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Year: 2010
Garlic extract induces intestinal P-glycoprotein, but exhibits noeffect on intestinal and hepatic CYP3A4 in humans
Hajda, J; Rentsch, K M; Gubler, C; Steinert, H; Stieger, B; Fattinger, K
Hajda, J; Rentsch, K M; Gubler, C; Steinert, H; Stieger, B; Fattinger, K (2010). Garlic extract induces intestinalP-glycoprotein, but exhibits no effect on intestinal and hepatic CYP3A4 in humans. European Journal ofPharmaceutical Sciences, 41(5):729-735.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:European Journal of Pharmaceutical Sciences 2010, 41(5):729-735.
Hajda, J; Rentsch, K M; Gubler, C; Steinert, H; Stieger, B; Fattinger, K (2010). Garlic extract induces intestinalP-glycoprotein, but exhibits no effect on intestinal and hepatic CYP3A4 in humans. European Journal ofPharmaceutical Sciences, 41(5):729-735.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:European Journal of Pharmaceutical Sciences 2010, 41(5):729-735.
Garlic extract induces intestinal P-glycoprotein, but exhibits noeffect on intestinal and hepatic CYP3A4 in humans
Abstract
Garlic extracts have been shown to decrease drug exposure for saquinavir, a P-glycoprotein andcytochrome P450 3A4 substrate. In order to explore the underlying mechanisms and to study the effectsof garlic on pre-systemic drug elimination, healthy volunteers were administered garlic extract for 21days. Prior to and at the end of this period, expression of duodenal P-glycoprotein and cytochrome P4503A4 protein were assayed and normalized to villin, while hepatic cytochrome P450 3A4 function andsimvastatin, pravastatin and saquinavir pharmacokinetics were also evaluated. Ingestion of garlic extractincreased expression of duodenal P-glycoprotein to 131% (95% CI, 105-163%), without increasing theexpression of cytochrome P450 3A4 which amounted to 87% (95% CI, 67-112%), relative to baseline inboth cases. For the erythromycin breath test performed, the average result was 96% (95% CI, 83-112%).Ingestion of garlic extract had no effect on drug and metabolite AUCs following a single dose ofsimvastatin or pravastatin, although the average area under the plasma concentration curve (AUC) ofsaquinavir decreased to 85% (95% CI, 66-109%), and changes in intestinal P-glycoprotein expressionnegatively correlated with this change. In conclusion, garlic extract induces intestinal expression ofP-glycoprotein independent of cytochrome P450 3A4 in human intestine and liver.
Accepted Manuscript
Title: Garlic extract induces intestinal P-glycoprotein, butexhibits no effect on intestinal and hepatic CYP3A4 in humans
Authors: Jacek Hajda, Katharina M. Rentsch, ChristophGubler, Hans Steinert, Bruno Stieger, Karin Fattinger
PII: S0928-0987(10)00342-8DOI: doi:10.1016/j.ejps.2010.09.016Reference: PHASCI 2143
To appear in: European Journal of Pharmaceutical Sciences
Received date: 1-4-2010Revised date: 21-9-2010Accepted date: 28-9-2010
Please cite this article as: Hajda, J., Rentsch, K.M., Gubler, C., Steinert, H., Stieger,B., Fattinger, K., Garlic extract induces intestinal P-glycoprotein, but exhibits no effecton intestinal and hepatic CYP3A4 in humans, European Journal of PharmaceuticalSciences (2010), doi:10.1016/j.ejps.2010.09.016
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
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Garlic extract induces intestinal P-glycoprotein, but exhibits no effect on
intestinal and hepatic CYP3A4 in humans
Jacek Hajda 1,2, Katharina M. Rentsch 3, Christoph Gubler 4, Hans Steinert 5, Bruno Stieger 1
and Karin Fattinger 6
1 Division of Clinical Pharmacology and Toxicology, University Hospital Zürich,
Switzerland, 2 Coordination Centre for Clinical Trials (KKS) Heidelberg, Germany, 3 Institute
of Clinical Chemistry, University Hospital Zürich, Switzerland, 4 Division of
Gastroenterology and Hepatology, University Hospital Zürich 5 Division of Nuclear
Medicine, Department of Medical Radiology, University Hospital Zurich, and 6 Department
of General Internal Medicine, Inselspital – University Hospital Bern and University of Bern,
Switzerland
Correspondence: Karin Fattinger Department of General Internal Medicine Inselspital - University Hospital Bern and University of Bern CH - 3010 Bern Switzerland Phone + 41 31 632 21 11, ask for 181 8009 Fax + 41 31 632 88 85 Email [email protected]
*Manuscript
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Abstract
