pharmacology of robenacoxib: a novel selective inhibitor of cyclooxygenase-2. J. vet. Pharmacol. Therap. 32,[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] This manuscript reports the results of preclinical studies in the rat with robenacoxib, a novel selective cyclooxygenase (COX)-2 inhibitor. Robenacoxib selectively inhibited COX-2 in vitro as evidenced from COX-1:COX-2 IC 50 ratios of 27:1 in purified enzyme preparations and >967:1 in isolated cell assays. Binding to COX-1 was rapid and readily reversible (dissociation t 1 ⁄ 2 << 1 min), whilst COX-2 binding was slowly reversible (t 1 ⁄ 2 = 25 min). In vivo, robenacoxib inhibited PGE 2 production (an index of COX-2 inhibition) in lipopolysaccharide (LPS)-stimulated air pouches (ID 50 0.3 mg ⁄ kg) and for at least 24 h in zymosan-induced inflammatory exudate (at 2 mg ⁄ kg). Robenacoxib was COX-1 sparing, as it inhibited serum TxB 2 synthesis ex vivo (an index of COX-1 inhibition) only at very high doses (100 mg ⁄ kg but not at 2-30 mg ⁄ kg). Robenacoxib inhibited carrageenan-induced paw oedema (ID 50 0.40-0.48 mg ⁄ kg), LPS-induced fever (ID 50 1.1 mg ⁄ kg) and Randall-Selitto pain (10 mg ⁄ kg). Robenacoxib was highly bound to plasma protein (99.9% at 50 ng ⁄ mL in vitro). After intravenous dosing, clearance was 2.4 mL ⁄ min ⁄ kg and volume of distribution at steady-state was 306 mL ⁄ kg. Robenacoxib was preferentially distributed into inflammatory exudate; the AUC for exudate was 2.9 times higher than for blood and the MRT in exudate (15.9 h) was three times longer than in blood (5.3 h). Robenacoxib produced significantly less gastric ulceration and intestinal permeability as compared with the reference nonsteroidal anti-inflammatory drug (NSAID), diclofenac, and did not inhibit PGE 2 or 6-keto PGF 1a concentrations in the stomach and ileum at 30 mg ⁄ kg. Robenacoxib also had no relevant effects on kidney function at 30 mg ⁄ kg. In summary, results of preclinical studies in rats studies suggest that robenacoxib has an attractive pharmacological profile for potential use in the intended target species, cats and dogs.(Paper

1 This manuscript presents the preclinical profile of lumiracoxib, a novel cyclooxygenase-2 (COX-2) selective inhibitor. 2 Lumiracoxib inhibited purified COX-1 and COX-2 with K i values of 3 and 0.06 mM, respectively. In cellular assays, lumiracoxib had an IC 50 of 0.14 mM in COX-2-expressing dermal fibroblasts, but caused no inhibition of COX-1 at concentrations up to 30 mM (HEK 293 cells transfected with human COX-1). 3 In a human whole blood assay, IC 50 values for lumiracoxib were 0.13 mM for COX-2 and 67 mM for COX-1 (COX-1/COX-2 selectivity ratio 515). 4 Lumiracoxib was rapidly absorbed following oral administration in rats with peak plasma levels being reached between 0.5 and 1 h. 5 Ex vivo, lumiracoxib inhibited COX-1-derived thromboxane B 2 (TxB 2 ) generation with an ID 50 of 33 mg kg À1, whereas COX-2-derived production of prostaglandin E 2 (PGE 2 ) in the lipopolysaccharidestimulated rat air pouch was inhibited with an ID 50 value of 0.24 mg kg À1 . 6 Efficacy of lumiracoxib in rat models of hyperalgesia, oedema, pyresis and arthritis was dosedependent and similar to diclofenac. However, consistent with its low COX-1 inhibitory activity, lumiracoxib at a dose of 100 mg kg À1 orally caused no ulcers and was significantly less ulcerogenic than diclofenac (Po0.05). 7 Lumiracoxib is a highly selective COX-2 inhibitor with anti-inflammatory, analgesic and antipyretic activities comparable with diclofenac, the reference NSAID, but with much improved gastrointestinal safety. British Journal of Pharmacology (2005) 144, 538-550. doi:10.1038/sj.bjp.0706078 Published online 17 January 2005 Keywords: Lumiracoxib; COX-2; cyclooxygenase-2 selective inhibitor; preclinical Abbreviations: AUC, area-under-curve of the concentration vs time curve; C max , maximum drug plasma concentration; CFA, complete Freund's adjuvant; 51 Cr-EDTA, chromium-51 labelled EDTA; COX, cyclooxygenase; D 30 , dose at which 30% inhibition was achieved; DMSO, dimethyl sulphoxide; F 0 , fraction of uninhibited enzyme at equilibrium; GI, gastrointestinal; HEK, human embryonic kidney; IL-1, interleukin-1; K i , inhibitor constant; k on , second-order rate constant representing speed at which an inhibitor binds to an enzyme; I, inhibitor concentration; LC/MS/MS, liquid chromatography/mass spectrometry/mass spectrometry; LPS, lipopolysaccharide; NSAID, nonsteroidal anti-inflammatory drug; O 2 , oxygen; PGE 2 , prostaglandin E 2 ; s, arachidonic acid concentration; t 1/2 , half-life; t opt , time to optimal velocity; TxB 2 , thromboxane B 2 ; V 0 , velocity in the absence of inhibitor; V obs , observed velocity in the presence of inhibitor; V opt , highest observed O 2 consumption velocity; V max , Michaelis-Menten constant for the maximal calculated velocity

