The use of a tissue glue comprising a stabilized fibrinogen preparation, which can be stored in the liquid or frozen state and comprises a chaotropic substance, and a thrombin preparation, for reducing or preventing tissue adhesions, is described.

One embodiment of the invention relates to the use of a tissue glue for reducing or preventing postoperative tissue adhesions, which is distinguished from known tissue glues for its improved antiadhesive properties.

It is known that in the development of tissue glues to date, priority has been given to the hemostatic or sealing activity (e.g., against loss of CSF) of the glue. These indications still account for a very considerable number of the uses of tissue glues nowadays.

Tissue glues are commercially available either as lyophilizates or as frozen preparations. However, after reconstitution or after defrosting, the products are stable in solution for only a few days, because with the highly concentrated fibrinogen-/Factor XIII-solutions, an aggregation and therefore an (for example, proteolytic) inactivation occurs, which makes any further use impossible.

Other tissue glues that have been described in the literature until now are not yet commercially available and are generally comprised of frozen or freeze-dried components that must be defrosted or dissolved prior to use. European patent 0,085,923, German patent application 196 17 369 and European patent application 0,856,317 describe the use of chaotropic agents or additives such as arginine or urea or their derivatives or derivatives of benzene, imidazole, pyrozol, furan, thiazole and purine, which generally improve the solubility of proteins. Chaotropic agents in this context are agents that reduce or destabilize the reciprocal effect between proteins or parts thereof and therefore reduce their tendency towards aggregation. However, with liquid storage, but especially also with storage in frozen state, the loss of Factor XIII-activity in the formulations described thus far was so high that in the presence of effective quantities of chaotropic agents, the Factor XIII-content often clearly drops after only a few weeks or months, often even below the detection limit.

For example, we have shown that in the formulations in accordance with European patent application 0,856,317, tranexaminic acid (AMCA), especially in the presence of chaotropic agents such as arginine and inorganic salts, clearly reduced the Factor XIII content in the course of storage at -20.degree. (see instant Table 1b, Batch 1). Storage at 4.degree. Celsius leads to an increase in viscosity in this formulation (Table 1 a, Batch 1), which also rules out a long-term storage. Thus, these formulations must be considered non-stable when considering the simultaneous stability of fibrinogen and Factor XIII. Formulations in accordance with DE 196 17 369 also indicate problems in maintaining Factor XIII-activity (see instant Table 1, Batch 2 and 2).

Another biological adhesive for human or animal tissue is known from the European patent specification 0,487,713. Said adhesive is stabilized in liquid form at low temperatures. This is supposed to be achieved because the preparation containing fibrinogen comprises at least one chaotropic agent in a concentration between approximately 0.3 M and 1 M and because the adhesive is liquid at the storage temperature.

Such fibrinogen concentrate typically comprises about 4 mmol tri-sodium citrate, 240 mmol NaCl, 80 mmol-amino caproic acid (EACA), 240 mmol glycine, 1% polysorbate, 0.6 grams/liter sodium caprolate, 0.5 mol urea, 2,000 KIE/ml aprotinin, if necessary, and a pH of 7.5. The stability was evaluated after only one month, which is very short for a therapeutic preparation. The Factor XIII-activity was not analyzed. J. Chabbat et al. reported about a fibrinogen concentrate that remains stable in liquid state at 4.degree. Celsius over a period of six months (J. Chabbat, M. Tellier, P. Porte and M. Steinbuch: Properties of a new fibrin glue stable in liquid state. Thromb. Res. 76: 525-533 (1994)). In addition to other formulation components, typically 60 mmol/liter NaCl, 20 mmol/liter EACA and 60 mmol/liter glycine, this concentration comprised 0.5 mol urea or 5% arginine (=0.29 mol). However, the Factor XIII-strength of this concentration was also not tested.

These liquid formulations, which were described in the European patent specification 0,487,713 and in the literature, are characterized in that the aggregation (polymerization) and thus the increase in viscosity of the concentrated fibrinogen component, is prevented or reduced at refrigeration temperatures. However, Factor XIII, an essential component of fibrinogen concentrates for fibrin glues, is inactivated to a greater or lesser degree under these conditions. In the formulations provided for storage in cooled state in accordance with European patent specification 0,487,713 or the related publication by Chabbat et al. (J. Chabbat, M. Tellier, P. Porte and M. Steinbuch: Properties of a new fibrin glue stable in liquid state. Thromb. Res. 76:525-533 (1994)), the instability of Factor XIII is therefore a significant problem that is not solved by the proposed formulations (see instant Table 1, Batches 4-5). Furthermore, the strength of chaotropic agents is relatively high at 0.3 to 1.0 mol/liter.

