Process for making dry and stable hemostatic compositions

The present invention relates to processes for making hemostatic compositions in storage-stable form.

Hemostatic compositions in dry storage-stable form that comprise biocompatible, biodegradable, dry stable granular material are known e.g. from <patcit id="pcit0001" dnum="WO98008550A"><text>WO 98/008550 A</text></patcit> or <patcit id="pcit0002" dnum="WO2003007845A"><text>WO 2003/007845 A</text></patcit>. 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.

<patcit id="pcit0003" dnum="US20080109002A1"><text>US 2008/0109002 A1</text></patcit> refers to devices and methods for mixing and extruding medically useful compositions, including a layered preparation of freeze dried thrombin and gelatin beads. <patcit id="pcit0004" dnum="EP1648867A1"><text>EP 1 648 867 A1</text></patcit> discloses a solution of thrombin, albumin, sodium acetate and sodium chloride which may be lyophilised. <patcit id="pcit0005" dnum="US4752466A"><text>US 4,752,466 A</text></patcit> discloses a PVP solution which is admixed with thrombin and then lyophilised. <patcit id="pcit0006" dnum="US5595735A"><text>US 5,595,735 A</text></patcit> also discloses thrombin being mixed in solution with PEG so as to obtain a solution and only then lyophilisation takes place.

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 "ready-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 solvation (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.

Therefore, the present invention provides a process for making a dry and stable hemostatic composition, said process comprising: <ol id="ol0001" compact="compact"><li>a) providing a first component comprising a dry thrombin preparation,</li><li>b) providing a second component comprising a dry preparation of a crosslinked gelatin,</li><li>c) providing said first component and said second component in a combined form in a final container, by filling said first component and said second component into said final container so as to obtain a dry mixture in said final container, and</li><li>d) finishing the 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.</li></ol>

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 ready-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.

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 thrombin preparation (the "thrombin component") and a second component comprising a dry preparation of a crosslinked gelatin (the "crosslinked gelatin 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 thrombin component may be provided which is reconstituted by a pharmaceutically acceptable (aqueous) diluent. The 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 "dry thrombin preparation" 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. 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 "ready-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./ml. The diluent is used in an amount to achieve the desired end-concentration in the "ready-to-use" composition.

The "crosslinked gelatin polymer" according to the present invention is known e.g. from <patcit id="pcit0007" dnum="WO9808550A"><text>WO 98/08550 A</text></patcit>. A "dry" hemostatic composition according to the present invention has only a residual content of moisture which may approximately correspond to 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. 80% 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 dry mixture of thrombin and a crosslinked gelatin is not trivial, because mixture has to be made in the dry form. Mixing the components in the soluble (suspended) form 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.

The "dry" crosslinked gelatin (the "polymer") and thrombin according to the present invention are usually provided with particle sizes of 0.1 to 5.000 µm. Usually, the thrombin particles used herein may be smaller than the polymer particles; thrombin particles preferably 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 1 to 100 µm, especially 5 to 50 µm; the polymer particles 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 and thrombin are therefore provided in granular form, especially in powder 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; dry thrombin powders according to the present invention preferably have a median size between 1 and 10 µm. Granuls include coarser granular materials that do not tend to form clumps except when wet.

Accordingly, in principle two embodiments for arriving at this aim can be used. The first principle includes mixing the two components in the solid state before filling the final container according to the present invention; alternatively, the components can be added successively into the final container. Mixing can then be achieved by agitation of the final container, if desired. Specifically when performing step c) in the process of the present invention it is preferred to use dry thrombin in powder form, i.e. with a medium diameter of preferably 1 to 10 µm (median size). Lyophilized preparations of thrombin may comprise larger particles, it may even be obtained as a discontinuous solid, porous phase (also depending on the technique thrombin has been lyophilized). If such a lyophilizate is crushed into particles, the particles obtained could have a broad particle size distribution. In such cases, it is advantageous to narrow the particle size distribution by milling the lyophilized thrombin to obtain a powder. For such milling, the milling techniques and instruments usually applied for milling proteinaceous lyophilized material is applied. A preferred particle size range of the thrombin particles (especially for spray-dried thrombin particles) are 0.1 to 500 µm, more preferred 0.5 to 100 µm, especially 1 to 50 µm. According to a specifically preferred embodiment, the first component contains thrombin obtained by spray drying, preferably by aseptic spray drying. The spray drying can be performed in any spray drying apparatus, especially for proteinaceous material, preferably, the apparatuses are sterilized immediately before bringing in the thrombin solution for spray drying. Preferably, the spray drying step, is followed by an agglomeration step, to create a thrombin powder.

According to the second principle, one of the dry components is provided in the final container and then the second component is added. This can be performed by placing one of the components in dry form into the final container and then add the second component. This, however, can also be performed by placing one of the components as a solution or suspension (or other wet preparation) into the final container, drying the component, preferably by lyophilization, and then - after completion of the drying process (in situ in the final container) - adding the other component in dry form. Preferably, thrombin is provided in the final container, then dried, preferably by lyophilization, and then combined with the second component.

Preferably, the process according to the present invention is carried out in an aseptic environment, especially the combination step in the final container (step c) 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, step d) comprises an EO (ethylene oxide) sterilization. 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 drying 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 my further contain comprise other ingredients, such as excipients. An "excipient" is an inert substance which is added to the solution, e.g. to ensure that 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.

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 base. 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 chains. 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.

Preferably, the crosslinked gelatin is a granular material. This granular material can rapidly swell when exposed to a fluid (i.e. the diluent) and in this swollen form is capable of contributing to a flowable paste that can be applied to a bleeding site. The crosslinked 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 (median size).

