Tralokinumab Effectively Disrupts the IL-13/IL-13Rα1/IL-4Rα Signaling Complex but Not the IL-13/IL-13Rα2 Complex.

Tralokinumab, a fully human mAb specifically targeting the IL-13 cytokine, has demonstrated clinical efficacy and safety in patients with moderate-to-severe atopic dermatitis. Tralokinumab binds IL-13 with high affinity, which prevents the interaction of IL-13 with IL-13Rα1 and subsequent signaling. Similarly, tralokinumab-bound IL-13 cannot bind to IL-13Rα2, a proposed decoy receptor that is reported to bind IL-13 with extraordinarily high affinity. It has however not been fully elucidated to what extent tralokinumab interferes with the endogenous regulation of IL-13 through IL-13Rα2. In this mechanistic study, we used biophysical, biochemical, and cellular assays to investigate the effect of tralokinumab on the interaction between IL-13 and IL-13Rα1 and IL-13Rα2, respectively, as well as the effects on IL-13Rα2-mediated IL-13 internalization. We demonstrate that IL-13Rα2 binds IL-13 with exceptionally high affinity and that tralokinumab is unable to displace IL-13 from IL-13Rα2. In contrast to this, tralokinumab is able to disrupt the IL-13/IL-13Rα1 and IL-13Rα1/IL-13/IL-4Rα complex. Furthermore, we demonstrate that whereas the IL-13/tralokinumab complex is unable to bind IL-13Rα2, any IL-13 that is not bound by tralokinumab (i.e., free IL-13) can be bound by IL-13Rα2 and subsequently internalized, regardless of the presence of tralokinumab. In summary, our study indicates that tralokinumab does not interfere with endogenous IL-13Rα2-mediated regulation of free IL-13.

Characterization of a novel non-steroidal glucocorticoid receptor agonist optimized for topical treatment.

Glucocorticoids (GCs) are commonly used topical treatments for skin diseases but are associated with both local and systemic side effects. In this study, we describe a selective non-steroidal glucocorticoid receptor (GR) agonist for topical use, LEO 134310, which is rapidly deactivated in the blood resulting in low systemic exposure and a higher therapeutic index in the TPA-induced skin inflammation mouse model compared with betamethasone valerate (BMV) and clobetasol propionate (CP). Selectivity of LEO 134310 for GR was confirmed within a panel of nuclear receptors, including the mineralocorticoid receptor (MR), which has been associated with induction of skin atrophy. Topical treatment with LEO 134310 in minipigs did not result in any significant reduction in epidermal thickness in contrast to significant epidermal thinning induced by treatment with BMV and CP. Thus, the profile of LEO 134310 may potentially provide an effective and safer treatment option for skin diseases compared with currently used glucocorticoids.

Calcipotriol/Betamethasone Dipropionate Foam Inhibits Th17 Cytokine Secretion and Improves Epidermal Barrier Markers in a Human Th17 Skin Inflammation Model.

INTRODUCTION: T-helper 17 (Th17) cytokines play a key role in the pathophysiology of psoriasis by driving inflammatory responses that lead to epidermal alterations. Markers of epidermal differentiation, including the proteins loricrin (LOR), filaggrin (FLG) and involucrin (IVL), are dysregulated in psoriatic skin. The fixed-dose combination of calcipotriol/betamethasone dipropionate (Cal/BD) foam and clobetasol propionate (CP) are widely used, effective topical treatments for psoriasis. In this study, we investigated the effects of Cal/BD foam and CP cream on Th17 cytokine secretion and epidermal differentiation using a human Th17 skin inflammation model (InflammaSkin®). METHODS: The fixed-dose combination Cal/BD foam and the CP cream were applied once and twice daily, respectively, onto the air-exposed epidermal surface of InflammaSkin cultures for 7 days. Th17 cytokine levels were measured in culture supernatants, and gene expression analysis and immunohistochemical staining for LOR, FLG and IVL were performed on the skin samples. RESULTS: Topical treatment with Cal/BD foam almost completely inhibited Th17 cytokine secretion and upregulated LOR and IVL expression, but not FLG expression, at the mRNA and protein levels. Topical treatment with CP cream significantly reduced Th17 cytokine levels, but to a lesser extent than Cal/BD foam, and did not improve expression of any of the epidermal differentiation markers. CONCLUSION: Compared with CP treatment, the fixed-dose combination Cal/BD foam showed a greater suppression of Th17 cytokine secretion and improved epidermal differentiation, resulting in an overall higher degree of improvement of the skin. These results support our understanding of the mechanisms behind the clinical efficacy observed for Cal/BD foam and of its use for long-term proactive treatment of psoriasis vulgaris.