Garlic extracts have been shown to decrease drug exposure for saquinavir, a P-glycoprotein
and cytochrome P450 3A4 substrate. In order to explore the underlying mechanisms and to
study the effects of garlic on pre-systemic drug elimination, healthy volunteers were
administered garlic extract for 21 days. Prior to and at the end of this period, expression of
duodenal P-glycoprotein and cytochrome P450 3A4 protein were assayed and normalized to
villin, while hepatic cytochrome P450 3A4 function and simvastatin, pravastatin and
saquinavir pharmacokinetics were also evaluated. Ingestion of garlic extract increased
expression of duodenal P-glycoprotein to 131% (95%CI, 105-163%), without increasing the
expression of cytochrome P450 3A4 which amounted to 87% (95%CI, 67-112%), relative to
baseline in both cases. For the erythromycin breath test performed, the average result was
96% (95%CI, 83-112%). Ingestion of garlic extract had no effect on drug and metabolite
AUCs following a single dose of simvastatin or pravastatin, although the average area under
the plasma concentration curve (AUC) of saquinavir decreased to 85% (95%CI, 66-109%),
and changes in intestinal P-glycoprotein expression negatively correlated with this change. In
conclusion, garlic extract induces intestinal expression of P-glycoprotein independent of
cytochrome P450 3A4 in human intestine and liver.
Key words: herbal medicines, drug interactions, MDR1, P-glycoprotein, pravastatin,
simvastatin, saquinavir.
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1. Introduction
Use of over-the-counter herbal medicines is increasing, while also becoming more common,
with one out of five patients currently using natural products (Navarro, 2009, King et al.,
2009). However, data on the efficacy and safety of herbal medicines are often incomplete
(O'hara et al., 1998, Bent, 2008). Furthermore, drug interactions between herbal medicines
and prescription or over-the-counter medications can be hazardous in some cases (Izzo and
Ernst, 2009). For example, St John’s Wort has been shown to decrease the efficacy of several
drugs, including oral contraceptives (Schwarz et al., 2003) and HIV protease inhibitors
(Piscitelli et al., 2000). In these cases, the induction of several cytochrome P450 drug
metabolizing enzymes and the intestinal efflux transporter P-glycoprotein (PGP) have been
detected (Dürr et al., 2000).
Both experimental data, and data from controlled clinical trials, suggest that garlic
preparations may lower lipid levels and have anti-hypertensive and anti-coagulant properties,
which could lower cardiovascular risk factors for patients (Stevinson et al., 2000, Ried et al.,
2008, Rahman, 2007, O'hara et al., 1998). The efficacy of garlic and its derivatives have been
linked to the generation of organosulfurs, such as monosulfides, polysulfides, and ajoenes, by
the body in response to the alliin contained in garlic (Iciek et al., 2009, Rose et al., 2005).
Therefore, alliin content is considered to be a critical component of garlic preparations.
Currently, there are three studies that have investigated the effects of garlic on CYP450 drug
metabolizing enzymes in humans (Gurley et al., 2002, Markowitz et al., 2003, Gurley et al.,
2005). Using debrisoquine, midazolam, caffeine, and chlozoxazone as test substrates, Gurley
et al. showed that garlic oil decreases CYP2E1 activity by 39% and 22% in young and elderly
volunteers, respectively, yet exhibits no effect on CYP1A2, 2D6, and 3A4 function (Gurley et
al., 2002, Gurley et al., 2005). Similarly, Markowitz et al. confirmed that garlic exhibits no
effect on CYP2D6 and CYP3A4 function in drug interaction studies of Kwai garlic
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supplements from Lichtwer Pharma and dextromethorphane or alprozolam (Markowitz et al.,
2003). In contrast, results from a clinical trial of 9 healthy volunteers demonstrated that 600
mg of garlic extract from GarliPure twice a day (b.i.d.) administered for 21 days decreased
the average area under the plasma concentration curve (AUC) and the average maximal
plasma concentration (Cmax) of saquinavir by 51% and 54%, respectively (Piscitelli et al.,
2002). The protease inhibitor, saquinavir, is a substrate of CYP3A4 and PGP (Plosker and
Scott, 2003), suggesting that induction of CYP3A4 and/or PGP by garlic extracts may
represent the underlying mechanism.
