The present invention relates to a solid composition useful for tissue gluing, tissue sealing and haemostasis consisting essentially of a) a carrier which has at least one of the following physical properties: elasticity module in the range of 5-100 N/cm, density of 1-10 mg/cm.sup.3, chamber diameter of more than 0.75 mm and less than 4 mm and/or having a chamber diameter average below 3 mm and evenly distributed and fixed upon said carrier, b) solid fibrinogen, and c) solid thrombin.The carrier is a biodegradable polymer such as a polyhyaluronic acid, polyhydroxy acid, e.g. lactic acid, glucolic acid, hydroxybutanoic acid, a cellulose, gelatine or collagen, such as a collagen sponge, e.g. a collagen sponge consisting essentially of collagen type I fibres. The fibrinogen and thrombin are preferably human, purified from a natural source, or transgenic or recombinant human fibrinogen and/or thrombin. In a preferred embodiment the composition does not comprise any antifibronolytic agent such as aprotinin, .epsilon.-aminocaproic acid or .alpha.2-antiplasmin.

Description of the Invention

The present invention relates to a solid composition consisting essentially of a carrier which has at least one of the following physical properties elasticity module in the range of 5-100 N/cm.sup.2, such as 10-50 N/cm.sup.2; density of 1-10 mg/cm.sup.3, such as 2-7 mg/cm.sup.3; chamber diameter of more than 0.75 mm and less than 4 mm and/or having a chamber diameter average below 3 mm and evenly distributed and fixed upon said carrier b) solid fibrinogen, and c) solid thrombin.

The composition may have two, three or all of the above mentioned physical properties. In presently preferred embodiments the carrier material is produced as described in DK PA 2001 00135 and further in the PCT application entitled "A method of preparing a collagen sponge, a device for extracting part of the collagen foam and an elongated collagen sponge" filed by Nycomed Pharma AS on 25 Jan. 2002 claiming priority from said application. 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. Thus, when the carrier is cut, the chambers are divided and cut to caverns. The solid fibrinogen and the solid thrombin is fixed to the carrier and most of it is present in the caverns thus providing a substantially even distribution of the solid thrombin and solid fibrinogen. Due to this and the fixation, it is possible to introduce substantial amounts of fibrinogen and thrombin on the carrier in contrast to the situation where liquid compositions of thrombin and fibrinogen are e.g. dropped or sprayed onto the material.

Preparation of Coated Carrier

The preparation of a coated carrier consists essentially of preparation of a suspension of the active ingredients even distribution of the suspension to the carrier drying of the coated carrier to a solid composition/fixation of the active ingredients to the carrier.

Preparation of a suspension with fibrinogen and thrombin comprises: providing a fibrinogen mixture of fibrinogen and an alcohol, such as ethanol providing a thrombin mixture of thrombin and an alcohol, such as ethanol mixing the fibrinogen mixture and the thrombin mixture so as to obtain said suspension.

At the step of providing the mixtures, the mixture may be homogenized or sieved obtaining a suspension containing fibrinogen and thrombin particles with the Folk Ward mean diameter of the particles being 25-100 .mu.m. The temperature is between 0.degree. C. and 12.degree. C.

The carrier may be a collagen carrier, such as a collagen sponge. The collagen sponge may fulfill at least one and preferably a plurality of the following criteria: pH-value between 5.0 and 6.0, lactic acid content at the most 5%, ammonium content at the most 0.5%, soluble protein content, calculated as albumin content, at the most 0.5%, sulphate ashes content at the most 1.0%, heavy metal content at the most 20 ppm, microbiological purity, at the most 10.sup.3 CFU/g, collagen content of 75 to 100%, density of 1 to 10 mg/cm.sup.3, elasticity module in the range of 5-100 N/cm.sup.2.

In a presently preferred embodiment, the collagen carrier is produced as described in DK PA 2001 00135. The physical properties of three examples of collagen carriers are provided in the table -- see Original Patent.

Even distribution of the suspensions is carried out either using a drip-on-device as disclosed in U.S. Pat. Nos. 5,942,278 and 6,177,126 or an applicator comprising at least one jet may be used for applying the suspension to the carrier. The jet applicator is forcing the suspension through the jet while the carrier and the jet are moved relatively 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 another 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.

Both systems are applying a volume of 0.08 ml-0.12 ml of suspension pr. cm.sup.2 carrier.