Thus, it can be noted that it was found in the analysis of the stability of fibrinogen/Factor XIII preparations as well as the viscosity of various known fibrinogen/Factor XIII-preparations in refrigerated state (0 to 10.degree. Celsius) or frozen state with subsequent storage in refrigerated state (0 to 10.degree. Celsius) that the previously described formulations do not lead to stable protein preparations. Either the fibrinogen or Factor XIII show a significant reduction in activity during the storage time, or the aggregation of fibrinogen leads to a viscous material that can no longer be applied (see instant Table 1, Batches 1 to 5).

Thus, one objective of the invention was to develop protein preparations that are liquid and stable over several months, or frozen and stable over several months following defrosting, in which the fibrinogen and/or Factor XIII are stabilized over months or years without any significant loss of effect.

The problem is solved with stabilized protein preparations that in comparison to the state of the art have the advantage that, in a first embodiment, not only fibrinogen but also Factor XIII are stabilized by the additives and that the content of chaotropic reactants can be reduced, or that, in a second embodiment, fibrinogen and Factor XIII are formulated separately and thus remain stable.

In one embodiment of the invention, this is achieved in that for frozen preparations and preparations that must be kept stable for several weeks or months following defrosting, a chaotropic agent corresponding to the definition provided here is used in a lower concentration to avoid the aggregation of fibrinogen, and that the concentration of inorganic salts is reduced and that, if necessary, an antifibrinolytic as well as other common additives and buffer substances are used. A fibrinogen preparation used for this purpose can also contain Factor XIII from the starter material as well as other plasma proteins, such as fibronectin and von Willebrand-Factor (vWF), or it can contain purified Factor XIII as an additive.

Aprotinin, lysine, .epsilon.-amino caproic acid (EACA), or p-aminomethylbenzoic acid (PAMBA) or their physiologically safe salts can be used as an antifibrinolytic. Studies on the influence of various antifibrinolytics have surprisingly shown that lysine, PAMBA or EACA do not have a negative effect on the activity of Factor XIII, while tranexaminic acid (AMCA) does. Therefore, with frozen fibrinogen/Factor XIII mixtures and also with fibrinogen/Factor XIII mixtures stored in liquid state, EACA or lysine can be used in place of AMCA. Other stabilizers can be used for Factor XIII, such as sodium citrate, amino acids and sugar.

Instead of the aforementioned protein preparations, which comprise Factor XIII as well as fibrinogen and their respective stabilizers, it is also possible to store both concentrations separately and only mix them with the thrombin-containing preparation immediately prior to using them as tissue glue. Therefore, another embodiment of the invention relates to a tissue glue that is comprised of a solution that contains the stabilized Factor XIII, a solution that contains the stabilized fibrinogen, and a solution that contains stabilized thrombin, which are provided separately in one packaging unit prepared to be used together. Another advantage of this is that the ratio of Factor XIII and fibrinogen can be changed and adapted to the specific situation as needed.

Preclinical or clinical uses of tissue glues for avoiding adhesion after surgical intervention have also been described in the past with varying success. Thus, H. Moro et al. reported inhibition of pericardial adhesions in a dog model (H. Moro, J. Hayashi, H. Ohzeki, T. Nakayama, O. Namura, K. Hanzawa and N. Yagi. Jap J Thor Cardiovasc Surg 47: 79-84,1999). H. Takeuchi et al. and P.A. De Iaco et al. also describe the successful use of tissue glues for avoiding or reducing adhesions on the horn of the rabbit uterus (H. Takeuchi, Y. Toyonari, N. Mitsuhashi and Y. Kuwabara. J Obstet Gynaecol 23: 479-484,1997; P.A. De Iaco, A. Costa, G. Mazzoleni, G. Pasquinelli, L.

Bassein and A. Marabini. Fertil Steril 62: 400-404, 1994). The reduction in peritoneal adhesions has likewise been described by S. Lindenberg et al. on use of tissue glues in a rat model (S. Lindenberg and J. G. Lauritsen. Annales Chirurgiae and Gynecologiae 73: 11-13, 1984). However, there have also been other authors who observe no reduction in adhesions on use of fibrin glues by comparison with an untreated control.

