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Biochemistry/Biophysics Group, Amgen Pharmacokinetics and Drug Metabolism, Seattle, Washington
(Received September 1, 2006; accepted November 3, 2006)
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Abstract |
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 Top Abstract Materials and MethodsResults and DiscussionReferences |
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The level of use combined with the increasing complexities of herbal remedies has led to a heightened awareness on the part of physicians and researchers with respect to the potential underlying herb-drug interactions (Fugh-Berman and Ernst, 2001
; Ioannides, 2002; Zhou et al., 2003). In the United States, drug interactions account for more than 100,000 deaths per year (Lazarou et al., 1998; Bachmann et al., 2003). Many drug interactions occur on both the pharmacokinetic and pharmacodynamic level, with alteration of cytochrome P450 (P450) activity often leading to the observed pharmacokinetic changes. Herbal extracts from garlic, St. John's wart, ginko, danshen and Zingiber aromaticum, to name only a few, have been shown to be modulators of P450 activity (Lo et al., 1992; Delgoda and Westlake, 2004; Subehan et al., 2005). P450-catalyzed metabolism has been shown to be inducible via the pregnane X receptor ligand hyperforin found in St. John's wart preparations (Obach, 2000; Komoroski et al., 2004), competitively inhibited by the diallyl sulfide component in garlic (Zhou et al., 2003), and susceptible to mechanism-based inactivation by the isoflavan glabridin, a derivative of the licorice root (Kent et al., 2002). In addition to P450-catalyzed metabolic pathways, additional non-P450-mediated catalysis routes such as xanthine oxidase have been shown to be prone to herb-drug interactions (Saruwatari et al., 2003). In light of the widespread use of and incomplete knowledge regarding the metabolic disposition of herbal remedies, more research, particularly around potential drug interactions, is urgently needed.
The primary goal of this study was to determine the extent of drug interactions with an herbal remedy composed of multiple herbs. To this end, a commonly marketed dietary supplement indicated for treatment of the common cold and containing a complex mix of eight herbal extracts and nine vitamins and minerals was examined for drug interactions with well documented P450 probes in human liver microsomes. Based upon initial observations, select components were isolated from the complex mixture by high-performance liquid chromatography and screened for individual P450 inhibitory potential.
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Materials and Methods |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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In vitro assays were performed in human liver microsomes using well defined probe substrates and incubation conditions (Walsky and Obach, 2004). Inhibition of P450 activity by the herbal tablet was assessed at an estimated gut concentration of 9.61 mg/ml (final incubation concentration). Assays aimed at determining the inhibitory potency of the individual components of the herbal mixture were conducted with synthetic standards at 0, 10, and 50 µM, with full IC50 curves generated as warranted.
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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Inhibition of cytochrome P450 activity by the herbal mixture was evaluated using human liver microsomes and well documented assay conditions (Walsky and Obach, 2004). At an estimated gut concentration of approximately 9.61 mg/ml (single tablet of herbal mixture = 4.8 g; intestinal volume 
500 ml), the mixture significantly inhibited the catalytic activities of CYP2B6 (bupropion hydroxylation) and CYP2C9 (tolbutamide 4-hydroxylation) to less than 10% of their control activities (Fig. 1). The activities of CYP2D6 (dextromethorphan demethylation) and CYP2E1 (chlorzoxazone hydroxylation) were inhibited to 24% and 29% of their control activities, respectively. CYP3A4 appeared to undergo moderate inhibition, whereas both 1-hydroxymidazolam and 6ß-hydroxytestosterone activities were reduced to approximately 60% of their controls. CYP2A6 (coumarin hydroxylation) activity appeared slightly inhibited (71% of control activity remaining), whereas CYP1A2 activity (phenacetin dealkylation) did not exhibit any signs of inhibition in the presence of the herbal mixture. It should be noted, however, that actual hepatic concentrations may be somewhat lower than the calculated gut concentration, if phenomena such as solubility, gut metabolism or efflux transporters play a role in the overall bioavailability of the herbal mixture.
The ability of the individual components of the herbal mixture to inhibit the activities of the more sensitive P450 isoforms CYP2B6, CYP2C9, and CYP2D6 was examined based on the overall inhibition of these three P450 isoforms by the herbal mixture. Using neat synthetic standards, the apparent rate of bupropion hydroxylation catalyzed by CYP2B6 was inhibited to various extents by all of the individual components tested except for chlorogenic acid (Fig. 2). Luteolin, indigo, and menthone, active components of lonicera, isatis root, and schizonepeta, respectively, exhibited the most potent inhibition of CYP2B6 activity. Only luteolin appeared to inhibit CYP2C9 activity, with no formation of 4-hydroxytolbutamide detected in the presence of 50 µM luteolin (IC50 < 1 µM; data not shown). CYP2D6-catalyzed dextromethorphan demethylation was reduced to approximately 40% of control activity by both luteolin and ß-caryophyllene.
The assessment of P450 inhibition by potential drug candidates in vitro is an essential activity for drug discovery departments across the pharmaceutical industry. Alternatively, not as much focus has been placed on the relevant information regarding the drug interaction potential of herbal supplements, a situation that may present itself when an herbal remedy is coadministered with a drug that is primarily cleared by a single metabolic route. The results in this report begin to describe the potential for complex drug interactions to occur when herbal remedies that may contain multiple P450 inhibitors, which may be used concomitantly, are used in conjunction with clinically prescribed drugs.
Although most of the active constituents tested as inhibitors of P450 activity in vitro would not be classified as potent inhibitors (observed IC50 values > 10 µM; data not shown), several drugs, such as the macrolide antibiotic erythromycin, have been shown to exhibit clinically relevant drug interactions even with relatively high IC50 values (Echizen et al., 1993). In addition, the possibility of synergistic or antagonistic effects can become a factor when more than one inhibitory agent is present in a system (Greco et al., 1995). Of further interest are compounds such as the flavanoid luteolin, previously shown to exhibit mixed-type inhibition of CYP1A2 (Kim et al., 2005), which in this work appears to inhibit the activity of multiple P450 isoforms with varying potencies. However, as most of the herbal products on the market today lack any mechanistic insight surrounding the metabolic fate and inhibitory potencies of their active components as well as any relevant clinical data, the potential for in vivo drug interactions with these products is widely unknown.
In conclusion, the data reported above suggest the potential for alternative medicines that contain multiple herbal constituents to produce complex drug interaction scenarios. Further evaluation of the inhibitory potency of various combinations of active components as well as the inhibition of multiple P450-catalyzed pathways by a single active ingredient is ongoing. Until further information surrounding the inhibition of P450 activity by herbal mixtures in vitro is obtained, and such data can be validated in a clinical setting, it is the opinion of the authors that such herbal remedies should be taken with caution, especially when the remedy is coadministered with other clinically prescribed drugs.
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Acknowledgments |
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Footnotes |
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ABBREVIATION: P450, cytochrome P450.
Address correspondence to: Robert S. Foti, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119. E-mail: rfoti{at}amgen.com
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) relative to the control activity (gray columns) of a probe substrate selective for the given P450 isoform.
, CYP2C9; light gray columns, CYP2D6) in human liver microsomes by the individual active ingredients (final incubation concentration = 50 µM) isolated from the herbal mixture.