TRENDS IN DERMATOPHARMACY - wohlrab-net.de

Trends in Clinical and Experimental Dermatology 

Editor: J. Wohlrab 

VOLUME 1 

TRENDS IN DERMATOPHARMACY 

- UPDATE 2002 - 

Volume Editors 

J. Wohlrab 

R.R.H. Neubert 

W.Ch. Marsch 

Shaker Verlag 

Aachen

Trends in Clinical and Experimental Dermatology 

Editor: J. Wohlrab 

Volume 1 

_______________________________________________________ 


- UPDATE 2002 - 

Volume Editors: 

Johannes Wohlrab 

Reinhard R.H. Neubert 

Wolfgang Ch. Marsch 

Shaker Verlag 

Aachen 2002

Dedicated to 

Prof.Dr.rer.nat.W.A.Wohlrab 

in honor of his 65 th birthday

Contents 

Cutaneous NO-Metabolism and its therapeutic Influence................. 1 

J.Wohlrab 

The value of high-frequency sonography in objectifying drug 

effects...................................................................................................... 25 

A.Unholzer, H.C.Korting 

Bioengineering of the skin: non-invasive methods for the 

evaluation of efficacy............................................................................. 32 

T.Gambichler, F.G.Bechara. M.Stücker, K.Hoffmann, P.Altmeyer 

The Repetitive Washing Test as a Model for the Evaluation of 

Barrier Creams...................................................................................... 47 

W.Gehring 

Impact of Topical Skin Care and Maintenance Products on 

Stratum corneum Barrier..................................................................... 52 

M.Gloor 

The Tandem Repeated Irritation Test (TRIT) – A New Method for 

the Evaluation of Barrier Creams........................................................ 58 

J.Spoo, W.Wigger-Alberti, S.Schliemann-Willers, A.Klotz, P.Elsner 

Atomic absorption spectrometry for the determination of physical 

sunscreen filters..................................................................................... 70 

G.Gottbrath, J.Grünefeld, C.C.Müller-Goymann 

Determination of Clobetasol Propionate in Extracts by Means of 

HPLC and LC-MS................................................................................. 80 

T.Hagemeister, M.Lienscheid, H.-J.Weigmann, J.Lademann, R.v.Pelchrzim, 

W.Sterry 

Analytical methods fort he determination of dermatopharmacokinetics................................................................................... 

90 

J.Lademann 

HPTLC and LC/MS in ceramide analysis........................................... 106 

K.Raith, H.Farwanah, R.Neubert 

HPLC-analysis for permeation studies of 5-aminolevulinic acid 

and its derivatives.................................................................................. 118 

A.Winkler, C.C.Müller-Goymann

Photoinduced Lesions in DNA – Detection using Micro-HPLC and 

Ion Trap MS........................................................................................... 129 

G.Zhang, M.Linscheid 

The Topical Application of Vitamin D3-Analogues in Psoriasis 

vulgaris................................................................................................... 137 

M.Fischer 

Trends of topical immunmodulatory Therapy................................... 145 

K.Jahn, R.H.H.Neubert, J.Wohlrab 

Trends of Treatment with Topical Glucocorticoids........................... 153 

R.Niedner 

The Topical Treatment of Atopic Dermatitis and Psoriasis with 

Vitamin B12........................................................................................... 158 

C.Stoerb, P.Altmeyer, R.Niedner, J.Hartung, M.Stücker 

Modern Vehicle Systems....................................................................... 167 

C.Huschka, J.Wohlrab 

Microemulsions as drug vehicles for dermal application.................. 181 

K.Jahn, R.H.H.Neubert, J.Wohlrab 

Transfersomes ® and their Application in Dermatopharmacy........... 186 

J.Lehmann, M.Rother 

Trends in the systemic antibiotic therapy of dermatological 

infectious diseases.................................................................................. 195 

N.H.Brockmeyer, A.Kreuter 

Systemic therapy with immunomodulatory drugs in dermatology.. 198 

M.Sticherling 

Systemic Therapy with Recombinant Antibodies in Dermatology... 209 

M.Lüftl 

Current state and perspectives of immunomodulation of the 

allergic contact dermatitis..................................................................... 220 

H.-D. Göring 

Therapy of Immediate Type Reactions................................................ 228 

W.Ch.Marsch 

In vitro Models for the detection of immunomodulating properties 

of pharmaceuticals................................................................................. 236 

G.Wichmann, I.Lehmann

Editors Preface 

Dermatopharmacy and –pharmacology have developed enormously over 

the past years, especially thanks to the rapid technical development of 

analytical, synthesis and diagnostic procedures. In addition, genetechnical 

and molecular-biological methods have brought innovation not 

only in systemic, but especially also in topical applications of medicinal 

and active substances. Modern vehicle systems have initiated a new age 

in galenics and make it possible to apply even high-molecular or hard-todissolve 

substances in therapeutic doses. 

With detailed knowledge of the structure and function of the skin, as 

well as the regulation of metabolic processes, there is an increasing 

merging of biological processes with pharmacological-therapeutic 

manipulations. 

Despite these positive developments, dermatopharmacy needs 

enthusiastic, engaged persons with a broad range of ideas, who will work 

out the basic scientific foundation, design the practical relationships and 

lead in the conception of an effective, marketable product. 

In this sense, I wish to thank all the authors of this book for their 

collaboration and innovative contributions. 

Johannes Wohlrab, MD 

Editor

Address of Volume Editors: 

Johannes Wohlrab, MD 

Department of Dermatology 

Martin-Luther-University Halle-Wittenberg 

Ernst-Kromayer-Str. 5-6 

D-06097 Halle (Saale) 

Germany 

Reinhardt Neubert, PhD 

Institut of Pharmaceutic Technology and Biopharmacy 


Wolfgang-Langenbeck-Str. 4 


Germany 

Wolfgang Ch. Marsch, MD 





Germany

Volume Editors Preface 

In March 2002, in honor of the 65 th birthday of our esteemed teacher, 

colleague and friend, Professor Wolfgang A. Wohlrab, we held the 

Congress “Trends in Dermatopharmacy” in Halle an der Saale 

(Germany). It was our particular intention to honor his many-sided 

academic life’s work. In nearly 3 decades of dedicated and responsible 

work in the areas of experimental dermatology and dermatopharmacy, 

Prof. Wohlrab has created an exceptional life’s work and stood firm 

against the sharp headwinds of social-political circumstances. The 

numerous publications and patents, the hordes of his students and an 

outstanding institution document both his performance and the quality of 

his work. 

We thank Wolfgang Wohlrab for his engagement, his friendship and his 

loyalty, which is characterized by profound academic and Christian 

principles. We wish him health and continued vitality for the coming 

years, without the pressures of daily, always enthusiastically-performed 

duties. 

We hope that this book will contribute with its number of excellent 

articles to the modern understanding of dermatopharmacy, as the 

Honoree would wish! 

J. Wohlrab, MD R.R.H. Neubert, PhD W.Ch. Marsch, MD

Cutaneous NO-Metabolism and its therapeutic Influence 1 

Cutaneous NO-Metabolism and its therapeutic 

Influence 

J. Wohlrab 

Universitätsklinik und Poliklinik für Dermatologie und Venerologie 

Martin-Luther-Universität Halle-Wittenberg 



Germany 

Arginine .......................................................................... 5 

• Chemistry of Arginine....................................................5 

• Arginine and its biochemical-metabolic importance......5 

• Place of Arginine in NO-metabolism............................7 

Transmembranous Transport of L-Arginine................. 14 

Therapeutic Influence of L-Arginine Metabolism ....... 15 

References..................................................................... 18 

In 1980, Robert Furchgott discovered rather by accident the 

endothelium-derived relaxing factor (EDRF) which has been 

recognized as a central messenger substance in the regulation of 

hemovascular perfusion. Furchgott was able to show that EDRF is 

produced by endothelial cells, has a potent vasodilatative effect and 

a very short half-life. Despite intensive efforts to chemically 

identify EDRF, it was not until 1986 that Salvador Moncada and 

Louis Ignarro independently identified EDRF as the gas nitric 

oxide. (NO). At least since the Nobel Prize for Physiology and 

Medicine was awarded to the American scientists Robert Furchgott 

(State University of New York), Louis Ignarro (University of 

California, Los Angeles) and Ferid Murad (University of Texas 

Medical School, Houston) in 1998, the biochemical importance of 

nitric oxide. (NO) in the regulation of hemo- and lymphovascular 

perfusion and in the initiation and maintenance of immunoprocesses 

has become widely known and the subject of intensive basic 

scientific research. The central position of NO in the pathogenesis 

of various dermatoses has been increasingly examined only in 

recent years. Knowledge of the pathogenetic relationships, 

especially in inflammatory skin conditions, has revealed new 

------------------------------------------------------------------------------------ 

Wohlrab J, Neubert R, Marsch W (eds): Trends in Dermatopharmacy 

In: Trends Clin Exp Dermatol, Aachen, Shaker, 2003, vol 1

2 Cutaneous NO-Metabolism and its therapeutic Influence 

aspects and also opened new therapeutic options in the treatment of 

these and other diseases. 

The amino acid arginine is a very promising active substance with 

respect to topical and possibly also systemic application thanks to 

its central metabolic involvement in cell metabolism. Arginine is 

the substrate for enzymatic synthesis of NO. Moreover, arginine has 

other important functions as a regulating starting material in the 

urea cycle, protein biosynthesis and creatin metabolism. 

Little information is available at present about the 

dermatopharmaceutical use of arginin. The activity of arginase, 

which is expressed especially strongly in the basal epidermal layers, 

is increased by L-arginin substitution and manganese ions acting as 

a cofactor. The result of this influence is increased urea synthesis in 

the keratinocytes. In healthy subjects, there is a reduction in 

transepidermal water loss and an increased in hydratation of the 

horny layer following topical application of preparations containing 

L-arginin. Application of an amphiphilic standard vehicle system 

containing L-arginin to patients with atopic dermatitis showed 

clinical effects comparable to equivalent systems containing urea, 

but without the irritative effect. The therapeutic efficacy of Larginin 

in subepidermal skin layers has not yet been examined. For 

this reason, clarification of interactions with relevant cutaneous cell 

systems (such as cutaneous microvascular endothelial cells), 

objectification of the penetration dynamics and kinetics from 

various vehicle systems, examination of the influence on vital 

perfusion models and examination of the effects in healthy subjects 

are prerequisite to therapeutic application to patients. 

The fundament of regulation of the cutaneous microcirculation is 

the basic tone of the small arteries, arterioles and venoles. This is 

mediated essentially by a basic innervation, realized by the 

vegetative nervous system. There is no direct nervous innervation in 

the capillary end pathways. Nonetheless an adrenergic effect due to 

diffusion of neurotransmitters or intercellular signal transduction in 

the sense of a stimulation system must be assumed. Moreover, 

numerous vasoconstricting transmitter substances are known. Under 

physiological conditions Endothelin-I is very important. NO acts as 

a vasodilatative competitor. The basic production of NO is probably 

realized by iNOS, that is by the inducible NOS-isoform. The 

purpose of these antagonistically-directed systems is to make 

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possible the need-oriented distribution of blood for nutrition of the 

tissues and for thermoregulation, to maintain a laminar blood flow 

and prevent sludge phenomena of erythrocytes and thrombocytes. 

Moreover, NO binds very rapidly to iron-containing hemoglobin in 

the cutaneous microcirculation because of the narrow spatial 

relationship between the endothelial cells (vascular wall) and 

erythrocytes (flow medium) compared to the macrocirculation, and 

is almost completely inactivated to nitrates. Nitrite and Snitrosothiol, 

which are formed in plasma, are largely absent here. 

However, penetration of NO into the cell depends on the diffusion 

rate with respect to NO/NO 2- , the oxidative saturation of Hb and on 

the redox potential. 

The process of inactivation of NO is specific to the endothelial cells 

and proportional to the expressed NO-concentration. It can thus be 

used as a comparison measure for NO-production. In human blood 

plasma in equilibrium there are about 3nM NO. With respect to the 

temporal dimension of synthesis, NO-production is estimated at 

about 4 pmol/min per mg protein or 0.8 pmol/min pro mg 

endothelial cells. Given a total weight of endothelial cells of 1.5 kg 

per person, about 1728 µmol NO/day, are produced by eNOS. 

In addition to the basic production of NO by iNOS, a rapid reaction 

capacity is important in order to adapt to the rapidly changing 

functional states. This is realized via the non-inducible, though 

activatable NOS forms (eNOS, nNOS). A mid-term or even longer 

period of elevated NO requirement can only be increased to adjust 

the basic equilibrium by synthesis of the protein. In addition to 

eNOS there is also an iNOS in microvascular endothelial cells, 

which causes increase in NO-concentration in mid- or long-term 

independent of the intracellular calcium content. 

Interesting in this connection is also the NO-production by 

granulocytes in the flowing medium blood and tissue-immigrating 

populations. It is known that NO also increases the expression of 

adhesion molecules (e.g. VCAM-1). This prepares a recruiting of 

blood cells, which is centrally important, especially in inflammation 

processes. The initial phase of this recruiting process is 

characterized by the rolling phenomon, in which the flow velocity 

of mononuclear cells slows until they adherent on the vascular wall. 

The NO-production of such activated granulocytes may also be 

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important for the regulation of the contraction state of regional 

vascular segments, under physiological conditions as well. 

The quantity consumed by guanylate-cyclase and thus for cGMPproduction 

represents only a small portion of the expressed NO. Of 

interest are especially the peroxynitrites, which form during 

reaction with superoxides. These enter directly and indirectly via 

NO2, NO2+, �OH in interaction with the tissue (tissue damage) and 

lead to endoluminal platelet adhesion and to increased vascular 

permeability. After induction of iNOS, high NO concentrations 

result. NO interacts thereby with thiol groups or so-called 

transition-metal-containing protein (TMCP). This interaction leads 

to functional influence of certain regulatory proteins or to induction 

of gene expression of cell-protective or apoptosis-inducing 

substances. The reaction patterns of various types of cell differ very 

markedly. In mesenchymal cells, apoptosis is elicited. In 

macrophages or endothelial cells, as well as lymphocytes, 

eosinophilic granulocytes or hepatocytes, antiapoptotic processes 

are observed. The knowledge that there is no uniform function 

pattern of NO-induced regulation processes, which depend 

decisively on the pathogenetic influence factors of a pathological 

functional state, is of great importance for the conception of 

therapeutic strategies. 

The NO-mediated effects on human skin are particularly 

proinflammatory and cytotoxic in nature. Ormerod et al. 

demonstrated that NO leads to increased expression of CD3, CD4, 

CD8, CD68, neutrophile elastase, ICAM-1, VCAM-1, 

nitrosotyrosin and p53 on relevant cells. Moreover, there is 

increased induction of apoptosis and reduction in the proportion of 

CD1a-positive Langerhans cells in the epidermis. The described 

effects depend on the NO-concentration and the application time. 

Thus, the cytotoxic effect is caused essentially by accumulation of 

p53 and subsequent apoptosis. High doses of NO lead paradoxically 

to a smaller increase in the number of macrophages and T-cells and 

give rise to the assumption of an immunosuppressive effect of 

higher NO-concentrations. 

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Arginine 

• Chemistry of Arginine 


The basic structure of arginine (2-Amino-5-guanidovalerianic acid) 

consists, like that of all amino acids, of an amino group (NH2), a 

carboxyl group (COOH) and a hydrogen atom bound to the 2 nd 

carbon atom. Arginine is a diaminomonocarbonic acid and, like 

lysine and histidine, carries an additional basic group in the side 

chain. It is easily soluble in water and a white crystalline powder at 

room temperature. The melting point is 238°C and the specific 

rotation at 20°C in sodium light is 27.4°. Of the more than 300 

known amino acids, 20 have been identified in animal proteins. All 

of them, except glycine, are found in the optical L-configuration. 

Arginine is a glucoplastic, non-essential amino acid and can be 

synthesized by human cells. 

H2N 

NH 

NH 

NH 2 

Fig. 1: Arginine (C6H15N4O2; Molweight 174.20 g/mol) 

However, functional conditions are known (e.g. shock, sepsis, 

growth), in which there may be a deficiency in endogenous 

synthesis or impaired distribution. For this reason, arginine is also 

referred to as a “conditionally essential” amino acid. 

• Arginine and its biochemical-metabolic importance 

In 1866, Schulze and Steiger first isolated arginine in crystalline 

form. 10 years later, its presence in animal tissues was proven for 

the first time. It was recognized that arginine has essential 

importance in connection with growth processes and in the 

regulation of nitrogen balance. 

O 

OH 

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L-arginine is synthesized intracellular by proteolyse and in the urea 

cycle from arginine succinate in the arginine succinase-reaction, or 

it is transported from extracellular to intracellular by a specific 

transport system. In addition to protein synthesis in human dermal 

microvascular endothelial cells (HDMEC) L-arginine is available as 

a substrate for two very important enzymatic reactions. 

A Nitric oxide (NO)-Synthase-Reaction 

In this reaction, NO and L-citrullin are formed from Larginine, 

catalytically controlled by NO-synthase. L-citrullin 

functions as a substrate for the citrate and urea cycle. 

B Arginase-Reaction 

The enzyme arginase converts L-arginine to urea and Lornithin. 

L-ornithin, in turn, may also be metabolized by 

ornithintranscarbomylase in the urea cycle to L-citrullin, or is 

available for polyamine synthesis. 

L-arginine is thus very important in the biochemistry of the organ 

skin. In forming NO, it contributes essentially to the regulation of 

hemo- and lymphovascular perfusion and thus makes a needappropriate 

distribution and recycling of nutrients and fluids 

possible. Moreover, it indirectly regulates the temperature balance. 

For the epidermis, L-arginine serves as a source of urea and thus 

largely determines the water-binding capacity of the Stratum 

corneum. The intracellular urea synthesized by arginase thus is of 

essential importance in the formation and maintenance of the 

epidermal barrier function. 

------------------------------------------------------------------------------------ 



UREA 

+ ORNITHINE 

Arginase 

NO- 

Synthase 


PROTEIN 

METABOLISM 

ARGININE 

CITRULLINE 

+ 

NITRIC OXIDE 

Fig. 2: Schema of Arginine Metabolism in the Skin 

• Place of Arginine in NO-metabolism 

CREATIN 

METABOLISM 

Fumarat Malat 

ARGININ - 

SUCCINAT 

Aspartat 

Oxal 

- azetat 

One of the most important regulation factors in the cardiovascular 

system is the gas nitric oxide. NO is formed along with L-citrullin 

by the enzyme NO-synthase (NOS) from L-arginine and rapidly 

metabolized after oxygenation to nitrates and nitrites and thus 

inactivated. There are three known isoforms of NOS. In addition to 

two membrane-bound, calcium and calmodulin-dependent forms 

(cNOS or niNOS), there is a cytosolic, calcium-independent form 

induced by endotoxins or inflammatory cytokines such as IL-1β or 

TNF-α (iNOS). The non-inducible isoforms can be found in 

neuronal (nNOS – in the brain also bNOS) and endothelial cells 

(eNOS) and the inducible form (iNOS) e.g. in macrophages. The 

intracellular calcium content is decisive for the regulation of the 

enzyme activity of cNOS. The iNOS, by contrast, contain the 

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calcium-binding protein calmodulin, but their control is not 

calcium-dependent. Increased activity is only possible by means of 

protein synthesis. All NOS-isoforms are specific for the L-isomer of 

arginine. They all occur in particulate (membrane-bound) and 

soluble (cytosolic) form. 

Fig. 3: Schema of Arginase Molecule 

By contrast, little is known about the regulation of the gene 

expression of NOS. A key function is ascribed to NO itself. It is 

assumed that NO contributes to the expression of redox-controlled 

transcription factors by changing the intracellular oxidationreduction 

(Redox) balance. To date, this regulation route could be 

demonstrated for the transcription factors NF-κB and activator 

protein 1 (AP-1). The potential of NO to develop both anti- and 

pro-oxidative effects explains the different reaction patterns in 

different cell types. 

The catalysed reaction by NOS of L-arginine to L-citrullin and NO 

proceeds in two steps. Both partial reactions are NADPH-, calcium- 

or. calmodulin- (except iNOS) and tetrahydrobiopterin (BH4)dependent. 

In an intermediate step, N-hydroxy-L-arginine is formed 

from L-arginine with formation of water and NADP+ (Fig. 4). The 

product stochiometry of L-citrullin and NO is 1:1. 

NADPH, O2, Tetrahydrobiopterin (BH4) and frequently also 

flavinadenosindinukleotid (FAD) and flavinmononukleotid (FMN) 

act as cofactors for all isoforms. The NADPH- and flavin-binding 

domain of cNOS is identical with the cytochrome P450-reductase. 

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The molecular weight of the monomers of the isoforms differs and 

is cited for iNOS at ca. 130kDa and for cNOS at 155kDa. 

Moreover, differences can be demonstrated between the soluble and 

particulate type. All NOS-isoforms have a binding site for Larginine, 

Hem, FAD, FMN, NADPH and calmodulin. The bNOS 

also has a PDZ binding domain and the eNOS a myristoylation 

(M)- and a caveolin (C)-binding domain. By comparison, a 

transmembranous binding site TMD) could be demonstrated for 

cytochrome P 450 reductase. 

L-arginine 

Ca 2 + / C a M / B H 4 

O 2 

H 2 O 

N AD P H N A DP + 

N-hydroxy-L-arginine 

F A D - F MN o x . F A D -FMN r e d . 

F e - H e m r e d . F e - Hem ox. 

Ca 2 + /CaM/BH4 

O2 

H2 O 

NADPH NADP + 

L-citrulline + NO 

Fig. 4: Metabolic steps of NO-Reaction 

The molecular-biological characterization of NOS shows largely 

homology between individual species with respect to the amino 

acid sequence (human eNOS and bovine eNOS; homology = 94%). 

In 1991, Bredt et al. cloned NOS for the first time. By means of 

gene mapping, the sequence was localized on the human 

chromosome 12q12-q24 and gene 12q24.3 identified by 

fluorescence-in-situ-hybridization (FISH). Later the eNOSsequence 

on 7q35-q36 and the iNOS-sequence on 17q11.2 were 

found. Moncada et al. found multiple copies of the gene-specific 

sequences in human genomes for iNOS, unlike bNOS and eNOS, 

which show monochromosomal localization. For this reason, 3 

subtypes (A-C) of iNOS are differentiated, of which the biological 

function is not yet completely understood. 

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bNOS 

eNOS 

iNOS (murin) 

iNOS (human) 

Cytochrome P450 

reductase 

PDZ Arg Hem CAL FAM FAD NADPH 

M C 500 1000 

TMD 

Fig. 5: Sequence homologies of the molecular isoforms of NOS 

compared to Cytochrome P450 Reductase. 

The varying physiological importance of NOS-isoforms was tested 

in knock-out mice. eNOS knock-out mice developed arterial 

hypertension due to elevation of peripheral resistance. iNOS-Gene 

knock-out mice, by contrast, showed an elevated susceptibility to 

viral, bacterial and parasitic infections and developed a higher 

number of malignant tumors. Moreover, a high-polymorph 

microsatellite marker has been identified in the human iNOSpromotor 

region, for which a strong positive allele association to 

the fetal form of cerebral malaria has been found. This observation 

underlines the assumption that the iNOS have an essential function 

in signal transduction within immunological regulatory processes. 

The distribution of the NOS-isoforms in human microvascular 

endothelial cells (HDMEC) is of decisive importance for the 

regulation of perfusion in the end-flow pathways. Especially with 

respect to complex biological functional processes, in which a 

different reactions of the macro- and microvascular system or 

individual function areas of the macro- and/or microvascularization 

appear meaningful or necessary (such as thermoregulation, shock, 

etc), different regulation mechanisms must be assumed. 

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Name Isotype Regulation 

bNOS Constitutive 

eNOS Constitutive 

iNOS-A Inducible 


Ca 2+ /Calmodulin, 

Stress 

Ca 2+ /Calmodulin, 

Stress, UV, 

HSP90 

Endotoxins, 

cytokines 

neuropeptides 

Predominant 

occurrence 

Brain, neuronal 

structures 

chromosomal 

ascription 

12q24.1– 

12q24.31 

Endothelial cells 7q35-7q36 

Macrophages 

neutrophils 

mesenchymal 

cells 

iNOS-B Unknown unknown 

iNOS-C Unknown unknown 

17q11.2 

17p11.2- 

17q11.2 

17p11.2- 

17q11.2 

Tab. 1: Chromosomal localization of gene sequences of the NOSisotypes 

in different regions of human chromosomes 

The question thus arises, whether this is primarily a calciumdependent 

process (eNOS), as in the case of macrohemovascular 

endothelial cells (e.g. HUVEC) or a calcium-independent process 

via increased concentrations of enzyme inductors (e.g. L-arginine), 

which causes an increase in the NOS activity via gene activation 

and increased expression of the protein (iNOS). Pathogenetic 

involvement of such regulation patterns must be discussed 

especially in chronic-inflammatory skin diseases. Moreover, it is 

known that about a 1000x higher (in the nmol/L range) NO 

expression prolonged up to several days compared to the output of 

constitutive NOS-isotypes (in the pmol/L range) can be expected 

due to induction of iNOS (for example by TNF-α, IFN-γ, IL-8, IL- 

1β) with a latency time of 6-8 hours. Our team examined the gene 

expression of eNOC and iNOS on HDMEC under the influence of 

L-arginine and the cofactors calcium and tetrahydrobiopterin. We 

could demonstrate a concentration-dependent increase in iNOSmRNA 

expression by L-arginine. The eNOS-mRNA expression 

was not influenced by L-arginine. 

Weitzberg et al. discovered a non-enzymatic synthesis route of NO 

in 1998. In this, NO is formed under particular conditions via 

reduction of anorganic nitrites. This non-enzymatic generation of 

NO occurs on the skin surface and is thought to be important for 

primitive biological processes, such as defence against bacteria and 

viruses. 

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Chemically, nitric oxide is a very simple molecule. It consists of a 

simple oxygen atom bound to a nitrogen atom. This trivial structure 

has made very extensive and detailed chemical characterization 

possible in the past few years. Three biochemical reactions of 

dissolved NO have been found to be basic to physiological 

regulatory processes. The first and probably most important 

reaction for in-vivo NO consumption is the irreversible and rapid 

formation of nitrate by binding to oxyhemoglobin (Hb) or 

oxymyoglobin. 

Hb-Fe 2+ -O2 + �NO → Hb-Fe 2+ OONO → Hb-Fe 2+ - 

+ NO3 

(Reaction 1) 

The second reaction is the binding of NO to iron-containing Hem of 

guanylate-cyclase or other proteins This is important for the 

activation of signal transduction pathways. 

Hem - Fe 2+ + �NO → Häm-Fe 2+ - NO (Reaction 2) 

The third reaction is the formation of peroxynitrite anions 

(ONOO - ). In this, superoxides (O2� - ) react irreversibly with NO. 

�NO + �O-O: - → ONOO - (Reaction 3) 

All of the reactions cited contribute together to the short half-life of 

NO in the hemovascular system. This remarkable variety of 

reactions is based on an unpaired electron in the so-called “highest 

occupied molecular orbital (HOMO)“, that is, on the outermost 

electron shell. 

.. .. 

. 

:O=N-N=O: 2 :N O: :N O: 

Dinitrogendioxide Nitric oxide Nitrosonium ion 

+ 

-1 e- Fig. 6: Chemical Reactivity of NO 

Nitric oxide is known as a toxic gas. It thus appears very surprising 

that NO controls important cellular processes. But the same 

chemical properties which are responsible for the toxicity of NO are 

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also responsible for the rapid and locally-limited effect of NO as a 

messenger substance. As a small-molecular hydrophobic gas, NO 

penetrates cell membranes more easily than molecular oxygen or 

carbon dioxide, and needs no receptor or transmembranous 

transport mechanism. NO distributes isotropically in the tissue. The 

diffusion coefficient (at 37°C) of NO in water is greater than that of 

O2, CO2 or CO. Due to the high reactivity and the resultant 

extremely short half-life, the biological effect of NO is locally 

limited. The concentration and the biological effect of NO 

decreases radially around the formation site by reaction with 

oxygen. Due to the unpaired electron, as described above, NO binds 

with high affinity to metallic cofactors of enzymes. Activation of 

the guanylate-cyclase has special importance for the vasoactive 

effect and leads to an increase in the intracellular messenger cGMP. 

This produces inactivation of the contractile elements in smooth 

muscle cells of the vascular walls and thus leads to vasodilatation. 

Moreover, nitrinergic neurotransmission in peripheral smooth 

muscle cells, inhibition of blood coagulation (thrombocyte 

aggregation and adhesion) and modulation of synaptic plasticity are 

mediated. 

Intracellular cGMP is catalyzed by the soluble isoform of guanylatcyclase 

(sGC) and has effects on transcription, mRNA translation 

and interacts directly with DNA. A posttranslational modification 

of proteins and inhibition of Fe-dependent enzymes are of central 

importance especially with respect to NO-metabolism. GTP is 

transformed by the key enzyme GTP-cyclohydrolase I to 

tetrahydrobiopterin (BH4), which is an essential cofactor for all 

NOS isoforms. 

Important facts are already known about the importance of NO for 

the microcirculation in the skin. However, it is still unclear whether 

the extensive knowledge gained on macrovascular NO-metabolism 

in recent years can be transposed to the cutaneous microcirculation. 

Some functional and morphological details make it appear that this 

is not entirely possible or permissible. 

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Transmembranous Transport of L-Arginine 

The transmembranous transport of the cationic amino acid Larginine 

occurs with L-ornithin and L-lysin in animal cells largely 

selectively via the stereospecific and sodium, calcium and pHindependent 

Y + -System. The activity of the Y + -system is controlled, 

tension-dependent, via hyperpolarization of the cell membrane and 

is very low under normal conditions. The opening of calciumdependent 

potassium channels thus leads to increased influx of Larginine. 

Moreover, the activity of the Y + -system is increased by 

elevated glucose, insulin or PGI2 concentrations as well as by 

Interleukin-1 (IL-1)- and tumor necrosis factor α (TNFα)-mediated 

release of endotoxin, and reduced by lysophosphatidylcholin (14), 

cationic proteins (15) or hypoxia (16). L-arginine transport is 

effectively inhibited by an excess of cationic arginine-analogues 

and by the eNOS-blockers L-N G -monomethyl-L-arginine (L- 

NMMA) and L-N G -(1-Iminoethyl)-L-ornithin-dihydrochloride (L- 

NIO). It remains unclear, however, whether the Y + -System is 

involved in the regulation of the activity of the NOS-isoforms 

and/or the arginase and the extent to which the extracellular 

arginine concentration influences the expression and activity of the 

Y + -System. Moreover, it is known from in-vitro studies on rat 

hepatocytes (FTO2B) and human vascular smooth muscle cells 

(VSMC) and in vivo- studies on rats that the genes CAT1 and 

CAT2, of which the expression is regulated in various ways 

transcriptional and post-transcriptional, are particularly important. 

Stimulation by lipopolysaccharides (LPS) causes expression of 

CAT1 mRNA and CAT2B mRNA in lung, heart and kidney cells of 

rats. CAT2A mRNA is expressed LPS-independently in the liver. 

Under physiological conditions, the CAT1 mRNA expression in 

liver cells is largely stable. By contrast, stimulation of cell growth, 

elevated insulin levels or application of glucocorticoids leads to 

induction of mRNA expression of CAT1. The molecular 

mechanisms of CAT-expression regulation are, however, totally 

unknown. 

Our team was recently able to prove hCAT1, hCAT2A and B as 

well as hCAT4 in human dermal microvascular endothelial cells 

(HDMEC) and in human native keratinocytes (NHEK) by RT-PCR 

using a gene-specific primer. The expression of the gene was 

------------------------------------------------------------------------------------ 




quantified cell-specifically by densitometric assessment. A high 

expression of hCAT1 and low expression of hCAT2 was found in 

both types of cells examined. By contrast, expression of hCTA4 of 

keratinocytes was considerably higher than in human dermal 

microvascular endothelial cells. Whether one may conclude an 

elevated functional transport capacity for cationic amino acids can 

only be speculated at the moment. Recently, it was demonstrated 

that the CAT3-gene, which had been assumed only in rats and mice, 

is also expressed in humans. In addition to this specific transport 

system, unspecific amino acid transporters are known which 

probably have no relevance for cationic amino acids under 

physiological conditions. Noteworthy in this connection are the A- 

(neutral amino acid transporter), ASC- and B 0+ -systems, which 

require an Na 2+ -gradient on the plasma membrane and the Lsystem, 

which is Na 2+ -independent. Unlike the Y + -system, they 

accept a broader range of substrates, like cationic and neutral amino 

acids. 

extrazellulär 

intrazellulär 

Y + -System 

L-Arginin 

L-Ornithin 

L-Lysin 

L-NMMA 

L-NIO 

Fig. 7: Schema of Y + -System 

+ 

Em 

L-Arginin 

Therapeutic Influence of L-Arginine Metabolism 

Directed influence of pathobiochemical processes requires precise 

knowledge of the physiological regulation mechanisms and 

functional processes. With respect to cutaneous NO-metabolism, 

------------------------------------------------------------------------------------ 




important changes of general pathological importance and also 

disease-specific regulatory deficits can be differentiated. The latter 

are not only dependent on the etiology and pathogenesis of the 

disease itself, but also involve individual pathological and phaseassociated 

differences. 

Intensive research into NO-metabolism has disclosed the following 

relationships as being of particular importance: 

1. cell type-specific expression and activity of the NOSisoforms 

2. cell type-specific expression and activity of the Y+-system 

3. cell type-specific expression and activity of other arginineconsuming 

enzymatic reactions 

4. concentration-time profile of L-arginine and NOS-cofactors 

5. concentration time profile of NOS-inhibiting metabolites 

From a dermatopharmaceutical point of view, two basic principles 

of action arise from the metabolic relationships: NO-induction and 

NO-repression. There are a number of possible indications for both 

action principles. However, special attention should be paid to the 

concentration-dependent effect of NO mentioned earlier. 

For the Induction of NO in the skin, two different procedures are 

possible. In addition to induction of the enzymatic synthesis of NO 

by NOS, NO can also be substituted non-enzymatically. Table 2 

shows practical, relevant examples of these types of reactions. 

Increase in NOS-Expression 

- L-Arginin 

- Tetrahydrobiopterin 

- Calcium 

- Nisoldipin (Dihydropyridin) 

- Oxygen deficiency 

- Carbon dioxide excess 

------------------------------------------------------------------------------------ 


In: Trends Clin Exp Dermatol, Aachen, Shaker, 2003, vol 1 

Non-enzymatic NO-Induction 

- Nitro compounds 

- Sodium nitrite 

- Isosorbitdinitrate 

- Na-nitroprusside 

- direct NO application 

Tab. 2: Examples of substances for therapeutic induction of NO


In practical applications, the following prescriptions have proven 

beneficial (Tab. 3). However, no studies validated according to 

GCP Guidelines are available. 

Prescriptions Indication(s) 

Sodium nitrite 

Ascorbic acid 

(alternative salicylic acid 

Basis Creme DAC 

Isosorbitdinitrate (ISDN) 


Sodium nitroprusside 


L-arginine-HCl 


0.1-0.5 

0.1-0.5 

(0.5) 

ad 10.0 

0.1 

ad 10.0 

0.1 

ad 10.0 

1.0 

ad 10.0 

Application: 

- 1-2x daily under occlusion where 

appropriate 

- separate application of the substances 

is possible 

Indication: 

- Mollusca contagiosa 

- kutane Leishmaniose 

- Tinea pedis 

Application: 

- 2x daily 

Indication: 

- Anal rhagades, fissures 

- Verrucae vulgares 

- Impaired wound healing 

Application: 

- 2x daily 

Indication: 

- Verrucae vulgares 

- Impaired wound healing 

Application: 

- 2-3x daily 

Indication: 

- Mollusca contagiosa 

- erectile dysfunction; aphrodisiac 

Tab 3: Examples for Prescriptions with practical Relevance 

The Blockade of NO in the skin can be realized both by inhibition 

of NO-synthesis and indirectly by inhibition of the substrate 

transport. It must be remembered that a number of already-known 

antiinflammatory drugs very sufficiently inhibit the expression or 

------------------------------------------------------------------------------------ 




activity of the NOS-isoforms directly or indirectly. Such 

mechanisms of action have been described particularly for 

ciclosporin and glucocorticoids. There is also evidence that the heat 

shock protein 90 (HSP90)-blocker geldanamycin leads to a 

therapeutically utile reduction of NO. Moreover, a number of socalled 

specific NOS-blockers have been described in the literature 

which have only rarely been clinically applied. The exceptions are 

some arginine-analogues like N-methyl-L-arginine acetate (NMA, 

L-NMA, L-NMMA) and N-nitro-L-arginine methylester (L- 

NAME). An antipsoriatic or antiinflammatory effect has been 

described for these substances after topical application. Blockade of 

NO-synthesis can also be realized by NADPH- and flavin- or 

tetrahydrobiopterin-antagonists, since the compounds are essential 

cofactors of NOS. Finally, Hem-binding nitro-compounds like 

azoles (antimycotic efficacy) should be cited, which inhibit NOsynthesis 

at least in vitro. 

Finally, it remains to be said that there are numerous situations for 

therapeutic application of substances which directly or indirectly 

influence NO-metabolism. Only future, GCP-validated therapeutic 

studies will show whether and in which diseases such experimental 

forms of therapy are effective and what place they will occupy in 

practical dermatopharmaceuticals. 

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The value of high-frequency sonography in objectifying drug effects 25 

The value of high-frequency sonography in 

objectifying drug effects 

A. Unholzer, H.C. Korting 

Ludwig-Maximilians-Universität München 

Klinik und Poliklinik für Dermatologie und Allergologie 

Frauenlobstr. 9-11 

D-80337 München 

Germany 

The development of high-frequency ultrasound........... 25 

The value of 20 MHz sonography in objectifying 

unwanted drug effects................................................... 26 


wanted drug effects....................................................... 28 

The value of 50 MHz and 100 MHz sonography in 

objectifying drug effects ............................................... 28 

References..................................................................... 29 

The development of high-frequency ultrasound 

In the 1970s, ultrasound was introduced into medicine. Frequencies 

around 3,5 MHz are used for sonographic imaging of the organs of 

the abdomen. In dermatology, high-frequency ultrasound, with 

frequencies of 15 MHz and above, is employed [1]. 

The first ultrasound devices used in dermatology were 15 MHz Amode 

devices. A-(amplitude-)mode systems handle the amplitudes 

of the echoes received in a one-dimensional way [2]. B-(brightness- 

)mode devices visualize the intensity of ultrasound echoes using a 

scale of grey-shades respectively so-called false colours and 

compute a sectional image of the skin [2]. The first 20 MHz B-scan 

systems – DUB 20 by Taberna pro medicum (Lüneburg, Germany) 

and Dermascan C by Cortex Technology (Hadsund, Denmark) – 

were introduced in 1987 [1]. 

------------------------------------------------------------------------------------ 



26 The value of high-frequency sonography in objectifying drug effects 


unwanted drug effects 

High-frequency ultrasound, in particular 20 MHz B-mode 

ultrasound, has proved to be a precise method for objectifying and 

quantifying both unwanted and wanted pharmacological effects on 

the skin [1]. Skin atrophy following long-term application of topical 

glucocorticoids is one of the most important unwanted drug effects 

in dermatology. Usually, it is preceded by a reversible reduction of 

total skin thickness, so-called preatrophy [3]. 20 MHz B-scan 

sonography enabled us and other working groups to demonstrate 

that nonhalogenated glucocorticoid C-17,21 double esters such as 

prednicarbate or hydrocortisone aceponate reduce total skin 

thickness less than conventional, halogenated glucocorticoids such 

as betamethasone 17-valerate or clobetasol 17-propionate [4-7]. The 

nonhalogenated glucocorticoid C-17,21 double esters exert an antiinflammatory 

activity comparable to that of betamethasone 17valerate 

[8]. Thus, the nonhalogenated glucocorticoid C-17,21 

double esters are topical glucocorticoids with an improved 

benefit/risk ratio [3, 9]. 

In several randomized, double-blind clinical trials, the skin thinning 

effect of prednicarbate 0.25 % cream, hydrocortisone 1 % cream 

and the medium-potent halogenated glucocorticoid mometasone 

furoate 0.1 % cream did not differ clearly from that of the base 

preparation, but was less than that with betamethasone 17-valerate 

0.1 % or clobetasol 17-propionate 0.05 % cream [4-6]. Similarly, 

prednicarbate 0.25 % ointment and hydrocortisone aceponate 0.1 % 

ointment reduced skin thickness not more than vehicle, whereas 

betamethasone 17-valerate ointment proved to be more 

atrophogenic [5]. 

These studies were performed with an explorative approach. In an 

explorative study, data are analyzed after the conduct of the clinical 

trial. In a confirmatory study, a null hypothesis is formulated before 

the clinical trial is performed. Recently, we compared the skinthinning 

effect of prednicarbate 0.25 %, betamethasone17-valerate 

0.1 % and mometasone furoate 0.1 % ointments to that of a the 

vehicle corresponding to the prednicarbate 0.25 % ointment in a 

double-blind, placebo-controlled randomized trial with a 

confirmatory approach [10]. Over a six week period, 24 healthy 

------------------------------------------------------------------------------------ 




volunteers non-occlusively applied 100 mg of each ointment twice 

daily to the volar aspects of their forearms. Total skin thickness was 

measured weekly using a 20 MHz B-scan ultrasound device. 

Change in total skin thickness relative 

to baseline 

0% 

-5% 

-10% 

-15% 

-20% 

-25% 

day 1 day 8 day 15 day 22 day 29 day 36 day 43 

Vehicle 

Prednicarbate 0.25 % 

Mometasone furoate 0.1 % 

Betamethasone 17-valerate 0.1 % 

Time 

Fig. 1: Time course of total skin thickness reduction, relative to 

baseline in percent, during treatment with three different topical 

glucocorticoid ointment preparations and a vehicle preparation. On 

day 36, total skin thickness was reduced by a mean of 1 % in test 

fields treated with vehicle; for prednicarbate, mometasone furoate 

and betamethasone 17-valerate, the relative decreases amounted to 

13 %, 17 % and 24 %, respectively. From [10], with permission. 

The percental decrease of total skin thickness in relation to baseline 

was calculated for each test area and for each time of measurement. 

The differences to baseline were compared intraindividually, and 

the null hypothesis H0: „Reduction of total skin thickness in the test 

areas treated with prednicarbate, mometasone furoate, 

betamethasone 17-valerate and the ointment vehicle is the same (µ1 

= µ2=µ3=µ4)“ was tested. Application of betamethasone 17valerate 

and mometasone furoate led to a decrease of total skin 

------------------------------------------------------------------------------------ 




thickness by 24 % and 17 %, respectively, on day 36 (figure 1). The 

skin thinning effects of betamethasone 17-valerate and mometasone 

furoate were clearly more marked than that of prednicarbate, the 

differences being statistically highly significant with p < 0.0001 and 

p = 0.0061, respectively. Prednicarbate ointment still reduced total 

skin thickness to a higher extent than its vehicle (13 % versus 1 % 

on day 36; p < 0.0001; figure 1). 

The value of 20 MHz sonography in objectifying wanted 

drug effects 

20 MHz B-mode ultrasound is appropriate not only for objectifying 

and quantifying unwanted drug effects, but also for objectifying 

certain wanted pharmacological effects on the skin. For example, 

the clinical improvement of atopic eczema during therapy can be 

confirmed sonographically: In this disease, total skin thickness is 

usually increased, and the upper part of the dermis appears echopoor, 

due to oedema and inflammatory infiltrate [11]. Topical 

application of a glucocorticoid, i. e. of mometasone furoate, for 14 

days leads to normalization of both total skin thickness and 

echodensity of the dermis [11]. 

In cutaneous radiation fibrosis, total skin thickness and the 

echodensity of the dermis are increased, and the extension of the 

subcutaneous fatty tissue is reduced [11]. In a patient suffering from 

cutaneous radiation fibrosis as a complication of radiation therapy 

on account of a mammary carcinoma, oral treatment with 

pentoxifyllin (3 x 400 mg per day) and vitamin E (1 x 400 mg per 

day) induced a reduction of fibrosis and a continuous decrease in 

total skin thickness from the sixth month onward, amounting to 25 

% of the previous skin thickness after six months and 40 % after 

eight months [12]. 

The value of 50 MHz and 100 MHz sonography in 

objectifying drug effects 

Total skin thickness, i. e. the thickness of dermis plus epidermis, 

can be determined precisely and reproducibly by means of 20 MHz 

B-mode ultrasound. However, the resolution of 20 MHz 

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sonography (200 µm to the side and 75-80 µm to the depth [2, 11]) 

is not high enough for imaging the comparatively thin epidermis 

[1]. This problem might be solved by the introduction of systems 

with higher frequency and thus higher resolution. In 1989, the 

working group around Hoffmann developed a 50 MHz B-scan 

ultrasound device with an axial and lateral resolution of 37.5 µm 

and 120 µm, respectively [13]. Their 100 MHz system, introduced 

in 1993, offers a resolution of 11 µm to the depth and 30 µm to the 

side [14]. These devices make sonographic imaging of the 

epidermis possible. Evidence from first applications in monitoring 

the course of psoriasis vulgaris during therapy suggests that both 50 

MHz and 100 MHz ultrasound are appropriate for the evaluation of 

drug effects on the skin [13-15]. 100 MHz sonography even allows 

quantification of changes in the stratum corneum after occlusive 

application of different topical preparations (e. g. petrolatum, waterin-oil 

emulsion, oil-in-water emulsion and water) to the 

dermatoglyphic-bearing skin of the fingertip [16]. It could be 

demonstrated that a higher water content leads to faster and more 

marked swelling of the stratum corneum [16]. 

In conclusion, 20 MHz B-scan sonography is a proven method for 

objectifying and quantifying drug effects in dermatology. 50 MHz 

and 100 MHz B-mode ultrasound are very promising new 

technologies. It remains to be seen whether they will prove to be 

superior to 20 MHz B-scan sonography in the evaluation of drug 

effects on the skin. 

References 

1 Unholzer A, Korting HC (2002) High-frequency ultrasound in 

the evaluation of pharmacological effects on the skin. Skin 

Pharmacol Appl Skin Physiol 15:71-84 

2 Hoffmann K, El Gammal S, Altmeyer P (1990) B-scan- 

Sonographie in der Dermatologie. Hautarzt 41:W7-W16 

3 Schäfer-Korting M, Korting HC, Kerscher MJ, Lenhard S 

(1993) Prednicarbate activity and benefit/risk ratio in relation to 

other topical glucocorticoids. Clin Pharmacol Ther 54:448-456 

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4 Korting HC, Vieluf D, Kerscher M (1992) 0.25% prednicarbate 

cream and the corresponding vehicle induce less skin atrophy 

than 0.1% betamethasone-17-valerate cream and 0.05% 

clobetasol-17-propionate cream. Eur J Clin Pharmacol 42:159- 

161 

5 Kerscher MJ, Korting HC (1992) Topical glucocorticoids of the 

non-fluorinated double-ester type. Lack of atrophogenicity in 

normal skin as assessed by high-frequency ultrasound. Acta 

Derm Venereol 72:214-216 

6 Kerscher MJ, Hart H, Korting HC, Stalleicken D (1995) In vivo 

assessment of the atrophogenic potency of mometasone furoate, 

a newly developed chlorinated potent topical glucocorticoid as 

compared to other topical glucocorticoids old and new. Int J Clin 

Pharmacol Ther 33:187-189 

7 Lévy J, Gassmüller J, Schröder G, Audring H, Sönnichsen N 

(1994) Comparison of the effects of calcipotriol, prednicarbate 

and clobetasol 17-propionate on normal skin assessed by 

ultrasound measurement of skin thickness. Skin Pharmacol 

7:231-236 

8 Schackert C, Korting HC, Schäfer-Korting M (2000) Qualitative 

and quantitative assessment of the benefit-risk ratio of medium 

potency topical corticosteroids in vitro and in vivo. 

Characterisation of drugs with an increased benefit-risk ratio. 