To further examine the effects of garlic on pre-systemic drug elimination, healthy volunteers
were administered garlic extract (GarliPure) b.i.d. over 21 days. Levels of PGP, also known
as MDR1, and CYP3A4 in duodenal biopsy samples were then evaluated, and hepatic
CYP3A4 function was assessed using the erythromycin breath test (EMBRT). Due
overlapping treatment indications, the probability of concurrent intake of garlic extracts and
statins is high. Therefore, the effects of garlic extract on the pharmacokinetics of a single dose
of simvastatin or pravastatin, i.e. a CYP3A4-dependent and -independent metabolized statin,
respectively, were evaluated. Furthermore, a pharmacokinetic study of a single dose of
saquinavir was performed to directly compare these results with those from a previous study
by Piscitelli et al. (Piscitelli et al., 2002).
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2. Methods
2.1. Materials
Garlic extract (GarliPure, 600 mg caplets from Natrol, CA, USA containing gamma-
glutamylcysteines 12’000 µg, 4’800 µg alliin, 3’900 µg sulfur and 3’8000 µg thiosulfinates
resulting in a allicin yield 3’600 µg), simvastatin (Zocor, 20mg), pravastatin (Selipran, 20
mg), and saquinavir (Fortovase, 200 mg) were purchased from their respective
manufacturers. 14C-erythromycin breath test kits were obtained from Metabolic Solutions Inc.
(Nashua, NH, USA).
2.2. Clinical study
This study protocol was approved by the ethics committee of the Canton of Zürich and the
Swiss Federal Office of Health (BAG). An open-label, randomized, cross-over design was
used with two study periods. Ten non-smoking, male Caucasian subjects were enrolled that
ranged in age from 24-38 y (mean 28.8 y), in body weight from 54.0-92.0 kg (mean 74.4 kg),
and in body mass index (BMI) from 19.8-25.5 kg*m-2 (mean 22.5 kg*m-2). Based on medical
histories, physical examinations, standard clinical chemistry and hematology analyses, all
subjects were considered healthy. The subjects were instructed to abstain from taking any
type of medication, including over-the-counter remedies and supplements, grapefruit,
caffeine, or alcohol-containing food or beverages for at least 3 d prior to the start of the study
and throughout the course of the study. Written informed consent was obtained from each
subject prior to participation.
Each study period consisted of 4 d. On the first morning, volunteers arrived at 7:30 AM after
fasting overnight. Blood samples were collected prior to the drug administration (8:00 AM)
and 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h thereafter. On the first and second days, volunteers
randomly received either a single dose (20 mg) of simvastatin or pravastatin, followed by
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standardized meals served 4 and 10 h after dosing. On the third day, volunteers received
breakfast (consisting of 300 ml unskimmed milk, 2 small bread rolls, 10 g of butter, 4 thin
slices of salami, 1 boiled egg and 25 g of corn flakes and contained 30 g of fat, 83 g of
carbohydrates and 25 g of proteins) and two hours later a single dose (1200 mg) of saquinavir.
Blood samples were collected just before saquinavir was administered, as well as 0.5, 1, 1.5,
2, 3, 4, 6, and 8 h after receiving saquinavir. Additional standardized meals were served 2 and
8 h after dosing.
All blood samples were immediately centrifuged following collection at 2500 rpm and 4°C
for 10 min, then stored at –20°C. On the fourth day, volunteers underwent a
gastroduodenoscopy under midazolam sedation. Seven biopsy samples of duodenal intestinal
mucosa were obtained. One biopsy was used for histopathologic examination, while the
others were immediately frozen in liquid nitrogen and stored at –70°C. In the afternoon of the
same day, an EMBRT was performed according to the manufacturer’s instructions.
Thereafter, all subjects received a garlic extract dose of 600 mg twice daily for 21 days. On
days 19-22, the same procedures described above for days 1-4 were repeated.
