present invention is made by mixing the gelatin and thrombin solution into a wet paste and lyophilizing the paste inside a single syringe. The gelatin matrix can be bulk-sterilized before by irradiation. Gamma or beta irradiation are suitable for sterilizing the gelatin. The sterile gelatin is hydrated in bulk with a sterile thrombin solution. This produces a wet paste, i.e., a paste without a free liquid phase. Preferably, this step is carried out in bulk by gradually dropping the gelatin granules into the constantly stirred thrombin solution and blending until a homogenous paste is formed or by simultaneously feeding the granules and the thrombin solution into a approximately configured extrusion machine where the two components are intimately blended into a flow-able paste which can then be directly dispensed into syringes for further processing.
The paste may then be filled into syringes to allow lyophilization inside the syringe.
Following freezing of the filled syringes, the slurry is lyophilized using a suitable lyophilization program. The syringe is closed inside the lyophilizer by pushing on the plungers initially set in the syringe. At the same step the lyophilized matrix is compacted to close to the volume which would be occupied by the gelatin granules alone, to minimize the amount of air mixed into the product upon reconstitution. The product is now ready for packaging with the diluent syringe, EO sterilization of the pouches, and storage.
Diluent Syringe
The diluent syringe contains an appropriate reconstitution medium for hydrating the product. It is can be coupled with the Floseal syringe either directly or by means of a connector. The diluent is transferred into the Floseal syringe, and the hydrated product is transferred back and forth between the coupled syringes repeatedly to generate a flow-able paste. The diluent syringe can be prepared e.g. by a process such as the following: the medium is sterile filtered and filled in suitable syringes (like Toppac syringes, Clearshot, . . . ); and, if necessary, end-sterilized by irradiation.
Gelatin Granulate
The gelatin granules bulk manufacturing is performed according to established methods (WO 98/08550 A; WO 2003/00785 A; etc.). The granules (“Floseal” granules' “Floseal” matrix) are sterilized by gamma irradiation. For preclinical sterilization the Floseal matrix is filled into Schott glass bottles of appropriate size.
The required irradiation dose at the current maximum bioburden level (1000 cfu/sample) is 25-40 kGy for the product in the final container. The bulk material is then stored at −20° C. for further manufacturing.
Floseal “In Syringe Lyo”
0.81 g of Floseal gelatin (associated with water and corresponding to 0.704 g of dry gelatin powder) are weighed in into a lyophilization syringe. The syringe plunger is then set just above the gelatin granules from the back. On the other side a Floseal thrombin syringe is filled with 4.0 ml thrombin 500 IE/ml. Then the syringes are connected and swooshed for at least 21 passes. After the last pass the product has to be inside the lyophilization syringe. The luer of this syringe is closed using the Floseal luer caps. The product undergoes lyophilization inside this syringe. For lyophilization the lyophilization program for thrombin STIM5 500 IU/ml is used. The syringes are placed in custom built racks made from stainless steel. The rack is built such that the luer cap of the syringe rests at the lower floor of the rack, while the finger rests of the syringes rest on the upper floor. This ensures maximum stability of the syringes during the lyophilization cake compaction step.
The dried product is compacted under vacuum by lowering the hydraulic “stoppering” plate of the lyophilizer and thereby pushing the plungers past the lyophilization holes and into the syringes. This closes the syringes and compacts the lyophilization cake such that it occupies as low a volume as possible after the lyophilization step. The level down to which the stoppering plates are lowered is limited by metal spacers which ensures that the syringes are compacted to the right level without putting undue pressure on the product/devices.
2. Effectiveness in the Porcine Liver Abrasion Model
The purpose of this study is to compare the effectiveness of the dry hemostatic composition according to the present invention with an established standard product (Floseal VH S/D; Baxter Healthcare) in the porcine liver abrasion model. Floseal VH S/D is a gelatin matrix that delivers thrombin to stop active bleeding within 2 minutes of application. This product requires a 2-step preparation, (1) reconstitution of thrombin and (2) hydration of the gelatin particles with the reconstituted thrombin. The product according to the present invention is designed to reconstitute the dry hemostatic composition in 1 step and is a major improvement to the 2-step preparation which is unfavorable when the product is needed quickly or in large quantities.
Porcine Liver Abrasion Model
Six female domestic pigs, mean weight of 55.0 kg (range 52.4-58.4 kg), are obtained from Oak Hill Genetics (Ewing, Ill.) and weighed at the time of surgery. Upon arrival, animals are quarantined for 6 days. At the time of surgery, all six pigs show no signs of clinical illness. Ear tags are used to identify animals and cross-referenced to assigned identification numbers. Animals are group housed in pens. Pigs receive water ad libitum and a standard pig diet once daily.
