TY - JOUR A1 - Radbruch, Moritz Jan Florian A1 - Pischon, Jeanette Hannah Charlotte A1 - Du, Fang A1 - Haag, Rainer A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Mundhenk, Lars A1 - Gruber, Achim T1 - Biodegradable core-multishell nanocarrier: topical tacrolimus delivery for treatment of dermatitis JF - Journal of controlled release : official journal of the Controlled Release Society and of the Japanese Society of Drug Delivery Systems N2 - Two challenges in topical drug delivery to the skin include solubilizing hydrophobic drugs in water-based formulations and increasing drug penetration into the skin. Polymeric core-multishell nanocarrier (CMS), particularly the novel biodegradable CMS (bCMS = hPG-PCL1.1K-mPEG(2k)-CMS) have shown both advantages on excised skin ex vivo. Here, we investigated topical delivery of tacrolimus (TAC; > 500 g/mol) by bCMS in a hydrogel on an oxazolone-induced model of dermatitis in vivo. As expected, bCMS successfully delivered TAC into the skin. However, in vivo they did not increase, but decrease TAC penetration through the stratum corneum compared to ointment. Differences in the resulting mean concentrations were mostly non-significant in the skin (epidermis: 35.7 +/- 20.9 ng/cm(2) for bCMS vs. 92.6 +/- 62.7 ng/cm(2) for ointment; dermis: 76.8 +/- 26.8 ng/cm(2) vs 118.2 +/- 50.4 ng/cm(2)), but highly significant in blood (plasma: 1.1 +/- 0.4 ng/ml vs 11.3 +/- 9.3 ng/ml; erythrocytes: 0.5 +/- 0.2 ng/ml vs 3.4 +/- 2.4 ng/ml) and liver (0.01 +/- 0.01 ng/mg vs 0.03 +/- 0.01 ng/mg). bCMS were detected in the stratum corneum but not in viable skin or beyond. The therapeutic efficacy of TAC delivered by bCMS was equivalent to that of standard TAC ointment. Our results suggest that bCMS may be a promising carrier for the topical delivery of TAC. The quantitative difference to previous results should be interpreted in light of structural differences between murine and human skin, but highlights the need as well as potential methods to develop more a complex ex vivo analysis on human skin to ensure quantitative predictive value. KW - drug delivery systems KW - core-multishell (CMS) nanocarriers KW - tacrolimus KW - topical drug delivery KW - dermal drug administration KW - penetration enhancement Y1 - 2022 U6 - https://doi.org/10.1016/j.jconrel.2022.07.025 SN - 0168-3659 SN - 1873-4995 VL - 349 SP - 917 EP - 928 PB - Elsevier CY - New York, NY [u.a.] ER - TY - JOUR A1 - Beckmann, Nadine A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Gulbins, Erich A1 - Nomellini, Vanessa A1 - Caldwell, Charles C. T1 - Burn injury impairs neutrophil chemotaxis through increased ceramide JF - Shock : injury, inflammation, and sepsis, laboratory and clinical approaches N2 - Infection is a common and often deadly complication after burn injury. A major underlying factor is burn-induced immune dysfunction, particularly with respect to neutrophils as the primary responders to infection. Temporally after murine scald injury, we demonstrate impaired bone marrow neutrophil chemotaxis toward CXCL1 ex vivo. Additionally, we observed a reduced recruitment of neutrophils to the peritoneal after elicitation 7 days after injury. We demonstrate that neutrophil ceramide levels increase after burn injury, and this is associated with decreased expression of CXCR2 and blunted chemotaxis. A major signaling event upon CXCR2 activation is Akt phosphorylation and this was reduced when ceramide was elevated. In contrast, PTEN levels were elevated and PTEN-inhibition elevated phospho-Akt levels and mitigated the burn-induced neutrophil chemotaxis defect. Altogether, this study identifies a newly described pathway of ceramide-mediated suppression of neutrophil chemotaxis after burn injury and introduces potential targets to mitigate this defect and reduce infection-related morbidity and mortality after burn. KW - Acid sphingomyelinase KW - Akt KW - burn injury KW - ceramide KW - CXCR2 KW - immune KW - dysfunction KW - neutrophil chemotaxis KW - PTEN Y1 - 2021 U6 - https://doi.org/10.1097/SHK.0000000000001693 SN - 1073-2322 SN - 1540-0514 VL - 56 IS - 1 SP - 125 EP - 132 PB - Lippincott Williams & Wilkins CY - Hagerstown, Md. ER - TY - JOUR A1 - Wigger, Dominik A1 - Schumacher, Fabian A1 - Schneider-Schaulies, Sibylle A1 - Kleuser, Burkhard T1 - Sphingosine 1-phosphate metabolism and insulin signaling JF - Cellular signalling N2 - Insulin is the main anabolic hormone secreted by 13-cells of the pancreas stimulating the assimilation and storage of glucose in muscle and fat cells. It modulates the postprandial balance of carbohydrates, lipids and proteins via enhancing lipogenesis, glycogen and protein synthesis and suppressing glucose generation and its release from the liver. Resistance to insulin is a severe metabolic disorder related to a diminished response of peripheral tissues to the insulin action and signaling. This leads to a disturbed glucose homeostasis that precedes the onset of type 2 diabetes (T2D), a disease reaching epidemic proportions. A large number of studies reported an association between elevated circulating fatty acids and the development of insulin resistance. The increased fatty acid lipid flux results in the accumulation of lipid droplets in a variety of tissues. However, lipid intermediates such as diacylglycerols and ceramides are also formed in response to elevated fatty acid levels. These bioactive lipids have been associated with the pathogenesis of insulin resistance. More recently, sphingosine 1-phosphate (S1P), another bioactive sphingolipid derivative, has also been shown to increase in T2D and obesity. Although many studies propose a protective role of S1P metabolism on insulin signaling in peripheral tissues, other studies suggest a causal role of S1P on insulin resistance. In this review, we critically summarize the current state of knowledge of S1P metabolism and its modulating role on insulin resistance. A particular emphasis is placed on S1P and insulin signaling in hepatocytes, skeletal muscle cells, adipocytes and pancreatic 13-cells. In particular, modulation of receptors and enzymes that regulate S1P metabolism can be considered as a new therapeutic option for the treatment of insulin resistance and T2D. KW - Insulin resistance KW - Type 2 diabetes KW - Sphingolipids KW - Hepatocytes KW - Adipocytes KW - Skeletal muscle cells Y1 - 2021 U6 - https://doi.org/10.1016/j.cellsig.2021.109959 SN - 0898-6568 SN - 1873-3913 VL - 82 PB - Elsevier Science CY - Amsterdam [u.a.] ER - TY - GEN A1 - Lang, Judith A1 - Bohn, Patrick A1 - Bhat, Hilal A1 - Jastrow, Holger A1 - Walkenfort, Bernd A1 - Cansiz, Feyza A1 - Fink, Julian A1 - Bauer, Michael A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Lang, Karl S. T1 - Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1(-/-) mice results in replication of HSV-1 and Asah1(-/-) mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1400 KW - immunology KW - infection KW - membrane fusion KW - phagocytosis KW - sphingolipids Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-515661 SN - 1866-8372 IS - 1 ER - TY - JOUR A1 - Lang, Judith A1 - Bohn, Patrick A1 - Bhat, Hilal A1 - Jastrow, Holger A1 - Walkenfort, Bernd A1 - Cansiz, Feyza A1 - Fink, Julian A1 - Bauer, Michael A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Lang, Karl S. T1 - Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease JF - Nature Communications N2 - Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1(-/-) mice results in replication of HSV-1 and Asah1(-/-) mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol. KW - immunology KW - infection KW - membrane fusion KW - phagocytosis KW - sphingolipids Y1 - 2020 U6 - https://doi.org/10.1038/s41467-020-15072-8 SN - 2041-1723 VL - 11 IS - 1 SP - 1 EP - 15 PB - Nature Publishing Group UK CY - London ER - TY - GEN A1 - Naser, Eyad A1 - Kadow, Stephanie A1 - Schumacher, Fabian A1 - Mohamed, Zainelabdeen H. A1 - Kappe, Christian A1 - Hessler, Gabriele A1 - Pollmeier, Barbara A1 - Kleuser, Burkhard A1 - Arenz, Christoph A1 - Becker, Katrin Anne A1 - Gulbins, Erich A1 - Carpinteiro, Alexander T1 - Characterization of the small molecule ARC39 BT - a direct and specific inhibitor of acid sphingomyelinase in vitro[S] T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM's catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1407 KW - sphingolipids KW - sphingomyelin KW - cerami-des KW - lipid metabolism KW - enzymology KW - lysosome KW - lysosomal hydrolases KW - acid ceramidase KW - bisphosphonates KW - functional inhibitors of acid sphin-gomyelinase KW - 1-aminodecylidene bis-phosphonic acid Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-516635 SN - 1866-8372 IS - 6 ER - TY - JOUR A1 - Naser, Eyad A1 - Kadow, Stephanie A1 - Schumacher, Fabian A1 - Mohamed, Zainelabdeen H. A1 - Kappe, Christian A1 - Hessler, Gabriele A1 - Pollmeier, Barbara A1 - Kleuser, Burkhard A1 - Arenz, Christoph A1 - Becker, Katrin Anne A1 - Gulbins, Erich A1 - Carpinteiro, Alexander T1 - Characterization of the small molecule ARC39 BT - a direct and specific inhibitor of acid sphingomyelinase in vitro[S] JF - Journal of Lipid Research N2 - Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM's catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo. KW - sphingolipids KW - sphingomyelin KW - cerami-des KW - lipid metabolism KW - enzymology KW - lysosome KW - lysosomal hydrolases KW - acid ceramidase KW - bisphosphonates KW - functional inhibitors of acid sphin-gomyelinase KW - 1-aminodecylidene bis-phosphonic acid Y1 - 2021 U6 - https://doi.org/10.1194/jlr.RA120000682 SN - 1539-7262 SN - 0022-2275 VL - 61 IS - 6 SP - 896 EP - 910 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Fink, Julian A1 - Schumacher, Fabian A1 - Schlegel, Jan A1 - Stenzel, Philipp A1 - Wigger, Dominik A1 - Sauer, Markus A1 - Kleuser, Burkhard A1 - Seibel, Jürgen T1 - Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis JF - Organic & biomolecular chemistry : an international journal of synthetic, physical and biomolecular organic chemistry N2 - Here were report the combination of biocompatible click chemistry of omega-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that omega-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, omega-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection. Y1 - 2021 U6 - https://doi.org/10.1039/d0ob02592e SN - 1477-0520 SN - 1477-0539 VL - 19 IS - 10 SP - 2203 EP - 2212 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Solger, Franziska A1 - Kunz, Tobias C. A1 - Fink, Julian A1 - Paprotka, Kerstin A1 - Pfister, Pauline A1 - Hagen, Franziska A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Seibel, Jürgen A1 - Rudel, Thomas T1 - A role of sphingosine in the intracellular survival of Neisseria gonorrhoeae JF - Frontiers in Cellular and Infection Microbiology N2 - Obligate human pathogenic Neisseria gonorrhoeae are the second most frequent bacterial cause of sexually transmitted diseases. These bacteria invade different mucosal tissues and occasionally disseminate into the bloodstream. Invasion into epithelial cells requires the activation of host cell receptors by the formation of ceramide-rich platforms. Here, we investigated the role of sphingosine in the invasion and intracellular survival of gonococci. Sphingosine exhibited an anti-gonococcal activity in vitro. We used specific sphingosine analogs and click chemistry to visualize sphingosine in infected cells. Sphingosine localized to the membrane of intracellular gonococci. Inhibitor studies and the application of a sphingosine derivative indicated that increased sphingosine levels reduced the intracellular survival of gonococci. We demonstrate here, that sphingosine can target intracellular bacteria and may therefore exert a direct bactericidal effect inside cells. KW - Neisseria gonorrhoeae KW - sphingosine KW - sphingolipids KW - sphingosine kinases KW - invasion KW - survival KW - click chemistry Y1 - 2020 U6 - https://doi.org/10.3389/fcimb.2020.00215 SN - 2235-2988 VL - 10 PB - Frontiers Media CY - Lausanne ER - TY - GEN A1 - Rancan, Fiorenza A1 - Volkmann, Hildburg A1 - Giulbudagian, Michael A1 - Schumacher, Fabian A1 - Stanko, Jessica Isolde A1 - Kleuser, Burkhard A1 - Blume-Peytavi, Ulrike A1 - Calderón, Marcelo A1 - Vogt, Annika T1 - Dermal Delivery of the High-Molecular-Weight Drug Tacrolimus by Means of Polyglycerol-Based Nanogels T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Polyglycerol-based