cj-13610 and Inflammation

Zileuton, a redox and iron chelator 5-lipoxygenase (5-LOX) inhibitor and, leukotriene receptor antagonists are presently used clinically in the long term treatment of asthma. Recent data implicate 5-LOX pathway in pain signaling. We report 5-LOX expression in the central nervous system (CNS) and analyze the pain efficacy of a new class of non redox, non iron chelating 5-LOX inhibitor. CJ-13610, 4-(3-(4-(2-methyl-1H-imidazol-1-yl) phenylthio) phenyl)-tetrahydro-2H-pyran-4-carboxamide, demonstrated antihyperalgesic activity in inflammatory pain models including the acute carrageenan model and the chronic inflammatory model using complete Freund's adjuvant. Following complete Freund's adjuvant stimulus leukotrieneB(4) concentration in the brain was elevated (9+/-1 ng/g, mean+/-S.E.M.) by about 3 times that of the control group (3+/-0.11, mean+/-S.E.M.). Hyperalgesia and leukotrieneB(4) concentration were both reversed following CJ-13610 treatment. Furthermore, we demonstrate CJ-13610 efficacy against osteoarthritis like pain using the rat medial meniscal transection model. CJ-13610 at oral doses of 0.6, 2 and 6 mg/kg/day reversed two modalities of pain in this model; tactile allodynia and weight bearing differential. Taken together, these data suggest that 5-LOX pathway and the leukotriene products are important mediators of pain.

Topics: Administration, Oral; Animals; Arachidonate 5-Lipoxygenase; Blotting, Western; Cell Line; Disease Models, Animal; Enzyme Inhibitors; Freund's Adjuvant; Humans; Hydroxyurea; Imidazoles; Immunohistochemistry; Inflammation; Leukotrienes; Lipoxygenase Inhibitors; Male; Osteoarthritis; Pain; Rats; Rats, Sprague-Dawley; Substrate Specificity; Sulfides

The 5-lipoxygenase (5-LOX) pathway has been associated with a variety of inflammatory diseases including asthma, atherosclerosis, rheumatoid arthritis, pain, cancer and liver fibrosis. Several classes of 5-LOX inhibitors have been identified, but only one drug, zileuton, a redox inhibitor of 5-LOX, has been approved for clinical use. To better evaluate the efficacy of 5-LOX inhibitors for pharmacological intervention, a rat model was modified to test the in vivo efficacy of 5-LOX inhibitors. Inflammation was produced by adding carrageenan into a newly formed air pouch and prostaglandins produced. While macrophages and neutrophils are present in the inflamed pouch, little 5-LOX products are formed. Cellular 5-LOX activation was obtained by adding calcium ionophore (A23187) into the pouch thus providing a novel model to evaluate the efficacy and selectivity of 5-LOX inhibitors. Also, we described modifications to the in vitro 5-LOX enzyme and cell assays. These assays included a newly developed fluorescence-based enzyme assay, a 5-LOX redox assay, an ex vivo human whole blood assay and an IgE-stimulated rat mast cell assay, all designed for maximal production of leukotrienes. Zileuton and CJ-13,610, a competitive, non-redox inhibitor of 5-LOX, were evaluated for their pharmacological properties using these assays. Although both compounds achieved dose-dependent inhibition of 5-LOX enzyme activity, CJ-13,610 was 3-4 fold more potent than zileuton in all-assays. Evaluation of 5-LOX metabolites-by LC/MS/MS and ELISA confirmed that both compounds selectively inhibited all products downstream of 5-hydroperoxy eicosatetraenoic acid (5-HPETE), including 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxoETE), without inhibition of 12-lipoxygenase (12-LOX), 15-lipoxygenase (15-LOX), or cyclooxygenase (COX) products. In the rat air pouch model, oral dosing of CJ-13,610 and zileuton resulted in selective inhibition 5-LOX activity from pouch exudate and ex vivo rat whole blood with similar potency to in vitro assay. These data show that the rat air pouch model is a reliable and useful tool for evaluating in vivo efficacy of 5-LOX inhibitors and may aid in the development of the next generation of 5-LOX inhibitors, such as the non-redox inhibitors similar to CJ-13,610.

Topics: Air; Animals; Arachidonate 5-Lipoxygenase; Biological Assay; Calcimycin; Carrageenan; Cell Line, Tumor; Chromatography, Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Activators; Enzyme-Linked Immunosorbent Assay; Humans; Hydroxyurea; Imidazoles; Immunoglobulin E; Inflammation; Ionophores; Leukotrienes; Lipoxygenase Inhibitors; Male; Mast Cells; Oxidation-Reduction; Rats; Rats, Inbred Lew; Reproducibility of Results; Sulfides; Tandem Mass Spectrometry