Introduction

Retinoids are known to influence keratinocyte differentiation and have proven to be effective in the treatment of a variety of disorders of keratinization (Zouboulis, 2001). The mechanism of action of retinoids, such as all-trans-retinoic acid (tretinoin), occurs through their interaction with nuclear retinoic acid receptors (RARs), leading to subsequent gene expression via retinoic acid response elements (Idres et al., 2002). The binding affinity of retinoids to various RAR subtypes (, , or ) is thought to contribute to their relative potency. RAR is the predominant subtype in human epidermis (Fisher et al., 1994).

Binding of tretinoin to RARs is modulated by the cellular retinoic acid binding proteins (CRABPs): CRABP I and CRABP II (Donovan et al., 1995). CRABP II is expressed in the skin and regulates nuclear levels of tretinoin by sequestration and/or promotion of its metabolism (Boylan and Gudas, 1992; Fiorella and Napoli, 1994). Acitretin, an aromatic synthetic retinoid, has relatively low affinity for RARs, but high affinity for CRABPs (Norris et al., 1994), and has been shown to induce a shift in tretinoin binding from CRABPs to RAR heterocomplexes (Tian et al., 1997). This suggests that acitretin causes a redistribution of endogenous tretinoin from cytoplasmic to nuclear compartments, thereby indirectly potentiating retinoid activity.

Oral administration of acitretin has been shown to provide therapeutic benefit for the treatment of psoriasis (Lebwohl et al., 2001; Lee and Koo, 2005). It has been marketed for clinical use since 1997 for the treatment of severe psoriasis, and its safety profile is well characterized. It is known to be teratogenic, which limits its use (Kistler and Hummler, 1985; Reiners et al., 1988; Gollnick, 1996).

The clinical efficacy of acitretin following topical application in treating cutaneous diseases is as yet unknown. Percutaneous absorption studies in animals as well as humans have demonstrated that topical application of acitretin can produce dermal concentrations that exceed those achieved with oral administration (Surber et al., 1991, 1993a, 1993b). Topical administration of acitretin may increase the local bioavailability at the site of action (i.e., diseased skin) while minimizing potential systemic exposure; therefore, it would potentially provide a safer treatment for keratinization disorders of the skin.

The main objective of this study was to investigate the potential of acitretin as a topical therapy. Therefore, acitretin activity was benchmarked against commercially available, clinically proven, topical retinoid products. Where possible, direct comparisons between acitretin and other retinoids were made using the same vehicle.

Reconstructed human epidermis (RHE) is a stratified culture that shares histological features that closely resemble human epidermis in vivo: basal layer and differentiating spinous, granular, and cornified layers (Poumay et al., 2004). Tretitoin has been shown to induce specific changes in tissue morphology and biomarker expression in RHE and serves as a benchmark for retinoid effects in this model (Rosdy et al., 1997). Moreover, these responses to tretitoin in RHE cultures have been shown to correlate with those observed in normal human skin ex vivo, thereby validating the use of RHE as a predictive model for the screening of retinoid activity (Bernard et al., 2002).

The Rhino mouse (RHJ/LeJ) is a strain of the hairless mouse, whose skin contains keratinized pilosebaceous structures (horn-filled utriculi). The Rhino mouse utriculi reduction assay is a well-characterized animal model for the evaluation of retinoid activity (Kligman and Kligman, 1979; Mezick et al., 1984). Topically applied retinoids, such as tretitoin, have been shown to reduce the size of utriculi in the Rhino mouse skin, and this effect is considered predictive of retinoid efficacy (Beehler et al., 1995).

The effects of topical acitretin in RHE and Rhino mouse models were investigated in comparison to topical retinoids with known clinical efficacy and are presented in this study.

Results

Reconstructed human epidermis

Hematoxylin and eosin

Untreated control cultures feature intact basal, spinous, granulous, and cornified cell layers, and the epidermal stratification is orthokeratotic, regular, and normal. Cells in the stratum basale were polarized vertically, and numerous keratohyalin granules were detectable in the stratum granulosum. The RHE cultures treated with acitretin, tretinoin, and tazarotene showed a loss of keratohyalin granules in the stratum granulosum (Figure 1), while vehicle-treated cultures were similar to untreated controls.

Figure 1.

