The inhaled glucocorticoid fluticasone propionate efficiently inactivates cytochrome P450 3A5, a predominant lung P450 enzyme

Abstract

Inhaled glucocorticoid (GC) therapy is a vital part of the management of chronic asthma. GCs are metabolized by members of the cytochrome P450 3A family in both liver and lung, but the enzymes are differentially expressed. Selective inhibition of one or more P450 3A enzymes could substantially modify target and systemic concentrations of GCs. In this study, we have evaluated the mechanism-based inactivation of P450 3A4, 3A5, and 3A7 enzymes by GCs. Among the five major inhaled GCs approved for clinical use in the United States, fluticasone propionate (FLT) was the most potent mechanism-based inactivator of P450 3A5, the predominant P450 enzyme in the lung. FLT inactivated P450 3A5 in a time- and concentration-dependent manner with K(I), k(inact), and partition ratio of 16 muM, 0.027 min(-1), and 3, respectively. In contrast, FLT minimally inactivated P450 3A4 and did not inactivate 3A7, even with a concentration of 100 muM. The inactivation of P450 3A5 by FLT was irreversible because dialysis did not restore enzyme activity. In addition, the exogenous nucleophilic scavenger GSH did not attenuate inactivation. The prosthetic heme of P450 3A5 was not modified by FLT. The loss of P450 3A5 activity in lung cells could substantially decrease the metabolism of FLT, which would increase the effective FLT concentration at its target site, the respiratory epithelium. Also, inactivation of lung P450 3A5 could increase the absorption of inhaled FLT, which could lead to high systemic concentrations and adverse effects, such as life-threatening adrenal crises or cataracts that have been documented in children receiving high doses of inhaled GCs.

Figure 1

Single time-dependent loss of testosterone 6β-hydroxylation activity of CYP3A4, 3A5 and 3A7 enzymes following incubation with DMSO (○) or 100 μM of beclomethasone dipropionate (□), budesonide (◇), flunisolide (△), FLT (●), and triamcinolone acetonide (+) in the presence of NADPH. The data represent the mean and standard deviations from three separate experiments, albeit most of the standard deviations are smaller than the data label. The lines with an asterisk indicate statistically significant (p < 0.01) slope differences in testosterone 6β-hydroxylation activity from the control.

Figure 2

Time- and concentration-dependent inactivation of P450 3A5 with increasing concentrations of FLT. Incubation and assay conditions were described under Materials and Methods. The concentrations of FLT were 0 (○), 1 (●), 2 (△), 5 (▲), and 10 (◇) μM. Control incubations were done in the presence of NADPH, but without FLT. The data shown represent the mean and standard deviations from three separate experiments. The slopes of the lines represent the observed first-order rate constants (k_obs) of the inactivation reaction at a given FLT concentration. The inset shows the double-reciprocal Kitz-Wilson plot for P450 3A5 inactivation. The K_I (obtained from the x-intercept) was 16.1 μM, and the maximal rate of inactivation k_inact (obtained from the y-intercept) was 0.027/min^-1.

Figure 3

Time- and concentration-dependent inactivation of P450 3A4 by increasing concentrations of FLT. Incubation and assay conditions were as described under Materials and Methods. The concentrations of FLT were 0 (○), 10 (◇), 20 (◆), 50 (□), and 100 (■) μM. Control incubations were done in the presence of NADPH, but without FLT. The data shown represent the means and standard deviations from three separate experiments. The slopes of the lines represent the observed first-order rate constants (k_obs) of the inactivation reaction at a specific FLT concentration. The inset shows the double-reciprocal Kitz-Wilson plot for P450 3A4 inactivation. The K_I (obtained from the x-intercept) was 3.5 μM, and the maximal rate of inactivation k_inact (obtained from the y-intercept) was 0.006/min^-1.

Figure 4

Partition ratio for FLT-mediated inactivation of P450 3A5 showing loss of enzyme activity as a function of the ratio of [FLT]/[P450 3A5]. The enzyme was incubated with increasing concentrations of FLT (0, 0.5, 1, 2, 3, 5, 10, 15, 20, 25 μM) for 30 min as described under Materials and Methods. Percent activity remaining after complete inactivation (30 min) was plotted against the molar ratio of FLT to P450 3A5. The partition ratio was obtained from the intercept of the linear regression line containing the lower inactivator concentrations and the straight line drawn from the higher inactivator concentrations. The partition ratio for FLT-mediated inactivation of P450 3A5 was found to be 3. The data points were the means of three separate experiments.

Figure 5

Potential bioactivation pathways to reactive electrophilic intermediates of FLT by P450 3A enzymes are illustrated. Nucleophilic residues of the proteins could add to the epoxides or diene to inactivate the enzymes. Structures of beclomethasone dipropionate, budesonide, flunisolide, fluticasone propionate, and triamcinolone acetonide are shown.