These were, inter alia, J. F. H. Gauwerki, J. Mann and G. Bastert, Arch Gynt.about.kol Obstet 247: 161 (1990) and V. A. C. Evrard, A. De Bellis, W. Boeckx and I. A. Brosens, Hum Reprodt 11: 1877-1880 (1996). The reports, which are partly contradictory, can probably be attributed to the fact that the antiadhesive effect which can be achieved with the products available is insufficiently large or leads consistently to ambiguous results.

Very recently, the possibility of using fibrin layers for avoiding adhesions has also been mentioned in the patent literature. International patent application WO 96/22115 describes a sheet-like material consisting of crosslinked fibrin employed for preventing adhesions but not itself having hemostatic properties. In another embodiment, this material is produced in situ and used as a second tissue glue layer without hemostatic properties on top of a first tissue glue with hemostatic activity. However, these methods are either impractical, because the fixing of such a fibrin film is difficult, or laborious, because two tissue glues must be employed in order to achieve both hemostatic activity and antiadhesive properties.

In addition, a preparation of fibrin or fibrinogen and a biocompatible or biodegradable polymer, which forms a viscous solution and has antiadhesive properties, is disclosed in international patent application WO 92/22312.

Therefore, another object of the present invention was to develop a tissue glue which, while having good hemostatic properties, shows improved results in reducing or preventing tissue adhesions and, moreover, does so without addition of polymers, which form viscous solutions and have antiadhesive properties.

Because of their great medical importance, considerable research has been directed in recent years to the further development and improvement of known tissue glues. This has also involved particular attention being paid to the improvement of the storability of tissue glues. Thus, German patent applications DE-A-198 53 033, DE-A-198 61 158 and DE-A 100 12 732 describe tissue glues and components thereof which are distinguished, inter alia, by particularly long storability in the liquid and/or frozen state. German patent application DE-A 100 12 732 is hereby incorporated by reference. Detailed investigation of these novel tissue glues has now shown that they also have other advantageous properties which open up additional and valuable possible uses thereof.

For example, it has emerged that these novel tissue glues have considerably improved antiadhesive properties without involving the need to accept losses of their hemostatic properties. The antiadhesive properties of the novel tissue glues are evident both on untreated wounds and on wounds treated with conventional tissue glues. In this context, the skilled artisan would understand that a conventional tissue glue is a hemostatic tissue glue that either does not have antiadhesive properties or if it does, they are inadequate to perform antiadhesion. It is particularly surprising in this connection that distinctly improved effects, by comparison with conventional tissue glues, in reducing or preventing tissue adhesions is also achieved when the aforementioned novel tissue glues are employed. These effects have been observed both in a typical animal model for investigating the reduction in adhesions, such as a lengthwise incision wound on the horn of the rabbit uterus, and on hemostatic use in a partial resection of the rabbit liver.

In one embodiment, the invention therefore relates to the use of a tissue glue comprising: a stabilized fibrinogen preparation, which can be stored in the liquid and/or frozen state and to which a chaotropic substance is added, and a thrombin preparation for reducing or preventing postoperative tissue adhesions.

It is also possible to add to the tissue glue a preparation containing coagulation Factor XIII if the latter is not present in sufficient quantity, so that it is used as 3-component glue. This is because fibrin crosslinking, which should be as complete as possible, can enhance the antiadhesive effect of a fibrin glue by the fibrin matrix being, for example, less amenable to fibrinolytic degradation. However, it is also possible to admix coagulation Factor XIII to the fibrinogen preparation from the outset, so that a 2-component glue is employed. In the case of a 3-component glue, the mixing ratio of the components fibrinogen, Factor XIII and thrombin can be chosen in a suitable way in order to achieve good mechanical properties of the glue. Examples of suitable mixing ratios include about 1:1:1 to about 10:1:1 or 10:1:2 or, in general, x:y:z where x.gtoreq.z.gtoreq.y.

As mentioned previously, the tissue glue used according to the invention comprises a chaotropic substance in the fibrinogen preparation. Examples of chaotropic substances that have proved suitable include arginine, guanidine, citrulline, urea and their derivatives or mixtures thereof. Chaotropic substances are generally added to the fibrinogen preparation in amounts of from about 0.1 to about 1.0 mol/l, for example in amounts of less than 0.5 mol/l.

