Acta Derm Venereol 2009; 89: 12–20

INVESTIGATIVE REPORT Expression of -regulated Genes in Lamellar Ichthyosis vs. Healthy Control Epidermis: Changes after Oral Treatment with *

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 cytochrome­ P450 (CYP26)-mediated catabolism of en- aetiology of LI is heterogeneous: the first discovered dogenous all-trans . 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 analogues () 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; , 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 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 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 centre clinical trial designed as a randomized, double-blind study comparing two doses of liarozole (75 mg/day and 150 mg/day) and placebo in 96 patients (at a ratio of 7:7:1) for 3 Table I. Patient’s characteristics, doses of liarozole and ichthyosis months followed by a washout period1. Both the clinical trial scores in the clinical trial and the biopsy study were approved by the local ethics com- mittee at Uppsala University and by the Swedish authorities, Age Body Affected Liarozole Ichthyosis score and were performed according to the ethical principles of the No. Sex (years) weight (kg) gene dose (mg) Baseline 1 month Declaration of Helsinki. The patients signed a written informed 1 F 25 55 TGM1 Placebo 35 27 consent at the beginning of the trial. 2a F 26 50 TGM1 150 34 26 Twelve patients with LI, 4 males and 8 females (all Caucasians) 3 F 48 75 TGM1 75 36 27 aged 18–69 years, were recruited via our genodermatosis centre. 4 M 24 78 TGM1 150 35 34 Seven of the patients carried TGM1 mutations (20), 4 had Ichthyin 5 M 28 85 TGM1 75 34 20 mutations (21), and one patient was negative for these two types 6 M 32 64 TGM1 150 35 32 of mutations but showed otherwise typical findings of LI (Table 7 M 65 55 TGM1 75 35 34 I). One patient (no. 8) did not consent to skin biopsy but in all 8b F 18 56 Ichthyin 150 20 1 other aspects completed the clinical trial according to the proto- 9 F 54 65 Ichthyin 75 23 1 col. Another patient (no. 11) first accepted skin biopsy but was 10 F 61 71 Ichthyin 75 28 12 c later excluded from the clinical trial because of a heart problem 11 F 69 63 Ichthyin – 21 NA detected at screening. Although patient no. 2 was not available for 12 F 66 83 ? 75 22 7 skin sampling at screening, she accepted biopsy after one month aThe baseline sample in this case was obtained first one month after of therapy and again after the washout period at the end of the stopping therapy, i.e. when the ichthyosis score had returned to pre- therapy value. bNo biopsies were obtained from this patient. 1Vahlquist et al., Liarozole effective in treatment of lamellar ichthyosis, poster cThis patient was excluded from the clinical trial but a baseline biopsy was abstract, IID2008 Meeting, Kyoto, J Invest Dermatol (2008), 128, S60 taken and used in the comparison with healthy controls. NA: not applicable. Acta Derm Venereol 89 14 E. Pavez Loriè et al. photometry. First-strand complementary deoxyribonucleic acid diluted 1/40, KRT2 (clone: Ks2.342.7.1, AbCam plc, Cambridge, (cDNA) synthesis was performed using 1.5 µg of total RNA as UK) diluted 1/800, CYP26B1 (clone: 1H6, AbNova, Taipei City, template. After synthesis was complete, the cDNA was diluted Taiwan) diluted 1/50, or a polyclonal rabbit anti-human CYP26A1 with dH2O to obtain a concentration of 10 ng cDNA/µl. antibody (Alpha Diagnostics, San Antonio, TX, USA), again diluted 1/50. Biotinylated horse anti-mouse IgG (H+L) (Vector Laboratories Inc.) served as secondary antibody for the KRT4 Quantitative real-time polymerase chain reaction and CYP26B1. A secondary biotinylated goat anti-rabbit antibody The number of transcripts was studied by quantitative real-time was used for CYP26A1 followed by 30 min incubation with Texas polymerase chain reaction (QRT-PCR) as previously described Red Avidin D (Vector Laboratories Inc.). KRT2 was detected by (23). The primers and probes were obtained from Applied incubating with FITC conjugated rabbit anti-mouse IgG (Dako­ Biosystems; the sequences are described in Table II. Triplicate Cytomation, Glostrup, Denmark) for 30 min. CRABPII was reaction tubes were set up for each sample and the 25 µl reaction visualized by using a previously described monoclonal antibody was performed in a MyiQ (BioRad Laboratories, Inc., Hercules, (clone: 5CRA3B3) (12) diluted 1/200, followed by incubation CA, USA). Simultaneous amplification of known amounts of with biotinylated horse anti-mouse and Texas Red Avidin D as a PCR product generated a standard curve for comparison. previously described (25). The slides were mounted with The expression of two housekeeping genes (cyclophilin A and Vectashield Hard Mounting Medium containing DAPI (Vector β-actin) was used to normalize the expression of the genes of Laboratories Inc.). The staining was analysed and evaluated using a interest, with the help of geNORM software (Center for Medical Zeiss Axio Imager Z1 microscope and AxioVision software (Zeiss, Genetics, Ghent University, Ghent, Belgium) (24). Göttingen, Germany) as previously described (6). The microscope was equipped with filters for FITC, Texas Red and DAPI. The Analysis of protein expression staining intensities were scored using a scale from zero (negative) to three (very intense staining) for all examined proteins. Two punch biopsies were obtained at each sampling. One was fixed in Lana’s fixative for 30 min and then snap-frozen (for CRABPII Determination of epidermal and stratum corneum thickness analysis), while the other was frozen immediately (for all other histological analysis). Sections (6 µm) were fixed in 100% ice- Sections from the punch biopsies were stained with Harris cold acetone for 5 min, blocked with 10% normal horse or rabbit Haematoxylin (Histolab Products AB). The length (L) and area serum for 15 min (both from Vector Laboratories Inc., Burlingame, (A) of the epidermis and stratum corneum of each section was Canada), and incubated overnight at 4°C with monoclonal anti­ measured using a 20× magnification and the microscope and bodies: KRT4 (clone: 6B10, Gentex, San Antonio, TX, USA) software as described above. Epidermal and stratum corneum thickness (Th) was calculated (according to Th = A/L) from these measurements. Table II. Polymerase chain reaction (PCR) primers (F: forward, R: reverse and P: probe). Sequences are given in 5’-3’orientation Statistics

