Patent Application
20050214277
The present invention relates to a suspension comprising fibrinogen, thrombin, an alcohol and optionally aprotinin. The invention further relates to a method for preparing such a suspension and to a method for coating a carrier with such a suspension. The carrier may be a collagen carrier, such as a collagen sponge. The invention further relates to a method of drying a coated carrier, in particular a collagen carrier coated with a suspension according to the invention, and thereby obtained coated collagen carrier having the active substances solidly fixated to the carrier.
The coated collagen carrier may be used as a ready-to-use absorbable composition for tissue gluing, tissue sealing and hemostasis consisting essentially of a carrier coated with solidly fixed components of fibrin glue: fibrinogen and thrombin. This fixed combination can be applied directly to e.g. a wound surface. Upon contact with blood, body fluids or physiological saline, the mechanism of this system mimics the final stage of the coagulation cascade, in which thrombin catalyzes the conversion of fibrinogen to fibrin and the activation of factor XIII to give XIIIa. Factor XIIIa, once formed, stabilizes the fibrin clot by covalent cross-linking.
Like a two-component adhesive, wound surface and carrier are glued together by polymerization. During this process, which lasts approximately 3 to 5 minutes, the coated collagen carrier of the invention is preferably pressed onto the wound area. The components of the composition of the invention are degraded enzymatically in about 4-6 months after application.
Commercial fibrin glues, that mimic the last step of the coagulation cascade, and consist of a highly concentrated fibrinogen solution to be mixed with a thrombin solution before application to the surgical wound, exist. These mixtures contain a fibrinolysis inhibitor, e.g. aprotinin or ε-aminocaproicacid, to prevent premature dissolution of the fibrin clot by the fibrinolytic enzyme plasmin. These two-component fibrin glues are valuable in various surgical procedures but may be washed away before hemostasis is achieved if the bleeding is heavy.
The two-component fibrin glues furthermore need some preparatory steps including thawing or dissolution. Thus, they are rather impractical and cumbersome to work with and experience is needed for successful use of these fibrin glues.
During the last decade numerous fibrin sealants became the methods of choice in surgery in a number of indications. However, in the majority of trials with fibrin glues a collagen fleece was additionally used to improve haemostatic and adhesive features, indicating their disadvantages and their restrained use by the surgeons.
Collagen has been used as a hemostatic agent since the late sixties. Collagen is the most frequent structural protein in all mammalians. The monomeric protein of approximately 300 kDa (tropocollagen) is covalently crosslinked at specific sites. The mature protein is therefore insoluble and forms characteristic fibrils with high tensile strength. Numerous sub-classes of collagen have been described, the most common of which is collagen type I, the main collagen type in skin, tendons, bones and cornea. Collagen is a fibrous protein consisting of a triple helix with a length of approximately 290 nm. Five of these triple helices (tropocollagen molecules) are staggered to form a microfibril with a diameter of approximately 3.6 nm. These microfibrils have polar and non-polar segments that are readily accessible for specific inter- and intrafibrillar interactions. Microfibrils are packed into a tetragonal lattice to form subfibrils with a diameter of about 30 nm. These subfibrils are then assembled into the collagen fibril, the basic unit of connective tissue, which has a diameter of several hundred nm and is therefore visible in the light microscope as a thin line.
Collagen may be used as a material for sealing wounds, possibly with a coating comprising a fibrin glue. Fibrin glues, i.e. the combination of fibrinogen, thrombin and aprotinin, have successfully been used therapeutically for many years for gluing tissues and nerves and for sealing surfaces when there is minor bleeding. One drawback of the fibrin glues has been that in case of major bleeding the glue is usually washed away before sufficient polymerization of fibrin has occurred. To overcome this problem surgeons have begun applying manually liquid fibrin glues to absorbable carriers such as collagen fleece.
Despite the impressive success of these combined applications this method has not been applied on a broad scale, due to some disadvantages. The preparation is relatively cumbersome, the method requires experience and skilled personnel, and the preparation is not readily available in cases of emergency, the time for preparation being in the range of 10 to 15 min. These factors stimulated the development of an improved product resulting in the development of a fixed combination of a collagen carrier covered with a coating of solid fibrinogen, solid thrombin and solid aprotinin as disclosed in EP 0 059 265.
The function of the collagen carrier disclosed in EP 0 059 265 is mainly that of a carrier which adsorbs and confers mechanical stability to the coagulation preparation with which it is coated.
A product that combines the hemostatic features of fibrin glue with the asset of collagen as a carrier has been developed and manufactured under the trademark TachoComb®. TachoComb® is a ready-to-use and easily applicable fixed combination of a collagen patch coated with the following active components of fibrin glue: human fibrinogen, bovine thrombin and bovine aprotinin.
TachoComb® has been sold since the early 1990s by Nycomed Pharma and has been used in clinical trials in Europe in more than 2500 patients. The product has furthermore been used in more than 700 patients in the Japanese clinical program in a large variety of indications such as liver and lung resections, surgery of the biliary tract, splenic, renal and pancreatic surgery, ENT surgery, gynaecological surgery, and vascular surgery. TachoComb® was found to be effective and safe.