Objective. All ␥-chain cytokines signal through JAK-3 and JAK-1 acting in tandem. We undertook this study to determine whether the JAK-3 selective inhibitor WYE-151650 would be sufficient to disrupt cytokine signaling and to ameliorate autoimmune disease pathology without inhibiting other pathways mediated by JAK-1, JAK-2, and Tyk-2.Methods. JAK-3 kinase selective compounds were characterized by kinase assay and JAK-3-dependent (interleukin-2 [IL-2]) and -independent (IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF]) cell-based assays measuring proliferation or STAT phosphorylation. In vivo, off-target signaling was measured by IL-22-and erythropoietin (EPO)-mediated models, while on-target signaling was measured by IL-2-mediated signaling. Efficacy of JAK-3 inhibitors was determined using delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA) models in mice.Results. In vitro, WYE-151650 potently suppressed IL-2-induced STAT-5 phosphorylation and cell proliferation, while exhibiting 10-29-fold less activity against JAK-3-independent IL-6-or GM-CSF-induced STAT phosphorylation. Ex vivo, WYE-151650 suppressed IL-2-induced STAT phosphorylation, but not IL-6-induced STAT phosphorylation, as measured in whole blood. In vivo, WYE-151650 inhibited JAK-3-mediated IL-2-induced interferon-␥ production and decreased the natural killer cell population in mice, while not affecting IL-22-induced serum amyloid A production or EPO-induced reticulocytosis. WYE-151650 was efficacious in mouse DTH and CIA models.Conclusion. In vitro, ex vivo, and in vivo assays demonstrate that WYE-151650 is efficacious in mouse CIA despite JAK-3 selectivity. These data question the need to broadly inhibit JAK-1-, JAK-2-, or Tyk-2-dependent cytokine pathways for efficacy.

A series of chromene derivatives was synthesized and evaluated for their in vitro and ex vivo 5-lipoxygenase (5-LO) inhibitory activity. These compounds were prepared by condensation of appropriate salicyl aldehydes with alpha, beta-unsaturated carbonyl compounds, followed by transformation to the corresponding hydroxamic acids or N-hydroxyureas. Placement of phenoxy or p-fluorophenoxy substituents at the 6 position of the chromene ring led to a dramatic increase in the in vitro potency as demonstrated by the guinea pig PMN 5-LO assay. Chromene hydroxamic acids, in general, behaved poorly in the ex vivo dog model. On the other hand, replacement of the hydroxamic acid function with N-hydroxyurea yielded potent and long-lasting 5-LO inhibitors in the dog model. In most cases, the oral efficacy of the chromene N-hydroxyureas correlated very well with their in vitro activity. Compounds 43 (CGS 23885) and 55 (CGS 24891) are among the most potent inhibitors prepared, showing IC50 values of 48 and 51 nM, respectively. The values for the duration of action (DA) for compounds 43 and 55 are 21 and 20 h, respectively, following intravenous (i.v.) administration of 1.0 mg/kg. In the oral (po) experiments, 43 and 55 have DA's of 14 and 15 h, respectively, at a 1.0 mg/kg dose. In both iv and po experiments, 43 and 55 showed sustained maximal inhibition (> 95%) at earlier time points. The oral ED50 values of 43 and 55 in the ex vivo dog model are 0.23 and 0.23 mg/kg, respectively, at 6.0 h, and 2.37 and 1.63 mg/kg, respectively, at 24 h. Compound 43, which inhibits sheep seminal vesicle cyclooxygenase (CO) with an IC50 value of 36 microM, was shown to be a selective 5-lipoxygenase inhibitor in the ex vivo study. These compounds compare favorably with zileuton (A-64077) in all the parameters examined.

N-Hydroxyureas based on the 1,4-benzodioxan template were prepared from appropriately substituted 1,4-benzodioxan-2-methanols as the key intermediates and evaluated in the in vitro guinea pig polymorphonuclear leukocyte 5-lipoxygenase (5-LO) assay for their 5-LO inhibitory activity. Placement of a 7-phenoxy or 7-p-fluorophenoxy substituent resulted in a dramatic increase in in vitro potency. Selected compounds were subsequently assayed in an ex vivo dog model of LTB4 synthesis at a dose of 1.0 mg/kg. The 7-phenoxy derivatives 16 and 17 showed modest duration of action (DA) in this dog model. The 6-regioisomers 21 and 22 were less potent. Replacement of the 7-phenoxy group of 16 with the p-fluorophenoxy moiety enhanced the DA dramatically. Compound 18 (CGS 25667), which had an IC50 value of 100 nM in the in vitro guinea pig 5-LO assay, had a DA of 8.5 h (zileuton, DA = 8.5 h) at the oral dose of 1.0 mg/kg. Optical antipodes (24, 26) of 18 were independently synthesized in high (> 95%) enantiomeric purity from commercially available optically active glycidyl tosylates and evaluated. In the in vitro assay, the 2S-(-)-enantiomer (24, CGS 25997, IC50 = 85 nM) was found to be twice as active as the 2R-(+)-counterpart (26, CGS 25998, IC50 = 180 nM). In the ex vivo experiment, 24, which dose dependently inhibited plasma 5-LO activity, was shown to be significantly longer acting than 26, with a DA of 8.4 h when dosed orally at 1.0 mg/kg.