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Title: "Quercetin and rutin reduced the bioavailability of cyclosporine from Neoral, an immunosuppressant, through activating P-glycoprotein and CYP 3A4" Author(s): Yu, Chung-Ping; Wu, Ping-Ping; Hou, Yu-Chi; Lin, Shiuan-Pey; Tsai, Shang-Yuan; Chen Chiung-Tong; Chao, Pei-Dawn
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1
Quercetin and rutin reduced the bioavailability of cyclosporine from
Neoral
®, an immunosuppressant, through activating P-glycoprotein
and CYP 3A4
Chung-Ping Yu†,∥
, Ping-Ping Wu‡,∥
, Yu-Chi Hou※, Shiuan-Pey Lin‡,
Shang-Yuan Tsai‡, Chiung-Tong Chen§, Pei-Dawn Lee Chao*,‡
†
School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung, Taiwan 404, ROC. ‡School of Pharmacy, China
Medical University, Taichung, Taiwan 404, ROC. ※Department of Medical Research, China Medical University Hospital, Taichung, Taiwan 404, ROC.§Institute of
Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan 350, ROC. ∥
These authors contributed equally to the study
Corresponding Author:
Prof. Pei-Dawn Lee Chao
School of Pharmacy, China Medical University, 91 Hsueh-Shih Rd, Taichung, Taiwan 40402, ROC.
Telephone and fax numbers: 886-4-22031028 E-mail: [email protected]
2
ABSTRACT
1
Quercetin and rutin are popular flavonoids in plant foods, herbs and dietary 2
supplements. Cyclosporine (CSP), an immunosuppressant with narrow therapeutic 3
window,is a substrate of P-glycoprotein (P-gp) and cytochrome P-450 3A4 (CYP3A4). 4
This study investigated the effects of quercetin and rutin on CSP pharmacokinetics 5
from Neoral®and relevant mechanisms. Rats were orally administered Neoral® with and 6
without quercetin or rutin. Blood CSP concentration was assayed by a specific 7
monoclonal fluorescence polarization immunoassay. The results showed that quercetin 8
and rutin significantly decreased the Cmax of CSP by 67.8% and 63.2%, and reduced the
9
AUC0-540 by 43.3% and 57.2%, respectively. The in vitro studies indicated that the
10
quercetin and rutin induced the functions of P-gp and CYP3A4. In conclusion, 11
quercetin and rutin decreased the bioavailability of CSP through activating P-gp and 12
CYP3A. Transplant patients treated with Neoral® should avoid concurrent consumption 13
of quercetin or rutin to minimize the risk of allograft rejection. 14
15
KEYWORDS:
bioavailability; cyclosporine; quercetin; rutin; P-gp; CYP 3A4
3
INTRODUCTION
1
Flavonoids are a group of natural polyphenols widely distributed in plants. Many 2
epidemiological studies showed that high flavonoid intake lowered the occurrences of 3
coronary heart disease and possibly cancer (1). In addition, their abilities to modulate 4
cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) draw more interests than 5
ever for the roles they play in drug interactions (2, 3). 6
Quercetin (chemical structure shown in Figure 1), an ubiquitous flavonoid, and its 7
glycosides are popular constituents in plant foods and medicinal herbs, such as onion, 8
grapefruit, strawberry, grape, ginkgo and St. John's wort (SJW). Quercetin has been 9
reported to exert numerous pharmacological activities, such as free radical scavenging 10
(4), TNF-alpha inhibition (5), and anticarcinogenic effects (6-9). Rutin (chemical 11
structure shown in Figure 1), a glycoside of quercetin, is more abundant than quercetin 12
in plants and has been used for improving intermittent claudication. Nowadays, 13
commercial products of dietary supplements containing rich rutin and quercetin are 14
easily purchasable in the markets and the recommended dose was 250-500 mg twice 15
per day. Rutin has been known to be hydrolyzed into quercetin in gut lumen and 16
thought to demonstrate similar bioactivities as quercetin (10, 11). A previous study 17
reported that quercetin was an inhibitor of CYP 3A4 in vitro (12), while conflicting 18
modulation effects of quercetin on P-gp, either inhibition or stimulation, had been 19
4 demonstrated in different models (12-14). 1
Cyclosporine (CSP) is an important immunosuppressant with narrow therapeutic 2
window. Clinically, supratherapeutic CSP blood level would cause adverse effects 3
including nephrotoxicity, hepatotoxicity and neurotoxicity. Conversely, subtherapeutic 4
blood level would cause allograft rejection in transplant patients (15). The metabolism 5
and transport of CSP were found to be associated with CYP3A4 and P-gp, respectively 6
(16, 17). Accordingly, any modulator of P-gp or CYP3A4 may alter the 7
pharmacokinetics and pharmacodynamics of CSP. 8
The original oil-based formulation Sandimmune® demonstrated unpredictable 9
absorption of CSP and resulted in an increased frequency of acute and chronic rejection 10
in patients with poor bioavailability. Subsequently, a new microemulsion dosage form 11
Neoral® was thus developed to cope with this problem (18, 19). The Neoral® 12
formulation has self-emulsifying properties, which is less dependent on bile salts for 13
absorption than Sandimmune (19-21). Compared with Sandimmune®, Neoral® 14
provides increased bioavailability as evident in increased area under the curve (AUC), 15
increased peak blood concentration (Cmax) and decreased time to peak blood
16
concentration (Tmax).
