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SELECTED ABSTRACTS FROM THE ETRS
FOCUS MEETING
Thursday 12 - Saturday 14 September 2002
Nice, France
Fibrin I, A New Single-Component
Fibrin Sealant
Ian Cottingham, PhD, PPL Therapeutics Ltd., Roslin Biocentre, Edinburgh
EH25 9PP, Scotland UK
E-mail: cottingham@ppl-therapeutics.com
Fibrin I is a new single-component fibrin sealant which is simple to prepare,
easy to use, safe and effective.
Fibrin I is made from virally-inactivated purified human fibrinogen processed
with a specific protease, Batrox-obin, which is subsequently removed.
Treatment with this well-defined enzyme, already used extensively for
human treatment of thrombotic disease, generates a strong stable fibrin
clot. However, this clot differs from thrombin clots in that it completely
redissolves in weak acid and can be freeze-dried to form a stable powder.
Different in vitro clot properties result because, unlike thrombin,
Batroxobin does not activate the cross-linking enzyme factor XIII. However,
in vivo, the Fibrin I clot is rapidly cross-linked by local factor
XIII activity and is thus functionally indistinguishable from a natural
clot.
For use as a surgical haemostat Fibrin I is reconstituted in weak acid
- a process taking less that one minute - and co-applied with a neutralising
buffer. On mixing Fibrin I instantly forms a gel at the wound site - without
the addition of thrombin - and does not tend to run off like traditional
sealants. Because of its rapid thrombin-independent polymerisation properties
Fibrin I is also effective at a lower protein level and works at 25% of
the concentration typically found in other fibrin sealants.
The simplicity of Fibrin I makes it safe and effective. The absence of
high thrombin doses found in other fibrin sealants means that there is
a minimal risk from malad-ministration and no risk of transmural thrombogenicity
in microvascular surgery or coagulopathy due to self-antibodies to bovine
contaminants. Furthermore the addition of exogenous protease inhibitors
such as aprotinin from bovine lung or tranexamic acid - which may not
be suitable for use during neurosurgery - is not necessary.
Fibrin I is a highly versatile material that promotes rapid and natural
wound healing. It can be co-applied with the neutralising buffer through
dual syringes or via air-driven spray devices including a high precision
microspray pen applicator suitable for intermittent use. In addition to
the easily reconstituted lyophillized powder PPL have also developed a
liquid formulation which is stable at room temperature. Fibrin I is even
effective as a dry powder applied directly to the wound site - potentially
as a component of a simple bandage. It is also possible to make a slow-setting
version of Fibrin I for use in surgical procedures which require alignment
of tissues before sealing.
In a pig liver model for evaluating haemostasis evaluation Fibrin I has
been shown to be a highly effective haemostat. It promotes rapid natural
wound healing with a minimal inflammatory response - even using human
Fibrin I in the pig. It is also effective in the prevention of post-surgical
adhesions as demonstrated in the rabbit uterine horn model. Fibrin I can
also be used as a vehicle for the delivery and maintenance of viable fibroblasts,
or other cells, to a wound site, thereby providing the opportunity for
advanced wound care applications.
Fibrin I is in pre-clinical development and PPL Therapeutics are seeking
a partner for world-wide commercialisation.
Design of a novel proteolysis
resistant VEGF165 variant
Gereon Lauer, Stephan Sollberg, Melanie Cole, Thomas Krieg, Sabine
A. Eming, Department of Dermatology, University of Köln, Köln,
Germany
A disturbed balance of proteolytic and anti-proteolytic activity characterizes
the hostile environment of a chronic wound and is considered a critical
mechanism in the pathology of impaired wound healing. Recently we demonstrated
that the proteolysis of VEGF165, one of the most potent angiogenic
mediators, is increased in chronic non-healing wounds versus normal-healing
wounds (J Invest Derm, 115: 12-8, 2000). VEGF165 degradation
resulted in significant loss of its angiogenic properties, indicating
that VEGF165 biological activity and VEGF165 -mediated
angiogenesis is restrained in non-healing wounds. SDS-PAGE analysis, protease-inhibitor
studies and protein sequencing of rVEGF165 degradation products
suggested that serine-proteases, specially plasmin, are responsible for
rVEGF165 degradation in the chronic wound environment. These
data prompted us to investigate whether VEGF165 can be proteolytic
stabilized by mutating the plasmin cleavage site. By a site directed mutagenesis
approach we generated a novel plasmin resistant VEGF165 variant,
which is stabilized when incubated in wound fluid obtained from non-healing
wounds. This type of modification would be expected to increase the period
that topically applied VEGF protein is active in the wound environment,
implicating a potential clinical application.
