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677 Human skin biopsy culture model maintains psoriasis disease features and demonstrates Pathway Engagement by Dexamethasone
ABSTRACTS | Pharmacology and Drug Development 672
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Palladium and platinum nanoparticles activate AHR and NRF2 in keratinocytes— Implications in vitiligo therapy G Tsuji1, M Ichihashi2 and M Furue3 1 Research and Clinical Center for Yusho and Dioxin, Fukuoka, Japan, 2 Department of Cosmetic Dermatology, Saisei-Mirai Clinic, Kobe, Hyogo, Japan and 3 Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Reactive oxygen species (ROS) production has a crucial role in vitiligo development. Recently, nanomedicine such as nanoparticle-based therapy has been attempted as a new approach for ROS-mediated pathophysiology. A palladium and platinum nanoparticle solution, PAPLAL (approved in Japan), reportedly exerts antioxidant activity in vivo and is effective in vitiligo therapy, but the mechanism is unknown. Aryl hydrocarbon receptor (AHR) and nuclear factor erythroid-2-related 2 (NRF2) regulate ROS production; thus, we hypothesized that PAPLAL activates AHR and NRF2. Normal human keratinocytes were treated with 5% PAPLAL. Electron microscopy revealed that PAPLAL was retained in the cytoplasm after 3 h of PAPLAL treatment. PAPLAL uptake was observed over a 24-h period. PAPLAL treatment induced (1) AHR and NRF2 nuclear translocation and (2) CYP1A1 and NQO1 upregulation, indicating AHR and NRF2 activation. AHR activation negatively regulates the STAT1 pathway including the IFN-g-CXCL10 axis, a key mechanism in vitiligo development; therefore, we examined effects of PAPLAL on the IFN-g-CXCL10 axis. PAPLAL treatment inhibited IFN-ginduced CXCL10 upregulation, which was cancelled by AHR knockdown. Furthermore, PAPLAL treatment restored IFN-g-induced NQO1 downregulation contributing to the attenuation of oxidative damage. Finally, the effectiveness of PAPLAL treatment in vivo was evaluated using TNCB-induced contact hypersensitivity (C57BL/6 mice), a model of CD8 T-cell mediated skin inflammation including vitiligo. Topical application of PAPLAL in the elicitation phase inhibited ear swelling with reduced epidermal IFN-g and CXCL10 expression. Accordingly, PAPLAL exhibited dual effects on AHR and NRF2 activation, inhibiting the IFN-g- CXCL10 axis and protecting cells from oxidative damage; these findings imply the potential of PAPLAL in vitiligo treatment.
Viral status of Merkel cell carcinoma impacts responses to small molecule inhibitors T Gelb1, D Urban2, K Daily1, Y Xiao1, M Shen2, M Hall2 and I Brownell3 1 NCI, NIH, Bethesda, MD, 2 NCATS Chemical Genomics Center, NIH, Rockville, MD and 3 Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer with limited treatment options. Approximately 80% of MCC tumors have Merkel cell polyomavirus (MCPyV) DNA integrated into the host genome, and viral oncogenes are thought to drive carcinogenesis. In contrast, MCPyV-negative (MCPyV-) tumors have higher rates of protooncogene mutations. Transcriptome differences between MCPyV+ and MCPyV- tumors further suggest divergent underlying pathologies. In order to elucidate the pathophysiologies underlying MCPyV+ and MCPyV- MCC and develop novel treatments for MCC, we conducted high-throughput cell viability (CellTiter-Glo) screening of mechanistically annotated drug libraries. We screened w4,500 compounds including the NCGC Pharmaceutical Collection of approved and investigational drugs and the oncology-focused MIPE (Mechanism Interrogation PlatE) library against six MCC cell lines (3 MCPyV+ and 3 MCPyV-). Using hierarchical clustering we found that viral status impacted pharmacological responses. For example, a subset of HDAC inhibitors were more potent in MCPyV+ than MCPyV- MCC cells. In contrast, a dual RasGAP/ERK inhibitor more potently reduced viability in MCPyV- relative to MCPyV+ MCC cells. Next, we tested drug-combinations for their ability to reduce viability (CellTiter-Glo) or increase apoptosis (Caspase-Glo 3/7) in MCPyV+ and MCPyV- MCC cell lines using dose-response matrix-screening. There was extensive overlap between the compound combinations that most synergistically killed MCPyV+ and MCPyV- MCC cells. Overall, these results indicate that viral status strongly impacts response to single-agents and that specific compounds can sensitize both MCPyV+ and MCPyV- MCC cells to killing. Moreover, these results suggest that there are both distinct and overlapping pathophysiologies underlying MCPyV+ and MCPyV- MCC.
