Effects of herbal supplements on the bioactivation of chemotherapeutic agents

Effects of herbal supplements on the bioactivation ofchemotherapeutic agentsGregory S. Gorman, Lori Coward, Adrienne Darby and Bethany Rasberry

McWhorter School of Pharmacy, Samford University, Birmingham, AL, USA

KeywordsCYP450; herbal supplements; inhibition;irinotecan; tamoxifen

CorrespondenceGregory S. Gorman, Associate Professor ofPharmacy, McWhorter School of Pharmacy,Samford University, 800 Lakeshore Drive,Birmingham AL 35229, USA.E-mail: [email protected]

Received October 4, 2012Accepted February 11, 2013

doi: 10.1111/jphp.12055

Abstract

Objectives The aim of this study was to investigate the impact of commerciallyavailable, over-the-counter herbal supplements (St John’s wort, black cohosh andginger root extract) on the metabolic activation of tamoxifen and irinotecan.Methods Co-incubation of each drug and supplement combination over arange of concentrations was conducted in human liver microsomes and thedecrease in the rate of active metabolite formation was monitored using high-performance liquid chromatography tandem mass spectrometry. Data was ana-lysed using non-linear regression analysis and Dixon plots to determine thedominant mechanism of inhibition and to estimate the Ki and IC50 values ofthe commercial supplements.Key findings The data suggest that black cohosh was the strongest inhibitortested in this study for both CYP450 and carboxyesterase mediated biotransfor-mation of tamoxifen and irinotecan, respectively, to their active metabolites. StJohn’s wort was a stronger inhibitor compared with ginger root extract fortamoxifen (CYP mediated pathway), while ginger root extract was a strongerinhibitor compared with St John’s wort for the carboxyesterase mediated pathway.Conclusions Commercially available supplements are widely used by patientsand their potential impact on the efficacy of the chemotherapy is often unknown.The clinical significance of these results needs to be evaluated in a comprehensiveclinical trial.

Introduction

The use of over-the-counter herbal supplements in theUnited States by adults aged 20 years and older, which fallunder the category of complementary and alternative medi-cines, has increased from 42% in 1988–1994 to 53% in2003–2006, and continues to climb.[1,2] As a subgroup of thispopulation, it has been reported that 51.6% of patients withcancer were also taking various supplements.[3] Additionally,over 72% of these cancer patients do not inform theirhealthcare provider about their supplement use.[4] Becausethe pharmacologically active components in the supple-ments are metabolised by the same enzyme systems as otherxenobiotic, the potential for drug–herbal supplement inter-actions in patients taking herbal supplements is significant.The risk of unwanted interactions is increased for chemo-therapeutic drugs because of the narrow therapeutic indexthat most of these drugs have. Therefore, a relatively small

change in systemic exposure of the drug as a result of anunwanted drug–herbal supplement interaction could resultin either subtherapeutic effects or undesired serious side-effects. This change in exposure is even more important forchemotherapeutic drugs that rely on metabolic biotransfor-mation to generate active metabolites to achieve efficacy.Two such chemotherapeutic agents that fall into this realmare tamoxifen and irinotecan.

Tamoxifen is one of the most prescribed cancer treat-ments and is also used as a chemopreventative for womenwho have significant risk factors for developing breastcancer.[5] As administered, tamoxifen is a prodrug havinglittle affinity for oestrogen receptors and therefore itmust be metabolized by CYP450 enzymes to generate itstwo chemoactive metabolites: 4-hydroxytamoxifen andN-desmethyl-4-hydroxytamoxifen (endoxifen).[6,7] These

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And PharmacologyJournal of Pharmacy

Research Paper

© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1014–10251014

mailto:[email protected]

active metabolites act as antagonists and compete with oes-trogen in the body for binding to the oestrogen receptor,thus inhibiting transcription of oestrogen-responsivegenes.[8] Irinotecan is a semisynthetic prodrug analogue ofthe natural alkaloid camptothecin and is often used in com-bination with other chemotherapeutic drugs to treat colonand colorectal cancers. Bioactivation of irinotecan to itschemoactive metabolite, SN-38 (a topoisomerase I inhibi-tor), occurs via a microsomal carboxyesterase mediatedpathway. The inhibition of topoisomerase I by SN-38 even-tually leads to inhibition of both DNA replication and tran-scription. Inhibition of either the CYP450 pathway (forpatients treated with tamoxifen) or the carboxyesterasemetabolism pathway (for patients treated with irinotecan)by herbal supplements could potentially result in reducedefficacy due to the reduction in the amount of the chem-oactive metabolites formed.

