AU2004201135A1 - Use of a fibrin adhesive for tissue regeneration - Google Patents

Description

P001 Section 29 Regulation 3.2(2)

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: Use of a fibrin adhesive for tissue regeneration The following statement is a full description of this invention, including the best method of performing it known to me us: 1 USE OF FIBRIN ADHESIVE FOR TISSUE REGENERATION The invention relates to a novel use of a fibrin adhesive for tissue regeneration.

It is known that in modern surgery fibrin adhesives are gaining increasingly in importance, since what is involved here is a highly tolerable and wound healing-promoting biomaterial. To date, fibrin adhesion is being employed for the hemostasis of heavily bleeding wounds in operations on parenchymatous, internal organs, in skin transplants, in emergency surgery on internal and external injuries, but especially also for the supportive sealing of sutures to avoid postoperative hemorrhages. In ENT and facial surgery, the fibrin adhesive is preferred for cosmetic reasons in the suture closure for the healing of external wounds. Moreover, the fibrin adhesive is increasingly employed in endoscopic surgery, for example for the hemostasis of gastric ulcers. The commercially available fibrin adhesives such as Beriplast® contain the clotting factors fibrinogen, thrombin and factor XIII which are obtained from human plasma as main constituents.

It is moreover known that the regeneration of liver tissue destroyed by toxic effects is particularly dependent on the hepatocyte growth factor (HGF). The production of HGFs can be increased by a system of different, activated proteins.

At the beginning of this activating cascade, thrombin appears to play a not insignificant role. Despite this, to date it has not been attempted to employ the thrombin contained in the fibrin adhesive for the treatment of liver damage. As the mortality from acute liver failure is up to 80%, there is considerable need for prophylactic and therapeutic measures with which acute liver failure in particular can be prevented.

In acute liver failure, two factors primarily determine the survival of the patient: the extent of the liver damage and the rate of hepatocyte regeneration(1). Hepatocyte regeneration can be markedly increased for example, after bile duct ligature(2). In this case, bile duct cells show a 17-fold increase in proliferation after two days, accompanied by a 4-fold increase in hepatocyte regeneration.

The invention now relates to the use of a fibrin adhesive for regeneration of the liver tissue, preferably in which a fibrin adhesive occlusion is formed by injection of a fibrin adhesive into the hepatobiliary system. This invention is based on the observation that a fibrin adhesive occlusion is reversible within 24 hours and leads to a marked proliferation of the bile duct cells. The increase associated therewith in the expression of the hepatocyte growth factor HGF and its receptor c-met leads to a rapid regeneration of the liver tissue. It was possible to show the success of the use according to the invention of a fibrin adhesive for the regeneration of the liver tissue by means of a hepatobiliary fibrin adhesive occlusion in thio-acetamide-induced acute liver failure. Moreover, additive factors mixed with the fibrin adhesive can thus become effective locally in the liver in high dose by means of a hepatobiliary occlusion and/or by direct injection.

The invention also relates to a method of regeneration of liver tissue including administering to a patient requiring such treatment a pharmaceutically effective amount of a fibrin adhesive.

It has also been shown that the regeneration of other tissues is also significantly improved by the intraparenchymatous administration of fibrin adhesive. Thus in a rat diabetes mode (streptozotocin), the glucose tolerance was significantly improved by a intraductal/intraparenchymatous administration of fibrin adhesive. This shows that insulin-producing cells can be activated by the action of the fibrin adhesive from precursors. Thus a completely novel approach for the treatment of diabetes mellitus presents itself.

In addition, it was possible in the case of acute lung failure of the rat to show that the oxygenation of the body is significantly improved by intrabronchial and intraparenchymatous administration of fibrin adhesive.

It was furthermore observed that the regeneration ability of the skin after raising skin lesions is considerably improved by the subcutaneous administration of fibrin adhesives. In addition, the elasticity of the skin was markedly increased at the same time. A novel field of employment can thus be created for the fibrin adhesive in plastic surgery and regeneration of the skin.

Additive factors mixed with the fibrin adhesive can then become active locally in high concentration in the pancreas, in the lung or the skin.

3 To demonstrate the efficacy of a fibrin adhesive for the regeneration of the liver tissue, the following experiments and measurements were carried out.

1st experiment The model of acute liver failure was induced in Sprague Dawley rates by means of thioacetamide(3). Two groups of six rats each were treated in the following manner here. One three successive days, the rats of group A received thioacetamide in doses of 500 mg/kg, 500 mg/kg and 250 mg/kg intraperitoneally at the same time of the day. After laparotomy under pentobarbital anesthesia, a slowly hardening fibrin adhesive (Beriplast®) was injected into the hepatobiliary system of animals of group B 24 hours before the start of thioacetamide administration.

