Zafirlukast Is a Dual Modulator of Human Soluble Epoxide Hydrolase and Peroxisome Proliferator-Activated Receptor γ.

Cysteinyl leukotriene receptor 1 antagonists (CysLT1RA) are frequently used as add-on medication for the treatment of asthma. Recently, these compounds have shown protective effects in cardiovascular diseases. This prompted us to investigate their influence on soluble epoxide hydrolase (sEH) and peroxisome proliferator activated receptor (PPAR) activities, two targets known to play an important role in CVD and the metabolic syndrome. Montelukast, pranlukast and zafirlukast inhibited human sEH with IC50 values of 1.9, 14.1, and 0.8 µM, respectively. In contrast, only montelukast and zafirlukast activated PPARγ in the reporter gene assay with EC50 values of 1.17 µM (21.9% max. activation) and 2.49 µM (148% max. activation), respectively. PPARα and δ were not affected by any of the compounds. The activation of PPARγ was further investigated in 3T3-L1 adipocytes. Analysis of lipid accumulation, mRNA and protein expression of target genes as well as PPARγ phosphorylation revealed that montelukast was not able to induce adipocyte differentiation. In contrast, zafirlukast triggered moderate lipid accumulation compared to rosiglitazone and upregulated PPARγ target genes. In addition, we found that montelukast and zafirlukast display antagonistic activities concerning recruitment of the PPARγ cofactor CBP upon ligand binding suggesting that both compounds act as PPARγ modulators. In addition, zafirlukast impaired the TNFα triggered phosphorylation of PPARγ2 on serine 273. Thus, zafirlukast is a novel dual sEH/PPARγ modulator representing an excellent starting point for the further development of this compound class.

Cellular analysis of the histamine H4 receptor in human myeloid cells.

The human histamine H4 receptor (H4R) is a Gαi/o-coupled receptor which is mainly expressed on hematopoietic cells. Accordingly, the receptor is implicated in the pathology of various diseases such as autoimmune disorders, bronchial asthma and pruritus. Due to complicated receptor pharmacology, the lack of a reliable antibody and limited availability of primary cells expressing the receptor the physiology of this receptor is still poorly understood. Therefore, we aimed to assess absolute receptor mRNA expression and functionality (intracellular Ca(2+) release) in various human myeloid cell types such as granulocytes, monocytes, macrophages and dendritic cells (DCs). This was put into context with the expression of the H1R and H2R. In addition, the influence of various inflammatory stimuli on H4R expression was investigated in macrophages and monocyte-derived DCs. We found that classically activated macrophages treated with pro-inflammatory stimuli down-regulated histamine receptor mRNA expression as did LPS and zymosan A matured monocyte-derived DCs. In contrast, alternatively activated macrophages (IL-4 or IL-13) upregulated H2R and H4R expression compared to controls. Consistent with existing literature, we found eosinophils to be the major source of the H4R. Since availability of primary eosinophils is limited, we developed a cell model based on the differentiated eosinophilic cell line EOL-1, in which H4R pharmacology and physiology may be studied.

N-Benzylbenzamides: A Novel Merged Scaffold for Orally Available Dual Soluble Epoxide Hydrolase/Peroxisome Proliferator-Activated Receptor γ Modulators.

Metabolic syndrome (MetS) is a multifactorial disease cluster that consists of dyslipidemia, cardiovascular disease, type 2 diabetes mellitus, and obesity. MetS patients are strongly exposed to polypharmacy; however, the number of pharmacological compounds required for MetS treatment can be reduced by the application of multitarget compounds. This study describes the design of dual-target ligands that target soluble epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). Simultaneous modulation of sEH and PPARγ can improve diabetic conditions and hypertension at once. N-Benzylbenzamide derivatives were determined to fit a merged sEH/PPARγ pharmacophore, and structure-activity relationship studies were performed on both targets, resulting in a submicromolar (sEH IC50 = 0.3 µM/PPARγ EC50 = 0.3 µM) modulator 14c. In vitro and in vivo evaluations revealed good ADME properties qualifying 14c as a pharmacological tool compound for long-term animal models of MetS.

"Disease modifying nutricals" for multiple sclerosis.

The association between vitamin D and multiple sclerosis has (re)-opened new interest in nutrition and natural compounds in the prevention and treatment of this neuroinflammatory disease. The dietary amount and type of fat, probiotics and biologicals, salmon proteoglycans, phytoestrogens and protease inhibitor of soy, sodium chloride and trace elements, and fat soluble vitamins including D, A and E were all considered as disease-modifying nutraceuticals. Studies in experimental autoimmune encephalomyelitis mice suggest that poly-unsaturated fatty acids and their 'inflammation-resolving' metabolites and the gut microflora may reduce auto-aggressive immune cells and reduce progression or risk of relapse, and infection with whipworm eggs may positively change the gut-brain communication. Encouraged by the recent interest in multiple sclerosis-nutrition nature's pharmacy has been searched for novel compounds with anti-inflammatory, immune-modifying and antioxidative properties, the most interesting being the scorpion toxins that inhibit specific potassium channels of T cells and antioxidative compounds including the green tea flavonoid epigallocatechin-3-gallate, curcumin and the mustard oil glycoside from e.g. broccoli and sulforaphane. They mostly also inhibit pro-inflammatory signaling through NF-κB or toll-like receptors and stabilize the blood brain barrier. Disease modifying functions may also complement analgesic and anti-spastic effects of cannabis, its constituents, and of 'endocannabinoid enhancing' drugs or nutricals like inhibitors of fatty acid amide hydrolase. Nutricals will not solve multiple sclerosis therapeutic challenges but possibly support pharmacological interventions or unearth novel structures.

Cysteinyl leukotriene-receptor-1 antagonists interfere with PGE2 synthesis by inhibiting mPGES-1 activity.

Because of their favourable safety profile and beneficial anti-inflammatory properties, the CysLT1 receptor antagonists (LTRA), montelukast, zafirlukast and pranlukast are approved for the treatment of asthma and are frequently prescribed as add-on therapeutics to reduce the amount of inhaled glucocorticoids and ß2-agonists. There is evidence that some of these anti-inflammatory properties might be of a secondary nature and therefore, unrelated to the CysLT1 antagonism. Here, we show that LTRA inhibit PGE2 formation in cytokine-stimulated Hela and A549 carcinoma cells and in lipopolysaccharide (LPS)-stimulated human leukocyte preparations (IC50∼20µM). Neither expression of enzymes involved in PGE2 synthesis nor arachidonic acid release and COX activities were inhibited by the compounds. In contrast, mPGES-1 activity was suppressed at low micromolar levels (IC50 between 2 and 4µM). This suppression was specific for PGE2 synthesis, since PGD2 and PGI2 levels in LPS-stimulated leukocyte preparations were not negatively affected. PGF2α levels were concomitantly inhibited, probably due to its direct synthesis from PGE2. Several major conclusions can be drawn from this study: (A) clinical trials investigating elevated doses of the compounds are helpful to confirm suppression of PGE2 synthesis in vivo; (B) studies investigating the role of CysLTs in cell culture or animal models of inflammation and cancer have to be reassessed carefully, if higher doses of LTRA were applied or serum levels in cell culture assays were low; and (C) LTRA may serve as new scaffolds for the development of potent, selective and well tolerated mPGES-1 inhibitors.