2.3. Pharmacokinetics
Plasma concentrations of simvastatin, simvastatin acid, pravastatin, pravastatin lactone, R-
416, and saquinavir were determined using liquid chromatography-mass spectrometry (LC-
MS) as described before (27). Except for saquinavir, the quantification limit was 0.01 µg/l,
between-day precision was < 10%, and the accuracy of detection ranged from 84.5 – 112%.
The corresponding values for saquinavir were 0.05 mg/l as quantification limit, < 6.2% for
between-day precision and a range of 99 – 100% for accuracy. Areas under the plasma
concentration-time curves up until the last quantifiable concentration (AUC0-n) were
calculated using the trapezoidal rule.
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The sample size was determined based on the estimates reported in a interaction study on the
effects of St. John’s wort on simvastatin exposure (Sugimoto et al., 2001). Based on a
expected mean difference for total AUC of 15.2 ng h/ml and a standard deviation of AUC
change of 14.9 ng h/ml, a sample size of 10 (=0.05, 2-tailed) results in a power of 0.81.
2.4. Western blot analysis
Frozen biopsy specimens were thawed and processed exactly as described in (8). Protein
concentrations of the homogenates were determined using the bicinchoninic assay protein
determination kit from Pierce (Rockford, IL, USA). SDS-polyacylamide geleectrophoresis
and Western blotting are described in (8, 28) and were performed with the following
monoclonal antibodies: C219 for the detection of MDR1 (Alexis, Lausen, Switzerland),
CYP3A4 (Gentest, Woburn, MA, USA) and villin (Chemicon, Temeluca, CA, USA). Blots
were developed with the Uptilight detection kit from Interchim, Montfuçon, France). After
development, films were scanned with a CAMAG TLC Scanner II (CAMAG, Muttenz,
Switzerland) for comparison of relative protein expression levels and normalization to villin.
2.5. Statistics
The geometric means of CYP3A4 and MDR1 ratios to villin protein were calculated from 3
individual biopsy specimens, and AUC0-n data and ERMBT results were compared before and
after treatment with garlic using the paired Wilcoxon signed-rank test. All calculations and
drawings were done in Microsoft Excel and (S)-Plus 6.1 for Windows (Insightful
Corporation, 2002).
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3. Results
3.1. Intestinal expression of P-glycoprotein/MDR1 and CYP3A4 protein and CYP3A4
function in liver
Ten male, healthy volunteers consumed garlic extract (2 doses of 600 mg per day) for 3
weeks, and intestinal biopsy specimens were collected before and after the garlic intake
period. PGP, CYP3A4, and villin were detected by western blot, and a representative analysis
is shown in Figure 1. For each volunteer, protein levels before and after garlic intake were
compared, and an increase in PGP expression can already be visually detected in 6 of the
volunteers (subjects B, C, E, F, H, and I). In contrast, paired samples for subjects A, D, and G
were difficult to compare due to differences in epithelial content (based on detection of villin
expression). Expression of CYP3A4 was largely unaffected by ingestion of garlic extract.
Based on densitometric analysis of western blot detection, and normalization to levels of
villin, 3 pairs of biopsy specimens collected before and after garlic intake were averaged for
each individual using geometric means (Fig. 2, left panel). Intestinal PGP protein levels were
found to increase after intake of garlic extract in 9 out of the 10 volunteers assayed. When the
PGP/villin ratio detected prior to garlic intake was set to 100%, the average PGP/villin
content after receiving garlic extract was 131%, with a 95% confidence interval (95% CI)
ranging from 105-163% (p = 0.0195). Similarly, CYP3A4 levels prior to and following intake
of garlic extract intake were normalized to villin, and the average CYP3A4/villin content after
garlic intake was 87%, with a 95% CI ranging from 67-112%. Ingestion of garlic extract also
exhibited no effect on the EMBRTs performed (Fig. 2), with the geometric mean of EMBRTs
performed before and after garlic intake having a ratio of 96%, with a 95% CI ranging from
83-112%. Thus, these data demonstrate a consistent and significant increase in intestinal PGP
protein in response to garlic extract, yet no difference in levels of CYP3A4 in the intestine
and CYP3A4 function in the liver.