No clinical complications related to the application of TachoComb® have been reported in the course of the clinical trials performed.
In WO97/37694 (Immuno France S.A.) it is disclosed in reference example 4 that when a collagen product or TachoComb® was used, there was no hemostasis leading to bleeding to death when TachoComb® was used in contrast to hemostasis within 5 minutes when a collagen product without a thrombin content prepared according to WO97/37694 was prepared.
In WO96/40033 the disadvantages of the bovine thrombin used in TachoComb® are emphasized in that the use of bovine or other species of thrombin can introduce harmful viral contamination and possible transmission of bovine diseases, such as bovine spongiform encephalitis.
US Pat. No. 6,177,126 B1 discloses a device and a process for the production of a material for sealing and healing wounds. The device comprises a container having, at its bottom part, two perforated plates which are movable relative to each other, so as to allow a suspension contained in the container to drip onto a carrier which is moving past the container under the bottom part thereof.
It is an object of the invention to provide an improved suspension which is suitable, e.g., for use as a coating for a collagen carrier, with the aim of providing a ready-to-use absorbable composition for tissue gluing, tissue sealing and hemostasis. It is a further object of the invention to provide a method for producing such a suspension. It is a still further object of the invention to provide an improved method of coating a carrier, such as a collagen carrier, with a suspension containing fibrinogen and thrombin. A further object of the invention is to provide a method of drying a wet coating of the suspension applied to a carrier, with the aim of ensuring a satisfactory fixation of the coating to the carrier. It is a still further object of the invention to provide a coated collagen sponge with a coating of fibrinogen and thrombin which efficiently mimics the final stage of the coagulation cascade, once the coated collagen sponge has been brought into contact with blood, body fluids or physiological saline. Further, it is an object of the invention to provide a coated collagen sponge with the above coating which has a sufficient fixation of the coating to the collagen sponge, i.e. a satisfactory low abrasion of the coating when submitted to mechanical impact.
In a first aspect the invention provides a suspension comprising fibrinogen, thrombin and alcohol, the suspension having been obtained by a method comprising:
Due to the physical property of the suspension, especially the sedimentation behaviour of the rather large particles in an alcohol, no standard liquid viscosity measure of the suspension is possible. Thus, an alternative method for providing viscosity measure has been implemented. Accordingly, the suspension may have a viscosity so that a volume of 90-120 ml of suspension, when influenced by gravity only, exits through a bottom opening of a container having:
In case of the container being made from steel, the container and the opening having the following dimensions:
The parameters and features of the suspension disclosed below in connection with the method of the second aspect of the invention also apply to the suspension of the first aspect of the invention.
In a second aspect the invention provides a method of preparing a suspension with fibrinogen and thrombin, comprising:
At the step of providing the fibrinogen mixture, the fibrinogen may be pre-micronized by a suitable method, e.g. sieving, to obtain particles having a Folk Ward mean diameter of 25-100 μm. The micronized fibrinogen may, for example, be stirred into the alcohol to obtain the fibrinogen mixture. At the step of providing the mixture, the fibrinogen may be also directly homogenized in an alcohol, preferably at a temperature between 0° C. and 12° C., such as between 2° C. and 8° C. The temperature may be lowered during homogenization. The step of mixing the fibrinogen mixture and the thrombin mixture may be carried out while stirring the suspension, whereby the stirring may be carried out at a temperature between 0° C. and 12° C., such as between 2° and 8° C.
The thrombin may comprise human thrombin, bovine thrombin, or recombinant thrombin, and the fibrinogen may comprise human fibrinogen or recombinant fibrinogen. The alcohol may be an organic alcohol, such as methanol, ethanol, propanol, isopropanol, such as an anhydrous organic alcohol, an anhydrous ethanol, an anhydrous propanol or an anhydrous isopropanol. Human fibrinogen may be supplied in a solid freeze-dried form.
In a third aspect the invention provides a method for coating a carrier with a suspension comprising fibrinogen and thrombin, wherein the suspension has been derived from a method comprising the steps of:
The carrier may be a collagen carrier, such as a collagen sponge. The collagen sponge may fulfil at least one and preferably a plurality of the following criteria:
The collagen sponge may be derived from a method comprising the steps of:
In the present context, the term “chamber diameter” should be understood as the largest straight-line wall-to-wall distance in a chamber, i.e. the largest diagonal straight-line distance of a chamber. The chambers may be of a polygonal shape, such as of an octagonal shape. It has been found that a chamber diameter of more than 0.75 mm and less than 4 mm, or a chamber diameter of at most 3 mm, renders the collagen sponge particularly useful for being coated with a suspension containing fibrinogen and thrombin. It has further been found that a coated collagen sponge prepared by the above method is air and liquid tight in the sense that, once the coated collagen sponge has been applied to a wound, it will not allow air or liquid to soak through the collagen sponge.
