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Keywords:

  • collagen;
  • fibroblasts;
  • glucocorticoids;
  • keratinocytes;
  • skin atrophy

Abstract

  1. Top of page
  2. Abstract
  3. Clinical aspects
  4. Cellular mechanisms of glucocorticoid-induced skin atrophy
  5. Molecular mechanisms of glucocorticoid-induced skin atrophy
  6. Determining atrophogenic potential
  7. Strategies to identify GCs and GC like drugs with a reduced atrophogenic potential
  8. Concluding remarks
  9. Acknowledgements
  10. References

Abstract:  Glucocorticoids (GCs) are highly effective for the topical treatment of inflammatory skin diseases. Their long-term use, however, is often accompanied by severe and partially irreversible adverse effects, with atrophy being the most prominent limitation. Progress in the understanding of GC-mediated molecular action as well as some advances in technologies to determine the atrophogenic potential of compounds has been made recently. It is likely that the detailed mechanisms of GC-induced skin atrophy will be discovered and in vitro models for the reliable prediction of atrophy will be established in the foreseeable future. This knowledge will not only facilitate safety profiling of established drugs but will also foster further drug discovery by improving compound characterization processes. New insights into GC modes of action will guide optimization strategies aiming at novel GC receptor ligands with improved effect/side effect profile.


Abbreviations:
ECM

extracellular matrix

GC

glucocorticoid

GR

glucocorticoid receptor

GRE

glucocorticoid response element

IL-1

interleukin 1

MMP

matrix metalloproteinase

MPA

methylprednisolone aceponate

PC

prednicarbate

SEGRA

selective glucocorticoid receptor agonists

TIX

therapeutic index

TNF-α

tumor necrosis factor alpha.

Clinical aspects

  1. Top of page
  2. Abstract
  3. Clinical aspects
  4. Cellular mechanisms of glucocorticoid-induced skin atrophy
  5. Molecular mechanisms of glucocorticoid-induced skin atrophy
  6. Determining atrophogenic potential
  7. Strategies to identify GCs and GC like drugs with a reduced atrophogenic potential
  8. Concluding remarks
  9. Acknowledgements
  10. References

Glucocorticoids (GCs) are the most widely used class of anti-inflammatory drugs. The first systemic treatment with GCs was performed by Hench and co-workers (1) in patients with rheumatoid arthritis in 1949. The introduction of topical hydrocortisone in the early 1950s provided great advantages over previously available therapies. It was triamcinolone acetonide, the first of the halogenated corticosteroids, that began a revolution that cumulated in the appearance of the potent and superpotent GCs. Such highly effective GC are, on the one hand, of great advantage in certain indications, localizations, and types of disease; however, they may have deleterious effects when used over longer periods or when improperly applied. The enthusiasm for these highly effective agents was at its peak during the 1960s and 1970s and, perhaps inevitably, the more potent GCs were often used inappropriately and indiscriminately. Adverse effects became apparent and the subsequent backlash of opinion against topical GCs created confusion (2). However, topical GCs are successfully used in the treatment of several common cutaneous diseases but their major limitation, their side effect potential, is still remaining. GCs have pleiotropic effects (Fig. 1) potentially causing several adverse reactions such as osteoporosis, growth retardation, cushingoid appearance, glaucoma, and others in particular after systemic high-dose and long-term application (3).

image

Figure 1. Most important clinical effects of glucocorticoids (GC). Examples for desired therapeutic effects as well as side effects of GCs on different organs are shown. CNS, central nervous system; HPA, hypothalamic pituitary axis [reprinted from The Lancet 365: 801–803, Copyright (2005), with permission from Elsevier (122)].

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Skin atrophy is the most important cutaneous side effect of GC therapy (4), where a major function of the skin, the formation of a permeability barrier between the external milieu and the organism, is compromised (5). GC-induced skin atrophy is characterized by a profound increase in transparency of skin, a cigarette-paper-like consistency accompanied by an increased fragility, tearing, bruising (‘steroid purpura’) (6), thin, shiny, and telangiectatic surface (7,8). (Fig. 2). The first original description of the phenomenon was published in 1963 by Epstein and co-workers (9) reporting five patients with atrophic striae in the groins, who had topically received Mycolog cream (containing triamcinolone acetonide, neomycin, gramicidin, and nystadin). Histopathologically, in GC-induced skin atrophy flat dermal–epidermal junctions (6), reduced thickness of the epidermis (8), decreased size of keratinocytes (10) and reduced number of fibroblasts (10,11), rearrangement of the geometry of the dermal fibrous network (12), and diminution of stratum corneum intercellular lipid lamellae (13) are found.

image

Figure 2. Corticoid-induced skin atrophy. Chronic topical steroid use has led marked thinning of the skin (image kindly provided by Charles Goldberg and Jan Thompson, University of California, San Diego).

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Besides GC-induced skin atrophy, other diseases like lichen sclerosus et atrophicus, atrohodermia vermiculata (14), morphea plana atrophica (15), or dermatoborrelioses may cause skin atrophy. The senile degenerative skin atrophy is common and occurs physiologically in aged skin. The clinical appearance of senile atrophy is similar to that of GC-induced skin atrophy (16). This review, however, focuses on topical GC-induced skin atrophy as the most important side effect and the major limitation of conventional topical GCs at the skin.