Biodrugs 13:267-277 

9 Schäfer-Korting M, Schmid M-H, Korting HC (1996) Topical 

glucocorticoids with improved risk-benefit ratio. Rationale of a 

new concept. Drug Safety 14:375-385 

10 Korting HC, Unholzer A, Schäfer-Korting M, Tausch I, 

Gassmueller J, Nietsch K-H (2002) Different skin thinning 

potential of equipotent medium-strength glucocorticoids. Skin 


11 Korting HC, Gottlöber P, Schmid-Wendtner M-H, Peter RU 

(1996) Ultraschall in der Dermatologie. Ein Atlas. Blackwell, 

Berlin 

12 Gottlöber P, Krähn G, Korting HC, Stock W, Peter RU (1996) 

Behandlung der kutanen Strahlenfibrose mit Pentoxifyllin und 

Vitamin E – Ein Erfahrungsbericht. Strahlenther Onkol 172:34- 

38 

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13 El Gammal S, Auer T, Popp C, Hoffmann K, Altmeyer P, 

Passmann C, Ermert H (1994) Psoriasis 

14 vulgaris in 50 MHz B-scan ultrasound – Characteristic features 

of stratum corneum, epidermis and dermis. Acta Derm Venereol 

Suppl 186:173-176 

15 El Gammal S, El Gammal C, Kaspar K, Pieck C, Altmeyer P, 

Vogt M, Ermert H (1999) Sonography of the skin at 100 MHz 

enables in vivo visualization of stratum corneum and viable 

epidermis in palmar skin and psoriatic plaques. J Invest 

Dermatol 113:821-829 

16 Vaillant L, Berson M, Machet L, Callens A, Pourcelot L, Lorette 

G (1994) Ultrasound imaging of psoriatic skin: A noninvasive 

technique to evaluate treatment of psoriasis. Int J Dermatol 

33:786-790 

17 Kaspar K, Vogt M, Ermert H, Altmeyer P, El Gammal S (1999) 

100-MHz-Sonographie zur Darstellung des Stratum corneum an 

der Palmarhaut nach Anwendung verschiedener Externa. 

Ultraschall in Med 20:110-114 

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32 Bioengineering of the skin: non-invasive methods for the evaluation of efficacy 

Bioengineering of the skin: non-invasive methods 

for the evaluation of efficacy 

T. Gambichler, F.G. Bechara, M. Stücker, K. Hoffmann, P. Altmeyer 


Ruhr-University Bochum 

Gudrunstr. 56 

D-44791 Bochum 

Germany 

Introduction................................................................... 32 

Bioengineering of different skin functions ................... 33 

• Skin hydration.............................................................. 33 

• Transepidermal water loss (TWL)............................... 34 

• Skin-pH........................................................................ 34 

• Skin surface lipids........................................................ 34 

• Skin color..................................................................... 35 

• Skin elasticity............................................................... 35 

• Transcutaneous pO2 and pCO2 and microcirculation .. 36 

Imaging methods in the investigation of the skin......... 36 

• Capillary microscopy and thermography..................... 36 

• Laser Doppler fluxmetry/scanning .............................. 37 

• Profilometry................................................................. 37 

• High-frequency sonography ........................................ 38 

• Optical coherence tomography (OCT) ........................ 38 

• Confocal laser scanning microscopy (CLSM)............. 40 

Conclusions................................................................... 41 

References..................................................................... 42 

Introduction 

Classic dermatology is based on the observation of characteristics 

that can be appreciated by sensory perception. Dermatologists learn 

to recognize and classify lesions to identify stereotypes that permit 

diagnosis. That is why many dermatologists have not previously 

seen the necessity to develop instruments to detect skin changes. 

Their particular methodology has led some dermatologists to the 

conviction that their perception is superior to that of any apparatus. 

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Bioengineering of the skin: non-invasive methods for the evaluation of efficacy 33 

Nowadays dermatologists have started to make use of methods and 

research tools to aid diagnosis. Increasingly sophisticated 

techniques in skin exploration in a non-invasive way supply 

biophysical parameters of skin characteristics detected by analog 

methods. In particular in the field of dermatopharmacological 

research functional and morphological changes of the skin have to 

be recorded accurately in order to study the effect of therapeutic 

agents on the skin [1-3]. In the following a brief overview of 

selected bioengineering methods used in dermatopharmacology is 

provided. 

Bioengineering of different skin functions 

• Skin hydration 

The measurement of skin hydration is based on the electrical 

impedance of the horny layer (corneometry). The impedance of the 

horny layer measured by two high-grade steel electrodes is 

reciprocally proportional to the water content of the horny layer 

[4,5]. The measured conductibility is directly converted into 

numeric values corresponding to the relative moisture of the horny 

layer. Skin hydration can also be assessed by high-frequency 

currents, infrared spectroscopy, and nuclear magnetic resonance. 

These procedures are however less common and much more 

expensive than simple corneometry. The methods mentioned above 

can be employed in the evaluation of hydrating and/or dehydrating 

effects of topical formulations as applied in dermatology and 

cosmetology. Corneometry is frequently used in the assessment of 

effectiveness and tolerability of various therapeutics and care 

products with regard to inflammatory diseases such as atopic 

eczema and psoriasis. Corneometry finds also broad application in 

cosmetology recording the effects of skin cleaning products and 

moisturizers. 

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• Transepidermal water loss (TWL) 

The evaporimetric measurement of TWL is based on the law of 

water diffusion. The probe measures the temperature and relative 

humidity. By that, the partial steam pressure in the air is determined 

in two different levels. The inference onto the water vaporization 

and/or the TWL occurs about the gradient of the partial pressure 

that is directly proportional to the degree of vaporization (6). The 

TWL reflects the function of the skin barrier. Analogous to 

corneometry, evaporimetry is a basic method for monitoring 

eczematous skin diseases (1,2). Both, the efficacy of skin protection 

products and irritative potential of creams and effects of skin 

cleaning products, can accurately be examined by evaporimetry. 

• Skin-pH 

Since the skin surface with its secretion and high water content 

corresponds to the qualities of a diluted solution, it is well suitable 

for direct skin pH measurements. The potentiometric assessment is 

based on electrochemical processes on the skin surface. The pH 

value is expressed as the logarithmically reciprocal value of the 

concentration of free hydrogen ions (1). Skin pH measurement is 

elementary in the evaluation of cosmetic products (e.g., soaps, 

shampoos, deodorants). 

• Skin surface lipids 

The quantitative determination of the skin surface lipids (sebum) is 

usually carried out via the spectrophotometric method. An opaque 

plastic film is pressed onto the skin and coated with sebum. The 

resulting increased transmission of the film is measured 

spectrophotometrically [1]. With a precision balance, measuring the 

absorbent fat content of a paper strip, being pressed on the skin 

before, the sebum content can also be determined (gravimetry). The 

assessment of the sebum content of the skin surface is 

predominantly used in cosmetology for skin type determination. 

The method is also employed in the evaluation of cosmetic products 

that have a potentially altering influence on the formation of skin 

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surface lipids. In the same way the effect of topical or systemic 

retinoids can be investigated (e.g., monitoring of acne vulgaris) [2]. 

• Skin color 

The colorimetry is based on the tristimulus analysis of light that is 

reflected from the skin. The colors are expressed in a threedimensional 

coordinate system with black-white axis (L*), greenred 

axis (a*), and yellow-blue axis (b*). Another method frequently 

used is based on spectrophotometric measurements of the relevant 

chromophores (melanin, hemoglobin). The diodes employed emit 

light at 568 nm and 655 nm onto the skin. The reflected light 

encounters the photodetector and the calculation of the melanin and 

erythema index is provided, respectively [7]. The measurement of 

color, in particular the erythema index, is often used during therapy 

to quantify the cutaneous findings of inflammatory diseases (e.g., 

atopic eczema, psoriasis). Correspondingly, adverse effects of 

topical and systemic therapeutics can be quantified [8,9]. 

• Skin elasticity 

The mechanical properties of the skin are influenced by maturation, 

aging, and various cutaneous disorders. A couple of methods for 

measurement of biomechanical qualities of the skin have been 

proposed [10]. Current devices induce measurable deformation of 

the skin by torsion, pressure, or suction. A common technique for 

the quantification of skin elasticity is based on the use of a vacuum 

pump that is put vertically on the skin surface. The deformation of 

the skin within a test area of three mm² is measured. The resultant 

deformation curve is characterized by an elastic, a visco-elastic and 

a viscous part. This method is particularly suitable for the 

monitoring of diseases that cause skin induration (e.g., 

dermatosclerosis) or hyperelasticity of the skin (e.g., Ehlers-Danlos- 

Syndrom). In addition, the efficacy of anti-aging products can be 

examined [11-13]. 

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• Transcutaneous pO2 and pCO2 and microcirculation 

In the quantification of the cutaneous pO2 and pCO2, both the 

systemic blood gases and the function of the nutritive vasculature of 

the skin is objectively assessed. The measurement of the pO2 is 

performed under constant temperature by means of a thermoelectrode 

with integrated cathode and anode. The aperture of the 

electrode is covered with a membran pervious to oxygen. Combined 

probes enable simultaneous measurement of the pO2 and the 

transcutaneous pCO2. Laser-doppler devices with an integrated 

video microscopic function are also employed. In addition, the O2reflection 

spectroscopy is used in the quantification of the 

cutaneous oxygen content per tissue unit. Therefore, a spectrum 

analysis of reflected light is used in examining the oxygenation of 

the hemoglobin. These methods are applied in 

dermatopharmacology, particularly in monitoring of arterial and 

venous circulation malfunctions [14]. 

Imaging methods in the investigation of the skin 

• Capillary microscopy and thermography 

The capillary microscopy is a direct technique in qualitative and 

quantitative investigation of the capillaries in the papillary dermis. 

An optical microscope (magnification: 50-200) equipped with 

specific spectral filters and a cold light source is used. Changes of 

microcirculation can also be measured indirectly by means of 

thermographical methods. The infrared thermography is employed 

in the measurement of the skin surface and enables selective 

determination of absolute temperature levels, averaged temperature 

over a whole skin site, and the distribution of temperature. Capillary 

microscopy and thermography are frequently used for the 

investigation of disorders of microcirculation as observed in 

collagenoses (e.g., scleroderma). Effects of vasoconstrictive 

substances (e.g., nicotine acid) can be well established with 

thermography [14]. 

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• Laser Doppler fluxmetry/scanning 

Laser Doppler flow analysis is one of the most commonly used 

method in the evaluation process of cutaneous microcirculation. 

The method is based on measuring the velocity of blood flow in the 

capillaries by Doppler effect. A laser beam (e.g., helium, neon) 

guided by an optical fiber interacts with skin structures. The 

stationary tissue components reflect the light at the same 

wavelength, whereas those in movement (e.g., erythrocytes) reflect 

it at a different wavelength. Measuring with unidimensional laser 

Doppler fluxmetry the blood flow is selectively assessed by means 

of a single probe, whereas laser Doppler scanning enables 

systematic measurement of a larger skin site. Thereby, a twodimensional 

perfusion pattern is given [14,15]. In 

dermatopharmacology laser Doppler techniques are frequently used 

in appraising the efficacy of topical and systemic therapeutics. 

Analogous to the classic circulation malfunctions (e.g., 

atherosclerosis, chronic venous insufficiency) disorders of 

microcirculation (e.g., Raynaud’s syndrome, dermatosclerosis, 

dermatomyositis) can be evaluated with these techniques out of 

diagnostic reasons and in monitoring therapy. In addition 

experimentally caused erythema can be quantified. In numerous 

studies on patients with chronic inflammatory skin diseases laser 

Doppler methods were employed in the assessment of therapeutic 

and pharmacological effects [16-20]. 

• Profilometry 

The surface of the skin is frequently described with the term 

"roughness". Apart from direct mechanical-electronical scanners, 

indirect optical procedures are used for the determination of skin 

roughness. Besides interferometry, transmission profilometry, 

holography, and laser profilometry are frequently employed today. 

By means of these methods the effectiveness of anti-aging 

substances (e.g., topical retinoids) can be quantified. Changes of 

skin roughness caused by cosmetic products, hormone-containing 

emollients, alpha-hydroxy acids, and peeling substances are 

investigated via profilometric methods. Furthermore laser 

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profilometry is performed in measuring the therapeutic outcome in 

inflammatory skin diseases such as psoriasis [21,22]. 

• High-frequency sonography 

The high-frequency sonography provides information concerning 

the spatial (axial and lateral) spreading of tumerous and 

inflammatory processes of the skin. Today, the 20 MHz 

sonographie is well established in dermatological clinics and skin 

research centers. The most important physical characteristics of 20 

MHz sonography include the skin depth of eight mm, the axial 

solution of 80 µm, and the lateral solution of 200 µm. Ultrasound 

transducers with higher medium frequencies (e.g., 50 MHz, 100 

MHz) offer on the one hand increasing resolution, on the other hand 

decreased depth. Thus, in particular morphology of the epidermis 

can be investigated with 50 MHz and 100 MHz scanners. However, 

only a small number of specialised centers are equipped with these 

instruments. High-frequency sonography of inflammatory diseases 

such as psoriasis vulgaris, lichen ruber planus, and diverse forms of 

eczema showes characteristic echo-poor areas in the dermis. It 

represents, both the acanthosis of the epidermis and the dermal 

infiltration[19,23,24]. Thus a distinction between acanthotic 

epithelium and subepithelial infiltration cannot be made. Evaluation 

of micromorphological structures and their differentiation does not 

succeed in 20 MHz sonography. Nevertheless this method helps to 

objectify therapeutic effects, being observed during the treatment of 

dermatosclerosis-like skin modifications. Changes of skin thickness 

and dermal echogenicity are of special interest in therapy 

monitoring [25-27]. Pharmacological effects of anti-aging 

substances can also be examined with the high-frequency 

sonography [23]. 

• Optical coherence tomography (OCT) 

OCT is a relatively new non-invasive imaging tool in the 

investigation of epidermal morphology. The procedure is based on 

the principle of interference of coherent light waves in a scattering 

medium (Michelson interferometry). Infrared light (830 and/or 

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1300 nm) of a short coherence length (≅ 15 mm) is divided into 

both a reference beam and a test beam. The reflected reference light 

is compared with the reflected light of the skin tissue. The skin 

depth is about one mm. Structures smaller than ten mm can be 

established with OCT. Instruments with a lateral resolution of four 

mm and an axial resolution of seven mm have already been used in 

dermatopharmacological studies [28,29]. Because of the high 

resolution of OCT, structures in the horny layer and the vital 

epidermis up to the papillary dermis can be studied precisely. 

Fig. 1: Optical coherence tomography (OCT) of a pigmented nevus 

on the forearm. Clear demarcation of the dermal papillae and rete 

ridges. 

Experimental investigations on dimethyl-sulfate-oxide (DMSO) 

revealed, that in spite of clinically hardly perceptible wheals, the 

OCT showed clearly hyporeflective areas in the epidermis. These 

areas most probably corresponded to a spongiosis. Accordingly, it 

has previously been demonstrated that this method is suitable to 

establish spongiotic and vesicular epidermal changes in patients 

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with clinically inapparent dishydrotic eczema. Furthermore typical 

histological changes of psoriasis such as thickening of the horny 

layer and acanthosis can be demonstrated and quantified by means 

of OCT. These observations have been confirmed by experimental 

studies on contact dermatitis. Furthermore, keratolytic effects on 

palmoplantar skin can also be evaluated accurately. Moisturising 

preparations (e.g., urea-containing emollients) enhance the waterbinding 

capacity of the horny layer, whose thickening can be shown 

by means of OCT. Atrophogenic effects can easily be quantified 

through this method [28,29]. 

• Confocal laser scanning microscopy (CLSM) 

CLSM represents a new non-invasive bioengineering tool, which is 

used in the investigation of both functional and morphological 

changes of the skin. It allows skin imaging of a virtual section on 

the basis of living tissue. The Vivascope 1000 (Lucid Inc., USA) is 

frequently employed for this purpose. The system uses a laser 

source with wavelength of 830 nm, an illumination power up to 20 

mW on the object, a water immersion objective and an imaging rate 

of 20 per second. The field of view shows 128µm × 260µm. Its 

lateral resolution is 0.4µm, the vertical resolution is roughly 1.9µm. 

The microscope allows imaging of the epidermis and papillary 

dermis on a cellular level (a maximum of 250 µm skin depth). 

Unlike the high-frequency sonography and OCT, the CLSM 

produces horizontal image sections. Due to the high resolution 

single cells and cell structures can be demonstrated (for example 

cell nuclei, pigmented granula, capillaries, blood flow, membraneprotein-complexes). 

This method was already validated in the 

course of comparative studies on histological section series. 

Epidermal thickness and thydration of all epidermal cell 

populations can be quantified with the CLSM. Additionally 

different cell types can be distinguished from each other and the 

evalutation of differentiation disorders of cells is possible. Both 

inflammatory and scleroderma-like skin changes can be 

demonstrated and quantified by means of CLSM (30-33). Apart 

from the great advances of CLSM in non-invasive skin tumor 

diagnostics this technique has an extremly high potential in the field 

of dermatopharmacological and cosmetic research. 

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Fig. 2: Dermal papilla with dilated capillaries and erythrocytes in 

psoriatic skin investigated by confocal laser scanning microscopy in 

vivo (CLSM) [K. Sauermann, Hamburg, Germany]. 

Conclusions 

This brief overview of selected bioengineering methods has 

provided an insight into possibilities and the broad spectrum of noninvasive 

methods in the skin investigation. The use of 

bioengineering is of great advantage in modern medicine, as the 

parameters investigated can accurately be quantified with noninvasive 

objective procedures and can be subsequently evaluated 

and stored electronically. In addition, morphological and functional 

skin parameters can be investigated which are inaccessible to 

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simple clinical investigation. In dermatopharmacology as well as 

cosmetology, bioengineering is therefore indispensable in 

objectifying the efficacy and side effects of topical and systemic 

agents. The techniques vary from relatively simple methods to 

sophisticated and cost-intensive methods. Today, in 

dermatopharmacology as well as cosmetology bioengineering 

represent established components in evaluation pharmacological 

effects on the skin. Some selected methods are suitable in the 

investigation of specific functional parameters of the skin (e.g., 

sebum, pH, moisture) as relevant in testing cosmetic products. 

Other methods provide objective data on morphological structures 

of the skin. Especially the currently established skin imaging tools 

enable a differentiated insight into structural and 

micromorphological skin changes. Prior to starting 

dermatopharmacological investigations the foreknowledge on 

possible effects as well as adverse effects of pharmacological 

substances used is essential for the correct choice of a research tool. 

The characteristically functional and/or morphological alterations of 

different skin diseases can precisely be examined with adequate 

methods under standardized conditions. Special attention needs to 

be turned to the experimental protocol, study sample, and test 

conditions [34]. Further improvement and progress in 

bioengineering, in particular skin imaging, is ongoing. The fusion 

of different techniques - for example - the implementation of laser 

Doppler techniques into high-frequency sonography systems, or a 

combination of OCT with high-frequency sonography – is in 

development. However, all recent developments and sophisticated 

methods cannot replace the dermatological expertise and 

physician’s intuition. 

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Dermatol 40:708-713 

33 Rajadhyaksha M, Gonzalez S, Zavislan JM, Anderson RR, 

Webb RH (1999) In vivo confocal scanning laser microscopy of 

human skin II: advances in instrumentation and comparison with 

histology. J Invest Dermatol 113:293-303 

------------------------------------------------------------------------------------ 




34 Serup J (1995) Bioengineering and the skin: standardization. 

Clin Dermatol 13:293-297 

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The Repetitive Washing Test as a Model for the Evaluation of Barrier Creams 47 

The Repetitive Washing Test as a Model for the 

Evaluation of Barrier Creams 

W. Gehring 

Hautklinik am Klinikum der Stadt Karlsruhe gGmbH 

Moltkestr, 120 

D-76133 Karlsruhe 

Germany 

Introduction................................................................... 47 

Principle of the repetitive washing test......................... 48 

Method .......................................................................... 48 

Emulsions as barrier creams ......................................... 49 

Modulation of an o/w-emulsion as a barrier cream by 

addition of glycerol and urea ........................................ 49 

Skin protection by Ectoin ............................................. 50 

Hydrating effect of Dexpanthenol ................................ 50 

Summary....................................................................... 50 

References..................................................................... 50 

Introduction 

The repetitive washing test shall be presented as a new method to 

evaluate barrier creams and to demonstrate the efficacy of 

moisturisers (1, 2). The method is based on the following 

procedure: Any washing procedure may lead to a disturbance of the 

barrier function, because tensides are able to emulgate barrier lipids 

and to disturb the bilamellary structure of the barrier lipids, which is 

important for the water binding capacity of the stratum corneum. 

This disturbance of the barrier lipids can be documented as an 

increase in transepidermal water loss and as a reduction in horny 

layer moisture. 

------------------------------------------------------------------------------------ 



48 The Repetitive Washing Test as a Model for the Evaluation of Barrier Creams 

Principle of the repetitive washing test 

The principle of the repetitive washing test is the standardisation 

and maximisation of toxic irritant influences of a washing 

procedure. Thus comparative studies can be performed in a state 

that is similar to the situation encountered under frequent exposure 

of the skin to cleansing agents. This test method is specially suited 

for the evaluation of barrier creams used to protect the skin from 

hydrophilic irritants. 

Method 

The repetitive washing test can be performed on one single day or 

on several consecutive days. The repeated washing procedures 

represent a maximisation process simulating a situation usually 

encountered in occupational dermatology when the skin is exposed 

intensively to cleansing agents. The test is performed on the volar 

side of both forearms using 0.01 n SLS (Fig 1). For the 

standardisation of the washing process the test persons use a foam 

rubber roll with a diameter of 3.5 cm and a length of 5 cm to which 

a handle is fixed. A piece of lead is fixed within the hollow handle 

to achieve a weight of the roll of 200 g. For the washing procedure 

the roll is soaked with the cleansing solution. Subsequently it is 

moved 50 times back and forth over the test area with the pressure 

of its own weight. Afterwards the forearms are rinsed shortly with 

lukewarm water to remove the residues of the tensides from the 

skin surface. Finally the forearms are carefully dried with a paper 

towel. 

To evaluate the effect of barrier creams the test preparations are 

applied directly before the washing procedure. A prevention or 

reduction of the increase in TEWL produced by the washing 

procedure documents the efficacy of a barrier cream. Often the 

decrease in TEWL is accompanied by an improvement of the 

Stratum corneum hydration. 

The efficacy of moisturisers is expressed as an increase in Stratum 

corneum hydration which can be demonstrated best if the test 

------------------------------------------------------------------------------------ 




preparations are applied after the washing procedure which causes a 

deficiency in the moisture content of the skin. 

Emulsions as barrier creams 

The application of emulsions alone produces an increase in skin 

hydration (Fig. 2). For this effect it is of minor importance which 

emulsion system is used. The water content determines the degree 

of moisturising. It makes no difference for the increase in Stratum 

corneum hydration whether the same amount of water is applied in 

a w/o or an o/w emulsion (3). For the effect as barrier cream, the 

situation is different. In a repetitive washing test with SLS it could 

be shown that only a w/o emulsion can form an efficient protective 

barrier (Fig. 3). There is no protective effect of an o/w emulsion 

against the washing procedure (Fig. 4). This is the reason for the 

basic demand for w/o systems as skin protection of sensitive skin 

against hydrophilic irritants. 

Modulation of an o/w-emulsion as a barrier cream by 

addition of glycerol and urea 

The addition of glycerol and urea to an o/w-emulsion doesn't 

improve only the hydration of the Stratum corneum (Fig. 5) but also 

the protective effect against cleansing agents (3). Thus, an excellent 

cosmetic acceptance can be combined with the formation of an 

efficient protective barrier equalling that produced by a w/o 

emulsion (Fig. 6 and 7). A combination of 5% urea and 5% glycerol 

also is of advantage concerning the protective effect. The practical 

importance of this observation for care and protection in sensitive 

skin has been confirmed by a study of Grunewald et. al. where the 

situation encountered in occupations where the skin is exposed to 

prolonged or repeated water contact was mimicked. In an in-use test 

with five daily washing procedures on eight consecutive days with 

intermitting application of an o/w emulsion containing 5% urea and 

5% glycerol an almost complete prevention of irritation as 

compared with unprotected skin could be demonstrated (4). 

------------------------------------------------------------------------------------ 



50 The Repetitive Washing Test as a Model for the Evaluation of Barrier Creams 

Skin protection by Ectoin 

For the substance ectoin a stabilising effect on membranes is 

expected. In a study which is not yet published we could 

demonstrate that pre-treatment with a micro-emulsion containing 

ectoin had a stabilising effect on the epidermal barrier function 

shown by a decrease of TEWL in a repetitive washing test. This 

observation justifies the use of ectoin for sensitive skin (Fig. 8). 

Hydrating effect of Dexpanthenol 

After the experimental destruction of the epidermal barrier function 

which was accompanied by a loss of Stratum corneum hydration in 

a repetitive washing test over 5 days, Dexpanthenol increased the 

skin's moisture content (5). In spite of the repetitive washing 

procedures the Stratum corneum hydration increased on the third 

day. This effect could clearly attributed to Dexpanthenol. On the 

fifth day of the repetitive washings the loss of hydration of the 

Stratum corneum was markedly reduced (Fig. 9). 

Summary 

Among various test methods, the repetitive washing test has shown 

its usefulness for the evaluation of the efficacy of barrier creams 

and their modulation. Furthermore the test can be applied to 

investigate the hydrating effects of certain substances. 

References 

1 Gehring W., Gloor M., Kleesz P.: Predictive Washing Test for 

Evaluation the Individual Eczema Risk. Contact Dermatitis 39, 

8-13, 1998 

2 Gehring W., Gloor M.: Der repetitive Waschtest als Modell zur 

Beurteilung von Hautschutzpräparaten am Beispiel einer 

dexpanthenolhaltigen Formulierung. Akt. Dermatogie, 27, 279- 

284, 2001 

------------------------------------------------------------------------------------ 




3 Bettinger J., Gloor M., Gehring W.: Influence of a pretreatment 

with emulsions on the dehydration of the skin by surfactants. Int. 

J. Cosm. Sci. 16, 53-60, 1994 

4 Grunewald A.M., Gloor M., Bettinger J., Gehring W., Kleesz P.: 

Barrier creams: Commercially available barrier creams versus 

urea- and glycerol- containing oil in water emulsions. 

Dermatosen 43, 69-74, 1995 

5 Gehring W., Gloor M., Der Effekt von Dexpanthenol bei 

experimentell geschädigter Haut. Z Hautkr. 76,1-7, 2001 

------------------------------------------------------------------------------------ 



52 Impact of Topical Skin Care and Maintenance Products on Stratum Corneum Barrier 

Impact of Topical Skin Care and Maintenance 

Products on Stratum Corneum Barrier 

M. Gloor 

Hautklinik am Klinikum der Stadt Karlsruhe gGmbH 

Moltkestr, 120 

D-76133 Karlsruhe 

Germany 

Preventive Skin Protection............................................ 53 

Regenerative Skin Protection........................................ 53 

A Case Apart: Aluminum Chloride .............................. 55 

References..................................................................... 55 

The structure of the human stratum corneum resembles a brick wall, 

with the offset between corneocyte layers conferring greater 

mechanical stability than the columnar structure of the horny layer 

found in some animal species. This brick wall structure causes 

substantial lengthening of the intercellular permeation pathway 

used by both outbound diffusion of water (transepidermal water 

loss) and inbound penetration of most topical drugs and harmful 

substances in crossing the stratum corneum barrier. The barrier 

function of the stratum corneum results from the lamellar structure 

of the stratum corneum intercellular lipids. These polar lipids are 

aligned so that the lipophilic ends and the hydrophilic ends are next 

to each other, resulting in multilamellar layers in which a lipophilic 

layer alternates with a hydrophilic layer. The hydrophilic layers are 

characterized by their water-holding capacity. The lipophilic layers 

can switch between a solid crystalline state, a gel crystalline state, 

and a liquid crystalline state. When in the solid crystalline state, 

skin lipids can prevent drug penetration. Some ingredients of 

topical skin products, glycerol in particular, can modify the stratum 

corneum lipid crystallization state, which, however, depends also 

on numerous other factors including temperature [1,2]. 

Atopic dermatitis is characterized by a change in barrier lipids. 

There is a relative lack of ceramides, above all of ceramide 1, 

thought to have a key role in maintaining stratum corneum barrier 

function. Reduced barrier regeneration after stratum corneum 

------------------------------------------------------------------------------------ 



Impact of Topical Skin Care and Maintenance Products on Stratum Corneum Barrier 53 

compromise appears to be a major underlying problem in atopic 

dermatitis sufferers. The physiological correlate of those 

biochemical changes is the known reduction in stratum corneum 

moisture content and the known increase in transepidermal water 

loss in atopic dermatitis patients [3]. Atopic dermatitis sufferers 

therefore feel a particular need for the use of skin care creams. The 

skin of older people poses different problems. While transepidermal 

water loss seems to be decreased with age, barrier regeneration after 

injury appears to be impaired in the skin of elderly people. The 

moisture content of the stratum corneum appears to be essentially 

unchanged with age [4]. Atopic dermatitis sufferers and, to a lesser 

extent, older people therefore require skin protection creams. A 

basic distinction is to be made between preventive and regenerative 

skin protection. 

Preventive Skin Protection 

Preventive skin protection has long been known. In fact, over three 

decades ago, lipophilic topical skin products had already been 

recognized to confer preventive skin protection against hydrophilic 

harmful substances, and, conversely, hydrophilic products had been 

considered to protect the skin against lipophilic harmful compounds. 

Recent research by the author has shown that preventive 

skin protection remains substantial over a treatment period of 

3 weeks. After longer treatment periods, however, there was a 

tendency for reduced preventive skin protection [5]. Also, W/O 

emulsions are known to be capable of preventing skin dehydration 

produced by washing [6]. 

Regenerative Skin Protection 

Regenerative skin protection has been known for a comparatively 

short period of time. Most studies along these lines have used 

glycerol, some urea, and one polyethylene glycol. 

Bettinger et al. [6] found that O/W emulsions containing glycerol or 

urea prevented skin dehydration caused by a single washing 

procedure although the same O/W emulsion without these active 

constituents was completely ineffective. In a study by Grunewald 

------------------------------------------------------------------------------------ 




et al. [7], healthy volunteers underwent standardized washings three 

times daily for one week. This treatment produced a substantial 

increase in transepidermal water loss (TEWL) as evidence of 

barrier compromise, a marked increase in laser Doppler readings as 

evidence of irritative hyperemia, and pronounced dehydration of the 

stratum corneum. Application of a 10% glycerol or 10% urea O/W 

emulsion after each washing prevented the decrease in corneometry 

readings and impressively reduced the TEWL and laser Doppler 

increases. In another study, Bettinger et al. [8] further investigated 

the mechanism of this effect on the example of glycerol. After 

producing stratum corneum damage either by exposure to sodium 

lauryl sulfate (SLS) or by tape stripping, these authors treated the 

test areas either with glycerol or with water under occlusive 

conditions. Treatment was followed by barrier function tests. Both 

the alkali resistance test and the SLS irritation test showed that 

glycerol treatment improved stratum corneum barrier function, as 

compared with water treatment. In a similar study along these 

lines, Fluhr et al. [9] produced barrier compromise by 3 days' treatment 

with SLS. This was followed by 4 days' treatment with 

glycerol products. TEWL was determined after one week with no 

treatment at all. Active-treated test areas were compared with 

untreated and vehicle-treated sites. Glycerol produced significant 

reductions in TEWL versus both vehicle and untreated. An 

intriguing finding of this study was that the effect of glycerol 

persisted for a full week beyond treatment. Taken together, the 

results of the studies presented above go to show that glycerol 

modulates barrier regeneration. 

Animal studies by Proksch et al. [10] suggest that barrier 

regeneration is controlled by TEWL. An increase in TEWL, 

associated with a flux of electrolytes, results in improved barrier 

regeneration. Proksch et al. observed this effect also in humans, 

while two other study groups failed to confirm this [11,12,13]. It 

has thus not been determined definitively whether the hygroscopic 

effect of glycerol is indeed the mechanism of action underlying its 

ability to improve barrier regeneration. Supportive evidence comes 

from studies with other hygroscopic substances (including urea and 

polyethylene glycol) which have also demonstrated barrierrestoring 

effects [14,15,16,17]. 

------------------------------------------------------------------------------------ 




Until recently, there was debate whether regenerative skin 

protection persists for prolonged treatment periods. Lodén et al. 

found that regenerative skin protection was no longer demonstrable 

after prolonged treatment with urea. In later studies, however, the 

same authors observed persistence of skin protection when using a 

cream containing urea, but they could not rule out that that effect 

might have been due to the vehicle [15,16]. Long-term glycerol 

treatment studies by the author demonstrated persistence of skin 

protection for a period of 6 weeks [5]. 

A Case Apart: Aluminum Chloride 

Five percent aluminum chloride solution occlusive film dressings 

reduce TEWL and hydrocortisone blanching but show no effect 

whatsoever in the SLS irritation test. These conflicting findings 

suggest an intriguing interpretation: While reducing TEWL and 

drug penetration from a topical skin product into the skin, 

aluminum chloride showed no protective effect in a standard skin 

irritation model, suggesting that aluminum chloride and/or any 

compounds it may form with cutaneous proteins are immediately 

destroyed by the surfactant SLS. This constellation shows a striking 

similarity to the situation in the skin of older people where TEWL 

and drug penetration are also reduced, while barrier compromise 

tests appear to produce greater irritation than in younger individuals 

[4]. 

References 

1 Forslind B (1996) New barrier models critically compared. Turk 

J Dermatopathol 5:35-43 

2 Melnik BC (1991) Disturbances of epidermal lipid metabolism 

and barrier function in atopic eczema. In: Ruzicka T, Ring J, 

Przybilla B (eds): Handbook of atopic dermatitis. Springer 

Verlag, Berlin, Heidelberg, New York, pp 296-305 

------------------------------------------------------------------------------------ 




3 Gloor M (2000) Externagrundlagen bei speziellen Indikationen. 

In: Gloor M, Thoma K, Fluhr J (eds) Dermatologische 

Externatherapie unter besonderer Berücksichtigung der 

Magistralrezeptur. Springer Verlag, Berlin, Heidelberg, New 

York, pp 133-151 

4 Gloor M (2001) Anforderungen an Kosmetika bei der 

Altershaut. SÖFW J 127:60-68 

5 Gloor M, Gehring W (2001) Increase in hydratation and 

protective function of horny layer by glycerol and a w/o 

emulsion: are these effects maintained during long-term use? 

Contact Dermatitis 44:123-125 

6 Bettinger J, Gloor M, Gehring W (1994) Influence of a 

pretreatment with emulsions on the dehydration of the skin by 

surfactants. Int J Cosm Sci 16:53-60 

7 Grunewald AM, Gloor M, Gehring W, Kleesz P (1995) Barrier 

creams-commercially available barrier creams versus urea- and 

glycerol containing oil-in water emulsions. Dermatosen 43:69- 

74 

8 Bettinger J, Gloor M, Peter C, Kleesz P, Fluhr J, Gehring W 

(1998) Opposing effects of glycerol on the protective function of 

the horny layer against irritants and on the penetration of hexyl 

nicotinate. Dermatology 197:18-24 

9 Fluhr JW, Gloor M, Lehmann L, Lazzerini S, Distante P, 

Berardesca E (1999) Glycerol accelerates recovery of barrier 

function in vivo. Acta Derm Venereol (Stockh) 79: 418-421 

10 Proksch E, Feingold KR, Mao Quiang M, Elias PM (1991) 

Barrier function regulates epidermal DNA synthesis. J Clin 

Invest 87:1668-1673 

11 Proksch E, Brasch J, Sterry W (1996) Integrity of the 

permeability barrier regulates epidermal Langerhans cell 

density. Br J Dermatol 134:630-638 

12 Kerkhof PCM van de, Mare S de, Arnold WP, Erp PEJ (1995) 

Epidermal regeneration and occlusion. Acta Derm Venereol 

(Stockh) 75:6-8 

13 Welzel J, Wilhelm KP, Wolff HH (1996) Skin permeability 

barrier and occlusion: No delay of repair in irritated human skin. 


------------------------------------------------------------------------------------ 




14 Gloor M, Wolnicki D (2001) Do polyethylene glycol gels have a 

protective effect on the skin. Contact Dermatitis 44:316-317 

15 Lodén M (1996): Urea containing moisturizers influence barrier 

properties of normal skin. Arch Dermatol Res; 288:103-107 

16 Lodén M, Andersson AC, Lindberg M (1999) Improvement of 

skin barrier function in patients with atopic dermatitis after 

treatment with a moisturizing cream (Canoderm®). Br J 

Dermatol 140:264-267 

17 Serup J (1992) A double blind comparison of two creams 

containing urea as the active ingredient. Acta Derm Venereol 

(Stockh) Suppl 177: 34-37 

------------------------------------------------------------------------------------ 



58 The Tandem Repeated Irritation Test (TRIT) 

The Tandem Repeated Irritation Test (TRIT) 

A New Method for the Evaluation of Barrier Creams 

J. Spoo 1 , W. Wigger-Alberti 1 , S. Schliemann-Willers 1 , A. Klotz 2 , P. Elsner 1 

1 

Department of Dermatology, Friedrich-Schiller-University 

2 

Stockhausen GmbH & Co. KG, Krefeld 

Erfurter Str. 35 

D-07740 Jena 

Germany 

Introduction................................................................... 58 

Patients and Methods .................................................... 60 

• Subjects........................................................................ 60 

• Protective cream (STOKODERM ®, Stockhausen, 

Krefeld, Germany)....................................................... 60 

• Procedure ..................................................................... 60 

• Evaluation methods ..................................................... 61 

• Statistics....................................................................... 62 

Results........................................................................... 62 

Discussion ..................................................................... 65 

References..................................................................... 66 

Introduction 

Much effort has been undertaken to develop valid methods for 

evaluation of the benefit of protective creams to prevent irritant 

contact dermatitis (ICD) which remains the most common 

professional skin disease. Since Suskind introduced the 'slide test' to 

evaluate protective creams in the 1950s [1] various in vitro and in 

vivo studies have been performed to investigate both the effects of 

irritants on skin barrier function and the benefit of protective 

creams under experimental conditions [1-8] All of these studies, 

however, are considered not to be close enough to real work place 

situations. 

In 1994, Frosch & Kurte introduced the repetitive irritation test 

(RIT) with cumulative irritation over a two-week period by 

standard irritants such as sodium lauryl sulfate (SLS), sodium 

------------------------------------------------------------------------------------ 



The Tandem Repeated Irritation Test (TRIT) 59 

hydroxide, lactic acid and toluene [9]. This model has been 

modified and used in many laboratories as a routine procedure as it 

was considered to be suitable for comparing protective creams 

simultaneously to non-pretreated control sites on the backs of 

volunteers [10]. Since short duration and easy application given in a 

one-week test using the forearm of healthy volunteers was 

considered highly desirable, a test model based on the RIT was 

developed for opitimizing the concentrations of irritants against 

which protective creams are tested, and for evaluating the necessary 

cumulative application time [11, 12]. A one-week period proved 

sufficient for evaluating the efficacy of protective creams against 

most irritants even if lower concentrations of irritants were used. 

A multicentre study subsequently was designed to standardize a test 

procedure for the evaluation of skin protective products. In this 

irritation study, a repeated short-time occlusive irritation test 

(ROIT) was evaluated [13]. Using 2 irritants (SLS and toluene, each 

applied twice daily for 30 min) the evaluation showed that 

significant results could be achieved with the 5-day protocol. 

It may be criticized that in all models presented the investigation of 

protective cream efficacy has been limited to the exposure of a 

single irritant only. The anionic surfactant SLS and the organic 

solvent toluene have mainly been used although repetitive contact 

to both hydrophilic and hydrophobic substances together or, more 

commonly, one after the other, is frequent in the workplace setting. 

We recently investigated concurrent application of SLS and toluene 

showing that a mixed application of these irritants induced 

significantly stronger reactions than those caused by twice daily 

application of each irritant on its own [14]. It is obvious that the 

additive effect is important for the use of protective creams in 

practice and the way they should be tested. 

Therefore, the sequential application of two irritants in the so-called 

tandem repeated irritation test (TRIT) was investigated in the 

present study to evaluate the benefit of a commercially available 

protective cream compared to non-pretreated control sites. 

------------------------------------------------------------------------------------ 




Patients and Methods 

• Subjects 

Twenty healthy non-preselected Caucasian volunteers (17 women 

and 3 men; aged 18 - 49 years, median 29 years,) without any skin 

diseases were included. Informed consent was obtained from all 

participants, and the study was approved by the local ethical 

committee. Subjects were allowed to bathe as usual, but were 

instructed to avoid direct application of detergents, moisturizers or 

emollients on their forearms during the 5 days of investigation. 

• Protective cream (STOKODERM ® ) 

Composition according to INCI declaration: aqua, octyl stearate, 

glyceryl stearate SE, glycerin, cetearyl alcohol, sodium 

bischlorophenyl sulfamine, isopropyl palmitate, glycol distearate, 

xanthan gum, ceteareth-6, phenoxyethanol, methylparaben, 

ethylparaben, propylparaben, butylparaben, ceteareth-25, fragrance. 

• Procedure 

The application area was the clinically normal skin of the medial 

volar forearms. The placement of test fields and arms (6 chambers 

on the right and left forearms) was randomized. Three test fields 

were treated with 0.05 ml of protective cream rubbed onto a skin 

area 2 cm in diameter with a gloved finger. The other test fields 

served as untreated controls. After 10 min pretreatment, the irritants 

were applied on all 6 premarked test sites on the forearms for 30 

min under occlusion (Finn Chambers, 12mm diameter, filling 

volume 0.05 ml; Epitest Ltd., Hyrlä, Finland). The volunteers were 

tested with 0.5% aqueous SLS (Sigma, St. Louis, MO) or undiluted 

toluene (E. Merck, Darmstadt, Germany). After removal of the 

patches the skin was cleaned with a dry paper tissue. A second 

exposure with 0.5% aqueous SLS or undiluted toluene was 

performed the same day after 3 h. Thus, 3 treatment combinations 

were investigated, resulting in a repeated irritation due to SLS/SLS; 

------------------------------------------------------------------------------------ 




toluene/toluene; and SLS/toluene and the pre-irritation application 

of Stokoderm on the respective test areas. Since in a previous study 

the exact chronological order of the irritants was shown not to have 

any effect on the degree of irritation [14] the combination 

toluene/SLS was not included in the present study. Using this 

scheme of application the volunteers were treated from day 1 to day 

4 (in each case at the same time of day). 

• Evaluation methods 

The study was carried out from October to November 2000. All 

visual scorings and bioengineering measurements to compare the 

intensity of reactions were performed daily before starting 

treatments (day 1-4) and on day 5 by the same observer under 

controlled environmental conditions. All measurements were 

carried out in an air-conditioned room (room temperature 20-22°C, 

relative humidity between 34% and 46%) after 30 min for 

equilibration. 

Clinical score graded for erythema, scaling and fissuring was 

recorded according to Frosch & Kligman [15]. 

Transepidermal water loss (TEWL) (expressed in g/m 2 h) was 

measured using an evaporation meter (Tewameter TM 210, 

Courage & Khazaka, Cologne, Germany). Measurements were 

taken according to the Guidelines of the Standardization Group of 

the European Society of Contact Dermatitis [16]. 

Instrumental colour measurements were taken with a Minolta 

Chromameter (CR-200, Minolta, Osaka, Japan) according to 

published recommendations [17]. The colour coordinates were 

expressed in the L*a*b* 3-dimensial colourimetric system. The a* 

value is the component of separation between red (positive value) 

and green (negative value) as a sensitive measure for quantifying 

erythema. 

Electrical capacitance, indicating the hydration level of the skin, 

was measured by a Corneometer CM 825 (Courage & Khazaka, 

Cologne, Germany) [18]. 

If the clinical score progressed to a severe degree (>5), exposure 

was discontinued. For these test areas, the maximal scores and the 

measured values for TEWL, chromametry, and capacitance 

------------------------------------------------------------------------------------ 




obtained on the day of discontinuance were used for the final 

calculation. 

• Statistics 

Statistical analysis was conducted with SPSS/PC+ (Version 10.0, 

SPSS, Chicago, ILL, USA). Data of visual scoring are presented as 

means ± SEM. TEWL, skin colour a*, and skin hydration 

(differences between baseline values and after irritation) were 

determined. As the data were not normally distributed the 

differences between means were checked for significance using the 

Wilcoxon test for paired data for the erythema score, the 

comparison of TEWL, skin capacitance, and skin colour. The 

chosen level of significance was p


SLS/SLS d5 Visual Score TEWL Chromametry Capacitance 

Control mean 1.95 20.92 3.14 -9.07 

SEM 0.25 3.34 0.75 2.15 

Stokoderm mean 0.95 12.15 1.12 -8.21 

SEM 0.18 1.74 0.41 2.07 

TOL/TOL d5 Visual Score TEWL Chromametry Capacitance 

Control mean 1.55 3.57 1.78 -14.33 

SEM 0.32 0.82 0.69 2.74 


SEM 0.22 0.33 0.53 2.97 

SLS/TOL d5 Visual Score TEWL Chromametry Capacitance 

Control mean 3.75 32.65 5.76 -14.27 

SEM 0.27 5.92 0.79 3.50 


SEM 0.34 2.44 0.84 2.10 

Table 1: Mean-values and standard errors of the mean (SEM) at 

day 5 for visual scoring (VS), transepidermal water loss (TEWL, 

g/m 2 h), skin redness (chromametry, a*) and skin hydration 

measured by capacitance (arbitrary units). Data are given for twice 

daily application of SLS, toluene and tandem irritation. 

∆ TEWL [g/m²h] 

30 

25 

20 

15 

10 

5 

0 

SLS/SLS 

Stokoderm/SLS/SLS 

* 

d2 d3 d4 d5 

Fig. 1: TEWL (mean ± SEM, n=20) after sequential application of 

SLS/SLS. On day 3, 4 and 5 the protective effect of Stokoderm on 

the irritation was statistically significant (*p


period that was slightly suppressed by Stokoderm (Fig. 2). A 

moderate benefit of the test product against toluene was also 

confirmed by the measurement of skin capacitance and 

chromametry while the visual score showed contrary results. 


5 

4,5 

4 

3,5 

3 

2,5 

2 

1,5 

1 

0,5 

0 

TOL/TOL 

Stokoderm/TOL/TOL 

d2 d3 d4 d5 


TOL/TOL. The protective effect of Stokoderm on the irritation was 

not statistically significant. 

Monitoring of the instrumental measurements and the visual score 

following sequential application of SLS/toluene showed that the 

induced reactions were significantly stronger than those caused by 

twice daily application of the single irritants SLS or toluene. 

Additionally, pretreatment with Stokoderm suppressed the irritant 

reaction presented by all measurements. The TEWL and the visual 

scoring indicated a significant benefit of the product tested (Fig. 3). 

------------------------------------------------------------------------------------ 




45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

SLS/TOL 

Stokoderm/SLS/TOL 

d2 d3 d4 d5 



SLS/TOL. On day 4 and 5 the protective effect of Stokoderm on the 

irritation was statistically significant (* p


instance, are repetitively exposed to water-based metal working 

fluids, neat oils, detergents and organic solvents. This emphasizes 

the significant practical consequences of an interaction between 

irritant chemicals. 

Stokoderm® has previously been shown to significantly suppress 

the irritation due to SLS and toluene as single irritants in an animal 

model. The irritants were applied daily for 2 weeks to shaved back 

skin of young guinea pigs and the cream was applied 2 h prior to 

and immediately after exposure to the irritants. Control animals 

were treated with the irritants only [26]. It is satisfying that our 

results confirm the protective effect of Stokoderm against the single 

irritants though the benefit against toluene was not significant in our 

test, which confirms the results for other creams. The animal study 

did, however, not assess combined irritation in a tandem model. 

To establish a new method to evaluate the benefit of protective 

creams we performed the study with SLS and toluene which have 

been used as standard irritants in various types of patch tests [2, 3, 

9-14, 20, 23, 26]. Our results show that the TRIT seems to have 

great potential in differentiating the efficacy of protective creams in 

a relevant experimental setting that is quite close to a workplace 

situation where detergents and organic solvents are the major 

irritants used not exclusively, but concurrently. Nevertheless, this 

model must be validated by field studies under actual usage 

conditions. Interactive profiles of further relevant irritants should be 

investigated with attention to professions where a multitude of 

hazardous substances may cause ICD. We hope that this model also 

proves useful in other hands to investigate both the effect of 

combinations of irritants to the skin and the way skin care products 

may prevent contact dermatitis. 

