Identification of diet-derived constituents as potent inhibitors of intestinal glucuronidation

Abstract

Drug-metabolizing enzymes within enterocytes constitute a key barrier to xenobiotic entry into the systemic circulation. Furanocoumarins in grapefruit juice are cornerstone examples of diet-derived xenobiotics that perpetrate interactions with drugs via mechanism-based inhibition of intestinal CYP3A4. Relative to intestinal CYP3A4-mediated inhibition, alternate mechanisms underlying dietary substance-drug interactions remain understudied. A working systematic framework was applied to a panel of structurally diverse diet-derived constituents/extracts (n = 15) as inhibitors of intestinal UDP-glucuronosyl transferases (UGTs) to identify and characterize additional perpetrators of dietary substance-drug interactions. Using a screening assay involving the nonspecific UGT probe substrate 4-methylumbelliferone, human intestinal microsomes, and human embryonic kidney cell lysates overexpressing gut-relevant UGT1A isoforms, 14 diet-derived constituents/extracts inhibited UGT activity by >50% in at least one enzyme source, prompting IC(50) determination. The IC(50) values of 13 constituents/extracts (≤10 μM with at least one enzyme source) were well below intestinal tissue concentrations or concentrations in relevant juices, suggesting that these diet-derived substances can inhibit intestinal UGTs at clinically achievable concentrations. Evaluation of the effect of inhibitor depletion on IC(50) determination demonstrated substantial impact (up to 2.8-fold shift) using silybin A and silybin B, two key flavonolignans from milk thistle (Silybum marianum) as exemplar inhibitors, highlighting an important consideration for interpretation of UGT inhibition in vitro. Results from this work will help refine a working systematic framework to identify dietary substance-drug interactions that warrant advanced modeling and simulation to inform clinical assessment.

Fig. 1.

Initial testing of milk thistle flavonolignans and other diet-derived constituents as inhibitors of 4-MU glucuronidation in HIMs (A and B) and HLMs (C and D) at 10 μM (green) and 100 μM (black) compared with vehicle control (0.1% methanol) (white). Hatched black bars denote the prototypic UGT inhibitor nicardipine (400 μM). Dashed lines denote 50% inhibition. Control activity was 4.4 ± 0.5 and 12 ± 0.5 nmol/min/mg microsomal protein for HIMs and HLMs, respectively. Bars and error bars denote means and S.D.’s, respectively, of triplicate incubations. *P < 0.05, 10 versus 100 μM (paired Student’s t test using untransformed data).

Fig. 2.

Initial testing of milk thistle flavonolignans/extracts and other diet-derived constituents as inhibitors of 4-MU glucuronidation in HEK293 cell lysates overexpressing UGT1A1 (A and B), UGT1A8 (C and D), or UGT1A10 (E and F) at 10 μM (green) and 100 μM (black) compared with vehicle control (0.1% methanol) (white). Hatched black bars denote the prototypic UGT inhibitor nicardipine (400 μM). Dashed lines denote 50% inhibition. Control activity was 13 ± 1.1, 20 ± 5.6, and 56 ± 7.0 nmol/min per mg microsomal protein for UGT1A1, UGT1A8, and UGT1A10, respectively. Bars and error bars denote means and S.D.’s, respectively, of triplicate incubations. *P < 0.05, 10 versus 100 μM (paired Student’s t test using untransformed data).

Fig. 3.

Representative UHPLC-MS/MS chromatograms for silybin A (SA; 481.1 → 125.1 m/z), silybin A monoglucuronides (SA-glucs; 657.1 → 481.1 m/z), silybin B (SB; 481.1 → 125.1 m/z), silybin B monoglucuronides (SB-glucs; 657.1 → 481.1 m/z), and internal standard (IS; 579.0 → 271.0 m/z). Retention times were 4.6 minutes (SA), 2.1 minutes (SA monoglucuronide 1), 2.9 minutes (SA monoglucuronide 2), 4.8 minutes (SB), 2.2 minutes (SB monoglucuronide 1), 3.3 minutes (SB monoglucuronide 2), and 2.6 minutes (IS, naringin). A background peak (3.1 minutes) was consistently present in the glucuronide traces that did not demonstrate time or concentration dependence. CPS, counts per second.

Fig. 4.

Michaelis-Menten plots for glucuronidation of silybin A (A) and silybin B (B) by HIMs (green) and HLMs (gray) recovered using the multiple depletion curves method. Symbols and error bars denote means and S.D.’s, respectively, of observed values. Curves denote model-generated values.

Fig. 5.

Michaelis-Menten plots for glucuronidation of silybin A by HIMs (A) and HLMs (B) and of silybin B by HIMs (C) and HLMs (D) recovered using glucuronide metabolite formation [peak area ratios (PAR) over time]. Monoglucuronide 1 (left column) and monoglucuronide 2 (right column) designations were based on UHPLC retention time. Symbols and error bars denote means and S.D.’s, respectively, of observed values. Curves denote model-generated values.

Fig. 6.

Impact of inhibitor depletion on the recovery of apparent IC₅₀ of silybin A (A) or silybin B (B) by pooled HIMs and of silybin B by pooled HLMs (C). Curves denote nonlinear least-squares regression of observed 4-MU depletion data versus nominal inhibitor concentration (black) or predicted inhibitor concentration at 10 minutes (dashed) or 20 minutes (dotted) using Phoenix WinNonlin (v. 6.3).