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3.2 Simvastatin kinetics
When average simvastatin plasma concentration time profiles were compared before and at
the end of 3 weeks of garlic extract intake, they were slightly higher after the ingestion of
garlic extract (Fig. 3, left panel). Accordingly, the mean AUC after garlic intake was 22.1 ±
25.7 µg h/l, compared to 11.2 ± 5.7 µg h/l at baseline. Large increases in the simvastatin
plasma concentrations after garlic extract intake for subjects A and F were largely responsible
for the overall increase in simvastatin levels detected (Fig. 3), resulting in a geometric mean
AUC ratio of 137%, with a 95% CI ranging from 88-212%. For the metabolite, simvastatinic
acid, its average plasma concentration time profile, AUC, and other pharmacokinetic
parameters were similar before and after garlic extract intake .(Fig. 3, Table 1). Hence, no
significant changes, in particular no decrease in the bioavailability of simvastatin, was
detected following garlic extract intake.
3.3. Pravastatin kinetics
Average plasma concentration time profiles for pravastatin, pravastatin lactone, and the active
metabolite, R-146, were similar before and at the end of the ingestion of garlic extract (Fig.
4). Also AUCs (Fig. 4) and pharmacokinetic parameters (Table 1) did not show any effects
from the ingestion of garlic extract. Accordingly, the geometric mean AUC ratios for
pravastatin, pravastatin lactone, and R-146 were 94% (95% CI, 65-136%), 84% (95% CI, 40-
174%), and 89% (95% CI, 68-115%), respectively. These results did not indicate any
clinically relevant effects from garlic intake on pravastatin pharmacokinetics.
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3.4. Saquinavir kinetics
When saquinavir concentrations were assayed following ingestion of garlic extract at 3 h and
4 h time points, the average saquinavir concentration was found to be lower compared to
baseline (Fig. 5, left panel). Furthermore, the ratio of AUC from 0 to 8 h following garlic
extract intake were found to decrease in 7 of the study subjects (A-C, E, H-J), and increase in
the other 3 subjects (D, F, G) (Fig. 5). Overall, the geometric mean AUC ratio decreased to
85% from baseline, with a 95% CI ranging from 66-109%. In contrast, the corresponding
values for terminal half-life slightly increased to 120%, with a 95% CI ranging from 94-
152%. Since PGP has been hypothesized to limit saquinavir bioavailability, and ingestion of
garlic extract was found to increase intestinal PGP in this study, linear regression analysis of
individual changes in saquinavir AUC, i.e. the corresponding AUC ratios before and after
garlic intake, was performed to explore the effects of changes in intestinal PGP content (Fig.
5). A negative correlation was identified, with an R2 of 0.467 (p = 0.029).
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4. Discussion
This is the first study, to our knowledge, that demonstrates that garlic extract intake can
increase intestinal expression of PGP. This result is consistent with the reduced bioavailability
of saquinavir previously observed following ingestion of garlic extract (Piscitelli et al., 2002).
This study also demonstrates that garlic extract exhibits no effect on intestinal expression of
3A4, does not change hepatic 3A4 function, and does not reduce the bioavailability of
simvastatin. These results are consistent with a previous report that showed no changes in the
pharmacokinetics of the CYP3A4 substrates, midazolam and alprazolam (Markowitz et al.,
2003, Gurley et al., 2005, Gurley et al., 2002). Therefore, our results demonstrate a
differential induction of PGP and CYP3A4 in response to garlic extract, suggesting that garlic
extract can induce intestinal PGP independent of effecting CYP3A4.
After 3 weeks of garlic extract intake, expression of intestinal PGP increased 31%, which is
consistent with the 37% increase detected for intestinal PGP after 2 weeks of ingesting St.
John’s Wort by 8 healthy volunteers (8), with the same western blotting procedures used
(Dürr et al., 2000). In previous studies of St John’s Wort digoxin interactions, increased
expression of PGP was associated with an 18-25% decrease in digoxin AUC (Johne et al.,
1999, Dürr et al., 2000). The decrease in the saquinavir AUC observed in this study was
slightly less at 15%, with a rather broad 95% CI of (-9)-34%. We hypothesize that this
considerably larger variability associated with saquinavir versus digoxin is due to the low
bioavailability of saquinavir (~12% for the soft gelatin capsules), and its food-dependent
absorption properties (Plosker and Scott, 2003). To facilitate the drug absorption of saquinavir
in this study, administration of the soft gelatin capsules was combined with breakfast.