CRABPII F: AGCAGAAGCTCCTGAAGGGA Data obtained for mRNA and epidermal thickness were log-trans- R: CCCATCGTTGGTCAGTTCTCT formed prior to statistical analysis. A paired Student’s t-test was P: AGGGCCCCAAGACCTCGTGGAC used to evaluate the statistical significance of differences between KRT4 F: GCAGCTAGATACCTTGGGCAA data obtained at the baseline visit and data obtained at the visit after R: CTTCATACTTAGTCTTGAAGTCCTCCAC 4 weeks of treatment. p-values obtained for mRNA gene levels P: CTGTCCTGCATGGTCTTCAGCTCAGACT were adjusted for multiplicity using the False Discovery Rate (26) KRT2 F: TCTTGTGGAGGATTATAAGAAG controlling procedure. Two-sided p-values less than or equal to R: GTAGGCATTGTCCACGTCCTTT 0.05 were considered to indicate statistical significance. For mRNA P: Acaaaatcattctcagcagctgtgcgctta and epidermal thickness measurements, results were expressed as CYP26A1* F: TCACTTACCTGGGGCTCTACC geometric mean % difference and 95% confidence intervals. R: ACTTGTTGTCTTGATTGCTCTTGC CYP26B1* F: CCGCTTCCATTACCTCCCGTTC R: CCACCGCCAGCACCTTCAG Results CYP2S1* F: GCAGATGGACGGTTCAGGAAG R: AGGGAGAAGGCTTGTAGGATGG Comparison of mRNA expression in ichthyosis vs. RALDH2* F: CAGACTTGGTGGAACGGGACAG R: AGCCCAGCCTGCGTAATATCG normal skin at baseline LRAT F: CTGGTCAATCACCTGGACGA R: CCGCGCCACCTCCTC To determine whether the patients’ mRNA values were P: TCCCTCCAGAAAAAGGCACTGCTCAA abnormal at baseline, a comparison was made with age- IL-1α* F: CGCCAATGACTCAGAGGAAGA and sex-matched healthy controls. As shown in Table R: AGGGCGTCATTCAGGATGAA TNF-α* F: TGGCCCAGGCAGTCAGA III, the mean mRNA values of the genes of interest R: GGTTTGCTACAACATGGGCTACA did not differ markedly between patients and controls, Cyclophilin F: TGCTGGACCCAACACAAATG with the exception of CRABPII mRNA, which was six R: TGCCATCCAACCACTCAGTC times higher in the patients (p < 0.001). There were no P: TTCCCAGTTTTTCATCTGCACTGCCA β-actin F: ctggctgctgaccgagg obvious correlations between the mRNA expressions R: gaaggtctcaaacatgatctgggt and the disease aetiologies (data not shown). P: cctgaaccccaaggccaaccg *SYBR Green I was included in the PCR master mix instead of probe. Effects of liarozole therapy KRT: keratin; LRAT: lecithin:retinol acyltransferase; RALDH2: retinal­ dehydrogenase-2; CYP26: cytochrome P450; IL-1α: interleukin 1α; TNF-α: Clinical findings. All patients completed the medica- tumour necrosis factor α; CRABPII: cellular retinoic acid binding protein II. tion according to the protocol. No overt systemic toxi-

