citraconic-acid and Psoriasis

Oxidative stress contributes to inflammatory skin diseases, including psoriasis. Monomethylfumarate (MMF) is an antipsoriatic agent with a poorly understood mechanism of action. In other cell types MMF increases the expression of nuclear factor erythroid-derived 2-like 2 (Nrf2), a transcription factor that regulates cellular antioxidant responses, to reduce oxidative stress like that observed in inflammatory disorders such as multiple sclerosis. We tested the hypothesis that MMF enhances Nrf2 activity in keratinocytes, thereby improving their capacity to counteract environmental stresses. We used Western analysis, immunofluorescence, and real-time quantitative reverse-transcription polymerase chain reaction to examine the effect of MMF on the expression of Nrf2 and its targets. We also measured intracellular reactive oxygen species (ROS) levels following MMF treatment. Our data show that MMF increased total and nuclear Nrf2 levels in primary mouse keratinocytes and enhanced mRNA expression of several Nrf2-downstream effectors, including heme oxygenase-1 and peroxiredoxin-6. Moreover, MMF treatment attenuated the generation of ROS following hydrogen peroxide treatment. On the other hand, the expression and membranous localization of aquaporin-3 (AQP3), a glycerol channel implicated in keratinocyte differentiation, was stimulated by MMF, which also enhanced keratinocyte glycerol uptake. The Nrf2 activator sulforaphane also increased AQP3 levels, suggesting that AQP3 expression may be regulated by Nrf2. We show for the first time that MMF stimulates Nrf2 and AQP3 expression and function/activity in keratinocytes. This effect may account, in part, for the previously observed ability of MMF to inhibit proliferation and inflammatory mediator production and promote differentiation in keratinocytes and to treat psoriasis.

Topics: Animals; Animals, Newborn; Aquaporin 3; Base Sequence; Cells, Cultured; Fumarates; Gene Expression; Keratinocytes; Maleates; Mice; NF-E2-Related Factor 2; Psoriasis; RNA, Messenger

Topics: Dimethyl Fumarate; Fumarates; Healthy Volunteers; Humans; Hydrolases; Indoleamine-Pyrrole 2,3,-Dioxygenase; Kynurenine; Leukocytes, Mononuclear; Maleates; Primary Cell Culture; Psoriasis

Monomethylfumarate (MMF) is thought to be the bioactive ingredient of the drug Fumaderm (Biogen Idec, Cambridge, MA), licensed in Germany since 1994 for the treatment of moderate-to-severe psoriasis. Psoriasis is a common inflammatory hyperproliferative skin disorder that involves cross-talk between different cell types, including immune cells and keratinocytes. Psoriatic lesions are characterized by hyperproliferation, aberrant differentiation, and inflammation, with the psoriatic cytokine network maintained by communication between immune cells and keratinocytes. Recently, there is increasing evidence regarding the pivotal role of keratinocytes in mediating the disease process, and these cells can be regarded as safe therapeutic targets. From the data available on human subjects treated with Fumaderm, MMF is an effective antipsoriatic agent with known effects on immune cells. However, little is known about its direct effects on keratinocytes. We hypothesized that MMF has direct antiproliferative, prodifferentiative, and anti-inflammatory effects on keratinocytes. Indeed, MMF dose-dependently inhibited [(3)H]thymidine incorporation into DNA, indicating a direct antiproliferative action on keratinocytes. MMF significantly increased the protein level of keratin 10, the early keratinocyte differentiation marker, and the activity of transglutaminase, a late differentiation marker. These results are consistent with an ability of MMF to promote keratinocyte differentiation and inhibit proliferation, thereby improving psoriatic lesions. In 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced keratinocytes, MMF significantly inhibited the expression of the proinflammatory cytokines, tumor necrosis factor-α (TNFα), interleukin-6, and interleukin-1α as well as the production of TNFα. Our results support the notion that MMF has direct antiproliferative, prodifferentiative, and anti-inflammatory effects on keratinocytes, highlighting its potential use as a multifactorial antipsoriatic agent.

