Nonclinical and human pharmacology of the potent and selective topical retinoic acid receptor-γ agonist trifarotene.

BACKGROUND: First- and third-generation retinoids are the main treatment for acne. Even though efficacious, they lack full selectivity for retinoic acid receptor (RAR) γ, expressed in the epidermis and infundibulum. OBJECTIVES: To characterize the in vitro metabolism and the pharmacology of the novel retinoid trifarotene. MATERIALS AND METHODS: In vitro assays determined efficacy, potency and selectivity on RARs, as well as the activity on the expression of retinoid target genes in human keratinocytes and ex vivo cultured skin. In vivo studies investigated topical comedolytic, anti-inflammatory and depigmenting properties. The trifarotene-induced gene expression profile was investigated in nonlesional skin of patients with acne and compared with ex vivo and in vivo models. Finally, the metabolic stability in human keratinocytes and hepatic microsomes was established. RESULTS: Trifarotene is a selective RARγ agonist with > 20-fold selectivity over RARα and RARß. Trifarotene is active and stable in keratinocytes but rapidly metabolized by human hepatic microsomes, predicting improved safety. In vivo, trifarotene 0·01% applied topically is highly comedolytic and has anti-inflammatory and antipigmenting properties. Gene expression studies indicated potent activation of known retinoid-modulated processes (epidermal differentiation, proliferation, stress response, retinoic acid metabolism) and novel pathways (proteolysis, transport/skin hydration, cell adhesion) in ex vivo and in vivo models, as well as in human skin after 4 weeks of topical application of trifarotene 0·005% cream. CONCLUSIONS: Based on its RARγ selectivity, rapid degradation in human hepatic microsomes and pharmacological properties including potent modulation of epidermal processes, topical treatment with trifarotene could result in good efficacy and may present a favourable safety profile in acne and ichthyotic disorders.

Atrophic scar formation in patients with acne involves long-acting immune responses with plasma cells and alteration of sebaceous glands.

BACKGROUND: Possible outcomes of acne lesions are atrophic scars, which may cause serious psychological distress. Current treatments for postacne scarring often require invasive procedures. Pathophysiological studies on acne scarring have only investigated the first week of papule life. OBJECTIVES: To study the pathophysiology of atrophic scar formation to identify molecular and cellular pathways that can lead to new therapies for the prevention of acne scarring. METHODS: Large-scale gene expression profiling and immunohistochemistry analysis were performed on uninvolved skin and papules in both scar-prone (SP) and non-scar-prone (NSP) patients with acne, at different time points. RESULTS: Gene expression and immunohistochemistry analyses showed a very similar immune response in 48-h-old papules in SP and NSP populations, characterized by elevated numbers of T cells, neutrophils and macrophages. However, the immune response only persisted in SP patients in 3-week-old papules, and was characterized by an important B-cell infiltrate. Transient downmodulation of sebaceous gland markers related to lipid metabolism was observed in 48-h-old papules in NSP patients, followed by normalization after 3 weeks. In contrast, in SP patients a drastic reduction of these markers persisted in 3-week-old papules, suggesting an irreversible destruction of sebaceous gland structures after inflammatory remodelling in SP patients with acne. CONCLUSIONS: Long-lived acne papules are characterized by a B-cell infiltrate. A relationship exists between the duration and severity of inflammation and the alteration of sebaceous gland structures, leading to atrophic scar formation in acne.

An innovative method to quantitate tissue integration of hyaluronic acid-based dermal fillers.

BACKGROUND/PURPOSE: Following intradermal injection, hyaluronic acid (HA)-based fillers tend to spread within the reticular dermis and to distribute between the dermal fibers. This biointegration is commonly measured qualitatively using histological methods. We developed a "toolbox" consisting of a visual scoring and a semi-automatic image analysis method using internal developed algorithm to quantitate the biointegration of Restylane® in histological sections. METHODS: Restylane® was injected intradermally in the abdominal skin of 10 healthy human subjects scheduled for abdominoplasty. The injections were performed either in vivo before surgery or ex vivo on samples taken post-surgery at different time points. The samples were processed for histology by visual scoring and image analysis using algorithms developed in Definiens to assess biointegration. RESULTS: The image analysis segmentation was accurate with <5% manual changes. Furthermore, the results calculated with the semi-automatic method were consistent with the visual scores obtained on injected human skin samples by means of a 5-grade photographic scale. A modified hematoxylin-eosin staining was found adequate to visualize both, the filler and the general morphology, on the same section. An excellent correlation was observed between the integration results obtained with PAS/Alcian Blue and HE-stained slides, allowing for a single staining in future studies. CONCLUSION: We developed a modified HE staining histological method and a new histomorphometric image analysis tool to quantitate biointegration of HA-based fillers in human skin. The results obtained in this study confirmed the known intermediate biointegration properties of Restylane®, thus validating these innovative methods.

