Abstract
Background5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.Methodology/Principal FindingsAnalogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC50 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC50 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC50 values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.ConclusionsCAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.
Highlights
The caffeic acid phenethyl ester (CAPE) structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis
Stimulation of polymorphonuclear leukocytes (PMNL) in the presence of exogenous arachidonic acid (AA) excludes the possibility that the test compounds might affect LT biosynthesis by blocking AA availability
In PMNL stimulated with thapsigargin in the presence of exogenous AA, only CAPE 1 and zileuton significantly decreased production of 5-LO products by 53% and 17%, respectively (Figure 3A)
Summary
Ethics Blood was obtained from health volunteer subjects after having obtained written consent. Blood was centrifuged at 3006 g for 5 min at room temperature, plasma was collected and erythrocytes were removed by dextran sedimentation. Following centrifugation on a lymphocyte separation medium cushion (density, 1.077 g/ml) (Wisent, St-Bruno, Qc, Canada) at 9006 g for 20 min at room temperature, PMNL (.96%) were obtained from the pellet after hypotonic lysis to remove residual erythrocytes. Samples were centrifuged at 10006 g for 10 min, the supernatant was diluted with 4 volumes of acidified water (acetic acid, 0.1% v/v) and applied onto a preconditioned octadecyl (C18) column. Columns containing samples were washed with 2 ml acidified water and 5LO products were eluted with 3 ml of methanol. After evaporation of solvents with nitrogen, products were suspended in 20% methanol and subjected to RP-HPLC analysis with diode array detection as previously described [26]. Total 5-LO products quantified represents the sum of LTB4, its trans isomers, 20COOH- and 20-OH-LTB4 and 5-hydroxyeicosatetraenoic acid
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