Evaluation of 1β-Hydroxylation of Deoxycholic Acid as a Non-Invasive Urinary Biomarker of CYP3A Activity in the Assessment of Inhibition-Based Drug–Drug Interaction in Healthy Volunteers

Abstract

In this study, we aimed to evaluate the utility of endogenous 1β-hydroxy-deoxycholic acid/total deoxycholic acid ratio (1β-OH-DCA/ToDCA) in spot urine as a surrogate marker of cytochrome P450 3A (CYP3A) activity in the assessment inhibition-based drug–drug interactions in healthy volunteers. This was accomplished through an open-label, three-treatment parallel-arm study in healthy male volunteers from Zimbabwe. Each group received itraconazole (ITZ; 100 mg once daily; n = 10), fluconazole (FKZ; 50 mg once daily; n = 9), or alprazolam (APZ; 1 mg once daily; n = 8) orally. Midazolam (MDZ), dosed orally and intravenously, was used as a comparator to validate the exploratory measures of CYP3A activity and the effects of known inhibitors. Urinary metabolic ratios of 1β-OH-DCA/ToDCA before and after CYP3A inhibitor treatment showed a similar magnitude of inhibitory effects of the three treatments as that measured by oral MDZ clearance. The maximum inhibition effect of a 75% reduction in the 1β-OH-DCA/ToDCA ratio compared to the baseline was achieved in the ITZ group following six once-daily doses of 100 mg. The correlations of the two markers for CYP3A inhibitor treatment were significant (rs = 0.53, p < 0.01). The half-life of urinary endogenous 1β-OH-DCA/ToDCA was estimated as four days. These results suggested that 1β-OH-DCA/ToDCA in spot urine is a promising convenient, non-invasive, sensitive, and relatively quickly responsive endogenous biomarker that can be used for CYP3A inhibition-based drug–drug interaction in clinical studies.

Development of a Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Analytical Method for Urinary Endogenous Substrates and Metabolites for Predicting Cytochrome P450 3A4 Activity

“…To date, several biomarkers of CYP3A4 activity have been identified in plasma and urine; however, the most suitable CYP3A4 biomarkers for evaluating its activity have not been conclusively identified. [19][20][21][22][23][24][25][26][27][28] Therefore, a clinically available high-throughput method is required to investigate the utility of CYP3A4 biomarkers.…”

“…DCA 3-sulfate cannot be metabolized by sulfatase owing to insufficient reaction buffer conditions and pH for mediating deconjugated reactions. 23,24) More importantly, phosphate buffer competitively inhibits sulfatase based on the structural similarity of phosphates with sulfates. 42) We considered several factors, such as pH, enzyme amounts, and reaction buffer, and could successfully establish a protocol for deconjugation reaction.…”

“…Sample preparation for urine analysis was performed based on a previously reported method, with several modifications. 23,24) A 100 µL aliquot of urine was added to 900 µL methanol containing the IS mixture (see above) for deproteinization. After the mixture was centrifuged at 15000 × g for 10 min at 4 °C, the supernatant was vacuum-dried at 40 °C, and the dried powder was dissolved in 100 µL of 0.1 M Tris-HCl (pH 5.0) containing hydrolysis enzymes (5 U choloylglycine hydrolase and 10 µL β-glucuronidase/arylsulfatase).…”

“…To date, the concentration ratios of 4β-hydroxycholesterol to cholesterol, 6β-hydroxycortisol (6β-OHC) to cortisol (C), and 1β-hydroxydeoxycholic acid (1β-OHDCA) to deoxycholic acid (DCA) in the plasma or urine have been correlated with the concentration of drugs metabolized by CYP3A4. [19][20][21][22][23][24][25][26][27][28] Notably, 1β-OHDCA has been reported as a CYP3A4-specific biomarker, although CYP3A4 and CYP3A5 often exhibit an overlap in substrate specificity. 22) As 4β-hydroxycholesterol has a relatively long half-life (approximately 17 d) compared with that of other biomarkers, 29) 6β-OHC and 1β-OHDCA may represent better indicators of the actual CYP3A4 activity at the time of sample collection.…”

“…For clinical applications, sample preparation for calibration curves was performed based on a previously reported method with several modifications. 23,24) Each calibration standard (CS) was prepared by diluting the stock solutions to 3, 10, 30, 100, 300, 1000, and 3000 ng/mL in water/ethanol (1 : 1, v/v). After adding a 100 µL aliquot of CS, samples were added to 900 µL of acetonitrile containing 2 ng cortisol-13 C 3 , 25 ng 6β-hydroxycortisol-2 H 4 , 10 ng deoxycholic acid-2 H 4 , and 20 ng cholic acid-2 H 4 as ISs; the mixture was vacuum-dried at 40 °C, and the dried powder was dissolved in 100 µL of 0.1 M Tris-HCl (pH 5.0) followed by the addition of 300 µL acetonitrile.…”