However, the variability observed with these studies was still too great to achieve statistical
significance, even with a treatment group of 10 volunteers. Although, the data do show a clear
and significant linear negative correlation between change in intestinal PGP expression and
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changes in the bioavailability of saquinavir following ingestion of garlic extract. The
intestinal PGP ratio calculated also explains the 46.7% variability associated with the
saquinavir AUC ratio, and thus confirms the results of previous studies that demonstrated that
the intestinal efflux transporter, PGP, limits the oral bioavailability of saquinavir (Plosker and
Scott, 2003).
A study by Piscitelli et al. reported that 3 weeks of garlic extract intake reduced the AUC for
orally administered saquinavir to 49.5% of pretreatment values with a 90% CI ranging from
30.8-79.3% (Piscitelli et al., 2002). In contrast, the AUC for saquinavir following 3 weeks of
garlic extract intake in this study was slightly less pronounced at 85%, with a 95% CI ranging
from 66-109%. In order to directly compare these two studies, the same garlic extract
preparation, dosage, saquinavir preparation, and treatment period were maintained in this
study. Several drugs with rather short half-lives were selected in order to allow single dose
pharmacokinetics to be evaluated. Therefore, a 3-day course of saquinavir in combination
with garlic extract was not performed. Mechanistically, we hypothesize that this preference in
the study design does not account for differences in the magnitude of the effects observed, but
rather, the observed differences are the result of large intersubject and intrasubject
variabilities in saquinavir absorption. Pravastatin is a well-characterized substrate of the
hepatic organic anion transporter, OATP1B1 (Ito et al., 2005). The absence of any observed
effects on pravastatin pharmacokinetics following 3 weeks of garlic extract intake, suggests
that the Garlipure form of garlic extract does not significantly influence the expression and
function of OATP1B1 in humans.
We used the EMBRT to test for changes in hepatic CYP3A4 function. However, it has been
previously reported that erythromycin is also a PGP substrate and that the concurrent
administration of a PGP inhibitor may increase hepatic erythromycin metabolism and result in
false positive EMBRTs, i.e. increases in the EMBRT without CYP3A4 induction (Frassetto et
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al., 2007, Kurnik et al., 2006). In our study, the focus was on PGP and 3A4 induction and no
change in EMBRT was observed. Since also the clearance of the CYP3A4 substrate
simvastatin was not increased after garlic extract intake, we are confident about the
conclusion that garlic extracts do not induce CYP3A4 in the liver is clinically valid despite of
the possible limitations of the EMBRT.
The nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor
(CAR), have been shown to regulate the expression of drug transporters and metabolizing
enzymes in liver and intestine, including CYP3A4 and MDR1 (Kohle and Bock, 2009, Di
Masi et al., 2009). The hyperforin contained in St. John’s Wort has been shown to be a strong
ligand of human PXR, with PXR-mediated induction considered to be the underlying
mechanism for the coordinated induction of CYP3A4 and MDR1 by St. John’s Wort (Tirona
and Bailey, 2006). Studies in rat further suggest that the diallyl sulfide contained in garlic is
also a structurally atypical phenobarbital-type inducer (Dragnev et al., 1995), and that diallyl
sulfide acts as an activator of rat CAR (Chang, 2009). Accordingly, activation of CAR by
diallyl sulfide might be responsible for the observed induction of MDR1 by garlic extracts.
However, it has also been suggested that CAR controls the expression of CYP3A4 as well
(Kohle and Bock, 2009, Di Masi et al., 2009). However, diallyl sulfide is mainly found in
garlic oils, but only minor amounts of diallyl sulfide have been found in garlic powder tablets
(Lawson et al., 1991). Therefore, it remains to be investigated why garlic increases MDR1 in
intestine, but does not similarly effect CYP3A4 in intestine and liver.
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In contrast with the results of this study, previous studies in rats have demonstrated that garlic
oil and its constituents, diallyl sulfide, diallyl disulfide, and diallyl trisulfide, increase mRNA
and protein levels of CYP3A1 and other CYP proteins (Wu et al., 2002, Dragnev et al., 1995).
In combination, these results suggest that there are interspecies differences in the regulation of
CYP3A orthologues between rodents and humans.
