Organ
of the
GD — Society for Dermopharmacy
 | Issue 2 (2004) |
Dermopharmacy News
GD-Symposium in Berlin on 25 November 2004
Reduction of Animal Experiments by Human Skin Models
Report by Sylvia Schreiber, Berlin
The main focus of a scientific symposium organized by the Gesellschaft für Dermopharmacy (Society for Dermopharmacy) was on alternative methods for animal experiments by means of biotechnologically produced human skin models in Berlin on 25 November 2004. Topicality and significance of this subject are reflected by profound changes in the cosmetics and chemicals policy of the European Union (EU), legally anchoring animal experiment-free methods for the assessment of the hazard potential of substances. The event under the scientific lead of professor Dr. med. Horst Spielmann and professor Dr. Monika Schäfer-Korting was organized in cooperation with the Bundesinstitut für Risikobewertung (BfR) (Federal Institute for Risk Assessment), Berlin, and the Free University Berlin and comprised approximately 100 representatives from industry, science, authorities, and practice having a high degree of expertise in this field.
Main emphasis of the symposium was on the question to what extent the potential of cutaneous in-vitro models suffices to predict the in-vivo situation in humans, thus creating the precondition of the reduction or abolition of animal experiments. Information as to experience gained with human skin models available so far as well as latest findings concerning the testing of the cutaneous resorption, irritation and sensitization has been exchanged. Moreover, requirements devised by the European Union (EU) and the Organization for Economic Co-operation and Development (OECD) relating to the experimental validation of animal experiment-free test methods and the status of the official approval of these methods have been presented.
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Immune-competent skin models
After opening of the symposium by the head of the GD-department Dermatopharmacology, Dr. Karl-Heinz Nietsch, Bad-Soden, Dr. Andreas Emmendörffer, Euroderm GmbH, Leipzig, informed about the works monitored by him regarding the set-up of an immune-competent skin model. Requisite for the establishing of a skin model to be applied for testing of the sensitizing effect by substances or preparations is the integration of dendritic cells.
Starting from first studies relating to the integration of monocytes in a model with dermal fibroblasts and keratinocytes initially the development of an immune-competent full-skin model has been envisaged. According to Emmendörffer’s statements, this project is nevertheless not continued owing to the fact that a model of the kind cannot be produced compliant to Good Manufacturing Practice (GMP). Instead, the development of an immune-competent epidermis model is now planned at which the epidermis originates from cells of the exterior hair follicle sheath and the integrated dendritic cells are derived from monocytes of the whole blood.
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Statements relating to the predictivity of this model are so far not feasible. Necessary is the availability of sufficient tests based on known sensitizing substances. Outstanding equally remained the question of metabolic capacity of the model in the discussion which has not been investigated to date that is however of major significance in particular for sensitizing substances.
Full-skin models in comparison
Dr. Klaus-Rudolf Schröder of Henkel, Düsseldorf, informed about his experience with different full-skin models. He compared the commercially available skin models SkinEthic® Full Thickness (SkinEthic® Laboratories, Nizza), EpiDerm™-FT 200 (MatTek Corporation, Ashland) and AST-2000 (CellSystems® Biotechnologie Vertrieb GmbH, St. Katharinen) as well as the three-dimensional skin models by the companies Henkel and Phenion concerning histology, immuno-histology and response to surfactants.
Histologically, all skin models showed an enlargement of the stratum corneum after storage over the period of several days. On the ninth day after delivery, a detachment or at least emergence of cracks between dermis and epidermis has been observed. Solely the models by Henkel and Phenion did not reveal this phenomenon. Moreover, besides the other immune markers, elastin present in native skin, could only be proven in the Henkel and Phenion model.
Significant variances between the different models also resulted concerning the cytokine distribution and gene expression after topical application of surfactants. Striking in this context is that not in a single model the difference known from human testing and practical application of washing surfactants as to the irritating effect by sodium lauryl sulphate and sodium lauryl ether sulphate could be found. This gives rise to question, as expressed in the discussion, the aptitude of the presently available skin models for comparative irritation tests of surfactants.