The step of applying the suspension to the carrier may be performed at an ambient temperature of 0°-12° C., such as at 1°-10° C., such as at 2°-8° C. Further, the step of applying the suspension to the carrier may be carried out in an ambient atmosphere with a relative humidity of 75-99%, such as 85-95%. A volume of 0.08 ml-0.12 ml of suspension is preferably applied to the carrier per cm2 of the coating surface. To ensure a homogeneous efficacy of the final coated carrier across its whole surface, the suspension is preferably distributed evenly over a given width of the coating surface so that the mass of fibrinogen per area unit of the coating surface varies at most 25%, such as at most 20%, such as at most 15%, such as at most 10%.
An applicator comprising at least one jet may be used for applying the suspension to the carrier, whereby the suspension is forced through the jet while the carrier and the jet are moved relative to each other. The applicator may comprise or be arranged near a conveyor belt, a stirring unit connected to a pump or a system of pumps or other supplying equipment, and a jet or a system of jets which moves transversely, e.g. at right angles to the conveyor belt. Depending on the specific characteristics of the media, the jet or the system of jets may have various shapes and sizes. The jet or the system of jets may be connected to the supplying equipment via tubes. The supplying equipment may promote the coating medium from the stirring unit to the jet systems. During the coating process the jet system may move across the carrier. In its waiting position it may hold on one side of the conveyor belt. The coating process may be initiated by a light barrier sensing the presence of a carrier on the conveyor belt, and may likewise be stopped by a light barrier signal. Such an applicator confers a relatively small dead volume, and it is easy to handle, including easy to clean. Furthermore, it confers the possibility to interrupt the coating process at any time, it is applicable in a relatively broad range of viscosities, and it confers a homogenous coating.
Alternatively, or additionally, an applicator comprising a container having a plurality of separate outlets may be used for applying the suspension to the carrier, whereby the suspension is forced from the container through the outlets onto the carrier. The latter type of applicator, in the form of a container having movable plates at its bottom, is disclosed in U.S. Pat. No. 6,177,126 B1, which is hereby incorporated by reference in its entirety. Due to the even distribution conferred by the devices of U.S. Pat. No. 6,177,126 B1, one of those devices are applied in a preferred embodiment of the invention. The carrier and the applicator are preferably moved relative to each other in a transport direction while the suspension is being applied to the carrier, whereby the rate of movement may be 0.025 m/s-0.05 m/s, such as 0.03-0.04 m/s. The flow rate of suspension applied to the carrier through the applicator may be 400-600 ml/min, such as 470-550 ml/min, such as 495-505 ml/min.
In a fourth aspect the invention relates to a method of drying a suspension of fibrinogen, thrombin and an alcohol applied as a wet coating on a coating surface of a carrier, the method comprising the step of submitting the coated carrier to a pressure below 1000 mbar, so as to obtain a dried coating surface on the carrier, so as to fixate the dried coating to the coating surface. By applying a vacuum and using the vacuum in the drying process, a low temperature (2-10° C.) and a high relative humidity (80-95%) may be kept, whereby the structure and the physical properties of the carrier, in particular a carrier in the form of a collagen, such as a collagen sponge, as well as of the fibrinogen and thrombin may be maintained.
The suspension may be obtained by:
During drying, the coated carrier may be submitted to the pressure at a temperature of 0° C.-12° C., such as 1° C.-10° C., such as 2° C.-8° C., and/or at a relative humidity of the surrounding atmosphere of 75-99%, such as 85-95%. A flow of air may pass across the coated carrier during drying so as to convey vapor away from the coated carrier.
In order for the drying to complete, the coated carrier is preferably kept at these conditions for a period of at least 1 hour, such as at least 2 hours, such as at least 4 hours.
Due to shrinkage, the area of the dried coating surface is smaller than the size of the area of the wet coating surface. In the method according to the invention, the area of the dried coating surface is at least 75% the size of the area of the wet coating surface, such as least 80%.
In order to keep the active components stable when the coated carrier is stored, the carrier and the dried coating surface together preferably have a water content not exceeding 12% by weight, such as not exceeding 8% by weight.
Any parameters and features of the suspension and the collagen sponge, including their methods of manufacture, discussed in connection with the other aspects of the invention, also apply to the method of the fourth aspect of the invention.
In a fifth aspect the invention relates to a coated collagen sponge with a coating of fibrinogen and thrombin, wherein the coated collagen sponge has been obtained by a method comprising the steps of:
The even distribution of the suspension over the coating surface improves the efficacy of the coated surface when applied, e.g. for tissue gluing, tissue sealing or hemostasis. The low abrasion of the coating ensures that the coated collagen sponge may be transported, grabbed by a surgeon's hands and/or by a surgical instrument and otherwise handled without loosening the dried suspension, i.e. the coating. The fibrinogen formulation may account for approximately 60-90% of the total weight of the coated collagen sponge. The formulation usually contains about 50-60% of weight of the following substances: salts, amino acids and albumin. Fibrinogen alone usually constitutes 40-50% of the formulation.
