Anton Stuetz

Topical Treatment of Basal Cell Carcinomas in Nevoid Basal Cell Carcinoma Syndrome with a Smoothened Inhibitor

Basal cell carcinoma (BCC) is a distinctive manifestation in nevoid basal cell carcinoma syndrome (NBCCS) patients. Both inherited and acquired mutations of patched 1 (PTCH1), a tumor-suppressor gene controlling the activity of Smoothened (SMO), are the primary cause of the constitutive activation of the Hedgehog (HH) pathway, leading to the emergence of BCCs in NBCCS. LDE225, a distinct, selective antagonist of SMO, showed potent inhibition of basaloid tumor nest formation and mediated regression of preformed basaloid tumors in organ cultures of skin derived from Ptch1 heterozygous knockout mice. In a double-blind, randomized, vehicle-controlled, intraindividual study, a total of 8 NBCCS patients presenting 27 BCCs were treated twice daily with 0.75% LDE225 cream or vehicle for 4 weeks. Application of 0.75% LDE225 cream was well tolerated and showed no skin irritation. Of 13 LDE225-treated BCCs, 3 showed a complete, 9 a partial, and only 1 no clinical response. Except for one partial response, the vehicle produced no clinical response in any of the 14 treated BCCs. Treatment with 0.75% LDE225 cream in NBCCS patients was very well tolerated and caused BCC regression, thus potentially offering an attractive therapeutic alternative to currently available therapies for this indication.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.

Inhibition of Chronic and Acute Skin Inflammation by Treatment with a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor

Although vascular remodeling is a hallmark of many chronic inflammatory disorders, antivascular strategies to treat these conditions have received little attention to date. We investigated the effects of a newly identified vascular endothelial growth factor (VEGF) receptor tyrosine-kinase inhibitor, NVP-BAW2881, on endothelial cell function in vitro and its anti-inflammatory activity in different animal models. NVP-BAW2881 inhibited proliferation, migration, and tube formation by human umbilical vein endothelial cells and lymphatic endothelial cells in vitro. In a transgenic mouse model of psoriasis, NVP-BAW2881 reduced the number of blood and lymphatic vessels and infiltrating leukocytes in the skin, and normalized the epidermal architecture. NVP-BAW2881 also displayed strong anti-inflammatory effects in models of acute inflammation; pretreatment with topical NVP-BAW2881 significantly inhibited VEGF-A-induced vascular permeability in the skin of pigs and mice. Furthermore, topical application of NVP-BAW2881 reduced the inflammatory response elicited in pig skin by UV-B irradiation or by contact hypersensitivity reactions. These results demonstrate for the first time that VEGF receptor tyrosine-kinase inhibitors might be used to treat patients with inflammatory skin disorders such as psoriasis.

Selective inhibition of vitamin D hydroxylases in human keratinocytes

Human keratinocytes convert 25(OH)D(3) to hormonally active 1alpha,25(OH)(2)D(3) and respond to its antiproliferative/prodifferentiating action in vitro and in vivo. Levels and activity of 1alpha,25(OH)(2)D(3) are short-lived. 1alpha,25(OH)(2)D(3) induces 24-hydroxylase (CYP24) that rapidly metabolizes the hormone, yielding a cascade of side-chain oxidized products and this eventually results in the loss of activity. Aiming at stabilizing the levels of active hormone, we have searched for potent, selective inhibitors of CYP24. Selective inhibition was crucial in order to avoid impairment of 1alpha,25(OH)(2)D(3) synthesis, catalyzed by 1alpha-hydroxylase - a related member of cytochrome P-450 (CYP) superfamily. We describe here the testing protocol, using primary human keratinocyte cultures as an appropriate source of CYP24 and 1alpha-hydroxylase, (3)H-25(OH)D(3) (at physiological concentrations) as substrate and sensitive HPLC techniques to analyze the complex metabolite profiles. Four hundred potential inhibitors were screened by this method; most of them were synthesized in our laboratory. These compounds (entitled azoles) were capable of direct binding to the heme iron and of additional interactions with other parts of the enzyme. In this paper, we present VID400 and SDZ 89-443, as first examples of powerful selective CYP24 inhibitors. As anticipated, these compounds increased the levels of 1alpha-hydroxylated products generated from (3)H-25(OH)D(3) and extended their lifetime. Importantly, blocking of 24-hydroxylation led to a switch in metabolism, namely to preferential conversion of 1alpha,25(OH)(2)D(3) to 1alpha,25(OH)(2)-3epi-D(3). As spin-off from our program, selective inhibitors of 1alpha-hydroxylase were also found (e.g. SDZ 88-357). Using (3)H-25(OH)D(3) as substrate in the absence of SDZ 88-357, CYP24 showed high preference for freshly generated 1alpha-hydroxylated metabolites over abundant 25(OH)D(3). In the presence of SDZ 88-357, we noticed a great increase in 24-hydroxylation of (3)H-25(OH)D(3). Besides their use as valuable tools in elucidating regulatory mechanisms, inhibitors of VD hydroxylases may give rise to novel therapeutic strategies, especially in defects of cell growth and differentiation.