Acta Derm Venereol 89 Liarozole in lamellar ichthyosis: effect on gene expression 15

Table III. Epidermal mRNA expression of genes of interest in Table IV. Thickness of stratum corneum and viable epidermis untreated patients with lamellar ichthyosis and age- and sex- after 4 weeks of liarozole treatment (expressed as percentage of matched healthy controls baseline value)

Mean mRNA expression Thickness 3 a Gene (Ratio to housekeeping gene ×10 ) n (% of baseline) 95% CI product Patients (n = 11) Controls (n = 12) p-value Stratum corneum 8 88 (67–117) NSb Viable epidermis 8 128 (88–188) NSb CRABPII 317000 46800 0.0006a KRT2 5700 4700 0.36 aOf a total of 10 patients, one did not accept punch biopsies and another KRT4 190 180 0.69 received placebo. Therefore only 8 patients were included in this analysis. CYP26A1 0.007 0.002 0.17 bStatistically non-significant p( > 0.05). CYP26B1 73 72 0.76 95% CI: 95% confidence interval. LRAT 12 3.8 0.09 RALDH2 171 198 0.55 IL-1α 71 62 0.57 was a tendency towards reduced thickness of stratum TNF-α 110 75 0.55 corneum and increased thickness of viable epidermis ap < 0.05 is indicated in bold figures. after one month of therapy, but the changes were not KRT: keratin; LRAT: lecithin:retinol acyltransferase; RALDH2: retinalde­ statistically significant and no clearly dose-dependent hydrogenase-2; CYP26: cytochrome P450; IL-1α: interleukin 1α; TNF-α: differences were noted (data not shown). tumour necrosis factor α; CRABPII: cellular retinoic acid binding protein II. Changes in mRNA expression. The mRNA expression of ten genes was studied in paired shave biopsies obtained city was noted. The ichthyosis score after one month at baseline and after one month of treatment. Figs 1–3 improved most dramatically in patients with Ichthyin show the individual results observed in liarozole-treated mutations (n = 3) or unknown aetiology (n = 1) who patients (n = 9) in relation to both the underlying genetic had initially a milder disease; the response in patients cause of LI and the dose of liarozole. The mRNA values with TGM1 mutations (n = 6) was less pronounced, of the placebo-treated patient changed only minimally and in some cases not clearly different from that in during the trial (data not shown). Table V shows the statis- the placebo-treated patient (see Table I). tical analysis of liarozole-induced changes in mRNA for Disclosure of the randomization code showed that 6 all genes described below and for all patients together. of the biopsied patients received 75 mg of liarozole per Effects on well-established biomarkers. From the day, 3 received 150 mg/day and one received placebo combined results presented in Fig. 1 and Table V it is (see Table I). The unintended skewed distribution of evident that: (i) CRABPII expression increased in 7 out subjects between the liarozole groups was partially of 9 patients on therapy, but the mean change compared due to one patient in the high-dose group declining a with baseline (+37%) was not statistically significant, repeated biopsy. Because of this distortion, statistical probably as a result of high pre-therapy levels of CRAB- analyses (see below) were performed on all liarozole- PII mRNA (see above); (ii) KRT2 expression decreased treated patients together, irrespective of the dose. in all patients (p < 0.003), a result consistent with pre- Analysis of epidermal thickness. Histological sec- vious findings in healthy skin exposed to topical ATRA tions from repeated punch biopsies were available in 8 (13) or talarozole (6); (iii) KRT4 expression increased liarozole-treated patients. As shown in Table IV, there in 7 patients, but decreased in one patient, explaining