Topics: Animals; Anti-Inflammatory Agents; Cell Differentiation; Cell Proliferation; Dose-Response Relationship, Drug; Endothelial Cells; Female; Fumarates; Gene Expression Regulation; Keratinocytes; Male; Maleates; Mice; Psoriasis; RNA, Messenger; Tumor Necrosis Factor-alpha

Mast cells modulate autoimmune diseases such as psoriasis and multiple sclerosis. Fumaric acid esters (FAEs) are widely used for the treatment of psoriasis, and dimethylfumarate (DMF) has recently been approved for multiple sclerosis. In this study, we analysed the cytotoxic effect of FAEs on human mast cells. Specifically, cell death was analysed in the human mast cell line HMC-1 and in primary cord blood-derived mast cells (CBMCs) after incubation with fumaric acid (FA), monomethylfumarate (MMF), DMF and calcium bis(monomethylfumarate) (Ca-MF). Our data show that only DMF potently induces apoptotic cell death in HMC-1 cells and CBMCs. DMF-mediated apoptosis was associated with increased expression of Bax and Bak and activation of caspase-9 and caspase-6. Interestingly, DMF also enhanced the sensitivity of CBMCs towards TRAIL- and dexamethasone-induced apoptosis. These findings demonstrate for the first time that DMF induces apoptosis of human mast cells, primarily via the mitochondrial apoptotic pathway. Our study contributes to the understanding of the beneficial effects of FAEs in autoimmune diseases and provides a rationale for exploiting FAEs for other diseases associated with mast cells.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Calcium; Caspase 6; Caspase 9; Cell Death; Cell Line; Dermatologic Agents; Dexamethasone; Dimethyl Fumarate; Etoposide; Fumarates; Humans; Interleukin-8; Maleates; Mast Cells; Methotrexate; Psoriasis; TNF-Related Apoptosis-Inducing Ligand

Density functional theory (DFT) calculations at B3LYP/6-31 G (d,p) and B3LYP/6-311+G(d,p) levels for the substituted pyridine-catalyzed isomerization of monomethyl maleate revealed that isomerization proceeds via four steps, with the rate-limiting step being proton transfer from the substituted pyridinium ion to the C=C double bond in INT1. In addition, it was found that the isomerization rate (maleate to fumarate) is solvent dependent. Polar solvents, such as water, tend to accelerate the isomerization rate, whereas apolar solvents, such as chloroform, act to slow down the reaction. A linear correlation was obtained between the isomerization activation energy and the dielectric constant of the solvent. Furthermore, linearity was achieved when the activation energy was plotted against the pKa value of the catalyst. Substituted-pyridine derivatives with high pKa values were able to catalyze isomerization more efficiently than those with low pKa values. The calculated relative rates for prodrugs 1-6 were: 1 (406.7), 2 (7.6×10(6)), 3 (1.0), 4 (20.7), 5 (13.5) and 6 (2.2×10(3)). This result indicates that isomerizations of prodrugs 1 and 3-5 are expected to be slow and that of prodrugs 2 and 6 are expected to be relatively fast. Hence, prodrugs 2 and 3-5 have the potential to be utilized as prodrugs for the slow release of monomethylfumarate in the treatment of psoriasis and multiple sclerosis.

Topics: Catalysis; Computer Simulation; Fumarates; Humans; Isomerism; Kinetics; Maleates; Models, Molecular; Multiple Sclerosis; Prodrugs; Psoriasis; Pyridines; Quantum Theory; Solvents; Thermodynamics

The aim of this study was to evaluate pharmacokinetic parameters of fumaric acid esters (FAE) in psoriasis patients for the first time. For this prupose new HPLC assays were developed. Additionally, physicochemical parameters of FAE were determined, allowing a better interpretation of the in vivo data. In vivo, monomethylfumarate (MMF) and monoethylfumarate (MEF) were detected after t (lag) = 120 min. T (max) and c (max) of MMF were 210 min and 11.2 microM, respectively, 210 min and 5.2 microM for MEF. The half-life of MMF was 38.7 min, and 25.4 min of MEF. The AUC(0-infinity) of MMF was 172 min microg ml(-1) and 63.6 min microg ml(-1) of MEF. Data display median of three subjects. No plasma levels of dimethylfumarate (DMF) or fumaric acid (FA) were detected. The evaluation of physicochemical parameters of FAE showed that only DMF fulfils the criteria of Lipinski's rule of five. The pKa of MMF was determined as 3.63. The data of this study provide evidence that DMF is most likely absorbed out of the duodenum into the presystemic circulation and is not completely hydrolysed to MMF before uptake as assumed by others.