Development and characterization of a human Th17-driven ex vivo skin inflammation model.

Skin models mimicking features of psoriasis-related inflammation are needed to support the development of new drugs in dermatology. Reconstructed skin models lack tissue complexity, including a fully competent skin barrier, and presence and/or diversity of immune cells. Here, we describe InflammaSkin®, a novel human Th17-driven ex vivo skin inflammation model. In this model, skin-resident T cells are in situ activated by intradermal injection of anti-CD3 and anti-CD28 antibodies and Th17 cell polarization is sustained by culture in a chemically defined medium supplemented with IL-1ß, IL-23 and TGF-ß for seven days. The acquired Th17 signature is demonstrated by the sustained secretion of IL-17A, IL-17AF, IL-17F, IL-22, IFN-γ, and to some degree IL-15 and TNF-α observed in the activated ex vivo skin inflammation model compared with the non-activated skin model control. Furthermore, expression of S100A7 and Keratin-16 by keratinocytes and loss of epidermal structure integrity occur subsequently to in situ Th17cell activation, demonstrating cellular crosstalk between Th17 cells and keratinocytes. Finally, we demonstrate the use of this model to investigate the modulation of the IL-23/IL-17 immune axis by topically applied anti-inflammatory compounds. Taken together, we show that by in situ activation of skin-resident Th17 cells, the InflammaSkin® model reproduces aspects of inflammatory responses observed in psoriatic lesions and could be used as a translational tool to assess efficacy of test compounds.

Ex vivo culture of lesional psoriasis skin for pharmacological testing.

BACKGROUND: Psoriasis is a chronic, inflammatory skin disorder resulting from a complex interplay between immune and skin cells via release of soluble mediators. While a lot is known about the molecular mechanisms behind psoriasis pathogenesis, there is still a need for preclinical research models that accuratelyreplicate the disease. OBJECTIVE: This study aimed to develop and characterize ex vivo culture of psoriasis skin as a model for pharmacological testing, where the immunological events of psoriasis can be followed. METHODS: Full thickness punch biopsies of lesional psoriasis skin were cultured in submerged conditions up to 144 h followingin situ T cell stimulation with rhIL-23 and anti-CD3 and anti-CD28 antibodies. The T cell mediated skin inflammation was assessed by gene and protein l analysis for a panel of inflammatory mediators. Tissue integrity and morphology were evaluated by histological analysis. RESULTS: T cell stimulation resulted in functional and psoriasis specificin situ activation of T cells. The expression levels of most of the proinflammatory mediators related to both immune and skin cells were comparable to these in freshly isolated tissue at 48 and 96 h of culture. Tissue integrity and morphology were sustained up to 96 h. Treatment with a corticosteroid reduced the expression of several pro-inflammatory cytokines and chemokines, whereas anti-IL-17A antibody treatment reduced the expression of the IL-17A downstream markers IL-8 and DEFB4. CONCLUSION: By preserving keyimmunopathological mechanisms of psoriasis, ex vivo culture of psoriasis skin can be used for the investigation of inflammatory processes of psoriasis and for preclinical drug discovery research.

Short-term transcriptional response to IL-17 receptor-A antagonism in the treatment of psoriasis.