Fig. 1. Individual mRNA expression of three well-known retinoid biomarkers before and after 4 weeks of liarozole treatment. The mRNA expression of: (A) cellular retinoic acid binding protein II (CRABPII); (B) keratin 2 (KRT2); and (C) KRT4 is shown. Circles denote patients with TGM1 mutation (n = 6), squares denote patients with Ichthyin mutation (n = 2), and triangles denote one patient with unknown mutations. The subjects who received the higher dose (150 mg) are represented by filled symbols and those who received the lower dose (75 mg) by open symbols. All values were normalized to the expression of the two housekeeping genes, cyclophilin and β-actin (using geNORM software). Only KRT2 was significantly affected p( < 0.05).

Acta Derm Venereol 89 16 E. Pavez Loriè et al.

Table V. Epidermal mRNA expression of retinoid biomarkers, Effects on retinoid metabolizing enzymes. Despite retinoid metabolizing enzymes and markers of inflammation after markedly increased CYP26A1 levels in some patients 4 weeks of liarozole treatment expressed as percentage of baseline value (n = 9) receiving the higher dose of liarozole (Fig. 2A) and a mean treatment level three times above baseline Gene mRNA expression False discovery rate (Table V), the change was not statistically significant product (mean % of baseline) 95% CI adjusted p-value (p < 0.077). Interestingly, patients with a strong induc- CRABPII 137 (96–196) 0.151 tion of CYP26A1 by liarozole also showed an increased KRT2 30 (19–47) 0.003a expression of CRABPII and KRT4 and a decreased KRT4 434 (135–1394) 0.067 expression of KRT2, consistent with the anticipated re- CYP26A1 427 (119–1535) 0.077 CYP26B1 142 (81–251) 0.289 sponse to raised epidermal ATRA levels during therapy CYP2S1 109 (81–146) 0.567 (results of comparisons not shown). LRAT 76 (22–269) 0.635 CYP26B1, LRAT, and RALDH2 mRNA did not change RALDH2 117 (74–185) 0.560 IL-1α 45 (12–169) 0.289 consistently during liarozole therapy (Fig. 2B–D and TNF-α 54 (38–78) 0.022a Table V). CYP2S1 mRNA was barely detectable both be- ap < 0.05 is indicated in bold. fore and after therapy (no individual values are shown). CI: confidence interval; KRT: keratin; LRAT: lecithin:retinol Effects on pro-inflammatory cytokines. Although acyltransferase; RALDH2: retinaldehydrogenase-2; CYP26: cytochrome both interleukin 1α (IL-1α) and tumour necrosis factor P450; IL-1α: interleukin 1α; TNF-α: tumour necrosis factor α; CRABPII: α (TNF-α) mRNA were, on average, reduced by 50% cellular retinoic acid binding protein II. during therapy, statistical analysis only showed sig- nificant reduction of TNF-α (Table V). In fact, three why a mean change of almost three times above baseline of the patients showed increased expression of IL-1α was not statistically significant (p < 0.067). KRT4 is not after liarozole (Fig. 3A). In a previous study on healthy detected in normal skin, but is highly expressed in the skin, topical talarozole was found to significantly down upper epidermis during topical treatment with talarozole regulate IL-1α, but not TNF-α expression (6). (6) or ATRA (13). The reason for the high baseline level Changes observed in the immunostaining patterns. of KRT4 mRNA in one patient (no. 12) is unclear; her The protein expression in skin biopsies obtained before baseline CRABPII and KRT2 values were unremarkable, and after liarozole treatment was studied by immuno­ making any inadvertent or unreported retinoid exposure fluorescence analysis using commercially available prior to our study highly unlikely. antibodies against common retinoid biomarkers. Fig. 4