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The impact of environmental pressures on cellular bioenergetics in ageing human skin MJ Jackson1, PA Bulsara2, JM Crowther2, X Wang2, DJ Moore2 and MA Birch-Machin1 1 Newcastle University, Newcastle upon Tyne, England, United Kingdom and 2 GlaxoSmithKline plc., Warren, NJ Exposure to environmental stressors causes an increase in oxidative stress at the skin resulting in damage to cellular and extracellular structures and components. Damage to cellular bioenergetics compromises aerobic oxidative phosphorylation (OXPHOS) and anaerobic glycolysis, decreasing ATP synthesis with profound effects on cell viability, growth and senescence. Aim: To validate and further develop an oxidative stress model in vitro with which to investigate and protect against the detrimental effects of oxidative stress on cell bioenergetics. Methods: The Seahorse XFe96 analyser was utilised to monitor oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as markers for OXPHOS and glycolysis respectively. Single injections of H2O2 were applied to a neonatal human dermal fibroblast (HDFn) cell line to determine a suitable dose to exhibit substantial oxidative damage. The established skin care agent, niacinamide, was applied as three 1mM injections at 30 minute intervals, with the initial injection coinciding with the single H2O2 dose. Results: 0.75mM H2O2 did not produce a sustained level of oxidative damage in absence of niacinamide. 1mM H2O2 caused sustained reduction in both OCR (41.5%) and ECAR (26.9%) with no innate recovery. OCR (112%) and ECAR (105%) demonstrated recovery in the presence of niacinamide. Conclusion: A dose of 1mM H2O2 induces a high level of oxidative stress on major cellular bioenergetic function. Niacinamide is confirmed to restore function. This model offers a technique with which to explore oxidative damage and its prevention with various stressors, cell types and treatments.
BET bromodomain inhibitor JQ1 decreases CD30 and CCR4 expression as well as proliferation of cutaneous T-cell lymphoma M Sugaya1, H Kamijo2, N Takahshi2, T Oka1, T Miyagaki2, Y Asano3 and S Sato3 1 Department of Dermatology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan, 2 University of Tokyo Graduate School of Medicine, Tokyo, Japan and 3 Department of Dermatology, University of Tokyo Graduate School of Medicine, Tokyo, Japan Bromodomain and external domain (BET) proteins regulate cell growth, proliferation, cell cycle, and differentiation in various cancers. Therefore, they have emerged as interesting targets. The effect of BET inhibitor on cutaneous T-cell lymphoma (CTCL), however, is yet to be known. Here, we examined the effect of BET inhibitor JQ1 on four cell lines (MyLa, SeAx, Hut78 and HH cells). CTCL cell lines were treated with JQ1 and cell number, cell cycle, frequency of apoptosis, and expression of CD25, CD30 and CCR4 on the cell surface were evaluated by flow cytometry. Cell surface molecules were also analyzed by quantitative RTPCR. JQ1 dose-dependently decreased the cell number of CTCL cells through G1 arrest. JQ1 induced senescence only in MyLa cells and apoptosis in SeAx, Hut78 and HH cells. It was consistent with the fact that p53, which induces senescence, is fully functional only in MyLa cells, but not in SeAx, Hut78, or HH cells. Moreover, JQ1 downregulated CD30 and CCR4 expression both on the cell surface and at mRNA levels. Thus, BET bromodomain inhibitor JQ1 may be useful for treatment of CTCL. Combination with an antibody therapy targeting CCR4 or CD30 may not be recommended because JQ1 decreases expression of the target molecules.
