Two flavonolignans from milk thistle (Silybum marianum) inhibit CYP2C9-mediated warfarin metabolism at clinically achievable concentrations

Abstract

Milk thistle (Silybum marianum) is a popular herbal product used for hepatoprotection and chemoprevention. Two commercially available formulations are the crude extract, silymarin, and the semipurified product, silibinin. Silymarin consists of at least seven flavonolignans, of which the most prevalent are the diastereoisomers silybin A and silybin B; silibinin consists only of silybin A and silybin B. Based on a recent clinical study showing an interaction between a silymarin product and the CYP2C9 substrate losartan, the CYP2C9 inhibition properties of silybin A and silybin B and corresponding regioisomers, isosilybin A and isosilybin B, were evaluated using human liver microsomes (HLMs), recombinant CYP2C9 (rCYP2C9) enzymes, and the clinically relevant probe, (S)-warfarin. Silybin B was the most potent inhibitor in HLMs, followed by silybin A, isosilybin B, and isosilybin A (IC(50) of 8.2, 18, 74, and >100 microM, respectively). Next, silybin A and silybin B were selected for further characterization. As with HLMs, silybin B was more potent than silybin A toward rCYP2C9 1 (6.7 versus 12 microM), rCYP2C9 2 (9.3 versus 19 microM), and rCYP2C9 3 (2.4 versus 9.3 microM). Using a matrix of five substrate (1-15 microM) and six inhibitor (1-80 microM) concentrations and HLMs, both diastereoisomers inhibited (S)-warfarin 7-hydroxylation in a manner described best by a mixed-type inhibition model (K(i) values of 4.8 and 10 microM for silybin B and silybin A, respectively). These observations, combined with the high systemic silibinin concentrations (>5-75 microM) achieved in a phase I study involving prostate cancer patients, prompt clinical evaluation of a potential warfarin-milk thistle interaction.

Fig. 1.

Structures of the four selected flavonolignans from milk thistle.

Fig. 2.

Inhibitory effects of selected flavonolignans on (S)-warfarin 7-hydroxylation activity in human liver microsomes. Human liver microsomes (0.1 mg/ml) were incubated with (S)-warfarin (4 μM) and flavonolignan (1, 10, or 100 μM; hatched, solid, and checkered bars, respectively) for 30 min. Reactions were initiated by the addition of NADPH (1 mM). (S)-Warfarin 7-hydroxylation activity in the presence of vehicle control (0.75% methanol, v/v) was 5.8 ± 0.1 pmol/min/mg microsomal protein. Bars and error bars denote means and S.D., respectively, of triplicate incubations. *, p < 0.05 versus flavonolignan at 10 μM; #, p < 0.05 versus flavonolignan at 1 μM (two-way ANOVA, followed by Tukey's test).

Fig. 3.

Dixon plots showing the inhibition of (S)-warfarin 7-hydroxylation by silybin A (A) and silybin B (B) in HLMs. HLMs (0.1 mg/ml) were incubated with (S)-warfarin (1–15 μM) and flavonolignan (1–80 μM silybin A or 1–50 μM silybin B) for 30 min. Reactions were initiated by the addition of NADPH (1 mM). Symbols denote means of duplicate incubations. Solid lines denote regression lines through values generated from a simple mixed-type inhibition model using WinNonlin (version 5.0.1).

Fig. 4.

Inhibitory effects of silybin A and silybin B on (S)-warfarin 7-hydroxylation activity in recombinant CYP2C9 enzymes. Recombinant enzymes (12.5 pmol/ml) were incubated with (S)-warfarin (4 μM) and a range of concentrations (1–100 μM) of silybin A (■) or silybin B (▴) for 30 (CYP2C9*1 and CYP2C9*2) or 60 (CYP2C9*3) min. Reactions were initiated by the addition of NADPH (1 mM). (S)-Warfarin 7-hydroxylation activity in the presence of vehicle control (0.75% methanol, v/v) was, respectively, 15 ± 1.3, 8.0 ± 0.4, and 3.6 ± 0.3 pmol/min/nmol of recombinant enzyme. Symbols and error bars denote means and S.D., respectively, of triplicate incubations. Solid (silybin A) and dashed (silybin B) curves denote nonlinear least-squares regression of observed values using WinNonlin (version 5.0.1).

Fig. 5.

IC₅₀ shift plot for silybin A and silybin B. Human liver microsomes (0.1 mg/ml) were incubated first with silybin A (squares) or silybin B (triangles) (0.1–1000 μM) in the presence (closed symbols) or absence (open symbols) of NADPH (1 mM). The primary reaction mixture was diluted 10-fold to initiate the secondary reaction, which contained NADPH (1 mM) and (S)-warfarin (4 μM). (S)-Warfarin 7-hydroxylation activity in the presence of vehicle control (0.75% methanol, v/v) was 1.5 and 1.6 pmol/min/mg microsomal protein in the absence and presence, respectively, of NADPH. The inset depicts the effects of the known CYP2C9 mechanism-based inhibitor, tienilic acid (circles) (1–500 μM). Symbols denote means of duplicate incubations. Open symbols denote observed values when NADPH was absent from the primary reaction mixture; solid symbols denote observed values when NADPH was present in the primary reaction mixture. Curves denote nonlinear least-squares regression of observed values using WinNonlin (version 5.0.1).