Fig. 3. Individual mRNA expression of two pro- Fig. 2. Individual mRNA expression of four enzymes involved in retinoid metabolism proteins inflammatory cytokines before and after liarozole therapy. before and after liarozole treatment. The diagrams correspond to: (A) cytochrome P450 The diagrams correspond to: (A) interleukin (IL)-1α; (CYP26)A1; (B) CYP26B1; (C) retinaldehydrogenase-2 (RALDH2); and (D) lecithin:retinol and (B) tumour necrosis factor (TNF)-α. (For other acyltransferase (LRAT). (For other explanations see Fig. 1). None of these enzymes of retinoid explanations see Fig. 1). Only TNF-α was significantly metabolism were significantly changed as a result of therapy. affected by the treatment with liarozole (p < 0.05).

Acta Derm Venereol 89 Liarozole in lamellar ichthyosis: effect on gene expression 17 shows examples of CRABPII, KRT2, KRT4, CYP26A1 therapy (see above), KRT2 protein expression was and CYP26B1 staining in normal skin, in ichthyotic skin slightly increased by liarozole. KRT4 was negative at baseline and after liarozole therapy, and in appropriate both in normal skin and in ichthyotic skin at baseline, controls. In accordance with the mRNA results (see but a faint staining of the granular layer appeared after Table III) and previous protein analysis (27), CRABPII liarozole therapy, and this co-localized with the staining was over-expressed in untreated ichthyotic epidermis in healthy skin exposed to ATRA. compared with normal epidermis, which was stained Figure 4 further shows that CYP26A1 expression, only in the stratum granulosum. No clear-cut increase which was confined to the basal layer in normal skin, in CRABPII staining was observed in liarozole-treated was increased in both intensity and extension in ichthy­ skin. KRT2, which was strongly expressed in the supra- otic epidermis before therapy, and decreased somewhat basal layers of normal epidermis, occurred in reduced after therapy. This was in sharp contrast to the increased amounts in ichthyotic skin at baseline. However, in CYP26A1 mRNA expression seen after therapy (see contrast to the strongly reduced mRNA levels after above). Finally, the weak and scattered CYP26B1

Fig. 4. Immunofluorescence staining of cellular retinoic acid binding protein II (CRABPII), keratin (KRT)2, KRT4, cytochrome P450 (CYP26)A1 and CYP26B1 in normal skin and in ichthyotic skin before and after liarozole treatment (patient number 6, Table I). The first row shows the expression of CRABPII protein in (a) normal and (b) ichthyotic skin before therapy showing staining of suprabasal layers, which did not change after therapy (c). A negative control omitting primary antibody is shown in (D). The second row shows examples of KRT2 expression in (e) normal human skin, (f) ichthyotic skin at baseline, and (g) ichthyotic skin after 4 weeks of liarozole (150 mg per day). A negative control is shown in (h). The third row shows negative expression of KRT4 in (i) normal skin and (j) the baseline skin biopsy; this is in contrast to (k) a faint positive staining of the stratum granulosum in liarozole-treated skin and (l) a strong staining of this layer in normal skin treated with all-trans retinoic acid for 4 days under occlusion (positive control). The fourth row represents CYP26A1, showing (m) staining of the basal layer in normal skin (which could be blocked by specific peptide), (n) a more diffuse staining of ichthyotic skin at baseline, (o) attenuation after liarozole therapy and (p) a negative control. The last row shows CYP26B1 staining which was (q) essentially negative in normal skin, and patchily positive in the epidermis and papillary dermis of ichthyotic skin both (r) at baseline and (s) after liarozole therapy. (T) is a negative control omitting the CYP26B1-specific antibody. Bar: 50µm. Dotted line: basal membrane zone.

