Acta Derm Venereol 2009; 89: 12–20 INVESTIGATIVE REPORT Expression of Retinoid-regulated Genes in Lamellar Ichthyosis vs. Healthy Control Epidermis: Changes after Oral Treatment with Liarozole* Elizabeth PaveZ LORIÈ1, Agneta GÅNEMO1, Marcel BORGERS2, Luc WOUTERS2, Stan BLOCKHUYS2, Lieve van DE PLASSCHE2, Hans TÖRMÄ1 and Anders VAHLQUIST1 1Department of Medical Sciences/Dermatology, Uppsala University, Uppsala, Sweden, and 2Barrier Therapeutics nv, Geel, Belgium Lamellar ichthyosis is a keratinization disorder caused Lamellar ichthyosis (LI) is a rare autosomal recessive by TGM1, Ichthyin and several other gene mutations. disorder of keratinization characterized by generalized A new treatment option is liarozole, which blocks the scaling and a variable degree of skin erythema. The cyto chrome P450 (CYP26)-mediated catabolism of en- aetiology of LI is heterogeneous: the first discovered dogenous all-trans retinoic acid. This study focuses on gene abnormalities were truncating mutations in TGM1, the expression of retinoid-related genes in ichthyotic encoding transglutaminase 1, which cross-links the cor- epidermis before and after treatment with oral liaro- nified cell envelope proteins in stratum corneum. Sub- zole. We first compared the mRNA expression of cellu- sequently, several other genes involved in the formation lar retinoic acid binding protein II (CRABPII), keratin of the epidermal lipid barrier have been implicated in the (KRT) 2 and 4, CYP26A1 and B1, and two markers of aetiology of LI, including Ichthyin, ALOX12B, ALOXE3, inflammation (interleukin-1α and tumours necrosis fac- and ABCA12 (for review see 1, 2). tor (TNF)-α) in shave biopsies from 11 genetically de- Treatment of LI comprises topical moisturizers, fined, untreated patients and 12 age- and sex-matched keratolytic agents and vitamin A analogues (retinoids) healthy controls, finding no overt differences between (2, 3). Oral therapy with synthetic retinoids is usually the groups, besides elevated CRABPII expression. We effective in ameliorating LI, but may be associated then studied the biomarkers before and after 4 weeks of with side-effects (for review see 1, 4). An alternative treatment with liarozole (75 or 150 mg/day), which pro- approach is to try to increase the endogenous level of duced a better therapeutic response in patients with Icht- all-trans retinoic acid (ATRA) in the skin by blocking hyin (n = 3) than in those with TGM1 (n = 6) mutations. its catabolism to less active vitamin A metabolites (5). A significant decrease in the mRNA expression of KRT2 Compounds with this propensity are called retinoic acid and TNF-α, and trends toward increased expression of metabolism blocking agents (RAMBAs). One such sub- KRT4 and CYP26A1 were observed in liarozole-treated stance, talarozole (also known as RambazoleTM, Barrier patients, consistent with an increased retinoid stimula- Therapeutics nv, Geel, Belgium) was recently shown by tion of epidermis. However, there were no dose-related us to affect the expression of retinoid-regulated genes responses and the results of the immunostaining did in healthy human skin (6). This is indirect proof of in- not always parallel the mRNA findings. The results sug- creased tissue levels of ATRA during talarozole therapy, gest that liarozole exerts a therapeutic effect in lamellar while ATRA per se cannot be quantified in small skin ichthyosis by mildly affecting the expression of retinoid- biopsies due to methodological limitations (Vahlquist, regulated genes in epidermis. Key words: retinoic-acid unpublished observation). metabolism blocking agents; keratin; vitamin A metabo- Yet another RAMBA, liarozole (LiazaTM, Barrier lism; cytochrome P450, skin disorders. Therapeutics nv, Geel, Belgium), has been shown to be effective as an oral and topical treatment of icht- (Accepted August 5, 2008.) hyosis and psoriasis (for review see 7). Although its Acta Derm Venereol 2009; 89: 12–20. effects on ATRA metabolism and expression of retinoid biomarkers have been studied in healthy human skin Anders Vahlquist, Department of Medical Sciences/ (8), the corresponding effects in LI epidermis have not Dermatology, University Hospital, SE-751 85 Uppsala, previously been studied in any detail. Sweden. E-mail: [email protected] Vitamin A metabolism in human keratinocytes involves esterification of retinol to fatty acyl esters and oxidation of retinaldehyde to ATRA; processes which are catalysed *Results from this study have been presented preliminarily at the International by lecithin:retinol acyltransferase (LRAT) and retinalde- Investigative Dermatology meeting in Kyoto, 2008 (Elizabeth Pavez Loriè et al. Altered expression of retinoid regulated genes during oral liarozole hydrogenase-2 (RALDH2 or ALDH1A2), respectively