Rat skin more permeable
than human skin
The second part of the scientific program was dedicated to the cutaneous resorption. Dr. med. vet. Armin O. Gamer, BASF AG, Ludwigshafen, informed of the testing of the dermal penetration of pesticides into human or animal skin. When deploying these substances the human skin is the most important way of exposition.
Risk assessment of pesticides is presently in the first instance based on data relating to the acute toxicity and dermal penetration of rat skin. Gamer explained by means of a comparison performed by the BASF to the skin penetration on rats and excised human skin that human skin is basically less permeable than rat skin. This fact should be taken into account when establishing a risk assessment by means of a mathematical correction.
Of problematic nature is likewise the missing standardization of the present in-vitro methods applied for penetration tests on human skin. Thus a recently published ring study revealed a very high variability both within one laboratory and also different laboratories. At present the question is analyzed whether this variability can be reduced by applying artificial human skin models.
Many ways lead through the skin
Skin penetration is looked upon in the pharmaceutical sector from a different viewpoint. Prerequisite for the dermal resorption of a medicinal agent is presumably the overcoming of the permeation barrier in the stratum corneum which comes about by interplay between the corneocytes and the lipid layers situated in-between. How this barrier can be overcome and influenced by the drug carrier has been explained by Dr. Ulrich F. Schäfer from the department Biopharmacy and Pharmaceutical Technology of the University Saar, Saarbrücken.
 Dr. Ulrich F. Schäfer, Saarbrücken, outlines how the skin barrier can be overcome by medicinal agents as well as the influence of medicinal agent carriers on the skin barrier. |
In view of its interference by carrier systems, the intra-cellular way out of the principally available penetration ways is considered to have a major significance. Thus it could be shown for example that the intra-cellular permeation of Ketoprofen by pre-treatment of human epidermis by means of semi-solid triglycerides is increased whereas a pre-treatment with Vaseline or wool wax alcohol ointment reduces the permeation. Other tests showed an influence of the water content by creams and ointments on the permeation of flufen amine acid. These effects are due to interactions of the vehicle with the skin lipids.
In contrast to the intra-cellular route, the trans-cellular way is only considered to play a minor role for the overall permeation in general. This way, however, is gaining in importance by using substances as urea causing a loosening effect on the corneocyte structure. The same applies to the follicular penetration path relevant despite its relatively low part on the diffusion surface mainly for nano-particular systems.
As his preceding speaker, Schäfer also outlined the necessity of standardized conditions for penetration studies. Already small deviations in the test set-up, as for example by impurities at the skin surface with sub-cutaneous lipids, may entail significant changes of the penetration behaviour.
 Scientific conference heads and moderators of the symposium were the members of the GD committee, professor Dr. Monika Schäfer-Korting, Institute for Pharmacy at the Free University Berlin and professor Dr. med. Horst Spielmann of the Bundesinstitut für Risikobewertung (BfR), Berlin (Federal Institute for Risk Assessment). |
Furtherance
of research projects
The scientific conference head professor Dr. Monika Schäfer-Korting also dealt with the influence of carrier systems on the cutaneous resorption in her lecture. As speaker in a research group under the name of “Innovative drugs and carrier systems” supported by the Deutsche Forschungsgemeinschaft (DFG) (German Research Society) she presented results concerning the penetration of different lipid-based preparations as solid lipid nano particles which have been charged with the glucocorticoids Prednicarbate and Betamethasonvalerate. By means of parelectric spectroscopy it could be shown that the active agents tested adsorb to a different extent to the surface of the particles thus showing a different penetration behaviour.
Moreover, Schäfer-Korting reported about the status of a multi-purpose project supported by the Bundesministerium für Bildung und Forschung (BMBF) (Federal Ministry for Education and Research) relating to the assessment of the cutaneous penetration and permeation by foreign objects with biotechnologically produced human skin models. The project objective is a considerable reduction or even abolition of animal experiments in the development and testing of industrial chemicals, pesticides and drugs.
Besides the Free University Berlin additional universitary institutions and industry partners take part in this project. First permeation data based on the use of skin models showed an improved reproducibility compared with the results from investigations performed on excised skin of humans or swine. As the support of the project has recently been prolonged by the BMBF, validation of the new method will start now.