An important step is the drying of a suspension of fibrinogen, thrombin and an alcohol applied as a wet coating on a coating surface of a carrier. An example of a method comprises the step of submitting the coated carrier to a pressure below 1000 mbar, so as to obtain a dried coating surface on the carrier and 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.degree. 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.

By the term "consisting essentially of" is meant that the three components are all essential and necessary for the invention. However, inessential additives such as calcium ions and a coloring marker such as riboflavin can also be present in the composition. The composition may further comprise other useful ingredients such as one or more pharmaceutical active substances which may e.g. be selected from the group consisting of antibiotic, such as antibacterial or antimycotic, and antineoplastic agents.

Although the carrier material is preferably a collagen sponge which comprises collagen type I material from mammalian, transgenic or recombinant sources, it may be produced by means of other types of collagen i.e. collagen type I, II, III, IV, VII and X. However, it is also envisaged that the carrier may be a biodegradable polymer such as a polyhyaluronic acid, polyhydroxy acid, e.g. lactic acid, glucolic acid, hydroxybutanoic acid, a cellulose, or gelatine.

In a preferred embodiment of the invention, the composition comprises a carrier which has one or more active sides wherein fibrinogen is present in an amount of 2-10 mg/cm.sup.2 such as 4.3-6.7 mg/cm.sup.2, preferably about 5.5 mg/cm.sup.2, and thrombin is present in an amount of 1.0-5.5 IU/cm.sup.2, preferably about 2.0 IU/cm.sup.2. The fibrinogen and/or thrombin is preferably human, e.g purified from a natural source by methods known to the person skilled in the art, or transgenic or recombinant human fibrinogen and/or thrombin produced by methods known to the person skilled in the art.

The prior art products such as TachoComb.RTM., Beriplast.RTM. and TissueSeal.RTM. all contain aprotinin or similar anti-fibrinolytic agents. Aprotinin can only be provided from a bovine source. It has been an object of the present inventors to develop a composition with an improved carrier material and only components of human, recombinant or transgene origin. Therefore, developments have been made with respect to the carrier material and it has been investigated whether it was possible to replace the bovine thrombin with human thrombin and to avoid the aprotinin. The present inventors have worked towards this goal through a two-step process.

First of all, TachoComb H has been developed as a follow-up product of TachoComb.RTM. with e.g. the bovine thrombin being replaced by human thrombin. Clinical experience with TachoComb H has been performed with regard to a number of therapeutic confirmatory (phase IIIa) clinical trials within the indications hemostasis, tissue gluing and tissue sealing. The yet unpublished results gained in these studies confirmed the efficacy and safety of TachoComb H in the control of blood and air leakage, thus serving as an adjuvant therapy to suturing in hemostasis, tissue gluing and tissue sealing during surgery. In particular, the efficacy of TachoComb H in achieving local hemostasis, expressed as a significant reduction in time-to-hemostasis compared to controls, was convincingly shown in vascular and liver surgery alike.

Also, it has been found that TachoComb H may be able to reduce pulmonary defects in size, resulting in a more rapid resolution of air leakage and may be useful in sealing severe pulmonary leaks and in emphysematous lungs.

However, TachoComb.RTM. and TachoComb H both comprise aprotinin as an integral part of the product. Aprotinin has been considered necessary to inhibit possible conversion of small amounts of plasminogen to plasmin in the fibrinogen component and to prevent premature lysis of the fibrin clot especially under hyperfibrinolytic conditions.

The present inventors devised new experiments in order to test this hypothesis that aprotinin was necessary. The in vitro experiments showed the antifibrinolytic protection of aprotinin in the clot and that TachoComb.RTM. without aprotinin (TachoComb S) was not dissolved within a very short time. Therefore stressful animal models were designed and TachoComb S was compared to TachoComb H to prove similar efficacy. In all models TachoComb H or S, respectively, was used as only means of hemostasis.

Four extensive experimental series have been performed in order to investigate the efficacy and histopathological pattern of the presently preferred embodiment of the present invention TachoComb S compared to TachoComb H. TachoComb S or TachoComb H were applied on the organs liver, spleen, pancreas or brain/meninges of dogs, pigs or rabbits. The experiments were designed in a way to resemble normal surgical conditions, severely stressful conditions and hyperfibrinolytic conditions.