17
Although our previous study had reported decreased bioavailability of 18
Sandimmune® by coadministration of quercetin in pigs and rats (22), this study 19
5
continued to access the effects of both quercetin and its glycoside rutin on the 1
pharmacokinetics of CSP from the new dosage form Neoral® in rats. Furthermore, in 2
vitro models including LS180 cell line and recombinant CYP3A4 isozyme were used to
3
identify the possible mechanisms of interaction. 4
5
MATERIALS AND METHODS
67
Chemicals and reagents. Cyclosporine (Neoral®, 100 mg/mL) was kindly provided by
8
Novartis (Taiwan) Co. Ltd. Rutin hydrate (purity 95 %), quercetin (purity 98 %), 9
glycofurol, rhodamine 123, sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), 10
3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton X-100, 11
verapamil and sulfatase (type H-1 from Helix pomatia) were purchased from Sigma (St. 12
Louis, MO, USA). Dulbecco's Modified Eagle Medium (DMEM), trypsin/EDTA, 13
nonessential amino acid, Hank's Buffered Salt Solution (HBSS), 14
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and Vivid® CYP450
15
screening kits were purchased from Invitrogen (Grand Island, NY, USA). TDx kit was 16
supplied by Abbott Laboratories (Abbott Park, IL, USA). Milli-Q plus water (Millipore, 17
Bedford, MA, USA) was used for all preparations. 18
Drug administration and blood collection. Eighteen Sprague-Dawley rats weighing
6
250-350 g were randomly divided into three groups. The rats were fasted for 12 h 1
before dosing and food was withheld for another 3 h. Water was supplied ad libitum. 2
CSP solution was prepared by diluting Neoral® with deionized water to afford a 3
concentration of 625 μg/mL. Quercetin and rutin were dissolved in glycofurol. CSP 4
(1.25 mg/kg) was given orally with and without an oral dose of 50 mg/kg of quercetin 5
and 110 mg/kgof rutin (equimolar with 50 mg/ kg of quercetin) in a parallel design. 6
Control rats received equal volume of glycofurol (1.0 mL/kg) as blank vehicle. CSP 7
was administered immediately after quercetin and rutin. Blood samples were withdrawn 8
via cardiopuncture at 20, 40, 60, 180, 300 and 540 min after dosing of CSP. 9
For all treatments described above, the blood samples were collected into small 10
plastic vials containing EDTA and assayed within 24 h. Water was supplemented to rats 11
by feeding with gastric gavage at specific time during experiment. One week was 12
allowed for washout. This animal study protocol has been approved by China Medical 13
University, Taichung, Taiwan (CMU95-79-N) and all animal experiments adhered to 14
‘‘The Guidebook for the Care and Use of Laboratory Animals’’ published by the 15
Chinese Society of Animal Science, Taiwan, R.O.C. 16
Quantitation of blood CSP concentration. CSP concentration in blood was measured
17
by a specific monoclonal fluorescence polarization immunoassay (Abbott, Abbott Park, 18
III, USA). Validation of calibration curve was conducted by testing three controls 19
7
before sample assay. Otherwise, a new calibration curve will be constructed if necessary. 1
The calibration range was 0.0 - 1500.0 ng/mL and the LLOQ was 25.0 ng/mL. 2
Cell line and culture conditions. LS 180, the human colon adenocarcinoma cell line,
3
was obtained from the Food Industry Research and Development Institute (Hsinchu, 4
Taiwan). Cells were cultured in DMEM medium supplemented with 10% fetal bovine 5
serum (Biological Industries Ltd., Kibbutz Beit Haemek, Israel), 0.1 mM nonessential 6
amino acid, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 292 μg/mL of 7
glutamine. Cells were grown at 37℃ in a humidified incubator containing 5 % CO2.