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 <patcit id="pcit0008" dnum="WO9808550A"><text>WO 98/08550 A</text></patcit> and <patcit id="pcit0009" dnum="WO2003007845A"><text>WO 2003/007845 A</text></patcit>. 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 will 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 of 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 an 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 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.

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 present 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).

Variant "Powder Mix" (step c)

The "powder mix" variant is made by mixing dry gelatin and lyophilized thrombin, and filling this into a single syringe. If applicable, the gelatin matrix is bulk-sterilized by irradiation (same as used for end sterilization of the product already on the market).

Variant "Thrombin in Syringe Lyo"

The "Thrombin in Syringe Lyo" is made by first lyophilizing the thrombin solution inside of the Floseal syringe, and then filling the gelatin granules on top of the lyophilized thrombin. If applicable, the gelatin matrix is bulk-sterilized by irradiation (same as used for endsterilization of the current product).

The thrombin solution is formulated and filled into stoppered Floseal syringes which allow evacuation of the inside of the syringe for container closure. The thrombin solution is freezed and lyophilized using a suitable lyophilization program.

The syringes are not stoppered in the back to allow filling of the gelatin powder from the back. The necessary amount of gelatin is filled onto the thrombin lyophilizate. The plunger is then set into the syringe body. The syringes are placed into a suitable rack inside of an evacuation chamber with movable plates for stoppering the syringes. The plates are moved down on the syringe plungers after evacuation closes the syringes. This also serves to compact the lyo cake such that it takes up little room inside of the syringe. The product is now ready for packaging with the diluent syringe, EPO 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 (<patcit id="pcit0010" dnum="WO9808550A"><text>WO 98/08550 A</text></patcit>; <patcit id="pcit0011" dnum="WO200300785A"><text>WO 2003/00785 A</text></patcit>; etc.). The granules ("Floseal" granules; "Floseal" matrix) are immediately 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.

step c: "powder mix"

Thrombin preparation

Sterile thrombin powder in this example is manufactured in two distinct ways: by milling lyophilized thrombin in an aseptic fashion or by aseptic spray drying.

Bulk Lyophilization with Subsequent Aseptic milling

Formulated Thrombin solution with 500 IU/ml thrombin, 50 g/l HSA and 4,4 g/l NaCl is transferred to a sterile lyophilization tray of appropriate size in the laminar flow hood, and the tray covered with sterile aluminum foil for transport into the lyophilization. The lyophilization tray is placed into the cleaned lyophilizer, and the lyophilization carried out with the appropriate program. After completed lyophilization the lyophilizer is vented using medical grade nitrogen, and the lyo tray is again covered with sterile aluminum foil.

For milling using the Retsch MM200 ball mill, the 25 ml screw-top milling vessels and the appropriate milling balls (12 mm) are sterilized by either autoclaving or alcohol immersion, and the sterile milling vessels are transported to the laminar flow hood. The milling vessels are filled with an appropriate amount of the lyophilized thrombin , and a sterile milling ball is added. The vessels are then closed with the screw-top and taken to the ball mill for milling. After milling the vessels are taken back to the laminar flow hood, where they are emptied with sterile spatulas into a sterile collection vessel (50 ml Falcon or glass bottle) and refilled for repeat milling.

Aseptic spray drying

For preclinical production, spray drying is carried out using a Büchi Spray drying setup with gas conditioning for the carrier gas.

Aseptic Thrombin and Gelatin powder filling into syringes

The two sterile powders are sequentially filled into the Floseal syringe on an analytical scale. The weight of the Floseal matrix is calculated as filling weight in g =70,4/solid content in %, ensuring that 704 mg of dry Floseal matrix are filled in the syringe. The weight of the thrombin powder, containing 2000 IU thrombin for 5 ml of Floseal final product, is 4 (ml) x 55 (mg/ml) = 220 mg. Filling is done with an accuracy of +/- 5 %, the limits have to be calculated for each batch of Floseal matrix.

First, the milled thrombin powder is filled into the sterile Floseal syringes that have been closed with the appropriate sterile luer cap and that are placed on an analytical scale using a small rack. The scale is tared, and the appropriate amount of thrombin powder is filled into the syringe using either a sterile manual spatula or a vibration spatula with the appropriate attachment. Next the scale is tared again, and the appropriate amount of Floseal matrix is filled into the syringe using either a sterile manual spatula or a vibration spatula with the appropriate attachment. The plunger is then set from the back to a position just at the top of the Floseal matrix, if necessary by slightly opening the luer plug, if necessary.

Alternatively the order of filling can be reversed, resulting in the thrombin powder being at the back of the syringe.

Alternatively, the two powders can be mixed before filling, eliminating the second filling step.

"thrombin in syringe lyo"

4,0ml thrombin 500IU/ml were filled in a lyophilization syringe, lyophilized and compacted under vacuum. Then the Floseal gelatin was weighed in into the syringe by filling it on top of the compacted thrombin. The syringe was closed and compacted again under vacuum. All steps of preparation were done under aseptic conditions.

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, Illinois) 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 study. 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 2:1 nitrogen to oxygen carrier. Pigs are intubated and ventilated at a rate of 10-15 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 (40 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 assessed 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 maintence 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 Evaluation

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 (c1/c2) 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.

Further animal experiments

A preclinical evaluation is performed to to compare in vivo efficacy of Floseal "Powder Mix", and Floseal "Thrombin In-Syringe Lyo" to Floseal VH in a very stringent (highly anticoagulated) 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 were surgically closed, and after 24 the animals are evaluated for re-bleeding.

All variants gave results in terms of time to hemostasis that are equivalent to or better than standard Floseal in this particular preclinical laboratory session.