The present findings suggest that phytochemical components in the milk thistle and black cohosh formulations investigated in this study did not significantly affect the pharmacokinetics of MDZ in humans, and therefore are not likely to pose a significant interaction risk with other CYP3A substrates. This interpretation is bolstered by the significant changes in MDZ pharmacokinetics observed following the administration of clarithromycin, a known CYP3A4 inhibitor, and rifampin, a recognized inducer of CYP3A4 expression. In addition, our results do not support previous in vitro findings that silymarin (i.e., silibinin A, silibinin B silidianin, silichristin, and taxifolin) inhibits CYP3A4 activity, at least not in the context of the recommended supplementation regimen used in the study. This discrepancy may stem from the fact that milk thistle flavanolignans are practically insoluble in water and that in vitro studies demonstrating an inhibitory effect of silymarin on CYP3A4 activity have utilized solubilizing agents (e.g., dimethylsulfoxide27,29,30
) to facilitate hepatocyte or microsomal membrane permeability. Depending on the in vitro model utilized, IC50
values for individual flavanolignans or silymarin extract have ranged from 25–250 μM,27–30
with some evidence that silibinin can act as a mechanism-based inhibitor of CYP3A.26
From the results of the present study, it would appear that local silymarin concentrations at intestinal enterocyte membrane interfaces were lower than the 25–250 μM necessary for in vitro inhibition of CYP3A,27–30
and the degree of mechanism-based inhibition, if any, was not comparable to that observed with clarithromycin.
The milk thistle product used in the present study was formulated with soybean oil, glycerin, and lecithin in a soft gelatin capsule, and upon disintegration the contents appeared to remain undissolved. Since flavanolignan serum concentrations were not measured, any indication as to their in vivo solubility and/or bioavailability status remains unknown. Nevertheless, bioavailability and dissolution characteristics for silymarin-containing products have been shown to vary widely. An evaluation of nine separate milk thistle products found that the amount of silibinin released over one hour into an aqueous buffered solution (pH 7.5, 37°C) ranged from 0–85%;45
while a comparative bioavailability study of three silibinin-containing dosage forms found that values for AUC and Cmax
varied among products by factors of 3 and 6, respectively.46
Moreover, several studies have demonstrated that silymarin-containing products exhibit especially poor bioavailability and drug-release properties when not formulated with solubility-enhancing agents like phosphatidylcholine and polyethylene glycol.45,47–49
The bioavailability and drug-release characteristics of silymarin is significantly enhanced when silibinin is complexed with phosphotidylcholine or formulated as lipid-containing microspheres.50–53
Unlike many European products, the majority of milk thistle supplements sold in the United States do not appear to incorporate these technologies.52
With the multitude of supplement brands available on the market, discrepancies in product content, formulation, dissolution, and bioavailability can be the bane of any clinical safety, efficacy, or herb-drug interaction study—a problem not limited to milk thistle. Formulation differences could explain the disparity between a recent study performed in India22
and several conducted in the United States12,19–21
regarding the effects of milk thistle on the disposition of CYP3A substrates. Rajnarayana et al. found that 9 days of silymarin administration (Silybon™) increased the clearance of metronidazole, a substrate of CYP2C9, CYP3A, and P-gp, by almost 30%.22
The authors concluded that induction of CYP and/or P-gp might have accounted for the observed effects. Conversely, milk thistle interaction studies conducted in the United States and Canada found no statistically significant changes in the pharmacokinetics of indinavir, a CYP3A/P-gp substrate,19–21
although slight reductions (8–18%) in indinavir AUC were noted in each study. Collectively, the above studies hint at a possible mild inductive effect of silymarin on CYP3A and/or P-gp. In the only in vitro examination of milk thistle components on CYP3A induction, Raucy, using a reporter gene assay for the human pregnane X receptor and promoter regions of CYP3A transfected in HepG2 cells, found no evidence that silymarin, when solubilized with dimethylsulfoxide (DMSO), could induce the enzyme.54
Furthermore, in a previous study utilizing MDZ, we found that 28 days of milk thistle supplementation had no significant effect on 1-hour HMDZ/MDZ ratios (a finding confirmed by the current results) implying that conventional silymarin formulations are devoid of any clinically relevant CYP3A modulatory effects.12
Taken together, our results and those reported previously suggest that, when compared to potent inducers and inhibitors of CYP3A, milk thistle flavanolignans pose no clinically significant risks for pharmacokinetic herb-drug interactions involving CYP3A substrates.