Noninvasive proteome analysis of psoriatic stratum corneum reflects pathophysiological pathways and is useful for drug profiling.

BACKGROUND: Protein expression is disturbed in the psoriatic stratum corneum (SC). Noninvasive methods for the description of pathophysiological changes and drug profiling in psoriasis are desirable. OBJECTIVES: Undertake large-scale noninvasive protein expression studies in psoriatic SC to identify biomarkers of pathophysiological processes and use them for drug profiling. METHODS: Psoriatic SC was harvested through repetitive tape-stripping. Nonlesional and lesional SC, as well as vehicle-treated and drug-treated lesional SC samples were collected. Protein extracts from nonlesional and lesional skin biopsies were used for comparison. Calcipotriol-betamethasone (CB) was used as a reference medication. Proteins extracted from pooled tape strips were quantified using mass spectrometry (MS), Western blotting, enzyme-linked immunosorbent assay and Luminex technologies. RESULTS: MS-based methods identified 140 proteins differentially expressed in psoriatic SC. Epidermis development, glycolysis, regulation of apoptosis, cytoskeleton organization and peptide cross-linking were modulated, all reflecting perturbed epidermal differentiation. Using antibody-based techniques, increased levels of sICAM1, of CXCL1- and CXCL8-attracting neutrophils, of CXCL10- and CCL4-attracting T helper (Th) 1 cells, and of CCL2- and CCL4-attracting monocytes and dendritic cells were observed. Quantification of the Th1 and Th17 markers tumour necrosis factor, interleukin (IL) 12B, IL17A and IL17F in lesional SC was successful, while the Th2 cytokines IL4, IL5 and IL13, not involved in the disease process, were not detected. The pruritic cytokine IL31 was detected in lesional SC. CXCL1, CXCL8, CXCL10 and sICAM were used to investigate disease remission, ranking three topical treatments according to their known clinical efficacy. CONCLUSIONS: Protein biomarker quantification in psoriatic SC detects key pathophysiological mechanisms and enables noninvasive drug profiling in translational medicine settings.

Antihypertensive drugs and the risk of incident rosacea.

BACKGROUND: Despite scarce evidence, use of calcium channel blockers is discouraged in patients with rosacea, whereas beta-blockers are recommended as an off-label treatment for erythematotelangiectatic rosacea. OBJECTIVES: To study the association of the use of calcium channel blockers, beta-blockers and other antihypertensive drugs with incident rosacea. METHODS: We conducted a matched case-control study of antihypertensive drugs and incident rosacea, using the U.K.-based General Practice Research Database. Cases had an incident diagnosis of rosacea recorded between 1995 and 2009. Each case was matched to one control on age, sex, general practice and years of history on the database before the index date. Drug use was stratified by timing (≤ or > 180 days before the index date) and duration (number of prescriptions) of drug exposure, in a multivariate conditional logistic regression model. RESULTS: Among 53 927 cases and 53 927 controls, we observed odds ratios (ORs) around unity for calcium channel blockers across all strata, with a slightly decreased OR of 0·77 (95% CI 0·69-0·86) for current users of dihydropyridine calcium channel blockers with ≥ 40 prescriptions. Among beta-blockers, atenolol and bisoprolol yielded slightly decreased ORs across all exposure strata, whereas propranolol revealed ORs around 1·0, irrespective of timing and duration of exposure. Neither angiotensin-converting-enzyme inhibitors nor angiotensin receptor blockers altered the relative rosacea risk. CONCLUSIONS: Our data contradict the prevailing notion that calcium channel blockers increase the risk of rosacea. Beta-blocker use was associated with a slightly decreased risk of rosacea, but the effect may be somewhat stronger in patients with erythematotelangiectatic rosacea.

The association between psychiatric diseases, psychotropic drugs and the risk of incident rosacea.