Both systemic as well as topical GC therapy can induce atrophy. Factors that determine the atrophogenic potential of a given topical GC are its potency, duration of therapy, frequency of application (17), and further unknown factors. Different formulations may also markedly influence both the clinical efficacy of a GC as well as its atrophogenic potential simply because of the differing potential of the vehicle to release the drug into the stratum corneum (18). The area where a GC is applied is an important factor contributing to the development of GC-induced skin atrophy. Atrophy is more common in areas of the body where humidity and occlusion results in greater penetration of the steroid such as groin and axillae (19). The face or intertriginous areas are more sensitive to corticosteroid penetration (20), whereas kapillitum, palmae, and plantae are less sensitive for GC-induced skin atrophy (21).

The predominant classification of topical GCs is based on their potencies (Table 1). Thus, class I GCs like hydrocortisone have weak effects (regarding both desired anti-inflammatory effects and side effects), whereas class IV GCs like clobetasol propionate have strong effects. More recently, the therapeutic index (TIX) has been introduced for topical GCs (21). The guideline is based on a concept as devised by Schackert et al. (22). The concept of the benefit risk ratio evaluation with topical GCs is based on both, in vitro and in vivo data, concerning both efficacy and safety (22–24). The TIX indicates the ratio of desired and adverse effects of several GCs. Since a TIX of 1–2 is defined as an equal relation of desired and adverse effects, a TIX of 2–3 indicates a GC with improved benefit/risk ratio. For example, mometasone furoate, a class III GC with a TIX of 2 has stronger desired effects than side effects. The TIX considers atrophogenicity (Table 1) and further side effects as well as a series of beneficial effects of a given GC and thus integrates its advantages and limitations (21). Local administration of metabolically unstable compounds and introduction of the prodrug principle represent major steps in the development of GCs with an improved benefit/risk ratio.

Table 1a.  Classification of glucocorticoids based on potency
ClassExamples
IHydrocortisone, hydrocortisone acetate, prednisolone
IIDexamethasone, hydrocortisone butyrate, methylprednisolone aceponate, prednicarbate, triamcinolone acetonide
IIIBetamethasone valerate, betamethasone dipropionate, desoximetasone, fluocinoide, mometasone furoate
IVClobetasol propionate

Over 300 papers dealing with GCs and skin atrophy are listed in Medline. They were included into a search to review the mechanisms underlying GC-induced atrophy. Such knowledge is of importance for the development of better (effective and predictive) preclinical methods to determine the atrophogenic potential of compounds and to guide optimization strategies aiming at novel GC receptor ligands with an improved effect/side effect profile.

Cellular mechanisms of glucocorticoid-induced skin atrophy

  1. Top of page
  2. Abstract
  3. Clinical aspects
  4. Cellular mechanisms of glucocorticoid-induced skin atrophy
  5. Molecular mechanisms of glucocorticoid-induced skin atrophy
  6. Determining atrophogenic potential
  7. Strategies to identify GCs and GC like drugs with a reduced atrophogenic potential
  8. Concluding remarks
  9. Acknowledgements
  10. References

GCs have profound effects on both anatomically distinct layers of the skin, the epidermis and the dermis that is connected by the basement membrane. The epidermis is constituted mainly of keratinocytes. Keratinocytes originate from basal proliferating cells attached to the basement membrane, whose daughter cells migrate into the upper layer of the epidermis undergoing a differentiation process. As keratinocytes die, they produce a cornified external membrane, the stratum corneum. The stratum corneum provides mechanical resistance and is embedded in a lipid-enriched extracellular matrix (ECM), which is responsible for the permeability barrier. Lamellar bodies secrete barrier lipids in the form of free fatty acids, cholesterol, and ceramides into the intercellular space between the cells of the stratum corneum and the underlying epidermal layer, the stratum granulosum, forming intercellular lipid layers. The free fatty acids are required as critical structural ingredients of the barrier (25). Cholesterol is critical for permeability barrier function (26). Ceramides are the principal repositories for some fatty acids (27). Taken together, lipids play a central role in normal skin physiology and thus impact of GCs on these molecules is of importance.

The dermis is mainly composed of fibroblasts. Their major function is production and delivery of components of connective tissue, like proteins of the dermal ECM. The basement membrane contains specialized structures, called anchoring complex, which ensure stability of connection and communication between these two tissue compartments. Keratinocytes and fibroblasts are known target cells of GC effects in skin (Fig. 3).

image

Figure 3. Cutaneous effects of glucocorticoid (GC) treatment. The proliferation and the extracellular matrix (ECM) protein synthesis of keratinocytes and fibroblasts are suppressed by GCs. The intercellular lipid layers are also reduced by GCs, caused by the decreased synthesis of epidermal lipids, like ceramides, cholesterol, and fatty acids. Thereby, the stratum corneum gets thinner, followed by an increased transepidermal water loss (blue arrow). The skin loses its barrier function, its tensile strength and elasticity caused by the water loss and the degraded ECM.

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Keratinocytes effects

GCs may decrease the size (10,28) and can suppress proliferation of keratinocytes (29). The reason for decreased cell size is not exactly known, but it is assumed that reduced biosynthesis of macromolecules of these cells plays an important role (30). A known cause of the antiproliferative effect of GCs is reduced mitotic rates of GC-treated keratinocytes (31,32). However, an accelerated keratinocyte maturation results in a thinning of the living epidermis due to the shorter epidermal cell life (28,33,34). Inconsistently, it has been shown that topical GCs inhibit the epidermal terminal differentiation (35–37). The decreased size and suppressed proliferation of keratinocytes may then result in structural defects of the epidermis, epidermal thinning, and especially thinning of the stratum corneum. The consequences are an increased permeability and an increased transepidermal water loss (10), which generally indicates disrupted barrier function of the skin (5,37).

