Monitoring UV-induced signalling pathways in an ex vivo skin organ culture model using phospho-antibody array.

We investigated UV-induced signalling in an ex vivo skin organ culture model using phospho-antibody array. Phosphorylation modulations were analysed in time-course experiments following exposure to solar-simulated UV and validated by Western blot analyses. We found that UV induced P-p38 and its substrates, P-ERK1/2 and P-AKT, which were previously shown to be upregulated by UV in cultured keratinocytes and in vivo human skin. This indicates that phospho-antibody array applied to ex vivo skin organ culture is a relevant experimental system to investigate signalling events following perturbations. As the identified proteins are components of pathways implicated in skin tumorigenesis, UV-exposed skin organ culture model could be used to investigate the effect on these pathways of NMSC cancer drug candidates. In addition, we found that phospho-HCK is induced upon UV exposure, producing a new candidate for future studies investigating its role in the skin response to UV and UV-induced carcinogenesis.

A topical treatment containing heat-treated Lactobacillus johnsonii NCC 533 reduces Staphylococcus aureus adhesion and induces antimicrobial peptide expression in an in vitro reconstructed human epidermis model.

Staphylococcus aureus colonization is thought to contribute to the pathophysiology of atopic dermatitis (AD). AD patients exhibit reduced levels of cutaneous antimicrobial peptides (AMPs), which may explain their increased susceptibility to infections. Using an in vitro reconstructed human epidermis (RHE) model, we sought to determine whether topical application of a non-replicating probiotic, heat-treated Lactobacillus johnsonii NCC 533 (HT La1), could inhibit S. aureus adhesion to skin and boost cutaneous innate immunity. We found that application of HT La1 suspension to RHE samples reduced the binding of radiolabelled S. aureus by up to 74%. To investigate a potential effect of HT La1 on innate immunity, we analysed the expression of nine AMP genes, including those encoding beta defensins and S100 proteins, following topical application of HT La1 in suspension or in a daily moisturizer lotion. Analysed genes were induced by up to fourfold in a dose-dependent manner by HT La1 in suspension and by up to 2.4-fold by HT La1 in the moisturizer lotion. Finally, using ELISA and immunohistochemical detection, we evaluated the expression and secretion of the AMPs hBD-2 and psoriasin and determined that both proteins were induced by topical HT La1, particularly in the stratum corneum of the RHE. These findings demonstrate that a topically applied, non-replicating probiotic can modulate endogenous AMP expression and inhibit binding of S. aureus to an RHE model in vitro. Moreover, they suggest that a topical formulation containing HT La1 could benefit atopic skin by enhancing cutaneous innate immunity and reducing S. aureus colonization.

Brimonidine displays anti-inflammatory properties in the skin through the modulation of the vascular barrier function.

BACKGROUND: Rosacea is a chronic inflammatory skin disease. Characteristic vascular changes in rosacea skin include enlarged, dilated vessels of the upper dermis and blood flow increase. Brimonidine is approved for symptomatic relief of the erythema of rosacea. It acts by selectively binding to α2-adrenergic receptors present on smooth muscle in the peripheral vasculature, resulting in transient local vasoconstriction. OBJECTIVES: To provide further evidence of the anti-inflammatory potential of brimonidine across preclinical models of skin inflammation and its ability to decrease the neutrophil infiltration in human skin after ultraviolet light exposure. METHODS: The anti-inflammatory properties of brimonidine through modulation of the vascular barrier function were assessed using in vivo neurogenic vasodilation and acute inflammatory models and a well-described in vitro transmigration assay. A clinical study assessed the neutrophil infiltration in human skin after exposure to UV in 37 healthy Caucasian male subjects. RESULTS: In vitro, brimonidine affects the transmigration of human neutrophils through the endothelial barrier by modulating adhesion molecules. In vivo, in the mouse, topical treatment with brimonidine, used at a vasoconstrictive dose, confirmed its anti-inflammatory properties and prevented leucocyte recruitment (rolling and adhesion) mediated by endothelial cells. Topical pretreatment with brimonidine tartrate 0.33% gel once a day for 4 days significantly prevented neutrophil infiltration by 53.9% in human skin after exposure to UV light. CONCLUSION: Results from in vitro, in vivo and from a clinical study indicate that brimonidine impacts acute inflammation of the skin by interfering with neurogenic activation and/or recruitment of neutrophils.

Squaramides as novel class I and IIB histone deacetylase inhibitors for topical treatment of cutaneous t-cell lymphoma.