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Fluorescence lifetime imaging microscopy (FLIM) as a novel method to assess skin residency of a fluorescent compound: experience with GSK2894512 A Alex1, S Frey1, H Angelene1, CD Neitzel2, J Li3, AJ Bower3, DR Spillman3, SA Boppart3 and Z Arp1 1 GSK, Collegeville, PA, 2 Carle Foundation Hospital, Urbana, IL and 3 University of Illinois at Urbana-Champaign, Urbana, IL Fluorescence Lifetime Imaging Microscopy (FLIM) is a non-invasive optical imaging method that can monitor chemical, morphologic, and metabolic changes in-vivo. FLIM was utilized as a novel endpoint in the evaluation of 2 different formulations of GSK2894512, an inherently fluorescent compound, being developed as a novel topical anti-inflammatory agent for atopic dermatitis and psoriasis. An open-label, 15 day study (# 201661) in 6 healthy adult males was conducted with 2 different formulations of 1% GSK2894512, Cream A and B. Both creams were applied for 7 days on opposite forearms followed by 8 days of post-treatment observation. FLIM assessments were collected for both creams on Days 1 to 15 with 3 biopsies collected only from the Cream B application area during the 7-day post-treatment period. GSK2894512 was clearly observable for both creams by FLIM monitoring. FLIM was able to detect a maximum penetration depth of 95 mm, and residency for up to 48 hrs posttreatment with Cream A and up to 24 hrs with Cream B. Analyses of GSK2894512 concentrations from the Cream B biopsies using LC-MS/MS agreed with FLIM observations. This study demonstrates the capability of FLIM as a novel non-invasive detection method to follow penetration and residency of GSK2894512 in healthy skin. FLIM allowed for the observation of GSK2894512 for both creams without the need for biopsy. These results suggest FLIM could be a viable alternative to skin biopsies without the usual patient discomfort and limitations, thereby enabling the direct measurement of skin distribution and true longitudinal monitoring.
S116 Journal of Investigative Dermatology (2017), Volume 137
Human skin biopsy culture model maintains psoriasis disease features and demonstrates Pathway Engagement by Dexamethasone S Huang1, S Grosskurth2, L Miller1, H Patel1, L Olson1, J Wetter1, R Edelmayer1, M Domanus1, C Miller1, P Honore3 and V Scott3 1 AbbVie Inc., North Chicago, IL, 2 AbbVie Inc, North Chicago, IL and 3 AbbVie, North Chicago, IL In drug discovery, early indication of efficacy or pathway engagement of potential clinical candidates in relevant disease models is extremely valuable. Although animal models of skin disease are useful, not all features of human skin such as the substantial differences in keratinocyte layers, are recapitulated in mouse models,. In collaboration with the AbbVie Clinical Pharmacology Research Unit, we have obtained skin biopsies from psoriasis patients to develop and characterize an ex vivo human skin biopsy culture model. The skin biopsies were cultured at the air-liquid interface under 95% O2 at 37 C to evaluate the effects of both culturing conditions and pharmacological treatment. Analysis of cytokine levels in conditioned media and biopsy gene expression profiling demonstrated that many features of psoriasis disease (such as elevated TNF, IL-17A and IL-23 release) are maintained in culture of lesion versus non-lesion biopsies over 24 hours. Effects of the reference anti-inflammatory compound Dexamethasone were evaluated in this model for pathway engagement. Transcriptomic and cytokine analyses of samples treated with Dexamethasone exhibited decreased inflammation, as well as previously reported agonistic effects on the glucocorticoid receptor NR3C1. The work presented here demonstrate the value of the explant human skin biopsy culture model for maintaining clinical features of psoriasis as well as for investigating pharmacodynamic pathway engagement, and potential efficacy of novel candidate therapeutics.
