| Description |
Asthma is a common disease that is most frequently treated with inhaled glucocorticoids which are used to decrease inflammation and mucus production in the airways. However, about 30% of asthma patients do not respond to treatment. A possible hypothesis for glucocorticoid insensitivity is increased metabolism of inhaled glucocorticoids by cytochrome P450 3A (CYP3A) enzymes, particularly in the lung. The objectives for this dissertation were to evaluate the metabolism of five inhaled glucocorticoids (budesonide, beclomethasone dipropionate, fluticasone propionate, triamcinolone acetonide, and flunisolide) by CYP3A enzymes, and to determine if treatment with glucocorticoids in lung cells induced CYP3A enzyme expression, further increasing the metabolism of glucocorticoids in the lung. All three CYP3A enzymes (CYP3A4, 3A5, and 3A7) metabolized the five glucocorticoids, but to varying degrees and with unique products. CYP3A4 and CYP3A5 were the most efficient at metabolizing the glucocorticoids; CYP3A7 had the lowest rates of metabolism. The most common metabolites produced by CYP3A enzymes with triamcinolone acetonide, budesonide, flunisolide, and beclomethasone dipropionate were 6β-hydroxylated and Δ6-dehydrogenated product, all of which are believed to be clearance metabolites. Investigation into the metabolism of beclomethasone dipropionate by A549 lung cells showed that a dehydrogenated P450-mediated metabolite, [M5], was produced, decreasing bioavailability of the active drug. It was also demonstrated that CYP3A5 mRNA was induced in A549 cells with glucocorticoid treatment. The induction of CYP3A mRNA was blocked when cells were co treated with esterase inhibitors and BDP, confirming the active metabolite, beclomethasone 17-monopropionate ([M1]), was mediating the induction of CYP3A5 mRNA, presumably through the glucocorticoid receptor (GR). CYP3A5 mRNA induction was also attenuated by inhibiting GR using the antifungal drug, ketoconazole, further supporting the hypothesis that glucocorticoids binding to GR was the mechanism of CYP3A5 induction in A549 cells. Additional experimentation with primary cells (NHBE, lobar, SAEC, BEAS-2B, and tracheal cells) demonstrated that only SAEC cells expressed CYP3A5. However, CYP3A5 mRNA was not induced in SAEC cells with glucocorticoid treatment despite extensive manipulation of cell culture conditions, such as removing hydrocortisone and utilizing charcoal-stripped FBS for treatment, which could have interfered with the mechanism observed in A549 cells. Overall, the collective results described in this dissertation support the hypothesis that increased metabolism of glucocorticoids in the lung could lead to decreased bioavailability of pharmacologically active drug, and that continued treatment with inhaled glucocorticoids could perpetuate the inefficacy by inducing CYP3A5 enzymes, potentially causing glucocorticoid insensitivity seen in patients. |