Ascomycins: promising agents for the treatment of inflammatory skin diseases

Ascomycin derivatives represent a novel class of anti-inflammatory macrolactams currently under development for the treatment of skin diseases. The main biological effect of ascomycins is an inhibition of the synthesis of both Th1 and Th2-type cytokines in target cells. Several compounds are being developed with SDZ ASM 981 being at the most advanced stage. It has high anti-inflammatory activity in animal models of skin inflammation and does not induce skin atrophy. Topical application of SDZ ASM 981 was shown to be effective in atopic dermatitis (AD), allergic contact dermatitis and also in psoriasis under semi-occlusive conditions. In patients with AD, SDZ ASM 981 cream led to consistently low systemic exposure even when applied on large areas of skin. SDZ ASM 981 overcomes the drawbacks of current topical therapies of inflammatory skin diseases as its safety profile is better than that of topical corticosteroids. Studies continue to investigate its efficacy and safety in the treatment of inflammatory skin diseases.

Mechanical and Metabolic Injury to the Skin Barrier Leads to Increased Expression of Murine β-Defensin-1, -3, and -14

Protection of the skin against microbiological infection is provided by the permeability barrier and by antimicrobial proteins. We asked whether the expression of murine β-defensins (mBDs)-1, -3, and -14-orthologs of human β-defensins hBD-1, -2, and -3, respectively--is stimulated by mechanically/physicochemically (tape stripping or acetone treatment) or metabolically (essential fatty acid-deficient (EFAD) diet) induced skin barrier dysfunction. Both methods led to a moderate induction of mBD-1 and mBD-14 and a pronounced induction of mBD-3 mRNA. Protein expression of the mBDs was increased as shown by immunohistology and by western blotting. Artificial barrier repair by occlusion significantly reduced the increased expression of mBD-14 after mechanical injury and of all three mBDs in EFAD mice, supporting an interrelationship between permeability and the antimicrobial barrier. mBD-3 expression was stimulated in vitro by tumor necrosis factor-α (TNF-α), and a neutralizing anti-TNF-α antibody significantly reduced increased mBD-3 expression after barrier injury in mouse skin, indicating that induction of mBD-3 expression is mediated by cytokines. The expression of mBD-14 was stimulated by transforming growth factor-α and not by TNF-α. In summary, we demonstrated upregulation of mBD1, -3, and -14 after mechanically and metabolically induced skin barrier disruption, which may be an attempt to increase defense in the case of permeability barrier dysfunction.

Pimecrolimus Enhances TLR2/6-Induced Expression of Antimicrobial Peptides in Keratinocytes

Calcineurin inhibitors are potent inhibitors of T-cell-receptor mediated activation of the adaptive immune system. The effects of this class of drug on the innate immune response system are not known. Keratinocytes are essential to innate immunity in skin and rely on toll-like receptors (TLRs) and antimicrobial peptides to appropriately recognize and respond to injury or microbes. In this study we examined the response of cultured human keratinocytes to pimecrolimus. We observed that pimecrolimus enhances distinct expression of cathelicidin, CD14, and human beta-defensin-2 and beta-defensin-3 in response to TLR2/6 ligands. Some of these responses were further enhanced by 1,25 vitamin D3. Pimecrolimus also increased the functional capacity of keratinocytes to inhibit growth of Staphylococcus aureus and decreased TLR2/6-induced expression of IL-10 and IL-1beta. Furthermore, pimecrolimus inhibited nuclear translocation of NFAT and NF-kappaB in keratinocytes. These observations uncover a previously unreported function for pimecrolimus in cutaneous innate host defense.

Discovery of topical calcineurin inhibitors and pharmacological profile of pimecrolimus

Using a newly developed model of allergic contact dermatitis in pigs, calcineurin inhibitors of the tacrolimus and ascomycin type were shown to have a highly anti-inflammatory action after topical application. These findings provided the first pharmacological evidence of the efficacy of this novel class of topical agents in the treatment of inflammatory skin diseases, and, thus, their potential to become the first alternative to corticosteroids in more than 40 years. As a result of a large research program into ascomycins, pimecrolimus (Elidel®, SDZ ASM 981) was selected for development due to its favorable pharmacology and safety profile, alongside tacrolimus (Protopic®, FK 506). In vitro, pimecrolimus inhibits the transcription and release of pro-inflammatory cytokines in T cells. Similar to the corticosteroids, betamethasone-17-valerate and dexamethasone, pimecrolimus is effective at nanomolar concentrations. Targeting mainly T cells, pimecrolimus has, however, a more specific mode of action. Moreover, in contrast to corticosteroids, pimecrolimus has no effect on Langerhans’ cells, the professional antigen- presenting dendritic cells of the skin that are crucial for local immunosurveillance. When applied topically, pimecrolimus exerts a high and selective anti-inflammatory activity in the skin, shows minimal percutaneous absorption, and has a low potential to affect systemic immunoreactions. Pimecrolimus cream 1% has proven to be well tolerated, safe, and highly effective in clinical studies in patients with atopic dermatitis.