Morphological comparison of RHE tissues treated topically with various retinoid formulations. RHE tissues were (a) untreated or treated with (b) vehicle, (c) acitretin 0.02% solution, (d) tretinoin 0.025% cream, or (e) tazarotene 0.1% cream, and incubated for 3 days at 37°C. Following this incubation period, tissues were fixed, sectioned, and stained with H&E. Representative micrographs from triplicate cultures are shown. sc, stratum corneum; sg, stratum granulosum; ss, stratum spinosum; sb, stratum basale. Bar=50 m.

Full figure and legend (204K)

Keratin 10

In untreated control cultures, keratin 10 (K10) is expressed in all suprabasal cell layers. K10 expression was lower in the stratum spinosum of acitretin-treated RHE cultures than in untreated controls. A similar decrease in K10 expression was observed in tretinoin- and tazarotene-treated tissues (Figure 2).

Figure 2.

K10 expression in RHE cultures treated topically with various retinoid formulations. RHE tissues were (a) untreated or treated with (b) vehicle alone, (c) acitretin 0.02% solution, (d) tretinoin 0.025% cream, or (e) tazarotene 0.1% cream, and incubated for 3 days at 37°C. Following this incubation period, tissues were fixed, sectioned, and stained for K10. Representative micrographs from triplicate cultures are shown. Bar=50 m.

Full figure and legend (188K)

Keratin 19

Keratin 19 (K19) is absent in untreated cultures. Treatment with acitretin induced significant expression of K19 in all viable cell layers (Figure 3), although the amount was slightly less than that observed in tretinoin- and tazarotene-treated tissues.

Figure 3.

K19 expression in RHE cultures treated topically with various retinoid formulations. RHE tissues were (a) untreated or treated with (b) vehicle alone, (c) acitretin 0.02% solution, (d) tretinoin 0.025% cream, or (e) tazarotene 0.1% cream, and incubated for 3 days at 37°C. Following this incubation period, tissues were fixed, sectioned, and stained for K19. Representative micrographs from triplicate cultures are shown. Bar=50 m.

Full figure and legend (193K)

Filaggrin

Filaggrin is normally expressed in the stratum granulosum. Acitretin-treated RHE cultures displayed a decreased expression of filaggrin, as did tretinoin- and tazarotene-treated cultures (Figure 4).

Figure 4.

Filaggrin expression in RHE cultures treated topically with various retinoid formulations. RHE tissues were (a) untreated or treated with (b) vehicle alone, (c) acitretin 0.02% solution, (d) tretinoin 0.025% cream, or (e) tazarotene 0.1% cream, and incubated for 3 days at 37°C. Following this incubation period, tissues were fixed, sectioned, and immunostained for filaggrin. Representative micrographs from triplicate cultures are shown. Bar=50 m.

Full figure and legend (175K)

The results described above demonstrate that acitretin modulated epidermal morphology and expression of specific biomarkers of keratinocyte differentiation in a manner similar to that of tretinoin and tazarotene.

Cytokine release

The release of IL-1 and IL-8 in the growth media of RHE cultures treated topically with 0.02% acitretin solution was approximately fourfold higher compared to untreated controls (P<0.01) (Figure 5). IL-1 and IL-8 release in response to tretinoin and tazarotene was 8- 10-fold higher than that in untreated controls, suggesting that acitretin may be less irritating than tretinoin and tazarotene. The cytokine release in vehicle-treated tissues was similar to that in untreated controls, indicating that the vehicle itself was not irritating.

Figure 5.

Release of IL-1 and IL-8 from RHE cultures treated topically with various retinoid formulations. Triplicate RHE cultures were treated with various retinoid formulations and incubated for 3 days at 37°C. At the end of this incubation period, concentrations of IL-1 and IL-8 in the media were determined by ELISA. Values are meanSD, ^*P<0.05 (t-test) compared to untreated control.

Full figure and legend (12K)

Rhino mouse model

Once-daily topical administration of 0.2% acitretin solution for 7 days caused a complete (100%) reduction in utricle diameter, which was similar to that observed in tretinoin- and tazarotene-treated mice (Figure 6 and Table 1). Treatment with a lower concentration of acitretin (0.02%) also resulted in a significant reduction of utriculi, but to a lesser degree. In females treated with 0.02% acitretin, utriculi diameter decreased from 90.418.5 m (sham control) to 2.15.2 m, whereas in males, utriculi diameter decreased from 123.012.9 to 67.614.2 m, representing reductions of 98 and 45% in females and males, respectively. Treatment with vehicle solution alone did not induce a significant reduction in utriculi, demonstrating that the effects of acitretin were specific. Moreover, utricles from skin biopsies taken from nontreated sites were similar to sham controls, indicating that the acitretin effect was local and unlikely to be a result of systemic absorption (Table 1).