St John’s wort (SJW), black cohosh (BC) and ginger rootextract (GRE) are commonly used herbal supplements thatmay be taken by patients while on a tamoxifen or irinotecanregimen to alleviate the side-effects of chemotherapy or asherbal therapies for other indications. SJW is from the plantspecies Hypericum perforatum and is available in variousforms including capsules, tablets, tinctures, teas and oil-based skin lotions. Its usual dose of 300 mg (standardized to0.3% hypericin extract), 3 times a day, is used for milddepression and mood disorders. BC comes from the plantfamily Ranunculaceae (Actaea racemosa) and is most com-monly available in capsule and liquid forms. A dose of100 mg (standardized to 1 mg triterpene glycosides) can betaken up to 3 times a day to treat hot flashes, night sweats,migraines and mood disturbances. GRE is obtained fromthe rhizomes of the plant Zingiber officinale and is commer-cially available as capsules, teas, chews or essential oils.Doses of 1000 mg (standardized to 5% ginger phenols), 3times a day, are used to improve digestion and to treatstomach upset and nausea. Each of these supplements hascomplex mixtures of compounds from various chemicalclasses that under in-vivo conditions may have synergisticeffects on inhibition of metabolic pathways.

The aim of this study was to investigate the potential formetabolic inhibition of chemotherapeutic agents by severalherbal supplements using an in-vitro system. While therehas been a significant amount of in-vitro research intodrug–herbal inhibition, the majority of it has been withindividual compounds from various supplements. In thiswork, we focused on the cumulative effect of all of the com-ponents in each of the commercially available preparations.To this end, our data may be more predictive of actual clini-cal exposure in which the combined effects of all the com-pound classes are represented in the reported qualitativeand quantitative values. The impact on CYP450 andcarboxyesterase activation pathways was evaluated by

co-incubation of the selected chemotherapeutic drugs withvarious supplements, allowing for qualitative assessments tobe made regarding the properties of the supplements interms of Ki and IC50.

Methods and Materials

Reagents

Tamoxifen and 4-hydroxytamoxifen were purchased fromSigma Chemical Co. (St Louis, MO, USA). Endoxifenand a-hydroxytamoxifen were purchased from TorontoResearch Chemicals (Ontario, Canada). Irinotecan was pur-chased from LC Labs (Woburn, MA, USA). SJW (BotanicChoice, 1000 mg/ml alcohol-free liquid extract), BC root(Botanic Choice, 100 mg/ml alcohol-free liquid extractstandardized for 1 mg triterpene glycosides, as 27-dexoyacetein) and GRE (Botanic Choice, 1000 mg/mlalcohol-free fluid extract) were purchased from Walgreens(Deerfield, IL, USA) as over-the-counter herbal supple-ments. Ammonium acetate (enzyme grade), acetonitrile,methanol (HPLC grade) and formic acid were obtainedfrom Fisher Scientific (Atlanta, GA, USA).

Treatments

The in-vitro reaction systems were comprised of humanliver microsomes (Xenotech LLC, Lenexa, KS, USA) at reac-tion mixture concentrations of 0.25 mg/ml for tamoxifenand 1 mg/ml for irinotecan. Microsomes were incubatedwith uridine 5′-diphosphoglucuronic acid (UDPGA) cofac-tor solution A (BD Biosciences, San Jose, CA, USA) at areaction concentration of 2 mM in deionised water alongwith solution B (BD Biosciences) containing 50 mM Tris-HCl, 8 mM MgCl2 and 25 mg alamethicin in deionisedwater. The final reaction concentrations of tamoxifen andirinotecan were dependent on the experiment. The reac-tions were initiated with the addition of Rapid StartNADPH regenerating solution at a reaction concentrationof 1 mM NADP(H), 5 mM glucose-6-phosphate and 1unit/ml of glucose-6-phosphate dehydrogenase (XenotechLLC). The in-vitro reaction mixtures were incubated at37°C in a shaking water bath for the appropriate timeperiod for each experiment. The reactions were quenchedwith ice-cold acetonitrile containing the internal standard(terfenadine, 10 ng/ml) and centrifuged at approximately21 000g for 5 min. The supernatant was transferred toautosampler vials for HPLC-MS/MS analysis.

Michaelis-Menten kinetics

Kinetic experiments were carried out using 10 concentra-tions of tamoxifen and 13 concentrations of irinotecan toyield final concentrations ranging from 0.5 mm to 200 mmfor tamoxifen and 0.5 mm to 238 mm for irinotecan. The

Gregory S. Gorman et al. Inhibition of prodrugs by supplements

© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1014–1025 1015

in-vitro reaction mixtures were incubated at 37°C in ashaking water bath for 15 min, then processed and analysedas described above.

Progress curves

Individual progress curves were obtained for tamoxifenusing the inhibitors SJW and GRE. Reaction mixtures wereincubated using six concentrations of SJW or GRE rangingfrom 0 to 10 mg/ml for each inhibitor. The reaction mix-tures were sampled at 12 time-points over the 50-min incu-bation period, then processed and analysed as describedabove.