References 

1 Suskind RR (1955) The present status of silicone protective 

creams. Indust Med Surg 24:413-416 

2 Boman A, Wahlberg JE, Johansson G (1982) A method for the 

study of the effect of barrier creams and protective gloves on the 

percutaneous absorption of solvents. Dermatologica 164:157- 

160 

------------------------------------------------------------------------------------ 




3 Mahmoud G, Lachapelle JM, Van Neste D (1984) Histological 

assessment of skin damage by irritants: its possible use in the 

evaluation of a 'barrier cream'. Contact Dermatitis 11:179-185 

4 Lodén M (1986) The effect of 4 barrier creams on the absorption 

of water, benzene, and formaldehyde into excised human skin. 


5 Treffel P, Gabard B, Juch R (1994) Evaluation of barrier creams: 

an in vitro technique on human skin. Acta Derm Venereol 74:7- 

11 

6 Grunewald A, Gloor M, Gehring W, Kleesz P (1995) Efficacy of 

skin barrier creams. In: Elsner P. Maibach HI. (Eds.) Irritant 

Dermatitis: New clinical and experimental aspects. Karger, 

Basel, p 187-197 

7 Fine Olivarius F, Hansen A B, Karlsmark T, Wulf H C (1996) 

Water protective effect of barrier creams and moisturizing 

creams: a new in vivo test method. Contact Dermatitis 35:219- 

225 

8 Zhai H, Willard P, Maibach HI (1998) Evaluating skinprotective 

materials against contact irritants and allergens. 


9 Frosch P J, Kurte A (1994) Efficacy of skin barrier creams (IV). 

The repetitive irritation test (RIT) with a set of 4 standard 

irritants. Contact Dermatitis 31:161-168 

10 Schlüter-Wigger W, Elsner P (1996) Efficacy of 4 commercially 

available protective creams in the repetitive irritation test (RIT). 


11 Wigger-Alberti W, Rougier A, Richard A, Elsner P (1998) 

Efficacy of protective creams in a modified repeated irritation 

test (RIT): methodological aspects. Acta Derm Venereol 78:270- 

273 

12 Wigger-Alberti W, Caduff L, Burg G, Elsner P (1999) 

Experimentally-induced chronic irritant contact dermatitis to 

evaluate the efficacy of protective creams in vivo. J Am Acad 


13 Schnetz E, Diepgen TL, Elsner P, Frosch PJ, Klotz AJ, Kresken 

J et al. (2000) Multicentre study for the development of an in 

vivo model to evaluate the influence of topical formulations on 

irritation. Contact Dermatitis 42: 336-343 

------------------------------------------------------------------------------------ 




14 Wigger-Alberti W, Krebs A, Elsner P (2000) Experimental 

irritant contact dermatitis due to cumulative epicutaneous 

exposure to sodium lauryl sulphate and toluene: single and 

concurrent application. Br J Dermatol 143:551-556 

15 Frosch PJ, Kligman AM (1979) The soap chamber test. A new 

method for assessing the irritancy of soaps. J Am Acad 


16 Pinnagoda J, Tupker R A, Agner T, Serup J (1990) Guidelines 

for transepidermal water loss (TEWL) measurement. A report 

from the Standardization Group of the European Society of 

Contact Dermatitis. Contact Dermatitis 22:164-178 

17 Fullerton A, Fischer T, Lahti A, Wilhelm KP, Takiwaki H, 

Serup J (1996) Guidelines for measurement of skin colour and 

erythema. A report from the Standardization Group of the 

European Society of Contact Dermatitis. Contact Dermatitis 

35:1-10 

18 Courage W (1994) Hardware and Measuring Principle: 

Corneometer. In: Elsner P, Beradesca E, Maibach HI (Eds.) 

Bioengineering of the Skin: Water and the Stratum Corneum. 

CRC Press, Boca Raton p 171-175 

19 Hogan DJ, Dannaker CJ, Lal S, Maibach HI (1990) An 

international survey on the prognosis of occupational contact 

dermatitis of the hands. Derm Beruf Umwelt 38:143-147 

20 Lachapelle JM (1996) Efficacy of protective creams and/or gels. 

In: Elsner P, Lachapelle JM, Wahlberg JE, Maibach HI (Eds.) 

Prevention of Contact Dermatitis. Karger, Basel p. 182-192 

21 Wigger-Alberti W, Elsner P (1998) Do barrier creams and 

gloves prevent or provoke contact dermatitis? Am J Contact 

Dermatitis 9:100-106 

22 Wigger-Alberti W, Elsner P (2000) Barrier Creams and 

Emollients. In: Kanerva L, Elsner P, Wahlberg JE, Maibach HI 

(Eds.) Handbook of Occupational Dermatology. Springer, Berlin 

Heidelberg New York p. 490-496 

23 Frosch P, Schulze-Dirks A, Hoffmann M, Axthelm I (1993). 

Efficacy of skin barrier creams (II). Ineffectiveness of a popular 

"skin protector" against various irritants in the repetitive 

irritation test in the guinea pig. Contact Dermatitis 29:74-77 

------------------------------------------------------------------------------------ 




24 Wigger-Alberti W, Maraffio B, Wernli M, Elsner P (1997) Selfapplication 

of a protective cream: pitfalls of occupational skin 

protection. Arch Dermatol 133:861-864. 

25 Diepgen TL, Coenraads PJ (2000) The epidemiology of 

occupational contact dermatitis. In: Kanerva L, Elsner P, 

Wahlberg JE, Maibach HI (Eds.) Handbook of Occupational 

Dermatology. Springer, Berlin Heidelberg New York p 4-16 

26 Frosch P, Schulze D, Hoffmann M, Axthelm I, Kurte A (1993) 

Efficacy of skin barrier creams (I). The repetitive irritation test 

(RIT) in the guinea pig. Contact Dermatitis 28:94-100. 

------------------------------------------------------------------------------------ 



70 Atomic absorption spectrometry for the determination of physical sunscreen filters 

Atomic absorption spectrometry for the 

determination of physical sunscreen filters 

S. Gottbrath, J. Grünefeld*, C.C. Müller-Goymann 

Technische Universität Carolo-Wilhelmina 

Institut für Pharmazeutische Technologie, 

* Institut für Pharmazeutische Chemie 

Mendelssohnstraße 1 

D-38106 Braunschweig 

Germany 

Introduction................................................................... 70 

Materials and Methods.................................................. 71 

• Ingredients of the formulation ..................................... 71 

• Tape stripping .............................................................. 71 

• Atomic absorption spectrometry (AAS)...................... 71 

• Sun protection factor measurement (SPF)................... 74 

• Preparation of formulation........................................... 74 

• Area analysis................................................................ 75 

• Transmission electron microscopy (TEM) .................. 76 

Results........................................................................... 76 

Discussion ..................................................................... 78 

References..................................................................... 79 

Introduction 

Microfine titanium dioxide is well established as an effective UVfilter. 

Besides sun protection potential the evaluation of titanium 

dioxide formulations has to include stability aspects of the 

formulations as well as penetration potential of the physical 

sunscreen filter into deeper layers of the stratum corneum. Tape 

stripping can be used to remove corneocytes layer by layer with an 

adhesive film and to determine the amount of titanium dioxide in 

these layers. The present contribution presents atomic absorption 

spectrometry for the determination of titanium dioxide particles 

removed with an adhesive film from the skin of the proband. 

Atomic absorption spectrometry is often used for trace 

------------------------------------------------------------------------------------ 



Atomic absorption spectrometry for the determination of physical sunscreen filters 71 

determination because of its high specificity, selectivity, and 

sensitivity. [1, 2, 3] 

Materials and Methods 

• Ingredients of the formulation 

Titanium dioxide predispersion Tioveil AQ N (Tioxide Specialties, 

Billingham, Great Britain), Synperonic PE/F 68 (Uniqema, 

Everberg, Belgium), Cetiol HE (Henkel, Düsseldorf, Germany), 

lecithin Phospholipon ® G 90 (Nattermann Phospholipid GmbH, 

Düsseldorf, Germany), propylene glycol (BASF, Ludwigshafen, 

Germany), ammonium sulfate (Caelo, Hilden, Germany), sulfuric 

acid (Merck, Darmstadt, Germany). Water was used in bistilled 

quality. 

• Tape stripping 

The formulation either with or without titanium dioxide was applied 

on the ventral side of the forearm, according to COLIPA standard in 

a concentration of 2 mg/cm 2 . After 45 minutes of equilibration the 

formulation was removed from the skin with a dry tissue. 10 strips 

were taken from the pretreated area and analysed by atomic 

absorption spectrometry in the case of titanium dioxide formulation. 

The strips taken from the placebo treated area were viewed with an 

inverted microscope to obtain the amount of corneocyte aggregates 

(see details in chapter: area analysis). 

• Atomic absorption spectrometry (AAS) 

Sample preparation 

The amount of titanium dioxide on the removed strips was 

determined by atomic absorption spectrometry. Each single strip 

was dissolved in concentrated sulfuric acid combined with 

ammonium sulfate at boiling temperature. Ammonium sulfate is 

responsible for the formation of titanium sulfato complexes. The 

complexation of titanium reduces the amount of titanium adsorbed 

------------------------------------------------------------------------------------ 




to the inner surface of the sample vials. After dilution and filtration 

with a cellulose acetate filter of a pore size of 0.2 µm (Sartorius 

AG, Göttingen, Germany) samples were analysed by atomic 

absorption spectrometry. 

Instrumentation 

Atomic absorption spectrometry uses the absorption of light to 

measure the concentration of gas phase atoms. The main 

components of an atomic absorption spectrometer are a radiation 

source, an atomisation cell and a device for wavelength selection 

and detection. 

The most common radiation source is the hollow cathode lamp 

(HCL). The HCL is a line source that emits radiation characteristic 

of a particular element. 

A Perkin Elmer 2380 (Perkin Elmer, Überlingen, Germany) atomic 

absorption spectrometer was used, equipped with an AS-40 

autosampler, a HGA-500 graphite furnace for vaporization of the 

titanium ions or atoms from the sample and a titanium hollow 

cathode lamp (L.O.T.-Oriel, Darmstadt, Germany). 

Using a graphite furnace in contrast to flame atom absorption 

spectrometry avoids interferences of the matrices. Furthermore very 

small sample quantities can be analysed. Samples are placed 

directly into the graphite furnace by the sampling capillary (sample 

amount 5-100 µl). The maximum temperature is 2700° C by 

electrical heating with 10 V. An optical temperature measuring unit 

is installed to control the temperature of the external wall of the 

graphite tube. Argon as protective gas is used to avoid burning of 

the graphite tube. The protective gas flows around the graphite tube 

externally and protects from unwanted oxidation. The internal gas 

flow is divided into two corresponding gas flows. They flow from 

the two endings to the center of the graphite tube and leave the 

graphite tube through the sample opening. The external gas flow is 

limited to a value of 900 ml/min. The internal gas flow is variable 

in the range from 0 to 300 ml/min. The transport of volatile 

compounds out of the graphite tube is executed by the internal gas 

flow. The inner surface of the graphite tube is grooved to avoid that 

the liquid sample runs out of the tube. The instrumental parameters 

are listed in Tab. 1. 

------------------------------------------------------------------------------------ 




Wavelength 364.3 nm 

Lamp current 21 mA 

Slit 0.2 nm 

Mode Peak height 

Sample volume 25 µl 

Tab. 1: Instrumental parameters 

Another important fact for the successful determination of titanium 

is the heating rate and heating time of the graphite furnace. The 

heating program should avoid splattering or boiling of the sample 

which may cause sample loss. The program was developed by 

Skipor et al. and was slightly modified. The heating program 

sequence is listed in Tab. 2. 

Temperature [°C] Ramp [sec] Hold [sec] Read 

130 30 30 

500 40 20 

1400 15 20 

2600 0 5 x 

2700 1 5 

20 1 45 

Tab. 2: Heating program sequence 

The first step of the heating program is responsible for drying the 

sample and to vaporize low boiling liquids from the sample. The 

chosen temperature depends on the boiling point of the solvent. For 

diluted aqueous samples a drying temperature in the range from 

100° C to 130° C is necessary. 

The Second step is responsible for the thermic pretreatment of the 

sample and to remove components of the matrix. The removal of 

components of the matrix that are more volatile than compounds of 

the interesting element avoids unspecific absorptions. To assure that 

all disturbing components leave the sample during the thermic 

pretreatment phase, the temperature and its duration have to be 

sufficient although an increased temperature for a longer period of 

time may lead to a loss of the interesting element. In this case the 

high temperature of 1400° C for thermic pretreatment was selected 

to remove all matrix compounds and for an undisturbed 

determination of titanium. 

------------------------------------------------------------------------------------ 




Third step is the phase of atomization of the sample. For titanium a 

minimum temperature of 2600° C is necessary. An increased 

temperature leads to higher sensitivity but causes a reduced lifetime 

of the graphite tube. The temperature of 2600° C is held for 5 

seconds. During atomization the internal gas flow is stopped to 

extend the duration of stay of the titanium atoms in the graphite 

tube. 

Fourth step is to anneal the graphite tube to eliminate the 

components of the sample remaining in the graphite tube. At the 

end of the heating program sequence the graphite tube is cooled 

down to a temperature of 20° C. 

Carbide generating elements like titanium provoke chemical 

interferences with components of the graphite tube which cause 

tailed signals and memory effects. To avoid formation of carbide 

compounds pyrocarbon coated graphite tubes were used. An 

instrumental background compensation was not necessary, because 

a blank sample showed no absorption. The blank sample was taken 

from a placebo treated area and processed following the same way 

as TiO2 containing strips. 

Limit of detection of this method was 5.8 ppb. [4, 5, 6] 

• Sun protection factor measurement (SPF) 

The sun protection factor of the titanium dioxide formulations was 

determined by a SPF-290 Analyzer (Optometrics, Leeds, Great 

Britain). For the in vitro measurement the formulations were 

applied on a Transpore ® tape (3M, Neuss, Germany) with a 

concentration of 2 mg/cm 2 . The attenuation of the radiation by the 

formulation on the Transpore ® tape was measured in the range of 

290-400 nm. 

• Preparation of formulation 

Tab. 3 shows the percentage composition (w/w) of the standard 

formulation. 

------------------------------------------------------------------------------------ 




Tioveil AQ N 12.5% 

Synperonic PE/F68 10.0% 

Cetiol HE 20.0% 

propylene glycol 8.0% 

water 49.5% 

Tab. 3: Composition of the standard formulation (micellar solution 

with dispersed titanium dioxide particles) 

The lipophilic and hydrophilic ingredients were heated to 60°C 

separately and homogenized with an ultra turrax (IKA-Werk, 

Staufen, Germany). Tioveil AQ N was stirred with an ultra turrax 

prior to use. 

Tab. 4 shows the percentage composition (w/w) of the liposomal 

formulation 

Phospholipon G 90 20.0% 

Tioveil AQ N 12.5% 

water 67.5% 

Tab. 4: Composition of the liposomal formulation. All components 

were heated to 60°C while being stirred on a magnetic stirrer. 

• Area analysis 

For determination of the amount of corneocytes removed with 

every single strip, the strips were viewed using an inverted 

microscope (Olympus IX50, Hamburg, Germany). 

The digital pictures taken from the strips were analysed using 

analySIS ® software (Soft Imaging System GmbH, Münster, 

Germany). The software allows the quantification of the strips in 

terms of the area covered with corneocytes. The knowledge of the 

amount of corneocytes removed with every strip is necessary for a 

more precise location of the titanium dioxide microparticles in the 

stratum corneum. 

------------------------------------------------------------------------------------ 




• Transmission electron microscopy (TEM) 

TEM was used for the visualization of titanium dioxide 

microparticle distribution. The sample was frozen at a temperature 

of –210° C and fractured (Balzers GmbH BAF 400, Wiesbaden, 

Germany) at a temperature of –100° C and 5x10 -6 bar. The fractured 

surface was shadowed with platinum/carbon of 2 nm thickness and 

with pure carbon for mechanical stabilization of the replica. 

Sulfuric acid was used for cleaning the replica. The replica on 

uncoated grids were viewed using a transmission electron 

microscope (Philips EM-300, Kassel, Germany). 

Results 

Two different formulations were compared in terms of sun 

protection factor and penetration into the stratum corneum. The first 

formulation was a micellar solution with a sun protection factor of 

7.6 (s.d.= 1.2) (n=3). 

The second formulation was a liposomal formulation with a sun 

protection factor of 3.1 (s.d.= 0.6) (n=3). Figure 1 shows the 

distribution of the titanium dioxide particles and the liposomes 

within the formulation. 

Fig. 1: TEM micrograph of the liposomal formulation (bar = 1 µm) 

------------------------------------------------------------------------------------ 




The picture shows multilamellar liposomes of sizes between 320 

and 1 µm and needle-shaped titanium dioxide microparticles with a 

length of 20 – 30 nm. 

titanium concentration [µg/cm2] 

14 

12 

10 

8 

6 

4 

2 

standard formulation 1. determination 

standard formulation 2. determination 

liposomal formulation 1. determination 

liposomal formulation 2. determination 

0 

0 1 2 3 4 5 6 7 8 9 10 11 

number of strip 

Fig. 2: Penetration from standard formulation and liposomal 

formulation 

Both the standard formulation and the liposomal formulation, with a 

concentration of 5% titanium dioxide each, were applied on the 

ventral side of the forearm of a male test person (30 years old). Fig. 

2 represents the AAS results from tape stripping. 

Plotting AAS results versus the number of strips shows an 

exponential decline. At strip 6 the titanium concentration 

approaches a value of 1.0 µg/cm 2 . After application of the standard 

formulation higher amounts of titanium dioxide can be removed 

with the first strips in comparison with the liposomal formulation. 

This result has to be considered together with the results from the 

area analysis of the placebo strips. The removed amount of 

corneocyte aggregates after treatment with the standard formulation 

was much higher compared to the liposomal formulation due to the 

adhesive power of the standard formulation. Fig. 3 assigns the 

amount of corneocyte aggregates to the titanium concentration of 

every strip. 

------------------------------------------------------------------------------------ 




number of strips 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

percental amount of removed corneocytes aggregates [%/10] or titanium concentration [µg/cm2] 

0 1 2 3 4 5 6 7 8 9 10 

standard formulation, AAS 

------------------------------------------------------------------------------------ 



standard formulation, area analysis 

liposomal formulation, AAS 

liposomal formulation, area analysis 

Fig. 3: Titanium concentration respectively corneocyte aggregates 

vs. number of strips 

Discussion 

To detect small amounts of titanium dioxide within deeper layers of 

the stratum corneum tape stripping in combination with AAS is a 

suitable analytical method. The proposed heating program causes 

no splattering or boiling of the analysed sample which may lead to a 

loss of the sample. For the evaluation of titanium dioxide penetrated 

into stratum corneum from different formulations the amount of the 

removed corneocyte aggregates has to be considered, too. This 

leads to a more precise localization of the titanium dioxide 

microparticles in the stratum corneum. Results reveal a higher 

amount of titanium dioxide in deeper layers of the stratum corneum 

after treatment with the standard formulation compared to the 

liposomal formulation. Hence, the effect of different formulations 

on the penetration potential of titanium dioxide microparticles is 

obvious.

References 


1 Lademann J, Weigmann HJ, Rickmeyer C, Barthelmes H, 

Schaefer H, Mueller G, Sterry W (1999) Penetration of titanium 

dioxide particles in a sunscreen formulation into the horny layer 

and the follicular orifice, Skin Pharmacol Appl Skin Physiol 

12:247-256 

2 Weigmann HJ, Lademann J, Meffert H, Schaefer H, Sterry W 

(1999) Determination of the horny layer profile by tape stripping 

in combination with optical spectroscopy in the visible range as 

a prerequisite to quantify percutaneous absorption, Skin 


3 Müller-Goymann CC, Bennat C, Grünefeld J (1998) Penetration 

von partikulären UV-Filtern in die Haut, Parfümerie und 

Kosmetik 5:24-26 

4 Skipor AK, Jacobs JJ, Schavocky J, Black J, Galante JO (1994) 

Determination of titanium in human serum by zeeman 

electrothermal atomic absorption spectroscopy, Atomic 

Spectroscopy 5/6:131-134 

5 Welz B, Sperling M (1997) Atomabsorptionsspektroskopie. 

Wiley-VCH, Weinheim 

6 Mason JT (1980) Quantitative determination of titanium in a 

commercial sunscreen formulation by atomic absorption 

spectrometry, Journal of Pharmaceutical Sciences 1:101-102 

------------------------------------------------------------------------------------ 



80 Determination of Clobetasol Propionate in SC Extraxts by Means of HPLC and LC-MS 

Determination of Clobetasol Propionate in SC 

Extraxts by Means of HPLC and LC-MS 

T. Hagemeister, M. Linscheid, *H.-J. Weigmann, *J. Lademann, 

*R. v. Pelchrzim, *W. Sterry 

Institut für Chemie, *Charité, Klinik für Dermatologie, Venerologie und 

Allergologie mit Asthmapoliklinik der Humboldt-Universität zu Berlin 

D-10098 Berlin 

Germany 

Abstract ......................................................................... 80 

Introduction................................................................... 81 

Materials and Methods.................................................. 82 

• The tape stripping protocol.......................................... 82 

• The extraction protocol................................................ 82 

• UV/VIS-Spectroscopic Measurements........................ 83 

• The HPLC method....................................................... 84 

• Mass Spectrometry ...................................................... 85 

Conclusion .................................................................... 88 

References..................................................................... 88 

Abstract 

The introduced LC/ESI-MS investigations were based on an 

isocratic HPLC method for the determination of Clobetasol 

propionate (detection limit of 0,15 mg/l [1]). The HPLC eluate was 

led directly to an ESI-Single Quad Mass Spectrometer. The 

characteristic protonated molecular ions [M+H] + of Clobetasol 

Propionate and the internal standard Betametasone-17-valerate were 

generated and the quantification was carried out in the (single ion 

mode SIM) and the multiple ion mode (MIM) respectively. The 

chromatographic separation by HPLC and the selective mass 

spectrometric detection of the target compounds allowed a reliable 

identification with a remarkable improvement of sensitivity 

(detection limit: 2.15 µg/l). 

------------------------------------------------------------------------------------ 



Determination of Clobetasol Propionate in SC Extraxts by Means of HPLC and LC-MS 81 

Introduction 

The synthetic corticosteroid Clobetasol propionate (CB), an analog 

of prednisolone, is the active component in some commercial 

creams for topical dermatologic use (Figure 1). The determination 

of the penetration behavior of the substrate in dependence of 

different creme formulations is of special pharmacological interest. 

Therefore the stratum corneum was sampled by tape stripping. Prior 

to extraction the number of corneocytes attached to the tape strip 

were measured by a UV/VIS-procedure [2]. Subsequently the 

quantitative measurement of Clobetasol propionate with HPLC- 

MS-SIM of an extract of the particular single tape was performed. 

For quantification the internal standard method was utilized. The 

possible correlation of the number of corneocytes with the amount 

of Clobetasol extracted from a single tapestrip admits the 

establishment of high resolution horny layer profiles. These are a 

reliable basis for the interpretation and comprehension of the 

bioavailability of dermatologic relevant substances. The tape strip 

investigations are closely related to the proposals outlined in [3], 

and given in the Draft Guidance for Industry (FDA) [4]. For 

additional and detailed information on these topics refer to 

reference [1]. 

O 

HO 

CH3 

F 

CH 3 

CH 2Cl 

C=O 

OCOCH 2CH 3 

CH 3 

O 

HO 

CH3 

F 

CH 3 

CH 2OH 

C=O 

OCO(CH 2) 3CH 3 

Fig.1: Clobetasol propionate (M=466g/mol) and the internal 

standard Betametason-17-valerate (M=476g/mol) 

CH 3 

------------------------------------------------------------------------------------ 





• The tape stripping protocol 

Three cremes were examined, namely Clobetasol Propionate 

Cream, Temovate Crème and Temovate Emollient. The stratum 

corneum removal from the drug treated skin site was performed by 

tape stripping with TESA tape (TESA film No. 5529, Beiersdorf, 

Hamburg, Germany) for which it was ascertained that during the 

HPLC measurements matrix peaks did not disturb the Clobetasol 

propionate (CB; MW: 466 g/mol) peak. Tape stripping was done at 

0.5, 2.0 and 6.0 hours after application. In brief, the treated skin site 

was tape stripped 21 times. The first tape strip was discarded due to 

potential residual drug contamination, and the tape strips 2-21 were 

submitted for successive chemical extraction and analysis by 

HPLC-MS. 

• The extraction protocol 

The tapes 2-21 (1.9 x 2 cm in size) corresponding to the application 

areas were solely placed in a test tube respectively. The adhesive 

layer was directed towards the inside of the test tube. 1500 µl of 

solvent (acetonitrile : water = 55 : 45) were pipetted in two steps 

into the tube, using a volume adjustable digital pipette (Eppendorf, 

500 - 1000 µL). 40 µl of the internal standard solution, containing 

6.68 mg/l Betamethasone-17-valerate (BM; MW: 476 g/mol), were 

added with a volume adjustable digital pipette (Eppendorf, 10 - 

100 µL). The test tube, 160 mm long, 16 mm in diameter, was 

closed with a ground glass stopper. A PTFE, disposal sleeve for 

ground joints, was used in combination with Parafilm "M" 

(Laboratory Film; American National Can) to prevent leaking. The 

tube was fixed on a shaker in the horizontal position and shaken for 

20 minutes. During this time the tube was turned twice to make sure 

that all parts of the tapes would be in contact with the solution. The 

solution was drawn from the test tube with a syringe and filtered 

through a nylon syringe filter (ISO - DISC N - 34; 3 mm Diameter 

------------------------------------------------------------------------------------ 




Nylon Membrane; 0.45 µm Pore Size; Supelco). An aliquot of the 

solution was analyzed by HPLC-MS. 

• UV/VIS-Spectroscopic Measurements 

The amount of the corneocyte aggregates removed with a single 

tape and the thickness of the horny layer vary considerably for 

different individuals [2]. Therefore a method is required that allows 

the correlation of the amount of the active substance (CB) on 

individual adhesive film strips to the depth profile of the horny 

layer. For the determination of that depth profile, a correct 

measurement of the amount of the corneocyte aggregates is 

mandatory. Optical spectroscopy in the visible spectral range fulfils 

the prerequisites. The particulate corneocyte aggregates reflect and 

scatter the light nearly wavelength independently. All other 

components on the tape strips act as absorbers with absorption 

bands in the UV range (Figure 2). 

absorbance (au) 

1,2 

1,1 

1,0 

0,9 

0,8 

0,7 

0,6 

0,5 

0,4 

0,3 

0,2 

0,1 

0,0 

1,23 

Clobetasol propionate 

Clobetasol Propionate Cream 

Temovate Cream 

Temovate Emollient 

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 

wavelength (nm) 

Fig. 2: UV/VIS-spectra of Clobetasol Propionate and the analysed 

cremes containing the substrate 

------------------------------------------------------------------------------------ 




The UV/VIS spectroscopic measurements were made with a 

spectrophotometer (Lambda 20, Perkin Elmer), modified to obtain a 

rectangular beam diameter of 10x10 mm 2 . The spectra from 240- 

500 nm were recorded. The transmission was corrected with a blank 

tape in the reference beam. The absorbance at 430 nm was taken as 

the measure for the number of the corneocyte aggregates on the 

tape to obtain tape strip resolved depth profiles correlated with the 

quantitative data of Clobetasol propionate. 

• The HPLC method 

Clobetasol propionate was determined using an HPLC method (for 

details of the extraction procedure refer to reference [4]). The 

HPLC system comprised a binary pump (HP 1100 ChemStation), a 

reversed phase column system (main column: 2 × 100 mm with a 

precolumn: 2 × 5 mm; Hypersil ODS 5 µm; Knauer Berlin; 

Germany) and a UV diode array detector (HP 1100 ChemStation). 

The solvent was acetonitrile / 1 mmol/l NH4CH3COO buffer (55 : 

45) with a flow of 0.2 ml/min. 20 µl of each sample was injected. 

The retention time of the drug was 5.07 + 0.04 min and for the 

standard BM was 3.83 + 0.02 min. It was ascertained that the peaks 

of interest were seperated from peaks of interfering substances like 

matrix of cream, tape and skin (figure 3). The detection wavelengh 

(240 nm) allowed the identification of Clobetasol propionate (CB) 

and of the internal standard Betametasone-17-valerate (BM) 

(detection limit of a HPLC based UV-detection: 0.16mg/l [4]). 

------------------------------------------------------------------------------------ 




Fig. 3: UV-Chromatogramm: Absorbance at 240nm wavelength. 

• Mass Spectrometry 

For the analysis a HPLC/single quad mass spectrometer equipped 

with an electrospray ion source (LC/MSD Hewlett Packard ® ) was 

used. The ion source parameters were optimized to maximum 

intensity of the selected molecular ions and minimal fragmentation. 

Therefore a drying gas flow of 10 l/min with a temperature of 310 

°C (N2; Purity ≥ 98 %) was established. 

------------------------------------------------------------------------------------ 




100 

80 

60 

40 

20 

0 

rel. 

Int. 

O 

HO 

CH3 

CH2Cl 

C=O 

CH3 

200 300 400 500 

Fig. 4: Mass spectrum of CB in positive ionisation mode. Inset 

structure corresponds to loss of ketene from sodiated molecular ion 

(m/z 447). 

Especially thermally induced fragmentations could be avoided by a 

decrease of the drying gas temperature. The [M+H] + of CB at m/z 

467 and BM at m/z 477 were chosen for quantification (Figure 4). 

Single ion monitoring was performed with low resolution and a 

dwell time of 109 msec. Beside the protonated molecular ions 

[M+H] + the sodium adducts [M+Na] + appear at m/z 489 for CB and 

at m/z 499 for BM. One single fragment of considerable intensity 

can be seen at m/z 447 (CB) and at m/z 457 (BM), respectively. 

That fragment is obviously generated by a fragmentation of the 

sodium adducts [M+Na] + . The ring A of the steroid structure is a 

candidate for the loss of ketene CH2=C=O (42 u). 

------------------------------------------------------------------------------------ 



F 

OCOCH2CH3 

CH3 

[M+Na] + -CH2CO 

447 

467 

[M+H] + 

[M+Na] + 

489 

505 

[M+K] + 

m/z

Norm. 


Int. 

2 4 6 8 10 12 14 16 18 min 

Fig. 5a: Single ion monitoring mass chromatograms of tapestrip 

extracts. 3 rd tapestrip (0,114mg/l), 

Rel. Int. 

2 4 6 8 10 12 14 16 18 min 

Fig. 5b: Single ion monitoring mass chromatograms of tapestrip 

extracts. 17 th tape strip (0,00695mg/l). Dotted line: SIM of internal 

standard BM (m/z 477), solid line: SIM of analyte CB (m/z 467). 

Signals in chromatogramm B are normalised. 

The LC/MS SIM detection strategy overcomes problems with 

chromatographic interference of background substances and with 

------------------------------------------------------------------------------------ 



A 

B


the moderate UV absorption of the target compounds (Figure 5). 

Consequently an enormous increase in sensitivity of the introduced 

LC-ESI-MS procedure was achieved: calibration function: 

y=0.488x-0.00486; fit: 0.998; detection limit: 2.15 µg/l. 

Conclusion 

The HPLC/ESI-MS method for the detection and quantification of 

steroids demonstrates the analytical power of the applied technique. 

The mass spectrometrical detection of the substrates of interest 

allows new comprehension of the complex behavior of 

dermatologic active substances because of the possibility to 

determine horny layer profiles of high resolution. This example 

underlines the success of the interdiciplinary collaboration of 

different techniques for the solution of analytical problems. 

References 

1 Weigmann H.-J, Lademann J, Pelchrzim R v, Sterry W, 

Hagemeister T, Molzahn R, Schäfer M, 

Linscheid M, Schaefer H, Shah V P (1999) Bioavailability of 

Clobetasol Propionate – Quantification of Drug Concentrations 

in the Stratum Corneum by Dermatokinetics using Tape 

Stripping. Skin Pharmacol Appl Skin Physiol (Switzerland) 

12:46-53 

2 Weigmann H-J, Lademann J, Meffert H, Sterry W (1999) 

Determination of the Horny Layer Profile by Tape Stripping in 

Combination with Optical Spectroscopy in the Visible Range as 

a Prerequisite to Quantify Percutaneous Absorption. Skin 

Pharmacol Appl Skin Physiol (Switzerland) 12:34-45 

3 Weigmann H-J, Lademann J, Meffert H, Sterry W (1997) 

Influence of sunscreen on the spectral energy distribution of 

ultraviolet radiation in human skin. In: Altmeyer P, Hoffmann 

K, Stuecker M (ed) Skin Cancer and UV Radiation. Springer- 

Verlag, Berlin Heidelberg New York, S 357-362. 

------------------------------------------------------------------------------------ 




4 Guidance for Industry, Topical Dermatological Drug Product 

NDAs and ANDAs - In Vivo Bioavailability, Bioequivalence, In 

Vitro Release, and Associated, U. S. Department of Health and 

Human Services, Food and Drug Administration, Center for 

Drug Evaluation and Research (CDER), June 1998. 

------------------------------------------------------------------------------------ 



90 Analytical methods for the determination of dermatopharmacokinetics 

Analytical methods for the determination of 

dermatopharmacokinetics 

J. Lademann 

Center of Experimental and Applied Cutaneous Physiology (CCP) 

at the Charité, Department of Dermatology, Humboldt-Universität 


Germany 

Introduction................................................................... 90 

Penetration measurements by tape stripping in 

combination with spectroscopic measurements............ 92 

Dermatopharmacokinetics of sunscreen components... 96 

Dermatopharmacokinetics of topically applied drugs .. 98 

Perspectives in dermatopharmacokinetics.................... 99 

Summary ..................................................................... 102 

References................................................................... 102 

Introduction 

The penetration kinetics of cosmetic products and drugs in or 

through the skin, the so-called dermatopharmacokinetics, has a 

significant influence on the action and protection properties of 

topically applied substances. 

The determination of the penetration kinetics and the penetration 

pathways of chemical compounds is still an object of intensive 

investigations. Different analytical in vitro and in vivo methods 

were used for this purpose (Figure 1). The investigations were 

carried out on human and animal skin. 

------------------------------------------------------------------------------------ 



in vitro 

• Franz Cell experiments [1,2] 

• Biopsy [3,4] 

• Tape stripping [5,6] 

Analytical methods for the determination of dermatopharmacokinetics 91 

Animal model 

Human tissue 

in vivo 

• Rad ioactive labeling [7,8 ] 

• Biopsy [9,10] 

• Tape stripping [11,12] 

• Surface recovery [13] 

• Biological/pharmacological response [14] 

• A utoradiography [15] 

• Fluorescence measurements [16] 

Fig. 1: Method for the investigation of dermatopharmacokinetics 

On the one hand, the in-vitro measurements have the advantage that 

both allergens and toxic substances can be investigated. On the 

other hand, it is sometimes difficult to correlate the in-vitro to the 

in-vivo measurements. 

In the present paper, the state of the art and the perspectives of invivo 

measuring methods used in dermatopharmacokinetics will be 

discussed. Some of the in-vivo methods frequently used for 

measuring the cutaneous absorption of substances are presented in 

the right column of Figure 1. 

Only the first three methods allow both a qualitative and 

quantitative analysis of the penetration of topically applied 

substances into the skin. The time factor must be taken into 

consideration for the determination of the penetration kinetics. This 

means, that the process must be analyzed either by on-line 

measurements or by taking samples from the same volunteer at 

different times. 

Although well-suited to penetration measurements, the method of 

applying radioactive labeled substances is unacceptable from an 

ethical point of view. This applies also to skin biopsies. 

Tape stripping is a non-invasive method only suited to analyze the 

stratum corneum. Regardless of this limitation, this method can be 

used for the investigation of dermatopharmacokinetics of cosmetic 

------------------------------------------------------------------------------------ 




products and topically applied drugs, because the horny layer 

mainly determines the barrier and reservoir function of the skin. 

The only disadvantage of this method is the insufficient 

reproducibility, as the penetration curves are related to the number 

of tape strips and not to the actual horny layer profile. The amount 

of corneocytes on the removed tapes depends on different factors, 

e.g. skin type of the volunteers, pretreatment of the skin, and the 

formulation of the substances applied. 

A new analytical method for penetration measurements was 

developed at the Center of Experimental and Applied Cutaneous 

Physiology (CCP). The method is based on the well-known tape 

stripping technique in combination with spectroscopic 

measurements that allow the determination of the amount of 

corneocytes on each tape strip removed [17]. 

Penetration measurements by tape stripping in 

combination with spectroscopic measurements 

During the tape stripping procedure, a transparent, adhesive film is 

pressed onto a defined surface area of the skin after application and 

penetration of topically applied substances. The next film is pressed 

onto the same area after having removed the tape from the skin. 

Using this procedure, the stratum corneum is removed in thin 

layers. The removed tape strips contain specific amounts of 

corneocytes and substances applied topically. 

The distribution of the corneocyte aggregates on different tape 

strips of the same series is presented in Figure 2. It is obvious that 

the corneocyte density is reduced with increasing numbers of tapes. 

------------------------------------------------------------------------------------ 




1. strip 12. strip 30. strip 

Fig. 2: Distribution of the corneocyte aggregates of different tape 

strips from the same series 

The spectra of the first tapes, removed from sunscreen treated and 

untreated skin surfaces, are compared in Figure 3. The absorption 

bands of the applied UVA and UVB filter substance removed with 

the corneocyte aggregates can be clearly recognized. The 

absorbance at wavelengths higher than 400 nm, which is only 

caused by the scattering properties of the corneocytes, is identical 

for both tapes. The value of the absorbance at 430 nm was used in 

the following for the characterization of the amount of corneocytes 

on the removed tapes. Because of the inhomogeneous distribution 

of the corneocytes on the removed tape strips, PERKIN ELMER 

developed a special spectrometer with a measuring spot of 1 cm 2 

for integral measurements. 

------------------------------------------------------------------------------------ 




ABSORBANCE 

1,4 

1,2 

1 

0,8 

0,6 

0,4 

0,2 

1. Strip 

sunscreen 

applied 

0 

230 248 270 295 326 364 412 474 

WAVELENGTH [nm] 

------------------------------------------------------------------------------------ 



UVB f ilter 

absorption 

UVA f ilter 

absorption 

1. strip 

untreated skin Inf luence of 

corneocyte 

aggregates 

Fig. 3: Spectra of the first tapes removed from a sunscreen treated 

and untreated skin surface 

The principle of the combined method is summarized schematically 

in Figure 4. Using the additional information concerning the 

amount of corneocytes on the removed tape strips, it became 

possible to relate the amount of substance detected on the single 

tapes to the actual horny layer profile. 

This standardized method allows reproducible measurements of the 

penetration kinetics of topically applied substances into the stratum 

corneum. 

Amount of corneocytes 

• Light scattering measurements 

• Staining techniques 

• Determination of the corneocyte 

density on the tape strips 

Amount of topically applied 

substances 

• HPLC 

• GC/MS 

• AAS 

• Spectrometry 

• Spectroscopy 

Tab. 1: Analytical methods frequently used for the determination of 

the amount of corneocytes and the concentration of the topically 

applied Substances on the removed tape strips


The influence of different factors, such as the applied formulation 

and the skin type of the volunteers can be investigated qualitatively 

and quantitatively [18]. The analytical methods frequently used for 

the determination of the amount of corneocytes and the 

concentration of the substances on the removed tape strips are 

summarized in Table 1. 

Amount of corneocytes 

0,2 

0,15 

0,1 

0,05 

0 

Extended method 

UV/VIS spectroscopy 

Horny layer profile 

1 2 3 4 5 6 7 8 9 10 

Tape number 

Amount of corneocytes [%] 

0 

100 

0 

Series of tapes 

Amount of active agent 

3,5 

Amount of active agent [%] 

3 

2,5 

2 

1,5 

1 

0,5 

0 

Traditional method 

UV/VIS spectroscopy, 

XRF, AAS, HPLC, GC/MS 

Concentration on 

the single tapes 

1 2 3 4 5 6 7 8 9 

Tape number 

1 

5 

10 

30 

40 

70 

100 

Fig. 4: Principle of the tape stripping method in combination 

with spectroscopic measurements 

Tape number 

------------------------------------------------------------------------------------ 




Dermatopharmacokinetics of sunscreen components 

The penetration profiles of two different filter substances 

EUSOLEX 6300 and PARSOL 1789 of the same sunscreen sample 

after a penetration time of one hour are presented in Figure 5. It is 

obvious that the filter substances penetrated identically and were 

located in the upper part of the stratum corneum [17]. The 

penetration kinetics can be determined by analyzing the penetration 

profiles of a topically applied substance at different times after 

application. In the following example, this is demonstrated for 

coated titanium dioxide microparticles used in sunscreens. The 

amount of corneocytes on the removed tapes was determined by 

spectroscopic measurements at 430 nm. 

The amount of TiO2 on the tapes was measured by x-ray 

fluorescence analysis. The TiO2 concentration was not analyzed on 

every tape, because of the expensive x-ray fluorescence 

measurements. The penetration profiles obtained 1 hour and 24 

hours after sunscreen application are shown in Figure 6. The filter 

substances are mainly located in the upper layers of the stratum 

corneum. 

After 24 hours the amount of TiO2 was reduced significantly, on 

account of the fact that the skin was covered with textiles during the 

night. Small amounts of titanium dioxide were detected in the lower 

part of the stratum corneum. These TiO2 microparticles were 

located in the follicle orifices [19]. 

------------------------------------------------------------------------------------ 



Horny layer thickness [%] 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 


44 ,3 

6,2 

0 

0 5 10 15 20 25 30 35 40 45 50 

UVB filter concentration [µg/cm 2 77 

] 

0,5 

1, 5 

1,0 

1 

5 

10 

20 

50 

Strip number 

Horny layer thickness [%] 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

0 5 1 0 15 20 2 5 30 35 40 45 50 

UVA filter conc en tratio n [µg/ cm 2 ] 

EUSOLEX 6300 PARSOL 1789 

Fig. 5: Penetration profiles of the VU filter substances EUSOLEX 

6300 and PARSOL of a sunscreen sample after a penetration time 

of one hour 

0 

RELATIVE 

HORNY 

LAYER 

THICKNESS 

[%] 

100 

0,04 

0,15 

2,8 

1,5 

0,26 

TITANIUM CONCENTRATION [µg/square centimeter tape] 

1 

5 

10 

15 

NUMBER RELATIVE 

OF HORNY 

TAPE LAYER 

STRIPPING THICKNESS 

[%] 

30 

40 

83 

0 

100 

18,6 

------------------------------------------------------------------------------------ 



0,5 

0,2 

2,7 

0, 7 

1 

5 

10 

20 

50 

77 

Strip number 

TITANIUM CONCENTRATION [µg/square centimeter tape] 

1 hour 24 hour 

Fig. 6: Penetration profiles of coated titanium dioxide obtained 

1 hour and 24 hours after sunscreen application 

0,04 

0,15 

2,8 

1,5 

0,26 

1 

5 

10 

15 

NUMBER 

OF 

TAPE 

STRIPPING 

30 

40 

83


Dermatopharmacokinetics of topically applied drugs 

Although the combined method of tape stripping and spectroscopic 

measurements is only applicable to the analysis of the penetration 

kinetics inside the stratum corneum, this method can be used for the 

investigation of the dermatopharmacokinetics of topically applied 

drugs passing through the skin. The differences in the penetration 

profiles of the steroid compound, clobetasol, applied at the same 

concentration in different formulations on the skin, are shown in 

Figure 7 [20]. The influence of the formulation on the amount of 

corneocytes removed with the single tapes is obvious in this case. 

These significant differences could not be made visible by the 

conventional tape stripping methods, which relate the determined 

concentration of the substances on the removed tape strips to the 

strip number instead of to the horny layer profile. 

The penetration profiles in Figure 7 clearly demonstrate the 

reservoir function of the stratum corneum. Clobetasol has different 

penetration kinetics depending on the type of formulation [21]. 

The blanching effect is the biological response of living tissue on 

the interaction with clobetasol. This effect, caused by the 

constriction of the blood vessels, correlates to the detected 

penetration kinetics. 

Rel horny layer thickness [%] 

0 

-20 

-40 

-60 

-80 

-100 

0 Concentration 1 2of clobetasolpropionate 3 [µg/cm 4 5 

2 40 

-80 

40 

50 

50 

60 

60 

0 ], 

85 

5 

-100 

0 Concentration 

1 

of 

2 

clobetasolpropionate 

3 

[µg/cm 

4 

79 

5 

correlated to the absorbance 430 nm 

2 ], 

correlated to the absorbance at 430 nm 

------------------------------------------------------------------------------------ 



1 

5 

10 

20 

30 

Tape number 

Fig. 7: Penetration profiles of clobetasol in different 

formulations 6 hours after application [21] 

Rel. horny layer thickness [%] 

0 

-20 

-40 

-60 

1 

5 

10 

20 

30 

Tape number


Perspectives in dermatopharmacokinetics 

The perspectives in dermatopharmacokinetics are directed to the 

development and application of online methods for penetration 

measurements. These methods are based on optical and 

spectroscopic measurements, which utilize the absorption or 

scattering properties of the topically applied substances. In the case 

of laser Doppler blood flow measurements, the influence of a 

topically applied substance on the blood flow is investigated 

dependent on penetration time. 

The most promising online methods are the attenuated totalreflection 

spectroscopy (ATR) [22], opto-acoustic spectroscopy, 

optical coherent tomography (OCT) [23, 24], and the laser scanning 

microscopy (LSM) [25, 26]. These methods are presented 

schematically in Figure 8. 

However all these methods still have limitations, especially 

concerning the insufficient depth resolution of the measurements 

inside the stratum corneum or on account of the non-quantitative 

character of the measurements. In several cases, the substances 

under investigation should be dye or isotope labeled [25, 22]. In 

other cases, the direct contact of the sensor head to the skin surface 

leads to occlusion effects [22,23]. 

The rapid technical progress, especially in the field of laser 

techniques and spectroscopic methods, will overcome these 

limitations in the near future. One example for this is the 

development and application of a dermatological laser scanning 

microscope [27], which permits the detection of substances in 

different depths of the stratum corneum and in different layers 

(Figure 9) of the living human skin, by fluorescent measurements. 

------------------------------------------------------------------------------------ 




ATR 

LSM 

Opto 

acoustics 

------------------------------------------------------------------------------------ 



OCT 

Laser Doppler 

measurements 

Fig. 8: Promising online methods for the determination of 

dermatopharmacokinetics 

Using the principle of two-phonon excitation, laser radiation in the 

red or near infrared spectral region will be applied in the future to 

LSM systems. In this way, it will also be possible, to investigate 

deeper skin layers with a high space resolution. 

As mentioned before, the dermatopharmacokinetics of topically 

applied substances can be analyzed by detection of the biological 

response. In the case of skin treatment with clobetasol, the observed 

blanching effect is caused by changes in the blood flow. By 

applying the method of optical coherent tomography (OCT), series 

of images obtained (Figure 10) can be used for the determination of 

the blood flow in different depths of the skin [28]. These results are 

in correlation with the penetration kinetics of steroids into the living 

skin.


Stratum corneum 

Stratum Basale 

Stratum Granulosum 

Papillary Dermis 

Fig. 9: Detection of substances in different layers of the living 

human skin by laser scanning microscopy measurements [27] 

Fig. 10: The blood flow in different depths of the skin can be 

determined by optical coherent tomography (OCT image, forearm) 

------------------------------------------------------------------------------------ 




Summary 

In summary, it can be established that tape stripping, in 

combination with the determination of the horny layer profile, is a 

suitable qualitative and quantitative in vivo method for the analysis 

of dermatopharmacokinetics of topically applied substances. The 

development of non-invasive online methods based on optical and 

spectroscopic measurements will expand and improve the 

determination of the penetration processes, thus permitting a new 

insight into penetration pathways. 