Acta Derm Venereol 89 18 E. Pavez Loriè et al.

Table VI. Scoring (0–3) of the protein expression in skin biopsies expression pattern has been noted in psoriasis lesions obtained before and after liarozole treatment. Data are presented a (25) also characterized by keratinocyte hyperprolife- as change in relation to pre-treatment value (n = 8 ) ration. Therefore, increased CRABPII expression may Protein Mean change p-value be secondary to epidermal hyperplasia in LI and not a CRABPII 0.5 0.124 reliable indicator of ATRA stimulation during liarozole KRT2 0.7 0.037b therapy. Indeed, neither CRABPII mRNA nor the pro- KRT4 0.5 0.051 tein levels changed significantly in our study. CYP26A1 –0.9 0.032b Interestingly, despite only marginal effects on CRAB- CYP26B1 Dermis 0.0 0.844 CYP26B1 Epidermis –0.2 0.629 PII expression, good clinical results were obtained after only 4 weeks of liarozole therapy, particularly in the aOf a total of 10 patients, one did not accept punch biopsies and another received placebo. Therefore only 8 patients were included in this analysis. patients with Ichthyin or other non-TGM1 mutations who bp < 0.05 is indicated in bold. had initially a somewhat milder ichtyosis than these with KRT: keratin; CYP26: cytochrome P450; CRABPII: cellular retinoic acid TGM1 mutations. Since none of these patients had pre- binding protein II. viously been given oral retinoids or RAMBAs, we do not know whether their superior therapeutic response is spe- staining in normal skin and at baseline did not increase cific for liarozole. Ichthyin is a putative transmembrane as a result of therapy. receptor that transports lipids to the lamellar bodies (LB) Table VI shows the mean changes in the immunofluore­ essential for the formation of a skin water barrier (28). scence scores observed in paired biopsy specimens Interestingly, functional deficiency of ichthyin results in before and after therapy. Epidermal CRABPII protein characteristic ultra-structural abnormalities of LBs that expression was not significantly altered by therapy, are not seen in patients with TGM1 mutations (21). In a whereas staining of KRT2 and KRT4 was up-regu- study of retinoic acid receptor γ null mice (29), similar lated (p = 0.037 and 0.051, respectively), and that of LB abnormalities were observed in murine skin, implying CYP26A1 was down-regulated (p = 0.032). a possible relationship between retinoid signalling and ichthyin shuttling of lipids in the maintenance of skin barrier function. In theory, it might be possible partially to Discussion compensate for the negative impact of ichthyin deficiency This is the first study exploring the mechanism of on LB function by increasing the retinoid activity in the action of RAMBA therapy in LI and attempting to skin. Further studies on a larger group of patients with correlate the therapeutic outcome to the underlying Ichthyin mutations are needed to test this hypothesis, genetic abnormalities in this complex skin disease. A although this may be problematic due to the rarity of such limitation of this study is the scarcity of patients with patients (only 7 cases are known in Sweden). LI and the fact that for ethical and practical reasons we Keratin protein expression has been studied previously could only include Swedish patients who were enrol- in ichthyosis patients treated with oral liarozole (30), but led in a multicentre clinical trial of two fixed doses of no corresponding mRNA data were available at the on- liarozole and who were also willing to accept repeated set of this study. Our results show that the transcription skin sampling. Two types of skin samples were ob- KRT2 and KRT4 changed relatively little after 4 weeks tained: punch biopsies and shave biopsies. The latter of oral liarozole treatment, especially when comparing type of biopsy is, in our experience, the gentlest way the effects with those of topical treatment with ATRA of producing an epidermis-enriched sample that can be (13) or talarozole (6) in healthy control skin. KRT2 is directly extracted for RNA. negatively regulated by retinoid stimulation of normal It is well-known that topical retinoids and RAMBAs skin (6, 13). The reason for the reduced KRT2 protein affect the expression of ATRA biomarkers such as expression in LI epidermis at baseline is unknown, but CRABPII, KRT4 and KRT2 in normal human epidermis this intrinsic abnormality may explain why no further (6, 10, 13). In contrast, little is known about the cor- reduction occurred after oral liarozole treatment. responding effects in LI skin, and after oral adminis- Belonging to the group of putative retinoid biomarkers tration of RAMBAs. At the outset of this study it was are the enzymes that regulate epidermal vitamin A meta- not even known if the expression of retinoid biomarkers bolism. For example, exposure of normal skin to topical is intrinsically abnormal in ichthyotic epidermis, thus ATRA results in increased retinol esterification (31), potentially affecting the response to liarozole therapy. which may serve as a feedback loop by which epidermal In fact, we found increased CRABPII mRNA levels in retinol is diverted from oxidation to ATRA. In the present many LI patients (Table III) and the protein was ex- study, we examined the mRNA expression of LRAT and pressed in all suprabasal cells instead of only stratum RALDH2, two enzymes which are rate-limiting in retinol granulosum (Fig. 4). Previously, increased CRABP esterification and retinal oxidation, respectively (9). No protein levels have been found by binding assay in LI significant changes were seen in the expression of these epidermis (27). A similar abnormal CRABPII protein enzymes in LI skin, which suggests that the ATRA levels