The following results were observed: the histological findings on the hepatocytes of the rats of group A showed a swelling of the nucleus with highly prominent nucleoli, and an extensive loss of parenchyma from the center of the lobe with more or less compact round-celled infiltration. In group B, the portal areas showed a slight edema and discrete round-cell infiltration, while the hepatocytes were distinguished by a noticeable lightening with isolated centrilobular individual cell necrosis.

The mortality of group A was 100% after five days, while all animals of group B survived. The conclusion is to be drawn from this that a hepatobiliary fibrin adhesive occlusion can prophylactically prevent the lethal thioacetamideinduced liver failure in the rat.

2 nd experiment SD rats each day received an injection of 100 mg/kg of thioacetamide intraperitoneally. On the seventh day, 20 animals were laparotomized under anesthesia, the bile duct was microsurgically dissected, cannulated and 0.2ml of Beriplast@ fibrin adhesive was subsequently injected. 20 animals were only laparotomized (control group). The continuation of the thioacetamide treatment was then carried out up to the 14 th day. On the 14 th day, laparotomy with removal of blood from the aorta was carried out and, after exsanguination of the animals had been carried out, organ removal. The determinations of glutamate oxalacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), gamma-glutamyl transferase (yGT), alkaline phosphatase, bilirubin, urea and creatinine were carried out using routine methods. For the immunohistochemical workup of the livers fixed in liquid nitrogen, these were cut into 5mrn thin preparations using a freezing microtome and two preparations in each case were mounted on Superfrost plus slides. Seven slides from each animal were prepared for staining and a further seven slides as a negative or isotype control.

Dilution stages: HGF-alpha: 1:200; HGF-beta: 1:100, c-met: 1:200.

The starting concentration in each case was 200pg/ml. The evaluation of immunohistochemical stains were carried out without knowledge of the group association in a semiquantitative manner using five graduations.

Results: Table 1 (data as mean values 1 SD) *p<0.005 GOT GPT yGT AP Bilirubin Control 40 6 19 9 3 0.9. 237 42 0.7 0.1 Occlusion 35 8 17 5 3 0.9 335 149 0.4 0.1* Table 2 (data as mean values 1 SD) *p<0.05 c-met HGF-alpha HGF-beta Control 3.33 0.62 2.67 1.18 0.33 0.47 Occlusion 3.73 0.44 3.73 0.44* 2.27 1.06* These experiments show that the hepatobiliary fibrin adhesive occlusion increases the expression of HGF in the thioacetamide-damaged liver. Application possibilities of the fibrin adhesive for the regeneration of liver tissue result from this.

References: 1. Lake J. and Sussman N. (1995) Editorial: Determining Prognosis in Patients with Fulminant Hepatic Failure: When you Absolutely, Positively Have to Know the Answer; Hepatology 21, 879-881 2. Polimeno Azzarone Zeng Panella Subbotin Carr B., Bouzahzah Francavilla Starzl T.E (1995) Cell Proliferation and Oncogene Expression after Bile Duct Ligation in the Rat: Evidence of a Specific Growth Effect on Bile Duct Cells; Hepatology 21, 1070-1078 3. Zimmermann Ch., Ferenci Pifl Ch., Yurdaydin Ebner Lassmann Roth H6rtnagl H. (1989), Hepatic Encephalopathy in Thioacetamide- Induced Acute Liver Failure in Rats: Characterization of an Improved Model and Study of Amino Acidergic Neurotransmission; Hepatology 9, 595-601.

Claims (9)

1. The use of a fibrin adhesive for the regeneration of the liver tissue.

2. The use as claimed in claim 1, wherein a fibrin adhesive occlusion is formed by injection of a fibrin adhesive into the hepatobiliary system.

3. The use as claimed in claims 1 and 2 wherein the expression of the hepatocyte growth factor (HGF) and of its receptor c-met is increased by the formation of a fibrin adhesive occlusion in the hepatobiliary system.

4. The use as claimed in any one of claims 1 to 3 wherein additive factors are additionally mixed with the fibrin adhesive and thus, as a result of a heptobiliary occlusion, can be effective locally in the liver in a high dose.

A method of regeneration of liver tissue including administering to a patent requiring such treatment a pharmaceutically effective amount of a fibrin adhesive.

6. The method of regeneration of liver tissue as claimed in claim 5 wherein a fibrin adhesive occlusion is formed by injection of a fibrin adhesive into the hepatobiliary system.

7. The method as claimed in claim 5 or 6 wherein the expression of the of the hepatocyte growth factor (HGF) and of its receptor c-met is increased by the formation of a fibrin adhesive occlusion in the hepatobiliary system.

8. The method as claimed in any one of claims 5 to 7 wherein additive factors are additionally mixed with the fibrin adhesive and thus, as a result of a heptobiliary occlusion, can be effective locally in the liver in a high dose.

9. The use of a fibrin adhesive for the regeneration of liver tissue substantially in accordance with experiments 1 and 2 as described. DATED this 18th day of March 2004 AVENTIS BEHRING GMBH WATERMARK PATENT TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA KJS/DMF