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Palladium and platinum nanoparticles activate AHR and NRF2 in keratinocytes— Implications in vitiligo therapy G Tsuji1, M Ichihashi2 and M Furue3 1 Research and Clinical Center for Yusho and Dioxin, Fukuoka, Japan, 2 Department of Cosmetic Dermatology, Saisei-Mirai Clinic, Kobe, Hyogo, Japan and 3 Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Reactive oxygen species (ROS) production has a crucial role in vitiligo development. Recently, nanomedicine such as nanoparticle-based therapy has been attempted as a new approach for ROS-mediated pathophysiology. A palladium and platinum nanoparticle solution, PAPLAL (approved in Japan), reportedly exerts antioxidant activity in vivo and is effective in vitiligo therapy, but the mechanism is unknown. Aryl hydrocarbon receptor (AHR) and nuclear factor erythroid-2-related 2 (NRF2) regulate ROS production; thus, we hypothesized that PAPLAL activates AHR and NRF2. Normal human keratinocytes were treated with 5% PAPLAL. Electron microscopy revealed that PAPLAL was retained in the cytoplasm after 3 h of PAPLAL treatment. PAPLAL uptake was observed over a 24-h period. PAPLAL treatment induced (1) AHR and NRF2 nuclear translocation and (2) CYP1A1 and NQO1 upregulation, indicating AHR and NRF2 activation. AHR activation negatively regulates the STAT1 pathway including the IFN-g-CXCL10 axis, a key mechanism in vitiligo development; therefore, we examined effects of PAPLAL on the IFN-g-CXCL10 axis. PAPLAL treatment inhibited IFN-ginduced CXCL10 upregulation, which was cancelled by AHR knockdown. Furthermore, PAPLAL treatment restored IFN-g-induced NQO1 downregulation contributing to the attenuation of oxidative damage. Finally, the effectiveness of PAPLAL treatment in vivo was evaluated using TNCB-induced contact hypersensitivity (C57BL/6 mice), a model of CD8 T-cell mediated skin inflammation including vitiligo. Topical application of PAPLAL in the elicitation phase inhibited ear swelling with reduced epidermal IFN-g and CXCL10 expression. Accordingly, PAPLAL exhibited dual effects on AHR and NRF2 activation, inhibiting the IFN-g- CXCL10 axis and protecting cells from oxidative damage; these findings imply the potential of PAPLAL in vitiligo treatment.
Viral status of Merkel cell carcinoma impacts responses to small molecule inhibitors T Gelb1, D Urban2, K Daily1, Y Xiao1, M Shen2, M Hall2 and I Brownell3 1 NCI, NIH, Bethesda, MD, 2 NCATS Chemical Genomics Center, NIH, Rockville, MD and 3 Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer with limited treatment options. Approximately 80% of MCC tumors have Merkel cell polyomavirus (MCPyV) DNA integrated into the host genome, and viral oncogenes are thought to drive carcinogenesis. In contrast, MCPyV-negative (MCPyV-) tumors have higher rates of protooncogene mutations. Transcriptome differences between MCPyV+ and MCPyV- tumors further suggest divergent underlying pathologies. In order to elucidate the pathophysiologies underlying MCPyV+ and MCPyV- MCC and develop novel treatments for MCC, we conducted high-throughput cell viability (CellTiter-Glo) screening of mechanistically annotated drug libraries. We screened w4,500 compounds including the NCGC Pharmaceutical Collection of approved and investigational drugs and the oncology-focused MIPE (Mechanism Interrogation PlatE) library against six MCC cell lines (3 MCPyV+ and 3 MCPyV-). Using hierarchical clustering we found that viral status impacted pharmacological responses. For example, a subset of HDAC inhibitors were more potent in MCPyV+ than MCPyV- MCC cells. In contrast, a dual RasGAP/ERK inhibitor more potently reduced viability in MCPyV- relative to MCPyV+ MCC cells. Next, we tested drug-combinations for their ability to reduce viability (CellTiter-Glo) or increase apoptosis (Caspase-Glo 3/7) in MCPyV+ and MCPyV- MCC cell lines using dose-response matrix-screening. There was extensive overlap between the compound combinations that most synergistically killed MCPyV+ and MCPyV- MCC cells. Overall, these results indicate that viral status strongly impacts response to single-agents and that specific compounds can sensitize both MCPyV+ and MCPyV- MCC cells to killing. Moreover, these results suggest that there are both distinct and overlapping pathophysiologies underlying MCPyV+ and MCPyV- MCC.