treatment of lamellar ichthyosis, J Invest Dermatol (2008), 128, S60). (for review see 9). The cellular level of ATRA is also The corresponding author is also the Editor-in-Chief of this journal; the regulated by cellular retinoic acid binding protein II article has been handled fully by one of the Associate Editors, who made (CRABPII), which is highly expressed in human epi- the decision for acceptance. dermis, especially after ATRA therapy (10). CRABPII Acta Derm Venereol 89 © 2009 The Authors. doi: 10.2340/00015555-0573 Journal Compilation © 2009 Acta Dermato-Venereologica. ISSN 0001-5555 Liarozole in lamellar ichthyosis: effect on gene expression 13 functions both as a “hydrophobic sink” for ATRA (11) and clinical trial when all her skin symptoms had relapsed; the latter as its shuttle to the cell nucleus where it binds to a family biopsy was considered analogous to baseline. All patients had moderate to severe LI, with ichthyosis scores of retinoid receptors all involved in gene regulation (12). ranging from 20 to 36 (maximal score 44). Seven patients had Accordingly, ATRA affects the transcriptional activity received acitretin previously (but not within one month prior of epidermal genes, such as those encoding CRABPII, to the trial), and all were using emollients on a regular basis keratin (KRT) 4 and KRT2 (13, 14). during the trial (cream application was avoided for > 10 h prior The catabolism of ATRA in epidermis is controlled by to clinical scoring and skin sampling). After randomization, the patients received tablets containing several cytochrome P450-dependent enzymes (namely liarozole (75 or 150 mg) or vehicle in identical-appearing capsu- CYP26A1, B1 and C1), which catalyse the formation les for 3 months. Vital signs, electrocardiogram (ECG), routine of less active 4-hydroxy and 18-hydroxy metabolites blood chemistry, urinalysis, and severity of skin symptoms were (15, 16), and are the intended targets for RAMBA checked before the trial and at monthly intervals during the trial. therapy. Recent studies have identified a retinoic acid The results of the laboratory tests were essentially negative, and will be reported separately. At baseline and after 4 weeks of treat- response element in the CYP26A1 gene (17), supporting ment, two punch biopsies (diameter 3 mm) and one superficial the existence of an auto-regulatory loop that controls shave biopsy, typically containing approximately 80% epidermis the cellular level of ATRA (18). Another cytochrome (from now on referred to as “epidermis”) (22), were obtained P450 enzyme, CYP2S1, was also shown to catabolize from the buttocks after infiltrating the skin with 1 ml of lidocaine- ATRA in epidermis (19), but its importance for ATRA adrenalin (Astra Zeneca, Södertälje, Sweden). The time interval between the samplings (1 month) was chosen so that early rather homeostasis is still uncertain. than late effects of liarozole should be highlighted. The main purpose of this study, performed in a sub- The punch biopsies were first placed in cold Histocon (Histo- set of patients with LI participating in a randomized lab Products AB, Gothenburg, Sweden) and then subsequently multi-centre trial examining the clinical effects of two snap frozen and stored at –70°C. The shave biopsies were im- different doses of oral liarozole1, was to investigate mediately frozen on dry ice and kept at –70°C until isolation of total ribonucleic acid (RNA) was performed. The blinding whether the epidermal expression of retinoid-related of the samples (by an independent employee) was performed biomarkers and metabolizing enzymes is affected in a before the start of the analysis and kept secret until data proces- manner consistent with the drug’s proposed mechanism sing was complete. of action. As a corollary, we compared the gene expres- Shave and punch biopsies from a group of 12 age- and sex- sion pattern in untreated ichthyotic skin vs. normal matched healthy individuals (average age: 41 years, male/fema- le: 5/7), most of whom had participated in a previous study (6), skin, and looked for differences in the clinical efficacy were used to establish the normal mRNA and protein expression of liarozole that could be related to the aetiology of profiles for the biomarkers. The control samples were analysed ichthyosis, e.g. TGM1 or Ichthyin mutations. in parallel with the patients' baseline samples. RNA isolation and reverse transcription Materials AND METHODS Isolation of total RNA and reverse transcription has been de- scribed in detail previously (23). In brief, RNA was prepared by Study design homogenizing the tissue in 1 ml Trizol (Invitrogen, Carlsbad, The patients included in this study were part of a larger multi- CA, USA), and the concentration was determined by spectro­­