Inhibition studies

Before conducting the inhibition studies, experimental con-ditions such as substrate concentrations, incubation timeand protein concentration were optimized to ensure thatinitial reaction rates for the metabolites of interest could bemeasured. Substrate concentrations were based on physi-ologically relevant doses as well as concentrations that werenear or below the measured Michaelis-Menten rate constant(Km) values from the Michaelis-Menten curves to ensuremeasurements were made over the linear range of thekinetic profile. Inhibitor concentrations used in the studywere selected from range-finding studies bracketing the fullrange of inhibition and did not exceed suggested physi-ological dosages.

Tamoxifen

Inhibition reactions with SJW were carried out in triplicateat three concentrations of tamoxifen (2, 5 and 10 mm) andsix concentrations of SJW ranging from 0 to 6 mg/ml in thefinal reaction mixture. The reaction mixtures were incu-bated for 20 min at 37°C, and then processed and analysedas described above. Reactions with BC were carried outusing three concentrations of tamoxifen (10, 20 and 30 mm)and eight concentrations of BC ranging from 0 to 4 mg/ml.Reactions with GRE were carried out using 20 mmtamoxifen and seven concentrations of GRE ranging from 2to 20 mg/ml. The reaction mixtures with BC and GRE wereconducted in triplicate and incubated for 30 min at 37°C,and then processed and analysed as described above.

Irinotecan

Inhibition reactions with SJW were carried out in triplicateusing three concentrations of irinotecan (10, 25 and 35 mm)and five concentrations of SJW ranging from 0 to 30 mg/mlin the final reaction mixture. Triplicate reactions with BCwere carried out using four concentrations of irinotecan (5,10, 25 and 35 mm) and six concentrations of BC rangingfrom 0 to 4 mg/ml in the final reaction mixture. Reactions

with GRE were carried out in triplicate using 5 mm irinote-can and five concentrations of GRE ranging from 0 to40 mg/ml in the final reaction mixture. The reaction mix-tures were incubated at 37°C in a shaking water bath for20 min, and then processed and analysed as describedabove.

Analytical methods

Chromatographic separation of each compound and itsmetabolites from the inhibition and kinetic experimentswas achieved using a Shimadzu HPLC system consisting oftwo Shimadzu LC20-AD pumps, SIL20-AC HT autosam-pler and a DGU-20A3-in-line degasser (Shimadzu ScientificInstruments, Columbia, MD, USA) with a Luna C18column (100 mm ¥ 2 mm, 5 mm particle size) (Phenom-enex, Torrance, CA, USA) and C18 SecurityGuard cartridgeboth maintained at ambient temperature. The mobile phaseconsisted of 5 mM ammonium acetate and acetonitrile,each fortified with 0.1% formic acid. For tamoxifen and itsmetabolites, the acetonitrile concentration began at 10% for1 min, then increased to 80% at a linear rate over 7 min,and was held for 1 min before returning to 10% andre-equilibrated for 3 min at a constant flow rate of 400 ml/min. For irinotecan, the acetonitrile concentration was heldat 10% for 1 min, then increased to 90% at a linear rate over6 min, held at 90% for 0.5 min, and then returned to 10%and re-equilibrated for 2.5 min at a constant flow rate of400 ml/min. Mass detection was accomplished with an ABSciex 4000 QTRAP triple quadrupole ion trap mass spec-trometer (AB Sciex, Foster City, CA, USA) equipped with anelectrospray ionization source operated at a potential of5 kV at 450°C operating in the multiple reaction monitor-ing (MRM) positive ion mode. Data was collected usingAnalyst 1.5.1 (Applied Biosystems, Foster City, CA, USA).The following mass transitions of the compounds andmetabolites were monitored: tamoxifen: m/z 372-72(parent), m/z 388-72 (4-hydroxytamoxifen, a-hydroxytamoxifen), m/z 374-58 endoxifen; irinotecan: m/z587-124 (parent), 393-349 (SN-38).

Characterization of the herbal supplements was con-ducted using a combination of liquid chromatographywith UV/visable detection (LC-UV/Vis) and liquidchromatography-tandem mass spectrometry (LC-MS/MS)for compositional analysis. Chromatographic separation ofSJW extract was achieved using an Chromenta KB-C18column (250 mm ¥ 4.6 mm, 5 mm particle size) (Colum-nex, New York, NY, USA) maintained at ambient tempera-ture. The mobile phase consisted of deionised water andacetonitrile. The acetonitrile concentration began at 5% for1 min, then increased to 90% at a linear rate over 39 min,and was held for 4 min before returning to 5% andre-equilibrated for 5 min at a constant flow rate 1 ml/min.