Fibroblasts effects

Antiproliferative effects of GCs on fibroblasts (29,38,39) are also caused by a reduced mitotic rate (38). Actually, Hammer and colleagues (40) demonstrated that dexamethasone is able to protect primary human fibroblasts against tumor necrosis factor alpha (TNF)-α-, UV- or ceramide-induced apoptosis. The cellular effects of GCs on fibroblasts result in the thinning of the dermis, which causes a decrease of tensile strength and elasticity of the skin. Moreover, fibroblasts play an important role in the synthesis of ECM proteins whose production is significantly interfered by GCs, as shown below.

Molecular mechanisms of glucocorticoid-induced skin atrophy

  1. Top of page
  2. Abstract
  3. Clinical aspects
  4. Cellular mechanisms of glucocorticoid-induced skin atrophy
  5. Molecular mechanisms of glucocorticoid-induced skin atrophy
  6. Determining atrophogenic potential
  7. Strategies to identify GCs and GC like drugs with a reduced atrophogenic potential
  8. Concluding remarks
  9. Acknowledgements
  10. References

Mechanisms of glucocorticoid action

GCs mediate their effects via many and diverse genomic and non-genomic mechanisms. After diffusion into the cell, they bind to the intracellular GC receptor (GR). Binding of ligands induces a conformational change in the GR molecule resulting in translocation of the activated GR into the nucleus (41). There, the GR is able to affect gene expression either positively (transactivation) or negatively (transrepression). The positive regulation of target genes is mediated by a specific binding of the activated GR as a homodimer to GC response elements (GREs) in the promoter or enhancer region of responsive genes (42), followed by an induction of gene transcription. Three types of GREs, which require slightly different GR conformations, are described: simple, composite, and tethering GREs. The activated GR binds as a sole sequence-specific DNA-binding protein, i.e. without recruitment of cofactors, to simple GREs. GR binding to composite GREs needs one or more non-receptor factors. For binding to tethering GREs, the GR is recruited through protein–protein interaction to another DNA-bound factor (43).

Transrepression effects of the receptor are mechanistically as variable as described for the transactivation effects. First, activated GR can bind to negative GREs (nGRE), leading to repression of gene transcription (44). Second, the GR binds to sequences that overlap the recognition sites of other essential transcription factors and interferes with their binding (45). Third, the GR interacts via protein–protein interaction with other transcription factors, e.g. activator protein-1, nuclear factor-κB, or Smad3, preventing an activation of transcription by these factors (46–48).

Extracellular matrix proteins effects

The tensile strength and elasticity of the skin decreases by reducing the synthesis and inducing the degradation of ECM proteins in fibroblasts. ECM proteins include collagens, proteoglycans, and elastin. Alterations of the dermis mainly result from effects of GCs on collagen turnover (49,50). Collagens as major components of ECM account for about 70% of the dry weight of skin, and constitute a highly specialized family of glycoporteins with at least 27 members. Collagens distribute tensile strength to the skin. The major skin collagen fibril, forming approximately 80% of the skin collagen, is type I collagen. The minor components are types III (10–15%), V (5%), and IV collagens (basement membrane) (51). Type I collagen is a heterodimer composed of three α-chains encoded by the tightly controlled COL1A1 and COL1A2 genes (52). The promoter regions of type I collagens contain neither GREs nor nGREs (53). It is described, however, that Smad proteins are essential for the induction of type I collagen transcription (54) and that GR can have a negative regulatory effect to Smad-activated gene transcription (55). GCs also decrease procollagen gene expression through a GRE (56). In contrast to the well-investigated mechanism of type I collagen regulation, the mechanism for regulation of the type III collagen gene is far less understood. GC treatment reduces type III collagen with a more devastating effect than with type I collagen. This reflects the situation with fibrillar collagen: Type III collagen is more sensitive to GC treatment compared with type I collagen (57,58). Although the content of type III collagen in the ECM is only approximately 10% of that of type I collagen, the drastic reduction of type III collagen gene expression in GC-treated animals implies both clinical and physiological importance (57). Little is known about the mechanism that results in regulation of collagens type IV and V in skin and the according effects of GCs on these proteins so far.

All in all, GCs may affect collagen synthesis indirectly by reducing prolyl hydroxylase activity (59,60) or by increasing the degradation of collagen mRNAs (50). Prolyl hydroxylase is an important rate-limiting enzyme of collagen synthesis (61) that catalyzes the formation of hydroxyproline in collagens by hydroxylation of proline residues in peptide linkages. In turn, hydroxyproline, a major component of collagen, helps providing stability to the triple-helical structure of collagen by forming hydrogen bonds. Owing to the fact that GCs reduce prolyl hydroxylase activity, GCs decrease hydroxyproline formation in vitro and in vivo (60,62,63).

Besides collagens, other ECM proteins are also affected by GCs (11,64). Proteoglycans are characterized by a core protein with one or more glycosaminoglycan side chains attached. Their major functions are retaining water and providing the self-assembly of collagens (fibrillogenesis). Decorin, e.g. is a proteoglycan that can inhibit collagen fibrillogenesis of both type I and type II collagens (65). The decorin mRNA expression is induced by GCs (66), whereas the available nucleotide sequence of the 5′-flanking region of the human decorin gene does not contain GREs required for activation of transcription by GCs (67). Such elements may, however, reside outside the currently known regulatory sequences. A second protein, elastin, represents the major protein of the elastic fibres forming approximately 2% of skin dry weight. Although the elastin gene contains a functional GRE in the promoter (68), a decrease of elastin synthesis was observed in GC-treated cultivated fibroblasts (69).