The results obtained in these four studies did not show any relevant difference between TachoComb S and TachoComb H. Both products behaved similarly with regard to haemostatic and wound sealing efficacy including severely stressful conditions like increased intraorgan pressure or hyperfibrinolysis induced by local r-tPA application.

It can be concluded that the preclinical program designed to evaluate the overall necessity of aprotinin as a component of TachoComb H has proven similar efficacy of TachoComb H and TachoComb S. Both products have been used successfully as only means of hemostasis, tissue gluing and tissue sealing under all experimental conditions. In the course of animal experiments, there were no undesirable tissue reactions. Consequently, aprotinin has been eliminated from the composition of the invention.

The composition of the invention is expected clinically to exert the same hemostatic, tissue gluing and tissue sealing properties as its predecessors and to have the same or an even more satisfactory safety profile. The absence of aprotinin which is presently only available from bovine sources adds safety against hypersensitivity reactions. In this regard it should be noted that antibodies against aprotinin occurred in three Japanese studies. No such immunological response is anticipated with a composition without aprotinin.

In a presently preferred embodiment, the invention relates to a composition for haemostasis, tissue sealing and tissue gluing which comprises a flexible carrier which has at least one of the following physical properties: elasticity module in the range of 5-100 N/cm.sup.2, such as 10-50 N/cm.sup.2; density of 1-10 mg/cm.sup.3, such as 2-7 mg/cm.sup.3; chamber diameter of more than 0.75 mm and less than 4 mm and/or having a chamber diameter average below 3 mm and which further comprises solid fibrinogen and solid thrombin and does not comprise any antifibronolytic agent such as aprotinin, .epsilon.-aminocaproic acid, or .alpha.2-antiplasmin, the solid fibrinogen and solid thrombin being fixed to the carrier in a manner so that the abrasion is less than 1.0 mg/cm.sup.2 when a sample of the coated material is shaken on a Vibrofix shaker at a frequency of about 1000 rpm for 2 minutes and if the coated carrier material is inserted into endoscopic equipment and thereafter removed, the material is substantially unchanged and has cast coating material less than 20% as an indication of the flexibility of the carrier and the solid adhesion of the solid fibrinogen and solid thrombin, and the material being substantially air tight and liquid tight and having an elasticity factor of at least 1.25 as determined by a test comprising fixation of the coated carrier to a Latex sheet, expansion of the Latex by pressure three times and at the third time measuring the area of the coated carrier at the highest point of Latex sheet expansion and comparing the expanded area of the coated carrier with the starting area of the coated area.

The preferred composition of the invention wherein the fibrinogen and thrombin are human, e.g. purified from a natural source or transgenic or recombinant human fibrinogen and thrombin, is the only non-bovine fibrin sealant available with a fixed combination of active components coated on a flexible carrier and has several advantages: Ready-to-use, no time consuming thawing or preparation procedure needed Easily applied directly onto most tissue and organ surfaces Endoscopic application possible No problems with the hemostatic running off or being rinsed off the target area Combination of the gluing effect of fibrin clotting and the mechanical support of the flexible carrier Highly flexible and withstanding heavy stretching and compression Effective hemostasis and tissue sealing within 3-5 minutes Favorable safety profile, i.e. no bovine components Biodegradable leaving only minor tissue scars Can be stored at +2.degree. C. to +8.degree. C. and will have an expected shelf life of 36 months, or at room temperature for a period of up to at least 2 years.

The reason to develop the preferred composition of the invention derives from a wish to get rid of the last bovine component in order to prevent any, even theoretical, risk of transmittance of diseases from cows to humans, including transmissible spongiform encephalopathies (TSEs). The active components fibrinogen and thrombin are thus of human origin, and bovine aprotinin, the inhibitor of the fibrinolytic enzyme plasmin, has been removed. Thus, the advantage of the preferred composition of the invention containing no bovine components is that the risk of transmitting diseases, including bovine spongiform encephalopathy (BSE), via bovine material has been eliminated.

Similarly to other fibrin glues, the composition of the invention works by reproducing the last step of the blood coagulation cascade. A mixture of fibrinogen and thrombin forms a fixed solid layer on the surface of the flexible carrier. Upon contact with fluids, e.g. a bleeding surface, body fluids or physiological saline, the components of the layer dissolve, diffuse into the cavities of the wound and start to react.