A series of squaramide-based hydroxamic acids were designed, synthesized and evaluated against human HDAC enzyme. Squaramides were found to be potent in the Hut78 cell line, but initially suffered from low solubility. Leads with improved solubility and metabolic profiles were shown to be class I, IIB and IV selective.

Discovery and process development of a novel TACE inhibitor for the topical treatment of psoriasis.

Targeting the TNFα pathway is a validated approach to the treatment of psoriasis. In this pathway, TACE stands out as a druggable target and has been the focus of in-house research programs. In this article, we present the discovery of clinical candidate 26a. Starting from hits plagued with poor solubility or genotoxicity, 26a was identified through thorough multiparameter optimisation. Showing robust in vivo activity in an oxazolone-mediated inflammation model, the compound was selected for development. Following a polymorph screen, the hydrochloride salt was selected and the synthesis was efficiently developed to yield the API in 47% overall yield.

Pharmacological Transdifferentiation of Human Nasal Olfactory Stem Cells into Dopaminergic Neurons.

The discovery of novel drugs for neurodegenerative diseases has been a real challenge over the last decades. The development of patient- and/or disease-specific in vitro models represents a powerful strategy for the development and validation of lead candidates in preclinical settings. The implementation of a reliable platform modeling dopaminergic neurons will be an asset in the study of dopamine-associated pathologies such as Parkinson's disease. Disease models based on cell reprogramming strategies, using either human-induced pluripotent stem cells or transcription factor-mediated transdifferentiation, are among the most investigated strategies. However, multipotent adult stem cells remain of high interest to devise direct conversion protocols and establish in vitro models that could bypass certain limitations associated with reprogramming strategies. Here, we report the development of a six-step chemically defined protocol that drives the transdifferentiation of human nasal olfactory stem cells into dopaminergic neurons. Morphological changes were progressively accompanied by modifications matching transcript and protein dopaminergic signatures such as LIM homeobox transcription factor 1 alpha (LMX1A), LMX1B, and tyrosine hydroxylase (TH) expression, within 42 days of differentiation. Phenotypic changes were confirmed by the production of dopamine from differentiated neurons. This new strategy paves the way to develop more disease-relevant models by establishing reprogramming-free patient-specific dopaminergic cell models for drug screening and/or target validation for neurodegenerative diseases.

In Vitro Safety Pharmacology Profiling of Topical α-Adrenergic Agonist Treatments for Erythema of Rosacea.

BACKGROUND: Topical α-adrenergic agonist therapy has been developed to treat the persistent erythema of rosacea patients. Brimonidine and oxymetazoline are both topical α-adrenergic agonists. OBJECTIVES: The objective of this in vitro safety pharmacology study was to compare the potential safety profiles of brimonidine and oxymetazoline. METHODS: Brimonidine and oxymetazoline underwent pharmacological profiling with a standard panel of 151 assays, including α-adrenergic receptors and 5-hydroxytryptamine (5-HT) receptors. A valvular interstitial cell (VIC) proliferation assay was performed with oxymetazoline hydrochloride. RESULTS: Brimonidine was highly selective for the α2 adrenergic receptors, specifically α2A, whereas oxymetazoline was found to be much less selective and was highly active against a wide range of targets. Negligible activity was observed with brimonidine at the 5-HT2B receptor, whereas oxymetazoline had significant 5-HT2B receptor agonist activity and caused proliferation of mitral VICs in vitro. CONCLUSION: As the 5-HT2B receptor is potentially involved in drug-induced valvulopathy, the benefit/risk ratio should be carefully considered, especially in patients with cardiovascular disease or other comorbidities.

Discovery and Characterization of CD12681, a Potent RORγ Inverse Agonist, Preclinical Candidate for the Topical Treatment of Psoriasis.

With possible implications in multiple autoimmune diseases, the retinoic acid receptor-related orphan receptor RORγ has become a sought-after target in the pharmaceutical industry. Herein are described the efforts to identify a potent RORγ inverse agonist compatible with topical application for the treatment of skin diseases. These efforts culminated in the discovery of N-(2,4-dimethylphenyl)-N-isobutyl-2-oxo-1-[(tetrahydro-2H-pyran-4-yl)methyl]-2,3-dihydro-1H-benzo[d]imidazole-5-sulfonamide (CD12681), a potent inverse agonist with in vivo activity in an IL-23-induced mouse skin inflammation model.

A new mechanism for prolactin processing into 16K PRL by secreted cathepsin D.