 Besides extensive information, the GD-Symposium also allowed sufficient opportunity for discussion and interdisciplinary interchange of ideas. |
Classification
of skin-sensitizing potential
The third part of the symposium dealt with methods for testing of skin sensitization. Dr. Manfred Liebsch from the Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergänzungsmethoden (ZEBET) (Central Office for the Recording and Assessment of Substitutional and Complementary Methods for Animal Experiments) established at the BfR, introduced a validation study supported by the European Centre for the Validation of Alternative Methods (ECVAM) dealing with the classification of skin-sensitizing potential which is presently performed under the management of ZEBET.
Due to the fact that no validated in-vitro tests for the test of skin sensitization were available before the study initiation, a pre-validation study based on five different in-vitro test systems has been carried out initially. In the course of this process it turned out that no test system available was sufficiently technically mature to perform a validation. After improvement of the test systems, finally three of them were formally validated, i.e. the commercially available epidermis models Episkin® (Episkin SNC, Lyon) and EpiDerm™ (MatTeK Corporation, Ashland) as well as the Skin Integrity Function Test (SIFT) at murine skin.
The study objective is to be able to classify hazardous substances in skin-irritating and non-skin irritating. In this aspect the two epidermis models would have supplied excellent and congruent predictions within the acceptance criteria defined beforehand. In contrast, the SIFT has to this effect achieved surprisingly poor results and is therefore no longer subject to testing.
The second phase of the study considers besides the viability of the skin also the release of the Interleukin-1-alpha as final product. Thus the informative capability of the epidermis models is to be improved and the possibility created to be able to classify also mildly sensitizing substances apart from skin-sensitizing and non-skin-sensitizing ones which is of particular importance for the safety assessment of cosmetic raw materials.
Testing of skin irritation in humans
As the animal protection legislation prohibits animal experimental tests of cosmetic ready-to-use products for a long time already, they are today similar to topical dermatotherapeutic agents predominantly tested in human trials to their local compatibility. An overview about the opportunities and limitations of the presently applied methods has been presented by the dermatologist professor Dr. med. Klaus P. Wilhelm, holder of the institute proDerm in Schenefeld near Hamburg.
For the routine test of the acute and cumulative irritation potential, procedures as the single and triple 24-hour-patch test, the Duhring-Chamber Test and the repetitive open application test are performed at 20 to 50 voluntary skin-healthy probands respectively. The sensitization hazard may, as far as ethically justifiable, be tested in the Human Repeated Insult Patch Test (HRIPT). Moreover, it may – according to the product category - be reasonable to also test the photo-toxicity, the photo-sensitization, the eye compatibility as well as the sensory irritation by means of methods especially developed to this aim.
Human skin models
in practical application
Dr. Jens-Olaf Eichler of Stockhausen GmbH, Krefeld, informed about a new field of application for three-dimensional human skin models. He investigated in a like model the regenerative effect of emulsions which are used in the occupational sector as skin protection agent for the prevention of irritatively conditioned contact eczema. Disadvantageous at this skin model in contrast to the proband test and the perfused organ model is a less marked barrier layer, major advantages however, are obtained by the structural similarity to human skin and the possibility to allow testing of active mechanisms and biochemical repair processes over longer periods.
Emulsions exemplarily tested enhanced the regeneration of the pre-damaged skin barrier by sodium lauryl sulphate. They reduced the cell lysis as well as the distribution of the inflammatory mediators Prostaglandin-E2 and Interleukin-1-alpha. As the emulsions tested in the skin model also showed regenerative effects in a repetitive occlusive irritation test at probands, performed parallel, the referee considers the new test method as qualified procedure for a targeted active agent and product development.
Methods for testing
of sensitization
Apart from the irritation, clinical importance is also attached to the sensitization of the skin by foreign matters. When developing new products, sensitizing substances should therefore be identified in preliminary stages. At the time being refined tests at animals are applied for this purpose, as professor Dr. Hans-Werner Vohr, Bayer Health Care AG Wuppertal reported - the advanced local lymph node test (LLNA) at the mouse ear. This improved method allows, in contrast to the classical LLNA, a distinction of irritating and sensitizing properties.