The suspension preferably has a water content of 20-80 mg/ml, such as 24-32 mg/ml. The thrombin content of the suspension may be 20-40 I.U./ml, such as 24-33 I.U./ml. In average, the thrombin content after coating may be 2-4 I.U./cm2 over the coating surface, such as 2.3-3.3 I.U./cm2. It may be desirable that the thrombin content does not exceed 5 I.U./cm2 at any location on the coating surface, or that it does not exceed 3.8 I.U./cm2 at any location on the coating surface.
The microbiological purity of the coated carrier preferably is at most 4 CFU/cm2, such as at most 2.25 CFU/cm2.
In a further independent aspect the invention relates to the use of the above-mentioned coated collagen sponge for tissue gluing, tissue sealing and hemostasis.
Preferred embodiments of the methods and products of the present invention are described below, cf. also
A suspension comprising fibrinogen, thrombin and alcohol may be produced by the method for producing a suspension according to the invention, as follows:
Fibrinogen is homogenized in a 100% ethanol at 2-8° C., resulting in a mixture of fibrinogen and alcohol, the mixture constituting approximately 80% of the volume of the final suspension volume. Then, riboflavin is added. The mixture is subsequently stirred in a closed vessel until further processing thereof.
Human or bovine thrombin is dissoluted with water for injection. The solution is added to a 35 fold amount of 100% ethanol at 0-8° C. The thereby achieved thrombin suspension is homogenized at 0-8° C. for 80-100 sec.
Before the fibrinogen and thrombin mixtures are mixed, an aprotinin solution and water for injection are added to the fibrinogen mixture. Then, the thrombin mixture is added to the fibrinogen mixture. Final volume of suspension is prepared by adding a 100% ethanol at 2-8° C.
Methods comprising the above steps are hereinafter referred to as “group I methods”.
As an alternative, the method for producing a suspension according to the invention, comprising fibrinogen, thrombin and alcohol, comprises the following steps:
The fibrinogen mixture is obtained by adding pre-micronized fibrinogen of a particle size of 35-80 μm Folk Ward mean diameter and riboflavin while stirring to a 94-97% ethanol at 2-8° C. The thereby resulting mixture of riboflavin and ethanol constitutes approximately 70-80% of the final suspension volume. The fibrinogen mixture is further stirred in a closed vessel until further processing thereof.
The thrombin mixture is obtained by adding thrombin to a 94-97% ethanol at −30° C. The thereby achieved thrombin mixture is homogenized for 80-100 sec. Alternatively, the thrombin mixture is obtained by solving thrombin in water for injection, and subsequently the thereby obtained thrombin solution is slowly added to 17-35 fold amount of 100% ethanol at −30° C.
The suspension is homogenized 80-100 sec.
UltraTurrax equipment by IKA may be used as homogenizing equipment.
The thrombin mixture is added to the mixture containing fibrinogen and riboflavin. A 94-97% ethanol at 2-8° C. is added.
Methods comprising the above steps are hereinafter referred to as “group II methods”.
The above group I and group II methods may result in a suspension according to the invention, preferably with the following characteristics:
In one embodiment of the method of drying according to the invention, collagen sponge strips are incubated at 2-8° C. at 80-91% relative humidity for 2-30 hours before coating of a carrier in the form of a collagen sponge. The applicator for applying the suspension to the collagen sponge is described above. Once coated, the collagen sponge strips are incubated at 2-8° C. and 80-90% relative humidity for 8-60 minutes. The coated collagen sponge strips are dried in a vacuum drying chamber at an air temperature of 2-8° C., 80-90% relative humidity. An air flow is passed over the collagen strips through an aspiration valve, at an air flow rate of 1,2-40 m3 per hour. A vacuum of 30-60 mbar is applied, i.e. an absolute pressure of approximately 970 mbar, depending upon atmospheric pressure, and the coated collagen strips are dried for 2-5 hours.
A measure of the viscosity of the suspension is obtained by use of one of the devices depicted in
The device shown to the right in
length of passage connected to the bottom opening: less than 5 mm.
Examples I-VI below illustrate various procedures for preparation of a coated collagen sponge with a coating of fibrinogen and thrombin according to the invention. The procedures include methods of preparing a suspension according to the invention, resulting, in the embodiments described below, in suspensions according to the invention. Further, methods for coating according to the invention and methods for drying according to the invention are applied.
In the present example, the suspension contains human fibrinogen formulation B and human thrombin formulation B.
A final suspension volume of 3500 ml was obtained by a group II method by applying the following quantities and parameters:
Fibrinogen mixture:
The fibrinogen mixture was stored for 20 hours at 2-8° C. while being stirred.
Thrombin mixture:
The thrombin mixture was stored for 18hours at −30° C.