Matrix metalloproteinases (MMP) are a family of at least 26 highly homologous Zn2+ endopeptidases that collectively cleave the constituents of the ECM. The gene expression of MMP-1 is repressed by the activated GR via protein–protein interaction with activator protein-1 (70). GCs repress the mRNA expression of MMP-3 (71) and MMP-8 (72) by interfering with the binding of the essential transcription factor AP-1, the major enhancer factor of the collagenase promoter. Other GC-sensitive genes in cutaneous cells (Table 2) may also be relevant for skin atrophy.

Table 2.  Genes in cutaneous cells regulated by glucocorticoids (GCs)
ProteinCellGeneRegulation by GCsReference
Type I collagenFibroblastCOL1A1Down(50,56,57,123,124)
COL1A2Down(124)
Type III collagenFibroblastCOL3A1Down(50,57,124)
Type IV collagenFibroblastCOL4A1Down(125)
Type V collagenFibroblastCOL5A2Down(124)
DecorinFibroblastDCNDown(66)
ElastinFibroblastHDE-1Down(126)
FibronectinFibroblastFNUp No (127,128) (127)
Matrix metalloproteinase-1 (MMP-1)KeratinocyteMMP1Down(70,72,129)
MMP-2 (type IV collagenase)KeratinocyteMMP2Down(129)
MMP-3 (stromelysin 1)KeratinocyteMMP3Down(71)
Tenascin-CFibroblastTN-20/1Down(130)
Tissue inhibitors of MMP-1 and 2KeratinocyteTIMP1, TIMP2Down(57)

Extracellular matrix lipids

In addition to the effects on proteins of ECM, GCs can also diminish the intercellular lipid layers of the upper epidermal layers that play an important role for the barrier function of the skin (13). GCs decrease the synthesis of epidermal lipids like ceramides, cholesterol, and fatty acids (10). It was demonstrated that increased transepidermal water loss correlates with the obvious decrease of lamellar bodies in the granular cell layers and with empty interstices at the stratum corneum – stratum granulosum interface in the GC-treated atrophogenic skin (73,74).

Although several activities of GCs can be related to the described cellular or molecular mechanisms, and despite long-standing efforts to understand the molecular causes of GC-induced skin atrophy, the mode of action that underlies this important side effect is still not entirely understood.

Determining atrophogenic potential

  1. Top of page
  2. Abstract
  3. Clinical aspects
  4. Cellular mechanisms of glucocorticoid-induced skin atrophy
  5. Molecular mechanisms of glucocorticoid-induced skin atrophy
  6. Determining atrophogenic potential
  7. Strategies to identify GCs and GC like drugs with a reduced atrophogenic potential
  8. Concluding remarks
  9. Acknowledgements
  10. References

Clinical assessment

The ultimate atrophogenic potential of GCs is determined in clinical studies. Since atrophy is potentially irreversible and obtaining skin biopsies represents an invasive method with certain risks (scars, infection), it is aimed to limit such investigations or at least to apply compounds to very small areas and to use non-invasive methods. There are many non-invasive methods to clinically investigate the atrophogenic potential of GCs. A current model to rank topical steroids is the vasoconstrictor assay (75). The skin-blanching assay is unique in that the methodology utilizes the localized vasoconstriction side effect following topical corticosteroid application. Besides the visual assessment with a dermoscop for applicability in quantifying vasoconstriction that follows corticosteroid application to the skin (76), several instrumental methods have been evaluated, like chromameter measurement or digital image analysis (18). Whereas this method is suited to characterize classical GCs (with no dissociation between effects and side effects), it is not suited for novel approaches, since it just measures a surrogate for the potency of GCs. However, several methods exist to measure the thinning of the skin directly, e.g. micrometer screw gauge, confocal laser microscopy, and ultrasound (10,77). With a screw gauge, the thickness of skin folds (two thicknesses of the skin) can be measured; however, it may include a varying proportion of subcutaneous tissue. Confocal laser scanning microscopy is a valuable tool for obtaining high-resolution images and three-dimensional reconstructions. Ultrasonography delivers a two-dimensional, cross-sectional view of the skin. A 20 MHz sonography, or in particular skin sonography at large, is the experimental approach of choice so far to quantify cutaneous atrophy in the context of topical GC application to the skin. The methodology has originally been developed by Marks (78). These two methods delineate one thickness of skin as well as they allow the selective measuring of epidermal or dermal thickness without any other tissues. All of these methods are capable of detecting skin atrophy induced by a potent topical corticosteroid.

For both research purposes and clinical evaluation, measurement of GC-induced skin barrier impairment is frequently applied. Here, evaporimetry is used to demonstrate transepidermal water loss. Stratum corneum lipid assessment is used to demonstrate reduction of ceramides, cholesterol, or free fatty acids (10).

All in all, assays for atrophogenicity in humans are burdensome and time-consuming, sometimes requiring 6 weeks of occlusive exposure and may be challenged for ethical reasons (12).

