USE OF FIBRIN ADHESIVE FOR TISSUE REGENERATION

UTILISATION D'UN ADHESIF A BASE DE FIBRINE POUR LA REGENERATION DE TISSUS

English Abstract

The use of a fibrin adhesive for the regeneration is described. In the case
of the treatment of liver damage, a fibrin adhesive occlusion is formed here
by injection of a fibrin adhesive into the hepatobiliary system or by direct
injection into the liver tissue. As a result of the fibrin adhesive occlusion
in
the hepatobiliary system, the expression of the hepatocyte growth factor
(HGF) is increased and thus the regeneration of the liver tissue is
promoted. The use of the fibrin adhevise by injection into lung, pancreas or
skin tissue for the treatment of lung failure and of diabetes mellitus and
also in plastic surgery is additionally described.

Claims

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claims:
1. The use of a fibrin adhesive for the regeneration of tissue.
2. The use as claimed in claim 1, wherein the fibrin adhesive causes
activation of insulin-producing cells as a result of injection into the
pancreas.
3. The use as claimed in claim 1, wherein the fibrin adhesive improves
the oxygenation of the body as a result of injection into the
intrabronchial tissue.
4. The use as claimed in claim 1, wherein the regeneration ability of
the skin is improved by subcutaneous injection of the fibrin
adhesive.
5. The use of a fibrin adhesive for the regeneration of the liver tissue.
6. The use as claimed in claim 5, wherein a fibrin adhesive occlusion
is formed by injection of a fibrin adhesive into the hepatobiliary
system.
7. The use as claimed in claims 5 and 6, 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.
8. The use as claimed in claims 5 to 7, wherein additive factors are
additionally mixed with the fibrin adhesive and thus, as va result of a
heptobiliary occlusion, can be effective locally in the liver in a high
dose.

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9. The use as claimed in claims 1 to 4, wherein additive factors mixed
with the fibrin adhesive can be locally active in a high dose in the
skin or in the lung.

Description

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CENTEON PHARMA GMBH 199812023-Ma 1196-C 31
Use of a 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 XII I 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.

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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, in which a fibrin adhesive occlusion is formed by injection
of
a fibrin adhesive into the hepatobiliary system and/or fibrin adhesive 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.
Moreover, it has now been shown that the regeneration of other issues 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 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 prsents
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.

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

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1st experiment
The model of acute liver failure was induced in Sprague Dawley rats by
means of thioacetamide (3). Two groups of six rats each were treated in
the following manner here. On 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 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 thioacetamide-induced liver failure in the rat.
2nd experiment
40 SD rats each day received an injection of 100 mglkg of thioacetamide
intraperitoneally. On the seventh day, 20 animals were laparotomized
under ether anesthesia, the bile duct was microsurgically dissected,
canulated and 0.2 ml 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

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14th day. On the 14th 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 5 ~m 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 200 ~g/ml. The evaluation of
immunohistochemical stains was 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 406 199 30.9 23742 0.70.1
Occlusion 35 17 5 3 0.9 335 149 0.4 0.1
8

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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.

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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 L., Azzarone A., Zeng Q.H., Panella C., Subbotin V., Carr
B., Bouzahzah B., Francavilla A., 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 P., Pifl Ch., Yurdaydin C., Ebner J.,
Lassmann H., Roth E., Hortnagl 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, 594-601