Recent in vitro findings demonstrate that, in the presence of DMSO, black cohosh extracts or individual triterpene glycosides can inhibit CYP3A.32
However, the results reported here, and earlier,12
indicate that, when compared to clarithromycin and rifampin, recommended doses of black cohosh triterpene glycosides do not affect MDZ disposition and are therefore not effective modulators of CYP3A in vivo. Whether this lack of effect is a function of dose, solubility, bioavailability, or a combination of factors remains to be seen. Like many botanical supplements, the pharmacokinetic profile of black cohosh’s constituent phytochemicals has not been adequately investigated. Only one group has described an attempt at measuring mercapturate conjugates of black cohosh constituents (fukinolic acid, fukiic acid, caffeic acid, and cimiracemate B) in the urine of women after consuming 256 mg of a standardized black cohosh extract, and none of the target conjugates were detected.55
This finding alone brings into question the cellular permeability and bioavailability of these specific phytochemicals. Accordingly, the available in vivo evidence would seem to render black cohosh as an unlikely source of clinically important herb-drug interactions.
Poor systemic bioavailability of phytochemicals, however, may not always be associated with a lack of CYP3A modulation. This is best illustrated with grapefruit juice. Grapefruit juice, a well recognized mechanism-based inhibitor of intestinal CYP3A4 produces significant reductions in the oral clearance of many CYP3A4 substrates, yet the two phytochemicals primarily responsible for this effect, bergamottin and 6′,7′-dihydroxybergamottin, do not attain measurable concentrations in the plasma.56
From the results of the present study, however, it would appear that concentrations of specific milk thistle and black cohosh phytochemicals were insufficient to elicit any clinically noticeable effects on CYP3A.
Of particular interest was the close agreement between MDZ pharmacokinetic parameters described here and those reported earlier by Gorski et al.39,40
Like Gorski, we too noted a sex-related difference in oral MDZ clearance with women exhibiting higher values than men.40
Unlike Gorski, however, we failed to observe a sex-related effect in the response of CYP3A to induction by rifampin39
or inhibition by clarithromycin.40
Our findings also support the utility of 1-hour HMDZ/MDZ ratios as a practical method for identifying herb-drug interactions that involve CYP3A induction or inhibition. Previously, this approach had been used to document CYP3A induction following St. John’s wort supplementation,13,14
and its inhibition by goldenseal.12
The method also demonstrated that an absence of change in mean phenotypic ratios following botanical supplementation could be interpreted as a lack of effect on CYP activity. Such was the case with Citrus aurantium, Ginkgo biloba
, Panax ginseng,
and saw palmetto extracts13,14,33
—the latter three examples being confirmed by other investigators using more conventional area-under-the-curve assessments.57–59
The list can now be extended to include milk thistle and black cohosh. Thus, a range of herb-mediated effects on CYP activity (e.g. induction, inhibition, or no effect) can be differentiated with single time-point phenotypic ratios. It must be emphasized, however, that single-time point phenotypic ratios simply provide estimates of probe drug clearance. The utility of these approximations have recently come under question, with some investigators finding the correlations unacceptable.38
We, however, found that the change, or lack thereof, in HMDZ/MDZ ratios provided a reasonable correlation with oral MDZ clearance (). Our aim is not to propose that traditional means of determining MDZ clearance can be supplanted by metabolic phenotypic ratios; however, we do believe they provide a less labor-intensive, more subject-friendly means for evaluating multiple CYP enzymes and multiple botanical supplements in vivo while using a limited blood-sampling scheme.12–14,33
In short, single-time point metabolic phenotypic ratios may serve as a cost-effective in vivo screening method for assessing potential CYP-mediated herb-drug interactions. Once identified, candidate herbs may be evaluated further by using more traditional pharmacokinetic approaches.
In conclusion, when compared to the effects of rifampin and clarithromycin, the specific brand of milk thistle and black cohosh supplements utilized in this study produced no significant changes in the disposition of midazolam, a clinically recognized CYP3A substrate. Accordingly, these two products appear to pose no clinically significant risk for CYP3A-mediated herb-drug interactions. However, given the inter-product variability in phytochemical content, potency, and formulation among botanical supplements, these results may not extend to regimens utilizing higher dosages, longer supplementation periods, or brands with improved dissolution and/or bioavailability characteristics.