Interaction of common azole antifungals with P glycoprotein

Abstract

Both eucaryotic and procaryotic cells are resistant to a large number of antibiotics because of the activities of export transporters. The most studied transporter in the mammalian ATP-binding cassette transporter superfamily, P glycoprotein (P-gp), ejects many structurally unrelated amphiphilic and lipophilic xenobiotics. Observed clinical interactions and some in vitro studies suggest that azole antifungals may interact with P-gp. Such an interaction could both affect the disposition and exposure to azole antifungal therapeutics and partially explain the clinical drug interactions observed with some antifungals. Using a whole-cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the abilities of the most widely prescribed orally administered azole antifungals to inhibit the function of this transporter. In a cell line presenting an overexpressed amount of the human P-gp transporter, itraconazole and ketoconazole inhibited P-gp function with 50% inhibitory concentrations (IC(50)s) of approximately 2 and approximately 6 microM, respectively. Cyclosporin A was inhibitory with an IC(50) of 1.4 microM in this system. Uniquely, fluconazole had no effect in this assay, a result consistent with known clinical interactions. The effects of these azole antifungals on ATP consumption by P-gp (representing transport activity) were also assessed, and the K(m) values were congruent with the IC(50)s. Therefore, exposure of tissue to the azole antifungals may be modulated by human P-gp, and the clinical interactions of azole antifungals with other drugs may be due, in part, to inhibition of P-gp transport.

FIG. 1.

Intracellular retention of DNR (A) or Rho (B) in G185 cells versus competing itraconazole concentration. Fluorescence intensity is expressed as relative fluorescence. The average number of cells per assay was 10,000. The function for the line through the data is the Hill equation: v = V_maxSⁿ/(K′ + Sⁿ). The parameters IC₅₀ and Hill coefficient (n; with standard deviation) are shown on the respective graphs. I_max, a percentage of the complete inactivation that occurred with vanadate; CSA, cyclosporine A.

FIG. 2.

Intracellular retention of DNR (A) or Rho (B) in G185 cells versus competing ketoconazole concentration. Fluorescence intensity is expressed as relative fluorescence. The average number of cells per assay was 10,000. The function for the line through the data is the Hill equation: v = V_maxSⁿ/(K′ + Sⁿ). The parameters IC₅₀ and Hill coefficient (n; with standard deviations) are shown on the respective graphs. I_max, a percentage of the complete inactivation that occurred with vanadate; CSA, cyclosporine A.

FIG. 3.

Intracellular retention of DNR (A) or Rho (B) in G185 cells versus competing fluconazole concentration. Fluorescence intensity is expressed as relative fluorescence. The average number of cells per assay was 10,000.

FIG. 4.

P-gp-mediated ATP hydrolysis rates in the presence of itraconazole (A), ketoconazole (B), and fluconazole (C). The data were fit to a hyperbola. For itraconazole, V_max was equal to 69 ± 1.5 nmol/min/mg of membrane protein, with K_m equal to 0.8 ± 0.2 μM. For ketoconazole, V_max was equal to 53 ± 7 nmol/min/mg membrane protein, with K_m equal to 8.6 ± 3 μM. For fluconazole, V_max was equal to 61 ± 2.3 nmol/min/mg membrane protein, with K_m equal to 2.5 ± 1 μM. V₀, initial velocity.