In vitro-/ ex vivo-Hautmodelle zur Erfassung der Reaktion auf externe Reize sowie der Wirkung einer antiinflammatorischen Behandlung

Dermatoses are wide-spread. Beside physical discomforts like pain and itches, often
psychosocial problems are caused and the quality of life of patients is strongly affected. In
addition, the treatment of dermatoses is still unsatisfying in many cases, as only symptoms
are considered and results are moderate. Researches in pathophysiology and therapeutics
are therefore of great need.
An important tool thereby are in vitro / ex vivo skin models. They allow to carry out
experiments under definite conditions and regardless of human subjects.
Penetrationexperiments with active ingredients and potentially toxic chemicals by means of
in vitro / ex vivo skin models are common nowadays. Also for measuring the irritant and
phototoxic effect as well as the allergic potential of substances and preparations such
models are established in the mean time. But by now, only few investigations make use of
both applications in parallel.
In vitro / ex vivo skin models potentially allow an integrated examination, whereas not only
galenical aspects but also attributes of the active substance of a pharmaceutical are
considered. The elaboration of a corresponding test procedure as a screening instrument for
research and development of dermatics will be of great value.
With regard to these purposes, the present study investigates the possibilities and limits of
three currently available in vitro / ex vivo skin models: SkinEthic®, Phenion FT® and
Natskin®. SkinEthic® and Phenion® FT are both in vitro reconstructed models of human skin
cells. Whereas SkinEthic® is an epidermis model, consisting exclusively of keratinocytes,
Phenion® FT contains epidermis and dermis (fibroblasts in collagen matrix). Natskin® is a
ready to use model consisting of excised human skin.
All three models were exposed to external stimuli, namely chemical irritants or UV irradiation.
Subsequently, betamethasone 17-valerate, a potent and widely documented glucocorticoid,
particularly designed for dermatological administration, was applied in different dosages. Due
to its anti-inflammatory efficacy, a reduction of the previously provoked skin irritation was
expected.
For measuring the reaction on the external stimuli as well as the effect of the antiinflammatory
treatment, cytokines (interleukin-1a, interleukin-6, tumour necrosis factor-a),
chemokins (interleukin-8), and other proteins (interleukin-1 soluble receptor type II,
interleukin-1 receptor antagonist, lipocortin-1), which are involved in the cutaneous
inflammatory response, were quantified. These mediators were analysed, on the one hand
on protein level by enzyme linked immunosorbent assay (ELISA) (in homogenised tissue and
secretion), and on the other hand on transcriptional level (mRNA) by real time-polymerase
chain reaction (RT-PCR). Additionally, skin viability was assessed by means of different
enzymatic and histological methods.
In parallel to its impact on the skin, the penetration / permeation of betamethasone 17-
valerate through all three models and after administration in two vehicles (ethanol -
phosphate buffer and oil-in-water emulsion) was compared.
The present study provides evidence that SkinEthic®, Phenion® FT, and Natskin® are
appropriate skin models for quantifying effects in response to UV irradiation (290-
400 nm, 20-250 mJ/cm2). However, the mode and extent of these effects vary from model to
model. Particularly, the expression profile of the SkinEthic® model differs from that of the
Phenion® FT model. While in the SkinEthic® model the interleukin-1a secretion increases
depending on the applied UV dose, it is not affected in the Phenion® FT model. For the
Phenion FT® model, however, interleukin-8 turned out to be a suitable indicator of irritation.
Depending on the UV dose, both in the tissue and in the culture medium an enhanced
interleukin-8 concentration could be detected. Nevertheless, in the SkinEthic® model
interleukin-8 was not a reliable indicator of cellular and pathophysiological processes at all.
Additionally, the viability was influenced differently by UV irradiation in the three tested skin
models. The SkinEthic® model proved to be most susceptible to UV induced tissue damage
(plasma membrane damage [LDH-test] and number of metabolic active cells [MTT-test]). The
Phenion® FT and the Natskin® model showed better robustness. Most likely, these different
reactions are due to the varying composition of the particular skin models. The presence of a
dermis (fibroblasts and connective tissue) and thus, effects due to the interaction of
keratinocytes and fibroblasts seemed to play an important role.
Furthermore, the irritant effect of sodium dodecyl sulphate (0.1 to 0.4%) as well as of
betamethasone 17-valerate in a usual dosage for topical therapy (2 x 10-3 M) could be
detected by means of an increased interleukin-1a secretion in the SkinEthic® model. Hence,
the present study shows for the first time the irritant effect of a glucocorticoid in an in vitro
reconstructed skin model.
Moreover, it could be shown, that the penetration / permeation of betamethasone 17-valerate
depended on one side on the attributes of the skin model and on the other side on the
characteristics of the vehicle. Betamethasone 17-valerate permeated markedly easier
through the in vitro reconstructed skin models SkinEthic® and Phenion FT® than through the
ex vivo excised Natskin® model. Regarding the vehicle, penetration / permeation of
betamethasone 17-valerate was not influenced by the totally incorporated quantity of the
active but by its concentration in the vehicle phase directly adjacent to skin.
Reduced interleukin-1a secretion and mRNA induction were perceptible in the UV exposed
SkinEthic® model after administration of betamethasone 17-valerate dosed 2 x 10-5 M and
2 x10-6 M, which, by tendency, indicated an anti-inflammatory effect. However, the Phenion®
FT and the Natskin® model showed no analogous reaction. This is in contrast to the
demonstrated presence of the active substance at the target location and to the fact, that the
pharmacological relation between the UV induced skin irritation and the chosen indicating
proteins on the one hand and the mechanism of action of glucocorticoids on the other hand
are described in the literature.
Possibly, this could be related to the incomplete composition of the skin models and
therefore insufficient representation of the in vivo situation, whereas, for a pharmacological
response, the cooperation of all naturally present skin components would be necessary. In
addition, the optimal experimental range to prove a corresponding impact is presumably
narrow. On one side, the intensity of the stimulus must be strong enough to provoke a
distinct effect, but no irreversible cell damage should be caused. On the other side, active
substance, dosage, vehicle, and point of treatment must be adjusted at best. In addition, the
strong intra- and interbatch variation, which is characteristic for the examined skin models,
rendered the definition of the optimal experimental range even more difficult.
The present study gives a proof of concept for the use of in vitro / ex vivo skin models to
measure the irritant effects induced by external stimuli, particularly of UV irradiation. But the
pharmacological response to a subsequent anti-inflammatory treatment may be the subject
of further investigations.