Swine are a well-accepted cardiovascular model and suitable for this type of stuffy. The multiple, large lobes of the liver allowed multiple lesions for direct comparisons of the different test items.
Anesthetics and Fluid Therapy
Pigs are medicated with Midazolam (0.3 mg/kg, IM) and masked-induced with Isoflurane in a 2:3 nitrogen to oxygen carrier. Pigs are intubated and ventilated at a rate of 1-15 breaths per minute. Anesthesia is maintained with Isoflurane in an oxygen carrier. Pigs receive a continuous rate infusion of warmed Lactated Ringer's Solution.
Liver Abrasion Procedure
A porcine liver abrasion model is used for this study. Six pigs are prepared with the goal that 120 lesions (4o per treatment group) are evaluated and sufficient to detect a difference in rates of 80 percent versus 40 percent with α=0.05 and power=90%. Each series is confided to either the medial, left lateral or right lateral lobe.
Each lesion series contain three 1 cm diameter, 3-4 mm deep liver abrasions created using a hand drill fixed with sandpaper. Bleeding is accessed and the lesion is randomly and blindly treated with reference or test article. Reference and test article is randomized using a random number generator. Each article is placed onto the lesion, held in place with damp gauze for 2 minutes and blindly assessed for hemostasis 2, 5 and 10 minutes following treatment. Excess reference or test article is irrigated away after the 5 minute assessment.
Heparinization Protocol
A baseline Activated Clotting Time (ACT) is taken and each pig receives a loading dose of heparin, 200 IU/kg. The ACT is assessed every 10 minutes until the ACT is at least 2 times baseline. If the ACT measures less than or near equal to 2 times baseline, the pig was treated with a bolus heparin dose, 75 IU/kg.
Once greater than 2 times baseline, ACT is measured every 20 minutes. If ACT measures less than or near equal to the target 2 times baseline, the pig is given a bolus dose of heparin, 40 IU/kg. If the ACT measures more than the target 2 times baseline, the pig is not treated or given a maintenance bolus dose of heparin, limited to no more than 2,000 IU/hour.
All heparin is given via a peripheral venous catheter. All blood samples are taken from a jugular catheter. Blood pressure and heart rate reference values are recorded at the time of ACT measurements.
Hemostasis is assessed at 0, 2, 5 and 10 minutes after the lesion series is created and treated, where 0 minutes refers to pre-treatment. Scores of 0, 1, 2, 3, 4, and 5 are assigned to no bleeding, ooze, very mild, mild, moderate, and severe; respectively. All three lesions are treated at approximately the same time to avoid difference in location and coagulation that may result from treating each independently. Blood from the lesion is blotted away following each assessment as necessary.
Measurements and Records
The ACT, hemostasis, blood pressure and heart rate are evaluated according to standard methods.
Statistical Analysis
The sampling unit for this study is the liver lesion site with 40 lesions per treatment group for a total of 120 lesions.
Multiple logistic regression is used to evaluate the treatment effect on bleeding score (0=no, 1=ooze, 2=very slight, 3=slight, 4=moderate, and 5=severe) at 2, 5, and 10 minutes post treatment. Independent variables includes treatment group, pig, liver lobe (medial, right or left) and initial bleeding score. The odds ratios for the effects of FB/FS, Lyo/FS, FB/Lyo, and their confidence intervals are computed at each time point post treatment.
The locations of lesions are not evenly distributed across the three lobes and pigs. The lobe effect is found to be not significant, and therefore the analyses are re-performed without this effect. The conclusions are based on the analyses without the lobe effect in the model.
Results:
The performance of the dry hemostatic composition according to the present invention is not significantly different from Floseal VH S/D at all time points. This shows that the production method according to the present invention and the 1 step reconstitution mode do not have negative impact on the performance of the composition but provide the desired advantage in practical handling thereby proving that the object of the present invention is solved.
A preclinical evaluation is performed to compare in vivo efficacy of Floseal “in-Syringe Lyo” to Floseal VH in a very stringent (highly anti-coagulated) model. This model consists of a 5 mm full-thickness liver puncture with 4 additional incisions radiating from the puncture defect in a cross-wise fashion. 6 animals are used per study group, these animals are heparinized to 4.000 I.U./kg. After the lesion is placed, reconstituted Floseal is applied, and for 2 min light pressure with wet gauze is applied. After this time primary hemostasis after is assessed. If primary hemostasis is not achieved, product is re-applied until hemostasis is achieved, or product (5 ml)/time (15 min) is exhausted. Primary endpoints are achievement of primary hemostasis (Yes/No) and time to hemostasis (min).
If primary hemostasis is achieved, the animals are surgically closed, and after 24 the animals are evaluated for re-bleeding.
The present composition give results in terms of time to hemostasis that are equivalent to or better than standard Floseal in this particular preclinical laboratory session.