Figure 6.

Utriculi of Rhino mice treated topically with various retinoid formulations. Rhino mice (n=6 per group) were dosed topically once daily for 7 consecutive days with (a) water, (b) vehicle alone, (c) acitretin 0.02% solution, (d) acitretin 0.2% solution, (e) tretinoin 0.025% solution, or (f) tazarotene 0.1% cream. Tissues were fixed, sectioned, and stained with H&E. Representative micrographs are shown. Bar=100 m.

Full figure and legend (280K)

Table 1 - Utricle diameter of Rhino mice treated topically with various retinoids.

Full table

Discussion

The cytotoxicity of various solvents in RHE cultures was investigated, and the dose of acitretin used in these experiments (0.02%) was based on its maximum solubility in a non-cytotoxic solvent, octyldodecanol (data not shown). The effects of the tretinoin 0.025% cream formulation on RHE cultures have been well characterized; thus, tretinoin served as a positive control (Bernard et al., 2002). The tazarotene formulation has not been previously demonstrated to induce effects in the RHE model, and served as an additional comparator control. Because tazarotene is a proprietary compound, the concentration and formulation used (0.1% cream) were based on its commercial availability.

Acitretin induced in vitro effects in RHE cultures which were comparable to those of tretinoin- and tazarotene-treated cultures. These effects included degranulation, induction of K19 expression, and reduction of K10 and filaggrin. K19 is a non-epidermal keratin that is absent in normal human epidermis. K10 is an early biomarker of epidermal differentiation and is normally expressed in all suprabasal layers (Poumay et al., 1999; Ekholm and Egelrud, 2000). Filaggrin is a marker of late-stage keratinocyte differentiation that is normally expressed in the stratum granulosum. Although the immunohistochemistry data presented are not quantitative, these in vitro results suggest that acitretin directly affects differentiating keratinocytes in a manner similar to other topical retinoids with well-established dekeratinizing effects.

The release of pro-inflammatory cytokines, IL-1 and IL-8, in RHE cultures is a well-established biomarker of acute irritation (de Brugerolle de et al., 1999; Medina et al., 2001; Tornier et al., 2006). Topical tretinoin and tazarotene are generally recognized as being clinically effective, but are known to be associated with dermal irritation, which may limit their use. In the RHE model, the acitretin solution appears to be less irritating than the two commercially available products tested (tretinoin and tazarotene creams), suggesting that a topical acitretin product, in a non-irritating vehicle, may strike a more optimal balance between efficacy and irritation in treating disorders of keratinization, while minimizing the risk of systemic adverse effects.

The Rhino mouse bioassay has frequently been used to evaluate the potential efficacy of topical retinoids. Commonly prescribed topical retinoids, such as tretinoin and tazarotene, have shown positive results in the Rhino mouse bioassay in their commercial formulations (Sakuta and Kanayama, 2005). The dermal toxicity of various solvents on Rhino mice was investigated, and the highest dose of acitretin used in these experiments (0.2%) was based on its maximum solubility in a non-toxic solvent, dimethyl isosorbide (data not shown). The lower dose of 0.02% acitretin was chosen in an attempt to demonstrate a concentration-dependent effect. Tretinoin was diluted in the same solvent as acitretin, thus allowing for a direct comparison. The concentration of tretinoin used (0.025%) was chosen on the basis of its clinically effective dose as well as the results of previously published studies using this model (Kligman and Kligman, 1979; Bernerd et al., 1991; Bouclier et al., 1991). Because tazarotene is a proprietary compound, the concentration and formulation used were based on its commercial availability. Tazarotene 0.1% has been previously shown to reduce utriculi diameter in Rhino mice (Sakuta and Kanayama, 2005).

Topical acitretin induced a localized, dose-dependent decrease in utricle diameter in Rhino mice, which was comparable to that of tretinoin- and tazarotene-treated animals. There was no significant increase in epidermal hyperplasia, indicating that dermal inflammation did not significantly contribute to utriculi reduction. Utricle reduction appeared to be sex dependent, which was surprising as this effect has not been reported in previous studies involving male and female Rhino mice (Bouclier et al., 1991). More studies are required to confirm this observation.