References 

1 Ogiso T, Iwaki M, Tanino T, Nishioka S, Higashi K, Kamo M 

(1997) In vitro skin penetration and degradation of enkephalin, 

elcatonin and insulin. Biol Pharm Bull. 20(1): 54-60 

2 Bartlett A, Brown M, Marriott C, Whitfield PJ (2000) The 

infection of human skin by schistosome cercariae: studies using 

Franz cells. Parasitology 121: 49-54 

3 Golan TD, Sigal D, Sabo,-E, Shemuel Z, Guedj D, Weinberger 

A (1997) The penetrating potential of autoantibodies into live 

cells in vitro coincides with the in vivo staining of epidermal 

nuclei. Lupus 6(1):18-26 

4 Kim ED, Lipshultz LI, (1997) Advances in the evaluation and 

treatment of the infertile man. World-J-Urol. 15(6): 378-93 

5 Elias PM, Matsuyoshi N, Wu H, Lin C Wang ZH, Brown BE, 

Stanley JR (2001) Desmoglein isoform distribution affects 

stratum corneum structure and function. J-Cell-Biol. 153(2): 

243-9 

6 Koch PJ, de Viragh PA, Scharer E, Bundman D, Longley MA, 

Bickenbach J, Kawachi Y, Suga Y, Zhou Z, Huber M, Hohl D, 

Kartasova T, Jarnik M, Steven AC, Roop DR (2000) Lessons 

from loricrin-deficient mice: compensatory mechanisms 

maintaining skin barrier function in the absence of a major 

cornified envelope protein. J-Cell-Biol. 151(2): 389-400 

------------------------------------------------------------------------------------ 




7 Goldman DL, Casadevall A, Zuckier LS (1997) 

Pharmacokinetics and biodistribution of a monoclonal antibody 

to Cryptococcus neoformans capsular polysaccharide antigen in 

a rat model of cryptococcal meningitis: implications for passive 

immunotherapy. J. Med. Vet. Mycol. 35(4): 271-8 

8 Jimbow K (1998) Development of targeted chemoradiotherapy 

for malignant melanoma by exploitation of metabolic 

pathway.Hokkaido Igaku Zasshi. 73(2): 105-10 

9 Traber MG, Rallis M, Podda M, Weber C, Maibach H.I, Packer 

L (1998) Penetration and distribution of alpha-tocopherol, alpha- 

or gamma-tocotrienols applied individually onto murine skin. 

Lipids. 33(1): 87-91 

10 Hurks HM, van der Molen RG, Luiting C, Vermeer BJ, Claas 

FH, Mommaas M (1997) Differential effects of sunscreens on 

UVB-induced immunomodulation in humans. J Invest Dermatol. 

109(6): 699-703 

11 Dreher F, Arens A, Hostynek JJ, Mudumba S, Ademola J, 

Maibach HI (1998) Colorimetric method for quantifying human 

Stratum corneum removed by adhesive-tape stripping. Acta 

Derm.Venereol. 78(3): 186-9 

12 Weigmann H-J, Lademann J, Meffert H, Schaefer H, Sterry W 



a prerequisite to quantify percutaneos absorption. Skin Pharm 

Appl Skin Phys. 12:34-45 

13 Koizumi T (2000) Kinetic analysis of drug disposition and 

biological response. Yakugaku Zasshi. 120(5): 445-54 

14 Touitou E, Dayan N, Bergelson L, Godin B, Eliaz M (2000) 

Ethosomes - novel vesicular carriers for enhanced delivery: 

characterization and skin penetration properties. J. Controlled 

Release. 65(3): 403-18 

15 Zellmer S, Reissig D, Lasch J (1998) Reconstructed human skin 

as model for liposome-skin interaction. J Controlled Release 

55(2-3): 271-9 

16 Rhee GJ, Woo JS, Hwang SJ, Lee YW, Lee CH (1999) Topical 

oleo-hydrogel preparation of ketoprofen with enhanced skin 

permeability. Drug Dev Ind Pharm. 25(6): 717-26 

------------------------------------------------------------------------------------ 




17 Weigmann H-J, Lademann J, Meffert H, Schaefer H, Sterry W 



a prerequisite to quantify percutaneos absorption. Skin Pharm., 

12:34-45. 

18 Weigmann H-J, Lademann J, v. Pelchrzim R, Meffert H, Sterry 

W (2000) Quantitative Methode zur Bestimmung des 

Penetrationsverhalten von Sonnenschutzmitteln und 

Arzneimitteln in Relation zum realen Hornschichtprofil, 

Aktuelle Dermatologie, 26,138-142 

19 Lademann J, Weigmann H-J, Rickmeier Ch, Barthelmes H, 

Schaefer H, Müller G, Sterry W (1998) Penetration of titanium 

dioxide microparticles in a sunscreen formulation into the horny 

layer and the follicular orifice. Skin Pharm., 12:34-45. 

20 Weigmann H-J Lademann J, v Pelchrzim R, Sterry W, 

Hagemeister T, Molzahn R, Schäfer M, Linscheid M, Schaefer 

H (1998) Bioavailability of Clobetasol propionate – 

Quantification of Drug concentrations in the stratum corneum by 

dermatopharmacokinetics using tape stripping. Skin Pharm., 

12:46-53 

21 v. Pelchrzim R. (2002) Dissertation, Bestimmung der 

Bioverfügbarkeit von Clobethasolproprinat in Abhängigkeit von 

verschiedenen Grundlagen. Humboldt-Universität zu Berlin, 

Med. Fakultät Charité 

22 Heise HM, Küppel L, Pittermann W, Butvina LN (2001) New 

tool for epidermal and cosmetic formulation studies by 

attenuated total-reflection spectroscopy, Fres J Anal Chem 271, 

753-57 

23 Jackle S, Gladkova N, Feldchtein F, Terentieva A, Brand B, 

Gelikonov G, Gelikonov V, Sergeev A, Fritscher S, Ravens A, 

Freund J, Seitz U, Soehendra S, Schrodern N (2000) In vivo 

endoscopic optical coherence tomography of the human 

gastrointestinal tract--toward optical biopsy. Endoscopy 32(10): 

743-9 

24 Puliafito CA, Hee MR, Schuman JS, Fujimoto JG (1995) 

Optical coherence tomography of diseases, SLACK 

Incorporated Grove Road, Thorofare NJ, 12-42 

------------------------------------------------------------------------------------ 




25 Meuwissen ME, Janssen J, Cullander C, Junginger HE, 

Bouwstra JA (1998) A cross-section device to improve 

visualization of fluorescent probe penetration into the skin by 

confocal laser scanning microscopy, Pharm-Res. 15(2): 352-6 

26 Lademann J, Otberg N, Richter H, Weigmann H-J, Lindemann 

U, Schaefer H, Sterry W (2001) Investigation of follicular 

penetration of topically applied substances, Skin-Pharm.14 17- 

22 

27 Product information OptiScan, Live Micro Imaging Lawrenz 

Meßtechnik GmbH Eschede, Germany 

28 SkinDex 300, ISIS optronics GmbH, Mannheim, Germany 

------------------------------------------------------------------------------------ 



106 HPTLC and LC/MS in ceramide analysis 

HPTLC and LC/MS in ceramide analysis 

K. Raith, H. Farwanah, R. Neubert 

Martin Luther University Halle-Wittenberg 

Institute of Pharmaceutics and Biopharmaceutics 

W.-Langenbeck-Str. 4 

D-06120 Halle (S.) 

Germany 

Abstract ....................................................................... 106 

Introduction................................................................. 107 

Materials and Methods................................................ 108 

• Extraction................................................................... 108 

• Thin-layer Chromatography ...................................... 108 

• Mass Spectrometry .................................................... 109 

Results and Discussion ............................................... 110 

• Thin-layer Chromatography ...................................... 111 

• LC/MS ....................................................................... 112 

• Tandem MS ............................................................... 113 

Conclusion .................................................................. 115 

Acknowledgement ...................................................... 116 

References................................................................... 116 

Abstract 

The investigation of the role of ceramides in maintaining the barrier 

function of stratum corneum has brought about the need of specific 

analytical methods. The article describes a combination of highperformance 

thin-layer chromatography using automated multiple 

development, with subsequent liquid chromatography-electrospraymass 

spectrometry for the profiling of ceramides. The fractionation 

of complex lipid extracts with the help of thin-layer 

chromatography facilitates a specific, sensitive and reliable 

analysis. Characteristic patterns of the molecular mass distribution 

in different ceramide fractions are easily obtainable. Additional 

electrospray tandem mass spectrometry using an ion trap mass 

spectrometer provides fragment ions which indicate the sphingoid 

------------------------------------------------------------------------------------ 



HPTLC and LC/MS in ceramide analysis 107 

base as well as the fatty acid moiety and therefore enables the exact 

assignment of ceramide structures. 

Introduction 

The stratum corneum lipids are known to play a decisive role in 

maintaining the barrier function of the skin against transepidermal 

water loss and penetration of substances from the environment [1]. 

It has been shown that a number of skin disorders, such as psoriasis, 

atopic dermatitis, ichthyosis or xerosis, entail changes in lipid 

composition [2-6]. Therefore, the analysis of the major stratum 

corneum lipid classes, particularly of the ceramides, is a 

prerequisite to a better understanding of these disorders and to their 

specific treatment. 

For the separation of the major stratum corneum lipid classes, i.e. 

ceramides, fatty acids, cholesterol and its esters, thin-layer 

chromatography (TLC) is the method of choice. Normal phase 

HPLC as a possible alternative [7] is less robust to matrix 

components from biological lipid extracts. Furthermore, detection 

problems result from lacking UV absorbance of the lipids and 

incompatibility of the used mobile phase to electrospray mass 

spectrometry, respectively. Reversed phase LC implies a different 

selectivity due to its susceptibility to chain length influences, 

therefore not allowing ceramide class separation according to 

number and position of the hydroxy functions. 

The automated multiple development high performance thin-layer 

chromatography (AMD-HPTLC) uses automated procedures for 

eluent mixing, chamber conditioning, development and drying 

steps. Furthermore, it is less time and solvent consuming and avoids 

band broadening due to the reconcentration effect [8]. 

The ceramides can be separated in at least seven previously 

described subclasses [9], which are known to consist of a certain 

type of sphingoid base (sphingosine or phytosphingosine) and a 

certain type of fatty acid (normal or α-hydroxy fatty acids), 

respectively. Each of these subclasses consists of a variety of 

species differing with respect to chain length. 

A further analysis of the composition of each ceramide subclass can 

be performed using mass spectrometry. For this purpose, the 

------------------------------------------------------------------------------------ 




separated bands can be recovered and submitted to mass 

spectrometry. Due to the nature of ceramides as polar lipids, 

electrospray ionization is the method of choice. In negative 

ionization mode the best sensitivity can be achieved. To handle 

small amounts of sample and to prevent ion suppression effects, a 

preceding liquid chromatography on reversed phase columns is 

advisable. 

The identification of the different ceramide species is possible using 

tandem mass spectrometry (MS/MS). This requires an ion trap, 

triple quadrupole or any other type of mass analyzer capable of 

performing MS/MS experiments. 


• Extraction 

For surface extraction purposes a cylindric glass beaker with 4 cm 

ID (contact area 12.56 cm 2 ) was filled with 8 ml n-hexane/ethanol 

2:1 (V/V). The open side was pressed tightly to a skin area at the 

inner forearm to prevent lateral leakage. The extraction time was 5 

min throughout. The extraction mixture was thereafter evaporated 

at 50 °C and the residue dried under a stream of argon. The dried 

lipids were then dissolved in 500 µl chloroform/methanol 1:1. 

• Thin-layer Chromatography 

The HPTLC plates were washed three times with 

chloroform/methanol 65:35 (V/V) before use. Sample application 

has been carried out automatically using an Automatic TLC 

Sampler 4 (Camag, Muttenz, Switzerland) at a dosage speed of 100 

µl/s. 15 samples were applied on each plate at a start line 8 mm 

from the bottom, including 6 lanes for skin lipid samples (each 6 µl) 

and 9 lanes for reference lipids ranging from 200 ng to 4 µg for 

each standard lipid. The band width was 8 mm with 4 mm distance 

to the neighbouring lane. 

The development of the plates has been carried out automatically 

using an AMD-2 apparatus (Camag, Muttenz, Switzerland). The 

------------------------------------------------------------------------------------ 




used AMD procedure included a 17 step gradient of decreasing 

polarity using ethanol, acetone, chloroform, ethylacetate and nhexane. 

After drying, the plates were dipped into an aqueous solution of 

10% CuSO4, 8% H3PO4 (V/V), and 5% methanol for 20 s. 

Afterwards, the plates were charred in a drying oven at 150°C for 

20 min. 

The developed and visualised plates were scanned from 7 cm to 

front using a TLC Scanner 3 (Camag, Muttenz, Switzerland). The 

measurement was performed in reflectance mode at a wavelength of 

546 nm. The slit dimensions were 4 x 0.2 mm at a scan speed of 20 

C hole sterolester 

Cho lest erol 

Ceramide 1 [EOS] 

Cerami de 2 [NS] 

C eramide 3 [NP] 

Ceramide 4 

Cerami de 5 [AS] 

C eramide 6 [AP] 

Ceramide 7 

Fa ty Acids 

Fig. 1: The AMD-2 apparatus Fig. 2: Stratum corneum lipid 

(Camag, Muttenz, Schweiz) lipid separation using AMD- 

HPTLC 

mm/s and a data resolution of 25 µm per step. Integration and 

quantification based on peak areas were performed using CATS 

software (Camag, Muttenz, Switzerland). To avoid experimental 

errors, individual curves were set up for each HPTLC plate. 

Quantitative results for all ceramides were related to Ceramide NP. 

• Mass Spectrometry 

Provided that a non-destructive detection is used or detection is 

limited to the margin zones of the plate [10], the bands 

corresponding to the ceramide subclasses can be recovered from the 

plate and submitted to mass spectrometry. Herefore, the loaded 

------------------------------------------------------------------------------------ 




silica fractions can be reextracted for 10 min with 

chloroform/methanol 1:1 assisted by ultrasound. After 

centrifugation the supernatant can be analysed directly by LC/MS. 

These analyses were performed using a Spectra System P 4000 LC 

pump equipped with an autosampler AS 3000, a membrane 

degasser SCM 1000 and a controller SN 4000 (ThermoFinnigan, 

San Jose, CA, USA). In ceramide analysis a reversed phase column 

Nucleosil ® 120 3C18 (125x2mm ID) (Macherey-Nagel, Düren, 

Germany) and a mobile phase consisting of 

methanol/tetrahydrofurane 97:3 was used. 

An ion trap mass spectrometer Finnigan LCQ (ThermoFinnigan, 

San Jose, CA, USA) equipped with an electrospray interface was 

used throughout. More details about the LC/MS method are given 

in literature [11]. Electrospray ionization was performed preferably 

in the negative ion full scan mode. Tandem mass spectra are 

obtained by collision-induced dissociation in the ion trap. A relative 

collision energy of 28 % was applied, which resulted in a decrease 

of the precursor ion to approx. 10 % relative intensity. 

Fig. 3. The LC/MS system described 

under Materials and Methods 

(ThermoFinnigan, San Jose, CA, USA). 

Results and Discussion 

------------------------------------------------------------------------------------ 



Fig. 4: The ion trap 

mass analyzer of the 

LCQ (cross-section). 

To obtain the results described below, using the methods of AMD- 

HPTLC and LC/MS, two strategies turned out to be practicable: 

firstly, lipid extraction of stratum corneum samples obtained by 

means of a Bligh-Dyer extraction. This approach requires a 

sufficient amount of stratum corneum (> 60 mg) and is therefore


limited to body areas were such an amount can be obtained, namely 

the plantar sole skin. The second approach is the in-vivo-surface 

extraction of stratum corneum lipids. The general advantage of 

surface extraction is that it is noninvasive and can be performed 

easily in vivo. However, one has to bear in mind that sebum lipids 

consisting mainly of triacylglycerol and wax esters and are 

coextracted with the stratum corneum lipids. Several solvent 

mixtures have been tested. In contrast to some literature reports 

[12], mixtures containing diethylether turned out to be 

impracticable, because the high steam-pressure caused solvent 

losses. Furthermore, many solvents including chloroform or acetone 

should not be used, for they are inducing pain and lasting skin 

damages. The chosen hexane/ethanol 2:1 mixture was a 

compromise between extraction efficiency and ethical aspects. 

If the analysis is exclusively aimed at the ceramides, a preceding 

solid phase extraction is advisable to separate the ceramides from 

other lipids [13]. 

• Thin-layer Chromatography 

An improved procedure for the separation of major stratum 

corneum lipids by means of AMD-HPTLC has recently been 

developed [14]. The first 11 steps were performed using mixtures of 

chloroform, ethanol and aceton. Thereafter followed 3 isocratic 

steps with chloroform. These steps allowed the separation of 

cholesterol sulfate, the various ceramide classes and cholesterol. 

For the separation of cholesterol, fatty acids, triacylglycerol, 

cholesterol esters, and squalene, 2 additional steps were required 

with a mixture containing n-hexane and ethylacetate followed by an 

isocratic hexane step. Before each step the plates were conditioned 

with a stream of 4 N acetic acid in order to focus the fatty acid 

bands and to achieve a better resolution in the ceramide fractions. 

This gradient enables base line separation of the lipid standards 

cholesterol sulfate, ceramide AP, ceramide AS, ceramide NP, 

ceramide NS, cholesterol, palmitic acid, triacylglycerol, cholesterol 

oleate, and squalene. Since the used ceramide AP is a semisynthetic 

substance containing a racemic 2-hydroxy fatty acid moiety, the 

corresponding spot is separated into 2 bands. 

------------------------------------------------------------------------------------ 




The developed AMD-HPTLC method has been applied to surface 

lipid extracts of human skin. The skin lipids were extracted in vivo 

as described under Materials and Methods. 

Lipid bands could be unequivocally identified by relating the Rf 

values to those of the standard lipids as well as profiles from 

previous studies [12,15,16]. In some cases, the Rf of the skin lipids 

was slightly larger. This results because the fatty acid as well as 

sphingoid base moieties in stratum corneum ceramides have longer 

chain lengths compared to the standards. In addition, the broad 

spectrum of chain lengths implies a more complex densitometric 

profile, although the resolution obtainable using AMD is still much 

better than with conventional manual procedures. The present 

protocol enables a good separation of all major stratum corneum 

and sebum lipids in 150 min with less solvent consumption 

(ethanol: 8 ml, acetone and ethylacetate: 6 ml each, n-hexane: 19 

ml, and chloroform: 81 ml). It can be emphasised, that particularly 

the separation of ceramides could be improved in comparison to 

previously reported AMD protocols [17]. Hence, the present 

procedure is suitable to be used for diagnostic purposes as well as 

for high-throughput analysis of the major stratum corneum lipids. 

The calibration curves for densitometric quantification are usually 

not linear and were fitted using a saturation function such as the 

Michaelis-Menten equation as described before [18]. 

Detailed comments on the results obtainable by AMD-HPTLC 

quantification are given in [14]. 

• LC/MS 

LC/MS enables a rapid and sensitive ceramide quantification, 

which is completed by structural information obtainable by tandem 

mass spectrometry. Unfortunately, the disturbing effects of the 

extremely complex matrix do not allow the direct quantitation of 

single ceramide species in the lipid extract. For this reason, stratum 

corneum ceramide profiling by ESI-MS and MS/MS without 

chromatographic separation was not successful (in contrast to the 

less complex intracellular ceramides), especially in view of the fact 

that suitable internal standards (deuterated ceramides) are not 

available. Therefore, we combined AMD-HPTLC and LC/MS. 

------------------------------------------------------------------------------------ 




The fractions containing Cer [NS], [NP], [AS] and [AP] were 

localized by comparison with the Rf values of the detected standard 

lipids at the edge of the plate, scraped from the plate, reextracted 

and analyzed using LC/MS. All measurements were acquired in full 

scan mode. Therefore, it has been possible to extract every mass-tocharge 

ratio from the total ion current, if evidence has been found 

for in average mass spectra. As a reference, “expected” molecular 

weights were calculated from the percentages of incidence, that 

Wertz et al. have presented for fatty acids and long chain bases in 

each of the ceramide classes [6]. Details are given in [10]. In 

accordance to the predicted data, the largest peaks have been 

detected at m/z= 706 and m/z= 678. Additional tandem mass 

spectrometry revealed that these peaks are caused mostly by 

ceramides formed by the combination of C-20-sphinganine with 

lignoceric and hexacosanoic acid, respectively. In the ceramide 

fractions [NP], [AS] and [AP] also a good correlation of the 

predicted and observed molecular weight distribution was found. 

In the described LC/MS approach, the calculation of the relative 

abundance of the most important ceramides in each fraction was 

performed in two ways. First, the peak areas of all distinguished 

peaks were calculated and related to the largest one. Second, the 

calculation refered to the relative abundance in a mass spectrum, 

which has been summed over a time range from 1 to 8 min 

retention time in LC. The first approach is more specific but less 

sensitive. In the case of several coeluting substances the peak 

integration becomes difficult. The second approach provides better 

sensitivity and reproducibility (3% RSD versus 5-10%) but sums 

the homologs with identical m/z. The molecular mass distribution 

of ceramides in the ceramide-2-fraction is obviously not a simple 

Gaussian distribution. 

• Tandem MS 

For a real species specific quantitation LC/MS/MS is needed. 

Whereas in positive mode in many cases only multiple stage MS 

(MS n ) enables an unequivocal identification, in negative mode this 

goal can be achieved yet in MS/MS. The parameters for ceramide 

------------------------------------------------------------------------------------ 




fragmentation have been reported in [19]. Figure 5 shows a 

negative ESI-Tandem mass spectrum of N-Stearoylsphinganine, a 

Cer[NS] species. Figure 6 illustrates the corresponding fragment 

structures. As can be seen, both the sphingoid base as well as the 

fatty acid moiety are directly accessible. By means of LC/MS/MS 

even homologs within a fraction with identical molecular mass and 

retention behaviour may be separately profiled. Electrospray 

tandem mass spectra of ceramides and the proposed structures of 

the fragment ions have been discussed in detail in [10] and [19], 

respectively. The exhaustive LC/MS/MS investigation of all 

possible ceramides (probably by far more than 1000) is a largescale 

task. It seems to be more useful to screen ceramide samples 

from different origins by LC/MS and use MS/MS as an additional 

tool, if significant differences in the molecular mass distribution are 

observed. 

It is also possible to perform equal MS/MS studies using a triple 

quadrupole mass spectrometer. Thereby the obtainable sensitivity is 

slightly better in Selected Ion Monitoring, but not so good in Full 

Scan, which may be disadvantageous in the analysis of complex 

samples including unknown substances. 

------------------------------------------------------------------------------------ 



Relative Abundance 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

Conclusion 

0 

26 5.4 

23 9.4 

300.3 

283.5 

30 8.4 

324.3 


200 250 300 350 400 450 500 550 600 

m/z 

Fig. 5: Negative ESI-Tandem mass spectrum of N- 

Stearoylsphinganine. 

Stearic acid 

m/z=283 

R O 

m/z=239 

R 

- 

Ceramide II [M-H] 

N-Stearoylsphinganine 

m/z=567 

m/z=265 

O 

R 

N 

H 2 

O 

m/z=300 

N 

OH 

O 

R 

OH 

OH 

N 

-H 2 O 

O 

m/z=308 

------------------------------------------------------------------------------------ 



518.6 

-CH 2 OH,-2H 

R 

53 4.5 

m/z=548 

R 

548.4 

m/z=518 

N 

H 

O 

567.5 

N 

O 

R 

m/z=324 

m/z=534 

Fig. 6: Fragment structure corresponding to Fig. 5. 

The described combined approach provides detailed information 

about stratum corneum lipid composition, particularly with respect 

to ceramide structures. However, the evaluation of all the 

obtainable data is not yet completed. These methods are capable of 

solving numerous analytical problems raising from dermatological 

studies. From the analytical point of view, the further development 

should be aimed to online coupling and automation. In future 

assays, TLC could be replaced by normal phase LC, e.g. with 

evaporative light scattering detection. After fraction collection the 

samples could be submitted to reversed phase LC/MS/MS. 

Thereafter, an online LC/LC/MS/MS approach could be developed. 

O


Acknowledgement 

We wish to thank Manuela Woigk for excellent technical 

assistance. 

References 

1 P.M. Elias, J. Goerke, D.S. Friend, J. Invest. Dermatol., 1977, 

69, 535. 

2 S. Motta, M. Monti, S. Sesana, R. Ghidoni and M. Monti, Arch. 

Dermatol. Res., 1995, 287, 691. 

3 A. di Nardo, P.W. Wertz, A.Giannetti and S. Seidenari, Acta. 

Derm. Venereol., 1998, 78, 27. 

4 C. Schmidt, N.S. Penneys, V.A. Ziboh, L. Leim, T. Schlossberg, 

J. Invest. Dermatol., 1977, 68, 206. 

5 K. Akimoto, N. Yoshikawa, Y. Higaki, M. Kawashima, G. 

Imokawa, J. Dermatol., 1993, 20, 1. 

6 P.W. Wertz, M.C. Miethke, S.A. Long, J.S. Strauss and D.T. 

Downing, J. Invest. Dermatol., 1985, 84, 410. 

7 H. Farwanah, R. Neubert, K. Raith, 26 th Int. Conference on 

HPLC and Related Technologies, Montréal, Canada, 03.- 

06.06.2002. 

8 F. Bonté, P. Pinguet, J.M. Chevalier, A. Meybeck, J. 

Chromatogr. B, 1995, 664, 311. 

9 K.J. Robson, M.E. Stewart, S. Michelsen, N.D. Lazo and D.T. 

Downing, J. Lipid Res., 1994, 35, 2060. 

10 K. Raith, S. Zellmer, J. Lasch, R.H.H. Neubert, Anal. Chim. 

Acta, 2000, 418, 167. 

11 K. Raith, R. Neubert, Anal. Chim. Acta, 2000, 403, 295. 

12 M. Ponec, E. Boelsma, A. Weerheim, Acta Derm. Veneoreol., 

2000, 80, 89. 

13 H. Farwanah, K. Raith, C. Huschka, W. Wohlrab, R. Neubert, 

Arch. Pharm. Pharm. Med. Chem., 2001, 334, Suppl. 2, 66. 

14 H. Farwanah, R. Neubert, S. Zellmer, K. Raith, submitted to 

Analyst, 2002. 

15 A.P.M. Lavrijsen, I.M. Higounec, A. Weerheim, E. Oestmann 

and E.E. Tuinenburg, Arch. Dermatol. Res., 1994, 286, 495. 

------------------------------------------------------------------------------------ 




16 S.A. Long, P.W. Wertz, J.S. Strauss and D.T. Downing , Arch. 

Dermatol. Res., 1985, 277, 284. 

17 S. Motta, M. Monti, S. Sesana, R. Caputo, S. Carelli and R. 

Ghidoni, Biochim. Biophys. Acta, 1993, 1182, 147. 

18 S. Zellmer, J. Lasch, J. Chromatogr. B, 1997, 691, 321. 

------------------------------------------------------------------------------------ 



118 HPLC-analysis for permeation studies of 5-aminolevulinic acid and its derivatives 

HPLC-analysis for permeation studies of 5aminolevulinic 

acid and its derivatives 

A. Winkler C.C. Müller-Goymann 

Technische Universität Carolo-Wilhelmina 


Mendelssohnstraße 1 

D-38106 Braunschweig 

Germany 

Introduction................................................................. 118 

Experimental methods................................................. 120 

• Materials .................................................................... 120 

• HPLC conditions ....................................................... 120 

• Derivatisation of ALA and ABE ............................... 121 

• In vitro permeation studies ........................................ 122 

• Stability of ALA and ABE during in vitro 

permeation ................................................................. 122 


Conclusion .................................................................. 126 

Acknowledgements..................................................... 126 

References................................................................... 126 

Introduction 

5-Aminolevulinic acid (ALA) is a precursor in the biosynthetic 

pathway of porphyrins, especially protoporphyrin IX (Pp IX), and 

heme [1]. Photodynamic therapy (PDT) uses ALA induced 

accumulation of Pp IX in abnormal cells to treat skin diseases or 

cancer of the bladder, oesophagus, and lung after topical or 

systemic application of exogenous ALA. The subsequent activation 

by light of an appropriate wavelength causes photodamage of the 

tumour by photochemical reaction [2, 3]. 

A major drawback of ALA is its high hydrophilicity as a 

hydrochloride which results in a low intercellular bioavailability. In 

order to increase lipophilicity and to improve the diffusion 

properties of ALA several derivatives of ALA were synthesized by 

Kloek et al. [4], one of which is 5-aminolevulinic acid-n-butyl ester 

------------------------------------------------------------------------------------ 



HPLC-analysis for permeation studies of 5-aminolevulinic acid and its derivatives 119 

(ABE). ABE induced higher Pp IX concentration in cultivated cells 

than ALA did. 

In order to investigate and compare the diffusion properties of ALA 

and ABE into human skin an in vitro model was developed using 

modified Franz cells. The separating membrane between donor and 

receiver was excised human stratum corneum which is the main 

barrier of human skin especially for hydrophilic substances. 

Furthermore artificial skin constructs (ASC) were used, being an 

alternative to excised human skin. ASC can be produced by 

standardised cultivation in highly reproducible quality [5, 6]. 

Quantification of the permeated amounts of either drug requires a 

sufficiently sensitive method. ALA and ABE concentrations which 

are likely to be sampled during in vitro permeation studies cannot 

be determined by UV detection with sufficient sensitivity, as has 

been demonstrated by [7]. The UV detection limit for a reversed 

phase ion pair chromatography on octadecyl silica with a mobile 

phase of methanol water containing 1-heptanesulphonic acid was 50 

µg/ml [7]. A colorimetrical determination of ALA based on the 

colour reaction of ALA-pyrrole with Ehrlich´s reagent also shows a 

low sensitivity [8, 9]. Sensitive methods using fluorescence 

detection after derivatisation of ALA with acetylacetone and 

formaldehyde [10] or o-phthaldialdehyde (OPA) [11] have been 

reported in combination with high performance liquid 

chromatography. OPA derivatisation was developed by Roth 

originally for amino acid analysis [12]. Primary amines react with 

OPA in the presence of thiol forming an instable fluorescent 

product [13]. Advantages of this method are a short reaction time of 

2 minutes and an adequate reactivity of the used reagents at ambient 

temperature opposite to the derivatisation with acetylacetone and 

formaldehyde which needs heating of the mixture for 10 min at 

100°C [10]. However amino acids released from stratum corneum 

and ASC during in vitro permeation studies react the same way. 

Hence, these substances have to be separated from ALA and ABE. 

Due to frequent sampling during permeation studies, HPLC 

separation of ALA and ABE from other amino acids should occur 

within time intervals as short as possible. 

------------------------------------------------------------------------------------ 




This report provides in vitro permeation studies, including an HPLC 

method for the quantification of ALA and ABE, respectively. ALA 

quantification has been adopted from Ho et al. [11] whereas ABE 

quantification combines both [11] and an amino acid quantification 

according to Graser et al. [14]. The methods from the literature are 

slightly modified to determine ALA or ABE and amino acids from 

stratum corneum or ASC simultaneously. 

Experimental methods 

• Materials 

ALA was provided by Medac GmbH, Wedel, Germany. ABE was 

synthesized according to Kloek et al. [4]. n-butanol and thionyl 

chloride were used to esterify ALA. n-butanol was purchased from 

Heraeus GmbH, Karlsruhe, Germany, thionylchloride from Merck 

KGaA, Darmstadt, Germany. ABE was purified by recrystallisation 

in diethylether purchased from Fluka, Neu-Ulm, Germany (all 

chemicals were reagent grade). 

Acetonitrile, methanol and acetic acid (all chemicals were HPLC 

grade) were purchased from J.T. Baker, VA Deventer, The 

Netherlands. OPA (HPLC quality) was purchased from Fluka, Neu- 

Ulm, Germany, mercaptoethanol (pro analysi) and all buffer 

substances (pro analysi) from Merck KGaA, Darmstadt, Germany. 

Boric acid (HPLC quality) was purchased from Carl Roth GmbH, 

Karlsruhe, Germany, absolute ethanol (HPLC quality) by Riedel de 

Haën, Seelze, Germany. Water was used in bidistilled quality. 

• HPLC conditions 

The HPLC system consisted of a fluorescence HPLC monitor RF- 

353 (Shimadzu, Kyoto, Japan) and a Spectroflow 400 solvent 

delivery system (Kratos Analytical, New Jersey, USA) equipped 

with a Rheodyne 7125 syringe loading sample injector (Rheodyne, 

Cotati, USA). The injector was fitted with a 20 µl loop. The 

analytical column (250 mm x 4.6 mm) was connected in line with a 

------------------------------------------------------------------------------------ 




guard column (10 mm x 4.6 mm). Both columns were packed with 

Hypersil ODS (particle size 5 µm) (Grom, Herrenberg, Germany). 

Eluation of ALA and ABE was performed with different mobile 

phases depending on different physicochemical properties of both 

drugs. ALA was eluted according to [9] with a mixture of an 

aqueous acetate buffer (22 mM) and methanol in the ratio of 7.5:5. 

The flow rate was 1.3 ml/min at ambient temperature. The mobile 

phase for ABE elution was prepared according to Graser et al. [14] 

who used this method for the determination of amino acids. An 

aqueous monobasic potassium phosphate buffer (12.5 mM, pH 7.2) 

was mixed with acetonitrile in the ratio of 5.5 to 5.0. ABE was 

isolated by isocratic elution at a flow rate of 1.5 ml/min at ambient 

temperature. 

• Derivatisation of ALA and ABE 

The derivatisation reagent was prepared according to Ho et al. [11]. 

27 mg OPA were dissolved in 500 µl ethanol. Subsequently 4.5 ml 

of a sodium borate buffer (0.4 M, adjusted to pH 9.5 with a 20 % 

sodium hydroxide solution) were added to this solution. At least 25 

µl mercaptoethanol were added. Prior to use the mixture was stored 

over night at room temperature. 

The derivatisation was performed according to Ho et al. [11], too. 

One hundred microliters of the derivatisation reagent were mixed 

with 100 µl of the sample. Aliquots from the receiver enriched with 

ABE after the permeation studies with ASC were diluted by ratio 

1:10 with phosphate buffer of pH 5.0 (Ph. Helv. 8) because of the 

high concentration of ABE. After a reaction time of exactly 2 

minutes 100 µl monobasic potassium phosphate buffer (0.1 M, pH 

4.0) were added to the mixture. Immediately afterwards 20 µl of 

this solution were injected onto the described column to avoid 

fading of fluorescence. Fluorescence was monitored with an 

excitation wavelength of 330 nm and an emission wavelength of 

418 nm. 

------------------------------------------------------------------------------------ 




• In vitro permeation studies 

In vitro permeation studies were performed with a modified Franz 

cell [15]. Excipial ® Fettcreme enriched with either 10 % (w/w) 

ALA or 10 % (w/w) ABE prior to the permeation studies served as 

donor. The donor was freshly prepared while mixing the drug with 

Excipial ® Fettcreme for 2.5 minutes using an Unguator ® (GAKO 

Konietzko GmbH, Bamberg, Germany). 

The receiver contained 4.5-6.0 ml of phosphate buffer of pH 5.0 

(Ph. Helv. 8) to guarantee for stability of ALA and ABE. At a pH 

above 5.2 an instability of ALA is recognized by an increasingly 

yellow colour and a decrease in pH of the solution due to formation 

of pyrazin derivatives [16-18]. The temperature of the receiver was 

maintained at 37 °C by a waterbath. 

Donor and receiver were separated by stratum corneum or ASC, 

respectively. Stratum corneum which was separated from human 

skin samples by trypsination was placed on a polycarbonate filter 

(Isopore ® membrane filters, type TMTP, 5.0 µm, Millipore, Ireland) 

for higher mechanical stability. ASC cultivated under standard 

conditions [5, 6] was used together with the polycarbonate 

membrane of the Transwell ® insert. 

• Stability of ALA and ABE during in vitro permeation 

For each substance stability was checked under the conditions of 

the in vitro permeation studies. While being stirred aqueous 

phosphate buffer solutions of pH 5.0 (Ph. Helv. 8) with ALA or 

ABE (40 µg/ml) were incubated at 37°C together with pieces of 

stratum corneum for 28 h or with pieces of ASC for 10 h, 

respectively (total diameter of stratum corneum and ASC was about 

1.5 cm each). Same solutions without stratum corneum or ASC 

were treated the same way as standards. Samples were taken as 

shown in figure 4. The concentration of each substance was 

analysed by HPLC as described. 

------------------------------------------------------------------------------------ 





The mobile phases of the HPLC methods from the literature [11, 

14] were optimised in order to improve separation of ABE or ALA 

from other amino acids. In the case of ABE a mobile phase of 

acetonitrile/ phosphate buffer in a ratio of 5.0:5.5 resulted in a 

retention time of about 11 minutes and sufficiently high sensitivity. 

With solvent systems of acetonitrile/ phosphate buffer in ratios of 

5.0:5.0 or 5.5:5.0 retention time decreased but the ABE peak 

interfered sometimes with other peaks. For quantification of ALA, a 

mobile phase of aqueous acetate buffer (22 mM) and methanol in 

the ratio of 7.5:5 yielded a separation superior to that with the 

mobile phase described by Ho et al. [11]. 

Representative HPLC chromatograms for ABE and ALA derived 

from permeation studies with stratum corneum are shown in figure 

1. Several peaks beside ABE are caused by amino acids and the 

derivatisation reagent, but no interference occurs between the drug 

and the other peaks. The reaction time for derivatisation of ALA 

and ABE and the running time of a single analysis are short enough 

for routine analysis of a large number of samples. About 5 runs 

could be performed per hour. 

The detection limit for both HPLC-methods is 0.100 µg/ml. The 

correlation coefficient for calibration in the range of 0.100 to 5.000 

µg/ml for ALA and 0.100 to 20.000 µg/ml for ABE was higher than 

0.999. This sensitive analysis of either drug is required for 

permeation studies with stratum corneum. The permeation profiles 

of ALA and ABE from Excipial ® Fettcreme across stratum 

corneum are shown in figure 3 (left). ALA or ABE could be proven 

in the receiver after 6 to 10 hours. The highest analysed ALA and 

ABE concentrations in the receiver were about 0.5 µg/ml and 4 

µg/ml, respectively, at the end of the permeation study. 

------------------------------------------------------------------------------------ 




Fluorecsence 

0,0010 

0,0005 

0,0000 

-0,0005 

Fluorescence 

ABE (11.03) 

0 2 4 6 8 10 12 

Time [min] 

Fluorescence 

0,0015 

0,0010 

0,0005 

0,0000 

-0,0005 

------------------------------------------------------------------------------------ 



ALA (8.3) 

-0,0010 

0 2 4 6 8 10 

Time [min] 

Fig. 1: HPLC chromatogram for ABE (left) and ALA (right) 

derived from permeation studies with stratum corneum; 

concentration of ABE: 0.42 µg/ml and ALA: 0.51 µg/ml 

0,020 

0,015 

0,010 

0,005 

0,000 

ABE (11.02) 

0 2 4 6 8 10 12 

Time [min] 

Fluorescence 

0,025 

0,020 

0,015 

0,010 

0,005 

0,000 

ALA (8.8) 

-0,005 

0 2 4 6 8 10 

Time [min] 

Fig. 2: HPLC chromatogram for ABE (left) and ALA (right) 

derived from permeation studies with ASC; concentration of ABE: 

30.57 µg/ml and ALA: 37.08 µg/ml 

Permeated amounts [µg/cm²] 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

0 500 1000 1500 2000 

Time [min] 

Permeated amounts [µg/cm²] 

7000 

6000 

5000 

4000 

3000 

2000 

1000 

0 

0 100 200 300 400 500 600 700 

Time [min] 

Fig. 3: Permeation of ALA (▲) and ABE (■) from Excipial ® 

Fettcreme enriched with ALA (10 % (w/w)) and ABE (10 % (w/w)) 

across stratum corneum (left) and ASC (right); ABE, n=6 for 

stratum corneum and 7 for ASC; ALA, n=9 for stratum corneum 

and 8 for ASC; graphs represent mean values and standard 

deviation [19]


The permeation profiles of ALA and ABE through ASC are shown 

in figure 3 (right). Permeability of ASC for both substances were 

much higher than for stratum corneum. Within the first 3 hours of 

the permeation studies permeated amounts of ALA and ABE 

increased rapidly. After 2 and 4-5 hours the ascent of the curve 

became linear, respectively. This rapid increase in permeated 

amounts agrees with the results of permeation studies with 

ibuprofen acid through ASC from literature [5]. Hence, each 

substance may be detected from the beginning of the permeation 

studies. Representative HPLC chromatograms for ABE and ALA 

derived from permeation studies with ASC are shown in figure 2. 

Resolution between ALA or ABE and other substances, which were 

released from ASC, were well defined. Higher amounts of either 

drug could be separated as well as small amounts as described 

above. Correlation coefficients for ALA and ABE calibrations in 

the range of 1-200 µg/ml were higher than 0.999. 

Concentration 

of ALA [µg/ml] 

Concentration 

of ABE [µg/ml] 

42 

40 

38 

0 400 800 1200 1600 

Time [min] 

44 

42 

40 

38 

0 400 800 1200 1600 

Time [min] 

Concentration 

of ALA [µg/ml] 

Concentration 

of ABE [µg/ml] 

42 

40 

38 

42 

40 

38 

0 100 200 300 400 500 600 

Time [min] 

0 100 200 300 400 500 600 

Time [min] 

Fig. 4: Stability studies for ALA and ABE (40 µg/ml) in phosphate 

buffer of pH 5.0 (Ph. Helv. 8) at 37°C; ALA (□, n=4) or ABE (○, 

n=4) was stirred in the presence of stratum corneum (left) or ASC 

(right); ▲: ALA or ABE was stirred without stratum corneum or 

ASC (n=2); graphs represent mean values and standard deviation 

Stability of ALA and ABE is guaranteed at the described conditions 

as shown in figure 4. Sample concentration of ALA or ABE did not 

decrease with time either in the presence of stratum corneum and 

ASC or in absence of stratum corneum and ASC. Therefore 

determination of either drug was not affected by substances which 

were released from stratum corneum or ASC. 

------------------------------------------------------------------------------------ 




Conclusion 

HPLC with precolumn OPA derivatisation is an important method 

for quantification of ALA and its derivatives besides other amino 

acids. ALA and ABE can be analysed with different mobile phases 

from 0.100 µg/ml upwards. Hence, sensitivity is sufficient for 

quantification of either drug from in vitro permeation studies with 

stratum corneum. Permeated amounts across ASC for both 

substances were higher than across stratum corneum. Stability 

studies for both substances show that sample concentration of ALA 

or ABE did not decrease at conditions of the permeation studies. 

Acknowledgements 

We would like to thank Medac GmbH, Wedel, Germany for the 

generous donation of ALA and Hans Karrer GmbH for the donation 

of Excipial ® Fettcreme. Dr. Flory (Hollwede Hospital, D- 

Braunschweig) is thanked for the donation of skin samples. The 

study was supported in part by Fonds der Chemischen Industrie. 

References 

1 Shemin D (1970) On the synthesis of heme. 

Naturwissenschaften 57: 185-190 

2 Peng Q, Berg K, Moan J, Kongshaug M, Nesland JM (1997) 5aminolevulinic 

acid-based photodynamic therapy: principles and 

experimental research. Photochem Photobiol 65: 235-251 

3 Szeimies R-M, Calzavara-Pinton P, Karrer S, Ortel B, 

Landthaler M (1996) Topical photodynamic therapy in 

dermatology. J Photochem Photobiol B 36: 213-219 

4 Kloek J, Beijersbergen van Henegouwen GMJ (1996) Prodrugs 

of 5-aminolevulinic acid for photodynamic therapy. Photochem 

Photobiol 64: 994-1000 

5 Specht C, Stoye I, Müller-Goymann CC (1998) Comparative 

investigations to evaluate the use of organotypic cultures of 

transformed and native dermal and epidermal cells for 

permeation studies. Eur J Pharm Biopharm 46: 273-278 

------------------------------------------------------------------------------------ 




6 Wassermann K, Müller-Goymann CC (2000) Standardized 

cultivation of artificial skin constructs for drug permeation 

studies. Arch Pharm Pharm Med Chem 333 (Suppl. 1) 34 

7 Lim CK, Rideout JM, Samson DM (1979) Determination of 5aminolevulunic 

acid and porphobilinogen by high-performance 

liquid chromatography. J Chromatogr 185: 605-611 

8 Mauzerall D, Granick S (1956) The occurrence and 

determination of δ-aminolevulinic acid and porphobilinogen in 

urine. J Biol Chem 219: 435-446 

9 Sun WM, Stein E, Gruen FW (1969) A single column method 

for the determination of urinary δ-aminolevulinic acid. Clin 

Chem 15: 183-189 

10 Tomokuni K, Masayoshi I, Hirai Y, Hasegawa T (1987) 

Optimized liquid-chromatographic method for fluorometric 

determination of urinary δ-aminolevulinic acid in workers 

exposed to lead. Clin Chem 33: 1665-1667 

11 Ho J, Guthrie R, Tieckelmann H (1986) Detection of δaminolevulinic 

acid, porphobilinogen and porphorins related to 

heme biosynthesis by high-performance liquid chromatography. 

J Chromatogr 375: 57-63 

12 Roth M (1971) Fluorescence reaction for amino acids. Anal 

Chem 43: 880-882 

13 Schneider HJ, Földi P (1986) Aminosäuren-Analyse, 

Gegenüberstellung konventioneller und neuester HPLC- 

Methoden-Teil I. GIT Fachz Lab 8: 783-786 

14 Graser TA, Godel HG, Albers S, Földi P, Fürst P (1985) An 

ultra and sensitive high-performance liquid chromatographic 

method for determination of tissue and plasma free amino acids. 

Anal Biochem 151: 142-152 

15 Franz TJ (1975) Percutaneous absorption. On the relevance of in 

vitro data. J Invest Dermatol 64: 190-195 

16 Novo M, Hüttmann G, Diddens H (1996) Chemical instability of 

5-aminolevulinic acid used in the fluorescence diagnosis of 

bladder tumours. J Photochem Photobiol B 34: 143-148 

17 Franck B, Stratmann H (1981) Condensation products of the 

porphyrin precursor 5-aminolevulinic acid. Heterocycles 15: 

919-923 

------------------------------------------------------------------------------------ 




18 Butler AR, George S (1992) The nonenzymatic cyclic 

dimerisation of 5-aminolevulinic acid. Tetrahedron 48: 7879- 

7886 

19 Winkler A, Müller-Goymann CC Comparative permeation 

studies for δ-aminolevulinic acid and its n-butyl ester through 

stratum corneum and artificial skin constructs. Eur J Pharm 

Biopharm in press (2002) 

------------------------------------------------------------------------------------ 



Photoinduced Lesions in DNA - Detection using Micro-HPLC and Ion Trap MS 129 

Photoinduced Lesions in DNA - Detection using 

Micro-HPLC and Ion Trap MS 

G. Zhang and M. Linscheid 

Humboldt University 

Department of Chemistry 

Brook-Taylor-Str.2 


Germany 

Introduction................................................................. 129 

Experimental............................................................... 130 


• Detection of UV-dimers .............................................130 

• Detection of 8-Oxo-deoxyguanosine..........................134 

Conclusions................................................................. 135 

References................................................................... 135 

Introduction 

Exposure to sunlight can cause lesions in skin DNA. The failure of 

the reparation of these UV-induced DNA damages is associated 

with mutagenesis and skin cancer[1][2]. Several of these lesions 

have been described already[3]. The UV portions of the sun light 

can induce different types of lesions depending on the energy and 

wavelength. UVB and UVC can directly excite DNA and cause 

pyrimidine nucleobases to form Cyclobutane dimers or (6-4) 

photoproducts. The longer wavelength radiation (UVA) can lead to 

oxidative DNA modifications, most importantly 8-oxo-dGuo, as the 

result of oxidative stress including hydroxyl radicals and singlet 

oxygen. 

To understand the biological consequences of both types, it is 

mandatory to command highly sensitive analytical methods. In the 

light of the extending ozone holes in both hemispheres, the risks 

associated with sun light exposure are increasing and skin cancer 

became one of the major causes for early deaths. 

In the last half century, many analytical methods have been 

developed for the detection of DNA photoaducts[3][4]. Recently, 

------------------------------------------------------------------------------------ 



130 Photoinduced Lesions in DNA - Detection using Micro-HPLC and Ion Trap MS 

HPLC-tandem mass spectrometry has been used to analyze both the 

photodimers and oxidative photoproducts of DNA[5][6]. We 

introduce here the use of micro-HPLC coupled with ion trap mass 

spectrometer to detect and characterize these photoproducts of 

DNA. Micro-HPLC-MS/MS can reduce the sample consumption 

and increase the sensitivity. 

Experimental 

Sample preparation: 0,5 mg/ml model compound TpT was 

irradiated with UVC (254nm). One 1mg/ml DNA sample was 

irradiated with UVC (254nm) and the other 1mg/ml DNA sample 

was irradiated with UVA (366nm). Then each of these two samples 

was enzymatically digested by Nuclease P1 and Phosphatase. 