Pimecrolimus permeates less than tacrolimus through normal, inflamed, or corticosteroid-pretreated skin

Abstract:  The permeabilities of normal human and normal, inflamed, or corticosteroid (CS) pretreated skin of young domestic pigs for pimecrolimus and tacrolimus were compared in vitro, using Franz‐type diffusion cells. The test articles were either used as 1.0% solutions or as the marketed formulations (Elidel 1% cream, Protopic 0.1%, and 0.03% ointment). In normal human skin, the permeation rate of pimecrolimus from the 1% cream was about sixfold lower than that of tacrolimus from 0.1% ointment and by a factor of 4.3 lower compared with tacrolimus from Protopic 0.03%. In pigs, sodium laurylsulfate‐induced irritant contact dermatitis resulted in significantly faster skin permeation of both drugs from applied solutions. The permeation rate for pimecrolimus was lower than that for tacrolimus. Thus, at 24 h, pimecrolimus concentrations in the receptor fluid were 2.8‐fold lower than the tacrolimus levels. Compared with normal porcine skin, permeation of drugs through hydrocortisone (1.0%)‐, mometasone (0.1%)‐, or clobetasol‐17‐butyrate (0.05%)‐pretreated skin was increased by factors of 3.6 (pimecrolimus, applied as 1% cream) and 1.7 (tacrolimus, applied as 0.1% ointment). In normal pig skin, the permeation rate of tacrolimus was found to be 11.2 times higher than that of pimecrolimus and 3.5‐ to 7.1‐fold higher in CS‐pretreated skin, independent of the potency of the CSs. The present in vitro data suggest that in patients with acute skin inflammation or after therapy with topical CSs, percutaneous absorption and, as a consequence, systemic drug exposure will be lower with Elidel 1% cream as compared with Protopic 0.1% and 0.03% ointment.

NVP-LDE225, a Potent and Selective SMOOTHENED Antagonist Reduces Melanoma Growth In Vitro and In Vivo

Melanoma is one of the most aggressive cancers and its incidence is increasing worldwide. So far there are no curable therapies especially after metastasis. Due to frequent mutations in members of the mitogen-activated protein kinase (MAPK) signaling pathway, this pathway is constitutively active in melanoma. It has been shown that the SONIC HEDGEHOG (SHH)-GLI and MAPK signaling pathway regulate cell growth in many tumors including melanoma and interact with each other in the regulation of cell proliferation and survival. Here we show that the SHH-GLI pathway is active in human melanoma cell lines as they express downstream target of this pathway GLI1. Expression of GLI1 was significantly higher in human primary melanoma tissues harboring BRAFV600E mutation than those with wild type BRAF. Pharmacologic inhibition of BRAFV600E in human melanoma cell lines resulted in decreased expression of GLI1 thus demonstrating interaction of SHH-GLI and MAPK pathways. Inhibition of SHH-GLI pathway by the novel small molecule inhibitor of smoothened NVP-LDE225 was followed by inhibition of cell growth and induction of apoptosis in human melanoma cell lines, interestingly with both BRAFV600E and BRAFWild Type status. NVP-LDE225 was potent in reducing cell proliferation and inducing tumor growth arrest in vitro and in vivo, respectively and these effects were superior to the natural compound cyclopamine. Finally, we conclude that inhibition of SHH-GLI signaling pathway in human melanoma by the specific smoothened inhibitor NVP-LDE225 could have potential therapeutic application in human melanoma even in the absence of BRAFV600E mutation and warrants further investigations.

Effect of pimecrolimus vs. corticosteroids on murine bone marrow‐derived dendritic cell differentiation, maturation and function

Abstract:  Pimecrolimus (SDZ ASM981) is a non‐steroid member of calcineurin inhibitors recently developed for the treatment of inflammatory skin diseases. In this study, we compared the effect of pimecrolimus and corticosteroids on the differentiation, maturation and function of murine bone marrow‐derived dendritic cells (BM‐DC). We added pimecrolimus at concentrations of 5–500 ng/ml or 0.5 ng/ml mometasone furoate at different timepoints to the BM‐DC culture and checked (i) the number of matured cells, (ii) the expression of activation markers, (iii) the release of cytokines and (iv) the stimulatory capacity of the resulting BM‐DC in vivo. Even at the highest concentration, pimecrolimus treatment resulted in only modest effects. In the pimecrolimus‐treated culture, we observed a decrease in the numbers of matured cells but no significant effects on the expression of activation markers. The release of some inflammatory cytokines was reduced, but the stimulatory capacity in vivo was not affected. In contrast, mometasone furoate has pronounced effects on BM‐DC at a concentration ten to 1000 times lower than those used with pimecrolimus. Furthermore, topical treatment of mice with clobetasole cream 0.05% resulted in almost complete depletion of splenic DC and a severe hyposplenia, while high‐dose oral pimecrolimus treatment did not show any effects on the spleen or on splenic DC. These results support that pimecrolimus, unlike corticosteroids, has little effects on dendritic cells. To the best of our knowledge, this is the first study of this type with use of BM‐DC.