In another embodiment of the invention, the properties of the aforementioned novel tissue glues are further advantageously influenced by addition of an antifibrinolytic agent. Examples of antifibrinolytic agents include aprotinin, c-aminocaproic acid (EACA), p-aminomethylbenzoic acid (PAMBA) or one of their physiologically tolerated salts or derivatives.

The fibrinogen preparation may additionally comprise as stabilizers: an inorganic salt or one or more physiologically tolerated salts of organic carboxylic acids, for example, citric acid or of lactic acid, or one or more amino acids or a mono- or disaccharide or a sugar alcohol or one of their mixtures.

In another embodiment of the invention, a beneficial effect on the antiadhesive properties of the claimed, improved fibrin glues can further be achieved by suitable purification methods, for example by reducing the plasminogen content of the fibrinogen component. Examples of possible methods of this type are immunoaffinity chromatography via coupled antibodies or affinity chromatography on amino group-containing supports. This invention therefore also encompasses, inter alia, fibrin glues with fibrinogen components whose plasminogen contents have been significantly reduced. For example, fibrinogen components may have a plasminogen to fibrinogen ratio of less than 1.8.times.10.sup.-4 (w/w), including less than 10.sup.-4 (w/w).

The Factor XIII preparation added to the tissue glue to be employed according to the invention may likewise be stabilized if it is not added to the previously stabilized fibrinogen. Therefore, in one embodiment of the invention, it may be advantageous to add to the Factor XIII preparation a stabilizer, for example, a physiologically tolerated salt of an organic di-, tri- or tetracarboxylic acid, such as citric acid, and, where appropriate, other stabilizers and/or buffer substances for Factor XIII.

Other stabilizers suitable in this connection include: a mono- or disaccharide or a sugar alcohol and/or one or more amino acids chosen from the group of glycine, glycylglycine, alanine, cysteine, histidine, glutamine or a physiologically tolerated salt of glutamic or aspartic acid and/or a reducing or oxidation-preventing agent and/or a surface-active substance.

Stabilizers are normally added in an amount of up to about 5% by weight of the Factor XIII preparation. Tissue glues of this type are described in German patent applications DE-A-198 53 033 and DE-A-198 61 158.

In another embodiment of the invention, the thrombin preparation present in the tissue glue employed according to the invention may contain a non-covalently binding inhibitor as stabilizer. Suitable substances for this purpose include, for example, compounds such as benzamidine or p-aminobenzamidine and other low to moderate affinity protease inhibitors. The addition of these low or moderate affinity inhibitors negligibly impairs the activity of thrombin in relation to substances such as fibrinogen. It is additionally possible, for stabilization purposes, to add to the thrombin preparation, besides a soluble calcium salt, sodium chloride, a sugar or a sugar alcohol and/or an amino acid or else the salt of a mono- or polycarboxylic acid and/or the salt of a mono- or polyhydroxy carboxylic acid or mixtures of said stabilizers.

The thrombin used for this purpose is prepared from the prothrombin obtained from plasma or from a plasma fraction. After an activation thereof to thrombin without addition of thromboplastin and, where appropriate, further processing steps, thrombin can be purified by a hydrophobic interaction chromatography and/or a cation exchange chromatography. This method is described in detail in German patent application DE-A-100 12 732.

In another embodiment of the invention, it may be advantageous in this connection for the tissue glue or its constituents also to be subjected to one or more methods for inactivating viruses.

It is possible to use as starting material for producing the individual components of the fibrin glue of the invention, apart from plasma fractions, recombinant proteins prepared by isolation from cell cultures or cell culture supernatants.

Claim 1 of 30 Claims

1. A method for reducing or preventing tissue adhesions comprising the application to a tissue of a tissue glue, wherein the tissue glue comprises: a fibrinogen preparation, and a thrombin preparation, wherein the fibrinogen preparation can be stored in the liquid or frozen state, wherein the fibrinogen preparation further comprises a chaotropic substance in the range of more than about 0.04 M to less than about 0.28 M, wherein the total concentration of water-soluble inorganic salts in the fibrinogen preparation is equal to or less than about 100 mmol/liter; and wherein the tissue glue reduces or prevents tissue adhesions.

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