HPLC condition: Column: 15 cm, 300 µm I.D., C18 RP, Flow rate: 

4 µl/min Eluent A: 0.01M ammonium acetate (pH 5), Eluent B: 

50% Methanol + 50% Eluent A Gradient: 100 %A to 95 %A for the 

first 15 minutes, 95 %A to 0 %A from 15 to 55 min. and isocratic 

100 %B from 55 to 60 min. Ion trap mass spectrometer: 

FINNIGAN Ion-trap LCQ Deca 


• Detection of UV-dimers 

Figure 1 shows the molecular structure of TpT, which is the most 

important source of UV-induced dimer in DNA. The main 

photoproducts of UVB and UVC are cis-syn TpT, trans-syn TpT 

and 6-4 TpT. 6-4 TpT is converted partly to its Dewar valence 

isomer upon exposure to UVA and UVB light (Figure 2). 

------------------------------------------------------------------------------------ 




Fig. 1: Thymidylyl- 3´-5´-Thymidylic acid (TpT) as the most 

important source for pyrimidine dimmer 

HO 

HO 

O 

O 

O 

O 

P O 

OH 

Cis-syn TpT OHTrans-syn 

TpT 

O 

O 

HN 

HN 

O 

O 

P 

OH 

O 

O 

N 

O 

N 

CH 3 

H 

CH 3 

H 

OH 

OH 

OH 

CH 3 

H 

O 

O 

N 

O 

N 

N 

NH 

O 

O 

6-4 TpT Dewar TpT 

HO 

HO 

O 

O 

O 

O 

P O 

O 

HN 

O 

O 

O P 

OH 

O 

O 

HN 

CH 

3 

OH 

Fig. 2: Structure of a few UV-induced TpT photoproducts 

OH 

------------------------------------------------------------------------------------ 



N 

O 

CH 3 

N 

H 

OH 

H 

CH 3 

H 

O 

O 

O 

N 

N 

NH 

N 

O 

O



100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

Unknown 

Cis-syn TpT 

m/z 545� m/z 447 

Dewar TpT 

m/z 545� 

m/z 432 

Unknown 

6-4 TpT 

m/z 545 � m/z 432 

Trans-syn TpT 

m/z 545� m/z 447 

Unknown 

0 10 20 30 40 50 

Time (min) 

------------------------------------------------------------------------------------ 



TpT 

m/z 545� m/z 419 

Fig. 3 Extracted single ion mass chromatogram (mass 545) of UVC 

irradiated TpT 

As described in the experimental part, our undamaged TpT sample 

was irradiated by 254 nm UVC light and the sample was then 

separated by micro-HPLC system and then analyzed by ion trap 

mass spectrometer. Ion-trap mass spectrometer was programmed to 

perform two scan events in the same run. One event was full scan 

from m/z 250-650, the other event was to monitor the fragment of 

m/z 545. Figure 3 is the extracted single ion mass chromatogram of 

mass 545 from the full scan mass chromatogram of UVC irradiated 

TpT. When we perform MS/MS experiment in negative mode of 

ion trap mass spectrometer, TpT and its several photoproducts have 

relatively clean fragment pattern, that is one dominant daughter ion 

peak and low background. In the negative mode, all TpT, Cis-syn 

TpT, Trans-syn TpT, 6-4 TpT and Dewar TpT have mass 545. Due 

to the loss of a thymine base, the major fragment peak of TpT is 

419. Cis-syn TpT and Trans-syn TpT have the same main daughter 

ion of m/z 447 after losing one 2-deoxyribose unit. 6-4 TpT and its 

Dewar valence isomer have also the same main daughter fragment 

peak at 432. This can be accounted for by the fragmentation of this 

saturated pyrimidine ring. The suspected losses of fragment from 

TpT and its 4 photodimers are circled in Figure 2. From Figure 3, 

we can see that those photodimers elute earlier than original


undamaged TpT. Cis-syn TpT and 6-4 TpT are two major 

photoproducts. Trans-syn TpT is produced in a smaller amount. 

Surprisingly, we found also trace Dewar TpT in the sample, this 

may be due to UVB impurity in UVC lamp. The structures of 

several minor substances remain unidentified. 

Figure 4 is the extracted single ion mass chromatogram of mass 545 

from the full scan chromatogram of enzymes digested UVC 

irradiated DNA sample. Undamaged TpT were completely digested 

to monomers and not detectable any more. Cis-syn and 6-4 TpT 

were still the main photoproducts. Trace Dewar TpT was found 

again in this DNA sample. Trans-syn TpT was not detected and it 

may be because the stereo restriction of thymine bases in DNA is 

less favor to the formation of trans-syn TpT. 

These results suggest that the substances of equal mass, but with 

slightly different structure can be separated by micro-HPLC. 

MS/MS data can provide structural information necessary to 

identify these substances. 


100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

Cis-syn TpT 

m/z 545� m/z 7 

Dewar TpT 

m/z 545� 

m/z 432 

6-4TpT 

m/z 545� m/z 432 

0 10 20 30 40 50 60 70 80 

Time (min) 

Fig. 4: Extracted single ion mass chromatogram (mass 545) of 

enzymes digested UVC irradiated DNA 

------------------------------------------------------------------------------------ 




• Detection of 8-Oxo-deoxyguanosine 

UVA in solar radiation generates reactive oxygen species and OH· 

radicals in the living cells . Within DNA, guanine base is the major 

target of the oxidative modification by these free radicals. 8-oxodGuo 

is the main oxidative DNA damage and is regarded as the 

biomarker of the oxidative stresses. Figure 4 is the single ion mass 

chromatogram (m/z 284) of enzymes digested UVA irradiated DNA 

sample in the positive electrospray mode. We ran two scan events in 

the same measurement. The first scan event was the SIM of m/z 

284. This is the [M+H] + of 8-oxo-dGuo. The second scan event was 

the MS/MS experiment. By losing one protonated 2-deoxyribose, 

the characteristic fragment peak of 8-oxo-dGuo is 168 (shown in 

Fig. 6) 

Fig. 5: Single ion mass chromatogram (m/z 284) of enzymes 

digested UVA irradiated DNA sample in the positive electrospray 

mode 

------------------------------------------------------------------------------------ 



Rel. 

abundant 


8-Oxodeoxyguanosine 

m/z 284 

Fig. 6: Positive electrospray ionization MS/MS spectrum of 8-Oxodeoxyguanosine 

(m/z 284) 

Conclusions 

Our results demonstrate that we can separate TT, cis-syn TT, transsyn 

TT and 6-4 TT, and to identify them using micro-HPLC 

coupled to an ion-trap mass spectrometer. Furthermore, the most 

important oxidative DNA damage caused by UVA-exposure, i.e., 8oxo-deoxyguanosine 

can be detected and structurally confirmed. 

References 

168,2 

H2N 

HO 

HN 

O 

N 

HO 

O 

N + 

H 

N 

OH 

m/z 168 

266,9 

185,7 194,0 203,8 224,0 

242,8 

153,2 181,3 221,4 235,6 

253,9 277,7 285,9 295,8 

1 Cleaver JE (1968) Deficiency in repair replication of DNA in 

xeroderma pigmentosum, Nature 218:652-656 

2 Cleaver JE (1970) DNA repair and radiation sensitivity in 

human (xeroderma pigmentosum) cells, Int. J. Radiat. Biol. 

18:557-565 

3 Cadet J, Vigny P (1990) Photochemistry of nucleic acids. In: 

Bioorganic Photochemistry, Volumn 1, Wiley, New York, S1- 

272 

4 Cadet J, Weinfeld M (1993) Detecting DNA damage, Anal. 

Chem. 65:675A-682A 

HO 

HO 

O + 

------------------------------------------------------------------------------------ 



H2N 

HN 

O 

N 

+ 

NH2 160 180 200 220 240 260 280 300 

NH 

O


5 Douki T, Court M, Sauvaigo S, Odin F, and Cadet J, (2000) 

Formation of the main UV-induced Thymine dimeric lesions 

within isolated and cellular DNA as measured by high 

performance liquid chromatography-tandem mass spectrometry, 

J. Biol. Chem. 275: 11678-11685 

6 Ravanat J, Duretz B, Guiller A, Douki T, and Cadet J, (1998) J. 

Chromatogr. B 715:349-356 

------------------------------------------------------------------------------------ 



The Topical Application of Vitamin D3-Analogues in Psoriasis vulgaris 137 

The Topical Application of Vitamin D3-Analogues 

in Psoriasis vulgaris 

M. Fischer 

Department of Dermatology and Venereology 


Ernst-Kromayer-Strasse 5-6 


Germany 

Introduction................................................................. 137 

Mode of action............................................................ 137 

Use in Psoriasis vulgaris............................................. 138 

References................................................................... 141 

Introduction 

A positive effect of vitamin D3 on psoriasis was first described in 

the 1930s, but this treatment concept was dropped because of 

unacceptable side effects, especially elicitation of hypercalcemias 

[1]. 50 years later, vitamin D3 was rediscovered thanks to a chance 

clinical observation. Further research in the following years led to 

the development of various locally-applicable vitamin D3analogues, 

of which calcipotriol and tacalcitol have become 

established in therapy. Moreover, the active form of vitamin D3 

(calcitriol = 1α,25-dihydroxycholecalciferol) is also available for 

the topical treatment of psoriasis. 

Mode of action 

Vitamin D3 (cholecalciferol) is converted by double hydroxylation 

to its active form 1,25-dihydroxycholecalciferol [2]. This 

metabolization occurs primarily in the liver and kidneys. However, 

studies on porcine skin have shown that keratinocytes may also 

form 1,25-dihydroxy vitamin D3 in considerable quantities [3]. 

1,25-dihydroxycholecalciferol and its analogues develop their effect 

via genomic and non-genomic mechanisms. The genomic effect 

------------------------------------------------------------------------------------ 



138 The Topical Application of Vitamin D3-Analogues in Psoriasis vulgaris 

proceeds via binding to the vitamin-D receptor (VDR), a member of 

the steroid receptor family. Activation of transcription is supported 

by the closely-related retinoid-X-receptor (RXR), which is 

considered a cofactor, with which the VDR forms a heterodimer 

[2]. This leads via acetylation of histones to exposure of individual 

DNA-segments, which can then be transcribed. The downregulation 

of proinflammatoy cytokines, like Interleukin 2 (IL-2) 

and of adhesion molecules is ascribed to this roughly-outlined 

mechanism of the genomic effect [4]. 

In addition to the genomic effect, an increase in intracellular 

calcium concentration induced by vitamin D3 analogues is seen as a 

non-genome-dependent effect. There is considerable evidence that 

an elevated intracellular calcium level promotes keratinocytic 

differentiation [5]. However, the actual importance of this 

observation remains unclear. 

Use in Psoriasis vulgaris 

Psoriasis vulgaris is an inflammatory dermatosis with a prevalence 

in western industrial countries of just under 3% [6]. It has been 

proven that the macro- and micromorphological changes observed 

during the course of the disease are mediated by T-lymphocytes [7]. 

Based on their cytokine pattern, these T-lymphocytes, which are 

found in lesional psoriatic skin, are ascribed to the Th1subpopulation. 

A number of different proinflammatory cytokines, 

such as Interferon gamma (IFN-δ) [8], Interleukin 2 (IL-2) and 

Tumor-Necrosing Factor alpha (TNFα), play a special role during 

the inflammation reaction in psoriasis [7]. Also, elevated serum 

levels and an intralesional increase in Interleukin 8 (IL-8) has been 

demonstrated in patients with psoriasis [9, 10]. As a consequence of 

this inflammation process, the elucidation of which is still only 

rudimentary, the typical hyperproliferation becomes manifest, with 

altered differentation as expressed histologically in 

hyperparakeratosis. 

Recent knowledge concerning the immunological background of 

psoriasis has led to novel treatment approaches, like the use of 

monoclonal chimeric antibodies to the various cytokines and their 

receptors [11, 12]. At the same time, it helps to explain the long- 

------------------------------------------------------------------------------------ 




known good efficacy of various therapeutics in psoriasis. Various 

possible sites of action become apparent for calcitriol and the 

vitamin D3 analogues. 

Alroy and colleagues [4] were able to demonstrate a downregulation 

of IL-2 by vitamin D3. Reduced transcription could be 

shown for δ-INF in both naive and activated Th1-cells [13]. The 

reduction of IL-8 proven for both calcipotriol [14] and tacalcitol 

[15] is cited as another favorable influence on the psoriasis-relevant 

mediators. In the study by Kang et al. it was accompanied by an 

increase in the immunosuppressive IL-10. Moreover, Lee et al. [16] 

found that calcipotriol exerts proliferation-inhibiting properties via 

inhibition of the Epidermal Growth Factor (EGF) receptors. Thus, 

vitamin D3 and its analogues have the basic capacity to act 

favourably on cytokine-regulation in psoriasis. The high number of 

articles published on this topic show that the trend in the treatment 

of psoriasis with vitamin D3-analogues lies in the progressive 

discovery of proliferation and anti-inflammatory influences. 

Both calcitriol and its analogues calcipotriol and tacalcitol are 

approved for treatment of mild and moderate cases of Psoriasis 

vulgaris. The good efficacy of all such preparations has been 

repeatedly confirmed in clinical studies. 

Calcipotriol is available as a cream, ointment and solution. The 

application quantity should not exceed 100g (ointment/cream) or 

60mL (solution) per week. The recommendations for Tacalcitol 

Ointment are similar. These limitations are aimed at prevention of 

hypercalcemia, but it must be noted that in patients with limited 

kidney function, hypercalcemia may occur even at lower quantities 

of active substance [1]. An elevation of the serum calcium level 

must be expected more frequently in application of Calcitriol 

Ointment, but this was limited in the various studies to isolated 

cases and was asymptomatic [17]. Erythema and irritative contact 

dermatitis occur as side effects in 4-26% of cases, but this led in the 

various studies to withdrawal of therapy in only about 4% of cases 

[1]. 

Although various studies could demonstrate better or comparable 

efficacy of calcipotriol vs. betamethason [18], dithranol [19] and tar 

[20, 21], the great potential of all vitamin D3-(analogues) lies in the 

excellent combination possibilities with various local and systemic 

antipsoriatica [22]. For example, Lebwohl et al. [23] could 

------------------------------------------------------------------------------------ 




demonstrate increased efficacy of calcipotriol in combination with 

halobetasol over the monosubstances alone. In general, a corticoidsparing 

effect can be attributed to both calcitriol and the vitamin 

D3-analogues. At the same time, combination with a corticosteroid 

can suppress a potential irritative contact dermatitis associated with 

the vitamin D3-analogues. 

A combination of calcipotriol with PUVA-therapy permitted 

reduction of the total UVA dose needed compared to PUVAtherapy 

alone [24]. Similar experience on synergistic effects is also 

available for UVB [25] and narrow-band UVB [26]. Although most 

studies on the combination of vitamin D3-analogues with UV-light 

were performed with calcipotriol, there is an increasing number of 

studies on calcitriol and tacalcitol which show similar results. Ring 

et al. [27] found a reduction of the UV dose by 34% for calcitriol, in 

addition to improved response to the combination therapy. This is 

very important, especially with respect to the possible induction of 

malignant tumors by UV therapy. In order to avoid inactivation of 

the vitamin D3-analogues by UV-radiation, the preparations should 

be applied after exposure or at least 2 hours prior to exposure [22]. 

Recent studies also indicate that vitamin D3-analogues exert a 

supportive effect on systemic antipsoriatica. In a placebo-controlled 

study, Grossman et al. [28] found synergistic effects of calcipotriol 

with Cyclosporin A. The placebo-arm with an active-free vehicle in 

local therapy showed lower response rates of the psoriasis lesions 

and a higher Cyclosporin dose compared to the group treated with 

calcipotriol. Van de Kerkhof et al. could also show dose-sparing 

effects with improved efficacy by the combination of retinoids with 

locally-applied calcipotriol [29]. Similar results also seem to apply 

for methotrexate, but there are no controlled studies as yet [22]. 

In monotherapy, vitamin D3-analogues have also been found 

beneficial when used in problem areas. Calcipotriol solution was 

developed for application to the hairy scalp. Exceptional efficacy on 

the scalp could be demonstrated in various prospective application 

observations on several hundred patients [30]. Moreover, there are 

also positive experience reports on the use of calcipotriol in the 

intertrigines [31]. Although the irritative potential of the vitamin 

D3-analogues is generally increased in skin folds, clinical 

experience has shown that the use can still be justified on the basis 

of good efficacy. Possible cutaneous irritations are of minor 

------------------------------------------------------------------------------------ 




importance in our own experience. However, no controlled studies 

have yet been performed on the use of vitamin D3-analogues in 

intertriginous areas. Another very promising use of vitamin D3analogues 

is nail psoriasis, which affects about 50% of all psoriasis 

patients. In a double-blind, placebo-controlled study, calcipotriol 

was effective and comparable to betamethason plus salicylic acid 

[32]. 

In addition to use in Psoriasis vulgaris, vitamin D3-analogues have 

also been successfully used in other skin diseases. There are 

positive reports for vitiligo, Pityriasis rubra pilaris, Acanthosis 

nigricans, various forms of Ichthyoses and Keratosis lichenoides 

chronica [33]. In contrast, no effect or even deterioration was 

observed after use in Morbus Darier and Acrodermatitis continua 

suppurativa [33, 34] 

In summary, it can be said that calcitriol, calcipotriol and tacalcitol 

are effective, safe preparations which are also excellent in 

combinations for the treatment of Psoriasis vulgaris. The 

therapeutic effect also appears applicable to a number of other skin 

diseases. 

References 

1 Fuhrmeister K (2001) Vitamin-D3-Analoga. In Korting HC, 

Sterry W (Eds.) Therapeutische Verfahren in der Dermatologie. 

Blackwell Wissenschafts-Verlag GmbH Berlin, S 139-146 

2 Jones G, Strugnell SA, DeLuca HF (1998) Current 

understanding of the molecular actions of vitamin D. Physiol 

Rev 78:1193-1231 

3 Bikle DD, Halloran BP, Riviere JE (1994) Production of 1,25 

dihydroxyvitamin D3 by perfused pig skin. J Invest Dermatol 

102:796-798 

4 Alroy I, Towers TL, Freedman LP (1995) Transcriptional 

repression of the interleukin-2 gene by vitamin D3: direct 

inhibition of NFATp/AP-1 complex formation by a nuclear 

hormone receptor. Mol Cell Biol 15:5789-5799 

5 Bikle DD (1996) 1,25(OH)2D3-modulated calcium induced 

keratinocyte differentiation. J Investig Dermatol Symp Proc 

1:22-27 

------------------------------------------------------------------------------------ 




6 Koo JY (1999) Current consensus and update on psoriasis 

therapy: a perspective from the U.S. J Dermatol 26:723-733 

7 Krueger JG (2002) The immunologic basis for the treatment of 

psoriasis with new biologic agents. J Am Acad Dermatol 46:1- 

23 

8 Fierlbeck G, Rassner G, Muller C (1990) Psoriasis induced at 

the injection site of recombinant interferon gamma. Results of 

immunohistologic investigations. Arch Dermatol 126:351-355 

9 Jiang WY, Chattedee AD, Raychaudhuri SP, Raychaudhuri SK, 

Farber EM (2001) Mast cell density and IL-8 expression in 

nonlesional and lesional psoriatic skin. Int J Dermatol 40:699- 

703 

10 Zheng M, Sun G, Cai S, Mrowietz U (1998) T-lymphocyte 

chemotaxis to IL-8 in patients with psoriasis in vitro. Chin Med 

J (Engl) 111:166-168 

11 Ogilvie AL, Antoni C, Dechant C, Manger B, Kalden JR, 

Schuler G, Luftl M (2001) Treatment of psoriatic arthritis with 

antitumour necrosis factor-alpha antibody clears skin lesions of 

psoriasis resistant to treatment with methotrexate. Br J Dermatol 

144:587-589 

12 Wohlrab J, Fischer M, Taube KM, Marsch WC (2001) 

Treatment of recalcitrant psoriasis with daclizumab. Br J 

Dermatol 144:209-210 

13 Staeva-Vieira TP, Freedman LP (2002) 1,25-dihydroxyvitamin 

D3 inhibits IFN-gamma and IL-4 levels during in vitro 

polarization of primary murine CD4+ T cells. J Immunol 

168:1181-1189 

14 Kang S, Yi S, Griffiths CE, Fancher L, Hamilton TA, Choi JH 

(1998) Calcipotriene-induced improvement in psoriasis is 

associated with reduced interleukin-8 and increased interleukin- 

10 levels within lesions. Br J Dermatol 138:77-83 

15 Fukuoka M, Ogino Y, Sato H, Ohta T, Komoriya K, Nishioka K, 

Katayama I (1998) RANTES expression in psoriatic skin, and 

regulation of RANTES and IL-8 production in cultured 

epidermal keratinocytes by active vitamin D3 (tacalcitol). Br J 


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16 Lee E, Jeon SH, Yi JY, Jin YJ, Son YS (2001) Calcipotriol 

inhibits autocrine phosphorylation of EGF receptor in a calciumdependent 

manner, a possible mechanism for its inhibition of 

cell proliferation and stimulation of cell differentiation. Biochem 

Biophys Res Commun 284:419-425 

17 Kowalzick L (2001) Clinical experience with topical calcitriol 

(1,25-dihydroxyvitamin D3) in psoriasis. Br J Dermatol 144 

Suppl 58:21-25 

18 Kragballe K, Barnes L, Hamberg KJ, Hutchinson P, Murphy F, 

Moller S, Ruzicka T, Van De Kerkhof PC (1998) Calcipotriol 

cream with or without concurrent topical corticosteroid in 

psoriasis: tolerability and efficacy. Br J Dermatol 139:649-654 

19 Wall AR, Poyner TF, Menday AP (1998) A comparison of 

treatment with dithranol and calcipotriol on the clinical severity 

and quality of life in patients with psoriasis. Br J Dermatol 

139:1005-1011 

20 Kaur I, Saraswat A, Kumar B (2001) Comparison of calcipotriol 

and coal tar in conjunction with sun exposure in chronic plaque 

psoriasis: a pilot study. J Dermatol 28:448-450 

21 Tham SN, Lun KC, Cheong WK (1994) A comparative study of 

calcipotriol ointment and tar in chronic plaque psoriasis. Br J 

Dermatol 131:673-677 

22 Lamba S, Lebwohl M (2001) Combination therapy with vitamin 

D analogues. Br J Dermatol 144 Suppl 58:27-32 

23 Lebwohl M, Yoles A, Lombardi K, Lou W (1998) Calcipotriene 

ointment and halobetasol ointment in the long-term treatment of 

psoriasis: effects on the duration of improvement. J Am Acad 


24 Kokelj F, Plozzer C, Torsello P (1997) Reduction of UV-A 

radiation induced by calcipotriol in the treatment of vulgar 

psoriasis with oral psoralen plus UV-A. Arch Dermatol 133:668- 

669 

25 Molin L (1999) Topical calcipotriol combined with 

phototherapy for psoriasis. The results of two randomized trials 

and a review of the literature. Calcipotriol-UVB Study Group. 

Dermatology 198:375-381 

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26 Ramsay CA, Schwartz BE, Lowson D, Papp K, Bolduc A, 

Gilbert M (2000) Calcipotriol cream combined with twice 

weekly broad-band UVB phototherapy: a safe, effective and 

UVB-sparing antipsoriatric combination treatment. The 

Canadian Calcipotriol and UVB Study Group. Dermatology 

200:17-24 

27 Ring J, Kowalzick L, Christophers E, Schill WB, Schopf E, 

Stander M, Wolff HH, Altmeyer P (2001) Calcitriol 3 microg g- 

1 ointment in combination with ultraviolet B phototherapy for 

the treatment of plaque psoriasis: results of a comparative study. 

Br J Dermatol 144:495-499 

28 Grossman RM, Thivolet J, Claudy A, Souteyrand P, Guilhou JJ, 

Thomas P, Amblard P, Belaich S, de Belilovsky C, de la 

Brassinne M, et al. (1994) A novel therapeutic approach to 

psoriasis with combination calcipotriol ointment and very lowdose 

cyclosporine: results of a multicenter placebo-controlled 

study. J Am Acad Dermatol 31:68-74 

29 van de Kerkhof PC, Cambazard F, Hutchinson PE, Haneke E, 

Wong E, Souteyrand P, Damstra RJ, Combemale P, Neumann 

MH, Chalmers RJ, Olsen L, Revuz J (1998) The effect of 

addition of calcipotriol ointment (50 micrograms/g) to acitretin 

therapy in psoriasis. Br J Dermatol 138:84-89 

30 Thaci D, Daiber W, Boehncke WH, Kaufmann R (2001) 

Calcipotriol solution for the treatment of scalp psoriasis: 

evaluation of efficacy, safety and acceptance in 3,396 patients. 

Dermatology 203:153-156 

31 Kienbaum S, Lehmann P, Ruzicka T (1996) Topical calcipotriol 

in the treatment of intertriginous psoriasis. Br J Dermatol 

135:647-650 

32 Tosti A, Piraccini BM, Cameli N, Kokely F, Plozzer C, Cannata 

GE, Benelli C (1998) Calcipotriol ointment in nail psoriasis: a 

controlled double-blind comparison with betamethasone 

dipropionate and salicylic acid. Br J Dermatol 139:655-659 

33 Sönnichsen N, Johannböcke R (2001) Calcipotriol: Neue 

Aspekte zu einer bewährten Substanz. Nichtpsoriatische 

Dermatosen. Der Deutsche Dermatologe 49:626-628 

34 Kokelj F, Plozzer C, Trevisan G (2001) Uselessness of topical 

calcipotriol as monotherapy for acrodermatitis continua of 

Hallopeau. Acta Derm Venereol 81:153 

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Trends of topical immunomodulatory Therapy 145 

Trends of topical immunomodulatory Therapy 

K. Jahn, R.H.H. Neubert, *J. Wohlrab 


* Universitätsklinik und Poliklinik für Dermatologie und Venerologie 


Wolfgang-Langenbeck-Straße 4 


Germany 

Trends of topical immunmodulatory Therapy............ 145 

References................................................................... 150 

The topical application of drugs is generally associated with a lower 

risk of side effects and a lower systemic exposure. The research and 

use of immunosuppressive drugs for topical application in chronic 

inflammatory skin diseases will be summarised in this paper. 

Ciclosporin A (CsA, Mr = 1202 g·mol -1 ) was the first T-cell active 

immunosuppressant approved for the systemic treatment of severe 

psoriasis and atopic dermatitis. Its use is limited due to the 

potentially systemic adverse effects such as arterial hypertension 

and nephrotoxicity. In order to reduce side effects after systemic 

application a topical formulation for skin disorders might be useful. 

In previous reports, conventional preparations (creams, ointments, 

solutions) containing different concentrations of CsA were 

examined for their topical use. Mostly, the ineffectiveness of the 

vehicles tested was explained with the drug’s inability to overcome 

the penetration barrier, stratum corneum [1]. 

The aim of our studies was the development of microemulsions for 

dermal application of ciclosporin A and the investigation of the 

penetration behaviour into human skin ex vivo using FRANZ-type 

diffusion cells. Microemulsions (ME) are special vehicle systems 

with a favourable solubilization capacity and penetration enhancing 

properties and should be used as drug delivery systems for topical 

application of CsA. 

------------------------------------------------------------------------------------ 



146 Trends of topical immunomodulatory Therapy 

concentration of CsA [mM] 

50 

40 

30 

20 

10 

concentration of CsA [µM] 

100 

80 

60 

40 

20 

0 

0 

Stratum corneum 

epidermis 

concentration of CsA [µM] 

------------------------------------------------------------------------------------ 



15 

10 

5 

0 

dermis 

permeated amount in acceptor solution [µg/cm 2 ] 

4 

3 

2 

1 

0 

acceptor 

microemulsion 

hydrophilic cream (O/W) 

lipophilic cream (W/O) 

Fig. 1: Concentration of CsA in different skin layers as well as 

permeated amount of CsA in the acceptor solution after penetration 

into excised human skin using a microemulsion, a hydrophilic 

cream (O/W), and a lipophilic cream (W/O). all formulations: 2 % 

(w/w) CsA, [ξ ± SD; n=3] 

The results of the penetration study from a microemulsion, a 

hydrophilic (O/W) and a lipophilic cream (W/O) after 30 minutes 

into excised human skin are presented in Fig. 1. As shown in this 

figure, only microemulsions are able to transport CsA through the 

viable skin layers into the acceptor solution. After application of the 

microemulsion approximately 20 % of the applied dose could be 

detected in the acceptor solution [2]. This fact is of special interest 

because the drug amount which permeated into the accector passed 

the viable skin layers. 

Furthermore, the clinical efficacy of the microemulsions containing 

CsA was evaluated in patients with chronic-plaque type psoriasis. A 

pilot study using the microplaque assay with a randomized, doubleblind 

and placebo-controlled design was performed. Erythema, 

infiltration and scaling were graded on a scale of 0-4 and laser- 

Doppler-flow and erythrometry were used to assess the efficacy of 

the formulations. After 14 days the antipsoriatic effect of one 

microemulsions was comparable to the positive controls calcipotriol 

and betamethasone-17-valerate cream. The results demonstrate that


microemulsions enable the penetration of CsA into human skin 

after topical application and the clearing of psoriatic lesions using 

the microplaque assay. 

Mycophenolate mofetil (MMF, Mr = 433,5 g·mol -1 ) is an 

immunosuppressive agent registered in combination with 

ciclosporin A and corticosteroids for the prevention of organ 

rejections after allogenic heart and kidney transplantations. It is the 

morpholinoethylester prodrug of mycophenolic acid (MPA). 

Recently, reports have been published concerning the use of MMF 

for the treatment of several autoimmune and inflammatory skin 

disorders such as psoriasis [3,4]. The systemic administration in 

dermal therapy is limited due to the side effects nausea, leucopenia, 

sepsis, and diarrhoea. The purpose of our studies was the 

investigation of the penetration behaviour of MMF into human skin 

ex vivo using FRANZ-type diffusion cells. Therefore, the drug was 

incorporated in an amphiphilic cream (2 %). 

The penetration of MMF from this formulation into excised human 

skin is shown in Fig. 2A. An interesting fact of these examinations 

was the detection of the active metabolite of MMF, MPA, in human 

skin, too. In vivo, MMF is rapidly converted to the active metabolite 

MPA by tissue and plasma esterases. The amounts of MPA arising 

from the metabolism of penetrated MMF are presented in Fig. 2B. 

After 30 minutes MPA was found in none of the skin layers. The 

detected amounts of MPA are very small because of limited enzyme 

capacity in excised human skin. Because of the limited enzyme 

capacity in excised human skin, a greater extent of metabolism will 

be expected in vivo. MPA was mainly detected in the viable 

epidermis and the upper dermis where the hydrolysing enzymes are 

located [5,6]. The clinical efficacy of this formulation after topical 

application in three patients with psoriasis could be demonstrated 

and was comparable to 0,1 % betamethasone-17-valerate cream [7]. 

The new class of immunmodulatory macrolactames such as 

tacrolimus and pimecrolimus (Ascomycin) may provide an 

alternative to glucocorticoids in topical treatment of psoriasis and 

atopic dermatitis. 

------------------------------------------------------------------------------------ 




% of applied dose 

8 A 

7 

6 

5 

4 

3 

2 

1 

0 

SC 

EP 

MMF 

DR 

4 B 

------------------------------------------------------------------------------------ 



3 

2 

1 

0 

30 min 

300 min 

1000 min 

SC EP 

MPA 

Fig. 2: Penetration of MMF from an amphiphilic cream into human 

skin ex vivo; (A) MMF expressed as % of applied dose, (B) 

metabolised amount of MPA, expressed as % of applied dose of 

MMF; SC = Stratum corneum, EP = epidermis, DR = dermis, 

[ξ ± SD; n=3] 

Tacrolimus (FK 506, Mr = 822 g·mol -1 ) is a hydrophobic 

substance, insoluble in water (< 1mg/ml), but soluble in ethanol and 

was first isolated from Streptomyces-species in 1984 [8]. The log 

PC (partition coefficient) between octanol and water is 2,74 [9]. Its 

mode of action is similar to ciclosporin A, but the drug exerts in 

vitro a 10 to 100 times higher immunosuppressive activity [10]. 

Tacrolimus is used in combination with glucocorticoids to prevent 

organ rejections after heart and kidney transplantations. A 

randomized, double-blind, multicenter study demonstrated, that 

tacrolimus ointment is effective in the treatment of atopic dermatitis 

[11]. In 2000, tacrolimus has been approved for the topical use in 

atopic dermatitis in Japan and the USA (Protopic ® ). It is available 

as an ointment containing 0,3 and 0,1 % tacrolimus, respectively. 

DR


The efficacy of tacrolimus after systemic administration in psoriasis 

patients has been proven in a multicenter study [12]. Contrary 

results have been published concerning the topical use of this drug 

in psoriasis. ZONNEFELD et al. performed a study with 70 patients. 

The tacrolimus ointment (0,3 %) was applied once daily under nonocclusive 

conditions. No statistically significant difference to 

placebo could be observed [13]. REMITZ et al. used the microplaque 

assay in order to evaluate topical tacrolimus formulations for 

treatment of chronic plaque-type psoriasis in 16 patients. In this 

study two tacrolimus ointments (0,3 %, with and without 

penetration enhancer), betamethasone and calcipotriol ointment 

were compared regarding their ability to clear psoriasis relative to 

the vehicle controls. As a result the tacrolimus ointment was 

effective under descaling and occlusion. The authors concluded that 

the drug generally penetrates into psoriatic skin. In future, 

formulations with penetration enhancing properties should be 

developed in order to use this substance for topical application in 

psoriasis [14]. 

The finding that tacrolimus does not cause skin atrophy may be an 

advantage in the treatment in comparison to topical glucocorticoids 

[15]. 

Pimecrolimus (syn. ascomycin, SDZ ASM 981, 

Mr = 810,5 g·mol -1 ) is a lipophilic substance, soluble in ethanol and 

dimethylsulfoxide, but slightly soluble in water (< 1 mg/ml) [16]. 

Different studies were performed in order to evaluate its efficacy 

after topical application in patients with atopic dermatitis [17,18]. A 

cream containing 1 % pimecrolimus was significantly better than 

placebo, but not as effective as 0,1 % betamethasone-17-valerate 

cream [19]. Pimecrolimus was well tolerated, caused no serious 

side effects except burning or feeling of warmth in the treated area 

and did not induce skin atrophy [20]. Therefore it can be regarded 

as an alternative to topical glucocorticoids. The substance was 

licensed in December 2001 in the USA under the product name 

Elidel ® as a cream and an ointment for treatment of patients with 

atopic dermatitis. MROWIETZ et al. examined the topical efficacy of 

1 % ascomycin in psoriasis under occlusion using the microplaqueassay. 

According to the study 1 % ascomycin was comparable to 

0,5 % clobetasol-17-propionate cream [21]. 

------------------------------------------------------------------------------------ 




The future will show if the wide use of the immunmodulatory 

macrolactames tacrolimus and pimecrolimus in atopic dermatitis is 

without any risks as expected. 

References 

1 Mrowietz U (1992) The enigma of Cyclosporin A treatment for 

psoriasis: Systemic efficacy versus topical Non-responsiveness. 

Acta Derm Venereol 72:321-326 

2 Wohlrab J, Neubert R, Jahn, K (2002) Patent application: 

Arzneiformulierung, enthaltend Ciclosporin A und deren 

Verwendung. PCT/EO 01/14749 

3 Haufs MG, Beissert S, Grabbe S, Schütte B, Luger TA (1998) 

Psoriasis vulgaris treated successfully with mycophenolate 

mofetil. Br J Dermatol 138:179-181 

4 Nousari HC, Sragovich A, Kimyai-Asadi A, Orlinsky D, Anhalt 

GJ (1999) Mycophenolate mofetil in autoimmune and 

inflammatory skin disorders. J Am Acad Dermatol 40:265-268 

5 Jahn K, Fischer A, Neubert RHH, Wohlrab J (2001) 

Investigation of the penetration behaviour of mycophenolate 

mofetil from a semisolid formulation into human skin ex vivo. J 

Pharm Pharmacol 53:1581-1587 

6 Plätzer M, Jahn K, Wohlrab J, Neubert RHH (2001) 

Quantification of mycophenolate mofetil in human skin extracts 

using high performance liquid chromatography-electrospray 

mass spectrometry. J Chrom B 755:355-359 

7 Wohlrab J, Jahn K, Plätzer M, Neubert R, Marsch W (2001) 

Topical application of mycophenolate mofetil in plaque-type 

psoriasis. Br J Dermatol 144:1263-1264 

8 Merck Index – an encyclopaedia of chemicals, drugs and 

biologicals (1996) Merck & Co. Inc., Whitehouse Station 12. 

Aufl. 

9 Lauerma AI, Surber C, Maibach HI (1997) Absorption of topical 

tacrolimus (FK 506) in vitro through human skin: comparison 

with cyclosproin A. Skin Pharmacol 10:230-234 

10 Ruzicka T, Assmann T, Homey B (1999) Tacrolimus – the drug 

for the turn of the millennium? Arch Dermatol 135:574-580 

------------------------------------------------------------------------------------ 




11 Ruzicka T, Bieber T, Schöpf E, Rubins A, Dobozy A, Bos JD, 

Jablonska S, Ahmed I, Thestrup-Pedersen K, Daniel F, Finzi A, 

Reitamo S (1997) A short-term trial of tacrolimus ointment for 

atopic dermatitis. New Engl J Med 337:816-821 

12 The European FK 506 Multicentre Psoriasis study group (1996) 

Systemic tacrolimus (FK 506) is effective for the tretament of 

psoriasis in a double-blind, placebo-controlled study. Arch 

Dermatol 132: 419-423 

13 Zonnefeld IM, Rubins A, Jablonska S, Dobozy A, Ruzicka T, 

Kind P, Dubertret L, Bos JD (1998) Topical tacrolimus is not 

effective in chronic plaque psoriasis. Arch Dermatol 134:1101- 

1102 

14 Remitz A, Reitamo S, Erkko P, Granlund H, Lauerma AI (1999) 

Tacrolimus ointment improves psoriasis in a microplaque assay. 


15 Reitamo S, Rissanen J, Remitz A, Granlund H, Erkko P, Elg P, 

Autio P, Lauerma AI (1998) Tacrolimus ointment does not 

affect collagen synthesis: results of a single-center randomized 

trial. J Invest Dermatol 111:396-398 

16 Grassberger M, Baumruker T, Enz A, Hiestand P, Hultsch T, 

Kalthoff F, Schuler W, Schulz M, Werner F-J, Winiski A, Wolff 

B, Zenke G (1999) A novel anti-inflammatory drug, SDZ ASM 

981, for the treatment of skin diseases: in vitro pharmacology. 


17 Harper J, Green A, Scott G, Gruendl E, Dorobek B, Cardno M, 

Burtin P (2001) First experience of topical SDZ ASM 981 in 

children with atopic dermatitis. Br J Dermatol 144:781-787 

18 Van Leent EJM, Gräber M, Thurston M, Wagenaar A, Spuls PI, 

Bos JD (1998) Effectiveness of the ascomycin macrolactam 

SDZ ASM 981 in the topical treatment of atopic dermatitis. 

Arch Dermatol 134:805-809 

19 Luger T, Van Leent EJM, Graeber M, Hedgecock S, Thurston 

M, Kandra A, Berth-Jones J, Bjerke J, Christophers E, Knop J, 

Knulst AC, Morren M, Morris A, Reitamo S, Roed-Petersen J, 

Schoepf E, Thestrup-Pedersen K, Van der Valk PGM, Bos JD 

(2001) SDZ ASM 981: an emerging safe and effective treatment 

for atopic dermatitis. Br J Dermatol 144:788-794 

------------------------------------------------------------------------------------ 




20 Queille-Roussel C, Paul C, Duteil L, Lefebvre M-C, Rapatz G, 

Zagula M, Ortonne J-P (2001) The new topical ascomycin 

derivative SDZ ASM 981 does not induce skin atrophy when 

applied to normal skin for 4 weeks: a randomised, double-blind 

controlled study. Br J Dermatol 144: 507-513 

21 Mrowietz U, Graeber M, Bräutigam M, Thurston M, Wagenaar 

A, Weidinger G, Christophers E (1998) The novel ascomycin 

derivative SDZ ASM 981 is effective for psoriasis when used 

topically under occlusion. Br J Dermatol 139:992-996 

------------------------------------------------------------------------------------ 



Trends of Treatment with Topical Glucocorticoids 153 

Trends of Treatment with Topical Glucocorticoids 

R. Niedner 

Klinik für Dermatologie 

Klinikum Ernst von Bergmann 

Charlottenstr. 72 

D-14467 Potsdam 

Germany 

Trends of Treatment with Topical Glucocorticoids ... 153 

References................................................................... 156 

Talking about trends we want to find out in which direction the 

theory and practice of glucocorticoids will go. Some things will be 

new, others are well known, but new details will lead the discussion 

in a new direction. Now, what is being discussed? 

Browsing through the Internet by using the keyword “cortisone”, I 

found a lot of remarks about the “dangerous“ use of corticoids. It is 

my impression that corticophobia is still a problem even though we 

have already been using topical corticoids (TCCs) of the 4 th 

generation for more than a decade. These TCCs are free from side 

effects. Are they really? 

TCCs of the 4 th generation are double esters of hydrocortisone or 

prednisolone which will be split by esterases, first into the active 

drug, and then in a second step within the epidermis into 

unesterified derivates. These derivates are not effect-free fragments 

which do no harm, they are still working like classical 

corticosteroids of the 1 st generation, either hydrocortisone or 

prednisolone. As weak TCCs they do not cause any or only mild 

atrophia of the skin [1], but when treating the face you have to be 

aware of perioral rosacea-like dermatitis. 

The pharmaceutical formulations of TCCs will influence their 

action. Transfersomes are special formulations which enable a 

better penetration of TCCs through the skin - they will be discussed 

in detail during this congress. But what about the “old“ 

formulations? TCCs are classified as weak or strong substances, but 

their corresponding formulation is not taken into account. 

------------------------------------------------------------------------------------ 



154 Trends of Treatment with Topical Glucocorticoids 

Regardless of their pharmaceutical composition as cream, ointment, 

lotion, solution etc. they are classified as having the same power – 

but that is not correct. Even the same active substance sold as 

different brands of ointment show marked differences (a factor of 

four) in their activity [2]. Furthermore, it has to be expected that 

these differences are greater when different formulations are used. 

To my knowledge only one pharmaceutical manufacturer takes 

account of this: fluticasone cream needs a tenfold concentration 

(0,05 %) of the active substance to approximate the activity of the 

ointment (0,005 %) [3]). Many years ago Lubach [4] proved this 

fact with other TCCs by vasoconstriction assays, but nobody took 

this into consideration. 

Tachyphylaxis of TCCs is a phenomenon which is barely 

understood but well-proven experimentally by vasoconstriction [5], 

DNA synthesis [6] and by histamine-induced wheal suppression 

[7]). Tachyphylaxis in general means a reduction or even cessation 

of the action of drugs, best known of indirect sympathomimetics 

which empty the reservoirs of noradrenalin. The corresponding 

action of the skin when treated with TCCs for some days is the 

reduction of blanching under experimental conditions. How often 

do we really observe tachyphylaxis of TCCs in practice, during 

therapy of atopic dermatitis or psoriasis? Miller and coworkers [8] 

looked for the clinical effects of betamethasone on psoriatic 

plaques. They treated 27 patients over a period of twelve weeks and 

registered the elevation, erythema, and scaling of the psoriatic 

plaques. These symptoms did not change and Miller consistently 

concluded that he was not able to prove tachyphylaxis of 

betamethasone in psoriasis. 

Absorption of TCCs is well known and the more powerful the 

corticoid is the more one has to be aware of adrenal suppressive 

effects, but modern topical corticoids are said not to have these 

effects. Visscher and coworkers [9] treated 12 healthy volunteers 

with hydrocortisone-17-butyrate (HCB) or mometasone furoate 

(MMF) under occlusion in a crossover study for 14 days. Both 

agents suppressed plasma cortisol concentrations, MMF more than 

HCB, but the ACTH test was normal in both, indicating that 

adrenocortical insufficiency had not developed. Comparable results 

were seen in treatment with methyprednisolone aceponate [1]. 

------------------------------------------------------------------------------------ 




The skin of children is much thinner than the skin of adults and 

absorption of corticoids is much better, so it is not surprising that 

the blood level of hydrocortisone (HC), externally applied to young 

children suffering from atopic dermatitis, was very high [10]. It is 

of great interest to know whether or not these findings correlate 

with a suppression of the endogenous hydrocortisone as a sign of 

disturbance of the hypothalamic-pituitary-adrenal (HPA) axis. 

Lucky and coworkers [11] treated children with 0.05 % desonide or 

2.5 % HC for four weeks. Neither HC nor desonide ointment 

compromised the HPA axis. Patel and coworkers [12] came to the 

same conclusion: when they checked children with atopic dermatitis 

who were treated under open conditions with mild TCCs like HC, 

clobetasone butyrate 0.05 %, betamethasone valerate 0.025 % or 

fluocinolone acetonide 0.00625 % for a long period of 6.5 years, 

neither the basal or peak, nor the increment and area-under-curve in 

plasma cortisol concentrations differed from controls, indicating 

normal adrenal sensitivity. 

Treatment with TCCs also means the application of corticosteroids 

to the nose or to the bowel – the latter will not be discussed here. 

Patients suffering from allergic rhinitis may need 

glucocorticosteroids because of severe symptoms. To avoid 

systemic effects topically (nasally) administered CCs are preferred. 

But is there really no CC absorption and regulation of the HPA 

axis? Wihl and coworkers [13] nasally applied two different TCCs 

(budesonid (BUD) and beclomethasone dipropionate (BDP)) with 

doses of 100, 200, 400, and 800 µg. None of the treatments 

influenced the plasma cortisol values, but cortisol in the urine was 

significantly lower after BUD 400 and 800 µg, but not after any 

dose of BDP. When TCC administration was repeated for four days, 

a dose-related decrease in urine-cortisol was observed in both 

drugs, BUD being twice as potent as BDP. These data demonstrate 

systemic effects from nasally administered TCCs. 

TCCs are applied by interval therapy in order to economize TCCs 

and in order to avoid side effects like atrophy. Lubach and 

coworkers [14] pretreated the skin with clobetasol propionate (CP) 

twice daily for 16 days and measured the atrophy. Thereafter, CPtreatment 

was continued every 5, 7, 10, or 14 days. The skin 

thickness was approximately the same when CP was applied every 

5 or 7 days, and it reached normal levels when applied every 10 

------------------------------------------------------------------------------------ 



156 Trends of Treatment with Topical Glucocorticoids 

days. The results demonstrate that atrophy must be expected from 

an intermittent maintenance therapy even when applied at rather 

long intervals. 

References 

1 Kecskés A, Heger-Mahn D, Kleine Kuhlmann R, Lange L 

(1993) Comparison of the local and systemic side effects of 

methylprednisolone aceponate and mometasone furoate applied 

as ointment with equal antiinflammatory activity. J am Acad 


2 Stoughton RB (1992) Copies of Innovator Formulations of 

Topical Clucocorticoids. In: Maibach HI, Surber C (eds.) 

Topical Corticosteroids. Karger, Basel, S 54-64 

3 Rote Liste 2001 (2001) Editio Cantor, Aulendorf 

4 Lubach D, Kietzmann M (1992) Dermatokortikoide – 

Pharmakologie und Therapie. Kohlhammer, Stuttgart Berlin 

Köln, S 96 

5 duVivier A, Stoughton RB (1975) Tachyphylaxis to the action 

of topically applied corticosteroids. Arch Dermatol 111:581-583 

6 duVivier A (1976) Tachyphylaxis to topically applied steroids. 