Topics: Adult; Duodenum; Fumarates; Half-Life; Humans; Hydrolysis; Male; Maleates; Middle Aged; Psoriasis

Nicotinic acid has been used for several decades to treat dyslipidemia. In mice, the lipid-lowing effect of nicotinic acid is mediated by the Gi coupled receptor PUMA-G. In humans, high (GPR109A) and low (GPR109B) affinity nicotinic acid receptors have been characterized. Here we identify monomethylfumarate as a GPR109A agonist. Monomethylfumarate is the active metabolite of the psoriasis drug Fumaderm. We show that monomethylfumarate activates GPR109A in a calcium based aequorin assay, cAMP assay and demonstrate competitive binding with nicotinic acid. We show that GPR109A is highly expressed in neutrophils and epidermal keratinocytes, and that its expression is increased in human psoriatic lesions. Our findings provide evidence that GPR109A is a target for the drug Fumaderm and suggest that niacin should be investigated to treat psoriasis in addition to its role in treating lipid disorders.

Topics: Cell Line; Dermatologic Agents; Dimethyl Fumarate; Fumarates; Humans; Hypolipidemic Agents; Maleates; Niacin; Psoriasis; Receptors, G-Protein-Coupled; Receptors, Nicotinic

Psoriasis vulgaris, a type-1 cytokine-mediated chronic skin disease, can be treated successfully with fumaric acid esters (FAE). Beneficial effects of this medication coincided with decreased production of IFN-gamma. Since dendritic cells (DC) regulate the differentiation of T helper (Th) cells, this study focussed on effects of monomethylfumarate (MMF, bioactive metabolite of FAE) on polarization of monocyte-derived DC. MMF-incubated, lipo-polysaccharide-stimulated DC (MMF-DC) produced dramatically (p<0.05) reduced levels of IL-12p70 and IL-10 (8+/-4% and 20+/-4%, respectively) compared to control DC. MMF-DC were mature. MMF affected polarization of DC irrespective of polarization factor(s) and ligands for the various Toll-like receptors used. Coculture of MMF-DC with naive and primed allogenous Th cells resulted in lymphocytes producing less IFN-gamma, i.e. 59% and 54% of that by the respective Th cells cocultured with control DC. IL-4 production by primed, but not naive Th cells cocultured with MMF-DC was decreased as compared to cocultures with control DC. IL-10 production by naive and primed Th cells cocultured with MMF-DC and control DC did not differ. In addition, MMF inhibited LPS-induced NF-kappaB activation in DC. Together, beneficial effects of FAE in psoriasis involve modulation of DC polarization by MMF such that these cells down-regulate IFN-gamma production by Th cells.

Topics: Antigens, CD; Cell Differentiation; Cell Polarity; Coculture Techniques; Dendritic Cells; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fumarates; Humans; Interleukin-10; Interleukin-12; Interleukin-8; Lymphocyte Activation; Maleates; Monocytes; Protein Subunits; Psoriasis; Th1 Cells; Tumor Necrosis Factor-alpha

