Treatment of hot flashes is the most common indication for black cohosh use, and occurrence of hot flashes is a serious clinical issue with TAM therapy, which often leads to poor compliance (Fisher et al., 1998
). At least two clinical trials examined effects of black cohosh extracts on TAM induced hot flashes with one finding no effect (Jacobson et al., 2001
) and another reporting significant efficacy of black cohosh (Hernandez Munoz and Pluchino, 2003
). Since women are continuing to use black cohosh, it is of interest to understand whether black cohosh supplements have any effect on TAM efficacy.
In this study we found that a 75% ethanolic extract of black cohosh has the potential to interfere with the metabolic conversion of TAM into active metabolite 4-OH TAM and N
-desmethyl-4-OH TAM (endoxifen) via inhibition of formation of N
-desmethyl TAM. Because of the dominant role of CYP3A4 and CYP2D6 in the metabolism of TAM, this study primarily focused on the identification of inhibitors of these two isoforms. Specific triterpene glycosides were found to inhibit CYP3A4 mediated metabolism of TAM. This class of compounds has been the major focus of phytochemical investigations of black cohosh for several decades, and numerous congeners have been isolated and characterized. Two recent in vitro
studies described weak inhibition of CYP3A4 by triterpenes (Huang et al., 2010
; Tsukamoto et al., 2005
), and our data for the major triterpenes are in agreement with these studies. Interestingly, 12
, which was found moderately active in this study, was determined to be only a weak inhibitor by Tsukamoto et al.
(Tsukamoto et al., 2005
). One possible explanation of this discrepancy lies in different experimental conditions used in the two studies. For example, Tsukamoto and coworkers used substrate concentration well above Km
, which effectively prevented competitive inhibitors from binding to the enzyme. In addition, they used 0.5% DMSO which is known to significantly inhibit CYP3A4, thereby masking inhibition by the triterpenes. Similarly, the study by Huang et al.
only mentions that organic solvent content was less than 1%, but did not specify the exact amount.
Our data indicate that triterpene glycosides are moderately potent competitive inhibitors, but with an apparent lack of structure-activity relationship. It is important to note that abundant triterpenes, such as the standardization markers 1
as well as the cogeneric 4
, were inactive against CYP3A4 and CYP2D6. This suggests that chemical standardization based on these abundant triterpene markers does not establish a quantitative measure for the extract’s potential to inhibit CYP3A4 or CYP2D6. Because all of the active triterpenoids identified in this study were minor constituents, one or a few individual compounds are unlikely to fully account for all the observed activity of the crude extract, suggesting the presence of additional active compounds. For example, the most abundant active compound was 12
which was present at a level of 1.6 mg/g of crude extract (Fabricant, 2006
). In the reaction mixture, this would amount to a concentration of 22 nM, which is below the observed Ki
The data from this study are in contrast with two clinical studies that reported no effect of black cohosh on the activity of CYP3A4 in vivo
(Gurley et al., 2006
; Gurley et al., 2005
), although one of the studies noted a small trend toward CYP3A4 inhibition by black cohosh (Gurley et al., 2005
). One obvious explanation for this discrepancy is differences in the types of extracts used. Although the in vivo
studies reported the content of major (inactive) marker triterpenoids such as 1
, the content of active CYP3A4 inhibitors in those extracts was not known, nor was activity of the crude extract tested in an in vitro
assay to establish possible in vitro
correlation. A recent pharmacokinetic study on 1
(van Breemen et al., 2010
) found that black cohosh triterpenes are orally bioavailable; however, the pharmacokinetics of the CYP3A4 active triterpenes has not been investigated to date. Thus, whether the extract investigated in the present study possesses in vivo
activity needs to be determined in a carefully designed clinical study.
Several in vitro
and in vivo
studies have addressed the potential of black cohosh to inhibit CYP2D6. An in vivo
study by Gurley et al.
found a small, but statistically significant inhibition of CYP2D6 (Gurley et al., 2005
). In a follow-up study by the same group, black cohosh was found to have no effect on CYP2D6 in vivo
(Gurley et al., 2008
). Recent in vitro
studies have provided conflicting reports with one reporting high (Ho et al., 2011
), one moderate (Huang et al., 2010
), and one no activity against CYP2D6 (Sevior et al., 2010
). The identification of protopine and allocryptopine as potent CYP2D6 inhibitors is an important new discovery in this study that may provide a possible explanation for apparent discrepancies among these previous studies. Many alkaloids, particularly those that possess methylenedioxyphenyl moiety, are potent (often mechanism-based) inhibitors of CYP2D6, yet the presence of this class of compounds in black cohosh has largely been overlooked,. Consequently, small differences in their quantities whether resulting from extraction procedures or growing conditions, can have profound effects on the observed biological activities of black cohosh extracts. It is also important to note that protopine and allocryptopine cannot fully account for the observed CYP2D6 activity and that other unidentified active compounds are still present.
When it comes to potential clinical significance of inhibition of TAM metabolism by black cohosh there are a number of issues to consider. First, it is important to note that in vitro
inhibition observed in this study may or may not translate into measurable in vivo
activity. Second, it has been shown that serum concentrations of TAM metabolites do not always predict clinical outcome (Bratherton et al., 1984
; Langan-Fahey et al., 1990
). The concentrations of active TAM metabolites that trigger hot flashes and those necessary for activity may or may not be the same or even linked. Thus, even if black cohosh inhibits formation of TAM metabolites in vivo,
the final clinical outcome of such interaction can be determined only in a carefully designed clinical study. Unfortunately, the two studies that examined effects of black cohosh on TAM induced hot flashes (Hernandez Munoz and Pluchino, 2003
; Jacobson et al., 2001
) did not measure the concentrations of active metabolites, thus it is not clear what effects, if any, black cohosh had on TAM metabolism. Finally, due to the polymorphic nature of CYP2D6, co-administration of CYP2D6 inhibitors with TAM may be more relevant for extensive and intermediate metabolizers than for poor metabolizers (Sideras et al., 2010
). Reduction in formation of α-OH TAM via inhibition of CYP3A4 by black cohosh may be a potentially beneficial aspect of this interaction. This metabolite has been implicated as a causative factor in increasing the risk of endometrial cancer during TAM therapy. Thus, black cohosh supplements might protect women from this side effect of TAM therapy.