Preclinical in vivo models

Many preclinical approaches and models determine the GC atrophogenic potential (Table 3) (79). Atrophogenic potential has been evaluated in mice (34,73,80,81), rats (63,82,83), pigs (84,85), and dogs (6). Castor and Baker (86) showed as early as 1950 that topical application of hydrocortisone to the skin of rats caused dermal thinning. The most frequently applied models in the pharmaceutical industry is the rat model of GC-induced skin atrophy. Here, hairless rats are treated over 19 days. Significant effects are observed with strong atrophogenic compounds as soon as after 8 days; however, detection of less pronounced but still significant effects requires prolonged treatment. Because of the high biological variation in the results, considerable group sizes (preferentially at least 10 animals per group) are needed (82). Consequently, the investigations are labor, time and cost intensive, and require large amounts of compounds. Unfortunately, no better method has yet been identified to reliably predict atrophy. As a major overall outcome parameter, skin thickness is determined over time using different methods either measured with a special gauge (34,80,82,83) or by counting the cell layers in skin biopsies (34) (Fig. 4). Measuring of skin-breaking strength of GC-treated skin is used as an additional parameter for skin atrophy (82). In addition, biochemical and histological analyses on skin biopsies are performed. Further tissue parameters can be assessed in biopsies of GC-treated skin, such as regression of sebaceous glands, size and number of horn-filled cysts, subcutaneous fat, and muscular layers (34,73). On the molecular level, various proteins like glucosaminoglycan, fibronectin, and collagens may be used as indicators of GC effects (63,81). Measurement of transepidermal water loss is also possible using skin biopsies (63,73). Besides the direct parameters of skin atrophy, systemic effects of topically administered compounds can also be measured such as weight loss of body, thymus, spleen, and adrenal glands (63,82,83).

Table 3.  Overview on preclinical in vivo models to determine the atrophogenic potential of glucocorticoids (GCs)
ModelRemarksReference
Mouse earTreatment over 11 days (daily). Thinning of mouse ear was used to assess the atrophogenic potential of five GCs. Well suited to measurement with a ratchet control micrometer; low standard errors, reproducible; reliable, non-invasive, safe.(80)
Dorsal skin of hairless mouseTreatment over 18 days (daily). Loss of volume of all cutaneous compartments was used to assess the atrophogenic potential of nine GCs. GC-induced atrophy is similar to that in human. Most sensitive histologic markers are the reduction of volume of sebocytes, cysts, dermal, and muscle thickness. Caliper measurement of skin thickness is relative insensitive.(34)
Mouse tailTreatment over 3 weeks (daily). Epidermal thinning of mouse tail was used to assess the atrophogenic potential of five GCs. All tested GCs caused significant thinning of the epidermis. Ranking similar to that in human. Difficult to quantifying epidermal atrophy.(131,34)
Flank skin of shaved Sprague–Dawley ratTreatment over 12 days (daily). The degree of atrophy was determined after the experimentby comparing the weights of skin plugs taken from GC-treated areas with contralaterallypaired control areas. Potencies in the dermal atrophy assay comparable directly with topical anti-inflammatory potencies in the rat. Skin thickness in the rat is markedly dependent on the phase of hair growth cycle.(63)
Dorsal skin of hairless ratTreatment over 19 days (daily). Skin thickness and skin-breaking strength was used to assess the atrophogenic potential of a classic GC compared with selective GR agonists. Well suited to measure skin thickness with a special gauge; low standard errors, reproducible, reliable, non-invasive, and safe.(82)
Flank skin of guinea pigsTreatment over 4 weeks (daily). Epidermal thinning was used to assess the atrophogenicpotential of six high-dosed GCs. All tested GCs caused thinning of the epidermis. Ranking similar to that in human. Relative insensitive to topical corticoids.(87,132)
Yucatan hairless micropigTreatment over 6 weeks (three times a week). Histological similarities during GC treatment between micropig and human skin were examined. GC-induced atrophy is similar to that in human. Differences: No thinning of the stratum corneum, no hyalinization of the dermis(84)
Dorsal skin of domestic pigTreatment over 7 weeks in pig (5 days a week) vs. 3 weeks treatment in human (twice daily). Epidermal thinning was used to assess the atrophogenic potential of a new GC compared with a group of other topically active GCs. GC-induced epidermal atrophy is similar to that in human.(74,85)
Dorsal skin of hairless descendants of Mexican hairless dogsTreatment over 4 weeks (daily). The severity of histological changes was used to assess the atrophogenic potential of different types of GCs. GC-induced atrophy and its time course observed is similar to that in human. No thinning of the stratum corneum. No rebound phenomenon after completion of GC treatment. Relative sensitive (dog skin became much thinner than human).(6)
image

Figure 4. Glucocorticoid (GC)-induced skin atrophy in rats. Dorsal skin of hrhr rats were treated daily for 19 days with vehicle (ethanol/isopropylmyristate (95:5 v/v)), 0.01% mometasone furoate (a class II GC), 0.01% clobetasol propionate (a class IV GC), or 0.3% prednisolone (a class I GC). (a) Histology of dorsal skin treated with vehicle (left) and mometasone furoate (right) are shown. Marked epidermal and dermal thinning is seen. H&E staining. Magnification: 60× (image kindly provided by Lars Röse, Schering AG, Berlin). (b) Thickness of the skin (mean ± standard deviation) measured with a specifically designed dial thickness gauge. (c) Skin-breaking strength after 19 days treatment measured with an apparatus developed by Schering AG, Berlin.