Psoriasis is a chronic inflammatory skin disease that can be successfully treated with a mixture of fumaric acid esters (FAE) formulated as enteric-coated tablets for oral use. These tablets consist of dimethylfumarate (DMF) and salts of monoethylfumarate (MEF) and its main bioactive metabolite is monomethylfumarate (MMF). Little is known about the pharmacokinetics of these FAE. The aim of the present study was to investigate the hydrolysis of DMF to MMF and the stability of MMF, DMF and MEF at in vitro conditions representing different body compartments.. DMF is hydrolyzed to MMF in an alkaline environment (pH 8), but not in an acidic environment (pH 1). In these conditions MMF and MEF remained intact during the period of analysis (6 h). Interestingly, DMF was hardly hydrolyzed to MMF in a buffer of pH 7.4, but was rapidly hydrolyzed in human serum having the same pH. Moreover, in whole blood the half-life of DMF was dramatically reduced as compared to serum. The concentrations of MMF and MEF in serum and whole blood decreased with increasing time. These data indicate that the majority of the FAE in the circulation are metabolized by one or more types of blood cells. Additional experiments with purified blood cell fractions resuspended in phosphate buffered saline (pH 7.4) revealed that at concentrations present in whole blood monocytes/lymphocytes, but not granulocytes and erythrocytes, effectively hydrolyzed DMF to MMF. Furthermore, in agreement with the data obtained with the pure components of the tablet, the enteric-coated tablet remained intact at pH 1, but rapidly dissolved at pH 8.. Together, these in vitro data indicate that hydrolysis of DMF to MMF rapidly occurs at pH 8, resembling that within the small intestines, but not at pH 1 resembling the pH in the stomach. At both pHs MMF and MEF remained intact. These data explain the observation that after oral FAE intake MMF and MEF, but not DMF, can be readily detected in the circulation of human healthy volunteers and psoriasis patients.

Topics: Blood Cells; Buffers; Dermatologic Agents; Dimethyl Fumarate; Drug Stability; Esters; Fumarates; Half-Life; Humans; Hydrogen-Ion Concentration; Hydrolysis; In Vitro Techniques; Intestine, Small; Maleates; Psoriasis; Serum; Stomach

Monomethylfumarate (MMF), the most active metabolite of the new antipsoriasis drug Fumaderm, stimulates an anti-inflammatory mediator profile in human leucocytes and inhibits the proliferation of keratinocytes. These effects of MMF on cells in vitro may in part explain the beneficial action of Fumaderm in patients. In addition, we have reported that MMF stimulates an increase in the intracellular free Ca2+ concentration ([Ca2+]i) and the cyclic adenosine monophosphate (cAMP) concentration in granulocytes and keratinocytes. Because Ca2+ and cAMP control many physiological cellular responses, including cell proliferation and inflammatory mediator production, the present study focused on the intracellular signal transduction pathway which links interaction between MMF and granulocytes with increases in [Ca2+]i and the cAMP concentration. The increase in [Ca2+]i in granulocytes after MMF depended both on extracellular Ca2+ and Ca2+ from intracellular stores. Ca2+ is essential for the increase in the cAMP concentration after stimulation with MMF. The results found for pharmacological inhibitors of various protein kinases suggest that a staurosporine-sensitive protein kinase different from protein kinase C (PKC) and protein kinase A is involved in the MMF-induced increase in [Ca2+]i in granulocytes. As MMF activated protein tyrosine kinase (PTK), and inhibition of this protein kinase partially reduced the increase in [Ca2+]i in granulocytes, PTK activity most likely is involved. In addition, activation of protein kinase histone 4 (PKH4) probably plays a part in the MMF-stimulated increase in [Ca2+]i in granulocytes as well. As MMF stimulated an increase in the GTP-ase activity of membranes and pertussis toxin (PTX) inhibited the increase in the [Ca2+]i and PKH4 activity of granulocytes stimulated by this compound, it is concluded that MMF activates PTX-sensitive G proteins. Competition binding studies with radiolabelled dimethylfumarate (DMF) and unlabelled DMF and MMF revealed the presence of specific binding sites for methylated fumarates on granulocytes. In summary, MMF binds to specific sites on the plasma membrane of cells. This interaction activates pertussis toxin-sensitive G proteins which then stimulate an increase in PTK and PKH4 activity. These protein kinases may regulate the rise in [Ca2+]i and the intracellular cAMP concentration. Elevated [Ca2+]i and intracellular cAMP concentration stimulate protein kinases that regulate transcription factors. A