Orally administered acitretin has been used for over 10 years in the treatment of severe psoriasis. Although acitretin has been proven to be effective, concerns about potential side effects associated with its systemic absorption have limited its use. Topical administration of acitretin may reduce the risk of systemic exposure, with a potential to increase local efficacy in the treated skin disease area. We demonstrated that acitretin can induce dekeratinizing effects in vitro and in vivo when applied topically; therefore, it may be effective in treating skin diseases involving abnormal keratinization.

Materials and Methods

Reconstructed human epidermis model

RHE cultures were purchased from SkinEthic Laboratories (Nice, France). The cultures were maintained in chemically defined growth medium MCDB 153, containing 5 g/ml insulin, 1.5 mM calcium chloride, 25 g/ml gentamycin, and 1 ng/ml EGF. The preparation and characterization of these cultures have been described previously (Rosdy et al., 1997). Briefly, a fully differentiated epidermis was obtained by culturing primary keratinocytes, isolated from normal human foreskin and cultured on an inert microporous polycarbonate filter at the air–liquid interface for 14 days. The following test articles were applied topically at 5 l/0.5 cm² to triplicate RHE tissues and incubated for 3 days at 37°C/5% CO₂: acitretin (Roche, Basel, Switzerland) 0.02% dissolved in octyldodecanol (Cognis, Cincinnati, OH; 0.02% was the maximal amount of acitretin soluble in octyldodecanol), tretinoin cream 0.025% (Ortho Dermatological, Skillman, NJ), and tazarotene cream 0.1% (Allergan, Irvine, CA). Nylon mesh (8 mm diameter) was deposited onto the RHE tissues before application to ensure even spreading of the test articles. Triplicate untreated tissues and acitretin vehicle (octyldodecanol)-treated tissues served as negative controls. This study was performed twice using n=3 per group, and the data presented are representative of the two studies.

Cytokine release

Release of IL-1 and IL-8 was quantified in the RHE culture medium using ELISA kits from R&D Systems (Minneapolis, MN).

Rhino mouse model

Male and female Rhino mice (RHJ/LeJ) were purchased from The Jackson Laboratory (West Sacramento, CA). All animal handling was conducted at JAX Services (West Sacramento, CA) in compliance with the NIH Guide for the Care and Use of Laboratory Animals and approved by The Jackson Laboratory IACUC. Animals were housed individually in static caging in a room with yellow lighting (>500 nm). The mice had access to acidified tap water (pH 2.8–3.2) and Purina Lab Diet 5LL4 ad libitum throughout the study. Thirty-six mice, 5–7 weeks of age, were treated once daily for 7 days with the following: acitretin (0.2 or 0.02%) dissolved in dimethyl isosorbide (DMI, Uniqema, New Castle, DE) containing 0.1% butylated hydroxytoluene (BHT, Sigma-Aldrich, St Louis, MO); tretinoin (0.025%) dissolved in DMI containing 0.1% BHT; or tazarotene (0.1%) cream (n=6 per group). Sham (water treated) and vehicle controls were included. Coban dressings were applied to the mice after dosing to prevent rub-off and oral ingestion of the test agents. On day 7, 5 hours post-dosing, the skin at the site of application was harvested and cut in half (rostral and caudal sections) for histological evaluation. Ventral tissue (non-SOA) was harvested and used as negative control.

Histology

RHE cultures were fixed in 10% formalin (Richard Allen Scientific, Kalamazoo, MI) and embedded in paraffin. Sections (4 m) were stained with H&E or using immunohistochemical staining techniques for K10, K19, or filaggrin. Antibodies and staining kits were obtained from Vector Laboratories (Burlingame, CA) and used according to the manufacturer's recommendations.

Rhino mouse skin specimens were fixed in alcohol formalin and embedded in paraffin. Three serial sections (4 m) spaced 100 m apart were taken and stained with H&E. Histology was carried out by IDEXX Laboratories (Sacramento, CA).

Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

We acknowledge Sao Kun (Gloria) Ao Ieong for her help with histology, Dr Marina Aliferis of SkinEthic Laboratories (Nice, France) for conducting the RHE studies, and Dr Kara Koehler of The Jackson Laboratory (Sacramento, CA) for conducting the Rhino mouse studies.