Cathepsins are lysosomal enzymes that were shown to release the antiangiogenic fragments 16K prolactin (PRL), endostatin, and angiostatin by processing precursors at acidic pH in vitro. However, the physiological relevance of these findings is questionable because the neutral pH of physiological fluids is not compatible with the acidic conditions required for the proteolytic activity of these enzymes. Here we show that cathepsin D secreted from various tissues is able to process PRL into 16K PRL outside the cell. To specifically target extracellular proteolysis, we used tissues from PRL receptor-deficient mice, which are unable to internalize PRL. As assessed by the use of specific inhibitors of proton extruders, we show that the proteolytic activity of cathepsin D requires local acid secretion driven by Na(+)/H(+) exchangers and H(+)/ATPase. Although it is usually assumed that cathepsin-mediated generation of antiangiogenic peptides occurs in the moderately acidic pericellular milieu found in malignant tumors, we propose a new mechanism explaining the extracellular activity of this acidic protease under physiological pH. Our data support the concept that secreted lysosomal enzymes could be involved in the maintenance of angiogenesis dormancy via the generation of active antiangiogenic peptides in nonpathological contexts.

Topical Treatment of Rosacea with Ivermectin Inhibits Gene Expression of Cathelicidin Innate Immune Mediators, LL-37 and KLK5, in Reconstructed and Ex Vivo Skin Models.

INTRODUCTION: Numerous intrinsic and extrinsic factors have been associated with the pathophysiology of rosacea, including dysregulation of innate immunity. A high level of cathelicidin antimicrobial peptides (e.g., LL-37) has been shown in the facial skin of patients with rosacea. Excessive production of both LL-37 and KLK5, the serine protease responsible for its cleavage, has been suggested to play a role in the pathophysiology of rosacea. Ivermectin 10 mg/g cream, indicated for the treatment of inflammatory lesions of rosacea, is reported to have dual anti-parasitic and anti-inflammatory properties. However, the exact mechanism of action of ivermectin cream in the treatment of rosacea is unknown. METHODS: This study aimed to evaluate the effect of ivermectin on the expression of KLK5 and the subsequent effect on the maturation process of cathelicidins. Experimental studies were performed either on normal human epidermal keratinocytes (NHEK), reconstructed human epidermis (RHE) or on human skin ex vivo stimulated with calcitriol (1α,25-dihydroxyvitamin D3), which is known to induce KLK5 and LL-37 expression. RESULTS: The results show that ivermectin is able to inhibit KLK5 and CAMP gene expression and protein secretion in NHEK cells stimulated with calcitriol. Those results were confirmed in 3D models of the skin (RHE and skin ex vivo). The anti-inflammatory effects of ivermectin were associated with an inhibition of IL-8, IL-6 and MCP-1 (CCL2) secretion from NHEK cells. CONCLUSIONS: These results suggest that ivermectin can prevent the inflammatory effects of rosacea triggered by abnormal LL-37 processing, through the inhibition of KLK5 gene expression in the epidermis. FUNDING: Nestlé Skin Health R&D.

Platelet-rich plasma respectively reduces and promotes adipogenic and myofibroblastic differentiation of human adipose-derived stromal cells via the TGFß signalling pathway.

Autologous fat grafting is a gold standard therapy for soft tissue defects, but is hampered by unpredictable postoperative outcomes. Fat graft enrichment with adipose-derived stromal cell (ASCs) was recently reported to enhance graft survival. Platelet-rich plasma (PRP) has also emerged as a biologic scaffold that promotes fat graft viability. Combined ASC/PRP fat grafting enrichment is thus a promising new regenerative medicine approach. The effects of PRP on ASC proliferation are well documented, but the impact of PRP on ASC differentiation has yet to be investigated in depth to further elucidate the PRP clinical effects. Here we analyzed the human ASC fate upon PRP treatment. PRP was found to sharply reduce the potential of ASCs to undergo differentiation into adipocytes. Interestingly, the PRP anti-adipogenic effect was accompanied by the generation of myofibroblast-like cells. Among the various factors released from PRP, TGFß pathway activators played a critical role in both the anti-adipogenic and pro-myofibroblastic PRP effects. Overall, these data suggest that PRP participates in maintaining a pool of ASCs and in the repair process by promoting ASC differentiation into myofibroblast-like cells. TGFß may provide an important target pathway to improve PRP clinical outcomes.