thermoresponsive nanogels (tNGs) have been shown to have excellent skin hydration properties and to be valuable delivery systems for sustained release of drugs into skin. In this study, we compared the skin penetration of tacrolimus formulated in tNGs with a commercial 0.1% tacrolimus ointment. The penetration of the drug was investigated in ex vivo abdominal and breast skin, while different methods for skin barrier disruption were investigated to improve skin permeability or simulate inflammatory conditions with compromised skin barrier. The amount of penetrated tacrolimus was measured in skin extracts by liquid chromatography tandem-mass spectrometry (LC-MS/MS), whereas the inflammatory markers IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). Higher amounts of tacrolimus penetrated in breast as compared to abdominal skin or in barrier-disrupted as compared to intact skin, confirming that the stratum corneum is the main barrier for tacrolimus skin penetration. The anti-proliferative effect of the penetrated drug was measured in skin tissue/Jurkat cells co-cultures. Interestingly, tNGs exhibited similar anti-proliferative effects as the 0.1% tacrolimus ointment. We conclude that polyglycerol-based nanogels represent an interesting alternative to paraffin-based formulations for the treatment of inflammatory skin conditions. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1339 KW - tacrolimus formulation KW - nanogels KW - skin penetration KW - drug delivery KW - human excised skin KW - Jurkat cells Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-473270 SN - 1866-8372 IS - 1339 ER - TY - JOUR A1 - Wetzel, Alexandra Nicole A1 - Scholtka, Bettina A1 - Schumacher, Fabian A1 - Rawel, Harshadrai Manilal A1 - Geisendörfer, Birte A1 - Kleuser, Burkhard T1 - Epigenetic DNA methylation of EBI3 modulates human interleukin-35 formation via NFkB signaling BT - a promising therapeutic option in ulcerative colitis JF - International journal of molecular sciences N2 - Ulcerative colitis (UC), a severe chronic disease with unclear etiology that is associated with increased risk for colorectal cancer, is accompanied by dysregulation of cytokines. Epstein-Barr virus-induced gene 3 (EBI3) encodes a subunit in the unique heterodimeric IL-12 cytokine family of either pro- or anti-inflammatory function. After having recently demonstrated that upregulation of EBI3 by histone acetylation alleviates disease symptoms in a dextran sulfate sodium (DSS)-treated mouse model of chronic colitis, we now aimed to examine a possible further epigenetic regulation of EBI3 by DNA methylation under inflammatory conditions. Treatment with the DNA methyltransferase inhibitor (DNMTi) decitabine (DAC) and TNF alpha led to synergistic upregulation of EBI3 in human colon epithelial cells (HCEC). Use of different signaling pathway inhibitors indicated NF kappa B signaling was necessary and proportional to the synergistic EBI3 induction. MALDI-TOF/MS and HPLC-ESIMS/MS analysis of DAC/TNF alpha-treated HCEC identified IL-12p35 as the most probable binding partner to form a functional protein. EBI3/IL-12p35 heterodimers (IL-35) induce their own gene upregulation, something that was indeed observed in HCEC cultured with media from previously DAC/TNF alpha-treated HCEC. These results suggest that under inflammatory and demethylating conditions the upregulation of EBI3 results in the formation of anti-inflammatory IL-35, which might be considered as a therapeutic target in colitis. KW - decitabine KW - DNMT inhibitor KW - EBI3 KW - inhibitory cytokines KW - interleukin-35 KW - TNF alpha KW - Ulcerative colitis Y1 - 2021 U6 - https://doi.org/10.3390/ijms22105329 SN - 1422-0067 VL - 22 IS - 10 PB - MDPI CY - Basel ER - TY - JOUR A1 - Samaha, Doaa A1 - Hamdo, Housam H. A1 - Cong, Xiaojing A1 - Schumacher, Fabian A1 - Banhart, Sebastian A1 - Aglar, Öznur A1 - Möller, Heiko Michael A1 - Heuer, Dagmar A1 - Kleuser, Burkhard A1 - Saied, Essa M. A1 - Arenz, Christoph T1 - Liposomal FRET assay identifies potent drug-like inhibitors of the Ceramide Transport Protein (CERT) JF - Chemistry - a European journal N2 - Ceramide transfer protein (CERT) mediates non-vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate-limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non-homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Forster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT-mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96-well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis. KW - enzyme assays KW - Forster resonance energy transfer (FRET) KW - liposomes KW - sphingolipids KW - transport proteins Y1 - 2020 U6 - https://doi.org/10.1002/chem.202003283 SN - 0947-6539 SN - 1521-3765 VL - 26 IS - 70 SP - 16616 EP - 16621 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Fayyaz, Susann A1 - Japtok, Lukasz A1 - Schumacher, Fabian A1 - Wigger, Dominik A1 - Schulz, Tim Julius A1 - Haubold, Kathrin A1 - Gulbins, Erich A1 - Völler, Heinz A1 - Kleuser, Burkhard T1 - Lysophosphatidic acid inhibits insulin signaling in primary rat hepatocytes via the LPA(3) receptor subtype and is increased in obesity JF - Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry and pharmacology N2 - Background/Aims: Obesity is a main risk factor for the development of hepatic insulin resistance and it is accompanied by adipocyte hypertrophy and an elevated expression of different adipokines such as autotaxin (ATX). ATX converts lysophosphatidylcholine to lysophosphatidic acid (LPA) and acts as the main producer of extracellular LPA. This bioactive lipid regulates a broad range of physiological and pathological responses by activation of LPA receptors (LPA1-6). Methods: The activation of phosphatidylinositide 3-kinases (PI3K) signaling (Akt and GSK-3ß) was analyzed via western blotting in primary rat hepatocytes. Incorporation of glucose into glycogen was measured by using radio labeled glucose. Real-time PCR analysis and pharmacological modulation of LPA receptors were performed. Human plasma LPA levels of obese (BMI > 30, n = 18) and normal weight individuals (BMI 18.5-25, n = 14) were analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Results: Pretreatment of primary hepatocytes with LPA resulted in an inhibition of insulin-mediated Gck expression, PI3K activation and glycogen synthesis. Pharmacological approaches revealed that the LPA3-receptor subtype is responsible for the inhibitory effect of LPA on insulin signaling. Moreover, human plasma LPA concentrations (16: 0 LPA) of obese participants (BMI > 30) are significantly elevated in comparison to normal weight individuals (BMI 18.5-25). Conclusion: LPA is able to interrupt insulin signaling in primary rat hepatocytes via the LPA3 receptor subtype. Moreover, the bioactive lipid LPA (16: 0) is increased in obesity. KW - Lysophosphatidic acid KW - Insulin signaling KW - Adipose tissue KW - Autotaxin KW - Hepatic insulin resistance KW - LPA(3) receptor subtype Y1 - 2017 U6 - https://doi.org/10.1159/000480470 SN - 1015-8987 SN - 1421-9778 VL - 43 SP - 445 EP - 456 PB - Karger CY - Basel ER - TY - JOUR A1 - Neuber, Corinna A1 - Schumacher, Fabian A1 - Gulbins, Erich A1 - Kleuser, Burkhard T1 - Mass Spectrometric Determination of Fatty Aldehydes Exemplified by Monitoring the Oxidative Degradation of (2E)-Hexadecenal in HepG2 Cell Lysates JF - Lipidomics N2 - Within the last few decades, liquid chromatography-mass spectrometry (LC-MS) has become a preferred method for manifold issues in analytical biosciences, given its high selectivity and sensitivity. However, the analysis of fatty aldehydes, which are important components of cell metabolism, remains challenging. Usually, chemical derivatization prior to MS detection is required to enhance ionization efficiency. In this regard, the coupling of fatty aldehydes to hydrazines like 2,4-dinitrophenylhydrazine (DNPH) is a common approach. Additionally, hydrazones readily react with fatty aldehydes to form stable derivatives, which can be easily separated using high-performance liquid chromatography (HPLC) and subsequently detected by MS. Here, we exemplarily present the quantification of the long-chain fatty aldehyde (2E)-hexadecenal, a break-down product of the bioactive lipid sphingosine 