BACKGROUND: IL-17 antagonists induce impressive clinical benefits in psoriasis, but it is unknown to what extent cellular and molecular psoriasis characteristics are suppressed by a clinically relevant dose/schedule of any IL-17-receptor antagonist. OBJECTIVE: We sought to examine the effects of the IL-17 receptor-A antagonist brodalumab, on clinical and molecular psoriasis features over a 12-week period. METHODS: A subset of patients (n = 116) enrolled in 3 phase-3 randomized clinical trials (AMAGINE -1 [Efficacy, Safety, and Withdrawal and Retreatment With Brodalumab in Moderate to Severe Plaque Psoriasis Subjects], -2 [P3 Study Brodalumab in Treatment of Moderate to Severe Plaque Psoriasis], and -3 [Efficacy and Safety of Brodalumab Compared With Placebo and Ustekinumab in Moderate to Severe Plaque Psoriasis in Subjects]) participated in a mechanistic substudy where punch biopsies were collected (lesional and nonlesional skin) between baseline and 12 weeks. This cohort included moderate-to-severe psoriasis patients treated with 140 mg (n = 46), 210 mg (n = 41) brodalumab, or placebo (n = 29). Key epidermal psoriatic features, including T-cell and dendritic cell subsets, were examined using immunohistochemistry. Treatment-induced changes in lesional skin gene expression profiles were evaluated using Affymetrix arrays. RESULTS: IL-17 receptor-A antagonism caused extensive improvements in clinical, histologic, and transcriptomic features of psoriasis. Cellular infiltrates (CD3+, CD8+, CD11c+, CD163+), markers of keratinocyte proliferation (Ki67+, KRT16), and inflammatory cytokines (IL-17A/C/F, IL-23A, IL-12B) decreased progressively, reaching close to nonlesional levels, paralleled by decreases in epidermal thickness. Psoriasis transcriptome gene expression improved ∼85% to 95% in responders whose psoriasis area severity index improved by 75% from baseline by week 12 (n = 63), compared with ∼30% to 65% in nonresponders (n = 12), while the residual disease genomic profile was 10% of the psoriasis transcriptome, which is less than for earlier generation drugs. IL-17-dependent gene expression, including keratinocyte genes, improved earlier and more extensively following brodalumab treatment compared with ustekinumab treatment (anti-IL-23/-IL-12). CONCLUSIONS: The clinically approved dose and schedule for brodalumab leads to nearly complete resolution of clinical, histologic, and transcriptomic features of psoriasis. Evidently, IL-17-induced release of keratinocyte-derived inflammatory mediators is a key driver of psoriasis pathogenesis.

Epigenetic control of IL-23 expression in keratinocytes is important for chronic skin inflammation.

The chronic skin inflammation psoriasis is crucially dependent on the IL-23/IL-17 cytokine axis. Although IL-23 is expressed by psoriatic keratinocytes and immune cells, only the immune cell-derived IL-23 is believed to be disease relevant. Here we use a genetic mouse model to show that keratinocyte-produced IL-23 is sufficient to cause a chronic skin inflammation with an IL-17 profile. Furthermore, we reveal a cell-autonomous nuclear function for the actin polymerizing molecule N-WASP, which controls IL-23 expression in keratinocytes by regulating the degradation of the histone methyltransferases G9a and GLP, and H3K9 dimethylation of the IL-23 promoter. This mechanism mediates the induction of IL-23 by TNF, a known inducer of IL-23 in psoriasis. Finally, in keratinocytes of psoriatic lesions a decrease in H3K9 dimethylation correlates with increased IL-23 expression, suggesting relevance for disease. Taken together, our data describe a molecular pathway where epigenetic regulation of keratinocytes can contribute to chronic skin inflammation.

Effects of glucocorticoids on stratum corneum lipids and function in human skin-A detailed lipidomic analysis.