Dr. Carsten Goebel, Wella AG Darmstadt, introduced a promising novel in-vitro method for the testing of the sensitizing potential of chemicals. By means of this method the activation of dendritic cells derived from human peripheral monocytes is tested. For the securing of the bio-availability the skin penetration of the test substance is analyzed parallel. Only the mutual recording of these two - for the development of an allergenic reaction important steps - leads to a method which could replace future animal experiments.
Targets and test models
for novel pharmaceuticals
An extensive overview about the complex defense mechanisms triggered in case of an activation of the immune system by pathogens in the skin has been given by professor Dr. med. Heinfried H. Radeke from the Institute for Pharmacology and Toxicology at Frankfurt University. He delineated by means of a video simulation how Langerhans cells get from the epidermis to the lymph node as sub-population of the dendritic cells after inflammatory activation. The detailed enlightenment of the signal paths of these processes allows the identification of novel pharmacological targets both for the area of dermatology and other fields of development.
Professor Dr. med. Wolf-Henning Boehncke of University Dermatological Clinic, Frankfurt Main, is engaged in animal models of psoriasis for testing of medicinal agents. Their retrieval is made considerably difficult by the complex phenotype, the polygenetic determination and the multi-factor genesis of the disease. Most established psoriasis animal models only represent individual aspects of the disease.
| Test
Results of Pharmaceutical Agents in the Psoriasis-SCID-Mouse Model in Comparison
to Clinical Experience | |||
| Substance | Therapy- Protocol (SCID) | Result SCID- Model | Clinical Experience |
Dexametha- | 4 weeks orally | Satisfactory effectivity | Only scarcely applied |
Cobetasol-17- | 3 weeks topically | Moderate
| Established for mild to medium-severe psoriasis |
Ciclosporin | 2
weeks | Satisfactory effectivity | Established for severe psoriasis |
1-apha,25-Dihydroxy- | 4 weeks intracutaneously | Satisfactory effectivity | Topical at mild to severe psoriasis |
Efalizumab | 2
Wochen | Satisfactory effectivity | Effective at medium-severe to severe psoriasis |
Troglitazone | 6 weeks orally | Significant
| 5 patients successfully treated (no longer available in the market due to liver toxicity) |
PS
519 (blockade | 4
weeks | Satisfactory effectivity | Analogous substances in phase III (not psoriasis) |
Efomycin
M | 4 weeks subcutaneously | Satisfactory effectivity | Bimosiamosis effectively in phase I/II |
Regulatory aspects of
animal-experiment-free test methods
Concluding, professor Dr. med. Horst Spielmann, head of the ZEBET at the Bundesamt für Risikobewertung (BfR) (Federal Office for Risk Assessment) reported about the regulatory demands on novel animal-experiment-free test methods for risk assessment. They have to be experimentally validated before being internationally recognized. For this aim; ZEBET elaborated a corresponding concept in cooperation with the European Validation Center (ECVAM) and continuatively with the OECD. Subsequently, firstly a prevalidation, then a validation and finally an independent evaluation (peer review) follows, associated with the test development.
| Sponsors
of the symposium Aventis Pharma Deutschland GmbH, Bad Soden BASF AG, Ludwigshafen Henkel KGaA, Düsseldorf |
This concept has already proven successful for the validation of in-vitro photo-toxicity tests and in-vitro embryo toxicity tests concluded under the lead of ZEBET. Successfully terminated were also validations of in-vitro tests with human skin models for the testing of corrosivity accepted by the EU and American authorities. Ultimately, the OECD for the first time included four validated animal-experiment free test methods into the catalogue of the official OECD-test methods in 2004. Spielmann pointed out that in-vitro toxicology has meanwhile developed towards a mature science.
World congress
on alternative methods
Secluding, the GD-department head Dr. Karl-Heinz Nietsch expressed his thanks to all persons involved and to the sponsors of the symposium for their engagement and invited to visit the 9th GD-Annual Meeting in Vienna on 14 and 15 March 2005. Moreover, he stressed the 5th World Congress on Alternatives & Animal Use in the Life Sciences taking place under mutual responsibility of professor Schäfer-Korting and professor Spielmann in Berlin in August 2005 (please refer to information below).
| Event
note 5th World Congress on Alternatives & Animal Use in the Life Sciences 21 - 25 August 2005, Estrel Convention Center, Berlin Information: www.ctw-congress.de/act 2005 |
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