Suspension:
Suspension characteristics:
Carriers in the form of collagen strips were coated with the suspension. First, 48 collagen sponge strips were pre-incubated in a cooling chamber, at the following conditions:
An applicator as disclosed in U.S. Pat. No. 6,177,126 B1 was used for coating the collagen sponge strips with the suspension.
The coated collagen sponge strips were dried as follows:
The coated strips were incubated for 15 minutes at a temperature of 5.2° C. and an absolute humidity of 4.8 g water per kg air.
The coated strips were then dried in a vacuum drying chamber at the following drying conditions:
The abrasion of the obtained coating on the collagen sponge strips was approximately 0.2 mg/cm2 when a sample of 1×5cm2 is shaken in a test-tube on a Vibrofix shaker at a frequency of 800-1200 rpm for 2 minutes.
In the present example, the suspension contains human fibrinogen formulation C and human thrombin formulation C.
A final suspension volume of 3500 ml was obtained by a group II method by applying the following quantities and parameters:
Fibrinogen mixture:
The fibrinogen mixture was stored for 20 hours at 2-8° C. while being stirred.
Thrombin mixture:
The thrombin mixture was stored for 18 hours at −30° C.
Suspension:
Suspension characteristics:
Carriers in the form of collagen strips were coated with the suspension. First, 48 collagen sponge strips were pre-incubated in a cooling chamber, at the following conditions:
An applicator as disclosed in U.S. Pat. No. 6,177,126 B1 was used for coating the collagen sponge strips with the suspension.
The coated collagen sponge strips were dried as follows:
The coated strips were incubated for 13 minutes at a temperature of 4.9° C. and an absolute humidity of 4.8 g water per kg air.
The coated strips were then dried in a vacuum drying chamber at the following drying conditions:
The abrasion of the obtained coating on the collagen sponge strips was approximately 0.2 mg/cm2 when a sample of 1×5cm2 is shaken in a test-tube on a Vibrofix shaker at a frequency of 800-1200 rpm for 2 minutes.
In the present example, the suspension contains human fibrinogen formulation B and human thrombin formulation B, and aprotinin.
A final suspension volume of 1000 ml was obtained by a group II method by applying the following quantities and parameters:
Fibrinogen mixture:
The fibrinogen mixture was stored for 20 hours at 2-8° C. while being stirred.
Thrombin mixture:
The thrombin mixture was stored for 16 hours at −30° C.
Suspension:
The total volume of thrombin mixture was added to the fibrinogen mixture.
A 100% ethanol at 2-8° C. was added to fill to the final suspension volume of 1000 ml.
Suspension characteristics:1. Ethanol concentration: 95%
Carriers in the form of collagen strips were coated with the suspension. First, 16 collagen sponge strips were pre-incubated in a cooling chamber, at the following conditions:
An applicator as disclosed in U.S. Pat. No. 6,177,126 B1 was used for coating the collagen sponge strips with the suspension.
The coated collagen sponge strips were dried as follows:.
The coated strips were incubated for 35 minutes at a temperature of 5° C. and a relative humidity of 85%.
The coated strips were then dried in a vacuum drying chamber at the following drying conditions:
The abrasion of the obtained coating on the collagen sponge strips was approximately 0.2 mg/cm2 when a sample of 1x5cm2 is shaken in a test-tube on a Vibrofix shaker at a frequency of 800-1200 rpm for 2 minutes.
In the present example, the suspension contains human fibrinogen formulation C and human thrombin formulation C.
A final suspension volume of 780 ml was obtained by a group II method by applying the following quantities and parameters:
Fibrinogen mixture:
The fibrinogen mixture was stored for 20 hours at 2-8° C. while being stirred.
The thrombin mixture was stored for 16 hours at −30° C.
Suspension:
Suspension characteristics:
Carriers in the form of collagen strips were coated with the suspension. First, 8 collagen sponge strips were pre-incubated in a cooling chamber at the following conditions:
An applicator as disclosed in U.S. Pat. No. 6,177,126 B1 was used for coating the collagen sponge strips with the suspension.
The coated collagen sponge strips were dried as follows:
The coated strips were incubated for 45 minutes at a temperature of 5° C. and a relative humidity of 85%.
The coated strips were then dried in a vacuum drying chamber at the following drying conditions:
The abrasion of the obtained coating on the collagen sponge strips was approximately 0.2 mg/cm2 when a sample of 1×5 cm2 is shaken in a test-tube on a Vibrofix shaker at a frequency of 800-1200 rpm for 2 minutes.
In the present example, the suspension contains human fibrinogen formulation A and human thrombin formulation A.
A final suspension volume of 3120 ml was obtained by a group I method by applying the following quantities and parameters:
Fibrinogen mixture:
Thrombin mixture:
Suspension:
The thrombin mixture was added to the fibrinogen mixture.
A 100% ethanol at 2-8° C. was added to fill to the final suspension volume.
Suspension characteristics:
Carriers in the form of collagen strips were coated with the suspension. First, 48 collagen sponge strips were pre-incubated in a cooling chamber, at the following conditions:
An applicator as disclosed in U.S. Pat. No. 6,177,126 B1 was used for coating the collagen sponge strips with the suspension.