1-phosphate (S1P), after derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone (DAIH) via isotope-dilution HPLC-electrospray ionization-quadrupole/time-of-flight (ESI-QTOF) MS. Moreover, we show that the addition of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC hydrochloride) as a coupling agent allows for simultaneous determination of fatty aldehydes and fatty acids as DAIH derivatives. Taking advantage of this, we describe in detail how to monitor the degradation of (2E)-hexadecenal and the concurrent formation of its oxidation product (2E)-hexadecenoic acid in lysates of human hepatoblastoma (HepG2) cells within this chapter. KW - (2E)-hexadecenal KW - (2E)-hexadecenoic acid KW - Sphingosine 1-phosphate KW - Derivatization KW - DAIH KW - EDC KW - Isotope-dilution KW - HPLC-ESI-QTOF Y1 - 2017 SN - 978-1-4939-6946-3 SN - 978-1-4939-6944-9 U6 - https://doi.org/10.1007/978-1-4939-6946-3_10 SN - 0893-2336 SN - 1940-6045 VL - 125 SP - 147 EP - 158 PB - Humana Press CY - Totowa ER - TY - JOUR A1 - Gerecke, Christian A1 - Edlich, Alexander A1 - Giulbudagian, Michael A1 - Schumacher, Fabian A1 - Zhang, Nan A1 - Said, Andre A1 - Yealland, Guy A1 - Lohan, Silke B. A1 - Neumann, Falko A1 - Meinke, Martina C. A1 - Ma, Nan A1 - Calderon, Marcelo A1 - Hedtrich, Sarah A1 - Schaefer-Korting, Monika A1 - Kleuser, Burkhard T1 - Biocompatibility and characterization of polyglycerol-based thermoresponsive nanogels designed as novel drug-delivery systems and their intracellular localization in keratinocytes JF - Nanotoxicology N2 - Novel nanogels that possess the capacity to change their physico-chemical properties in response to external stimuli are promising drug-delivery candidates for the treatment of severe skin diseases. As thermoresponsive nanogels (tNGs) are capable of enhancing penetration through biological barriers such as the stratum corneum and are taken up by keratinocytes of human skin, potential adverse consequences of their exposure must be elucidated. In this study, tNGs were synthesized from dendritic polyglycerol (dPG) and two thermoresponsive polymers. tNG_dPG_tPG are the combination of dPG with poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)) and tNG_dPG_pNIPAM the one with poly(N-isopropylacrylamide) (pNIPAM). Both thermoresponsive nanogels are able to incorporate high amounts of dexamethasone and tacrolimus, drugs used in the treatment of severe skin diseases. Cellular uptake, intracellular localization and the toxicological properties of the tNGs were comprehensively characterized in primary normal human keratinocytes (NHK) and in spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (HaCaT). Laser scanning confocal microscopy revealed fluorescently labeled tNGs entered into the cells and localized predominantly within lysosomal compartments. MTT assay, comet assay and carboxy-H2DCFDA assay, demonstrated neither cytotoxic or genotoxic effects, nor any induction of reactive oxygen species of the tNGs in keratinocytes. In addition, both tNGs were devoid of eye irritation potential as shown by bovine corneal opacity and permeability (BCOP) test and red blood cell (RBC) hemolysis assay. Therefore, our study provides evidence that tNGs are locally well tolerated and underlines their potential for cutaneous drug delivery. KW - Drug delivery KW - nanoparticles KW - particle characterization KW - keratinocytes KW - nanotoxicology Y1 - 2017 U6 - https://doi.org/10.1080/17435390.2017.1292371 SN - 1743-5390 SN - 1743-5404 VL - 11 SP - 267 EP - 277 PB - Routledge, Taylor & Francis Group CY - Abingdon ER - TY - GEN A1 - Döge, Nadine A1 - Schumacher, Fabian A1 - Balzus, Benjamin A1 - Colombo, Miriam A1 - Hadam, Sabrina A1 - Rancan, Fiorenza A1 - Blume-Peytavi, Ulrike A1 - Kleuser, Burkhard A1 - Bodmeier, Roland A1 - Vogt, Annika T1 - Particle- based formulations and controlled skin barrier disruption have a signifi cant impact on the delivery and penetration kinetics of dexamethasone as assessed in an ex vivo microdialysis T2 - Journal der Deutschen Dermatologischen Gesellschaft N2 - Preclinical assessment of penetration not only in intact, but also in barrier‐disrupted skin