BACKGROUND: Topical glucocorticoids (GCs) are known to induce atrophy of human skin including thinning of epidermal and dermal compartments by influencing keratinocyte proliferation and synthesis of extracellular matrix proteins. GCs are also known to reduce skin barrier integrity but little is known about the changes in lipid composition in human skin following topical administration of GCs. OBJECTIVE: This study investigated the effects of GCs on stratum corneum (SC) function and lipid profile of human skin in vivo. METHOD: Over a period of 4 weeks, 16 healthy volunteers were treated on the forearms once daily with topical clobetasol proprionate (CP), betamethasone diproprionate (BDP) or vehicle. One day after last application (Day 29) SC lipids were collected by tape stripping and analysed by a high sensitivity liquid chromatography-mass spectrometry method. Gene expression was analysed in skin biopsies. The full skin, epidermal and SC thickness were assessed by ultrasound, optical coherence tomography and confocal microscopy, respectively, and barrier integrity was assessed by measuring transepidermal water loss (TEWL). RESULTS: Compared to vehicle controls, GCs induced significant alterations in SC lipid profiles. CP caused a reduction in 98 lipids of 226 analysed while BDP treatment only resulted in a significant change of 29 lipids. Most pronounced changes occurred among long chain, ester-linked, ceramide classes while other ceramide classes were much less affected. Almost the complete profile of triacylglycerols (TGs) was significantly decreased by CP while more modest changes were observed in free fatty acids. Topical GCs reduced the thickness of skin layers and increased TEWL. GC treatment also induced changes in expression of genes coding for extracellular markers and enzymes involved in lipid synthesis. CONCLUSIONS: This study shows a reduction in specific SC lipid classes following topical GC treatment of human skin and contributes to the characterisation of the barrier disruption in human skin induced by topical steroids.

Supersaturation of Calcipotriene and Betamethasone Dipropionate in a Novel Aerosol Foam Formulation for Topical Treatment of Psoriasis Provides Enhanced Bioavailability of the Active Ingredients.

INTRODUCTION: Previous studies have demonstrated the superior efficacy of a novel aerosol foam formulation of fixed combination calcipotriene 0.005% (Cal) and betamethasone dipropionate 0.064% (BD), compared with the ointment formulation. The aim of this study is to ascertain whether enhanced bioavailability of the active ingredients due to supersaturation and/or occlusive properties can explain the observed greater clinical efficacy. METHODS: Solubility and evaporation experiments were conducted to examine the abilities of Cal/BD aerosol foam ingredients to create a supersaturated environment. Optical microscopy, Raman imaging and X-ray powder diffraction were used to examine the physical state of Cal and BD in the formulations after application, and determine whether a supersaturated state remained stable for clinically relevant time periods. In vitro skin penetration and ex vivo biomarker assays were conducted to compare the skin penetration and bioavailability of Cal and BD from the aerosol foam and ointment formulations, respectively. Occlusive properties were examined via transepidermal water loss. RESULTS: Solubility studies showed that Cal and BD solubility increased with increasing dimethyl ether (DME) content. Both active ingredients are completely dissolved in the final aerosol foam formulation. DME rapidly evaporates after spraying, and the amount was reduced to 0.5% of the initial amount after 2 min. This led to the formation of a supersaturated environment, where Cal and BD crystals were absent for at least 26 h after application. Cal/BD aerosol foam had significantly greater in vitro skin penetration and had increased bioavailability compared with Cal/BD ointment. Both formulations effectively occluded the skin. CONCLUSION: A stable supersaturated solution of Cal/BD in the aerosol foam leads to increased bioavailability and explains the improved clinical effect when compared to the Cal/BD ointment. FUNDING: The studies included in the paper are all conducted by LEO Pharma A/S or CROs on behalf of LEO Pharma A/S.

Distinct molecular signatures of mild extrinsic and intrinsic atopic dermatitis.

Atopic dermatitis (AD) is a common inflammatory skin disease with underlying defects in epidermal function and immune responses. In this study, we used microarray analysis to investigate differences in gene expression in lesional skin from patients with mild extrinsic or intrinsic AD compared to skin from healthy controls and from lesional psoriasis skin. The primary aim was to identify differentially expressed genes involved in skin barrier formation and inflammation, and to compare our results with those reported for patients with moderate and severe AD. In contrast to severe AD, expression of the majority of genes associated with skin barrier formation was unchanged or upregulated in patients with mild AD compared to normal healthy skin. Among these, no significant differences in the expression of filaggrin (FLG) and loricrin at both mRNA and protein level were found in lesional skin from patients with mild AD, despite the presence of heterozygous FLG mutations in the majority of patients with mild extrinsic AD. Several inflammation-associated genes such as S100A9, MMP12, CXCL10 and CCL18 were highly expressed in lesional skin from patients with mild psoriasis and were also increased in patients with mild extrinsic and intrinsic AD similar to previous reports for severe AD. Interestingly, expression of genes involved in inflammatory responses in intrinsic AD resembled that of psoriasis more than that of extrinsic AD. Overall, differences in expression of inflammation-associated genes found among patients with mild intrinsic and extrinsic AD correlated with previous findings for patients with severe intrinsic and extrinsic AD.