Arch Dermatol 112:1245-1248 

7 Singh G, Singh PK (1986) Tachyphylaxis to topical steroid 

measured by histamine-induced wheal suppression. Int J 


8 Miller JJ, Roling D, Margolis D, Guzzo C (1999) Failure to 

demonstrate therapeutic tachyphylaxis to topically applied 

steroids in patients with psoriasis. J Am Acad Dermatol 41:546- 

549 

9 Vissher HW, Ebels JT, Roders GA, Jonkman JGH (1995) 

Randomised crossover comparison of adrenal suppressive 

effects of dermal creams containing glucocorticosteroids. Europ 

J Clin Pharmacol 48:123-125 

10 Wester RC, Maibach HI (1992) Percutaneous absorption in 

diseased skin. In: Maibach HI, Surber C (eds.) Topical 

Corticosteroids. Karger, Basel, S 128-141 

------------------------------------------------------------------------------------ 




11 Luckey AW, Grote GD, Williams JL, Tuley MR, Czernielewski 

JM, Dolak TM, Herdorn JH, Baker MD (1997) Effect of 

Desonide Ointment, 0.05 %, oh the Hypothalamic-Pituitary- 

Adrenal Axis of Children with Atopic Dermatitis. Cutis 59:151- 

153 

12 Patel L, Clayton PE, Addison GM, Price DA, David TJ (1995) 

Adrenal function following topical steroid treatment in children 

with atopic dermatitis. Brit J Dermatol 132:950-955 

13 Wihl J-A, Andersson K-E, Johansson S-A (1997) Systemic 

effects of two nasally administered glucocorticosteroids. Allergy 

52:620-626 

14 Lubach D, Rath J, Kietzmann M (1955) Skin Atrophy Induced 

by Initial Continuous Topical Applicaton of Clobetasol 

Followed by Intermittent Application. Dermatology 190:51-55 

------------------------------------------------------------------------------------ 



158 The Topical Treatment of Atopic Dermatitis and Psoriasis with Vitamin B12 

The Topical Treatment of Atopic Dermatitis and 

Psoriasis with Vitamin B12 

C. Stoerb, P. Altmeyer, *R. Niedner, **J. Hartung, M. Stücker 

Clinic for Dermatology and Allergology of the Ruhr University Bochum 

St. Josef Hospital 

*Clinic for Dermatology; Clinical Centre Ernst von Bergmann Potsdam 

** Institute for Statistics; Dortmund University 

Gudrunstr. 56 


Germany 

Introduction................................................................. 158 

Trial Medication and Design ...................................... 159 

• Study Population and Investigational Plan................ 159 

• Modified PASI Score................................................. 160 

• Modified SASSAD Score.......................................... 160 

• 20 MHz Sonography.................................................. 161 

Results......................................................................... 161 

• Primary Efficacy Parameter: PASI Score.................. 161 

• Primary Efficacy Parameter: SASSAD Score ........... 161 

• Sonographic Efficacy Parameters.............................. 162 

• Tolerability ................................................................ 163 

Discussion ................................................................... 163 

• Mode of Action of the External Vitamin B12 

Therapy...................................................................... 164 

References................................................................... 165 

Introduction 

In these two placebo-controlled and double-blind clinical trials, the 

efficacy of a vitamin B12 cream in atopic dermatitis and psoriasis 

was assessed by means of an intra-individual left/right comparison. 

Vitamin B12 is a crystalline powder, dark red and odourless, used 

for therapeutic purposes in the form of cyanocobalamin and/or 

hydroxocobalamin acetate. These two forms represent pro-drugs, 

which are converted in the body into the active forms of methyl- 

and 5-adenosylcobalamin. Vitamin B12 has a wide therapeutic 

range, toxic effects or symptoms of overdosage as well as 

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The Topical Treatment of Atopic Dermatitis and Psoriasis with Vitamin B12 159 

indications of a teratogenic or mutagenic potential are not known. 

Adverse reactions reported in isolated cases with parenteral use 

consisted of eczematous or urticarial drug reactions [1,2]. 

It was demonstrated already some time ago, that despite the size of 

the molecule, the skin is permeable to vitamin B12 [3] and that skin 

cells express the transcobalamin II required for the intracellular 

transport of vitamin B12 [4]. 

In view of the poor systemic bioavailability of vitamin B12 (rapid 

elimination of up to 90% of a single dose of 1 mg [2]), it is not 

surprising that systemic administration gave no evidence of a 

reliable therapeutic effect of systemic vitamin B12 in psoriasis 

[5,6,7]. 

Trial Medication and Design 

The vitamin B12-containing investigational drug, with the 

concentration of the active ingredient Cyanocobalamin DAB being 

0.07%, contained the following excipients: avocado oil DAC, citric 

acid DAB, distilled water, methyl glucose sesquistearate INCI and 

potassium sorbate DAB. As the investigational drug is of a light 

pink colour due to the vitamin B12, the placebo was made 

indistinguishable by adding the colorant E122 azurubin. 

Determined by a randomisation list, the patients applied the vitamin 

B12-containing active preparation to the affected skin areas of one 

body half and the placebo preparation to the contralateral side twice 

daily, the dosage depending on the extent of the affected skin areas 

and the severity of the disease symptoms. 

• Study Population and Investigational Plan 

The studies were conducted as placebo-controlled, double-blind, 

prospective and randomised phase III clinical trials with intraindividual 

left/right-comparison to assess the efficacy and 

tolerability of vitamin B12 cream in atopic dermatitis and psoriasis. 

Trial centres were the Clinic for Dermatology and Allergology of 

the Ruhr University Bochum at St. Josef Hospital in Bochum and 

the Clinic for Dermatology at Clinical Centre Ernst von Bergmann 

in Potsdam. 

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A total of 51 patients (21 female, 30 male, age 49.4 ± 10.7 years) 

were enrolled for psoriasis and 47 patients (28 female, 19 male, age 

33.6 ± 14.1 years) for atopic dermatitis. The treatment duration was 

8 weeks in both trials. Apart from the baseline and final 

examination, each patient had interim examinations after 2, 4 and 6 

weeks. 

The primary efficacy parameters were a modified PASI (Psoriasis 

Area and Severity Index) score [8] in psoriasis and a modified 

SASSAD (Six Area Six Sign Atopic Dermatitis) score [9] in atopic 

dermatitis. The secondary efficacy parameters included an 

investigator's and patient's global assessment of efficacy and, 

additionally in psoriasis, the thickness and density of the echolucent 

area of a reference plaque on each body half by 20 MHz 

sonography [10,11]. The efficacy parameters were determined at all 

examination dates. 

Drug safety variables in both trials were the recorded adverse 

events, investigator's and patient's global assessments of tolerability 

and a patient's assessment of the cream formulation with regard to 

feeling of the skin after application, odour, colour and effects on 

clothing. The safety parameters were determined at all interim 

examinations and the final examination, the patient's assessment of 

the cream formulation was performed at the final examination only. 

• Modified PASI Score 

The modified PASI score used in this trial records the severity of 

the three psoriasis symptoms desquamation, erythema and 

infiltration as well as the size of the affected skin areas. The 

modification consists in halving the weighting of the test area sizes 

due to the left/right comparison and the exclusion of the head from 

the assessment. 

• Modified SASSAD Score 

The SASSAD score includes the six atopic dermatitis symptoms 

dryness/desquamation, itching, erosion, lichenification, erythema 

and infiltration, whose severity and extent on the affected skin areas 

are assessed on an ordinal scale analogous to the PASI score. The 

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modification of the original score consists in replacing the symptom 

"exudation" with the more typical "infiltration" [12]. 

• 20 MHz Sonography 

High-frequency sonography is increasingly gaining importance in 

dermatological diagnostics. For the 20 MHz technique, the 

thickness and density of an epidermal echolucent aera, which is 

characteristic for psoriatic plaques, have been found to be the most 

sensitive parameters of the response profile during therapy [10]. 

Sonographic examination during the healing process of the plaque 

reveals a decrease in thickness accompanied by an increase in 

density. 

Results 

• Primary Efficacy Parameter: PASI Score 

The modified PASI score of the body halves treated with the 

investigational drug decreased with marked statistical significance 

compared to the body halves treated with placebo (p


period of 8 weeks would result in further alleviation of symptoms to 

the point of complete remission. 

Fig. 1: Treatment effect phase III trial psoriasis: Significantly more 

marked decrease of PASI under vitamin B12 cream compared to 

placebo. 

• Sonographic Efficacy Parameters 

With comparable baseline values, both forms of treatment revealed 

a continuously progressive reduction in the thickness of the 

echolucent areas throughout the entire treatment period. This effect 

was highly significantly more pronounced for the plaques treated 

with vitamin B12 cream than for the placebo-treated plaques 

(p


Fig. 2: Treatment effect phase III trial atopic dermatitis: 

Significantly more marked decrease of SASSAD under vitamin B12 

cream compared to placebo. 

• Tolerability 

Compared to other antipsoriatic drugs like vitamin A derivatives 

(tazaroten), vitamin D3 analogues (calcipotriol), tar preparations or 

anthralin, vitamin B12 cream caused no skin irritations at all. This 

good tolerability enhances the patients' compliance. 

Discussion 

Based on the hitherto available trials with vitamin B12 cream, the 

fields of application may be defined as follows: The non-irritant 

vitamin B12 cream means a therapeutic progress for patients with 

easily irritable skin (skin types I und II according to Fitzpatrick 

[13]). Furthermore the preparation is probably very suitable for the 

use in children and problematic intertriginous skin areas like 

armpits, groins or the buttocks' cleft. 

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To increase the efficacy of vitamin B12 cream in psoriasis, a 

preliminary treatment with keratolytic agents like salicylic acid 

could be useful, as the keratolysis facilitates the penetration of the 

active principle into the skin. After the intensive therapy of an acute 

psoriatic or eczematous episode, the after-treatment with vitamin 

B12 cream could maintain the therapeutic success and prolong the 

relapse-free interval. It would be possible as well to use vitamin B12 

cream in the combination resp. rotation therapy together with other 

external therapeutics for a higher efficiency of topical treatment. 

• Mode of Action of the External Vitamin B12 Therapy 

Vitamin B12 is involved in numerous biochemical processes in the 

human body, as it affects nucleic acid synthesis, especially in 

haematopoiesis and other cell maturation processes. The established 

indications of vitamin B12 to date are exclusively the prevention and 

therapy of clinical vitamin B12 deficiencies like hyperchromic 

macrocytic megaloblastic anaemia and funicular myelosis [1,2]. 

Recent research gave indications for an immunological action of 

vitamin B12 in the cytokine network of T-cell mediated 

inflammation. It was demonstrated that vitamin B12 is able to induce 

T-suppressor-cells [14] and to inhibit dose-dependently the 

production of interferon-gamma and interleukin-6 by Tlymphocytes 

[15] in vitro. Moreover, vitamin B12 is an effective 

scavenger of nitric oxide (NO) [16,17]. NO, e.g. produced by the 

iNOS (inducible nitric oxide synthase) in inflammatory dermatoses, 

is a part of the vasodilatory component of inflammation and able to 

stimulate keratinocyte proliferation [18]. Both atopic dermatitis and 

psoriasis are associated with an increase in cytokine formation and 

NO generation due to over-expression of iNOS [19,20], psoriasis 

additionally is characterised by an up to tenfold acceleration of 

epidermipoiesis. As NO-donor stimulated keratinocyte proliferation 

can be antagonised by vitamin B12 in vitro [18] and the topical 

application of a nitric oxide synthase inhibitor led to marked 

improvement in atopic dermatitis [21], the probable mode of action 

of topical applied vitamin B12 is the inhibition of cytokine 

formation and the binding of NO (thus restraining its influence on 

keratinocyte proliferation) in inflammative skin diseases. 

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References 


1 Bundesanzeiger vom 29.03.1989; Jg. 41, Nr. 59: 1614 

2 Europäisches Arzneibuch; Band II: 1366 (1975); Deutscher 

Apotheker-Verlag Stuttgart, Govi-Verlag GmbH Stuttgart 

3 Howe EE, Dooley CL,Geoffroy RF, Rosenblum C (1967): 

Percutaneous absorption of vitamin B12 in the rat and guinea pig, 

J Nutrition 92: 261-266 

4 Fràter-Schröder M, Porck HJ, Erten J, Müller MR, Steinmann B, 

Kierat L, Arwert (1985): Synthesis and secretion of the human 

vitamin B12-binding protein, transcobalamin II, by cultured skin 

fibroblasts and bone marrow cells Biochim Biophys Acta 845: 

421-427 

5 Baker H, Comaish JS (1962): Is vitamin B12 of value in 

psoriasis?, Br Medical J ii: 1729-1730 

6 Sneddon IB (1963): Vitamin B12 in psoriasis; Br Medical J; iii: 

328 

7 MacLennan A, Hellier FF (1961): The treatment time in 

psoriasis, Br J Dermatol 73: 439-444 

8 Fredriksson T, Pettersson U (1978): Severe psoriasis – oral 

therapy with a new retinoid; Dermatologica 157: 238-244 

9 Berth-Jones J (1996): Six area, six sign atopic dermatitis 

(SASSAD) severity score: a simple system for monitoring 

disease activity in atopic dermatitis; Br J Dermatol 135 (Suppl 

48): 25-30 

10 Hoffmann K, Dirschka T, Schwarze H, el-Gammal S, Matthes 

U, Hoffmann A, Altmeyer P(1995): 20 MHz sonography, 

colorimetry and image analysis in the evaluation of psoriasis 

vulgaris; J Dermatol Science 9: 103-110 

11 Bangha E, Elsner P (1996): Evaluation of topical antipsoriatic 

treatment by chromametry, visiometry and 20-MHz ultrasound 

in the psoriasis plaque test; Skin Pharmacology 9: 298-306 

12 Hanifin JM (1989): Standardized grading of subjects for clinical 

research studies in atopic dermatitis: Workshop report; Acta 

Derm Venereol 144: 28-30 

13 Fitzpatrick TB (1988): The validity and practicality of sunreactive 

skin types I through VI; Arch Dermatol 124: 869-871 

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14 Sakane T, Takada S, Kotani H, Tsunematsu T (1982): 

Effects of methyl-B12 on the in vitro immune functions of human 

T lymphocytes; J Clin Immunol 2: 101-109 

15 Yamashiki M, Nishimura A, Kosaka Y (1992): Effects of 

methylcobalamin (vitamin B12) on in vitro cytokine production 

of peripheral blood mononuclear cells; J Clin Lab Immunol 37: 

173-182 

16 Greenberg SS, Xie J, Zatarain JM, Kapusta DR, Miller MJ 

(1995): Hydroxocobalamin (vitamin B12a) prevents and reverses 

endotoxin-induced hypotension and mortality in rodents: Role of 

nitric oxide; J Pharmacol Exp Therap 273: 257-265 

17 Danishpajooh IO, Gudi T, Chen Y, Kharitonov VG, Sharna VS, 

Boss GR (2001): Nitric oxide inhibits methionine synthase 

activity in vivo and disrupts carbon flow through the folate 

pathway; J Biol Chem 276: 27296-27303 

18 Clark JE, Green CJ, Motterlini R(1997): Involvement of the 

heme oxygenase-carbon monoxide pathway in keratinocyte 

proliferation; Biochem Biophys Res Com 241: 215-220 

19 Rowe A, Farrell AM, Bunker CB (1997): Constitutive 

endothelial and inducible nitric oxide synthase in inflammatory 

dermatoses; Br J Dermatol 136: 18-23 

20 Ormerod AD, Weller R, Copeland P, Benjamin N, Ralston SH, 

Grabowski P, Herriot R (1998): Detection of nitric oxide and 

nitric oxide synthases in psoriasis; Arch Dermatol Res 290: 3-8 

21 Morita H, Semma M, Hori M, Kitano Y (1995): Clinical 

application of nitric oxide synthase inhibitor for atopic 

dermatitis; Int J Dermatol 34: 294-295 

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Modern Vehicle Systems 

C. Huschka, J. Wohlrab 

Modern Vehicle Systems 167 

Universitätsklinik und Poliklinik für Dermatologie und Venerologie der 



D-06097 Halle/S. 

Germany 

Modern Vehicle Systems............................................ 167 

References................................................................... 178 

The application of common vehicles like ointments, creams, gels or 

emulsions is only possible to a limited degree for several externallyapplied 

active substances, since the skin barrier cannot be crossed. 

In recent years, alternatives have been sought in intensive research. 

Thus, modern, colloidal vehicle systems, like liposomes and their 

further developments such as the DMS ® System, nanoparticles 

(nanoemulsions), multiple emulsions or microemulsions are gaining 

importance. Among the so-called problem drugs in topical therapy 

are, for example, substances with low solubility in the vehicle, since 

uptake in the skin is only possible in the dissolved state. Likewise, 

drugs with pronounced hydrophilic properties need the support of 

galenic excipients, since their affinity to the lipophilic structures of 

the external skin layer is very low. The application of colloidal 

vehicle systems is can also be used for substances which tend to be 

unstable, for example in the form of hydrolysis, photosensitivity or 

oxidation. 

An overview of the activities of the last ten years in the area of 

research of colloidal vehicle systems for dermal application is 

found in Fig. 1. This patent search discloses a marked trend in 

patent applications for liposomal formulations which peaked in 

2001 at more than 300 applications. By contrast, the number of 

patent applications for nanoparticles and microemulsions appears 

small. However, there is also an increasing tendency here. 

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168 Modern Vehicle Systems 

Individual colloidal vehicle systems are described in some detail 

and recent developments are discussed below. An overview of the 

basic structures of these vehicles is shown in Fig. 2. The liposomes 

are vesicles in the nanometer range, which are constructed of one or 

more double layers. Their structure is similar to that of a cell 

membrane. Lecithin from chicken egg yolk or soy is usually used as 

the basic building block for the liposomes. The use of non-ionic 

tensides is also common. Technological procedures like extrusion, 

mixing and also high-pressure homogenisation are used for 

manufacture [Lichtenberg and Barenholz 1988]. 

Aqueous suspensions of nanoparticles or nanoemulsions can be 

produced using high-pressure homogenization. For this, carrier 

lipids, like triglycerides, are dispersed in the aqueous phase and 

stabilized by means of tensides, such as phospholipids. When the 

particle core produced is fluid, they are usually termed 

nanoemulsions, although they may be termed solid lipid 

nanoparticles (SLN ® , Lipopearls ® Nanopearls ® ) [Dingler and Gohla 

2002, de Vringer and de Ronde 1995]. If a polymer or polymerbuilder 

is used instead of carrier lipids, nanoparticles with a shell or 

fixed matrix can be formed. For this, technologies of coacervation 

or solvent evaporation are used [Quintanar-Guerrero et al. 1998]. 

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Fig. 1: Patient applications for colloidal vehicle systems for topical 

application 

Fig. 2: Basic structures of colloidal vehicle systems 

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The basic building blocks of microemulsions may be micelles of 

various types. Due to their properties, these systems appear clear to 

opalescent. Microemulsions are usually made of a mixture of 

tenside and cotenside, a lipophilic and hydrophilic component (Fig. 

3) These microemulsions form in a specific mixture without 

applying energy, such as shearing strength, which means they are 

simple to produce. Microemulsions have several advantages over 

the other vehicles. They possess an excellent dissolution capacity 

for poorly-soluble substances and have very good penetration 

properties. They are able to reversibly reduce the barrier function of 

the skin. It must also be emphasized that these systems are 

thermodynamically stable and have a good spreading capacity, 

which is attributable to the low-viscous properties [Osborne et al. 

1991, Tenjarla 1999]. From a dermatological point of view, the 

frequently quite high tenside content of microemulsions must be 

viewed as a disadvantage, since it is associated with the occurrence 

of skin irritations. For this reason, a possible minimization of the 

tenside content and the selection of components well-tolerated by 

the skin should be given particular attention in the development of 

new microemulsion systems for dermal application. Thus far, 

application of microemulsions to the skin have been made mostly 

for the purpose of transdermal application of drugs [Alvarez- 

Figueroa and Blanco-Mendez 2001]. Experience over the past years 

has shown that these systems are also excellently suited for specific 

topical therapy [Schmalfuß 1997]. Depending on the composition 

of the ME, lipophilic or hydrophilic molecules can be enriched 

specially in the horny layer and the living layers of the skin. Thus 

effective concentration-time profiles can be achieved in the 

epidermis using these special formulations. Interesting 

developments for various indications are presented below from the 

numerous studies of microemulsions for topical application. 

The studies by Baroli et al. show that the use of microemulsions can 

improve the topical availability of 8-methoxypsoralen (8-MOP), 

the active substance for PUVA-therapy. In addition to 

isopropylmyristate and water as vehicles, several microemulsions of 

various compositions were tested. A selection of skin-tolerated 

tensides was made in producing the microemulsions. The active 

substance 8-MOP was applied in the saturated systems and the 

penetration and permeation of the active determined 8 hours after 

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application. Compared to the references IPM and water, the 

microemulsions stand out in their potency to promote penetration 

and permeation. With a specific composition of the vehicle, 

particularly high concentrations of the active could be attained in 

the skin, thus minimizing systemic stress. As a resumé of the 

studies, application of 8-MOP in a microemulsion can be 

recommended for local treatment of psoriasis [Baroli et al. 2000]. 

The use of non-steroidal antiphlogistics for external therapy of 

musculoskeletal or antirheumatic diseases is widespread, in order 

to avoid the typical side effects of oral administration of nonsteroidal 

antiphlogistics. The vehicle system used is decisive for the 

biological activity of the applied active substance. Usually, gel 

vehicles are selected for application, but they do not always 

guarantee optimal efficacy. 

Using piroxicam as an example, Dalmora et al. could demonstrate 

that improvement in the efficacy of piroxicam can be achieved 

when microemulsions are used as vehicle systems. In an 

inflammation model in rats, granuloma was first induced by 

implanting cotton pellets. The rats’ defence reaction was to be 

stopped by application of the antiphlogistic. The penetrationpromoting 

effect of the microemulsion was particularly clear in 

these studies, since the externally-applied microemulsions could be 

shown to have the same efficacy (reduction of the granuloma mass) 

as the subcutaneously-applied microemulsions. Thus, efficient 

enriching of piroxicam in tissue could be guaranteed by the use of 

special microemulsions [Dalmora et al. 2001]. 

Hydrophilic active substances are generally considered problem 

drugs for topical application. The topical availability of these 

actives can usually be increased with the help of colloidal vehicle 

systems. In the case of the hydrophilic active substance Biotin, 

microemulsions of various compositions were produced and the 

penetration of the active in human skin ex vivo determined after a 

short (30 min) and prolonged (300 min) application time. An O/W 

emulsion (Hydrophilic Ointment containing water DAB) was used 

as the reference vehicle, since Biotin can penetrate the skin better 

from this vehicle than when it is applied in water-in-oil type 

emulsion (such as lanolin-alcohol ointment containing water, 

DAB). While the microemulsion alone was only as effective as the 

standard in form of an O/W emulsion, double the quantity of Biotin 

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could be introduced after the addition of cholesterol. To our 

knowledge, this is due to the fact that cholesterol is capable of 

opening hydrophilic domains in the horny layer for the 

transportation especially of hydrophilic substances. The active 

substance quantity thus introduced in the horny layer of human skin 

could be diffused off from this depot into the living skin layers 

[Huschka et al. 2001]. 

Drawing conclusions from a number of studies on microemulsionsn 

and their topical applications, the following aspects can be rated as 

positive. The systems are simple to produce and show excellent 

stability. Microemulsions possess a high degree of dissolution 

capacity, which supports the application of problem drug 

substances. They have low viscosity and low surface tension and 

thus can be easily spread on the skin. Dermal and transdermal 

applications can be recommended. The high tenside content must be 

considered a disadvantage, since it is associated with the risk of 

intolerance 

Liposomes are another colloidal vehicle system for topical 

application. In the past twenty years, intensive work has been done 

to improve the topical availability of various active substances by 

using liposomes. Since then, especially the penetration-promoting 

effect of liposomes has been intensively studied for a range of 

vitamins, for steroidal and non-steroidal antiphlogistics, 

antimycotics and antibiotics and in addition to many other 

substances for macromolecules. Current examples of research in the 

area of topical application of liposomal formulations are discussed 

below. 

Improvement of the topical efficacy of the gyrase inhibitor 

enoxacin was the objective of studies by Fang et al. The 

accumulation of the active in the skin of hairless/nude? mice was 

determined after 48 hours. A pure enoxacin solution was used as the 

reference vehicle. As comparison, enoxacin was worked into 

various liposomal systems. The various vehicle systems produced 

different results with respect to the penetrating quantity of active 

substance. While liposomes of dimyristoylphosphatidylcholine 

(DMPC) and the combination of DMPC and cholesterol led to a 

reduction of active penetration, increased quantities of enoxacin 

could be found in the skin when lecithin of chicken egg yolk and of 

soy was used. The use of pure niosomes harvested from the vesicle 

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former SPAN ® (Sorbitan fatty acid ester) produced the greatest 

penetration-promoting effect [Fang et al. 2001]. 

An increasing number of articles is being published in the literature 

about a reduction of the damaging effects of UV-light. Among 

these are the articles by Yarosh [Yarosh 2001, Yarosh et al. 2001]. 

They are all aimed at the repair of UV-induced damage to DNA by 

the effect of a bacterial endonuclease. An essential requisite for the 

efficacy of this enzyme is the transport in intact liposomes into the 

living epidermis. The positive accomplishment of this idea could be 

confirmed by electronic microscopic images. 

Exposure to too much UV-light causes damage to the living 

epidermis, which is expressed by typical signs of inflammation. 

One signal for excessive light exposure can be found in the 

expression of IL-10. This interleukin could be 

immunohisotochemically proven in samples of damaged skin. 

There was no signal in skin treated with T4 endonuclease V 

(T4N5). A negative control, in which inactivated enzyme in 

liposomes was applied, disproved the efficacy of the vehicle alone 

[Wolf et al. 2000]. 

Patients with the genetically-caused disease Xeroderma 

pigmentosum tend to develop actinic keratoses or basal cell 

carcinoma after contact with UV-light. In order to prevent this, 

patients with this disease were treated for a year with T4N5liposomes. 

Compared to placebo, the sequelae could be greatly 

reduced. Thus, therapy or prophylaxis with enzyme-loaded 

liposomes appears meaningful in cases of UV-induced damage 

[Yarosh et al. 2001]. 

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Fig. 3: Properties and components of microemulsions 

Fig. 4: Nanoparticles with 5-ALA in the treatment of basal cell 

carcinomas 

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The following aspects can be summarized from the studies 

discussed: Liposomes are diverse in their structure and efficacy. 

Thanks to their specific structure, liposomes can enter into intensive 

interactions with the lipids in the horny layer. Thus, liposomal 

systems can provide for penetration enhancement of a number of 

molecules. Even for active substances with low topical availability, 

liposomal systems offer a possibility of enabling transdermal 

effects. The proprio-effect of blank liposomes (only phospholipids 

without active substance load), expressed by increased hydratation 

of the epidermis, should not be ignored. Although the manufacture 

itself of the vehicle can be considered simple, there is a need to 

product liposomes with long-term stability. Simply-constructed 

liposomes (such as those from pure lecithin) tend to be unstable 

(release of the active due to structural loss, fusion of the vesicle). 

Nanoparticles are colloidal vehicle systems, with dimensions in the 

lower nanometer range. High-pressure homogenization has become 

established among the possibilities of producing the particles from 

stabilized triglycerides [Lippacher et al. 2001]. A review of the 

published activities shows an increasing frequency of reports on the 

use of solid lipid nanoparticles (SLN ® ) for skin-care therapy. 

Several vitamins and other skin-care substances propagated also for 

the treatment of damaged skin or skin requiring special care, are 

worked into these particles [Bernadesca et al. 2001, Jenning et al. 

2000]. The use of nanoparticles under the aspect of controlled 

release of the active should be mentioned. Such developments are 

among the goals in the application of glucocorticoids [Maia et al. 

2000]. 

Improved penetration of the photosensitizer 5-Aminolaevulinic acid 

(5-ALA) was the object of the following study. Photodynamic 

therapy with nanoparticles containing 5-ALA was performed in 

patients with basal cell carcinoma, in order to better transport the 

active substance to the site of action. The active concentration in the 

particles was 10%. After single application for 6 hours with 

subsequent light exposure, efficacy of therapy could be 

demonstrated after 6 months in 15% of the patients (Fig. 4). Costs 

could be saved by reducing the quantity of active substance used (in 

cream vehicles, 20% is the usual concentration). 

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Fig. 5: Electronic-microscopic images of nanopartikels of starch 

acetate 

Fig. 6: Penetration of Biotin from starch acetate nanoparticles in 

human skin ex vivo That efficacy is attained is explained as an 

occlusive effect of uniform covering of the skin surface by the 

particles [Hürlimann et al. 1998]. 

------------------------------------------------------------------------------------ 




Physical filters have been used for several years to reduce the effect 

of sunlight on human skin, also to replace chemical filter substances 

(absorber) to reduce side effects. With the further development of 

micronized particles down to real nanoparticles of titanium dioxide, 

a new quality has been attained. The miniaturization of the particles 

lets the unattractive effect of whitening, such as happens after 

application of micronized partles, disappear. The smaller particles 

are invisible to the human eye and, thanks to their denser packing 

on the skin surface, also provide improved protection. The excellent 

spread of the particles on the surface means that even the smallest 

pores are reached, so that trouble spots are avoided and the effect 

optimized. 

In addition to the lipids, polymers can also be used in the 

production of solid particles. Using a lipophilic acetylated starch, 

nanoparticles with porous structure can be produced (solvent 

evaporation technique) and these loaded with the active substance 

Biotin (Fig. 5). Then the penetration of Biotin in human skin was 

traced [Huschka et al. 2001]. Differences in the extent of 

penetration were observed depending on the tenside used. Lipid and 

polymer systems brought more Biotin into the horny layer than the 

standard (Water-containing Hydrophilic Ointment DAB) (Fig. 6). 

Observed in a short application time, the affinity of the positivecharged 

particles produced with the emulgator benzalconium 

chloride was higher than the negative-charged particles (sodium 

dodecylsulfate). This may be attributed to the more intensive 

interactions with the negative-charged skin surface. 

The following aspects summarize again in brief the main points on 

nanoparticles. Nanoparticles can be easily produced in large scale 

from a technological point of view. Overall, most particulate 

systems are stable. With respect to dermal application, lipid 

particles are particularly well tolerated. By contrast, attention must 

be paid to a residual content of toxic monomers in several polymer 

particles. Due to the minimal surface of the particles, they are able 

to completely cover the skin surface. Thus occlusion is attained and 

the hydratation of the horny layer is increased. These are, therefore, 

systems which can be recommended for dry or aged skin. 

Moreover, the use of pure particles as light protection is possible. In 

addition, nanoparticles can be easily worked into other bases. The 

uptake of active substances can be realized, depends however on 

------------------------------------------------------------------------------------ 




the physicochemical properties of the particle. Nanoparticles can 

protect the active ingredient against external influence and thanks to 

their matrix are able to control the release of the incorporated 

substances [Wissing and Müller 2001]. 

References 

1 Alvarez-Figueroa MJ, Blanco-Mendez J.: Transdermal delivery 

of methotrexate: iontophoretic delivery from hydrogels and 

passive delivery from microemulsions. 

Int J Pharm 2001; 215 (1-2): 57-65 

2 Baroli B, Lopez-Quintela MA, Delgado-Charro MB, Fadda AM, 

Blanco-Mendez J: Microemulsions for topical delivery of 8methoxsalen; 

J Control Release 2000; 69 (1): 209-218 

3 Berardesca E, Barbareschi M, Veraldi S, Pimpinelli N.: 

Evaluation of efficacy of a skin lipid mixture in patients with 

irritant contact dermatitis, allergic contact dermatitis or atopic 

dermatitis: a multicenter study; Contact Dermatitis 2001; 45 (5): 

280-285 

4 Dalmora ME, Dalmora SL, Oliveira AG: Inclusion complex of 

piroxicam with beta-cyclodextrin and incorporation in cationic 

microemulsion. In vitro drug release and in vivo topical antiinflammatory 

effect. Int. J Pharm 2001; 222 (1): 45-55 

5 Dingler A, Gohla S.: Production of solid lipid nanoparticles 

(SLN): scaling up feasibilities.; J Microencapsul 2002; 19 (1): 

11-16 

6 Hürlimann AF, Hänggi G, Panizzon RG.: Photodynamic therapy 

of superficial basal cell carcinomas using topical 5aminolevulinic 

acid in a nanocolloid lotion; Dermatology 1998; 

197 (3): 248-254. 

7 Huschka C, Wolf R, Raith K, Neubert R, Wohlrab W. Relevance 

of topical application of biotin containing formulations. In 

Bonnekoh B, Gollnick H. (eds.) Dermatological treatment: novel 

clinical and experimental approaches 2002 (in press) 

8 Jenning V, Gysler A, Schafer-Korting M, Gohla SH.: Vitamin A 

loaded solid lipid nanoparticles for topical use: occlusive 

properties and drug targeting to the upper skin; Eur J Pharm 

Biopharm. 2000; 49 (3): 211-218. 

------------------------------------------------------------------------------------ 




9 Lichtenberg D, Barenholz Y.: Liposomes: preparation, 

characterization, and preservation; Methods Biochem Anal 

1988; 33: 337-462 

10 Lippacher A, Muller RH, Mader K.: Preparation of semisolid 

drug carriers for topical application based on solid lipid 

nanoparticles; Int J Pharm 2001 Feb 19; 214 (1-2): 9-12. 

11 Fang JY, Hong CH, Chiu WT, Wang YY.: Effect of liposomes 

and niosomes on skin permeation of enoxacin; Int J Pharm 2001; 

219 (1-2): 61-72. 

12 Maia CS, Mehnert W, Schafer-Korting M. Related Articles: 

Solid lipid nanoparticles as drug carriers for topical 

glucocorticoids; Int J Pharm 2000; 196 (2):165-167 

13 Osborne DW, Ward AJ, O'Neill KJ.: Microemulsions as topical 

drug delivery vehicles: in-vitro transdermal studies of a model 

hydrophilic drug; J Pharm Pharmacol 1991; 43 (6): 450-454 

14 Quintanar-Guerrero D, Allemann E, Fessi H, Doelker E.: 

Preparation techniques and mechanisms of formation of 

biodegradable nanoparticles from preformed polymers; Drug 

Dev Ind Pharm 1998 (12): 1113-1128 

15 Schmalfuß, U: Untersuchungen zur Modulation der Penetration 

eines hydrophilen Arzneistoffes aus Mikroemulsionssystemen in 

humane Haut unter ex vivo-Bedingungen; 1997; Dissertation, 

Math.-Nat.-Tech. Fak., Martin-Luther-Universität Halle- 

Wittenberg 

16 Tenjarla S.: Microemulsions: an overview and pharmaceutical 

applications; Crit Rev Ther Drug Carrier Syst 1999;16 (5): 461- 

521 

17 de Vringer T, de Ronde HA. : Preparation and structure of a 

water-in-oil cream containing lipid nanoparticles; J Pharm Sci 

1995; 84 (4): 466-472 

18 Wolf P, Maier H, Müllegger RR, Chadwick CA, Hofmann- 

Wellenhof R, Soyer HP, Hofer A, Smolle J, Horn M, Cerroni L, 

Yarosh D, Klein J, Bucana C, Dunner K, Potten CS, 

Hönigsmann H, Kerl H, Kripke ML.: Topical treatment with 

liposomes containing T4 endonuclease V protects human skin in 

vivo from ultraviolet-induced upregulation of interleukin-10 and 

tumor necrosis factor-alpha; J Invest Dermatol 2000; 114 (1): 

149-156 

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19 Yarosh DB: Liposomes in investigative dermatology; 

Photodermatol Photoimmunol Photomed 2001; 17 (5): 203-212 

20 Yarosh D, Klein J, O'Connor A, Hawk J, Rafal E, Wolf P: Effect 

of topically applied T4 endonuclease V in liposomes on skin 

cancer in xeroderma pigmentosum: a randomised study. 

Xeroderma Pigmentosum Study Group; Lancet 2001; 357: 926- 

929 

21 Wissing SA, Muller RH.: Solid lipid nanoparticles (SLN)--a 

novel carrier for UV blockers; Pharmazie 2001; 56 (10): 783- 

786 

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Microemulsions as drug vehicles for dermal application 181 

Microemulsions as drug vehicles for dermal 

application 

K. Jahn, R.H.H. Neubert, *J. Wohlrab 

Institut für Pharmazeutische Technologie und Biopharmazie, 

* Universitätsklinik und Poliklinik für Dermatologie und Venerologie der 


Wolfgang-Langenbeck-Straße 4 

D-06120 Halle 

Germany 

Microemulsions for dermal use .................................. 181 

Summary..................................................................... 183 

References................................................................... 184 

Microemulsions (ME’s) are isotropic, transparent or slightly 

opalescent, thermodynamically stable and low viscous systems. 

They are quaternary systems consisting usually of a hydrophilic and 

a lipophilic phase stabilized by a surfactant and a cosurfactant. 

ME’s can be manufactured easily, simply by stirring the 

components together. The spontaneous formation without energy 

input and the thermodynamic stability belong to their special 

features and are caused by the dramatic decrease of the interfacial 

tension between the hydrophilic and the lipophilic phase by mixing 

the components. Consequently, a characteristic microstructure of 

small droplets is formed. Alternating surfactant and cosurfactant 

molecules surround the internal phase. The main distinction 

between crude emulsions and ME’s is their particle diameter in the 

range of 10 to 200 nm. This property is the reason for the long-term 

stability without phase separation. These systems show structural 

similarities to micelles and inverse micelles. They represent highly 

dynamic structures due to the fluctuating surfaces as a consequence 

of the forming and deforming processes. Just as crude emulsions 

they can be divided into two types: w/o (water-in-oil) and o/w (oilin-water). 

The formation is dependent on the oil-to-water ratio and 

the physicochemical characteristics of the surfactant and 

cosurfactant used. Bicontinuous structures are also possible [1,2,3]. 

------------------------------------------------------------------------------------ 



182 Microemulsions as drug vehicles for dermal application 

Table 1 shows possible administration routes of microemulsions 

and some drug examples (literature data). 

administration 

route 

oral 

------------------------------------------------------------------------------------ 



drugs 

Ciclosporin A [4] (Sandimmun ® Optoral), hydrophilic peptides 

[5] 

Ophthalmic timolol [6], pilocarpine [7] 

parenteral flurbiprofen [8], diazepam [9] 

dermal/transdermal azelaic acid [10], local anaesthetics [11], ß-blockers [12] 

Tab. 1: Possible administration routes of microemulsions 

Microemulsions for dermal use 

In pharmacy, ME’s have been attracted great interest as dermal 

drug delivery systems to improve percutaneous penetration of 

problematical drugs. The dermal application of drugs can be 

advantageous in the treatment of several dermatologic diseases, in 

order to minimize side effects after systemic application as well as 

to avoid the hepatic first-pass metabolism or the destruction and 

inactivation of drugs following oral application. 

Numerous in-vitro release and penetration studies have been 

reported by several authors to assess the suitability of ME’s for 

dermal and transdermal application. Currently, the therapeutic use 

of these systems for dermal drug delivery is limited due to the high 

content of emulsifiers. In future, it is necessary to create vehicles 

containing surfactants with low irritative potential and good skin 

acceptability. It is possible to overcome this problem if non-ionic 

and polymeric surfactants will be preferred. 

Microemulsions of o/w-type are the more suitable vehicles for 

lipophilic drugs, whereas the w/o-type seems to be the better one 

for hydrophilic drugs. 

Generally, hydrophilic drugs have difficulties to pass lipophilic 

membranes. Therefore, the penetration-enhancing activities of w/o- 

ME’s can facilitate the drug diffusion into the skin. Lipophilic


drugs are mostly problematical regarding their penetration rate. Due 

to the physicochemical properties they enter into the lipophilic 

stratum corneum forming a drug depot, but they are not capable of 

diffusing in deeper skin layers. Incorporating such drugs into o/w- 

ME’s can lead to a greater penetration extent. 

An example for the dermal use of microemulsions containing an 

extremely lipophilic compound, ciclosporin A, is shown in another 

contribution to this book („Trends of topical immunomodulatory 

therapy“). 

Advantages of microemulsions for dermal use 

- favourable solubilization capacity for slightly soluble drugs 

- exert penetration enhancing properties due to their structure and 

composition 

- dermal or transdermal effect of applied drugs 

- Transport of drugs, which are problematical regarding their 

penetration behaviour, into human skin 

- reversible modification of the main penetration barrier Stratum 

corneum 

Summary 

Microemulsions are due to their favourable solubilization capacity 

suitable vehicles for slightly soluble drugs. In contrast to 

conventional preparations (creams and ointments) the dermal 

application of ME’s leads to a deeper and extended penetration of 

incorporated drugs into the skin. Further advantages of 

microemulsions are the thermodynamic stability, ease of 

manufacturing and spontaneous formation. In future, the interest 

has to be focused on the development of microemulsions with 

pharmaceutically-acceptable compounds. 

For further information see several reviews and book chapters 

[1,2,3,13,14]. 

------------------------------------------------------------------------------------ 



184 Microemulsions as drug vehicles for dermal application 

References 

1 Attwood D (1994) Microemulsions. In: Kreuter, J. (ed.) 

Colloidal drug delivery systems. Marcel Dekker, New York p 

31-71 

2 Lawrence MJ (1996) Microemulsions as drug delivery vehicles. 

Curr Opin Colloid Interface Sci 1:464-471 

3 Tenjarla S (1999) Microemulsions: An overview and 

pharmaceutical applications. Crit Rev Ther Drug Carrier Syst 

16(5):461-521 

4 Kovarik JM, Mueller EA, van Bree JB, Tetzloff W, Kutz K 

(1994) Reduced inter- and intraindividual variability in 

cyclosporine pharmacokinetics from a microemulsion 

formulation. J Pharm Sci 83:444-446 

5 Constantinides PP, Lancester CM, Marcello J, Chiossone DC, 

Orner D, Hidalgo I, Smith PL, Sarkahian AB, Yiv SH, Owen AJ 

(1995) Enhanced intestinal absorption of an RGD peptide from 

water-in-oil microemulsions of different composition and 

particle size. J Contr Rel 34: 109-116 

6 Gasco MR, Gallarate M, Trotta M, Bauchiero L, Gremmo E, 

Chiappero O (1989) Microemulsions as topical delivery 

vehicles: ocular administration of timolol. J Pharm Biomed Anal 

7:433-439 

7 Haße A, Keipert S (1997) Development and characterization of 

microemulsions for ocular application. Eur J Pharm Biopharm 

43:179-183 

8 Park K-M, Kim Ch-K (1999) Preparation and evaluation of 

flurbiprofen-loaded microemulsion for parenteral delivery. Int J 

Pharm 181:173-179 

9 Trotta M, Gasco MR, Carlotti ME (1990) Study on an o/w 

microemulsion carrying diazepam. Acta Technol Legis 

Medicamenti 1:137-148 

10 Gasco MR, Gallarate M, Pattarino F (1991) In vitro permeation 

of azelaic acid from viscosized microemulsions. Int J Pharm 

69:193-196 

11 Krause SA (2001) Entwicklung und Charakterisierung von 

Mikroemulsionen zur dermalen Applikation von Arzneistoffen, 

PhD Thesis, Martin-Luther-University Halle-Wittenberg, 

Germany 

------------------------------------------------------------------------------------ 




12 Kemken J, Ziegler A, Mueller BW (1991) Investigations into the 

pharmacodynamic effect of dermally administered 

microemulsions containing ß-Blockers. J Pharm Pharmacol 

43:679-684 

13 Lawrence MJ, Rees GD (2000) Microemulsion-based media as 

novel drug delivery systems. Adv Drug. Del Rev 45: 89-121 

(2000) 

14 Jahn K, Krause A, Janich M, Neubert RHH (2002) Colloidal 

Drug Carrier Systems. In: Bronaugh RL, Maibach HI (eds) 

Topical absorption of dermatological products. Marcel Dekker, 

New York p 483-493 

------------------------------------------------------------------------------------ 



186 Transfersomes® and their Application in Dermatopharmaceutics 

Transfersomes ® and their Application in 

Dermatopharmaceutics 

J. Lehmann, M. Rother 

IDEA AG 

Frankfurter Ring 193 a 

D-80807 München 

Germany 

What are Transfersomes ® ?.......................................... 186 

Therapeutic Applications for the Transfersome ® 

Technology.................................................................. 189 

Glucocorticoids: Triamcinolone acetonide in 

Transfersomes ® ........................................................... 189 

Conclusion .................................................................. 193 

Acknowledgement ...................................................... 193 

References................................................................... 193 

What are Transfersomes ® ? 

Transfersomes ® are ultraflexible lipid vesicles designed to deliver 

drugs through the skin barrier [1]. Their membrane consists of a 

minimum of two components with different solubility. This enables 

Transfersomes ® to rearrange the components in the membrane 

according to any external force exerted on them, e.g., the hydration 

gradient, to make the membrane locally much more adaptable and 

better suited to accommodate to ambient stress (Fig. 1, upper right 

panel). This allows Transfersomes ® to cross pores that have a 

diametre smaller than their own average diametre. 

In order to characterize the ability of different lipid vesicles to 

penetrate a semi-permeable barrier an in vitro assay was developed. 

The method consists of applying a pressure on a suspension of 

mixed lipid vesicles on one side of a barrier with 20 nm to 50 nm 

pores. As a control, water is pressed through the pores to determine 

maximum transfer ability. Such an assay can clearly differentiate 

between the liposomes with a stiff membrane that only pass through 

a nano-porous barrier under pressure of >1 MPa, and the highly 

------------------------------------------------------------------------------------ 



Transfersomes® and their Application in Dermatopharmaceutics 187 

shape-adaptable Transfersomes ® that start penetrating such a barrier 

at pressures around 0.1 MPa (Fig. 1). As an additional parameter 

vesicle integrity is checked by analysing the size of the vesicles that 

have passed the barrier. In case of liposomes, the original size is 

significantly reduced suggesting passage after vesicle 

fragmentation. In contrast, Transfersomes ® maintain their original 

size. This proves that the selected mixed lipid vesicles cross pores 

in a barrier due to their extremely high membrane adaptability. 

Penetration rel. to water [%] 

10 2 

10 1 

10 0 

10 -1 

10 -2 

10 -3 

Water 

Pressure [MPa] 

r V /r Pore = 3.5 

Transfersomes ® 

0 0.2 0.4 0.6 0.8 

Liposomes 

Fig. 1: In vitro assay for determining membrane adaptability of 

lipid vesicles. The flexibility of Transfersome ® membranes is much 

higher than that of liposome membranes, resulting in pore 

penetration ability of the former that is similar to that of water. 

Upper right panel: schematic representation of Transfersome ® 

membrane composition and deformation during entry in a confining 

pore. Lower right panel: schematic representation of liposome 

membrane composition and stiffness. rel.: relative; rV/rPore: relative 

vesicle/pore size ratio. 

In vivo, the driving force for Transfersome ® motion across the skin 

is the transepidermal water gradient. With increasing depth in the 

skin the water content of the stratum corneum as the outermost skin 

------------------------------------------------------------------------------------ 




region increases from approximately 15% to nearly 75% [2]. After 

an epicutaneous application Transfersome ® suspensions first dry to 

the vesicles´ solubility limit. The hydrophilic vesicles are 

consequently attracted by the higher water content in the inner skin. 

This drives Transfersomes ® through the stratum corneum through 

the hydrophilic pores between cells that are opened by the vesicles 

themselves. Aguilella et al. [3] estimated the corresponding pore 

radius to be approximately 20 nm. 

Fig. 2: Left: Schematic drawing of mammalian epidermis, revealing 

the existence of cell clusters and of potential inter-cluster or intercellular 

(intra-cluster) pathways in the stratum corneum. Right: 

Murine stratum corneum as viewed with confocal laser scanning 

microscopy from the top after epicutaneous treatment with 

fluorescently labelled Transfersomes ® [4]. The Transfersome ® - 

associated label stains intra- as well as inter-cluster pathways. 

Using fluorescently labelled Transfersomes ® and confocal laser 

scanning microscopy (CLSM) the morphology of the stratum 

corneum was investigated by Schätzlein and Cevc [4]. This 

revealed the existence of cell clusters, shown in the left panel of 

Fig. 2, and of inter-cluster as well as intra-cluster pathways shown 

in the right panel of Fig. 2. The inter-cluster pathways are marked 

intensively by the dye. The intra-cluster pathway is highlighted in 

the superposition of pictures taken at several depths in the stratum 

------------------------------------------------------------------------------------ 




corneum and projected on top of each other. Inter-cluster pathways 

appear as thick bands and inter-cellular (intra-cluster) pathways as 

thin-bands. Lipid aggregates with stiffer membranes only label the 

widest pathways between the cells and the intercellular space in the 

top layers of the stratum corneum [1]. 