In conclusion, this study demonstrates a differential induction of PGP versus CYP3A4 in
human intestinal samples from healthy volunteers, where garlic extract increased intestinal
expression of PGP independent of CYP3A4. Based on these results, decreased bioavailability
of the PGP substrate, saquinavir, would be predicted in the presence of garlic extract. In
addition, garlic extract did not exhibit any effects on hepatic CYP3A4 and OATP1B1
function, and exhibited no pharmacokinetic interaction with simvastatin and pravastatin.
Therefore, garlic extract represents an herbal medicine that can be safely combined with
statins for the treatment of hypercholesterolemia.
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Acknowledgements
The study was supported by the Swiss National Science Foundation Grant, 3200B0-109352
and in part by an unrestricted grant from Bristol-Myers Squipp.
Conflict of Interest / Disclosure
The authors declare no conflict of interest.
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Figure Legends
Figure 1: Expression of intestinal P-glycoprotein and CYP3A4 protein before and after
ingestion of garlic extract for three weeks. Ten healthy male volunteers ingested garlic extract
for 3 weeks, with duodenal biopsy specimens collected before and at the end of the study
period. Homogenates (100 μg) of individual biopsy specimens were incubated with antibodies
against either P-glycoprotein or CYP3A4, visualized by chemiluminescence. Membranes
were then stripped and probed with antibodies against villin. A representative blot is shown
from the 3 sets of biopsy specimens analyzed.
Figure 2: Ratios of P-glycoprotein/MDR1 and CYP3A4 to villin, and EBMRTs performed.
Intestinal P-glycoprotein (left panel) and CYP3A4/villin (middle panel) expression ratios for
subjects A-K were determined using densitometric analysis of western blots and are given as
the geometric means of 3 individual duodenal specimens obtained before and after garlic
intake. EMBRTs (right panel) were performed before treatment (control) and after treatment
(garlic). In each panel, the bold black line corresponds to the geometric mean value before
and after garlic intake, whereas the thin gray lines give the individual values for subject A to
K.
Figure 3: Effects of garlic extract ingestion on simvastatin pharmacokinetics. Left panel:
Plasma concentration-time profiles (mean ± SEM) for subjects A-K for simvastatin (triangles)
and simvastatin acid (circles) before (open symbols) and after 3 weeks of garlic extract intake
(filled symbols). Right panels: Comparison of simvastatin AUC profiles for the after
administration of simvastatin or simvastatin acid alone (controls in each case) versus each in
combination with garlic intake for subjects A-K. The bold black line corresponds to the
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geometric mean values, whereas the thin gray lines give the individual values for subject A to
K.
Figure 4: Effects of garlic extract ingestion on pravastatin pharmacokinetics. Left panel:
Plasma concentration-time profiles (mean ± SEM) for subjects A-K for pravastatin (triangles),
pravastatin lactone (circles), and R-416 (insert), before (open symbols) and after 3 weeks of
garlic extract intake (filled symbols). Right panels: Comparison of AUC ratios for pravastatin,
pravastatin lactone, and R-416 before and after garlic intake for subjects A-K. The bold black
line corresponds to the geometric mean values, whereas the thin gray lines give the individual
values for subject A to K.
Figure 5: Effects of garlic extract ingestion on saquinavir pharmacokinetics. Left panel:
Saquinavir plasma concentration-time profiles (mean ± SEM) for subjects A-K before (open
symbols) and after 3 weeks of garlic extract intake (filled symbols). Middle panel:
Comparison of saquinavir AUC ratios before and after garlic intake for subjects A-K. The
bold black line corresponds to the geometric mean values, whereas the thin gray lines give the
individual values for subject A to K. Right panel: Correlation of changes in saquinavir AUCs
and intestinal PGP expression, with data points representing the individual values for subjects
A-K, with the best-fit line generated by linear regression analysis (R2 = 0.467, p = 0.029) .