The coated collagen sponge strips were dried as follows:
The coated strips were incubated for 10 minutes at a temperature of 6.5° C. and a relative humidity of 90%.
The coated strips were then dried in a vacuum drying chamber at the following drying conditions:
The abrasion of the obtained coating on the collagen sponge strips was approximately 0.2 mg/cm2 when a sample of 1×5 cm2 is shaken in a test-tube on a Vibrofix shaker at a frequency of 800-1200 rpm for 2 minutes.
In the present example, the suspension contains human fibrinogen formulation A and bovine thrombin formulation, and aprotinin.
A final suspension volume of 16720 ml was obtained by a group I method by applying the following quantities and parameters:
Fibrinogen mixture:
The fibrinogen mixture was stored for 21 hours at 2-8° C. while being stirred.
Thrombin mixture:
Suspension:
The thrombin mixture was added to the fibrinogen mixture.
Suspension characteristics:
Carriers in the form of collagen strips were coated with the suspension. First, 288 collagen sponge strips were pre-incubated in a cooling chamber, at the following conditions:
An applicator as disclosed in U.S. Pat. No. 6,177,126 B1 was used for coating the collagen sponge strips with the suspension.
The coated collagen sponge strips were dried as follows:
The coated strips were incubated for 10 minutes at a temperature of 6.5° C. and a relative humidity of 89%.
The coated strips were then dried in a vacuum drying chamber at the following drying conditions:
The abrasion of the obtained coating on the collagen sponge strips was approximately 0.2 mg/cm2 when a sample of 1×5 cm2 is shaken in a test-tube on a Vibrofix shaker at a frequency of 800-1200 rpm for 2 minutes.
In a coated collagen sponge containing human fibrinogen, bovine thrombin and aprotinin, the stability of the coating suspension was investigated for the duration of a coating process of 7 hours under environmental conditions of the production rooms, e.g. at 2-8° C.:
Each active ingredient was assayed at different sampling times. The results are shown in table I below.
The results show satisfactory stability for all three components.
Reference is made to
Comparison of coated Nycomed sponge (TachoComb S) with other carrier products coated identically as TachoComb S.
Adhesion of the Layer
Procedure
An area of 2×4.5 cm2 of each carrier was coated with TachoComb S coating suspension. The amount of coating suspension corresponded to TachoComb specification (5.5 mg fibrinogen/cm2). The samples were dried.
2. A sample of 1×4 cm2 was prepared of each coated carrier.
3. The adhesion of the layer was tested as follows
Method Description
Apparatus
Analytical balance (measurement precision 0.5 mg)
Vibrofix shaker combined with fixation device
Ruler with millimeter graduation
Stop-watch, scalpel, tubes of 2 cm internal diameter with stopper
Procedure
The procedure and calculation for determining abrasion are described above.
Comment
All carriers except Nycomed collagen sponge are not flexible after coating. The sample has to be cut out very cautiously. If it is cut out by using a pair of scissors a lot of the coating material will flake off because the layer in itself is rigid. Ethisorbe patch showed almost no connection with the coating material at all. When shaken a little bit, all of the coating peels off like a “carpet”.
The difference between Nycomed collagen sponge and the other carrier materials shows quite clearly.
Elasticity of the Moistened Coated Carrier
Procedure
1. Coating of Different Carriers
An area of 2×4.5 cm2 of each carrier was coated with TachoComb S coating suspension. The amount of coating suspension corresponded to TachoComb specification (5.5 mg fibrinogen/cm2). The samples were dried.
2. A sample of about 5-7 cm2 was prepared of each coated carrier. The exact starting area of the dry sample was determined.
3. The sample was moistened and put on an elastic Latex sheet fixed to a special equipment as described in detail under the heading “procedure”. Then pressure was put on the Latex sheet which expanded. After 2 times of expansion and relaxation the sheet is expanded for a third time. The area of the carrier was measured at the highest expansion point.
Method Description
Apparatus/Chemicals
Peristaltic pump (IKA PA-SF)
Pressure buffering bottle (3 outlets)
VDO manometer (0-250 mbar)
Glass funnel (Ø opening 1:30 mm, opening 2:15 mm)
Silicone tubings and clamps, Latex gloves (Semper med), scalpel, ruler with millimeter graduation, scissors
Physiological saline
Procedure
The following equipment is connected air tight to the three outlets of the pressure buffering bottle via silicone tubings:
A double sheet of about 8×8 cm2 is cut from a Latex glove. This sheet is fixed airtight to the glass funnel/opening 1.
About 5-7 cm2 coated area are cut out of the coated carrier using a scalpel.
The area of the sample is measured (starting area). The coating of the sample is moistened with saline and placed on the Latex sheet. Then it is pressed to the Latex sheet manually for about 1 min.