## Claims

1. A process for making a dry and stable hemostatic composition, said process comprising:
a) providing a first component comprising a solution of a coagulation inducing agent;
b) providing a second component comprising a dry preparation of a biocompatible polymer suitable for use in hemostasis;
c) mixing said first component and said second component under conditions effective to form a wet paste while preventing degradation of the second component by said first component in a final container or mixing said first component and said second component under conditions effective to form the wet paste while preventing degradation of the second component by said first component and transferring said wet paste into a final container, wherein the wet paste is without a free liquid phase;
d) freezing and lyophilizing said paste in said container thereby obtaining a dry and stable hemostatic composition comprising said first and said second component in lyophilized form; and
e) finishing said dry and stable hemostatic composition in said final container to a storable pharmaceutical device containing said first component and said second component in a combined form as a finished dry and stable hemostatic composition, wherein:
the first component comprises thrombin,
the mixing step comprises mixing the first component and the second component at a temperature within a range from about 2° C. to about 15° C., and
a contact time between the first component and the second component in the wet paste before lyophilizing is within a range from five minutes to six hours,
85% (weight/weight) or less of the wet paste is the solution of the coagulation inducing agent,
the finishing step comprises sterilizing the dry and stable hemostatic composition in said final container,
said thrombin has an initial activity prior to the lyophilizing step, and
said finished dry and stable hemostatic composition has the capability to retain at least 90% of said thrombin activity after 24 months storage in dry form at room temperature.

2. The process according to claim 1, wherein step c) is performed under aseptic conditions.

3. The process according to claim 1, wherein step c) is performed by extrusion.

4. The process according to claim 1, wherein step d) comprises an ethylene oxide sterilization step of application of an ionizing irradiation.

5. The process according to claim 1, wherein said first component contains thrombin in a CaCl2 solution.

6. The process according to claim 1, wherein said first component contains thrombin in a concentration of 10 to 10,000 I.U./ml.

7. The process according to claim 1, wherein a syringe is used as said final container.

8. The process according to claim 7, wherein said syringe is a syringe finished together with a diluent syringe with a pharmaceutically acceptable diluent for reconstituting said dry and stable hemostatic composition.

9. The process according to claim 1, wherein said first component comprises human thrombin.

10. The process according to claim 1, wherein said first component comprises recombinant human thrombin.

11. The process according to claim 1, wherein said biocompatible polymer suitable for use in hemostasis contains a protein selected from the group consisting of gelatin, soluble collagen, albumin, hemoglobin, fibrinogen, fibrin, casein, fibronectin, elastin, keratin, and laminin, and derivatives or combinations thereof.

12. The process according to claim 1, wherein said biocompatible polymer suitable for use in hemostasis contains a polysaccharide selected from the group consisting of glycosaminoglycans, starch derivatives, cellulose derivatives, hemicellulose derivatives, xylan, agarose, alginate, and chitosan, and derivatives or combinations thereof.