Gregory S. Gorman et al.Inhibition of prodrugs by supplements


The photodiode array detector was scanned over a wave-length range of 190–400 nm and the compounds weredetected at 254 nm. Peak identification was based on rela-tive retention time and mass spectral analysis of thedetected peaks. Chromatographic separation of the GREwas achieved using an Aquasil C18 column (100 mm ¥2 mm, 5 mm particle size) (Thermo Fisher, Waltham, MA,USA) and C18 SecurityGuard cartridge both maintained atambient temperature. The mobile phase consisted of deion-ised water and acetonitrile. The acetonitrile concentrationbegan at 5% for 1 min, then increased to 90% at a linearrate over 39 min, and was held for 4 min before returning to5% and re-equilibrated for 5 min at a constant flow rate of400 ml/min. Mass detection was accomplished with the elec-trospray ionization source operating in the negative ionmode at a potential of -4.6 kV at 450°C. Neutral loss scansof 136 or 194 were obtained over a range of m/z 250–350for the detection of shogaols and gingerols, respectively.Chromatographic separation of the BC extract was achievedusing an Aquasil C18 column (100 mm ¥ 2 mm, 5 mm par-ticle size) (Thermo Fisher) and C18 SecurityGuard car-tridge both maintained at ambient temperature. The mobilephase consisted of deionised water and acetonitrile. Theacetonitrile concentration began at 5% for 1 min, thenincreased to 80% at a linear rate over 29 min, and was heldfor 5 min before returning to 5% and re-equilibrated for5 min at a constant flow rate of 400 ml/min. Mass detectionwas accomplished with the electrospray ionization sourceoperating in the negative ion mode at a potential of -4.5 kVat 450°C employing MRM, precursor and neutral loss scans.

Data analysis

Estimates of Vmax and Km values were made using standardMichaelis-Menten enzyme kinetic plots for monophasicreactions. Observed biphasic kinetics were fitted to a two-enzyme model (Equation 1) and solved using non-linearregression analysis to generate values for Km and Vmax foreach isoform:

VV S

K S

V S

K Smax

m

max

m

= ×+

+ ×+

1

1

2

2

(1)

where S is the substrate concentration, and subscripts 1 and2 represent Vmax and Km values for high affinity and lowaffinity activity, respectively.

In the case where non-competitive inhibition wasobserved to be the dominant contributor, Dixon plots wereused to determine the estimated IC50 value of the supple-ment since the Ki value cannot be obtained from this plotfor a non-competitive or uncompetitive inhibitor. Wherecompetitive inhibition was determined to dominate, theKi value was determined from the Dixon plot and theIC50 was calculated for each substrate concentration using

the Cheng-Pursoff equation for competitive inhibition(Equation 2).

IC K Ki m50 1= +( )[ ]S (2)

where S is the substrate concentration and Km is theMichaelis-Menten rate constant.

In the case of significant contributions from more thanone type of inhibition where the mechanism was indetermi-nate from the Dixon plot, the IC50 values were estimated byplotting inhibitor concentration versus percent change inmetabolite formation rate and fitting the data using a non-linear regression to an equation using reaction velocitiesexpressed as a ratio (Rv) (Equation 3):

Rv C C ICmaxA A A= − × +( )( )100 1 ( ) ( )E (3)

where Emax is the estimated maximum fractional inhibition,A is an exponent, and IC is the inhibitor concentration pro-ducing 50% of the maximum decrement. This was used todetermine the inhibitor concentration producing a 50%decrease in the rate of metabolite formation in the absenceof the inhibitor.[9] In Equation 3, the estimated inhibitorconcentration producing a reaction velocity of 50% of thecontrol (i.e. IC50) was calculated according to Equation 4.

IC IC A50 2 1 1= −( )maxE (4)

The Kruskal-Wallis test (one-way non-parametric analysis)was used to perform statistical analysis of the data while aDunn’s post-test was used to compare pairs of groupmeans. Unless otherwise noted, the level of significance forall statistical evaluations was 5% or P < 0.05. All curvefitting and statistical analysis were carried out using Graph-Pad Prism 4.03 (GraphPad Software, Inc, La Jolla, CA, USA)

Results

Kinetics of metabolite formation

Determination of the Vmax and Km values for the CYP450mediated metabolism of tamoxifen to its active metabolites4-hydroxytamoxifen and endoxifen, and the carboxyesterasemediated conversion of irinotecan to SN-38 were con-ducted using a single lot of pooled human liver microsomes(Figure 1). Biotransformation of tamoxifen to its activemetabolites was observed to follow traditional Michaelis-Menten kinetics, generating a Km of 41.2 mm and a Vmax of16.7 pmol/min per mg protein for 4-hydroxytamoxifen and9.7 mm and 0.59 pmol/min per mg protein for endoxifen,over a range of substrate concentrations from 0.5 to200 mm. The conversion of irinotecan to SN-38 via a car-boxyesterase mediated pathway did not reach enzyme satu-ration over the concentration ranges tested (0.5–238 mm).



