Licofelone, a Balanced Inhibitor of Cyclooxygenase and 5-Lipoxygenase, Reduces Inflammation in a Rabbit Model of Atherosclerosis

Abstract

Licofelone, a dual anti-inflammatory drug that inhibits 5-lipoxygenase (LOX) and cyclooxygenase (COX) enzymes, may have a better cardiovascular profile that cycloxygenase-2 inhibitors due to cycloxygenase-1 blockade-mediated antithrombotic effect and a better gastrointestinal tolerability. We examined the anti-inflammatory effect of licofelone on atherosclerotic lesions as well as in isolated neutrophils from whole blood of rabbits compared with a selective inhibitor of COX-2, rofecoxib. We also assessed the antithrombotic effect of licofelone in rabbit platelet-rich plasma. For this purpose, 30 rabbits underwent injury of femoral arteries, and they were randomized to receive 10 mg/kg/day licofelone or 5 mg/kg/day rofecoxib or no treatment during 4 weeks with atherogenic diet in all cases. Ten healthy rabbits were used as controls. Neutrophils and platelets were isolated from peripheral blood of rabbits for ex vivo studies. Licofelone reduced intima/media ratio in injured arteries, the macrophages infiltration in the neointimal area, monocyte chemoattractant protein-1 (MCP-1) gene expression, and the activation of nuclear factor-B in rabbit atheroma. Moreover, licofelone inhibited COX-2 and 5-LOX protein expression in vascular lesions. Rofecoxib only diminished COX-2 protein expression and MCP-1 gene expression in vascular atheroma. Prostaglandin E 2 in rabbit plasma was attenuated by both drugs. Licofelone almost abolished 5-LOX activity by inhibiting leukotriene B4 generation in rabbit neutrophils and prevented platelet thromboxane B 2 production from whole blood. Licofelone reduces neointimal formation and inflammation in an atherosclerotic rabbit model more markedly than rofecoxib. This effect, together with the antiplatelet activity of licofelone, suggests that this drug may have a favorable cardiovascular profile.

Chronic arthritis aggravates vascular lesions in rabbits with atherosclerosis: A novel model of atherosclerosis associated with chronic inflammation

“…To initiate atherosclerosis, rabbits were fed a hypercholesterolemic diet throughout the study, consisting of 2% cholesterol and 6% peanut oil (Letica, Barcelona, Spain). Two weeks after starting the diet, an endothelial lesion was induced in both femoral arteries by the intravascular instillation of gaseous nitrogen under general anesthesia (26)(27)(28)(29). AIA was induced as previously described, with some modifications that were intended to produce a severe phenotype (20,(30)(31)(32).…”

“…The images of the preparations were digitized with a BH-2 microscope (Olympus) connected to a video camera device (28,29). The size of the plaque and vessel lumen and the intima-media thickness ratio were measured in the selected regions.…”

“…Tissues were counterstained with hematoxylin. The area stained (per mm 2 of tissue) was assessed on digital photomicrographs and expressed as a percentage (28,29). The evaluation was performed at the site of maximal stenosis, and IgG isotype served as the negative control.…”

“…Total RNA from the femoral arteries, PBMCs, or synovial membranes was extracted with TRIzol reagent, and 1 g was reverse-transcribed with a High Capacity cDNA kit (Applied Biosystems, Foster City, CA) following the manufacturer's recommendations. The complementary DNA sequences of rabbit CCL2 and COX-2 obtained from GenBank were used to design Assays-on-Demand with the Primer Express software (Applied Biosystems) (29). The expression of eukaryotic 18S ribosomal RNA (VIC/TAMRA Probe; Applied Biosystems) was used as an endogenous control.…”

“…To identify macrophages in femoral lesions, a monoclonal antibody against rabbit macrophages (RAM11; Dako, Glostrup, Denmark) was used as described previously (28,29), and it was detected with biotinylated goat anti-mouse IgG (1:200; Amersham, Arlington Heights, IL). Development was done with avidin-biotin complex/horseradish peroxidase (Dako) and 3,3Ј-diaminobenzidine tetrahydrochloride (Dako).…”