Therapeutic Applications for the Transfersome ® 

Technology 

The Transfersome ® technology offers a wide range of treatment 

opportunities [5]. The molecules to be delivered may vary from 

small ones like glucocorticoids and antimycotics up to 

macromolecules. For optimum performance however each 

Transfersome ® formulation must be specifically designed according 

to its intended use, the target indication, the target tissue, and the 

drug molecule to be transported. The most obvious application is 

Transfersome ® usage for the treatment of skin diseases like 

psoriasis, mycoses, keratoses, etc.. In addition, Transfersomes ® are 

also suitable for non-invasive treatment of local indications like 

pain in the joints or muscle soreness. This is due to the fact that 

such carriers are uniquely able to transport the drug into the deep 

subcutaneous tissues [6]. This route of administration is especially 

interesting for non-steroidal anti-inflammatory drugs as it helps to 

avoid side effects caused by systemic application like bleeding of 

the gastrointestinal tract. A third area of use for Transfersomes ® is 

the non-invasive, systemic application of molecules that currently 

require an injection as route of administration like insulin or 

interferon. 

Glucocorticoids: Triamcinolone acetonide in 

Transfersomes ® 

Preclinical data obtained in the arachidonic acid-induced mouse ear 

oedema model [7] suggest that only one tenth of the triamcinolone 

acetonide (TAC) dose applied in Transfersomes ® compared to the 

dose applied in commercially available lotion or cream 

formulations is needed. 

------------------------------------------------------------------------------------ 




Based on the preclinical data, a monocentric, placebo-controlled, 

observer- and subject-blinded clinical trial was conducted [8]. The 

primary objective was to determine the bioequivalence between a 

ten-fold lower TAC dosage in Transfersomes ® and a commercially 

available cream and ointment. The test fields were randomly 

distributed on the skin of the back of 20 healthy volunteers, using 

several drug doses to investigate the equipotency. All formulations 

were applied epicutaneously in a non-occlusive way. The UVBinduced 

erythema test was used for intra-individual comparison. 

2.5 µg/cm² TAC in Transfersomes ® proved to be equipotent in 

erythema suppression to 25 µg/cm² TAC in a cream and an 

ointment. Equipotency was statistically significant with p=0.01 and 

p


of skin thickness. The results of this monocentric, placebocontrolled, 

observer-blinded trial with randomized test field 

distribution on the inner forearms and intraindividual comparison 

are based on the data obtained from 25 healthy volunteers. Both 

formulations studied were applied epicutaneously in a nonocclusive 

way. After twice daily applications of either 2.5 µg/cm² 

TAC in Transfersomes ® or 25 µg/cm² TAC cream for 6 weeks, 76% 

of the test fields treated with TAC cream showed atrophy defined as 

a reduction in skin thickness of at least 20% compared to baseline. 

In contrast, only 24% of the test fields treated with TAC in 

Transfersomes ® fulfilled this criterion. This criterion was only 

reached by one subject treated with placebo Transfersomes ® . After 

one week there was already a significant reduction in skin thickness 

between the TAC containing formulations and placebo 

Transfersomes ® (p


Table 2 shows the three most frequently reported adverse events: 

skin atrophy, telangiectasias and dry skin. For skin atrophy the 

highest reports were seen in those fields treated with TAC cream, 

while the rates were substantially lower for the Transfersome ® 

formulation containing the equipotent dose of TAC and for the 

placebo Transfersomes ® . The same effect was observed for 

telangiectasias which were considered signs of skin atrophy. Dry 

skin was reported to a similar level in all treatment groups. 

skin reduction relative to control + SD 

15% 

10% 

5% 

0% 

-5% 

-10% 

-15% 

-20% 

-25% 

-30% 

-35% 

-12.1% 

significance of a simple difference: 

p=0.007 ** 

p=0.007 ** 

at least a twofold superiority of TAC 

p=0.74 n.s. 

------------------------------------------------------------------------------------ 



-21.1% 

2.5 µg/cm² TAC TAC I (2.5 µg/cm²) Transfersomes Volon A Creme 

® 25 µg/cm² TAC cream 

Fig. 3: Skin thickness reduction relative to untreated control fields 

after twice daily treatment with equipotent doses of triamcinolone 

acetonide (TAC) in two different formulations, TAC in 

Transfersomes ® , and TAC in commercial cream. Ultrasound data 

obtained after 6 weeks of treatment revealed a significant difference 

in skin thickness reduction (Wilcoxon test of difference). SD: 

standard deviation. 

The majority of the reported adverse events occurred towards the 

end of the treatment phase (weeks 5 and 6) and were mainly due to 

the design of the study which was aimed at inducing measurable 

atrophy. As neither the duration of treatment nor the dosing 

frequency match the customary clinical practice, most of the 

adverse events are of limited relevance for the routine use of TAC.

Conclusion 


Non-invasive drug delivery with Transfersomes ® may improve the 

efficiency, applicability, safety, and/or targetability of transdermal 

transport. Lower amounts of drug are often needed for dermal 

targets as compared to oral or conventional topical formulations. As 

a consequence, a decrease in side effects and a better tolerability of 

the administered drug can be expected. Administration of drugs in 

Transfersomes ® reduces high initial plasma peak levels typically 

induced by subcutaneous injection. Furthermore, a depot effect in 

the skin can be created in order to reduce the frequency of 

application, thereby improving patient satisfaction and compliance. 

Acknowledgement 

We thank Prof. Dr. D. Abeck, Dr. H. Fesq, A. Glöckner, and I. 

Erdmann for conducting the two clinical trials in the Klinik und 

Poliklinik für Dermatologie und Allergologie am Biederstein, 

Technische Universität München, Germany. Many thanks also to 

Dr. U. Vierl for the schematic drawing of the stratum corneum 

morphology. 

References 

1 Cevc G (1996) The skin: a pathway for systemic treatment with 

patches and lipid-based agent carriers. Adv Drug Deliv Rev 18: 

349-378 

2 Warner RR, Myers MC, Taylor DA (1988) Electron probe 

analysis of human skin: determination of the water concentration 

profile. J Invest Dermatol 90: 218-224 

3 Aguilella V, Kontturi K, Murtomäki L, Ramírez P (1994) 

Estimation of the pore size and charge density in human cadaver 

skin. J Control Release 32: 249-257 

4 Schätzlein A, Cevc G (1998) Non-uniform cellular packing of 

the stratum corneum and permeability barrier function of intact 

skin: a high-resolution confocal laser scanning microscopy study 

using highly deformable vesicles (Transfersomes) Br J Dermatol 

138: 583-592 

------------------------------------------------------------------------------------ 




5 Cevc G (1997) Drug delivery across the skin. Exp Opin Invest 

Drugs 6: 1887-1937 

6 Cevc G, Blume B (2001) New, highly efficient formulation of 

diclofenac for the topical, transdermal administration in 

ultradeformable drug carriers, Transfersomes. Biochim Biophys 

Acta 1514: 191-205. 

7 Young JM, Spires DA, Bedord CJ, Wagner B, Ballaron SJ, de 

Young LM (1984) The mouse ear inflammatory response to 

topical arachidonic acid. J Invest Dermatol 82: 367-371 

8 Fesq H, Lehmann J, Glöckner A, Erdmann I, Theiling K, Rother 

M, Ring J, Cevc G, Abeck D (2002) Improved risk-benefit ratio 

for topical triamcinolone acetonide in Transfersomes ® in 

comparison to equipotent cream and ointment. Clin Pharmacol 

Ther, submitted. 

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Trends in the systemic antibiotic therapy of dermatological infectious diseases 195 

Trends in the systemic antibiotic therapy of 

dermatological infectious diseases 

N. H. Brockmeyer, A. Kreuter 


Ruhr-University Bochum 

Gudrunstr. 56 


Germany 

Systemic antibiotic therapy has acquired an important position in 

dermatological routine. An awareness of the spectrum of 

appropriate antibiotics, new substances and their characteristic 

resistance profiles plays a major role in deciding upon which 

therapeutic substances to employ. The choice of an antibiotic is also 

determined by its characteristic spectrum of activity as well as its 

profile of interaction with other substances. 

For this reason, new trends in the systemic antibiotic therapy of 

dermatological infectious diseases have to take into account newer 

therapeutic strategies and modalities, epidemiological developments 

as well as newly appearing antibiotic substances and pathogens. 

The most frequent indications for the systemic application of 

antibiotics in dermatology include erysipelas, impetigo, furuncles, 

carbuncles as well as wound, ulcer or soft tissue infections. As a 

rule, established substances are resorted to when choosing the 

antibiotic; for example cephalosporin with staphylococci, 

penicillins for streptococci, doxycycline or penicillin for borrelia, 

and tetracycline, doxycycline or minocycline for the therapy of acne 

and rosacea. Microorganisms were already shown to develop 

resistance to antibiotics immediately after they were introduced in 

the 1940s. By 1948, most strains of S.-aureus in British hospitals 

were resistant to penicillin. At the latest, the appearance of hospitalrelated 

infections due to Methicillin-resistant S.-aureus strains 

(MRSA) at the end of the 1960s emphasised the far-reaching 

importance of multiresistant strains. The resulting choice of second 

class substances, i.e. those with a lower efficacy, resulted again in 

the selection of pathogens that developed resistance. Because of this 

vicious cycle it has become increasingly more difficult to treat 

------------------------------------------------------------------------------------ 



196 Trends in the systemic antibiotic therapy of dermatological infectious diseases 

hospital-related infections effectively. One of the most important 

mechanisms underlying the development of resistance in 

nosocomial pathogens is the appearance of antibiotic inactivating 

enzymes. Other modifications involve the permeability and binding 

structures of the cellular wall, as well as alterations in the target of 

the antibiotic’s action. 

Since the introduction of modern antituberculosis agents, 

tuberculosis has become a well treatable disease with a cure rate of 

over 90% (provided that therapy is adequate and the pathogens are 

sensitive). Resistance towards antituberculotic agents is no rarity. 

Multiple resistance is the most problematic form, involving 

resistance to both isoniazid and rifampicin. In Germany it occurs in 

approx. 2 % of cases wherever individuals are treated inadequately 

or therapy is prematurely interrupted. Internationally, a 

monoresistance rate of approx. 10 % can be expected in nonpretreated 

cases, more frequently against isoniazid than rifampicin. 

With every case of assumed medication resistance, treatment should 

be performed with 4 agents until the results of resistance tests are 

available, and in cases of suspected multiresistance, 5 

antituberculotic agents should be given. The final antituberculotic 

treatment should then be performed with 4-5 resistance-corrected 

substances until at least 18 months after sputum conversion. 

No infectious disease has displayed such a drastic increase in 

incidence over the last few years as syphilis. The number of new 

syphilis infections has increased particularly significantly amongst 

HIV-positive individuals in major cities (Hamburg 1997: 19 cases, 

1999: 71 cases; Cologne 1997: 13 cases, 2001: 69 cases). In the 

USA, a similarly dramatic increase in new infections has been 

recorded following the decrease that characterised the 1980s. The 

incidence is approximately 10-fold higher than it is in other 

industrial countries. In some developing countries in Africa and 

Asia, according to information provided by the WHO, the incidence 

of syphilis exceeds 360/100,000 inhabitants. Experience has shown 

that in cases of concomitantly-existing HIV-infection, the risk of 

neurosyphilis appearing is particularly high. For this reason, even in 

the absence of neurological or psychiatric symptoms, penicillin 

therapy should also be carried out in AIDS-patients (as is also the 

case with neurosyphilis) with close surveillance and control 

examinations. The generation time for Treponema pallidum is 

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Trends in the systemic antibiotic therapy of dermatological infectious diseases 197 

approx. 30 hours. The minimum duration of uninterrupted 

bactericidal therapy is about 8 days. According to recommendations 

from the German Society of Sexually Transmitted Diseases, 

syphilis should be treated for an adequate period with intramuscular 

clemizole-penicillin. Benzathine-penicillin, which in the USA is 

usually employed presumably for patient-compliance and to prevent 

premature therapeutic interruption, only enters the cerebrospinal 

fluid in part. Although the accompanying meningitis occurring in 

the second phase can be treated, levels required to treat neurological 

syphilis can not be reached. Here, the intracellularly persisting 

pathogens can only be reached poorly by the antibiotics. In 

addition, the reproduction time of the pathogen is prolonged in this 

phase. Whether comparably high levels can be reached in the CSF 

with doxycycline, tetracycline or erythromycin is questionable and 

currently the subject of pharmacological investigation. 

With cyclic intravenous antibiotic therapy, a new efficient 

therapeutic concept has been established for chronically recurring 

erysipelas. For this purpose penicillin G is applied intravenously 

over a period of 10 days every three months. A mechanical 

lymphatic drainage is also performed. In a clinical study within our 

clinic, newly occurring episodes of erysipelas could be prevented in 

more than 90 % of all patients. A significant reduction in 

inflammation parameters such as antistreptolysin-titres, BSG as 

well as C-reactive protein was also brought about. It would 

therefore appear that elimination of pathogen from the interstitial 

and lymphatic spaces is possible in the case of erysipelas through 

application of a longer-term antibiotic treatment. 

The current expansion of medication resistance and new infections 

from bacterially –induced skin infections has made it vital for 

clinically active dermatologists to have knowledge about the correct 

administration of currently available systemic antibiotics. 

------------------------------------------------------------------------------------ 



198 Systemic therapy with immunomodulatory drugs in dermatology 

Systemic therapy with immunomodulatory drugs 

in dermatology 

M. Sticherling 

Klinik und Poliklinik für Hautkrankheiten 

Abteilung für Klinische und Experimentelle Dermatologie 

Universität Leipzig 

Stephanstr. 11 

D-04103 Leipzig 

Germany 

Introduction................................................................. 198 

Adjuvant drugs............................................................ 201 

Immunosuppressive drugs .......................................... 201 

Immunomodulatory drugs with suppressive activity.. 202 

Immunomodulatory drugs without suppressive 

activity......................................................................... 203 

References................................................................... 205 

Introduction 

The progress within experimental and clinical immunology over the 

last decades has grossly changed our attitudes towards 

pathogenesis, diagnostic procedures and therapeutic approaches to 

various inflammatory diseases. As a result of these advances, new 

drugs have been developed and made available under 

pathogenetically orientated aspects [1-4]. They may be opposed to 

drugs which are clinically used based on either empirical, partly 

only anecdotical knowledge of their effectiveness. Thus, current 

requirements of evidence based medicine for therapeutic 

approaches are often lacking. Following our advances in 

immunological knowledge, however, in many instances the mode of 

action of such drugs can now be explained at least in parts as 

exemplified for glucocorticosteroids or thalidomide. 

Inflammatory processes are resulting from the interaction of various 

cellular and humoral parameters and are based on either antigendriven 

or non-specific mechanisms. Among cellular components, 

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Systemic therapy with immunomodulatory drugs in dermatology 199 

apart from antigen-presenting cells and interacting lymphocytes of 

both T- and B-cell origin, various resident and migratory cells are 

involved to result in the tissue inflammatory reaction characteristic 

for the individual diseases. These interactive effects are mediated 

by cellular proteins like cell surface receptors and adhesion 

molecules. In addition to these parameters, cellular functions are 

modulated by various cytokines at both systemic and local level 

resulting in a network of inflammatory processes which is 

intricately regulated. 

Within modern concepts of inflammatory diseases, T-helper cell 

dysbalance has been addressed very extensively over the last years 

and resulted in the definition of TH1 and TH2 mediated 

responses.[1] A shift of immunological responses to either side is 

regarded as pathogenetically relevant. Resulting from these ideas, 

current therapeutic interventions are targeting such dysbalances by 

shifting the T-cell response with appropriate drugs. 

With these aspects in mind, immunomodulatory therapy can be 

defined as counteracting the dysbalances which can be found at the 

various steps of this immunological network (table 1). This may be 

effected by targeting pathogenic immune cells and their activation 

or by interfering with the cytokine network. The intention is to only 

target those elements which are pathogenetically relevant within the 

progression of the inflammatory response, however sparing other 

parts of the immune system which are necessary for normal 

immune responses. Currently available drugs fulfil these criteria to 

a variable extent by more or less specifically targeting single or 

multiple parameters. 

Following this concept, immunomodulatory drugs can be divided 

into those with prominent immunosuppressive or cytotoxic activity, 

those with both immunomodulatory and immunosuppressive and 

thirdly those with immunomodulatory activity only (table 2). As a 

result of current concepts of immunological dysbalance, the effects 

of modern immunomodulatory drugs are often referred to as 

immunodeviation, a term which describes the effects of the 

modulation of functional activity rather than physical eradication of 

the cells involved. Examples for each of these groups will be given 

within the context of this contribution without, however, special 

referral to individual diseases and specific therapeutic regimens. 

------------------------------------------------------------------------------------ 




Cellular targets Cell surface molecules Humoral targets 

T-cells Cell surface receptors 

(T-cell receptor, MHCantigens, 

costimulatory cell 

surface molecules) 

------------------------------------------------------------------------------------ 



Cytokines 

B-cells Adhesion molecules Chemokines 

Macrophages/Antigen-presenting 

cells 

Resident tissue cells 

(e.g.fibroblasts, endothelial 

cells) 

Other inflammatory mediators 

(e.g. lipid mediators, 

complement factors) 

Tab. 1: Cellular and humoral targets of immunomodulatory 

treatment 

Immunosuppressive /cytotoxic drugs 

• Cyclophosphamide 

• 

Immunomodulatory drugs with suppressive activity 

• Corticosteroids 

• Azathioprine 

• Methotrexate 

• Leflunomide 

• Mycophenolate mofetile 

• Ciclosporin 

• Macrolactames 

• UVB/PUVA 

Immunomodulatory dugs without suppressive activity 

• Fumaric acid derivatives 

• Intravenous immunoglobulins (IVIG) 

• Immunobiologicals 

- fusion proteins 

- monoclonal antibodies 

- cytokines 

Tab. 2: Systemic immunomodulatory drugs

Adjuvant drugs 


Different immunomodulatory agents are often referred to as 

adjuvant drugs which refers to their supportive or additive action 

when combined with corticosteroids. Due to their delayed clinical 

effects, they usually have to be combined with corticosteroids in the 

initial phase of the disease. In mild disease or after stopping steroids 

they may however be used as monotherapy. Apart from e.g. 

azathioprine, methotrexate and intravenous immunoglobulins which 

will be dealt with below, chloroquin and hydroxychloroquin are 

used as basic adjuvant drugs for the treatment of rheumatic 

disorders including their skin manifestations as well as lichen 

rubber [5-7]. Thalidomide has recently been reintroduced into 

clinical practice apart from lepra and is now used for chronic 

inflammatory, especially granulomatous skin diseases like 

cutaneous lupus erythematosus. Modulation of TNFα release from 

macrophages were shown to be a major activity exerted by this drug 

[8-11]. Similarly dapsone [12,13] and colchizine can very 

effectively used alone or in combination with corticosteroids by 

modulating cytokine release as well as leukocyte functions. The 

clincial effectiveness of antibiotics beyond their antibiotic activity 

underlines immunological effects and warrants their use as adjuvant 

drugs in diseases like bullous pemphigoid (tetracycline) and 

scleroderma (penicilline) [6]. 

Immunosuppressive drugs 

A representative of this group relevant within dermatology is 

cyclophosphamide which may be used in high dose and low dose 

regimens depending on both disease entity and activity [14,15]. It 

grossly interferes with immunological processes by targeting all 

proliferative cells. Among these, cells like T and B cells are 

involved in the inflammatory processes and should be the main 

target. Accordingly, a range of side effects is seen due to the 

involvement of other cells e.g. from the hematopoetical system and 

gastrointestinal tract. However, in many clincial cases, alternative 

drugs with equal effectiveness are missing and thus warrant the use 

of cyclophosphamide. 

------------------------------------------------------------------------------------ 




Immunomodulatory drugs with suppressive activity 

Allocation to this group can be made for glucocorticosteroids, 

azathioprine, methotrexate, ciclosporin and macrolactame 

antibiotics [1,4,16] as well as photochemotherapy regimens 

(PUVA) [17]. A gross range of effects on different cells apart from 

those immunologically relevant can be found for corticosteroids 

[14,18-20]. The resulting distinct side effects often restrict their 

short- and long-term use in certain patient groups. However, until 

today they are widely used as first-line treatment for diverse 

inflammatory disorders due to their timely effects. This is in 

contrast to other immunomodulatory drugs which may need several 

days to weeks to be effective. Accordingly, for dose-sparing and 

minimizing long-term side effects glucocorticosteroids are usually 

combined with other drugs which are referred to as adjuvant drugs 

and induce their effects by both immunosuppressive and nonimmunosuppressive 

action (see above). In cases of low disease 

activity or after tappering corticosteroids they may even be used as 

single drug. The classical immunosuppressive drug combined with 

corticosteroids is azathioprine [21,22] which is able to suppress 

lymphocyte activities by interfering with the DNA metabolism. The 

effects of methotrexate [23-25] at doses used in dermatology are 

beyond cytotoxicity. Functional modulation of lymphocyte activity 

by modulation of IL-1ß, IL-6 and TNFα-receptor expression as 

well as modulation of chemotaxis, migration and adhesion of 

polymorphonuclear leukocytes seem to be main effects [25]. 

Ciclosporin and macrolactame antibiotics like tacrolimus (FK506) 

and pimecrolimus represent drugs targeting distinct subsets of 

immunologically relevant cells [1-4]. They have been developed 

and extensively used in transplantation medicine before being 

introduced into dermatology for immunologically mediated 

diseases. Their effects have been pin-pointed to intracytoplasmic 

specific binding proteins which are part of the signaling cascade to 

interfere with cytokine expression at the the DNA level. Despite 

these very specific mechanisms of action, distinct side-effects on 

other cell systems and organs are seen and often restrict their 

clinical use. Though originally found within the group of 

macrolactame antibiotics, their immunological effects are beyond 

antibiosis. This highlightens possible effects on inflammatory and 

------------------------------------------------------------------------------------ 




immunological processes by other, classical antibiotics like 

penicilline, tetracyclines and minocycline which have been shown 

to modulate granulocyte and lymphocyte functions. Though their 

effectiveness in inflammatory processes is suggested by clinical 

experience, they have, however, been studied only to a limited 

extent. 

Another group of immunosuppressive drugs which before 

introduction into dermatology have extensively been used in other 

specialities are represented by mycophenolate mofetil (MMF) 

[26,27] and leflunomide [27-29]. Both interfere with DNA 

replication by antagonizing purin and pyrimidine metabolism 

respectively and thus modulate proliferation and functional activity 

of lymphocytes. MMF has already been successfully used in 

psoriasis vulgaris and autoimmune bullous skin diseases, especially 

bullous pemphigoid and pemphigus. Its comparability to established 

drugs in respect to efficacy is currently under evaluation in clinical 

studies. Both drugs do however represent alternatives in cases of 

poor response, contraindication or side-effects of established 

treatment modalities. 

Immunomodulatory drugs without suppressive activity 

Within this group, both novel and well-established drugs are found 

and include fumaric acid derivatives, cytokines, intravenous 

immunoglobulins as well as the expanding group of 

immunobiologicals. Apart from monoclonal antibodies, fusion 

proteins, receptor proteins and peptide fragments a number of 

agents are contained within this group which are still in 

experimental studies, do however indicate very interesting 

therapeutic perspectives. These drugs either induce effects 

indirectly by modulating the expression of certain cytokines or 

activation parameters. Alternatively, cytokines or proteinaceous 

mediators involved are directly applied as therapeutic agents 

themselves. 

Fumaric acid derivatives have so far been only used for 

dermatological diseases, mainly psoriasis [3,30,31]. Apart from 

inhibiting both lymphocyte proliferation and differentiation of 

dendritic cells mainly through NF-KB dependent processes, 

induction of apoptosis, a shift of TH1 responses characteristic for 

------------------------------------------------------------------------------------ 




psoriasis to TH2 responses could be demonstrated. These effects 

indicate the effectiveness of fumarates beyond psoriasis and will 

have to be demonstrated in the near future. 

Similarly, two compounds which have so far been used successfully 

in dermatology for local application, may be available soon for 

systemic application. Vitamin D derivatives [1,4] have been 

shown to modulate immunological parameters apart from 

keratinocye differentiation as does imiquimod [32] which induces 

different cytokines like interferons, IL-6, 8 and 10 when used 

locally. Systemic application of these compounds or derivatives 

with less pronounced side-effects beyond the indications studied so 

far in dermatology may be expected very soon. 

Different cytokines have been studied clinically for their 

effectiveness in dermatological diseases such as interferons and 

interleukins 2, 10 and 12 [1,2,16]. Their main action is regarded as 

deviating immunological dysbalances in either direction, of 

decreased or increased immunological responsiveness [33]. 

However, effectiveness has in many clinical studies been below that 

expected from earlier experimental data. 

Though intravenous immunoglobulins (IVIG) have successfully 

been used for diverse, pathogenetically very heterogenous diseases 

they are at the same time licensed only for a limited number of 

diseases [34-36]. This is reflected by a wide range of 

immunological and inflammatory effects which have been studied 

both in vitro and in vivo and are differentially involved within the 

different diseases. They include specific and non-specific effects on 

both humoral and cellular parameters like binding and inhibiting 

cytokines, adhesion molecules and complement factors or 

modulating macrophage and lymphocyte functions. The low rate of 

side-effects and good clinical effectiveness is counteracted by high 

costs and short-term effects. Currently, clinical use of IVIG is 

recommended only in combination with other immunomodulatory 

drugs [36]. 

The polyclonal mixture of antibodies against diverse antigens as 

contained in IVIG is reduced to single and defined antigens in 

preparations of monoclonal antibodies [37,38]. They are directed 

against proteinaceous structures which are involved in the 

pathogenesis of distinct diseases. Clinical experience shows that 

such antibody approaches is still not entirely successful either in 

------------------------------------------------------------------------------------ 




respect to total and long-standing clearance of disease or clearance 

in all patients. Possibly an “oligoclonal” mixture of defined 

antibodies will in the future increase the effectiveness of such 

regimens. Antibody and mediator approaches have been combined 

in some fusion proteins (LFA3-TIP or etanercept) and will have to 

be further evaluated in clinical studies. Long-term consequences on 

the immune system regarding incidence of tumors as well as 

allergological and immunological diseases have to be closely 

monitored as therapeutic approaches are made in chronic or 

recidivating diseases which may necessitate repetitive or constant 

long-lasting application of these drugs. 

Dissection of immunological parameters from a complex, 

multifactorial system in an experimental setting demonstrated both 

their relevance or irrelevance as well as their primary or secondary 

involvement within the pathogenesis of individual diseases. 

Experimental concepts which focus on single or few parameters 

regarded pathogenetically relevant have been established in basic 

immunology. Transfered to clinical immunology, many of these 

approaches have been disappointing in the complex system of 

human diseases. Based on these concepts, however, oligofactorial 

approaches by targeting a limited set of defined factors may in the 

future allow successful treatment of inflammatory diseases within 

dermatology and beyond. 

References 

1 Krueger JG (2002) The immunologic basis for the treatment of 

psoriasis with new biologic agents. J Am Acad Dermatol 46: 1- 

23 

2 Luger T (2001) Treatment of immune-mediated skin diseases: 

future perspectives. Eur J Dermatol 11: 343-347 

3 Mrowietz U (2001) Advances in systemic therapy for psoriasis. 

Clin Dermatol 26: 362-367 

4 Dutz JP, Ho VC (1998) Immunosuppressive agents in 

dermatology. An update. Dermatol Clin 16: 235-251 

5 Ochsendorf FR, Runne U (1996) Chloroquine: consideration of 

maximum daily dose (3.5 mg/kg ideal body weigth) prevents 

retinopathy. Dermatology 192: 382-383 

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6 Sapadin AN, Fleischmajer R (2002) Treatment of scleroderma. 

Arch Dermatol 138: 99-105 

7 Sticherling M (2001) Chronic cutaneous lupus erythematosus. 

In: Hertl M (ed) Autoimmune Disease of the Skin – 

Pathogenesis, diagnosis, management. Springer Wien, New 

York: 337-364 

8 Georgala S, Katoulis AC, Hasapi V, Koumantaki-Mathioudaki E 

(1998) Thalidomide treatment for hypertrophic lupus 

erythematosus. Clin Exp Dermatol 23: 141 

9 Kyriakis KP, Kontochristopoulos GJ, Panteleos DN (2000) 

Experience with low-dose thalidomide therapy in chronic 

discoid lupus erythematosus. Int J Dermatol 39: 218-222 

10 Ordi-Ros J, Cortes F, Cucrull E, Mauri M, Bujan S, Vilardell M 

(2000) Thalidomide in the treatment of cutaneous lupus 

refractory to conventional therapy. J Rheumatol 27: 1429-1433 

11 Warren KJ, Nopper AJ, Crosby DL (1998) Thalidomide for 

recalcitrant discoid lesions in a patient 

12 with systemic lupus erythematosus. J Am Acad Dermatol 39: 

293-295 

13 Glied M, Rico MJ (1999) Treatment of autoimmune blistering 

diseases. Dermatol Clin 17: 431-440 

14 Wozel G (1996) Dapson - Pharmakologie, Wirkmechanismus 

und klinischer Einsatz. Thieme Stuttgart, New York 

15 Becker L, Bastian B, Wesselmann U, Karl S, Hamm H, Bröcker 

EB (1998) Paraneoplastic pemphigus treated with 

dexamethasone/cyclophosphamide pulse therapy. Eur J 

Dermatol 8: 551-553 

16 Fleischli ME, Rachel H, Valek BS, Pandya AG (1999) Pulse 

intravenous cyclophosphamide therapy in pemphigus. Arch 

Dermatol 135: 57-61 

17 Thoma-Uszynski S, Hertl M (2001) Novel therapeutic 

approaches in autoimmune skin disorders. In: Hertl M (ed) 

Autoimmune Disease of the Skin – Pathogenesis, diagnosis, 

management. Springer Wien, New York: 337-364 

18 Gasparro FP (2000) Photodermatology: progress, problems and 

prospects. Eur J Dermatol 10: 250-4 

19 Werth VP (1993) Management and treatment with systemic 

glucocorticoids. Adv Dermatol 8: 81-101 

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20 Genant HK, Cooper C, Poor G, Reid I, Ehrlich G, Kanis J, 

Nordin BE, Barrett-Connor E, Black D, Bonjour JP, Dawson- 

Hughes B, Delmas PD, Dequeker J,Ragi Eis S, Gennari C, 

Johnell O, Johnston CC Jr, Lau EM, Liberman UA, Lindsay R, 

Martin TJ, Masri B, Mautalen CA, Meunier PJ, Khaltaev N 

(1999) Interim report and recommendations of the World Health 

Organization Task-Force for Osteoporosis. Osteoporos Int 10: 

259-64 

21 Joly P, Roujeau JC, Benichou J, Picard C, Dreno B, Delaporte E, 

Vaillant L, D'Incan M, Plantin P, Bedane C, Young P, Bernard 

P, The Bullous Diseases French Study Group (2002) A 

comparison of oral and topical corticosteroids in patients with 

bullous pemphigoid. N Engl J Med 346: 321-7 

22 Anstey A (1996) Management of immunobullous disorders: the 

clinical significance of interindividual variation in azathioprine 

metabolism. Clin Exp Dermatol 21: 247-248 

23 Tan BB, Lear TJ, Gawkrodger DJ, English JS (1997) 

Azathioprine in dermatology: a survey of current practice in the 

UK. Br J Dermatol 136: 351-355 

24 Jeffes EW 3 rd , McCullough JL, Pittelkow MR, McCormick A, 

Almanzor J, Liu G (1995) Methotrexate therapy of psoriasis: 

differential sensitivity of proliferating lymphoid and epithelial 

cells to the cytotoxic and growth –inhibitory effects of 

methotrexate. J Invest Dermatol 104: 184-188 

25 Bottomley WW, Goodfield MJ (1995) Methotrexate for the 

treatment of discoid lupus erythematosus. Br J Dermatol 133: 

655-656 

26 Cronstein BN (1996) Molecular therapeutics. Methotrexate and 

its mechanism of action. Arthritis Rheum 39: 1951-60 

27 Jayne D (1999) Non-transplant uses of mycophenolate mofetil. 

Curr Opin Nephrol Hypertens 8: 563-567 

28 Furst DE (1999) Leflunomide, mycophenolic acid and matrix 

metalloproteinase inhibitors. Rheumatology (Oxford) 38: 14-18 

29 Kurtz ES, Bayley SC, Arshad F, Lee AA, Przekop PA (1995) 

Leflunomide: an active antiinflammatory and antiproliferative 

agent in models of dermatologic disease. Inflamm Res 44: 187- 

188 

30 Nousari HG, Anhalt GJ (2000) Bullous pemphigoid treated with 

leflunomide. Arch Dermatol 136: 1204-1205 

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31 Altmeyer P, Matthes U,Pawlak F, Hoffmann K, Frosch PJ, 

Ruppert P (1994) Antipsoriatic effect of fumaric acid 

derivatives. Results of a multicenter double-blind study in 100 

patients. J Am Acad Dermatol 30: 977-981 

32 Mrowietz U, Christophers E, Altmeyer P (1999) Treamtent of 

severe psoriasis with fumaric acid esters: scientific background 

and guidelines for therapeutic use. Br J Dermatol 141: 424-429 

33 Sauder DN (2000) Immunomodulatory and pharmacologic 

properties of imiquimod. J Am Acad Dermatol 43: S6-11 

34 Thivolet J, Nicolas JF, Kanitakis J, Lyonnet S, Chouvet B 

(1990) Recombinant interferon alpha 2a is effective in the 

treatment of discoid and subacute cutaneous lupus 

erythematosus. Br J Dermatol 122: 405-409 

35 De Vita S, Ferraccioli GF, Di Poi E, Bartoli E, Bombardieri S 

(1996) High dose intravenous immunglobulin therapy for 

rheumatic diseases: clinical relevance and personal experience. 

Clin Exp Rheumatol 14: S85-92 

36 Jolles S, Hughes J, Whittaker S (1998) Dermatological uses of 

high-dose intravenous immunglobulin. Arch Dermatol 134: 80- 

86 

37 Sacher RA (2001) Intravenous immunoglobulin consensus 

statement. J Allergy Clin Immunol 108: 139-146 

38 Gelfand EW (2001) Antibody-directed therapy: Past, present and 

future. J Allergy Clin Immunol 108: 111-116 

39 Isaacs JD (2001) From bench to bedside: discovering rules for 

antibody design, and improving serotherapy with monclonal 

antibodies. Rheumatol 40: 724-738 

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Systemic Therapy with Recombinant Antibodies in Dermatology 209 

Systemic Therapy with Recombinant Antibodies 

in Dermatology 

M. Lüftl 


University Hospital of Erlangen 

Hartmannstr. 14, 

D-91052 Erlangen 

Germany 

Introduction................................................................. 209 

Recombinant antibodies – “tailor-made” therapeutics211 

Treatment of skin diseases with monoclonal antibodies 

- clinical studies .......................................................... 213 

Inflammatory skin diseases......................................... 213 

Neoplastic skin diseases ............................................. 214 

Limitations .................................................................. 215 

Outlook ....................................................................... 215 

References................................................................... 216 

Introduction 

In contrast to high-dose intravenous immunoglobulin therapy (a 

mixture of polyclonal antibodies) that unspecifically blocks the 

immune system, treatment with recombinant monoclonal antibodies 

leads to a selected deletion or inhibition of single targets. 

Antibodies, e.g. immunoglobulins mediate the humoral arm of 

specific immune responses. The primary functions of antibodies are 

to bind specifically to antigen and bring about the inactivation or 

removal of the offending protein, toxin, microbe or cell. All 

immunoglobulins have the basic structure of two heavy and two 

light chains structurally divided into domains (Fig. 1). Lytic 

antibodies mediate their effector functions after binding to the 

specific antigen via the Fab fragments by recruiting FcR-positive 

cells and/or complement components in turn to remove and/or 

destroy the target through opsonization, anitibody dependent 

------------------------------------------------------------------------------------ 



210 Systemic Therapy with Recombinant Antibodies in Dermatology 

cellular cytotoxicity (ADCC), or complement-mediated 

cytotoxicity. Non-lytic antibodies act by blocking the function of 

the target. 

Fv 

Fab 

Fc 

Fig. 1: Structure of an Immunoglobulin: Each immunoglobulin is 

structurally divided into domains of variable (VL, VH) or constant 

(CL, CH1-3) regions. The Fab region comprises VL/CL and 

VH/CH1, the Fc terminus comprises CH2/CH3. The actual antigenbinding 

site is formed by hypervariable loops (complementaritydetermining 

regions, CDR) in the Fv part (VH and VL) 

Attempts to an antibody therapy date back to the early 1900s in 

patients with cancer, but progress was stalled until discovery in 

1975 of a way to produce mouse monoclonal antibodies in vitro by 

hybridoma technology (14). These early attempts drew attention to 

the potential benefits and limitations of mouse anti-human 

monoclonal antibody therapy. Early trials with these antibodies in 

patients with cancer, however, found only short lived responses. 

Major reasons for the therapeutic failure were shortened circulating 

half-life, limited delivery to or penetration into tumor sites, 

inadequate recruitment of host leukocyte bearing constant region 

receptors (Fc) and, most important, production of neutralizing antimouse 

antibodies in treated patients. 

------------------------------------------------------------------------------------ 




Recombinant antibodies – “tailor-made” therapeutics 

During the past two decades, there has been progress in overcoming 

many of the above mentioned obstacles due to advances in 

molecular and protein biology techniques (Table 1). 

• Reduction of the immunogenicity so that antibody therapy can be 

used over extended periods 

• Addition of extrinsic effector functions or modification of intrinsic 

functions 

• Increase of antibody specificity, affinity and avidity 

• Increase of half-life 

• Generation of novel specificities, using phage antibody technology 

• Production of stable recombinant antibodies at high levels in a costeffective 

manner 

Tab. 1: Advantages of recombinant antibodies 

Genetic engineering offers the possibility to create “designer” 

therapeutics, since each antibody domain is encoded by different 

exons, and recombinant antibodies can be built by genetically 

fusing the desired exons together. Introducing nucleotide sequences 

of human immunoglobulin genes into the genes encoding mouse 

monoclonal antibodies results in “humanized” chimeric monoclonal 

antibodies containing human Fc region sequences, and antigenbinding 

region consisting of frameworks derived from mouse 

sequences (17). Since even humanized chimeric monoclonal 

antibodies often provoke antibodies that nullify the therapeutic 

benefit of engineered immunoglobulins, a way of making fully 

human antibodies has been sought and were found in recombinant 

DNA technology using bacteriophages containing a library for a 

vast number of variable regions (Fv) with high affinity for virtually 

any desired antigen. The modular arrangement of antibody domains 

(VH and VL bind antigen, while the Fc domains mediate effector 

functions) has allowed for a “mix and match” approach to designing 

antibody-based therapeutics (Table 2). Thus, functional domains 

------------------------------------------------------------------------------------ 




providing specific antigen-binding or effector functions can be 

exchanged between antibodies, expressed as separate units with 

biological activity (antibody fragments) or used as building blocks 

to construct novel fusion proteins (combination of an Ig with a 

toxin, a radionuclide or an effector protein) and bispecific 

antibodies (one arm of the Fab part binds the target, the other Fab 

arm binds the effector cell). 

Type Description and selected example 

Rodent monclonal Ab 

Chimeric 

“humanized” Ab 

Fully “humanized” 

Ab 

Ab fragments 

Fusion Proteins 

Bispecific Ab 

------------------------------------------------------------------------------------ 



Ig produced by rodent B-cells 

Not in clinical use any more 

Framework of the Ig human; variable 

region in part rodent 

Infliximab (anti-TNF-α) (4) 

Genetically engineered “human” Ig 

D2E7 (anti-TNF-α) (12) 

Neutralizing Ab fragments 

CDP870 (Fab anti-TNF-α) 

clinical trial, not yet published 

Combination of an Ig with a toxin, a 

radionuclide or an effector protein 

Etanercept (16) 

One arm of the Fab part binds the 

target, the other arm binds the effector 

cell. 

MDX-H210 (anti-HER2 x CD64) (11) 

Tab. 2: Antibody types and examples in clinical usage.


Treatment of skin diseases with monoclonal antibodies - 

clinical studies 

It was not the intention of the author to review the numerous 

clinical studies that have been performed with antibodies. Here are 

given examples that emphasize certain themes only. 

Inflammatory skin diseases 

Most data concerning systemic antibody therapy in dermatology are 

gained in psoriasis and psoriatic arthritis. Although no autoantigen 

has yet been defined in psoriasis, there is probably enough 

circumstantial evidence to categorise this disease as autoimmune in 

nature, and there is direct evidence of the involvement of T cells. 

Systemic antibody treatment approaches in psoriasis may be 

divided as either T cell targeting or cytokine modulating (Table 3). 

Most convincing in the treatment of severe psoriasis and proven by 

randomized placebo controlled double blind multicenter studies is 

blockade of the proinflammatory cytokine TNF-α with an 

impressive ability to substantially improve skin manifestation 

(including pustular lesions) and arthritis (4, 16, 18, 27). 

T cell targeting 

Cytokine modulating 

OKTcdr4a (anti CD4) (8) Infliximab (anti-TNF-α) (4) 

Efaluzimab (hu1124, anti CD11a) (7, 20) 

Daclizumab (anti CD25) (15) 

Alefacept (LFA-3/IgG1 fusion protein) (6) 

CTLA-4-Ig fusion protein (1) 

Etanercept (TNF-receptor fused 

with the Fc domain of human 

IgG1) (16) 

Tab. 3: Clinical studies using monoclonal antibodies in the 

treatment of psoriasis 

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Several other inflammatory, immune-mediated skin diseases have 

been shown to be receptive to antibody therapy including GvHD, 

vasculitis and Behcet’s disease (3, 22, 25) as well as autoimmune 

bullous disease (5). Case reports for CD4 Ab, CD40-ligand Ab, 

CTLA-4-Ig, complement-5 Ab (23, 26) indicated benefit in the 

treatment of systemic lupus erythematosus. 

An intriguing new treatment approach of allergic disorders twentyfive 

years after the discovery of immunoglobulin E offers a 

humanized, non-anaphylactogenic antibody against IgE. RhuMAb- 

E25 (omalizumab) is a novel anti-IgE antibody that is directed 

against the receptor-binding domain of IgE. This binding is specific 

towards free IgE thereby preventing it from attaching to the mast 

cell and its subsequent activation. Initial studies demonstrated 

attenuation of the early and late asthmatic responses when anti-IgE 

was administered to asthmatic subjects (2). 

Neoplastic skin diseases 

Progress in the treatment of cancer with antibody based therapies 

has been slower than for their application in immunosuppression. 

One reason is that immune reactivity against tumor cells requires 

more than blocking of receptor function, as the primary goal is cell 

death. 

Clinical trials have been performed using monoclonal antibodies to 

treat patients with cutaneous lymphomas. Two antibodies 

recognizing lymphocyte differentiation antigens have gained much 

attention in the recent years. One is the anti CD20 antibody 

rituximab which induced partial remission in patients with 

otherwise uncontrollable cutaneous B-cell lymphomas (9, 10). 

CD20 expression is usually retained in more than 90% of B-cell 

non-Hodgkin lymphoma, including cutaneous B-cell lymphoma. 

The other is CAMPATH-1H (anti CD52) which has been rarely 

used for B-cell lymphomas but more frequent in advanced T-cell 

lymphomas (19). 

In patients with advanced squamous cell carcinoma of the head and 

neck efficacy of a chimeric anti-epidermal growth factor receptor 

monoclonal antibody, cetuximab, in combination with radiation 

therapy has been reported (21) and some therapeutic and 

immunologic benefit following R(24) anti-GD3 monoclonal 

------------------------------------------------------------------------------------ 




antibody therapy was achieved in 37 patients with metastatic 

melanoma (13). 

Limitations 

Recombinant, humanized monoclonal antibody therapy can be 

regarded as safe resulting in only minor toxicity. However, 

anaphyllaxis, or other hypersensitivity type symptoms may require 

treatment break off. Since chimeric antibodies may induce 

neutralizing host antibodies, tachyphyllaxis has to be considered. 

Some antibodies with strong immunosuppressing properties like 

TNF blocking agents may induce exacerbation of infections. 

Special attention has to be devoted to intracellular bacteria 

(tuberculosis, listeriosis) or fungal infections. CAMPATH-1H 

induced rapid depletion of both B cells and T cells, resulting in an 

even more profound immunosuppression. Interestingly, patients 

with actinic skin damage developed invasive squamous cell 

carcinomas shortly after initiation of anti-TNF therapy (24), 

although clinical trials preceding approval by the Food and Drug 

Administration did not show any increase in the incidence of 

malignancies. 

Outlook 

More than 25 years after the revolutionary description of 

monoclonal antibody production via hybridoma technology, 

monoclonal antibodies have fully entered the clinical arena as new, 

unique, and important components of the medical armamentarium 

for the treatment of a variety of diseases. In fact, some of these 

antibodies can be regarded as major breakthroughs in the treatment 

of such disorders as psoriasis/psoriatic arthritis and non-Hodgkin 

lymphoma. It is virtually certain that this initial set of agents is but 

the first wave of what will become a broad array of therapeutics. 

Continuous improvement of the antibody design or creation of 

small-molecule mimetics (for example single chain Fv fragments) 

with similar activity will soon permit even better clinical results. 

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References 

1 Abrams JR, Kelley SL, Hayes E, Kikuchi T, Brown MJ, Kang S, 

Lebwohl MG, Guzzo CA, Jegasothy BV, Linsley PS, Krueger 

JG (2000). Blockade of T lymphocyte costimulation with 

cytotoxic T lymphocyte-associated antigen 4-immunoglobulin 

(CTLA4Ig) reverses the cellular pathology of psoriatic plaques, 

including the activation of keratinocytes, dendritic cells, and 

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220 Current state and perspectives of immunomodulation of the allergic contact dermatitis 

Current state and perspectives of 

immunomodulation of the allergic contact 

dermatitis 

H.-D. Göring 

Hautklinik und Immunologisches Zentrum 

Kühnauer Str. 24 

D-06846 Dessau 

Germany 

Current state and perspectives of immunomodulation of 

the allergic contact dermatitis ..................................... 220 

References................................................................... 224 

The immunomodulating therapy of the allergic contact dermatitis 

includes the conservative topical and systemic glucocorticosteroid 

application. Systemic immunosuppression, desensitization and 

tolerance induction are only in the early stage of their introduction 

in clinical practice and are studied above all in animals 

experiments. Glucocorticoids influence - during concrete case of 

illness – the existing inflammatory process of allergic contact 

dermatitis by their vasoconstrictoric, anti-inflammatory, 

cellmembran-stabilising and anti-proliverative effects. Because of 

their immunomodulating effects they slow down the further 

immunologically steered disease process (table 1). Potent effective 

topically applied glucocorticoids like clobetasol propionate inhibit 

the DNCB-contact dermatitis on pigs, while the less potent 

flucocinolone acetonide shows no effect [17]. Different 

glucocorticoids have different points of attack. Hydrocortisone 

leads, after the stimulation of antigen in vitro, to a reduced 

expression of MHC class II molecules and of the costimulatory 

molecules B 7.1 and B 7.2 as well as of the Langerhans cell (LC)specific 

CD 83 and to IL-12 secretion inhibition and reduced T cell 

proliferation [3]. Beta-methasone valerate under comparable 

experimental conditions supports the expression of MHC class II 

molecules and costimulatory molecules B 7.1 and B 7.2 as well as 

IL-2R (CD25) on the LC, but disturbs their allostimulatory activity 

and inhibits the dendritic cell (DC)-40- as well as the MHC class I 

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Current state and perspectives of immunomodulation of the allergic contact dermatitis 221 

molecule-expression [19]. The responsible contact allergen should 

be identified by epicutaneous testing and if possible should be 

avoided (allergen avoidance). A desensitization (hyposensitization) 

might be considered if the avoidance of the contact allergen is 

impossible. Experiments of desensitization have a long history. In 

1946 Chase [5] achieved a temporary gradual subsiding of DNCBcontact 

dermatitis if DNCB was fed to appropriately sensitized 

guinea pigs. In 1968 Polak and Turk [20] only succeeded in this in 

animal experiments about chromate allergy after feeding subletal 

doses of chromate. Among patients with nickel contact allergy a 

desensitization with 5 mg nickel sulfate once a week for a period of 

7 weeks led to a reduction of the number of circulating 

lymphocytes, which reacted against nickel. Skin tests and the 

clinical appearance remained unchanged [2]. 