Evaluation of irinotecan data with an Eadie-Hofstee plot(Figure 2) clearly shows a biphasic response, suggesting atwo-enzyme model. Non-linear regression analysis using atwo-enzyme equation (Equation 1) generated values for Km

and Vmax for each isoform. The high affinity isoform had anaverage Km value of 1.3 mm and a Vmax of 2.2 pmol/min permg protein, while the low affinity isoform had a Km value of128.8 mm and a Vmax of 9.2 pmol/min per mg protein. Thepossibility of mechanism-based inactivation of CYP medi-

ated transformations of tamoxifen was investigated usingprogress curve analysis and was only detected in theco-incubations of tamoxifen with GRE (Figure 3). The sta-tistical analysis of the data in Figure 3 showed a significantdifference between the various inhibitor concentrations ineach group. Additionally, pre-incubation of each supple-ment in the human liver microsomes in-vitro reactionsystem for 15 min before substrate addition did not resultin any decrease of inhibition for either tamoxifen or irinote-can, further suggesting that the supplements are not mecha-nism based inhibitors (data not shown).

Composition of herbal supplements

Figures 4–6 show the UV/Vis and extracted ion chromato-grams for each of the herbal preparations used in theseexperiments. For the SJW preparation (Figure 4), quercetinwas determined to be the most abundant single component.Other components detected were consistent with previouslypublished results.[10,11] GRE (Figure 5) was found to containmainly a mixture of gingerols and shogaols. Other compo-nents previously reported in the literature for variousherbal preparations, which include gingerdiols, paradols,dehydro and hydroxy gingerols and shogaols, were notdetected in the preparation used in these experiments.[12]

The preparation of BC (Figure 6) used in this studywas similar to those previously reported in the literature,with the exception of formonoetin, fukinolic acid andcimicifugic acid A and B not being present in ourpreparation.[13–15]

Effects of potential inhibitors

Inhibition was determined by co-incubating increasingconcentrations of each supplement with different

Form

atio

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ate

4-h

ydro

xyta

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-38

(pm

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Km = 41.2 μM

Tamoxifen (μM)

Vmax/2

Km = 9.7 μM

Vmax/2

Vmax(a)

(b)

(c)

Vmax

Vmax = 16.7 pmol/min per mg protein

Vmax = 0.59 pmol/min per mg protein

150 200

0 50 100

Tamoxifen (μM)

150 200

0

60

50

40

30

20

10

050 100

Irinotecan (μM)150 200

Figure 1 Kinetic characterization of tamoxifen and irinotecanmetabolism with human liver microsomes using conventionalMichaelis-Menten plots. (a) Tamoxifen to 4-hydroxytamoxifen; (b)tamoxifen to endoxifen; and (c) irinotecan to SN-38. Each data pointrepresents the average of triplicate determinations.

Fitted High Km enzyme contribution

Fitted Low Km enzyme contribution

V (

pm

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in/m

g p

rote

in)

0

25

20

15

10

5

00.2 0.4 0.6 0.8 1 1.2 1.4

V/[S] (μM)

Measured enzyme activity

Figure 2 Eadie-Hofstee plot showing the biphasic response formetabolism of irinotecan to SN-38. The linear interpolation of the highand low affinity carboxyesterase isoforms were used to graphicallydetermine the individual Km and Vmax of each isoforms from non-linearcurve fitting of V versus [S] data to a two-enzyme Michaelis-Mentenmodel. Each point represents the average of triplicate determinations.



concentrations of tamoxifen or irinotecan and quantifyingthe change in the rate of metabolite formation. Data fromthese reactions were evaluated using Dixon plots to initiallyevaluate the type of inhibition and quantitative values such

as Ki and IC50. Figure 7a and 7b shows the resulting Dixonplots for the inhibition of the biotransformation oftamoxifen to 4-hydroxytamoxifen with SJW and BC, respec-tively, while Figure 7c and 7d shows the same plots for theconversion of irinotecan to SN-38. The convergence of thelines on the x-axis from the Dixon plots for tamoxifen andirinotecan with SJW and for tamoxifen with BC suggeststhat non-competitive inhibition is likely the dominantmechanism. For irinotecan with BC, the lines convergeabove the x-axis, suggesting that competitive inhibition isthe dominant mechanism. For the inhibition reactions oftamoxifen and irinotecan with GRE, our data suggest thatthere were multiple major contributions from differenttypes of inhibition occurring and thus the type of inhibi-tion was indeterminate from the Dixon plots (data notshown). Statistically significant differences were foundbetween the various inhibitor concentrations of each treat-ment group, with the exception of the 5 and 10 mm inhibi-tor concentrations in Figure 7a. However, a statisticallysignificant difference was found between these concentra-tions and the 2 mm concentration. Figure 8 shows the effectof GRE on the bioactivation of tamoxifen and irinotecan,respectively. Table 1 lists the major types of observed inhibi-tion along with either the measured Ki or IC50 values fortamoxifen and irinotecan. The conversion of tamoxifen toendoxifen was relatively low even in the absence of potentialsupplement inhibitors, and was found to be consistently ator below the methods lower limit of quantitation (0.1 ng/ml) and highly variable in reactions with the inhibitors pro-hibiting accurate quantification. Therefore our workfocused only on the 4-hydroxy metabolite for tamoxifen.