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In the past, rats were shaved for atrophy assessment (63,80,83); however, because of the unreliability of haired skin responses (87,88), the use of hairless animals such as hr/hr rats (82) is currently preferred.

The advantages of in vivo preclinical compared with clinical tests are reduced variability and higher cost efficiency and in particular ethical aspects. An evident limitation of many in vivo models is, however, their limited predictivity for the situation in humans. In comparison with humans, the hairless rat seems to be more sensitive to atrophy following topical GC treatment. Anatomical differences of rodent and human skin such as the high number of skin appendages and the absence of a papillary dermis in rodents clearly might influence pharmacokinetics (80). Thus, absorption of topically applied compounds in rodent skin can exceed human skin by 10 times. Anti-inflammatory activities of GCs also differ in rats and humans (89), and, since anti-inflammatory potencies and atrophogenic potential usually closely correlates in the classical GCs, the relative dermal atrophy potencies are also expected to vary between species (63).

Preclinical in vitro models

The beauty of in vitro test systems in general is that they are fast, inexpensive, and feasible with limited amounts of compounds. They are, therefore, highly attractive for pharmaceutical companies, especially in early drug discovery projects. They also allow medium or even high throughput compound screening. Indeed, there are approaches that might to some extent allow to determine the atrophogenic potential of GCs in vitro (Table 3). Classically, those tests assess proliferation of keratinocytes and fibroblasts. Previous reports indicate that, dependent on the experimental model, GCs might either favor or inhibit division of fibroblasts, depending on the working group [reviewed in (90)]. An explanation for the differences observed among in vitro experiments could be that GCs also indirectly affect fibroblast proliferation by controlling the synthesis or actions of various factors produced by other cell types. Yet, more recent studies show only antiproliferative effects of GCs on primary human fibroblasts (29,38,91,92) and HaCaT cells, a human keratinocyte cell line (92). Similarly, effects of GCs on collagen synthesis in fibroblasts vary from stimulating (91) to inhibiting (93). However, the limitation of in vitro models is that the isolated cells are kept under non-physiological conditions that comprised of (i) difference between growth medium and tissue fluid, (ii) presence of the cells in a ‘monolayer’ and bathing in a large excess of growth medium, (iii) absence of inter-tissue interactions, and (iv) absence of many humoral factors acting in vivo (29,38,39).

So far, no validated in vitro test system exists that allows the prediction of skin atrophy. Much research remains to be done to establish reliable in vitro models to determine GC-induced atrophy.

Future models

An in vitro model with high predictivity for atrophogenicity of substances would be favorable for the characterization of novel substances in pharmaceutical development. Using in vitro models high sample throughput with little substance consumption and rapid experimental procedures may be accomplished. An adequate test system would be a benefit for the pharmaceutical industry. Monolayer or commercially available skin equivalents are potential candidates, which need to be validated. The advantages of skin equivalents over monolayer systems or animal experiments are (i) the human origin of the cells, (ii) the possible interactions of keratinocytes and fibroblasts, and (iii) reduced variability of read out parameters. Skin equivalents are proven alternatives to traditional animal testing, like dermal corrosion, and skin irritation tests (94). Such models could be used to identify surrogate markers for skin atrophy, e.g. by microarray technology. In parallel, experiments are required to identify GC-sensitive genes in GC-treated skin or cells and to verify data obtained in the different test systems.

With regard to animal models, experimental procedures may be optimized toward reduced treatment times and definition of more predictive read out parameters. Despite progress in biophotonics that will lead to more accurate and sensitive test systems, measurement of sensitive atrophy surrogate markers, once identified, may be useful.

Strategies to identify GCs and GC like drugs with a reduced atrophogenic potential

  1. Top of page
  2. Abstract
  3. Clinical aspects
  4. Cellular mechanisms of glucocorticoid-induced skin atrophy
  5. Molecular mechanisms of glucocorticoid-induced skin atrophy
  6. Determining atrophogenic potential
  7. Strategies to identify GCs and GC like drugs with a reduced atrophogenic potential
  8. Concluding remarks
  9. Acknowledgements
  10. References

Progress in early, sensitive and predictive determination of the atrophogenic potential will have impact on the identification and development of novel drugs. The atrophogenic potential of existing topical GC varies to some degree; however, the pro-atrophogenic potential of a given conventional GC in general closely correlates with its anti-inflammatory potency (95). To selectively address both the inflammation and atrophy components, one may consider strategies based on pharmacokinetics, i.e. temporal, histological, and spatial aspects, including those related to drug delivery, or strategies that involve pharmacodynamics, i.e. mechanistic aspects and modes of action that result from intrinsic pharmacological properties of a compound. Both types of approaches should ultimately favor suppressive effects on the relevant immunocytes, such as T cells, Langerhans cells, or various subpopulations of dendritic cells over those on keratinocytes and fibroblasts.

Optimizing treatment regimen

A relatively trivial approach to minimize GC-mediated atrophy is to simply limit the period of skin exposure by intermittent treatment schedules. Some reduction of skin atrophy can thereby be achieved even when strongly atrophogenic GCs are applied. This is indicated by reduced decrease of procollagen I and III under intermittent usage of topical hydrocortisone (96). Yet, to improve the overall outcome of anti-inflammatory treatment, interruption might not be appropriate. Rotational treatment with topical GC and an alternative regimen, e.g. topical calcineurin inhibitors, topical vitamin D3, or retinoids may, therefore be a better choice. Thus, continuous immunosuppression may be achieved by addressing complementary anti-inflammatory pathways (97). Other strategies include intermittent use of the GC only every second day (tandem therapy) (98–100) or intermittent treatment twice a week (101,102) or use of very potent GCs only for a very few days with subsequent switch to compounds from weaker classes as soon as a response is observed (103,104).

