A Systematic Approach to Evaluate Herb-Drug Interaction Mechanisms: Investigation of Milk Thistle Extracts and Eight Isolated Constituents as CYP3A Inhibitors

Abstract

Despite increasing recognition of potential untoward interactions between herbal products and conventional medications, a standard system for prospective assessment of these interactions remains elusive. This information gap was addressed by evaluating the drug interaction liability of the model herbal product milk thistle (Silybum marianum) with the CYP3A probe substrate midazolam. The inhibitory effects of commercially available milk thistle extracts and isolated constituents on midazolam 19-hydroxylation were screened using human liver and intestinal microsomes. Relative to vehicle, the extract silymarin and constituents silybin A, isosilybin A, isosilybin B, and silychristin at 100 mM demonstrated >50% inhibition of CYP3A activity with at least one microsomal preparation, prompting IC 50 determination. The IC 50 s for isosilybin B and silychristin were ∼60 and 90 mM, respectively, whereas those for the remaining constituents were >100 mM. Extracts and constituents that contained the 1,4-dioxane moiety demonstrated a >1.5-fold shift in IC 50 when tested as potential mechanism-based inhibitors. The semipurified extract, silibinin, and the two associated constituents (silybin A and silybin B) demonstrated mechanism-based inhibition of recombinant CYP3A4 (K I , ∼100 mM; k inact , ∼0.20 min 21) but not microsomal CYP3A activity. The maximum predicted increases in midazolam area under the curve using the static mechanistic equation and recombinant CYP3A4 data were 1.75-fold, which may necessitate clinical assessment. Evaluation of the interaction liability of single herbal product constituents, in addition to commercially available extracts, will enable elucidation of mechanisms underlying potential clinically significant herb-drug interactions. Application of this framework to other herbal products would permit predictions of herb-drug interactions and assist in prioritizing clinical evaluation.

Mechanism-Based Inhibitory and Peroxisome Proliferator-Activated Receptor α–Dependent Modulating Effects of Silybin on Principal Hepatic Drug-Metabolizing Enzymes

“…A panel of previous studies performed in vitro demonstrated that silybin could directly inhibit the enzyme activities of some P450s and UGTs (Venkataramanan et al, 2000;Sridar et al, 2004;Brantley et al, 2013). However, most of the in vivo studies indicated that silybin has negligible effects on DMEs.…”

Identification of Diet-Derived Constituents as Potent Inhibitors of Intestinal Glucuronidation

“…Incubation mixtures (150 ml total volume) consisted of HIMs (0.2 mg/ml), HLMs (0.4 mg/ml), or UGT1A-overexpressing HEK293 cell lysates (0.05, 0.025, or 0.02 mg/ml for UGT1A1, UGT1A8, and UGT1A10, respectively); 4-MU [100 mM (HIMs and HLMs) or 75 mM (HEK293 lysates)]; dietary constituent (10 or 100 mM) or the prototypic UGT inhibitor nicardipine (400 mM) (Lapham et al, 2012); bovine serum albumin (0.05%); alamethicin (50 mg/mg protein; mixtures containing HIMs and cell lysates); saccharolactone (100 mM); and Tris-HCl buffer supplemented with magnesium chloride (5 mM). The selected dietary constituent concentrations were based on a reasonable approximation of the anticipated range of concentrations in the gut (Brantley et al, 2010(Brantley et al, , 2013 and were used to plan subsequent IC 50 determination experiments (see below). HIMs and cell lysates were activated by incubating with alamethicin on ice for 15 minutes.…”

“…Silibinin, a semipurified milk thistle extract composed of a 1:1 mixture of silybin A and silybin B , was selected previously as a model herbal product perpetrator of dietary substance-drug interactions (Brantley et al, 2013(Brantley et al, , 2014b. Microsomal intrinsic clearances of silybin A and silybin B were determined to assess the impact of inhibitor depletion on the recovery of apparent IC 50 using pooled HIMs and HLMs.…”

“…Interaction risk is compounded by the relative lack of regulatory oversight to guide dosing recommendations, safety assessment, and chronic use of dietary substances. As proposed, examination of isolated constituents would facilitate development of systematic approaches (Brantley et al, 2013). A systematic evaluation of isolated dietary substance constituents as inhibitors of intestinal UGTs, including gut-specific isoforms, has not been reported.…”

Inhibition of Human Aldehyde Oxidase Activity by Diet-Derived Constituents: Structural Influence, Enzyme-Ligand Interactions, and Clinical Relevance

“…These results provide a first step toward developing in silico models to predict the human AO inhibitory activity of a xenobiotic solely The final aim of this study was to identify dietary constituents as potential perpetrators of clinically relevant AO-mediated interactions with conventional medications. Static models, used routinely to predict the risk of drug-drug interactions, have been extended to dietary substance-drug interactions (Zhou et al, 2004;Brantley et al, 2013). For example, [I]/K i is a simple metric used to predict the magnitude of an interaction in vivo, where [I] is the concentration of inhibitor in the systemic circulation.…”