Acta Derm Venereol 89 Liarozole in lamellar ichthyosis: effect on gene expression 19 attained during liarozole therapy usually remain below Acknowledgements the threshold value needed to activate any auto-regula- The valuable technical assistance of Mrs. Kristina Hymnelius, tory mechanisms in the epidermal retinol metabolism. Inger Pihl-Lundin and Eva Hagforsen at the Department of However, slightly increased ATRA levels must have Dermatology, Uppsala University is gratefully acknowledged. occurred during liarozole therapy, since a few other reti- We would also like to thank Dr Braham Shroot, Barrier Thera- peutics, for his valuable comments on the manuscript, and Drs noid biomarkers in the epidermis reacted in accordance Niklas Dahl, Maritta Hellström-Pigg and Johanna Dahlquist, with the RAMBA concept. For instance, CYP26A1 Department of Genetics and Pathology, Uppsala University, mRNA tended to increase in a dose-dependent manner for making the TGM1 and Ichthyin mutation analysis. The during liarozole therapy (see Fig. 2). Of course the ex- study was funded by Barrier Therapeutics and the Swedish pression of this and other CYP26 enzymes is of special Research Council. interest, because they are the actual targets for liarozole Conflict of interest: The study was partially financed by Barrier therapy, namely inhibition of CYP26-mediated 4-hydroxy­ Therapeutics, nv. None of the academics (EPL, AG, HT, AV) in this study have any conflict of interest to declare. lation of ATRA (15, 32–34). Somewhat surprisingly, several LI patients displayed increased protein staining of CYP26A1 before therapy and reduced staining after REFERENCES therapy (see Fig. 4N and O), despite increased CYP26A1 mRNA mean levels during therapy (see Table V). The 1. DiGiovanna JJ. Retinoid treatment of the disorders of cor- poor correlation between CYP26A1 mRNA and protein nification. In: Vahlquist A, Duvic M, editors. Retinoids and carotenoids in dermatology. 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