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Table 1: Simvastatin and pravastatin kinetics before and after garlic extract intake
Parameter Baseline Garlic Geometric mean ratio (95% CI)
Simvastatin
AUC0-24h (ug . h/L) 11.2 5.7 22.1 25.7 1.37 (0.88, 2.12)
CL/F (L/h) 2349 1340 2146 1660
t½ 4.92 0.93 5.00 1.90 1.02 (0.81, 1.27)
tmax (h) 1.4 0.7 1.5 0.9
Cmax (ug/L) 3.85 1.99 5.25 6.32
Simvastatin acid
AUC0-24h 7.1 3.2 9.1 5.2 1.25 (0.80, 1.93)
Pravastatin
AUC0-24h (ug . h/L) 41.8 24.1 35.4 15.5 0.94 (0.65, 1.36)
CL/F (L/h) 834 928 722 440
t½ 3.75 0.66 3.57 1.11 0.92 (0.5, 1.13)
tmax (h) 1.0 0.5 1.1 0.4
Cmax (ug/L) 18.8 12.2 14.2 6.7
R-416
AUC0-24h 3.6 1.1 3.3 1.3 0.84 (0.40, 1.74)
Pravastatin lactone
AUC0-24h 26.5 17.4 28.7 23.1 0.89 (0.68, 1.15)
AUC, area under the plasma concentration-time curve; Cmax, peak plasma concentration; tmax, time of peak plasma concentration; t1/2, terminal half –life. Results at baseline and after garlic intake are given as mean SD. The last column gives the point estimate and the 95% CIs of the geometric mean ratio of the results observed after garlic intake and at baseline.
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Table 2: Saquinavir kinetics before and after garlic extract intake
Parameter Baseline Garlic Geometric mean ratio (95% CI)
Saquinavir
AUC0-8h (ug . h/L) 1888 578 1698 882 0.85 (0.66, 1.09)
CL/F (L/h) 73.6 38.7 91.1 57.7
t½ 1.33 0.22 1.67 0.71 1.20 (0.94, 1.52)
tmax (h) 3.1 0.6 2.6 0.6
Cmax (ug/L) 729 231 659 287
AUC, area under the plasma concentration-time curve; Cmax, peak plasma concentration; tmax, time of peak plasma concentration; t1/2, terminal half –life. Results at baseline and after garlic intake are given as mean SD. The last column gives the point estimate and the 95% CIs of the geometric mean ratio of the results observed after garlic intake and at baseline.
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Figure 1:
A B C D E F G H I K positive control PGP
CYP3A4 Garlic
Villin
PGP
Villin
CYP3A4
- + - + - + - + - + - + - + - + - + - + positive control PGP
CYP3A4
Subject No.
Figure(s)
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Figure 2:
A AB
B
C
C
D
DE
EF
FG
G
H
H
I
I
K
K
PG
P/v
illin
0.0
0.5
1.0
1.5
2.0
2.5
Control Garlic
A
A
B
B
C
C
D
D
E
E
F
F
G G
H
H
I
I
K
K
CY
P3A
4/vi
llin0.
00.
20.
40.
60.
81.
01.
2
Control Garlic
A
A
B
BC
CDD
E
E
F
F
G
GH
H
I
IK
K
EM
BR
T0.
00.
51.
01.
52.
02.
5Control Garlic
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Figure 3:
Time (h)
Sim
vast
atin
/ Si
mva
stat
in a
cid
[ug/
l]
0 4 8 12 16 20 24
01
23
45
6
A
A
B
B
C CD DE
EF
F
G G
H
HI
I
KK
AU
C s
imva
stat
in [u
g.h/
l]0
2040
6080
Control Garlic
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Figure 4:
A
A
B
B
C
C
D
D
E
E
F
F
G
G
H
H
I
I
K
K
AU
C p
rava
stat
in [u
g.h/
l]0
2040
6080
Control GarlicTime (h)
Pra
vast
atin
and
lact
one
[ug/
l]
0 4 8 12 16 20 24
05
1015
20
Time (h)
R-4
16 [u
g/l]
0 4 8 12 16 20 24
05
1015
20
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Figure 5:
A
A
B
B
C
C
D
D
E
E
F
F
G
G
H
H
I
I
K
K
AUC
saq
uina
vir
01
23
4
Control GarlicTime (h)
Saq
uina
vir [
mg/
l]
0 2 4 6 8
0.0
0.2
0.4
0.6
0.8
A
BC
D
E
F
G
HI
K
PGP garlic / baselineA
UC
saq
uina
vir g
arlic
/ ba
selin
e0.5 1.0 1.5 2.0
0.0
0.5
1.0
1.5