Calcipotriol and betamethasone dipropionate exert additive inhibitory effects on the cytokine expression of inflammatory dendritic cell-Th17 cell axis in psoriasis.

BACKGROUND: Psoriasis vulgaris is characterised by epidermal hyper-proliferation and infiltration of immune cells including dendritic cells (DCs) and T cells. The inflammation is driven by a complex interplay between immune and skin cells involving interleukin (IL)-17A, IL-23 and TNF-α as key drivers. The calcipotriol/betamethasone dipropionate two-compound fixed combination product is widely used for topical treatment of psoriasis. However, the mechanism behind its high efficacy has not been elucidated in detail. OBJECTIVE: Here, we investigated and compared the immune modulatory effects of betamethasone, calcipotriol and the combination in ex vivo cultures of psoriatic skin and in vitro cultures of primary human cells that recapitulate key cellular activities of psoriatic inflammation. METHOD: The immune modulatory effect of the treatments on psoriatic skin and on in vitro differentiated Th1/Th17 cells, Tc1/Tc17 cells, monocyte-derived inflammatory dendritic cells and primary keratinocytes was assessed by a panel of inflammatory and phenotypic related transcription factors and cytokines. The expression was evaluated by both gene and protein analysis. RESULTS: Compared to vehicle control or mono-treatments, the effect of calcipotriol/betamethasone combination was significantly better in inhibiting the secretion of IL-17A and TNF-α in psoriatic skin. Additionally, the two components showed additive inhibitory effects on secretion of IL-23 and TNF-α by DCs, of IL-17A and TNF-α by both CD4(+) and CD8(+) T cells and reduced inflammatory responses in Th17-stimulated keratinocytes. Furthermore, calcipotriol was found to enhance IL-10 secretion in psoriatic skin and in human T cells, to induce secretion of type 2 cytokines by T cells and, lastly, to significantly modulate the differentiation of DCs and T cells. CONCLUSIONS: In summary, we demonstrate a unique and supplementary immune modulatory effect of calcipotriol/betamethasone combination on TNF-α and IL-23/Th17 immune axis, supporting the superior clinical efficacy of the combination product compared to the respective mono-treatments in psoriasis patients.

Calcipotriol counteracts betamethasone-induced decrease in extracellular matrix components related to skin atrophy.

The calcipotriol/betamethasone dipropionate fixed-combination gel is widely used for topical treatment of psoriasis vulgaris. It has been hypothesized that calcipotriol counteracts glucocorticoid-induced skin atrophy which is associated with changes in the extracellular matrix (ECM). To elucidate the combined effects of calcipotriol and betamethasone on key ECM components, a comparative study to the respective mono-treatments was carried out. The effect on collagen I synthesis, matrix metalloproteinase (MMP) secretion, and hyaluronic acid (HA) production was investigated in primary human fibroblast and keratinocyte cultures as well as in a human skin explant model. We show that calcipotriol counteracts betamethasone-induced suppression of collagen I synthesis. Similarly, calcipotriol and betamethasone have opposing effects on MMP expression in both fibroblasts and keratinocytes. Moreover, calcipotriol is able to restore betamethasone-impaired HA synthesis in keratinocytes and prevent betamethasone-induced epidermal thinning in minipigs upon treatment with the calcipotriol/betamethasone gel. In summary, our results show for the first time in primary human skin cultures that calcipotriol reduces early signs of betamethasone-induced skin atrophy by modulation of key ECM components. These results indicate that the calcipotriol component of the fixed-combination gel counteracts the atrophogenic effects of betamethasone on the skin.

