Effect of voriconazole and other azole antifungal agents on CYP3A activity and metabolism of tacrolimus in human liver microsomes.

Azole antifungal agents are known to inhibit cytochrome P450 3A (CYP3A) enzymes. Limited information is available regarding the effect of voriconazole on CYP3A activity. We examined the effect of voriconazole on CYP3A activity in human liver microsomes as measured by the formation of 6ß-hydroxytestosterone from testosterone. We also evaluated the interaction between voriconazole and tacrolimus, an immunosuppressive drug, using human liver microsomes. The effect of voriconazole on CYP3A activity and tacrolimus metabolism was compared to that of other azole antifungal agents. CYP3A4 activity and the metabolism of tacrolimus were measured in the absence and in the presence of various concentrations of voriconazole (0-1.43 mM), fluconazole (0-1.63 mM), itraconazole (0-14 µM) and ketoconazole (0-0.19 µM). At a concentration of 21.2 ± 15.4 µM and 29.8 ± 12.3 µM, voriconazole inhibited the formation of 6ß-hydroxytestosterone from testosterone and the metabolism of tacrolimus by 50%, respectively. The rank order of inhibition of 6ß-hydroxytestosterone formation from testosterone and the metabolism of tacrolimus, is ketoconazole > itraconazole > voriconazole > fluconazole. Our observations suggest that voriconazole at clinically relevant concentrations will inhibit the hepatic metabolism of tacrolimus and increase the concentration of tacrolimus more than two-fold. Close monitoring of the blood concentrations and adjustment in the dose of tacrolimus are warranted when transplant patients receiving tacrolimus are treated with voriconazole.

Bile acid signaling pathways increase stability of Small Heterodimer Partner (SHP) by inhibiting ubiquitin-proteasomal degradation.

Small Heterodimer Partner (SHP) inhibits activities of numerous transcription factors involved in diverse biological pathways. As an important metabolic regulator, SHP plays a key role in maintaining cholesterol and bile acid homeostasis by inhibiting cholesterol conversion to bile acids. While SHP gene induction by increased bile acids is well established, whether SHP activity is also modulated remains unknown. Here, we report surprising findings that SHP is a rapidly degraded protein via the ubiquitin-proteasomal pathway and that bile acids or bile acid-induced intestinal fibroblast growth factor 19 (FGF19) increases stability of hepatic SHP by inhibiting proteasomal degradation in an extracellular signal-regulated kinase (ERK)-dependent manner. SHP was ubiquitinated at Lys122 and Lys123, and mutation of these sites altered its stability and repression activity. Tandem mass spectrometry revealed that upon bile acid treatment, SHP was phosphorylated at Ser26, within an ERK motif in SHP, and mutation of this site dramatically abolished SHP stability. Surprisingly, SHP stability was abnormally elevated in ob/ob mice and diet-induced obese mice. These results demonstrate an important role for regulation of SHP stability in bile acid signaling in normal conditions, and that abnormal stabilization of SHP may be associated with metabolic disorders, including obesity and diabetes.

Determination of 13-O-demethyl tacrolimus in human liver microsomal incubates using liquid chromatography-mass spectrometric assay (LC-MS).

A simple, sensitive and specific liquid chromatography coupled electrospray ionization mass spectrometric (LC/ESI/MS) method for the determination of 13-O-demethylated metabolite (MI), one of the major metabolites of tacrolimus has been developed. The assay uses 32-demethoxyrapamycin (IS) as the internal standard; ethyl acetate as extraction solvent; a Hypersil-Keystone Beta Basic-18 reversed-phase column; and a gradient mobile phase of consisting 0.1% formic acid in water and methanol-acetonitrile (3:49, v/v). Mass detection is performed on a single quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface and operated in a positive ionization mode. MI in the microsomal incubates was quantitated by computing the peak area ratio (MI/IS) analyzed in single ion monitoring (SIM) mode (m/z: 804 and m/z: 901 for MI and IS, respectively). Precision of the assay was determined by calculating the intra-run and inter-run variation at three concentrations (15, 25, 80 ng/ml); the intra run relative standard deviation (R.S.D.) was less than 10% and ranged from 5.0 to 8.3%; and the inter-run R.S.D. was less than 10% and ranged from 4.6 to 9.6%. The limits of detection was 2 ng/ml. This assay has been used to evaluate the effect of three human immunodeficiency virus (HIV) protease inhibitors on the metabolism of tacrolimus in human liver microsomes.