BACKGROUND: Psychological conditions, such as traumatic events or stress, have been discussed controversially as aetiological factors for rosacea. OBJECTIVES: To assess the association between diagnosed depression, other affective disorders or schizophrenia and subsequent incident rosacea. We further aimed at evaluating the possible role of various psychotropic drugs within this association. METHODS: We conducted a matched case-control study of psychiatric diseases and incident rosacea, stratified by exposure to various psychotropic drugs, using the UK-based General Practice Research Database. Cases had a first diagnosis of rosacea recorded between 1995 and 2009. Each case was matched to one control on age, sex, general practice and years of history on the database. RESULTS: A history of depression or other affective disorders was not associated with an increased risk of developing rosacea; lithium was the only antidepressant drug that significantly altered this association. Current long-term use of lithium was associated with a decreased odds ratio (OR) of 0·58 [95% confidence interval (CI) 0·38-0·88] among people without a schizophrenia diagnosis (with or without affective disorders), compared with people not exposed to lithium. Patients with diagnosed schizophrenia revealed a decreased rosacea risk (OR 0·71, 95% CI 0·60-0·91), independent of antipsychotic drug use. CONCLUSIONS: Depression or other affective disorders were not associated with incident rosacea, whereas patients with schizophrenia were at a decreased risk of this skin disease in our study population. The materially decreased risk of rosacea among people with chronic lithium exposure may lead to new insights into the pathomechanism of rosacea.

A study on the epidemiology of rosacea in the U.K.

BACKGROUND: Rosacea is a chronic facial skin disease of unclear origin. Epidemiological data are scarce and controversial, with reported prevalences ranging from 0·09% to 22%. To our knowledge, incidence rates have not been quantified before. OBJECTIVES: In this observational study we quantified incidence rates of diagnosed rosacea in the U.K. and described demographic characteristics and the prevalence of ocular symptoms in patients with rosacea. We compared lifestyle factors such as smoking and alcohol consumption between patients with rosacea and controls. METHODS: Using the U.K.-based General Practice Research Database, we identified patients with an incident diagnosis of rosacea between 1995 and 2009 and matched them (1:1) to rosacea-free control patients. We assessed person-time of all patients at risk and assessed incidence rates of rosacea, stratified by age, sex, year of diagnosis and region. RESULTS: We identified 60,042 rosacea cases and 60,042 controls (61·5% women). The overall incidence rate for diagnosed rosacea in the U.K. was 1·65 per 1000 person-years. Rosacea was diagnosed in some 80% of cases after the age of 30 years. Ocular symptoms were recorded in 20·8% of cases at the index date. We observed a significantly reduced relative risk of developing rosacea among current smokers (odds ratio 0·64, 95% confidence interval 0·62-0·67). Alcohol consumption was associated with a marginal risk increase. CONCLUSIONS: We quantified incidence rates and characteristics of patients with rosacea diagnosed in clinical practice in a large epidemiological study using primary care data from the U.K. Smoking was associated with a substantially reduced risk of developing rosacea.

Gene expression profiling in psoriatic scalp hair follicles: clobetasol propionate shampoo 0.05% normalizes psoriasis disease markers.

BACKGROUND: Clobetasol propionate shampoo is effective and safe in treatment of scalp psoriasis (SP). Gene expression profiling of psoriatic skin biopsies led to the identification of numerous disease-related genes. However, it remained unknown whether the gene expression profile of hair follicles of SP patients was also affected. OBJECTIVES: To determine whether psoriasis-related genes are differentially regulated in the hair follicles of SP patients and whether the modulation of these genes can be correlated with clinical severity scores. METHODS: A single arm, open study was conducted in three centres. SP patients received daily treatment with clobetasol propionate shampoo. At Baseline, Weeks 2 and 4, investigators assessed clinical severity parameters and collected scalp hair follicles in anagen phase. Total RNA extracted from hair follicles was used to determine the expression level of 44 genes, which were reported previously to be upregulated in the skin of psoriasis patients. RESULTS: RNA of good quality and sufficient quantity was obtained from hair follicles of psoriasis patients and healthy volunteers (HV). The expression level of 10 inflammation-related genes was significantly increased in psoriatic hair follicles. The patient's exploratory transcriptomic score, defined as the mean fold modulation of these 10 genes compared with HV, correlated with clinical severity scores. Clobetasol propionate shampoo was effective in decreasing both the exploratory transcriptomics and the clinical severity scores. CONCLUSION: Hair follicles of SP patients are affected by the inflammatory process. The change in the expression level of inflammation-related genes correlates with the severity of the disease.

Gene expression profiles in psoriasis: analysis of impact of body site location and clinical severity.