Among guinea pigs with a contact allergy against urushiol (poison 

oak/ivy) only a combination of oral and epicutaneous allergen 

application led to desensitization. Under exclusive oral application 

the desensitization was incomplete and under exclusive 

epicutaneous application fully failed [16]. Parthenium 

hysterophorus is the most common reason for airborne contact 

dermatitis in northern India. Oral application led in 70% of the 

patients to a clinical improvement, in 30% it led to an exacerbation 

[13]. Belvisto [4] comes to the conclusion that according to current 

state of knowledge desensitization is no alternative to conventional 

therapy of allergic contact dermatitis. In animal experiments it is 

possible to achieve tolerance to nickel by oral application before 

percutaneous sensitization with nickel compounds [25]. A long time 

tolerance however requires a continuous oral or intraperitoneal 

NiCl(2)-application. By spleen- and lymph node cells of orally 

tolerized donors tolerance is transferable. The donors showed no 

increased IL-2 production. A persisting suppressor cell activity is 

assumed [1]. These findings support the clinical observation that the 

oral intake of nickel from tooth braces among children could have 

led to a lower sensitization rate against nickel by later earpiercing 

than for juveniles who did not have any tooth brace treatment [15]. 

Cyclosporin A causes an inhibition of the transcription of IL-2, IL- 

3, IL-4 and other pro-inflammatory cytokines that lead to a 

retardation of the auto- and parakrine stimulation of T-lymphocytes 

and macrophages [6]. Besides it also leads to an inhibition of the 

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Fas ligands mediated Keratinocyte (KC)-apoptosis. This apoptosis 

is caused in the allergic contact dermatitis by IFN-gamma from 

activated T-cells. IFN-gamma supports the expression of Fas 

ligands on the KC-surface and makes them sensitive for apoptosis. 

The same effect on the suppression of apoptosis - in animal 

experiments - have intravenous injected immunoglobulines (IVIG) 

as well as dexamethasone and the macrolides rapamycin and 

tacrolimus, the later by the systemic and topical application [24]. 

Under systemic application cyclosporin A and tacrolimus suppress 

the early mastcell-dependent and late inflammatory phase of the Tcell 

mediated contact sensitization. However the topical application 

of cyclosporin has no effect, while tacrolimus suppresses the early 

phase more clearly than the late phase [10]. Tacrolimus suppresses 

the allergic contact dermatitis in animal experiments and for human 

beings. Applicated in form of oinments on the skin, the expression 

of IL-2R, B 7.1, CD40 and MHC class II and I molecules is 

suppressed, not however of B 7.2. Only freshly from the skin 

isolated LC exprime the FK 506 (tacrolimus) binding protein 

(FKBP) [10]. During the LC maturing process in the lymph node it 

is lost. Under topical application tacrolimus suppresses the 

stimulating function 100 times more than beta-methasone valerate 

[19]. In different clinical studies tacrolimus has proven its efficacy 

against the allergic contact dermatitis of human beings [18]. 

A new immunosuppressive agent is the macrolid ascomycin. The 

ascomycin macrolactam derivative SDZ ASM 981 suppresses in 

vitro the proliferation of T cells after antigen stimulation, regulates 

the Th1-cytokine production (IL-2,IFN-gamma) and the Th2cytokine 

production (IL-4, IL-10) down after antigen stimulation 

and suppresses the liberation of pre-modulated anti-inflammatory 

mediators out of mast cells [12]. ABT-281, a macrolactam 

ascomycin analogon inhibits likewise the synthesis of cytokines of 

Th1 and Th2 cells and suppresses under topical application as a 0,3 

and 1 per cent solution in aceton and olive oil the DNCB-induced 

allergic contact dermatitis on the pig up to 90% in comparison to 

50% under topical application of the potent glucocorticoid 

clobetasol propionate [17]. Besides contact allergenes UVB-light 

also induces activated KC to IL-10 production [22]. This probably 

explains why it comes to an antigen specific tolerance instead of a 

contact sensitization in animal experiments after applying a strong 

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contact sensitizating substance onto a skin area which was only 

shortly before irradiated with UVB. IL-10 also inhibits the IFNgamma-mediated 

macrophage activation and the IL-12 production 

by macrophages. This suppresses the type IV reaction [22]. 

IL-10 inhibits the expression of the costimulatory molecules B 7.1 

and B 7.2 on LC. Is the costimulatory signal missing, it does not 

come to a clonal activation but to clonal inactivation of the T cells 

the so-called clonal anergy. Besides it comes to a check of IL-12 

production and to reduced T cell proliferation. IL-10 contribution 

before antigen stimulation leads to an inhibition of the Th1- and 

Th2-cytokine production [3]. Because of that LC lose their ability 

to stimulate Th1 cells. In the end the cytokine pattern of the 

epidermis is changed in such a way that pro-inflammatory 

regulation circuits are interrupted and a sensitization can no longer 

take place. Therefore IL-10 changes potent immunostimulating LC 

into tolerance introducing LC [22]. Epidermal nerves can be found 

in anatomical connection to LC. They contain the neuropeptide 

calcitonin gene-related peptide (CGRP). The local application of 

CGRP leads to an moderation of the contact sensitization. CGRP 

inhibits the expression of the costimulatory molecule B 7.2 on LC 

[23]. The antigen presenting function of the LC is regulated down 

by CGRP. UV-irradiation leads to a reduction of the LC-density in 

the epidermis. Beyond this UV-irradiation leads to a CGRP 

liberation out of sensoric neurons and supports together with NO 

the local immunosuppression. The CGRP content of the epidermis 

decreases about 2 hours after the irradiation (UV-dosis 0,5-2,0 

J/cm²) and reaches its minimum 6-12 hours later [11]. A further, 

however not yet applied possibility to check the allergic contact 

dermatitis is represented in the neuropeptide vasoactive intestinal 

peptide (VIP) and the structural related pituitary adenylate cyclase 

activating polypeptide (PACAP). They inhibits the expression of 

the costimulating molecules B 7.1 and B 7.2, the TNF-alpha, IL-12 

and IL-10 production [9]. The biochemical produced 

intramembraneous amino-acid sequence of the T-cell receptor chain 

(TCR mimic peptides) inhibits the interaction between Tlymphocytes 

and LC. In vitro it comes to the suppression of CD4+ 

and CD8+ cell-proliferation. TCR-mimic peptides block, under 

topical application, the ear swelling in mice as an expression of the 

failing contactallergic reaction, if chronologically applicated before 

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the contact allergen. TCR-mimic peptides neutralize the T cell 

mediated immunoreaction [8]. 

Further immunological possibilities exist to intervene in a highly 

specific way in the induction phase of the type IV reaction. Thus, 

the activity of IL-1 beta can be neutralized by monoclonal 

antibodies and hence the further course of the sensitization is 

prevented [7]. Antibodies against IL-1 and TNF-alpha inhibits their 

production and liberation in the skin, whereby a sensitization 

against haptene is impeded or prevented [14]. 

Natural IL-1R-antagonists bind on the IL-1R (receptor block) by 

which the receptor released signal and hence the forming of 

cytokine is prevented. The use of receptor antagonists is equally 

imaginable than this of soluble receptor molecules, which intercept 

the cytokine before it binds to the receptor. Here already exists a 

prototype of the soluble TNF-alpha-receptor. In vitro-models of 

specific cytokine-antagonists, transgene experimental animals, 

which exprime certain cytokines and others, whose certain 

cytokines are switched off (“gene-knock-out”-technology), already 

exist [21]. It is interesting that IVIG contains a series of factors 

which can interfere in the antigen presentation and the sensitization 

process as well as the release phase of type IV reaction: CD4- and 

MHC class II molecules, antibodies against TCR, IL-1 as well as 

TNF-alpha and MHC class II molecules, IL-1R-antagonists, soluble 

TNF-alpha receptors. IVIG can regulate pro-inflammatory 

cytokines down and check the Fas ligands mediated apoptosis [26]. 

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Tolerance to nickel: oral nickel administration induces a high 

frequency of anergic T cells with persistent suppressor activity. 

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2 Bagot M, Terki N, Bacha S, Moyse D, Suck C, Revuz J (1999) 

Per os desensitization in nickel contact eczema: a double-blind 

placebo-controlled clinico-biological study. Ann Dermatol 

Venereol 126: 502-504 

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3 Bellinghausen I, Brand U, Steinbrink K, Enk AH, Knop J, 

Saloga J (2001) Inhibition of human allergic T-cell responses by 

IL-10-treated dendritic cells: differences from hydrocortisonetreated 

dendritic cells. Allergy Clin Immunol 108: 242-249 

4 Belsito DV (2000) The diagnostic evaluation, treatment, and 

prevention of allergic contact dermatitis in the new millennium. 

Allergy Clin Immunol 105: 409-420 

5 Chase MW (1946) Inhibition of experimental drug allergy by 

prior feeding of the sensitizing agent. Proc Soc Exp Biol Med 

61: 257-259 

6 Diasio RB, Lo Buglio, AF: Immunmodulators: 

immunsuppressive agents and immunstimulants. In: Hardman, 

JG, bid,LE, Molinoff, PB, Ruddori RW, Goodman Gilman A 

(eds.) Goodman & Gilman’s The pharmacological basis of 

therapeutics. edn 9. New York Mc Grawhil1996, p. 1291-1308 

7 Enk AH, Katz SI (1992) Early molecular events in the induction 

phase of contact sensitivity. Proc Nat Acad Sci 89: 1398-1402 

8 Enk AH, Knop J (2000) T cell receptor mimic peptides and their 

potential application in T-cell-mediated disease. Int Arch 

Allergy Immunol 123: 275-281 

9 Ganea D, Delgado M (2001) Neuropeptides as modulators of 

macrophage functions. regulation of cytokine production and 

antigen presentation by VIP and PACAP. Arch Immunol et 

Therapiae Exp 49: 101-110 

10 Geba GP, Ptak W, Askenase PW (2001) Topical tacrolimus and 

cyclosporin A differentially inhibit early and late effector phases 

of cutaneous delayed-type and immunoglobulin E 

hypersensitivity. Immunology 104: 235-242 

11 Gillardon F, Moll I, Michel S, Benrath J, Weihe E, Zimmermann 

M (1995) Calcitonin gene-related peptide an nitric oxide are 

involved in ultraviolet radiation-induced immunosuppression. 

Eur J Pharmacol 293: 395-400 

12 Grassberger M, Baumruker T, Enz A, Hiestand P, Hultsch T, 

Kalthoff F, Schuler W, Schulz M, Werner FJ, Winiski A, Wolff 

B, Zenke G (1999) A novel anti-inflammatory drug, SDZ ASM 

981, for the treatment of skin disease: in vitro pharmacology. Br 

J Dermatol 141: 264-273 

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13 Handa S, Sahoo B, Sharma VK (2001) Oral hyposensitization in 

patients with contact dermatitis from Parthenium hysterophorus. 

Contact Dermatitis 44: 279-182 

14 Hauser C (1992) Current topics in Immunodermatology. 

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15 Hoogstraten IMW, Andersen KE, von Blomberg BME et al. 

(1991) Reduced frequency of nickel allergy upon oral nickel 

contact at an early age. Clin Exp Immunol 85: 441-445 

16 Ikeda Y, Yasuno H, Sato A, Kawai K (1998) Oral and 

epicutaneous desensitization in urushiol contact dermatitis in 

guinea pigs sensitized by 2 methods of different sensitizing 

potency. Contact Dermatitis 39: 286-292 

17 Mollison KW, Fey TA, Gauvin DM, Kolano RM, Sheets MP, 

Smith ML, Pong M, Nikolaidis NM, Lane BC, Trevillyan JM, 

Cannon J, Marsh K, Carter GW, Or YS, Chen YW, Hsieh GC, 

Luly JR (1999) A macrolactam inhibitor of T helper type 1 and 

T helper type 2 cytokine biosynthesis for topical treatment of 

inflammatory skin diseases. J Invest Dermatol 112: 729-738 

18 Nasr IS (2000) Topical tacrolimus in dermatology. Clin Exp 

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19 Panhans-Gross A, Novak N, Kraft S, Bieber T (2001) Human 

epidermal Langerhans‘ cells are targets for the 

immunosuppressive macrolide tacrolimus (FK506). J Allergy 

Clin Immunol 107: 345-352 

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administration of sensitizers in guinea pigs with contact 

sensitivity to inorganic metal compounds. I. The induction of 

immunological unresponsiveness in already sensitized animals. 

Clin Exp Immunol 3: 245-251 

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Venerologie. Lehrbuch und Atlas. Springer, Berlin Heidelberg, 

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Brocker EB, Blaser K, Addis CA (2001) Targeting keratinocyte 

apoptosis in the treatment of atopic dermatitis and allergic 

contact dermatitis. J Allergy Clin Immunol 108: 839-846 

25 Van der Burg CKH, Bruynzeel DP, Vreeburg KJJ et al. (1986) 

Hand eczema in hairdressers and nurses: a prospektive study. 

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228 Therapy of Immediate Type Reactions 

Therapy of Immediate Type Reactions 

W.Ch. Marsch 

Universitätsklinik und Poliklinik für Dermatologie und Venerologie 




Germany 

Therapy of Immediate Type Reactions....................... 228 

References................................................................... 234 

An allergic immediate-type reaction (Type I, Gell and Coombs) is 

an acute inflammation in preexisting sensitization. It is usually 

elicited locally or regionally at the site of allergen contact by 

mediators of sessile, allergen-specific immunoglobulin E-bearing 

mast cells and the functions of secondary immigrating eosinophile 

granulocytes. This may result primarily or even secondarily in a 

systemic effect of released mediators up to and including shock 

symptoms of life-threatening character (anaphylaxis). The 

immediate-type reaction is essentially determined initially by the 

dominating preformed and stored mast-cell mediator histamine 

via acute release by degranulation. The allergic pathomechanism is 

based on a coating established on the mast-cell surface by allergenspecific 

IgE-molecules, which elicit the signal to degranulation of 

the mast cells by bridging of two such molecules with a specific 

antigen, such as the birch pollen epitope. Mast cell degranulation 

and thus release of the preformed mediator histamine determines 

the early phase of the Type-I allergic reaction, which begins 

seconds or at the latest 30 – 60 minutes after allergen contact. The 

visible reaction of the skin is characterized by a dermis/coriumedema 

(wheal), erythema (flare) and localized irritation (itch), that 

is, Urticaria. In addition to mast cells, the - in its nature similar - 

basophilic leukocytes in blood circulation are also involved. Mast 

cell degranulation leads to synthesis in the membrane of secondary 

mast cell mediators, such as tryptase, leukotrien C4, prostaglandin 

D2, serotonin, thromboxan A2, and particularly the platelet- 

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Therapy of Immediate Type Reactions 229 

activating factor (PAF) and the tumor-necrosis-factor-alpha. The 

former are essentially proinflammatory mediators with vascular 

activity, like especially the dilatation-eliciting serotonin. PAF and 

TNF-alpha act as chemo-attractors for eosinophilic granulocytes, 

which mature in bone marrow then enter the blood-vascular system 

and migrate to the site of mast cell degranulation at postcapillary 

venules into the area of acute inflammation. This secondary 

immigration of eosinophilic granulocytes determines the late 

phase of the Type-I allergic reaction, which predominates about 

6 to 24 hours after specific antigen contact. The eosinophilic 

granulocyte contains preformed basic proteins (dominant: major 

basic protein, MBP) as well as eosinophilic cationic protein (ECP), 

further eosinophil-derived neurotoxin (EDN), also enzymes like 

arylsulfatase for breakdown of leukotrien C4, and histaminase 

(breakdown of histamine), phospholipase (breakdown of PAF), also 

collagenase and myeloperoxidase, but no lysozyme. Essential is the 

realization that specific cytokines (such as Interleukin 3, Interleukin 

5, GM-CSF) lead to longer lifetime (persistence) of the eosinophilic 

granulocytes (normal: 2 – 3 days, here possibly up to 14 days), 

while on the other hand leading to activation of their effector 

functions by increasing the formation of oxygen radicals and lipid 

mediators secondarily formed on the cytomembranes. In inflamed 

tissue, longer-lived and functionally-active eosinophilic 

granulocytes are thus present. The type-1 late-phase is 

pathophysiologically characterized by erythematous swelling, in the 

nasal mucosa by blockage of nasal respiration (nasal congestion). 

Which such Type I-Allergy diseases are dermatologically more or 

less relevant? These are diseases oriented to the target organs skin 

and mucosa and their occupation with mast cells: on the external 

skin (integument) within the upper layer of the dermis (corium), the 

conjunctiva of the eyes, the mucosae of the nasopharynx, the 

oropharynx, the larynx, the bronchial epithelium and the 

subepithelial tunica propria of the gastrointestinal tract. Allergy 

refers to a state of existing sensitization or sensitizations to one or 

more allergens, while the condition “Allergy” and additionally 

clinical signs of inflammation form the basis for an allergic 

disease. Measured on the different target tissues and organs cited, 

the following relevant Type I allergic diseases are the central focus 

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of our diagnostics (mostly allergen identification) and multibased 

therapy modes (allergen withdrawal, antiallergic 

pharmacotherapeutics, hyposensitization): 

Allergic Rhinoconjunctivitis (seasonal: mostly pollen; perennial: 

mostly in-door allergens like dust mites and animal epithelia). The 

rhinitis can manifest as rhinorrhoe (“runners“), “sneezers“ or 

blocked nasal respiration (“blockers“). The development of chronic 

sinusitis and/or bronchial asthma (“level change”) may be promoted 

by repetitive or persistent allergic inflammation (“allergic 

cascade“). 

Asthma bronchiale allergicum (usually elicited by animal 

epithelia, but also by occupational exposition to aeroallergens, 

seasonal also “concurrent asthma“ in pollen-related 

rhinoconjunctivitis), 

Urticaria, particularly the special form: Contact urticaria of the oral 

mucosa (“oral contact urticaria syndrome“ to kernal and stone fruits 

among patients allergic to birch pollen). Here, evidence that a 

urticaria can be elicited non-immunologically by non-Ig-Emediated 

mast cell degranulation is important (=pseudo-allergic 

reaction/intolerance reaction, e.g. to acetylsalicylic acid and other 

analgesics, food colorants, aromas, dextrane). The illness category 

“Urticaria“ includes a number of clinically different diseases (such 

as diverse physical forms of urticaria, including chronic-recurrent 

urticaria with a broad spectrum of eliciting, often even unknown 

causes). Frequently histamine is the dominant mediator, but some 

urticaria forms are not infrequently refractory to substances with 

only an antihistamine effect, and thus apparently characterized by a 

different spectrum of mediators. 

Immunological Food Intolerances (Food allergy) of the intestinal 

mucosa with the predominant symptoms of increased intestinal 

motility and diarrhea. Here, too, clinically identical pseudoallergic 

mechanisms are frequent, due to food-dependent histamine excess 

and intolerances. 

Hymenopter-toxin Allergy (wasps, bees), which is associated with 

a considerable risk of death, is important and there are 

exceptionally good therapeutic-preventive possibilities. 

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The allergological therapy must first examine in connection with 

the history and possibly targeted allergen exposition 

(rhinomanometry, bronchial provocation test) which of the 

sensitizations determined in the test procedures is currently acute 

for the patient (relevance testing), and can thus be considered the 

cause of the defined allergic disease. This is the basis for attempts 

at allergen avoidance. Hyposensitization (Hymenopter toxins, 

pollen, dust mites, animal epithelia) is considered to be a 

therapeutic method with very definite prophylactic effect. 

Pharmacotherapy with defined antiallergic Substances focuses 

on the following therapeutic goals: 1. Mediator-receptor blockade 

for histamine and leukotriens, 2. “Mast cell stabilization“ (release 

of histamine becomes more difficult) and reduction of secondary 

de-novo-synthesis of lipid mediators, 3. Blocking the immigration 

of eosinophilic granulocytes (“Eosinophilic Migration“). 

1. Histamine-1-Receptor Antagonists: The first generation 

antihistamines had a low receptor selectivity, namely a broadened 

spectrum of receptor binding, and thus acted on muscariniccholinergic, 

alpha-adrenergic, serotoninergic and tryptaminergic 

receptors, and because they are lipophil and pass the blood-brain 

barrier, also had an effect on intracerebral H-1-receptors. The 

pharmacological advances in antihistamines of the second 

generation are due to improved receptor selectivity (minimization 

of side effects, especially great reduction or lack of sedation), the 

receptor affinity (reduction of both dose and administration 

intervals) and hydrophilic character of the active substances 

(reduction of passage through the blood-brain barrier). 

Several examples of preparations of the 2 nd generation and their 

active metabolites (3 rd generation) with the main indications 

urticaria (acute and chronic-recurrent forms, some physical urticaria 

forms, such as sweat urticaria/cholinergic urticaria) and allergic 

rhinoconjunctivitis are given below: 

Fexofenadin (Telfast®): a further development of Terfenadin 

(Teldane®) has no cardiotoxic risk (QT-prolongation, ventricular 

fibrillation in AV-block patients). 

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Mizolastin (Zolim®): the substance has additional mast cellstabilizing 

and antiinflammatory/leukotrien antagonistic effects. It 

inhibits 5-lipooxygenase and reduces the afflux of eosinophilic 

granulocytes and thus the intensity of the late-phase reaction. 

Cetirizin (Zyrtec®,Alerid®): The substance is a mixture of 

stereoisomers (racemate), accumulates rapidly in tissues following 

oral administration (onset of action after 15-20 minutes) and has a 

special partial effect on eosinophilic granulocytes. Cetirizin 

apparently blocks their transmural cell passage at the selectin level 

of the endothelial adhesion molecule cascade and thus the 

eosinophil immigration into the inflammatory interstitial tissue. It 

also reduces the allergen-related ICAM-1 expression on epithelial 

cells of the rhinopharynx and thus the allergic inflammation 

cascade. The favorable prophylactic-preventive long-term action of 

reduction of secondary asthma development by Cetirizin in atopic 

children may possibly be based on this (Early Treatment of the 

Atopic Child :ETAC Study). 

Levocetirizin (Xusal®) is a new development which represents the 

active L-enantiomer of Cetirizin. The stereoisomeric mirror-image 

D-(dextro)-enantiomer is, by contrast, not active. Levocetirizin is a 

very selective and long-acting antagonist of peripheral H1receptors, 

which is twice as potent as the Cetirizin racemate. It can 

be given in a lower daily dose (5 mg vs. 10 mg Cetirizin) and is still 

found to be more effective than its “predecessor“ with respect to 

clinical items like reduction of the wheal area. 

One very important advantage of both substances is their almost 

completely unchanged renal excretion and in any event their lack 

of hepatic metabolization via the Cytochrome-P-450-Isoenzyme 

system. This is why there is no medication interaction with danger 

of, for example, accumulation. 

Desloratadin (Aerius®), the active metabolite of the precursor 

preparation Loratadin(Lisino®), has an even greater affinity than 

the precursor for peripheral H1-receptors and increased selectivity, 

which is why sedation and anticholinergic effects are entirely 

missing. Neither substance is metabolized via the CYP3A4isoenzyme, 

but alternatively via the isoenzyme CYP2D6, it has 

(unlike the very potent antihistamine Ebastin/Ebastel®, for 

example) no risk of interaction with active substances like 

erythromycin and ketoconazol with the consequence of cardiac 

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arrhythmias. Both also have a broad antiinflammatory effect. 

Desloratadin inhibits the release of inflammatory mediators from 

mast cells after stimulation of degranulation: except preformed 

histamine, tryptase, prostaglandin D2 and leukotrien C4, especially 

interleukines 3,6,8 and TNF-alpha. 

The following are cited as special cases: 

Levocabastin (Livocab®) and Azelastin (Allergodil®) are topically 

applicable as eyedrops and nasal spray, are fast-acting substances 

with very low resorption, and have thus very little systemic effect. 

Doxepin is a tricyclic antidepressive with potent H-1-receptor 

affinity and is applicable for serious physical forms of urticaria, e.g. 

cold urticaria. A topical application form has recently become 

available (Xepin®-Creme). 

2. Leukotrien-Receptor Antagonist: Montelukast (Singulair®). 

This substance blocks cysteinyl-leukotriens, also reduces the 

immigration of eosinophilic granulocytes in a histamine-dominated 

inflammation area. The substance can be used in combination with 

potent H-1 receptor antagonists, probably has a special indication 

for non-allergic physical forms of urticaria, which usually have a 

different mediator spectrum than conventional urticarial, histaminedominated 

inflammatory reactions. Montelukast is especially 

suitable for inflammations with eosinophile-rich tissues thanks to its 

inhibition of eosinophile migration. 

As a substance with anti-inflammatory action, Montelukast is used 

these days in long-term combination therapy (beta-2sympathicomimetics, 

glucocorticoids) in Asthma bronchiale where 

it is replacing the less effective cromons (DNCG,Neocromil), 

which, like ketotifen (Zaditen®) stabilize mast cells. 

The pharmacological trend has thus brought forth a broad palette 

of active substances with the following specific pharmacodynamic 

properties for optimized therapy of clinically variable and targetorgan-specific 

allergic immediate-type reactions: 

1. very strong-binding and receptor-specific Histamine-1 receptorantagonists 

with only slight or even no sedative side effects, 

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2. their additional potency with respect to “mast cell stabilization” 

including in the sense of reduction of the secondary de-novosynthesis 

of inflammatory lipid mediators and inhibition of the 

secondary immigration of eosinophilic granulocytes with 

proinflammatory action, 

3. antagonists of other mediator receptors, for example with respect 

to leukotriens (Montelukast), 

4. rapid and effective action of topical H-1-receptor antagonists on 

the nasal mucosa and ocular conjunctiva, 

5. non-metabolizing active substances (Cetirizin, Levocetirizin) 

with no risk of drug interaction or metabolization via an 

alternative p-450-isoenzyme (Loratadin, Desloratadin) with no 

critical interaction with azoles and macrolid-antibiotics, 

6. greater administration comfort (1x pro die ) thanks to longlasting 

action but with no essential danger of accumulation. 

Modern therapy of allergic immediate-type reactions in organvariant 

disease patterns thus makes use of potent active substances 

with antihistamine and anti-inflammatory properties and a favorable 

risk/benefit ratio. 

References 

1 Ellis AK, Day JH (2000) Second- and third-generation 

antihistamines. Dermatol Therapy 13:327-336. 

2 Kroegel C, Mock B, Reissig A, Hengst U, Machnik A, Henzgen 

M (2001) Therapie des Asthma bronchiale im Erwachsenenalter. 

Z ärztl Fortb Qualsich (ZaeFQ) 95:699-706. 

3 Llanes S, Grant A (2000) Comparison of the potency of 


4 Passalacqua G, Bousquet J, Bachert C, Church MK et al (1996) 

The clinical safety of H1-receptor antagonists. An EAACI 

position paper. Allergy 51:666-675. 

5 Sabroe RA, Greaves MW (2000) Chronic idiopathic urticaria 

and its management. Dermatol Therapy 13:384-391. 

6 Salmun LM (2002) Antihistamines in late-phase clinical 

development for allergic disease. Expert Opin Investig Drugs 

11:259-273. 

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7 Simons FE, Simons KJ (1999) Clinical pharmacology of new 

histamine H1 receptor antagonists. Clin Pharmacokinet 36:239- 

352. 

8 Spencer CM, Faulds D, Peters DH (1993) Cetirizine. A 

reappraisal of its pharmacological properties and therapeutic use 

in selected allergic disorders. Drugs 46:1055-1080. 

9 Walsh GM (2000) The anti-inflammatory effects of the secondgeneration 


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236 In vitro Models for the detection of immunomodulating properties of pharmaceuticals 

In vitro Models for the detection of 

immunomodulating properties of pharmaceuticals 

G. Wichmann, I. Lehmann 

Junior Research Group Environmental Immunology 

Department of Human Exposure Research and Epidemiology 

UFZ-Centre for Environmental Research Leipzig-Halle GmbH 

Permoserstr. 15 

D-04318 Leipzig 

Germany 

In vitro Models for the detection of immunomodulating 

properties of pharmaceuticals ..................................... 236 

References................................................................... 245 

The immune system is an integrated body system that is comprised 

of various organs, tissues, cells, and cell products which mediate 

host defense against microorganisms (e.g. bacteria, fungi, parasites, 

or viruses), foreign substances (e.g. bacterial or other toxins), or 

pathogenic cells (e.g. neoplasms). Protective immune responses 

depend on the successful orchestration of individual responses by a 

vast array of effector cell types including T- and B-lymphocytes, 

NK cells, monocytes and macrophages, dendritic cells, and 

granulocytes. These effector cell types arise from precursor cells 

that originate from either hematopoietic or primary lymphoid 

organs such as the bone marrow and thymus. Analysis of the 

activation, growth, proliferation and differentiative processes by 

which mature cells become effector cells (e.g. naive-toeffector/memory 

B-cell or T-cell conversions or monocyte-tomacrophage 

transitions), has led to a deepening understanding of 

the cellular and molecular mechanisms which underlie immunity 

and inflammation. Because of its central importance in providing 

host defense, and its intercommunication with other body systems 

in health and disease states, it is necessary to investigate (side-) 

effects of various noxious agents (e.g. chemicals, toxins, and 

pharmaceuticals) on the immune system. As a result, numerous in 

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In vitro Models for the detection of immunomodulating properties of pharmaceuticals 237 

vivo experimental animal model systems have been developed to 

gain crucial information and a better understanding of the 

physiological basis of immunity and inflammation and on the basis 

of the action of pharmaceuticals in this context. In addition, many 

in vitro models of the immune system have been established using a 

vast array of tissue and cell culture systems. These in vitro systems 

permit high-resolution identification and experimental scrutiny of 

the cellular and molecular mechanisms that underlie immunity and 

inflammation and how noxious agents affect them. 

Disadvantages of in vitro test systems Advantages of in vitro test systems 

• no assessment of systemic influences 

• insufficient assessment of organ-specific effects 

• no assessment of complex toxic effects, as in the 

reproduction toxicology and testing for cancerogenity 

• no assessment of chronic effects 

• no assessment of the reversibility or healing of toxic 

effects 

• no assessment of the interactions between different 

tissues, organs and organ systems 

• insufficient consideration of pharmacokinetic and 

metabolic aspects 

• no official acceptance for the toxicological evaluation of 

medicaments 

• no international official acceptance for the toxicological 

classification and indication of chemical materials and 

preparing 

• controlled test conditions 

• elimination of systemic influences 

• check of a large number of different 

test systems (cells or tissues) per 

dose possible 

• time-dependent repeated and/or 

simultaneous samplings possible 

• quick and approves of testing 

• small quantities of test chemicals 

necessary 

• small quantities of arrears results 

after the experiments 

• human cells and tissues can be 

tested 

• reduction of the number of experi- 

ments with animals (especially 

vertebrates) 

Tab. 1: Disadvantages and advantages of in vitro test systems in 

the toxicology (according to [7], modified). 

In recent years many substances have been described to have 

immunotoxic or immunomodulating properties and are therefore 

toxicologically relevant. Immunotoxic and immunomodulating 

substances are members of different structural classes of chemicals. 

An overview of some xenobiotics with immunotoxic properties is 

given in table 2. All of the effects of interactions of xenobiotics 

with the immune system described below were shown in animals 

and man. The consequences of the exposure to immunotoxic 

substances and pharmaceuticals are various. The immune system is 

able to react with these substances or their metabolites and thereby 

could be activated or suppressed. Many of the immunotoxic 

substances are highly or moderate electrophilic reagents or could be 

activated by a variety of metabolic systems to reactive 

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intermediates. These electrophilic chemicals are able to bind on 

macromolecules like nucleic acids and/or proteins and affect their 

biological function. In general, electrophilic substances not only 

affect the immune system but also other organs or organ systems, 

especially those with a higher proliferative activity. 

Pharmaceuticals 

Neomycin 

Procaine 

Sulfonamide 

Penicillin 

Benzocaine 

Thiuram 

Cobalt sulfate 

Inducers of allergies Immunosuppressors 

p-Phenylenediamine 

Hydroxybenzoic acid ester 

Nickel sulfate 

Potassium dichromate Formaldehyde 

PCDD and other dioxins 

PCB and PCDF 

Benzo[a]pyrene and other PAHs 

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Azathioprine 

Cyclosporin 

Glucocorticoids 

Cyclophosphamide 

Chlorambucil 

Carmustine 

Tab. 2: Immunotoxic xenobiotics. The known immunotoxic 

relevant substances can be distinguished by their chemical 

properties and their mode of action into at least two groups 

(according to [8], modified). 

On one hand, immunotoxic and immunosuppressive effects could 

occur and cause organ failure and toxicity in general. Because such 

general toxic effects affect cells with higher proliferating and/or 

metabolic activity to a higher degree than resting or fully 

differentiated cells, the consequences of general toxic effects on the 

immune system are greater than those effects on other body 

systems. The resulting suppression of immune responses leads to 

impaired resistance against opportunistic pathogens like viruses, 

fungi, and bacteria and to a higher risk of oncogene expression and 

the development of neoplasms and cancer. On the other hand, 

inadequate activation by pharmaceuticals or other substances bears 

the risk of hyperreactivity of the immune system. The exceeding 

immune reactions caused by immunologic dysregulation are 

responsible for the different forms of allergic diseases. Allergies are 

divided into four types (type I to type IV). The different types of 

allergies differ in their time course and the cell types and immune 

reactants involved in the immunologic reactions underlying this 

specific type of allergy. Xenobiotics are able to interfere with the 

immune system and to trigger all four types of allergies. 

Furthermore, hyperreactivity against chemicals, pharmaceuticals,


and other substances related to the environment are able to trigger 

immunologic dysregulation. These immune reactions which seem to 

be out of the physiologic normal regulatory control are often aimed 

at structures of the own body and therefore have the potency to 

trigger autoimmune reactions and autoimmune diseases. 

Additionally, environmental pollutants and diverse pharmaceuticals 

have the ability to interact specifically with transcription factors or 

are (after binding to specific intracellular receptors) part of 

transcription-factor complexes (as shown for the interaction of 

polycondensed aromatic hydrocarbons [PAHs], e.g. 

benzo[a]pyrene, with the aromatic hydrocarbon receptor [AhR] and 

the aromatic hydrocarbon receptor nuclear translocator [ARNT]) 

and act on the expression of immune regulatory relevant genes 

[1][2]. The result of such interactions could also be suppression or 

activation of expression of the respective gene(s). Furthermore, 

diverse xenobiotics act on the intracellular level of calcium ions and 

affect signal transduction [3][4]. In summary, the interactions of 

immunomodulating substances with the immune system might 

occur at different checkpoints of immune regulation on the cellular 

and sub-cellular level. The resulting effects might be further 

amplified and hence lead to pathological situations. Thus the 

exposure to immunotoxicants and immunomodulators bears a lot of 

risks. Therefore, the detection of immunomodulating potencies of 

such substances is very important. 

In vitro test systems in the form of cell and tissue cultures are used 

for a long time in pharmacological research. Animal experiments 

and later on clinical investigations are only carried out following 

effect checks of a new medicine material in in vitro systems. 

Oppositely to this in the toxicology in vitro tests are only used for 

specific questions, e.g. in mutagenicity testing or to get special 

information (or supplemental information) for a better basis for risk 

assessment. In vitro models for the detection of immunomodulating 

properties are used for the estimation of effects of pharmaceuticals 

or other chemicals potentially relevant for human beings on specific 

immune parameters, e.g. cytokine production, cytotoxicity or 

apoptosis. In vitro models for the detection of immunomodulating 

properties of pharmaceuticals or other chemicals make use of 

isolated and in vitro cultured cells of immunological origin. These 

cells used in the in vitro models are from different sources. On one 

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hand, the used indicator cells are primary cell cultures derived from 

immune organs of animals, mainly mice or rats, or from the 

peripheral blood of animals or humans. On the other hand, 

established cell lines or T- and B-cell clones are used for the testing 

of chemicals for their immunomodulating properties. In these tests 

the used immune cells are stimulated with polyclonal activators like 

lipopolysaccharide (LPS) or the mitogens phytohemagglutinin 

(PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). 

Another way is the use of stimulating antibodies against the T-cell 

receptor (TCR) or its signal-transducing components (the CD3 

complex and associated intracellular proteins), the B-cell receptor 

(BCR) or other proteins involved in the immunological stimulation 

of immune cells which are expressed on the surface of the cellular 

membrane, e.g. CD28 in the case of T cells, or CD40 in the case of 

B cells and dendritic cells (DC). Furthermore, there are models 

which frequently involve the use of primary cell cultures and 

established cell lines that depend upon the presence of (a) particular 

cytokine(s) for their growth or survival or that respond to a given 

cytokine. Fig. 1 shows a flowchart of a useful procedure for the 

detection of immunomodulating properties of pharmaceuticals and 

other substances. We use this test system in our laboratory for the 

detection of immunomodulating effects of a variety of 

environmental pollutants including volatile organic compounds 

(VOC), microbial VOC, mycotoxins, pesticides and polycondensed 

aromatic hydrocarbons. In our system freshly prepared peripheral 

blood cells of healthy donors are used. These cells (which serve as 

indicators for the effects of a given chemical on immune responses) 

were adjusted to a useful number of cells per volume (in the 

majority of cases 1 to 5 x 10 6 cells per ml) of an appropriate cell 

culture medium (mostly RPMI1640 supplemented with fetal calf 

serum or autologous serum) and seeded into the wells of sterile cell 

culture plates (24, 48, or 96 wells per plate) or tubes. The chemicals 

to be tested are solved in phosphate buffered saline (PBS) or – if 

they are not hydrophilic enough (which is a common problem) – in 

appropriate solvents like ethanol, methanol, dimethyl sulfoxide 

(DMSO), or acetonitrile, depending on their solvability. Following 

this the chemicals are serial diluted and then applied into the cell 

cultures at least in triplicates. After an appropriate culturing time 

the exposure to the chemicals is terminated. 

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Preparation of immune cells 

Adjustment to an appropriate 

number of cells/ml 

Seeding into culturing plates 

Application of immune 

stimulators 

Adjustment to a final number of 1 

x 10 6 cells/ml 

Solving of the compound to be 

tested (appropriate solvent) 

Serial dilution (same solvent) 

Application of individual 

dilutions (or solvent controls) into 

the cell culture 

Culturing under appropriate 

conditions (37°C, humidified 

atmosphere with 5% CO2) Harvesting of culture superna- 

Measurement of 

cell viability and/or 

proliferation using 

the MTT assay or 

the incorporation of 

[ 3 H]thymidine 

Harvesting of cells at useful 

endpoints 

Fixing of cells 

Fluorescence labeling of cells 

(phenotyping, expression of 

activation markers) 

Flowcytometric analysis in FACS 

tants and storage under appropriate 

conditions until analysis 

Measurement of cytokine and/or 

immunoglobulin concentrations 

with ELISA 

Preparation of mRNA 

Analysis of mRNA with RT-PCR 

Permeabilization of cells 

Fluorescence labeling of cells 

(intracellular cytokine staining 

and phenotyping) 

Fig. 1: Flowchart of a useful strategy for the testing of 

pharmaceutical compounds and other chemicals in immunological 

in vitro models. 

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By doing this it is crucial to analyze the in vitro immune response at 

different time-points that are matching the individual kinetic of the 

respective parameter. One common strategy includes washing steps 

after a short-time exposure of the cells to noxious agents to remove 

their remaining part outside the cells. This is then followed by the 

culturing of the remaining cells for a period of time sufficient for 

the expression of relevant proteins. Other strategies omit on such 

washing procedures and additional culturing times and analyze the 

direct effects of the agents on immune parameters during the 

exposure at different endpoints. 

There are different possibilities for the subsequent steps in the 

analysis of those effects caused by the chemicals. The first one, 

which is usually applied to all in vitro models for the estimation of 

immunotoxic effects, is the estimation of cell-viability and/or 

proliferation/activation of immune cells in response to mitogens. 

Estimations of cell-viability and/or activation of immune cells are 

often done using the MTT assay. The MTT assay is a quantitative 

colorimetric assay for cell survival and proliferation based on the 

ability of live cells to take up the pale yellow compound MTT (3- 

(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide) and 

to be converted by the mitochondrial succinate-tetrazolium 

reductase system to form a blue formazan dye [5]. The amount of 

formazan dye produced is proportional to the number of viable 

cells. Beside this, the proliferation of mitogene stimulated 

lymphocytes is mostly measured by the incorporation of 

[ 3 H]thymidine or 5-Bromodesoxyuridine into the DNA during 

replication before mitosis. 

Another possibility of detection of immunomodulating effects is 

harvesting of cell-free supernatants that is then followed by the 

measurement of e.g. cytokine concentrations by ELISA or in 

bioassays. The remaining cells can then be analyzed either on their 

content on mRNAs of different immune relevant genes or the 

presence of relevant protein components of signal transduction 

pathways. Another way is the analysis of cell surface markers 

(immune-phenotyping) of the former exposed cells, e.g. combined 

with the analysis of cytokine expression using the method of 

intracellular cytokine staining. Other models analyze the prior 

exposed cells for their ability to trigger cytolysis of target cells and 

------------------------------------------------------------------------------------ 




thus to act either as cytotoxic T cells (CTL) or as natural killer 

(NK) cells. 

One common problem is the use of appropriate controls for the 

verification of the effects observed under the influence of noxious 

agents that were applied in solvents. In such cases controls with 

identical concentrations of solvent are needed and the effects have 

to be calculated according to these controls. Nevertheless, it has to 

be taken into account that the data for immune responses obtained 

under the influence of solvents are principally a result of complex 

interactions of both the pharmaceutical and solvent with the 

immune cells and their biological activity. As an example, DMSO 

is an anti-inflammatory substance and acts at concentrations above 

5 % v/v in cultures on the expression and the quaternary structure of 

tumor-necrosis factor alpha and on its biological effects [6]. With 

this in mind, results for some immunologic effects obtained in those 

in vitro systems containing DMSO in higher concentrations have to 

be interpreted very carefully. 

One special topic is the assessment of the data obtained with the 

different techniques used for the analysis of immune parameters 

resulting after testing in the in vitro model. Each of the different 

tests used to measure the resulting effects in the in vitro model 

assays one or more special parameter(s) and offers answers only to 

those questions to which the test is addressed. Furthermore, the 

individual tests are suitable to a different degree for the 

measurement of the individual effect. The MTT assay, for instance, 

is suitable for the measurement of cell viability (or cytotoxic 

effects) based on the activity of the mitochondrial succinatetetrazolium 

reductase system and therefore depends on the number 

of viable cells as well as on their respiration activity. However, the 

MTT assay gains data about cytotoxic effects. It is very useful to 

have these data for a comparison with those concentrations of the 

test substance affecting other immunologic parameters. Useful 

insights into the immunotoxic or immunomodulating properties of a 

substance can be obtained by measurements of cytokine 

concentrations in culture supernatants extended by measurements of 

cytokine-mRNA expression in the former exposed cells and the 

analysis of cytokine production by flowcytometry. The use of 

flowcytometry (which means the measurement of fluorescencelabeled 

cells in a fluorescence-activated cell sorter [FACS]) for 

------------------------------------------------------------------------------------ 




phenotyping and intracellular cytokine-staining (ICS) of immune 

cells offers the possibility of detection of effects on different cell 

populations (e.g. subsets of T cells) on the single cell level. The ICS 

data therefore provide additional information on the targeted cell 

populations and the effects of noxious agents on different T-cell 

subsets. 

The measurement of culture supernatant concentrations of 

cytokines like tumor necrosis factor-alpha (TNF-α), interleukin 

(IL)-1β, and IL-6 with immunoassays or bioassays offers the 

possibility to detect pro-inflammatory potencies of a given 

substance. These three cytokines are known as pro-inflammatory 

cytokines and are involved in inflammation processes, regulation of 

acute phase reactions and show pyrogenic activity. Furthermore, 

these cytokines are growth and differentiation factors for B- and Tlymphocytes. 

Other cytokines are immune regulators that 

predominantly act on immune cells themselves. Such cytokines are 

interferon-gamma (IFN-γ), IL-2, IL-4, IL-13, and IL-5. The 

measurement of these cytokines offers the possibility to detect 

immunomodulating properties of a given substance targeting 

immune regulation. The balance of these cytokines and the balance 

of the producing T-cell subsets is crucial in immune regulation. 

Dysregulation affecting the balance of IFN-γ and IL-4 is thought to 

be related to autoimmunity and allergy. The measurement of IFN-γ 

and IL-4 in culture supernatants is therefore very suitable to gain 

information on the immunomodulatory activity of a given 

substance. Another way to measure cytokine concentrations is the 

use of bioassays. Generally the biological responses induced by 

cytokines show saturation kinetics which can be used to quantitate 

their amounts from dose-response curves. It should be noted that 

cytokine assays frequently measure only one single aspect of many 

biological activities of a given cytokine. Potential complex 

interactions among cytokines must also be considered when 

interpreting data obtained from cytokine bioassays. Furthermore, 

there are often differences between measurements of cytokine 

concentrations in bioassays and immunoassays (e.g. ELISA). This 

is thought to be related in some cases to the presence of different 

antigenic forms (including complexes with soluble receptors or 

monomeric forms of cytokines) that cannot be detected in 

bioassays. Because there are additional difficulties in the 

------------------------------------------------------------------------------------ 




measurement of culture supernatants containing the cytokines to be 

measured as well as the substance to be analyzed in the in vitro 

model (that eventually may further have the ability to act on the 

indicator cells used for the cytokine measurement in the bioassay) 

we suggest to avoid bioassays for cytokine measurements in such a 

background. 

In general, in vitro systems and analytical methods like those 

presented here are suitable for the analysis of immunomodulating 

effects of a broad spectrum of substances including 

pharmaceuticals, antibiotics and toxins [9]. This system as well as 

other comparable in vitro systems are able to show 

immunomodulating potentials of the test compound and therefore 

can serve as an inexpensive tool for the detection of 

immunomodulating properties. In this context arises the question 

about the transmissibility of in vitro data on the situation in vivo and 

further on their relevance for integrity of the whole organism and 

health. Since in vitro systems only indicate the potentials of a given 

substance under these artificial conditions (see [6]), it remains to be 

shown in further investigations that the observed effects are also 

real in vivo. 

References 

1 Reyes H, Reisz-Porszasz S, Hankinson O (1993) Identification 

of the Ah receptor nuclear translocator protein (Arnt) as a 

component of the DNA binding form of the Ah receptor. 

Science 256: 1193-1195. 

2 Safe S, Krishnan V (1995) Cellular and molecular biology of 

aryl hydrocarbon (Ah) receptor-mediated gene expression. Arch 

Toxicol Suppl 17: 99-115. 

3 Archuleta MM, Schieven GL, Ledbetter JA, Deanin GG, 

Burchiel SW (1993) 7,12-dimethylbenz[a]anthracene activates 

protein-tyrosine kinases Fyn and LCK in the HPB-ALL human 

T-cell line and increases tyrosine phosphorylation of 

phospholipase C-γ 1, formation of inositol 1,4,5-triphosphate, 

and mobilization of intracellular calcium. Proc Natl Acad Sci 

USA 90: 6105-6109. 

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4 Mounho BJ, Davila DR, Burchiel SW (1997) 

Characterization of intracellular calcium responses produced by 

polycyclic aromatic hydrocarbons in surface-marker defined 

human peripheral blood mononuclear cells. Toxicol Appl 

Pharmacol 145: 323-330. 

5 Mosmann T (1983) Rapid colorimetric assay for cellular 

growth and survival: application to proliferation and cytotoxicity 

assay. J Immunol Methods 65: 55-63. 

6 De Groote D, Grau GE, Dehart I, Franchimont P (1993) 

Stabilisation of functional tumor necrosis factor-alpha by its 

soluble TNF receptors. Eur Cytokine Netw 4: 359-362. 

7 Hockertz S (1994) Immunsystem. In: Marquardt H, Schäfer SG 

(Hrsg) Lehrbuch der Toxikologie. Spektrum Akad Verl, S 257- 

270. 

8 Spielmann H (1994) In-vitro-Methoden. In: Marquardt H, 

Schäfer SG (Hrsg) Lehrbuch der Toxikologie. Spektrum Akad 

Verl, S 814-820. 

9 Wichmann G, Herbarth O, Lehmann I (2002) The mycotoxins 

citrinin, gliotoxin and patulin affect rather interferon-γ than 

interleukin-4 production of human blood cells. Environ 

Toxicol 17 (3). 

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VOLUME 1 


- UPDATE 2002 - 

Volume Editors 

J. Wohlrab 

R.R.H. Neubert 

W.Ch. Marsch

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