Optimizing pharmacokinetics and biotransformation

Rather than limiting the periods of exposure, selective addressing of target cells and tissues may be a more straightforward approach to achieve immunomodulation while avoiding atrophy.

The major players in atopic dermatitis, psoriasis, and allergic contact dermatitis are T cells. Those may initially be activated by antigen-presenting cells of the skin such as Langerhans cells, plasmocytoid dendritic cells, inflammatory dendritic epidermal cells, or macrophages. Atrophy, on the other hand, is caused mainly by effects on dermal fibroblasts and epidermal keratinocytes. Yet, after decades of GC research, the ostensive lack of potent anti-inflammatory but non-atrophogenic GR ligands clearly illustrates the challenge to hit the respective target cells differentially. This may partly be ascribed to the fact that, under inflammatory conditions, T cells and antigen-presenting cells are present and acting in both epidermis and dermis. Thus, a spatial separation of atrophy and inflammation is virtually not possible.

Whatsoever, atrophogenic effects of dermal fibroblasts may overweigh those of those epidermal keratinocytes, whereas epidermal T cells and antigen-presenting cells may significantly contribute to skin inflammation.

This perception underlies efforts to optimize the physico-chemical properties of topical GC towards high lipophilicity and high molecular size. Increased lipophilicity (log P > 3) may facilitate partition into the skin, whereas increased molecular weight (> 500 Da) may slow down transdermal permeation into the bloodstream, providing skin-selective treatment, especially of the upper skin layers (105,106). Retention of active drug in the tissue with comparably high inflammatory activity but relatively low contribution to atrophy may therefore lead to a superior drug profile.

This principle might explain the favorable benefit/risk ratio, e.g. of prednicarbate (PC; prednisolone 17-ethyl-carbonate, 21-propionate), a topical GC with reduced atrophogenic potential. It is assumed that the advantage of PC relies on its rapid hydrolyzation to prednisolone 17-ethylcarbonate in the epidermis, a metabolite with high GR affinity and anti-inflammatory activity (107). PC as well as this metabolite permeate the skin slowly; thus, most of the drug is decomposed before lower skin levels, the dermis with its more atrophy-sensitive fibroblasts, is ever reached. Similarly, the active metabolite of mometasone furoate was found to have higher GR-binding affinities in the epidermis than in the dermis (108).

Aiming at increased drug permeation into the skin and reduced risk of systemic side effects, optimization of PC parameters as well as the prodrug-drug-antedrug concept has extensively been pursued in topical GC development, as exemplified by PC or methylprednisolone aceponate (MPA). The prodrug properties, achieved by, e.g. esterification, generally also increase lipophilicity and therefore facilitate drug uptake into the stratum corneum (109). The prodrug may have reduced or no pharmacologic activity. Within the skin, the prodrug becomes activated by ester cleavage through cutaneous esterases or spontaneous hydrolysis, as in the case of MPA or the inhaled GC budesonid (110). To prevent the body from systemic GC-mediated effects, the active drug requires biotransformation into an inactive or significantly less active metabolite (antedrug) in the systemic circulation or, with some protraction, even within the skin. This principle is widely accepted and applied. Screening for metabolically unstable corticoids, e.g. those that contain metabolically labile carboxamide moieties on the side chain or at the C-16 position of prednisolone (111) is therefore a standard approach.

Overall, the improved physico-chemical properties and biotransformation are highly rewarding regarding reduction of systemic side effects and may also lessen a compound's atrophogenic potential. Yet, because of the obvious colocalization of atrophy-prone fibroblasts and keratinocytes with the various kinds of immunocytes in the dermis and the epidermis, both its use to counter atrophy and its effect on the pro-inflammatory milieu in the deeper skin layers is logically limited.

More sophisticated adaptations of biotransformation approaches are required to achieve intracutaneous cell type-specific activation or deactivation. PC, for instance, is metabolized and activated to only a minor degree in fibroblasts (about 1%/h), whereas it is rapidly and almost completely activated in keratinocytes in vitro (112). This example shows that cell type-specific properties can in principle be exploited. However, it is obviously not representing the perfect solution, since both cell types account for atrophy and should ideally deal with the drug similarly. Differential metabolization of the compound should clearly be sought to affect immunocytes vs. keratinocytes and fibroblasts.

So far, however, findings of this kind are fortuitous, and stringent strategies to rationally exploit such differences are not established.

In addition, keratinocytes and fibroblasts may contribute also to the pro-inflammatory cascade, e.g. by releasing stimulatory cytokines such as TNF-α and interleukin 1 (IL-1) (113,114). Complete barring of these cell populations from GC action may therefore not be appropriate.