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The impact of environmental pressures on cellular bioenergetics in ageing human skin MJ Jackson1, PA Bulsara2, JM Crowther2, X Wang2, DJ Moore2 and MA Birch-Machin1 1 Newcastle University, Newcastle upon Tyne, England, United Kingdom and 2 GlaxoSmithKline plc., Warren, NJ Exposure to environmental stressors causes an increase in oxidative stress at the skin resulting in damage to cellular and extracellular structures and components. Damage to cellular bioenergetics compromises aerobic oxidative phosphorylation (OXPHOS) and anaerobic glycolysis, decreasing ATP synthesis with profound effects on cell viability, growth and senescence. Aim: To validate and further develop an oxidative stress model in vitro with which to investigate and protect against the detrimental effects of oxidative stress on cell bioenergetics. Methods: The Seahorse XFe96 analyser was utilised to monitor oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as markers for OXPHOS and glycolysis respectively. Single injections of H2O2 were applied to a neonatal human dermal fibroblast (HDFn) cell line to determine a suitable dose to exhibit substantial oxidative damage. The established skin care agent, niacinamide, was applied as three 1mM injections at 30 minute intervals, with the initial injection coinciding with the single H2O2 dose. Results: 0.75mM H2O2 did not produce a sustained level of oxidative damage in absence of niacinamide. 1mM H2O2 caused sustained reduction in both OCR (41.5%) and ECAR (26.9%) with no innate recovery. OCR (112%) and ECAR (105%) demonstrated recovery in the presence of niacinamide. Conclusion: A dose of 1mM H2O2 induces a high level of oxidative stress on major cellular bioenergetic function. Niacinamide is confirmed to restore function. This model offers a technique with which to explore oxidative damage and its prevention with various stressors, cell types and treatments.
BET bromodomain inhibitor JQ1 decreases CD30 and CCR4 expression as well as proliferation of cutaneous T-cell lymphoma M Sugaya1, H Kamijo2, N Takahshi2, T Oka1, T Miyagaki2, Y Asano3 and S Sato3 1 Department of Dermatology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan, 2 University of Tokyo Graduate School of Medicine, Tokyo, Japan and 3 Department of Dermatology, University of Tokyo Graduate School of Medicine, Tokyo, Japan Bromodomain and external domain (BET) proteins regulate cell growth, proliferation, cell cycle, and differentiation in various cancers. Therefore, they have emerged as interesting targets. The effect of BET inhibitor on cutaneous T-cell lymphoma (CTCL), however, is yet to be known. Here, we examined the effect of BET inhibitor JQ1 on four cell lines (MyLa, SeAx, Hut78 and HH cells). CTCL cell lines were treated with JQ1 and cell number, cell cycle, frequency of apoptosis, and expression of CD25, CD30 and CCR4 on the cell surface were evaluated by flow cytometry. Cell surface molecules were also analyzed by quantitative RTPCR. JQ1 dose-dependently decreased the cell number of CTCL cells through G1 arrest. JQ1 induced senescence only in MyLa cells and apoptosis in SeAx, Hut78 and HH cells. It was consistent with the fact that p53, which induces senescence, is fully functional only in MyLa cells, but not in SeAx, Hut78, or HH cells. Moreover, JQ1 downregulated CD30 and CCR4 expression both on the cell surface and at mRNA levels. Thus, BET bromodomain inhibitor JQ1 may be useful for treatment of CTCL. Combination with an antibody therapy targeting CCR4 or CD30 may not be recommended because JQ1 decreases expression of the target molecules.