8
The medium was changed every other day and cells were subcultured when 80 % to 90 9
% confluency was reached. 10
Cell viability assay. The effects of quercetin, rutin, verapamil and DMSO on the
11
viability of LS 180 cells was evaluated by MTT assay (23). Cells were seeded into a 12
96-well plate. After overnight incubation, the tested agents were added into the wells 13
and incubated for 72 h, then 15 μL of MTT (5 mg/mL) was added into each well and 14
incubated for additional 4 h. During this period, MTT was reduced to formazan crystal 15
by live cells. Acid-SDS (10 %) solution was added to dissolve the purple crystal at the 16
end of incubation and the optical density was detected at 570 nm by a microplate reader 17
(BioTex, Highland Park, Winooski, VT, U.S.A.). 18
Effects of quercetin and rutin on P-gp activity. The transport assay of rhodamine 123
8
was modified from a previous method (24). Briefly, LS 180 cells (1×105) were cultured
1
in each well in a 96-well plate. After overnight incubation, the medium was removed 2
and washed three times with ice-cold PBS buffer. Rhodamine 123in HBSS (1 μM, 100 3
μL) was added into each well and incubated at 37℃. After 1-h incubation, the 4
supernatants were removed and washed for three times with ice-cold PBS. Then, 5
quercetin, rutin, verapamil (as a positive control of P-gp inhibitor) and DMSO were 6
added to correspondent wells and incubated at 37℃. After 4-h incubation, the medium 7
was removed and the cells were washed three times with ice-cold PBS. Subsequently, 8
100 μL of 0.1 % Triton X-100 was added to lyse the cells, and the fluorescence was 9
measured with excitation at 485 nm and emission at 528 nm.To quantitate the content 10
of protein in each well, 10 μL of cell lysate was added to 200 μL of diluted protein 11
assay reagent (Bio-Rad, Hercules, CA, U.S.A.) and the optical density was measured at 12
570 nm. The relative intracellular accumulation of rhodamine 123 was calculated by 13
comparing with that of control. 14
Preparation and characterization of serum metabolites of rutin. In order to mimic
15
the molecules interacting with CYP 3A in enterocytes, the serum metabolites of rutin in 16
rats were prepared and characterized. Rutin was orally administered at 250 mg/kg to 17
rats fasted overnight. Blood was collected via cardiopuncture at 30 min after dosing. 18
After coagulation, the serum was vortexed with 3-fold volume of methanol. After 19
9
centrifuging at 10,000 g for 15 min, the supernatant was concentrated in a rotatory 1
evaporator under vacuum to dryness. To the residue, appropriate volume of water was 2
added to afford a solution with 10-fold serum concentration, which was divided into 3
aliquots and stored at -80℃ for later use. 4
A portion of the metabolite solution was characterized following a method 5
reported previously (25). Briefly, 200 μL serum sample was mixed with 100 μL 6
sulfatase (containing 100 units/mL of sulfatase and 3560 units/mL of β-glucuronidase), 7
50 μL ascorbic acid (200 mg/mL) and incubated at 37°C for 1 h under anaerobic 8
condition. After hydrolysis, the serum was acidified with 0.1N HCl and partitioned with 9
ethyl acetate (containing 6,7-dimethoxycoumarin as internal standard). The ethyl 10
acetate layer was evaporated under N2 to dryness and reconstituted with an appropriate
11
volume of methanol prior to HPLC analysis. On the other hand, blank serum was 12
vortexed with 3-fold volume of methanol to prepare deproteinized specimens with 1/8- 13
and 1/4- fold serum concentrations as controls for comparison with correspondent 14
specimens of serum metabolites of rutin. 15
Effects of serum metabolites of rutin on CYP3A4 activity. Vivid® CYP450
16
screening kits (Invitrogen, Carlsbad, CA, U.S.A.) was used to evaluate the effect of 17
serum metabolites of rutin on the activity of CYP3A. All the procedures were 18
performed according the manual provided by the manufacturer. Briefly, after incubating 19
10
serum metabolites of rutin (1/4- and 1/8- fold serum concentration) or deproteinized 1
blank serum specimen with CYP450 recombinant BACULOSOMES®,
2
glucose-6-phosphate and glucose-6-phosphate dehydrogenase in 96-well black plate at 3
room temperature for 20 min, a specific CYP3A substrate (Vivid® BOMR) and NADP+
4
were added and incubated at room temperature for another 30 min. At the end of 5
incubation, ketoconazole was added to stop the reaction and the fluorescence was 6
measured with excitation at 530 nm and emission at 590 nm. 7
Data analysis. The pharmacokinetic parameters of cyclosporine were calculated using
8
noncompartment model with the aid of WINNONLIN (version 1.1, SCI software, 9
Statistical Consulting, Inc., Apex, NC). The peak blood concentrations (Cmax) were
10
obtained from experimental observation. The area under the serum concentration-time 11
curve (AUC0-t) was calculated using trapezoidal rule to the last point. The statistical
12
software SPSS was used for analyzing the differences among treatments by using 13
ANOVA for three groups and unpaired Student’s t-test for two groups. Statistical 14
significance level was set at p < 0.05. 15
16
RESULTS
17The blood profiles of CSP in rats administered Neoral® with and without 18
quercetin (50 mg/kg) and rutin (110 mg/kg) are shown in Figure 2 and the 19
11
pharmacokinetic parameters of three treatments are listed in Table 1. The results 1
showed that quercetin and rutin significantly decreased the Cmax of CSP by 67.8% and
2
63.2%, and reduced the AUC0-540 by 43.3% and 57.2%, respectively.