Optimizing formulation

Besides physicochemistry and biotransformation, drug interaction with physical parameters such as skin hydration and formulation properties may also significantly influence atrophy. The importance of delivery issues becomes apparent, e.g. when PC is applied under occlusion. Its superiority regarding induction of atrophy is then abrogated (115). Use of transferosomes, highly flexible lipid vesicles, may on the other hand favorably modulate skin atrophy (116). The clinical use of liposome encapsulation of betamethasone dipropionate has been analyzed in Phase II trial in atopic eczema with an outcome supporting the concept (117). Another approach might involve the use of nanoparticles. These solid lipid particles might also favor epidermal drug delivery and retention vs. dermal delivery (118). It is moreover very tempting to speculate whether such drug-loaded particles would be engulfed by cutaneous phagocytes. If so, this might offer an appropriate way to direct anti-inflammatory compounds straight to crucial immunocytes such as macrophages.

Optimizing pharmacodynamics

The most ambitious and most promising approach to separate beneficial effects and side effects of GR ligands addresses differential molecular modes of action that might underlie immunosuppressive and pro-atrophogenic properties. Such mechanisms do obviously not obey clear-cut rules since GR ligands may activate and suppress gene activation in several different ways. There is evidence, however, that transactivation of GC responsive genes overweighs in side effect induction. In contrast, immunomodulation seems to primarily depend on GC-mediated transrepression, i.e. silencing of pro-inflammatory genes (119). First non-steroidal GR ligands with dissociated properties, termed selective GR agonists (SEGRA) have recently been identified (82,120,121). Such compounds show great potential to reduce side effects of GC although being equipotent regarding immunosuppression. To date, it is not unequivocally clear whether transactivation-dependent molecular events are also crucial for skin atrophy (see above). Rats, however, that were topically treated with a SEGRA compound showed significantly reduced skin atrophy when compared with prednisolone-treated animals. Dissociated GR ligands therefore bear great potential to achieve an improved benefit/risk ratio with respect to atrophy and immunomodulation, whereas epidermal immunocytes may significantly contribute to skin inflammation.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Clinical aspects
  4. Cellular mechanisms of glucocorticoid-induced skin atrophy
  5. Molecular mechanisms of glucocorticoid-induced skin atrophy
  6. Determining atrophogenic potential
  7. Strategies to identify GCs and GC like drugs with a reduced atrophogenic potential
  8. Concluding remarks
  9. Acknowledgements
  10. References

Skin atrophy is the most frequent and important side effect of topical GC therapy, with major consequences because it is irreversible and the thinned skin is fragile and has a diminished barrier function. For over 30 years, the effects of GCs on skin have been investigated showing reduced proliferation of GC target cells, keratinocytes and fibroblasts, disturbed metabolism of ECM proteins, and also in perturbed synthesis of skin lipids. Many approaches and models determine a GCs atrophogenicity in vivo such as measuring skin thickness in hairless rats or mice. Determining the atrophogenic potential of GCs with in vitro models is challenging and not validated. In fact, inconsistent data regarding the influence of GCs on the proliferation and collagen synthesis of keratinocytes and fibroblasts in general has been reported. Thus, in addition to the ongoing fundamental research of GC-induced skin atrophy, it is important to establish a reproducible and consistently in vitro model for determining the atrophogenic potential of GCs. So far, GC-sensitive regulation of only one parameter that of ‘type I collagen’ was examined. It is possible that analyzing the regulation of various ECM proteins gives better information about the atrophogenicity of GCs. Potentially artificial skin could become the future in vitro model for determining the atrophogenicity of GCs.

An insight into the molecular mechanisms of GC-mediated actions opens avenues for the development of novel GR ligands with an improved therapeutic index. Experiences with established topical GCs are instructive as they show that pharmacokinetic optimization may improve the ratio of immunomodulation vs. induction of atrophy. Unfortunately, inflammatory target cells and atrophy-related keratinocytes and fibroblasts reside in the same histological compartments. Complete dissociation by spatial discrimination is therefore not possible. Deducing the well-known prodrug concept, it may therefore be rewarding to design GR ligands that undergo either cell type-specific enzymatic activation in immunocytes, or selective inactivation in fibroblasts and keratinocytes. Alternatively, it is promising to exploit different molecular mechanisms that putatively underlie immunosuppression and atrophy as suggested by the concept of SEGRAs. The ideal topical drug would probably unite superior physicochemical, pharmacokinetic, metabolic, and pharmacodynamic properties. Further progress in understanding the molecular mechanisms of GC-induced skin atrophy and establishment and validation of better models for determining the atrophogenic potential will help us to achieve this.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Clinical aspects
  4. Cellular mechanisms of glucocorticoid-induced skin atrophy
  5. Molecular mechanisms of glucocorticoid-induced skin atrophy
  6. Determining atrophogenic potential
  7. Strategies to identify GCs and GC like drugs with a reduced atrophogenic potential
  8. Concluding remarks
  9. Acknowledgements
  10. References

We thank Dr Howard I. Maibach (San Francisco, California) and Dr Monika Schäfer-Korting for helpful discussions and critical reading of the manuscript. We thank Dr Julie Lucas for correcting the language. We are grateful to Dr Frank Buttgereit (Berlin, Germany), and Dr Charles Goldberg and Jan Thompson (San Diego, California), as well as Dr Lars Röse (Berlin, Germany) for kindly permitting reproduction of the Figs 1, 2, and 4a, respectively.

References

  1. Top of page
  2. Abstract
  3. Clinical aspects
  4. Cellular mechanisms of glucocorticoid-induced skin atrophy
  5. Molecular mechanisms of glucocorticoid-induced skin atrophy
  6. Determining atrophogenic potential
  7. Strategies to identify GCs and GC like drugs with a reduced atrophogenic potential
  8. Concluding remarks
  9. Acknowledgements
  10. References
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