BACKGROUND: Psoriasis is characterized by symmetry of plaques and modulation of multiple genes within those plaques. OBJECTIVES: We compared gene expression profiles of plaques of psoriasis at different anatomical sites for both symmetrical and asymmetrical disease to ascertain whether the same genes were expressed. METHODS: Gene expression profiles were analysed in biopsies from lesional and uninvolved skin from two groups of patients with either predominantly symmetrical or truncal plaques of psoriasis vulgaris, and from normal skin of healthy volunteers. Genomic analyses were performed using cDNA array and kinetically monitored reverse transcriptase-initiated polymerase chain reaction (kRT-PCR) approaches. A cluster of genes upregulated in involved psoriasis skin as compared with normal skin was identified using each of these two technologies. RESULTS: Clustering of patients based on their gene expression profile did not reveal any correlation with family history of psoriasis, age at onset or association of psoriasis with arthritis. There was no difference in gene expression profile between the type (symmetrical vs. truncal) or location (left vs. right side of body) of psoriatic plaques. Gene expression profiles of involved psoriatic skin analysed by kRT-PCR analysis did correlate with both global (Psoriasis Area and Severity Index) and local (erythema, desquamation and plaque elevation) clinical severity. CONCLUSIONS: These results indicate that it may be feasible to analyse the molecular effects of pharmacological agents on psoriatic skin in 'minizone' protocols, that the obtained data can be correlated with clinical severity and that plaques of psoriasis in the same individual express the same genes.

Activation function 2 in the human androgen receptor ligand binding domain mediates interdomain communication with the NH(2)-terminal domain.

Activation function 2 in the ligand binding domain of nuclear receptors forms a hydrophobic cleft that binds the LXXLL motif of p160 transcriptional coactivators. Here we provide evidence that activation function 2 in the androgen receptor serves as the contact site for the androgen dependent NH(2)- and carboxyl-terminal interaction of the androgen receptor and only weakly interacts with p160 coactivators in an LXXLL-dependent manner. Mutagenesis studies indicate that it is the NH(2)-/carboxyl-terminal interaction that is required by activation function 2 to stabilize helix 12 and slow androgen dissociation critical for androgen receptor activity in vivo. The androgen receptor recruits p160 coactivators through its NH(2)-terminal and DNA binding domains in an LXXLL motif-independent manner. The results suggest a novel function for activation function 2 and a unique mechanism of nuclear receptor transactivation.

The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways.

The nuclear receptor (NR) coactivator TIF2 possesses a single NR interaction domain (NID) and two autonomous activation domains, AD1 and AD2. The TIF2 NID is composed of three NR-interacting modules each containing the NR box motif LxxLL. Mutation of boxes I, II and III abrogates TIF2-NR interaction and stimulation, in transfected cells, of the ligand-induced activation function-2 (AF-2) present in the ligand-binding domains (LBDs) of several NRs. The presence of an intact NR interaction module II in the NID is sufficient for both efficient interaction with NR holo-LBDs and stimulation of AF-2 activity. Modules I and III are poorly efficient on their own, but synergistically can promote interaction with NR holo-LBDs and AF-2 stimulation. TIF2 AD1 activity appears to be mediated through CBP, as AD1 could not be separated mutationally from the CBP interaction domain. In contrast, TIF2 AD2 activity apparently does not involve interaction with CBP. TIF2 exhibited the characteristics expected for a bona fide NR coactivator, in both mammalian and yeast cells. Moreover, in mammalian cells, a peptide encompassing the TIF2 NID inhibited the ligand-induced AF-2 activity of several NRs, indicating that NR AF-2 activity is either mediated by endogenous TIF2 or by coactivators recognizing a similar surface on NR holo-LBDs.

TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors.

Nuclear receptors (NRs) act as ligand-inducible transcription factors which regulate the expression of target genes upon binding to cognate response elements. The ligand-dependent activity of the NR activation function AF-2 is believed to be mediated to the transcription machinery through transcriptional mediators/intermediary factors (TIFs). We report here the cloning of the 160 kDa human nuclear protein TIF2, which exhibits all properties expected for a mediator of AF-2: (i) it interacts in vivo with NRs in an agonist-dependent manner; (ii) it binds directly to the ligand-binding domains (LBDs) of NRs in an agonist- and AF-2-integrity-dependent manner in vitro; (iii) it harbours an autonomous transcriptional activation function; (iv) it relieves nuclear receptor autosquelching; and (v) it enhances the activity of some nuclear receptor AF-2s when overexpressed in mammalian cells. TIF2 exhibits partial sequence homology with the recently isolated steroid receptor coactivator SRC-1, indicating the existence of a novel gene family of nuclear receptor transcriptional mediators.