13. The process according to claim 1, wherein said biocompatible polymer suitable for use in hemostasis contains a polymer selected from the group consisting of polyacrylates, polymethacrylates, polyacrylamides, polyvinyl resins, polylactide-glycolides, polcaprolactones, polyoxyethlenes, and derivatives and combinations thereof.

14. The process according to claim 1, wherein said biocompatible polymer suitable for use in hemostasis contains a crosslinked polysaccharide, a crosslinked protein, a crosslinked non-biologic polymer, or mixtures thereof.

15. The process according to claim 1, wherein said biocompatible polymer suitable for use in hemostasis is a granular material.

16. The process according to claim 1, wherein said biocompatible polymer suitable for use in hemostasis is a cross-linked gelatin.

17. The process according to claim 1, wherein said final container further contains an amount of a stabilizer effective to inhibit modification of the polymer when exposed to sterilizing radiation, wherein the stabilizer is selected from the group consisting of ascorbic acid, sodium ascorbate, other salts of ascorbic acid, and an antioxidant.

18. The process according to claim 1, wherein the mixing step comprises mixing the first component and the second component at a temperature of about 4° C.

19. The process according to claim 1, wherein the mixing step comprises mixing the first component and the second component at a temperature within a range from about 2° C. to about 10° C.

20. The process according to claim 1, wherein the mixing step comprises mixing the first component and the second component at a temperature within a range from about 2° C. to about 6° C.

21. The process according to claim 1, wherein the mixing step comprises mixing the first component and the second component at a temperature of about 6° C.

22. The process according to claim 1, wherein the contact time between the first component and the second component during the mixing and before the lyophilization is within a range from five minutes to two hours.

23. The process according to claim 1, wherein the contact time between the first component and the second component during the mixing and before the lyophilization is within a range from five minutes to 30 minutes.

24. The process according to claim 1, wherein the dry and stable hemostatic composition has a moisture content from 0.5% to 5%.

25. The process according to claim 1, wherein less than 80% (weight/weight) of the wet paste is the solution of the coagulation inducing agent.

26. The process according to claim 1, further comprising sterilizing said first component and sterilizing said second component before mixing.

27. The process according to claim 1, wherein step e) comprises compacting the dry and stable composition.

28. The process according to claim 1, wherein step c) comprises gradually dropping the second component into a constantly stirred solution of the coagulation inducing agent.

29. The process according to claim 1, wherein step c) comprises simultaneously feeding the second component and the first component into an extrusion machine wherein the first component and the second component are blended into the wet paste.

30. The process according to claim 1, wherein the finished dry and stable hemostatic composition contains 500 to 5,000 I.U. thrombin.

31. The process according to claim 1, wherein the finished dry and stable hemostatic composition contains 50 to 5,000 I.U. thrombin.

32. The process according to claim 1, wherein the finished dry and stable hemostatic composition contains 100 to 1,000 I.U. thrombin.

33. The process according to claim 1, wherein said first component contains thrombin in a concentration of 500 I.U./ml.

34. The process according to claim 33, wherein said thrombin has a concentration of 450 IU./ml or more after reconstitution of the finished dry and stable hemostatic composition after 24 months storage in dry form at room temperature.

35. The process according to claim 1, wherein said finished dry and stable hemostatic composition has the capability to retain at least 95% of said thrombin activity after said 24 months storage.

36. The process according to claim 1, wherein 60% to 80% (weight/weight) of the wet paste is the solution of the coagulation inducing agent.

37. The process according to claim 1, wherein step c) comprises mixing said first component and said second component in the final container.

38. The process according to claim 1, wherein step c) comprises transferring said wet paste into the final container.

39. The process according to claim 38, wherein transferring said wet paste into the final container comprises extruding said wet paste into the final container.

40. The process according to claim 38, wherein step c) comprises repeated transferring of said first component and said second component between two syringes.

41. The process according to claim 1, wherein step d) comprises sterilizing with an ionizing radiation.

42. The process according to claim 1, wherein step d) comprises an ethylene oxide sterilization step.