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Fluorescence lifetime imaging microscopy (FLIM) as a novel method to assess skin residency of a fluorescent compound: experience with GSK2894512 A Alex1, S Frey1, H Angelene1, CD Neitzel2, J Li3, AJ Bower3, DR Spillman3, SA Boppart3 and Z Arp1 1 GSK, Collegeville, PA, 2 Carle Foundation Hospital, Urbana, IL and 3 University of Illinois at Urbana-Champaign, Urbana, IL Fluorescence Lifetime Imaging Microscopy (FLIM) is a non-invasive optical imaging method that can monitor chemical, morphologic, and metabolic changes in-vivo. FLIM was utilized as a novel endpoint in the evaluation of 2 different formulations of GSK2894512, an inherently fluorescent compound, being developed as a novel topical anti-inflammatory agent for atopic dermatitis and psoriasis. An open-label, 15 day study (# 201661) in 6 healthy adult males was conducted with 2 different formulations of 1% GSK2894512, Cream A and B. Both creams were applied for 7 days on opposite forearms followed by 8 days of post-treatment observation. FLIM assessments were collected for both creams on Days 1 to 15 with 3 biopsies collected only from the Cream B application area during the 7-day post-treatment period. GSK2894512 was clearly observable for both creams by FLIM monitoring. FLIM was able to detect a maximum penetration depth of 95 mm, and residency for up to 48 hrs posttreatment with Cream A and up to 24 hrs with Cream B. Analyses of GSK2894512 concentrations from the Cream B biopsies using LC-MS/MS agreed with FLIM observations. This study demonstrates the capability of FLIM as a novel non-invasive detection method to follow penetration and residency of GSK2894512 in healthy skin. FLIM allowed for the observation of GSK2894512 for both creams without the need for biopsy. These results suggest FLIM could be a viable alternative to skin biopsies without the usual patient discomfort and limitations, thereby enabling the direct measurement of skin distribution and true longitudinal monitoring.
S116 Journal of Investigative Dermatology (2017), Volume 137
Human skin biopsy culture model maintains psoriasis disease features and demonstrates Pathway Engagement by Dexamethasone S Huang1, S Grosskurth2, L Miller1, H Patel1, L Olson1, J Wetter1, R Edelmayer1, M Domanus1, C Miller1, P Honore3 and V Scott3 1 AbbVie Inc., North Chicago, IL, 2 AbbVie Inc, North Chicago, IL and 3 AbbVie, North Chicago, IL In drug discovery, early indication of efficacy or pathway engagement of potential clinical candidates in relevant disease models is extremely valuable. Although animal models of skin disease are useful, not all features of human skin such as the substantial differences in keratinocyte layers, are recapitulated in mouse models,. In collaboration with the AbbVie Clinical Pharmacology Research Unit, we have obtained skin biopsies from psoriasis patients to develop and characterize an ex vivo human skin biopsy culture model. The skin biopsies were cultured at the air-liquid interface under 95% O2 at 37 C to evaluate the effects of both culturing conditions and pharmacological treatment. Analysis of cytokine levels in conditioned media and biopsy gene expression profiling demonstrated that many features of psoriasis disease (such as elevated TNF, IL-17A and IL-23 release) are maintained in culture of lesion versus non-lesion biopsies over 24 hours. Effects of the reference anti-inflammatory compound Dexamethasone were evaluated in this model for pathway engagement. Transcriptomic and cytokine analyses of samples treated with Dexamethasone exhibited decreased inflammation, as well as previously reported agonistic effects on the glucocorticoid receptor NR3C1. The work presented here demonstrate the value of the explant human skin biopsy culture model for maintaining clinical features of psoriasis as well as for investigating pharmacodynamic pathway engagement, and potential efficacy of novel candidate therapeutics.