Cathepsin D processes human prolactin into multiple 16K-like N-terminal fragments: study of their antiangiogenic properties and physiological relevance.

16K prolactin (PRL) is the name given to the 16-kDa N-terminal fragment obtained by proteolysis of rat PRL by tissue extracts or cell lysates, in which cathepsin D was identified as the candidate protease. Based on its antiangiogenic activity, 16K PRL is potentially a physiological inhibitor of tumor growth. Full-length human PRL (hPRL) was reported to be resistant to cathepsin D, suggesting that antiangiogenic 16K PRL may be physiologically irrelevant in humans. In this study, we show that hPRL can be cleaved by cathepsin D or mammary cell extracts under the same conditions as described earlier for rat PRL, although with lower efficiency. In contrast to the rat hormone, hPRL proteolysis generates three 16K-like fragments, which were identified by N-terminal sequencing and mass spectrometry as corresponding to amino acids 1-132 (15 kDa), 1-147 (16.5 kDa), and 1-150 (17 kDa). Biochemical and mutagenetic studies showed that the species-specific digestion pattern is due to subtle differences in primary and tertiary structures of rat and human hormones. The antiangiogenic activity of N-terminal hPRL fragments was assessed by the inhibition of growth factor-induced thymidine uptake and MAPK activation in bovine umbilical endothelial cells. Finally, an N-terminal hPRL fragment comigrating with the proteolytic 17-kDa fragment was identified in human pituitary adenomas, suggesting that the physiological relevance of antiangiogenic N-terminal hPRL fragments needs to be reevaluated in humans.

Characterization of human knee and chin adipose-derived stromal cells.

Animal study findings have revealed that individual fat depots are not functionally equivalent and have different embryonic origins depending on the anatomic location. Mouse bone regeneration studies have also shown that it is essential to match the Hox code of transplanted cells and host tissues to achieve correct repair. However, subcutaneous fat depots from any donor site are often used in autologous fat grafting. Our study was thus carried out to determine the embryonic origins of human facial (chin) and limb (knee) fat depots and whether they had similar features and molecular matching patterns. Paired chin and knee fat depots were harvested from 11 subjects and gene expression profiles were determined by DNA microarray analyses. Adipose-derived stromal cells (ASCs) from both sites were isolated and analyzed for their capacity to proliferate, form clones, and differentiate. Chin and knee fat depots expressed a different HOX code and could have different embryonic origins. ASCs displayed a different phenotype, with chin-ASCs having the potential to differentiate into brown-like adipocytes, whereas knee-ASCs differentiated into white adipocytes. These results highlighted different features for these two fat sites and indicated that donor site selection might be an important factor to be considered when applying adipose tissue in cell-based therapies.

Transcriptional Analysis of Vitiligo Skin Reveals the Alteration of WNT Pathway: A Promising Target for Repigmenting Vitiligo Patients.

Vitiligo affects 1% of the worldwide population. Halting disease progression and repigmenting the lesional skin represent the two faces of therapeutic challenge in vitiligo. We performed transcriptome analysis on lesional, perilesional, and non-depigmented skin from vitiligo patients and on matched skin from healthy subjects. We found a significant increase in CXCL10 in non-depigmented and perilesional vitiligo skin compared with levels in healthy control skin; however, neither CXCL10 nor other immune factors were deregulated in depigmented vitiligo skin. Interestingly, the WNT pathway, which is involved in melanocyte differentiation, was altered specifically in vitiligo skin. We demonstrated that oxidative stress decreases WNT expression/activation in keratinocytes and melanocytes. We developed an ex vivo skin model and confirmed the decrease activation of the WNT pathway in human skin subjected to oxidative stress. Finally, using pharmacological agents that activate the WNT pathway, we treated ex vivo depigmented skin from vitiligo patients and successfully induced differentiation of resident stem cells into pre-melanocytes. Our results shed light on the previously unrecognized role of decreased WNT activation in the prevention of melanocyte differentiation in depigmented vitiligo skin. Furthermore, these results support further clinical exploration of WNT agonists to repigment vitiligo lesions.

Vasoconstriction and anti-inflammatory properties of the selective α-adrenergic receptor agonist brimonidine.