Discussion

The goal of this study was to determine the potential ofseveral commonly used and readily available (over-the-counter) supplements for inhibiting bioactivation ofchemotherapeutic drugs to their active metabolites. Thestudy was designed to take into account not just the role ofthe known or suspected active ingredients in the supple-ments but the full complement of compounds that a patientwould be exposed to in taking the supplement. The selec-tion of the supplements and chemotherapeutic agents wasbased on the population groups most likely to be eitheralready taking the supplement or initiate taking the supple-ment to help alleviate one or more of the side-effects associ-ated with chemotherapy. SJW is most widely known fortreating depression which is highly correlated to a cancerdiagnosis, but it is also used to treat anxiety and obsessive-compulsive disorder.[16] SJW has been shown to inhibit mul-tiple CYP450 enzymes in vitro including CYP2D6, 3A4 and2C9.[17] Three individual compounds found in SJW arebelieved to be the active inhibitors, namely hyperforin,

1/V

(p

mo

l/min

per

mg

pro

tein

)1/

V (

nm

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Tamoxifen with ginger

Tamoxifen with SJW

Time (min)0

11109876543210

1.75

1.50

1.25

1.00

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0.00

10 20 30 40 50

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(a)

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No inhibitor1 mg/ml

No inhibitor2 mg/ml4 mg/ml6 mg/ml8 mg/ml10 mg/ml

1.5 mg/ml2 mg/ml4 mg/ml6 mg/ml10 mg/ml

Time (min)

Figure 3 Plots of progress curves for tamoxifen incubated in humanliver microsomes with increasing concentrations (0–10 mg/ml) of StJohn’s wort (SJW) and ginger. (a) Lines show no time-dependent inhi-bition; lines are linear best fit. (b) Lines show increasing inactivationwith time and inhibitor concentration, indicating mechanism-basedinactivation for ginger. Each point represents the average of triplicatedeterminations.

Time (min)0.0

1 23

4

5

6

254 nM

5.0 10.0 15.0 20.0 25.0 30.0 35.0 min

Ab

sorb

ance

(m

AU

)

mAU

250

0

Figure 4 HPLC chromatogram of St John’s wort liquid extract diluted1 : 10 with deionised water. Identifications were assigned based onmass spectrometry data (not shown) and relative retention times. 1,Rutin; 2, hyperoside; 3, isoquercitrin; 4, quercetrin; 5, quercetin; and 6,biapigenen.



hypericin and I3,II8-biapigenin, however it is very likelythat other compounds in SJW may contribute to metabolicinhibition as well.[18] Dosages of SJW can vary by prepara-tion of individual products, however for the preparation of

SJW used in this study the suggested dose was 1000 mg(1 ml) in a small volume of water 3 times a day. BC is oftenused for premenstrual tension, perimenopausal indications,menopause and other gynaecological indications.[16] Clinical

Time (min)

2.0 × 107

4.8 × 105

Inte

nsi

ty (

cou

nts

/s)

Time (min)

Neutral loss scan of 194

Neutral loss scan of 136(a)

(b)

10 20 30

8-shogaol(m/z 303)

6-shogaol(m/z 275)

8-gingerol(m/z 321)

6-gingerol(m/z 293)

40

10 20 30 40

Inte

nsi

ty (

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nts

/s)

Figure 5 Negative ion LC-MS/MS chromatograms of ginger root liquid extract diluted 1 : 10 with deionised water using neutral loss scans over amass range of m/z 250–350. (a) Neutral loss scans of 136 show the detection of shogaols. (b) Neutral loss scans of 194 show the detection ofgingerols.

Time (min)10 20 30 40

10.0

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(a)

(b)

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/s)

Figure 6 (a) MRM chromatograms of black cohosh: 1, caffeic acid; 1*, caffenic acid isomers (m/z 179-135); 2, ferulic acid; 2*, ferulic acid isomers(m/z 193-139); 3, isoferulic acid. (b) Precursor ion scans of m/z 131 from black cohosh: 1, cimiracemoside A, 1*, cimiracemoside isomers; 2, cim-icifugoside H2 ; 3, 27-deoxyactein; 3*, deoxyactein isomer; 4, cimicifugoside M ; 4*, cimicifugoside isomer.



studies have shown that BC inhibits CYP2D6 in healthy vol-unteers treated with 1090 mg, twice daily, each capsulestandardized to 0.2% triterpene glycosides,[19] while datafrom the same investigator in another study showed no

inhibition for CYP3A4.[20] The bioactive components in BChave been identified as triterpene glycosides, acetein,27-deoxyactein, cimiracemoside A and the isoflavone, for-mononetin.[21] The suggested dose and frequency for the

St John's wort (mg/ml)–6 –4 –2 0

–1 0

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5 μM

10 μM

10 μM

10 μM

10 μM

25 μM

25 μM

35 μM

35 μM

5 μM

30 μM

20 μM

4.03.53.02.52.01.51.00.50.0

Black cohosh (mg/ml)

St John's wort (mg/ml)

Black cohosh (mg/ml)

Tamoxifen → 4-hydroxytamoxifen

Tamoxifen → 4-hydroxytamoxifen

Irinotecan → SN-38

Irinotecan → SN-38

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rote

in)