Clinical and objective measurement
of skin hardness in hypertrophic scars during silicone gel sheeting treatment
M. Romanelli, R.M. Semeraro, A. Magliaro, D. Mastronicola, S. Siani,
Department of Dermatology, University of Pisa, Italy
Introduction
Hypertrophic scars and keloids are major medical problems. Topical silicone
gel sheeting is widely used in the management of scarring although its
exact mode of action is still largely unclear.
Objective
We conducted a prospective analysis of the safety and efficacy of silicone
gel sheets in patients attending our tissue repair unit for immature hypertrophic
scars related to trauma or surgical intervention. Our purpose was to measure
skin hardness in treated sites and to compare clinical assessment with
objective measurement.
Methods
We mapped the clinical extension of twenty-eight scars with a validated
skin scoring system for hardness and measured objectively the same sites
and control areas with a durometer, before, during and after six months
of treatment with silicone gel sheets.
Results
We observed a significant reduction in skin hardness of eighteen scars
(72%) at the end of the study period. These results correlated well with
the skin scoring system but with a more significant accuracy (p < 0.002)
for the objective method. The silicon gel sheet was well tolerated and
accepted by the patients without evidence of side effects.
Conclusions
Our data support the use of silicone gel sheeting in hypertrophic scars
and provide a useful and simple techniques to monitor skin hardness during
treatment.
Mechanistic insights and possible
clinical applicability of non-viral liposomal gene therapy
Marc Jeschke MD PhD, University of Regensburg, Department of Surgery,
Franz-Joseph-Strauss-Allee 11, D-93053 Regensburg, Germany.
E-mail: Mcjeschke@hotmail.com
Non-viral gene therapy has several advantages over viral gene transfer
methods, one of which being that the liposomal constructs can be administered
repeatedly without causing an immune response or tachyphylaxia. Hence,
non-viral liposomal cDNA gene transfer represents an unique therapeutic
approach for several pathophysiolgic states. Despite broad therapeutic
possibilities relatively few studies examined mechanisms after non-viral
liposomal gene transfer. There are still some questions regarding cellular,
molecular and physiologic mechanisms unanswered. To answer these questions
we constructed a vector with a cytomegalovirus driven promoter containing
the sequence for insulin-like growth factor-I (IGF-I) or keratinocyte
growth factor (KGF) and defined in multiple studies cellular and physiologic
responses in-vivo. As a model we used an acute dermal and epidermal
wound. Immunological assays, northern and western blotting, histological
and immunohistochemical techniques were used to determine molecular mechanisms
and physiologic responses after gene transfer. We could show that after
liposomal gene transfer transfection was found in rapidly diving cells
in the granulation tissue. Furthermore, cDNA gene transfer increased mRNA
and protein expression of the administered growth factor. Using biological
assays we found that the cell recognizes the exogenous gene leading to
similar responses as the endogenous gene. Physiologic efficacy was determined
by measuring dermal and epidermal regeneration, including wound re-epithelialization,
collagen deposition and morphology, proliferation and apoptosis and different
growth factor concentrations. In summary, we found that exogenous cDNA
gene transfer exerts same intracellular responses as the endogenous gene.
This finding has great therapeutic potential, because the exogenous form
of non-viral liposomal cDNA leads to similar responses as the endogenous
physiologic protein, being more effective and showing no adverse side-effects.
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