BACKGROUND: The facial erythema of rosacea is recognized as the most prevalent and most difficult manifestation of rosacea to treat. A recent approach in patients with rosacea has been to reduce this erythema through vasoconstriction of cutaneous blood vessels by selectively targeting α2-adrenergic receptors with brimonidine. OBJECTIVE: To further investigate the pharmacodynamic profile of brimonidine, its vasoconstrictive effects and its anti-inflammatory properties. METHODS: The potency for the α1A, α1B, α2A, α2B and α2C receptors of brimonidine was measured, as well as performing a large target profiling study in order to determine the target selectivity profile of brimonidine. The vasoconstrictive effects of brimonidine were measured using ex vivo wire myography and human skin biopsy neuroinflammation models. The anti-inflammatory properties of brimonidine were measured using two in vivo mice ear inflammation models. RESULTS: Brimonidine was found to be highly selective for the α2A adrenoreceptor (EC50 0.45nM) over the other α-adrenoreceptors. Additionally, the large target profiling study demonstrated the high selectivity of brimonidine with minimal off-target effects. The ex vivo wire myography model showed that brimonidine is a potent vasoconstrictor of human subcutaneous vessels with a diameter of less than 200µm (EC50 0.4nM). The ex vivo human skin biopsy neuroinflammation model demonstrated that brimonidine completely inhibited vasodilation induced by capsaicin. Both in vivo mouse ear inflammation models highlighted that brimonidine inhibited ear edema (up to 76%) when compared to vehicle. CONCLUSION: The selectivity, vasoconstrictive and anti-inflammatory properties of brimonidine that have been described in these studies are in agreement with the benefits observed with this compound in the treatment of facial erythema in rosacea.

Structure-based discovery of allosteric modulators of two related class B G-protein-coupled receptors.

Despite the availability of X-ray crystal structure data for several members of the G-protein-coupled receptor (GPCR) superfamily, structure-based discovery of GPCR ligands has been exclusively restricted to class A (rhodopsin-like) receptors. Herein we report the identification, by a docking-based virtual screening approach, of noncompetitive ligands for two related class B (secretin-like) GPCRs: the glucagon receptor (GLR) and the glucagon-like peptide 1 receptor (GLP-1R). Starting from a knowledge-based three-dimensional model of the GLR, a database of 1.9 million commercially available drug-like compounds was screened for chemical similarity to existing GLR noncompetitive antagonists and docked to the transmembrane cavity of the GLR; 23 compounds were then selected based on protein-ligand interaction fingerprints, and were then purchased and evaluated for in vitro binding to GLR and modulation of glucagon-induced cAMP release. Two of the 23 compounds inhibited the effect of glucagon in a dose-dependent manner, with one inhibitor exhibiting the same potency as L-168 049, a reference noncompetitive GLR antagonist, in a whole-cell-based functional assay. Interestingly, one virtual hit that was inactive at the GLR was shown to bind to GLP-1R and potentiate the response to the endogenous GLP-1 ligand.

Cellular basis for bimatoprost effects on human conventional outflow.

PURPOSE: Bimatoprost is a widely used ocular hypotensive agent to treat glaucoma. It lowers intraocular pressure in humans by increasing both pressure-independent (uveoscleral) and pressure-dependent (conventional) aqueous humor outflow. The present study specifically examines bimatoprost effects on the cells that populate human outflow tissues. METHODS: The authors tested for prostamide receptor activation in primary cultures of human trabecular meshwork (TM), Schlemm's canal (SC), and ciliary smooth muscle (CSM) cells using cellular dielectric spectroscopy (CDS). RESULTS: The authors observed that bimatoprost produced an immediate and concentration-dependent increase in cell monolayer impedance for TM, SC, and CSM cells with EC(50) values of 4.3, 1.2, and 1.7 nM, respectively; corresponding to decreased cell contractility. Notably, in TM, SC, and CSM cells, bimatoprost was approximately equipotent to the selective FP receptor agonists fluprostenol and 17-phenyl PGF(2α). Bimatoprost effects were insensitive to cholera toxin and pertussis toxin but were abolished by phorbol 12-myristate 13-acetate pretreatment, suggesting Gq-involvement in cell signaling. The effects of bimatoprost on TM and SC cells were inhibited by the prostamide receptor antagonist AGN211334, with IC(50) values of 1.2 and 3.3 µM, respectively. Interestingly, AGN211334 behaved as an apparent inverse agonist in CDS assays involving TM cells but as a neutral prostamide antagonist with SC cells. CONCLUSIONS: Taken together, results suggest that bimatoprost specifically activates receptors in both cell types of the human conventional outflow pathway to modify intraocular pressure. However, only TM cell monolayers appear to have autocrine, or agonist-independent, receptor signaling that is sensitive to a prostamide receptor antagonist.