is important to explore the surplus value of novel drug delivery systems, which can be specifically designed for diseased skin. Here, we characterized physical and chemical barrier disruption protocols for short‐term ex vivo skin cultures with regard to structural integrity, physiological and biological parameters. Further, we compared the penetration of dexamethasone (Dex) in different nanoparticle‐based formulations in stratum corneum, epidermis and dermis extracts of intact vs. barrier‐disrupted skin as well as by dermal microdialysis at 6, 12 and 24 hours after topical application. Dex was quantified by liquid‐chromatography ‐ tandem‐mass spectrometry (LC‐MS/MS). Simultaneously, we investigated the Dex efficacy by interleukin (IL) analysis. Tape‐stripping (TS) and 4 hours sodium lauryl sulfate 5 % (SLS) exposure were identified as highly effective barrier disruption methods assessed by reproducible transepidermal water loss (TEWL) changes and IL‐6/8 increase which was more pronounced in SLS‐treated skin. The barrier state has also a significant impact on the Dex penetration kinetics: for all formulations, TS highly increased dermal Dex concentration despite the fact that nanocrystals quickly and effectively penetrated both, intact and barrier‐disrupted skin reaching significantly higher dermal Dex concentration after 6 hours compared to Dex cream. The surplus value of encapsulation in ethyl cellulose nanocarriers could mostly be observed when applied on intact skin, in general showing a delayed Dex penetration. Estimation of cytokines was limited due to the trauma caused by probe insertion. In summary, ex vivo human skin is a highly interesting short‐term preclinical model for the analysis of penetration and efficacy of novel drug delivery systems. Y1 - 2017 SN - 1610-0379 SN - 1610-0387 VL - 15 SP - 182 EP - 182 PB - Wiley CY - Berlin ER - TY - JOUR A1 - Balzus, Benjamin A1 - Sahle, Fitsum Feleke A1 - Hönzke, Stefan A1 - Gerecke, Christian A1 - Schumacher, Fabian A1 - Hedtrich, Sarah A1 - Kleuser, Burkhard A1 - Bodmeier, Roland T1 - Formulation and ex vivo evaluation of polymeric nanoparticles for controlled delivery of corticosteroids to the skin and the corneal epithelium JF - European journal of pharmaceutics and biopharmaceutics : EJPB ; official journal of the International Association for Pharmaceutical Technology N2 - Controlled delivery of corticosteroids using nanoparticles to the skin and corneal epithelium may reduce their side effects and maximize treatment effectiveness. Dexamethasone-loaded ethyl cellulose, Eudragit® RS and ethyl cellulose/Eudragit® RS nanoparticles were prepared by the solvent evaporation method. Dexamethasone release from the polymeric nanoparticles was investigated in vitro using Franz diffusion cells. Drug penetration was also assessed ex vivo using excised human skin. Nanoparticle toxicity was determined by MTT and H2DCFDA assays. Eudragit® RS nanoparticles were smaller and positively charged but had a lower dexamethasone loading capacity (0.3–0.7%) than ethyl cellulose nanoparticles (1.4–2.2%). By blending the two polymers (1:1), small (105 nm), positively charged (+37 mV) nanoparticles with sufficient dexamethasone loading (1.3%) were obtained. Dexamethasone release and penetration significantly decreased with decreasing drug to polymer ratio and increased when Eudragit® RS was blended with ethyl cellulose. Ex vivo, drug release and penetration from the nanoparticles was slower than a conventional cream. The nanoparticles bear no toxicity potentials except ethyl cellulose nanoparticles had ROS generation potential at high concentration. In conclusion, the nanoparticles showed great potential to control the release and penetration of corticosteroids on the skin and mucus membrane and maximize treatment effectiveness. KW - Dermal delivery KW - Dexamethasone KW - Ethyl cellulose KW - Eudragit (R) RS KW - Ocular delivery KW - Polymeric nanoparticle Y1 - 2017 U6 - https://doi.org/10.1016/j.ejpb.2017.02.001 SN - 0939-6411 SN - 1873-3441 VL - 115 SP - 122 EP - 130 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Giulbudagian, Michael A1 - Hönzke, Stefan