1/V

(p

mo

l/min

per

mg

pro

tein

)1/

V (

pm

ol/m

in p

er m

g p

rote

in)

Figure 7 Dixon plots for the inhibition reactions of tamoxifen to 4-hydroxytamoxifen with St John’s wort (SJW) (a) and black cohosh (b), and iri-notecan to SN-38 for St John’s wort (c) and black cohosh (d). Each point represents the average of triplicate determinations.



preparation of BC used in this study was 100 mg (1 ml) in asmall volume of water 8 times a day. GRE has been used fordyspepsia, gastroparesis and nausea, and aqueous extractsof bulk spices (25 mg/ml) have been shown to significantlyinhibit CYP2D6, 2C19 and 3A4 in in-vitro studies.[22] Activecomponents in ginger include phenylpropanoids, sesquiter-penes and gingerols,[23] and their pharmacokinetic proper-ties in humans have been previously studied.[24] Thesuggested dose of GRE used in this study was 1000 mg(1 ml) in a small volume of water 3 times a day. While theactual effectiveness of each of these supplements is oftendebated, there is potential for adverse drug–herbal supple-ment interactions when the supplement is taken in con-junction with a chemotherapeutic regimen. Tamoxifen,a selective oestrogen receptor modulator, was chosenbecause its chemotherapeutic effects for both treatment andprevention of breast cancer depends on CYP450 mediated

metabolic conversion to its active metabolites.[25–27] Thetwo chemoactive metabolites for tamoxifen are 4-hydroxytamoxifen and endoxifen, which have a 30- to 100-fold increased potency compared with the parent drug.[28,29]

The standard chemotherapeutic dosing regimen fortamoxifen in breast cancer is 20 mg a day for 5 years.[30] Iri-notecan, a topoisomerase I inhibitor[31,32] was also selectedas a probe compound because its metabolite SN-38 isactivated by a carboxyesterase pathway[33,34] rather thanCYP450, thereby providing data for supplement interactionwith non-CYP450 mediated activation pathways. Themaximum tolerated oral dose for irinotecan as a singleagent in patients with active solid tumour malignancies is60 mg/m2 per day.[35]

One difficulty in analysing the data from these experi-ments is that each supplement contains multiple inhibitors,functioning via different inhibition mechanisms, and theseinhibitors are present over a broad range of concentrationsin the supplement. Additionally, the composition of thecomponents in the supplements may also vary as a functionof product preparation or seasonal and geographical origin.Therefore, our results are considered to be estimates ofkinetic values and suggestive of what could occur in a clini-cal setting. Simple in-vitro inhibition studies often use asingle compound as the inhibitor in the presence of aspecific CYP450 probe substrate, and incubations areconducted at various inhibitor and substrate concentra-tions. Data from these experiments are evaluated usingvarious methods, including non-linear regression analysis,Lineweaver-Burke, Eadie-Hofstee and Dixon plots, to deter-mine the type of inhibition occurring, as well as Ki andIC50 values.[36–45] For a single inhibitor and probe substrate,inhibition is typically limited to a single mechanistic typeand can typically be accurately identified.[36,38,40,41,46] For theinhibition experiments in our studies, the potential inhibi-tors evaluated were composed of many different chemicalentities over a diverse concentration range, and therefore itis unlikely that the individual compounds contributing tothe overall observed inhibition are doing so by the samemechanism (e.g. competitive, non-competitive, uncompeti-tive). Rather, it is more likely that the observed inhibitionmechanism contains components from multiple types ofinhibition mechanisms. This leads to two primary mecha-nisms of inhibition in our studies. The first is where multi-ple types contribute to the observed inhibition but one typedominates, and the second is where more than one typecontributes significantly, resulting in mixed inhibition. Inthe case of the former, the data is evaluated as more of asingle type of inhibition, whereas in the latter case the com-bined impact on the reduction of reaction velocity is evalu-ated. Cross comparison of reduction in velocity where asingle inhibition mechanism dominates was made andcomparable results were obtained (data not shown).

20 μM Tamoxifen → 4-hydroxytamoxifen

5 µM Irinotecan → SN-38

IC50 = 8.3 mg/ml

IC50 = 25.5 mg/ml

Ginger root extract (mg/ml)

Ginger root extract (mg/ml)

1.5 15

Rea

ctio

n v

elo

city

(%

of

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tro

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eact

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80

70

60

50

40

30

20

10

0

100

90

80

70

60

50

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30

20

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Figure 8 The effect of ginger root extract on the conversion oftamoxifen to 4-hydroxytamoxifen (a) and irinotecan to SN-38 (b). Reac-tion velocity is expressed as a percentage of the control velocity withno inhibitor added. Each point represents the average of triplicatedeterminations. The curve and IC50 values are based on non-linearregression analysis of the average data points.