Using the peristaltic pump the Latex sheet is expanded by putting on a pressure of about 70 mbar. This is repeated twice with relaxation of the Latex sheet afterwards. At the third expansion the area (length and width) of the coated carrier is measured at the highest point of Latex sheet expansion.
Calculation:
Comment:
The elasticity of the moistened Collagen sponge Nycomed (TachoComb) is one of the important characteristics of the product. Elasticity is essential in thoracic and abdominal surgery. After gluing the carrier should be able to follow for example expansion and relaxation movements of the lungs or intestines. Especially Ethisorb® showed no elasticity at all. It detached from the coating immediately. Coated Willospon® Spezial and Opraskin® showed structural defects during the test.
Use of Coated Carrier in Endoscopic Surgery
Procedure:
1. Coating of Different Carriers
An area of 2×4 cm2 of each carrier was coated with TachoComb S coating suspension. The amount of coating suspension corresponded to TachoComb specification (5,5 mg fibrinogen/cm2). The samples were dried.
2. The handling of the coated carrier samples for use in endoscopic surgery and the loss of coating due to this handling are documented by digital photo-equipment.
Method Description
Apparatus
Endodock: Endoscopic tool designed for the use of TachoComb® in endoscopic surgery (see
Procedure
Picture series taken of each carrier:
Comment
TachoComb (coated equine collagen sponge/Nycomed) in endoscopic surgery is the most demanding application of the product. TachoComb is inserted into an endoscopic equipment. The tube of this equipment is generally 10-13 mm in diameter. To be inserted into the tube TachoComb is flattened and then wrapped around a guiding “pin” and then inserted carefully into the tube. Therefore the connection of the coating to the carrier and within itself has to be strong but the product has to stay flexible enough in dry condition to be bent and rolled up. When brought to the site of the surgery TachoComb is carefully pulled out of the tube. Then it has to be unwrapped and placed to the wound surface. This often requires some adjustments. Therefore adhesion of the layer to the carrier should be strong enough to withstand this handling.
Results
The results are seen from the enclosed
As Ethisorb® is a very rigid carrier the adhesion of the coating is very bad. Therefore coated Ethisorb® lost almost all of the coating in this investigation. Compared to coated collagen sponge of Nycomed all the other investigated carriers have a flat surface to be coated. Therefore the coating lies like a “flat carpet” on the carrier. This leads to a rather unflexible structure of the dry coated carriers. Bending or rolling up often breaks the coating in itself.
After insertion of the coated carriers into the tube of the endoscopic equipment and the unfolding of the sample afterwards all carriers except collagen sponge of Nycomed lost quite a lot of the coating so that large areas are left without coating material.
The structure and texture of Nycomed collagen sponge is the basis of the high flexibility of TachoComb in dry or moistened conditions. Nycomed collagen sponge is foamed and has polygonal chambers inside. On the surface these chambers are cut to caverns. These caverns enlarge the coating surface. During coating the coating suspension is distributed evenly onto the structured surface. During the drying the solution containing both fibrinogen and thrombin is fixed as solids into the caverns. Therefore TachoComb can be cut to desired sizes and can be inserted into endoscopic equipment with only a small loss of coating material or no loss at all.
The high flexibility of dry TachoComb is a big advantage compared to all other investigated coated carriers.
Manufacture of Collagen Sponge
The collagen sponge referred to in the present text may be manufactured by a method as generally illustrated in
It has been found that the successful coating of a collagen sponge with a fibrin glue preparation depends on the texture of the collagen sponge. It is thus desirable to provide a method of producing a collagen sponge with a certain texture, in particular with the aim of making the collagen sponge suitable for coating with a fibrin glue preparation, so as to obtain a material for healing and sealing wounds. It is further desirable to provide a method of producing a collagen sponge having improved physical characteristics in relation to prior art sponges, in the sense of improved humidity, elasticity, density and elasticity modulus. It is further desirable to provide a method for preparing a collagen sponge which is air and liquid tight in the sense that, once the collagen sponge is applied to a wound, it will not allow air or liquid to soak through the collagen sponge.
Thus, the method of preparing the collagen sponge may comprise the steps of:
In the present context, the term “chamber diameter” should be understood as the largest straight-line wall-to-wall distance in a chamber, i.e. as the largest diagonal straight-line distance of a chamber. The chambers may be of a polygonal shape, such as of an octagonal shape.
It has been found that a chamber diameter of more than 0.75 mm and less than 4 mm, or a chamber diameter average of at most 3 mm, renders the collagen sponge particularly useful for being coated with a fibrin glue preparation. Preferably, the collagen gel has a dry mass in the range of 2-20 mg dry mass per 1 g gel, such as 4-18 mg, such as 5-13 mg, such as 6-11 mg per 1 g gel. The dynamic viscosity of the collagen gel is preferably 2-20 Ncm, such as 4-10 Ncm, such as 6-8 Ncm. The collagen sponge preferably has a water content of not more than 20%, such as 10-15%, such as about 18%. The elasticity modulus of the collagen sponge is preferably in the range of 5-100 N/cm, such as 10-50 N/cm, and the density of the sponge is preferably 1-10 mg/cm3, such as 2-7 mg/cm3.