Humanized mouse model of skin inflammation is characterized by disturbed keratinocyte differentiation and influx of IL-17A producing T cells.

Humanized mouse models offer a challenging possibility to study human cell function in vivo. In the huPBL-SCID-huSkin allograft model human skin is transplanted onto immunodeficient mice and allowed to heal. Thereafter allogeneic human peripheral blood mononuclear cells are infused intra peritoneally to induce T cell mediated inflammation and microvessel destruction of the human skin. This model has great potential for in vivo study of human immune cells in (skin) inflammatory processes and for preclinical screening of systemically administered immunomodulating agents. Here we studied the inflammatory skin response of human keratinocytes and human T cells and the concomitant systemic human T cell response.As new findings in the inflamed human skin of the huPBL-SCID-huSkin model we here identified: 1. Parameters of dermal pathology that enable precise quantification of the local skin inflammatory response exemplified by acanthosis, increased expression of human ß-defensin-2, Elafin, K16, Ki67 and reduced expression of K10 by microscopy and immunohistochemistry. 2. Induction of human cytokines and chemokines using quantitative real-time PCR. 3. Influx of inflammation associated IL-17A-producing human CD4+ and CD8+ T cells as well as immunoregulatory CD4+Foxp3+ cells using immunohistochemistry and -fluorescence, suggesting that active immune regulation is taking place locally in the inflamed skin. 4. Systemic responses that revealed activated and proliferating human CD4+ and CD8+ T cells that acquired homing marker expression of CD62L and CLA. Finally, we demonstrated the value of the newly identified parameters by showing significant changes upon systemic treatment with the T cell inhibitory agents cyclosporine-A and rapamycin. In summary, here we equipped the huPBL-SCID-huSkin humanized mouse model with relevant tools not only to quantify the inflammatory dermal response, but also to monitor the peripheral immune status. This combined approach will gain our understanding of the dermal immunopathology in humans and benefit the development of novel therapeutics for controlling inflammatory skin diseases.

Psoriasis drug discovery: methods for evaluation of potential drug candidates.

INTRODUCTION: Psoriasis is a complex disease with several clinical subtypes, as well as variations in body location and severity. Many patients suffering from psoriasis now benefit from the increased understanding of the pathogenesis of the disease, which in turn drives translational efforts to test new therapeutic concepts in the clinic. However, a multitude of treatment options is currently needed to satisfy patient needs. AREAS COVERED: This review describes the drug discovery platform in relation to psoriasis with special emphasis on how the major disease mechanisms of psoriasis can be studied in experimental in vitro and in vivo settings. The value of using humanized models and experimental clinical studies is highlighted. EXPERT OPINION: The successful development of novel therapies requires a translational approach to develop and implement the best preclinical and experimental clinical models and analytical tools that capture the various biological aspects of the disease. There is a need for more advanced in vitro skin models that contain the relevant cellular constituents as well as a need for careful validation of relevant in vivo models for psoriasis.

IL-23-mediated epidermal hyperplasia is dependent on IL-6.

Psoriasis is a chronic inflammatory skin disease primarily driven by Th17 cells. IL-23 facilitates the differentiation and induces complete maturation of Th17 cells. Lesional psoriatic skin has increased levels of IL-23 and recent studies show that intradermal injections of IL-23 induce a psoriasis-like skin phenotype in mice. We have now characterized the IL-23-induced skin inflammation in mice at the molecular level and found a significant correlation with the gene expression profile from lesional psoriatic skin. As observed in psoriasis, the pathogenesis of the IL-23-induced skin inflammation in mice is driven by Th17 cells. We demonstrate a dramatic upregulation of IL-6 mRNA and protein after intradermal injections of IL-23 in mice. Using IL-6(-/-) mice we show that IL-6 is essential for development of the IL-23-elicited responses. Despite producing high levels of IL-22, IL-6(-/-) mice were unable to express the high-affinity IL-22 receptor chain and produced minimal IL-17A in response to intradermal injections of IL-23. In conclusion, we provide evidence for the critical role played by IL-6 in IL-23-induced skin inflammation and show that IL-6 is required for expression of IL-22R1A.