A1 - Bergueiro, Julián A1 - Işık, Doğuş A1 - Schumacher, Fabian A1 - Saeidpour, Siavash A1 - Lohan, Silke A1 - Meinke, Martina A1 - Teutloff, Christian A1 - Schäfer-Korting, Monika A1 - Yealland, Guy A1 - Kleuser, Burkhard A1 - Hedtrich, Sarah A1 - Calderón, Marcelo T1 - Enhanced topical delivery of dexamethasone by beta-cyclodextrin decorated thermoresponsive nanogels JF - Nanoscale N2 - Highly hydrophilic, responsive nanogels are attractive as potential systems for the topical delivery of bioactives encapsulated in their three-dimensional polymeric scaffold. Yet, these drug carrier systems suffer from drawbacks for efficient delivery of hydrophobic drugs. Addressing this, β-cyclodextrin (βCD) could be successfully introduced into the drug carrier systems by exploiting its unique affinity toward dexamethasone (DXM) as well as its role as topical penetration enhancer. The properties of βCD could be combined with those of thermoresponsive nanogels (tNGs) based on dendritic polyglycerol (dPG) as a crosslinker and linear thermoresponsive polyglycerol (tPG) inducing responsiveness to temperature changes. Electron paramagnetic resonance (EPR) studies localized the drug within the hydrophobic cavity of βCD by differences in its mobility and environmental polarity. In fact, the fabricated carriers combining a particulate delivery system with a conventional penetration enhancer, resulted in an efficient delivery of DXM to the epidermis and the dermis of human skin ex vivo (enhancement compared to commercial DXM cream: ∼2.5 fold in epidermis, ∼30 fold in dermis). Furthermore, DXM encapsulated in βCD tNGs applied to skin equivalents downregulated the expression of proinflammatory thymic stromal lymphopoietin (TSLP) and outperformed a commercially available DXM cream. Y1 - 2017 U6 - https://doi.org/10.1039/c7nr04480a SN - 2040-3364 SN - 2040-3372 VL - 10 IS - 1 SP - 469 EP - 479 PB - Royal Society of Chemistry CY - Cambridge ER - TY - GEN A1 - Pischon, Hannah A1 - Radbruch, Moritz A1 - Ostrowski, Anja A1 - Schumacher, Fabian A1 - Hoenzke, Stefan A1 - Kleuser, Burkhard A1 - Hedtrich, Sarah A1 - Fluhr, Joachim W. A1 - Gruber, Achim D. A1 - Mundhenk, Lars T1 - How Effective Is Tacrolimus in the Imiquimod BT - Induced Mouse Model of Psoriasis? T2 - The journal of investigative dermatology Y1 - 2017 U6 - https://doi.org/10.1016/j.jid.2017.09.019 SN - 0022-202X SN - 1523-1747 VL - 138 IS - 2 SP - 455 EP - 458 PB - Elsevier CY - New York ER - TY - JOUR A1 - Zoicas, Iulia A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Reichel, Martin A1 - Gulbins, Erich A1 - Fejtova, Anna A1 - Kornhuber, Johannes A1 - Rhein, Cosima T1 - The forebrain-specific overexpression of acid sphingomyelinase induces depressive-like symptoms in mice JF - Cells N2 - Human and murine studies identified the lysosomal enzyme acid sphingomyelinase (ASM) as a target for antidepressant therapy and revealed its role in the pathophysiology of major depression. In this study, we generated a mouse model with overexpression of Asm (Asm-tg(fb)) that is restricted to the forebrain to rule out any systemic effects of Asm overexpression on depressive-like symptoms. The increase in Asm activity was higher in male Asm-tg(fb) mice than in female Asm-tg(fb) mice due to the breeding strategy, which allows for the generation of wild-type littermates as appropriate controls. Asm overexpression in the forebrain of male mice resulted in a depressive-like phenotype, whereas in female mice, Asm overexpression resulted in a social anxiogenic-like phenotype. Ceramides in male Asm-tg(fb) mice were elevated specifically in the dorsal hippocampus. mRNA expression analyses indicated that the increase in Asm activity affected other ceramide-generating pathways, which might help to balance ceramide levels in cortical brain regions. This forebrain-specific mouse model offers a novel tool for dissecting the molecular mechanisms that play a role in the pathophysiology of major depression. KW - Smpd1 KW - acid sphingomyelinase KW - forebrain KW - depressive-like behavior KW - anxiety-like behavior KW - ceramide Y1 - 2020 VL - 9 IS - 5 PB - MDPI CY - Basel ER -