Topics: Binding Sites; Binding, Competitive; Calcium; Cell Membrane; Cells, Cultured; Cyclic AMP; Dimethyl Fumarate; Fumarates; Furocoumarins; Genistein; Granulocytes; GTP Phosphohydrolases; GTP-Binding Proteins; Humans; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Isoflavones; Maleates; Protein Kinase C; Protein-Tyrosine Kinases; Psoriasis; Signal Transduction; Staurosporine

Although the effectiveness of systemic antipsoriatic treatment with fumaric acid esters has been proven, their mode of action is still not understood. Recent results indicate their potency in inducing cytokine production in stimulated T cells. Since monocytes and their cytokines are also considered to be of pathogenic importance in psoriasis, we investigated the effect of monomethylfumarate (MMF) on proinflammatory (TNF-alpha, IL-12) and antiinflammatory (IL-10, IL-1RA) cytokine production by peripheral blood mononuclear cells (PBMC) and separated monocytes. In 24-h PBMC cultures from both psoriatic patients (n = 6-13) and healthy volunteers (n = 7-9), MMF at 100 microM induced secretion of TNF-alpha, IL-10, and IL-1RA. Kinetics of IL-10 protein and mRNA expression indicated de novo production. Moreover, MMF significantly augmented endotoxin-induced synthesis of TNF-alpha, IL-10 and IL-1RA. In contrast, no influence on IL-12 secretion was found. Similar effects of MMF in purified monocytes indicated these cells to be responsible for aberrant cytokine formation. Furthermore, enhanced expression of costimulatory molecules after MMF stimulation confirmed monocyte activation. Multiple restimulation with fumaric acid esters in vitro, however, and immunomonitoring in a patient during Fumaderm initial therapy suggested that initial monocyte activation is followed by subsequent deactivation associated with an antiinflammatory response. Our results may explain the well-known effects of therapy with fumaric acid esters. Thus, initial treatment is often accompanied by adverse effects which may be caused by MMF-induced TNF-alpha formation. The change in the IL-10/IL-12 balance as a result of elective induction of IL-10, however, may have antipsoriatic activity by diminishing type-1/proinflammatory cytokine over-expression and the antigen-presenting capacity of monocytes/macrophages, and by upregulation of IL-1RA.

Topics: Cells, Cultured; Cytokines; Fumarates; Humans; Maleates; Monocytes; Psoriasis; RNA, Messenger

Type 2 cytokines are thought to have a protective role in psoriasis vulgaris by dampening the activity of T helper 1 (Th1) lymphocytes. The aim of the present study was to determine the effect of monomethylfumarate (MMF), the most active metabolite of the new anti-psoriatic drug Fumaderm, on the production of cytokines and the development of Th subsets. MMF was found to enhance interleukin (IL)-4 and IL-5 production by CD2/CD8 monoclonal antibody-stimulated peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Maximal effects of MMF were found at a concentration of 200 microM and resulted in tenfold enhanced levels of IL-4 and IL-5 production. MMF did not affect the levels of IL-2 production, interferon (IFN)-gamma production or proliferative T cell responses in these cultures. Similar effects of MMF were observed in cultures of purified peripheral blood T cells indicating that this compound can act directly on T cells. MMF did not influence cytokine production by purified CD4+CD45RA+ (unprimed) T cells, but greatly enhanced IL-4 and IL-5 production without affecting IFN-gamma production by purified CD4+CD45RO+ (primed) T cells. Furthermore, MMF also augmented IL-4 and IL-5 production in established Th1/Th0 clones that were stimulated with CD2/CD28 monoclonal antibody. Finally, when PBMC were challenged with Mycobacterium tuberculosis that typically induces Th1 recall responses with strong IFN-gamma secretion, MMF again appeared to induce high levels of IL-4 and IL-5 secretion while IFN-gamma production was unaffected. These results may be relevant for the development of therapeutic regimens designed to correct inappropriate Th1 subset development in immunopathologic conditions.

Topics: Animals; Fumarates; Humans; Immunologic Memory; Interleukin-4; Interleukin-5; Leukocyte Common Antigens; Lymphocyte Activation; Maleates; Psoriasis; Sheep; Th2 Cells