It has been found that a collagen sponge prepared by the above method is air and liquid tight in the sense that, once the collagen sponge is applied to a wound, it will not allow air or liquid to pass through the collagen sponge. Liquids are absorbed in the sponge. This effect is primarily achieved due to the fact that the step of mixing air into the collagen gel provides a collagen sponge which has a three-dimensional structure with stacked chambers separated and substantially totally enclosed by walls of collagen material, in contradiction to those known collagen sponges which have a fiber structure.
The collagen gel may comprise material of different types, such as type I, II or III from mammalian, transgenic or recombinant sources, but all other types of collagen can be used. The collagen may comprise material from tendons selected from the group consisting of equine tendons, human tendons, and bovine tendons. The collagen gel may additionally or alternatively comprise recombinant collagen material.
The collagen content of the isolated parts of sponge is preferably 50% -100% related to dry mass of the sponge, such as 75% -100%, such as 80% -100%, such as 85% -100%, such as 90% -100%, such as 92-100%, such as 92-98%, such as 93-97%, such as 94% -96%.
The step of preparing the collagen gel preferably comprises the steps of:
The steps of storing, peeling, removing protein, reducing of germ content, and swelling aim at purifying the raw material, whereas the step of homogenizing aims at obtaining the collagen in the form of a gel.
The step of reducing of germ content preferably comprises adding an acid, such as an organic acid, such as lactic acid to the tendons. Further, an organic solvent, such as an alcohol, such as ethanol is preferably added to the tendons. Further, the step of swelling of the tendons preferably comprises adding lactic acid to the tendons. The lactic acid used may be a 0.40-0.50% lactic acid, such as a 0.45% lactic acid.
The step of swelling of the tendons may comprise storing the tendons at a temperature of 4° C. to 25° C., such as a temperature of 10° C. to 20° C., for a period of 48 to 200 hours, such as a period of 100 to 200 hours.
The step of homogenizing the swelled tendons is preferably carried out so as to obtain a particle size of collagen gel fragments, i.e. fiber balls, with a diameter of 0.8-1.2 cm, such as approximately 1 cm. Further, the physical characteristics of the collagen gel are preferably as stated above. The appropriate characteristics may for example be achieved by performing the step of homogenizing the swelled tendons by means of a toothed disk mill or adequate homogenization equipment.
The step of mixing air into the collagen gel preferably comprises the steps of:
At least some of the collagen gel separated from the collagen foam in the fractionizing channel may be led back to the mixer. In that case, the ratio between the amount of collagen gel which is led back to the mixer from the fractionizing channel and the amount of fresh collagen gel led to the mixer is preferably between 0.1 and 0.5. The step of separating collagen gel and collagen foam preferably comprises the steps of:
In a preferred embodiment of the method, a temperature of 15° C. to 40° C., such as 20° C. to 25° C., is maintained in the fractionizing channel.
Subsequent to mixing air into the collagen gel, the collagen foam may be homogenized for a period of 2 to 4 minutes.
Prior to the step of drying the collagen foam and subsequent to the step of mixing air into the collagen gel, a neutralizer may be added to the collagen foam, and the collagen foam is preferably neutralized in order to arrive from a pH-value of, usually, between 2.5 and 3.5 to a pH-value in the collagen foam between 6.5 and 8.5. A neutralized comprising an ammonia solution may be used, and the collagen foam is preferably neutralized for a period of 5-30 hours, such as 10-20 hours, such as approximately 24 hours.
Prior to the step of drying the collagen foam, the collagen foam is preferably filled into a drying container in such a way that substantially no air is drawn into the foam while filling.
The step of drying preferably comprises drying at a temperature between 15° C. and 60° C., such as between 200 and 40° C., for a period of 50-200 hours, such as 100-150 hours, so as to obtain a dry collagen sponge. The drying may be performed at a pressure slightly under atmospheric pressure, such as at a pressure of between 700 and 900 mbar, such as approximately 800 mbar.
The collagen sponge produced by the above method preferably fulfils at least one of the following criteria:
The step of isolating parts of collagen sponge may comprise dividing the collagen sponge into a plurality of parts by cutting. The parts obtained may be shaped in any desirable form, such as conical, cylindrical, including cylindrical with an annular cross-section, rectangular, polygonal, cubic, and flat sheets or they may be transformed into a granulate by an appropriate granulating method etc.
As it is apparent from the above, the collagen sponge may be produced by a method comprising the steps of:
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2001 00135 | Jan 2001 | DK | national |
This is a Continuation Application of U.S. patent application Ser. No. 10/054,889, filed Jan. 25, 2002, which claims priority from Provisional Application Serial No. 60/263,699, filed Jan. 25, 2001.
| Number | Date | Country | |
|---|---|---|---|
| 60263699 | Jan 2001 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10054889 | Jan 2002 | US |
| Child | 10990528 | Nov 2004 | US |
