Itraconazole-mediated inhibition of calcium entry into platelet-activating factor-stimulated human neutrophils is due to interference with production of leukotriene B4

Abstract

SummaryThe primary objective of this study was to probe the involvement of leukotriene B4 (LTB4) in itraconazole (0·1-5 mM)-mediated inhibition of Ca 2+ uptake by chemoattractant-activated human neutrophils. Following exposure of the cells to platelet-activating factor (PAF, 200 nM), LTB4 was measured by immunoassay, while neutrophil cytosolic Ca 2+ concentrations were determined by a fura-2/AM-based spectrofluorimetric procedure. Activation of neutrophils was accompanied by an abrupt and sustained (for about 1 min) elevation in cytosolic Ca 2+ which was associated with increased generation of LTB4, both of which were attenuated significantly by itraconazole at 0·5 mM and higher. The inhibitory effect of the anti-mycotic on Ca 2+ uptake by PAF-activated cells was mimicked by an LTB4 antibody, as well as by LY255283 (1 mM) and MK886 (0·5 mM), an antagonist of LTB4 receptors and an inhibitor of 5Ј-lipoxygenaseactivating protein, respectively, while addition of itraconazole to purified 5Ј-lipoxygenase resulted in inhibition of enzyme activity. A mechanistic relationship between itraconazole-mediated inhibition of LTB4 production and Ca 2+ influx was also supported by the observation that pulsed addition of purified LTB4 to PAF-activated neutrophils caused substantial restoration of Ca 2+ uptake by cells treated with the anti-mycotic. Taken together, these observations suggest that the potentially beneficial anti-inflammatory interactions of itraconazole with activated neutrophils result from interference with production of LTB4, with consequent attenuation of a secondary LTB4-mediated wave of Ca 2+ uptake by the cells.

Discovery of a Novel Dual Fungal CYP51/Human 5-Lipoxygenase Inhibitor: Implications for Anti-Fungal Therapy

“…In order to investigate whether the enzyme concentration was approaching the IC 50 value, we compared our IC 50 values of two high potency 5-LOX inhibitors to that in the literature. Setileuton displayed an IC 50 value of 60±6 nM, in good agreement with the literature value of 45±10 nM, and zileuton displayed an IC 50 value of 560±80 nM, in good agreement with the literature value of 500±100 nM [2], [13]. The solvent isotope effect of the inhibitor IC 50 was investigated utilizing the same conditions and methods as stated above.…”

“…However, it has been proposed that part of its effectiveness is due to its anti-inflammation activity, since it also weakly inhibits 5-LOX [12]. The anti-inflammatory activity of ketoconazole has also been seen for itraconazole, a similar anti-fungal therapeutic [13], which suggests a common theme for effective dandruff agents, dual anti-fungal/anti-inflammatory targeting. Nevertheless, the potency for ketoconazole and itraconazole against 5-LOX is poor, with IC 50 values greater than 50 µM for both molecules, which indicates a potential for improvement in their anti-inflammatory activity [12], [13].…”

Immunomodulatory activity of itraconazole in lung

“…The precise mechanism(s) by which ITC exerts its effects on the immune system and whether the corresponding changes have clinically significant results remain unclear. One possible mechanism is the ability of ITC to inhibit cytochrome P450 enzyme systems [23]. ITC has been shown to inhibit CYP450 enzyme 5-lipooxigenase dosedependently, interfering with the synthesis of leukotriene B4 (LTB4), the major product of arachidonic acid metabolism, which has been implicated as a potent mediator of inflammatory processes and immunoregulation [23].…”

“…One possible mechanism is the ability of ITC to inhibit cytochrome P450 enzyme systems [23]. ITC has been shown to inhibit CYP450 enzyme 5-lipooxigenase dosedependently, interfering with the synthesis of leukotriene B4 (LTB4), the major product of arachidonic acid metabolism, which has been implicated as a potent mediator of inflammatory processes and immunoregulation [23]. In addition, it has been demonstrated in primary cultures of human hepatocytes that some azolic drugs are antagonists of the human glucocorticoid receptor (GR), which belongs to the family of steroid/thyroid receptors.…”

Interactive inhibitory effects of formoterol and montelukast on activated human neutrophils

“…PKA in turn accelerates restoration of Ca 2+ homeostasis and downregulation of pro-inflammatory activities following exposure of the cells to receptor-linked, Ca 2+ mobilising stimuli by several mechanisms, including: phosphorylative inactivation of phospholipase C [28]; inactivation of inositol triphosphate receptors on intracellular Ca 2+ stores [29]; upregulation of the Ca 2+ sequestering/resequestering endo-membrane Ca 2+ -ATP [30]; and inhibition of p38 MAP kinase, resulting in interference with the activation of 5-lipoxygenase [31] and attenuation of an autocrine, LTB 4 -mediated secondary wave of Ca 2+ uptake by the cells [32].…”