A phase 2 multicentre randomized controlled comparative efficacy and safety study of Tiscover and AS210 in chronic (arterio-)venous ulcers

Chronic wounds (venous-, arterio-venous and diabetic foot- ulcers) represent a
major problem in our society (Grey et al. 2006; Vileikyte 2001). These ulcers
occur with high incidence and exist for long periods of time despite standard
therapy creating a large financial burden to society (Matricali et al. 2006;
Mekkes et al. 2003) and also a large personal burden to the patients involved
(Phillips et al. 1994; Vileikyte 2001). prevalence numbers vary between 1 and
5%, increasing with age. Characteristics of ulcers include a loss of skin or
underlying tissue and resistance to conventional types of treatment. Wound
healing stagnates resulting in ulcers remaining open for prolonged periods (>12
weeks * 50 years recurring). In western societies approximately 1% of the total
health care budget is spent on wound care. Most leg ulcers are located between
on the lower leg and are caused by venous insufficiency (45*60%), arterial
insufficiency (10*20%), diabetes (15*25%) or combinations of these (10*15%).

Standard therapies for ulcer treatment are described by the
Kwaliteitsinstituut voor de Gezondheidszorg CBO (Guidelines ulcus cruris) and
are co- written by dedicated top medical specialist in the field. The Dutch
guidelines are in accordance with European and American guidelines. In short
the standard wound care consists of wound cleaning, creation of an optimal
(moist) wound environment, and adequate compression therapy.
More advanced wound care therapies which are infrequently available require
hospitalization and/or operation. These advanced treatments include autograft
(punch biopsies, split skin) and negative pressure therapy (VAC®).

Until now the more advanced treatments (as mentioned above) do not results in
high success rates in healing a therapy resistant ulcer. For these reasons new
advanced therapies, such as application of living skin substitutes are being
developed. The advantage of living skin substitutes above acellular dressings
is now widely accepted: they provide an immediate cover but above all they
continuously secrete a cocktail of cytokines, chemokines and growth factors
which promote wound healing. These factors improve wound healing by stimulating
angiogenesis, granulation tissue formation and epithelialisation.

We have developed an autologous full thickness skin substitute for healing
therapy resistant chronic wounds. The autologous skin substitute (Tiscover)
consists of an acellular dermail matrix and both cultured autologous
fibroblasts and keratinocytes forming an epidermal and dermal layer, resembling
histology of normal healthy skin.

The advantage of autologous cell constructs is that they are not rejected. They
serve as an immediate cover for the wound and incorporate in the wound bed, so
continuously secrete growth factors and cytokines to accelerate wound closure.
Additionally the advantage of any cultured skin substitute above a native
autograft is that less donor skin is required.

Since 2004 our first generation cultured skin substitute, Tiscover I, has been
applied to a variety of chronic wounds in the hospitalized and out-patient
(multi-centre) setting.
EU regulation require skin transplants using autologous cell culture to comply
with the DG Sanco *Tissue Directives* which regulates the quality and safety of
advanced tissue products.
As of January 1ste 2009 the situation of non harmonized EU regulation, with
respect to market authorization which allows for free trade among member
states, was dissolved. This resulted in a amendment of the pharmaceutical
directive 2001/83/EU with regards to Advanced Therapy Medicinal Products
regulation.
In order to comply with the new National and European legislation (regarding
GMP, GCP, labelling, packaging) Tiscover I has been adjusted with extensive
change control procedures. The culture ingredients not in line with legislation
have been omitted or replaced by clinical grade or GMP grade products. This has
resulted in a second generation full thickness autologous skin substitute,
known as second generation Tiscover.

Determine the safety and efficacy of Tiscover compared to AS210 (acellular
donor dermis, construct Tiscover is cultured on) for the treatment of chronic,
therapy resistant (arterio-) veneus leg/foot ulcers in an out patient setting.

A prospective, multi centre, open randomized controlled phase II study in which
patients with hard to heal (chronic) therapy resistant (arterio-) venous
leg/foot ulcers are treated in an out patient setting with Tiscover (test
group) or AS210 (control group). Tiscover (autologous cultured skin substitute)
is constructed from small skin biopsies (diameter 3 mm) from unaffected skin as
described previously (Gibbs et al, 2006). The required number of skin biopsies
will be sufficient to culture Tiscover patches to cover 125% of the total wound
surface.
In week -2 keratinocytes and fibroblasts are isolated from the skin biopsies
and seeded into As210. The autologous skin cells and As210 are co-cultured for
approximately 3 weeks, after which Tiscover is ready for application.
The protocol for treatment of ulcers with Tiscover of AS210 consists of two
consecutive applications.
First appliction week 0: minimal approximally 1/4 of the total ulcer surface is
covered with Tiscover or AS210.
Week 1: After 1 week all Tiscover or AS210 is removed. The complete ulcer
surface is covered with new Tiscover or AS210, which will be held in place
until ingrowth.

Tiscover of AS210 are, after both applications, covered and held in place with
wound dressings and bandages. Each patient is seen weekly for wound care,
cleaning, debridement and bandage changes until complete wound closure or until
the end of the study at 26 weeks. After complete closure patients are seen for
follow up at 3 and 6 months to assess possible ulcer recurrence.

The burden for the participating patient is minimal (S. Gibbs et. al., 2006).
The study protocol consists of standard wound care which is supplemented with
application of Tiscover or AS210. Patients receive ambulatory standard wound
care during their weekly out patient wound control visits. The evaluation of
the wounds and dressing changes will take approximately 30 minutes instead of
the usual 12 minutes. Our experience from case studies and the multicenter
trial with Tiscover I, consisting of cultured autologous skin cells seeded into
a human acellular dermis, strongly support the statement that burden to the
patient is minimal. Patients experience minor inconvenience during removal of
biopsies and blood. Biopsies are removed under local anaesthesia.
A possible risk is intolerance or an allergic reaction to Tiscover or AS210.
However, until now over 100 patients have been treated with Tiscover I in pilot
studies, case studies as well as in a multicenter trial, without showing any
related adverse events. Over 10 hospitals and out patient clinics have been
participating without reporting any serious adverse reactions.
Compared to the culture procedure used for first generation Tiscover, for
second generation Tiscover most of the culture medium ingredients have now been
omitted and some have been replaced according to a validated change control
process, which reduces the already minimal risk to adverse events even more.
These validated controlled changes have resulted in a second genration Tiscover
which now complies with EU legislation (in place since January 1st 2009) for
ATMP.
We anticipate that the experimental treatment will activate the inert ulcer
wound bed resulting in decrease in ulser size and wound closure. This will
result in a new ambulant treatment for hard to heal leg ulcers.