The polymerization process produces a strong adhesion between wound surface and carrier patch. During the time required for gluing, i.e. 3 to 5 minutes, the composition of the invention should preferably be pressed gently onto the wound surface. The carrier patch provides mechanical support that allows tamponage of the wound. The patch keeps the coagulation components in place when wounds are bleeding profusely and prevents potential re-bleeding. The mechanism of action involves the conversion by thrombin of fibrinogen into fibrin by splitting off peptides. Fibrin monomers polymerize spontaneously into fibrin strands forming a viscous and elastic clot, which glues the carrier patch to the wound surface. The fibrin matrix subsequently serves as scaffolding for fibrinoblast migration (FIG. 8, see Original Patent).

Like a two-component adhesive, wound surface and carrier are glued together by polymerization. The mechanical stability of the carrier patch adds a tamponade effect to the hemostatic effect of fibrin clotting. Further, the active substances are only present on the carrier surface facing the wounded area and by virtue of the tamponade effect and the gentle pressure they do not diffuse through the carrier. Consequently, and in contrast to the situation when using most fibrin glues, there is no adhesion between the wounded area covered with the composition of the invention and other organs or parts thereof when the composition of the invention has been used.

Unlike cyanoacrylate and gelatine-resorcin-formaldehyde (GRF) glues, which are highly histotoxic to parenchymatous tissue, the solid composition of the present invention is physiologically degraded and replaced by tissue within weeks or months after application mainly via two mechanisms: 1. The fibrin clot is degraded partly by fibrinolysis and partly by cellular phagocytosis. 2. The carrier is degraded layer by layer by absorptive granulation tissue and converted into a pseudo-capsule consisting of endogenous connective tissue.

The composition of the invention is useful for hemostasis, tissue gluing and tissue sealing, in particular in surgical intervention in the gastrointestinal system, such as the esophagus, stomach, small intestine, large intestine, rectum, on parenchymal organs, such as liver, spleen, pancreas, kidneys, lungs, adrenal glands, thyroid and lymph nodes, cardiovascular surgery, thoracic surgery including surgery on the trachea, bronchi or lungs, surgical interventions in the ear, nose and throat (ENT) area including dental surgery, gynaecological, urological, bone (e.g. spongiosa resection), and emergency surgery, neurological surgery, lymphatic, biliary, and cerebrospinal (CSF) fistulae, and air leakages during thoracic and pulmonal surgery. The present invention thus also relates to the use of the described compositions for the above purposes.

It should be emphasized that the composition of the invention is substantially air tight and liquid tight which is the reason for the product being particularly useful to treat lymphatic, biliary, and cerebrospinal (CSF) fistulae, and air leakages during pulmonary and thoracic surgery. Further, due to the product being substantially liquid tight, it is highly useful in surgery of highly bleeding organs such as the liver and spleen, and for surgery e.g. in the gastrointestinal channel.

The product of the invention is to be applied when bleeding, or lymphatic, biliary, air or CSF leakage cannot be controlled with conventional methods or when these methods would yield unfavorable results.

The carrier is preferably a collagen sponge, fleece or patch which terms are used synonymously in the present specification and claims. The components collagen, fibrinogen and thrombin are preferably of mammalian origin. Preferably, the solid components are of human origin. The collagen, fibrinogen and thrombin may either be purified from a natural source or recombinant or transgenic human fibrinogen and/or thrombin.

A presently preferred source of collagen is equine. In order to prevent virus transmission due to contamination with equine viruses that are pathogenic to humans by virtue of the collagen patch, appropriate selection of source material and inactivation of potentially pathogenic agents by the manufacturing process is important as precautionary measures.
 

Claim 1 of 2 Claims

1. A carrier coated with 4.3-6.7 mg fibrinogen/cm.sup.2 and 1.5-2.5 I.U. thrombin/cm.sup.2, wherein said carrier has a strength of adhesion of about 84-110 mbar, said carrier having at least one of the following physical properties: elasticity module in the range of 5-100 N/cm.sup.2, density of 1-10 mg/cm.sup.3, a chamber diameter of more than 0.75 mm and less than 4 mm, and a chamber diameter average below 3 mm; wherein said fibrinogen and said thrombin are solid fibrinogen and solid thrombin evenly distributed and fixed upon said carrier.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.