Initial evaluation of the data using Dixon plots suggestedthat the commercial preparations of SJW and BC used inthis study inhibited the bioactivation of tamoxifen (medi-ated by CYP2D6, 3A4 and flavin-containing monooxygen-ase)[44] predominately by a non-competitive mechanism,and that BC is a much stronger inhibitor of tamoxifenmetabolism than SJW (Table 1). Recently, it has been shownthat at 50 mg/ml, a 75% ethanolic extract of BC inhibitedthe formation of 4-hydroxytamoxifen by 66.3% and eighttriterpenes identified did so via competitive inhibition ofCYP3A4, with IC50 values of 5.1 mm or less.[47] From inhibi-tion reactions of tamoxifen with GRE, the Dixon plot sug-gests that significant contributions to the overall inhibitionwere being made by multiple mechanisms, thus resulting inan indeterminate or mixed-type mechanism. In order tocalculate the IC50 for GRE, the measured reaction velocitiesin the presence of the inhibitor were expressed as a ratio ofthe control velocity (velocity in the absence of the inhibitor)and analysed by non-linear regression (Equation 3) and theIC50 value calculated from Equation 4.[9] The IC50 value forGRE was determined to be greater than that measured forBC or SJW, thereby suggesting that it is a weaker inhibitorrelative to SJW and BC for tamoxifen metabolism (Table 1).Data for irinotecan showed SJW to primarily follow a non-competitive mechanism and to be a weaker inhibitor of thecarboxyesterase enzymes relative to CYP450 enzyme fortamoxifen metabolism, although significant inhibition wasstill observed for the biotransformation of irinotecan toSN-38. Consistent with our in-vitro results, clinical data haspreviously shown that SJW (900 mg/day p.o. for 18 days)reduces plasma levels of SN-38 in patients treated with iri-notecan by 42%.[48] In another study, significant decreases inplasma levels and intracellular accumulation of both iri-notecan and SN-38 were found.[49] In experiments with BC,a competitive mode of inhibition was observed and the cal-culated IC50 values for both the low and high affinity CEisozymes were much lower than that for SJW, suggestingthat BC is a more potent inhibitor of CE activation for iri-notecan than SJW. BC has also been shown to function as a

mixed competitive ligand and agonist of serotonin recep-tors.[50] Inhibition reactions of irinotecan with GRE alsoshowed indeterminate or mixed inhibition mechanisms asobserved with tamoxifen. GRE was determined to be a lesspotent inhibitor of tamoxifen compared with BC and SJW,and was also less potent when inhibiting CE enzymes ascompared with CYP450 enzymes.

This work attempted to determine if metabolic inhibitionusing an in-vitro system could be observed throughco-incubation of chemotherapeutic drugs with supple-ments, and to make qualitative observations regarding theproperties of the inhibitions. Furthermore, this workattempted to estimate relative differences in the ability ofthe supplements to achieve inhibition for chemotherapeuticdrugs that rely on the formation of pharmacologicallyactive metabolites for efficacy. While these data cannot bereadily extrapolated to predict clinical outcomes, they dosuggest that concomitant use of these supplements withchemotherapeutic regimens containing tamoxifen or iri-notecan could possibly result in a sub-chemotherapeuticresponse due to the inhibition of metabolism to activemetabolites.

Conclusions

The increased use of supplements continues to present thepotential for adverse drug–herbal supplement interactionsin clinical settings. Because supplements are often complexmixtures of many different pharmacologically active com-pounds, their overall mode of enzyme inhibition is a com-bination of several types, but may be dominated by a singlemechanism. SJW, BC and GRE have different degrees ofimpact on the bioactivation of chemotherapeutic drugs viainhibition of both CYP450 and carboxyesterase enzymepathways. BC was observed to be the most potent inhibitorof the bioactivation of both tamoxifen and irinotecan, whileGRE and SJW were observed to be more potent for CYP450mediated pathways of tamoxifen than carboxyesterase acti-vation pathways of irinotecan.

Table 1 Estimated inhibition values of herbal supplement with chemotherapeutic prodrugs

Substrate St John’s wort Black cohosh Ginger root extract

Tamoxifen Non-competitive Non-competitive Indeterminatea

IC50 = 3.6 mg/ml IC50 = 0.52 mg/ml IC50 = 8.3 mg/mlIrinotecan Non-competitive Competitive Indeterminatea

IC50 = 49.4 mg/ml Ki = 1.62 mg/ml IC50 = 25.5 mg/mlHigh Km Low Km

IC50 (5 mM) = 1.69 4.74IC50 (10 mM) = 1.75 7.85IC50 (25 mM) = 1.95 17.2IC50 (45 mM) = 2.10 23.4

aMultiple contributions.



Declarations

Conflict of interest

The Author(s) declare(s) that they have no conflicts ofinterest to disclose.

Funding

Funding for this research was provided by the Pharmaceuti-cal Sciences Research Institute and the McWhorter Schoolof Pharmacy at Samford University.

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Effects of herbal supplements on the bioactivation of chemotherapeutic agents