3
To explore the possible involvement of P-gp in the observed pharmacokinetic 4
interaction, LS 180 was used for transport assay employing a typical P-gp substrate 5
rhodamine 123. MTT assay showed that incubation of quercetin (50 μM) and rutin (50 6
μM) with LS 180 for 72 h exerted no significant influences on cell viability. In 7
transport assay, the accumulation of rhodamine 123 in LS 180 cells measured after 4-h 8
incubation with tested agents are shown in Figure 3. The positive control verapamil at 9
100 μM significantly increased the intracellular accumulation of rhodamine 123 by 10
54.1%, whereas DMSO at 0.5 % (v/v) did not show significant influence. Quercetin at 11
10 and 50 μM significantly decreased the intracellular accumulation of rhodamine 123 12
by 29.6 and23.6 %, and rutin at 10 and 50 μM significantly decreased the intracellular 13
accumulation of rhodamine 123 by 19.5 and 31.8 %, respectively. 14
Characterization of rutin metabolites in the serum specimen showed that the 15
major molecules were quercetin glucuronides/sulfates in a concentration of 3.4 16
nmol/mL. The effects of rutin metabolites at 1/8- and 1/4- fold serum concentrations 17
on CYP3A activity are shown in Figure 4. As a positive control, ketoconazole at 10 18
μM significantly decreased CYP3A activity by 90.8 %. Contrary to the effect of 19
12
ketoconazole, rutin metabolites at 1/8- and 1/4- fold serum concentration significantly 1
increased CYP3A4 activity by 208.0 and 194.0 %, respectively, when compared to 2
those of correspondent concentration of deproteinized blank serum specimen. 3
4
DISCUSSIONS
5The use of botanical products as antioxidant supplements is on the rise among the 6
global population in recent decades. Although the safety profile of many botanical 7
products is promising, accumulated evidences showed significant interactions with 8
critical medicines, which can place individual patients at great risk. This study found 9
that the oral bioavailability of CSP from Neoral® was significantly decreased by 10
quercetin and rutin, which might result in subtherapeutic blood level of CSP and pose 11
transplant patients to a non-negligible hidden risk of allograft rejection. 12
Owing to the poor solubility of quercetin and rutin in water, glycofurol was used to 13
prepare the oral dosing solution in this study. We previously found that CSP 14
bioavailability was markedly reduced in second dose administration of Neoral® in rats 15
(22), a protocol of parallel design was thus conducted. The result of this study showing 16
that quercetin markedly decreased the bioavailabilityof CSP from Neoral® was in good 17
agreement with that reported for the oil-based Sandimmune® (26). In regard to rutin - 18
CSP interaction, this is the first report to demonstrate that rutin likewise reduced the the 19
13
bioavailabilityof CSP. Being a glycoside of quercetin, rutin has been known to be 1
hydrolyzed to quercetin in gut lumen and then presented as quercetin 2
sulfates/glucuronides in the circulation, which was the same as the metabolic fate of 3
quercetin (27-29). Therefore, that equimolar doses of rutin and quercetin conferred 4
comparable interaction with CSP can be accounted for by their metabolic relevance. 5
P-gp has been recognized to play an important role in the barrier function of the 6
intestine and drug - drug interactions (30). To explore the possible involvement of P-gp 7
in these interactions, transport assay of rhodamine 123 was conducted by using LS 180 8
cells. The MTT assay of LS 180 showed that quercetin and rutin below 50 μM did not 9
affect the cell viability, indicating that the cells were normal throughout the experiment 10
period. As shown in Figure 2, contrary to verapamil (a positive control of P-gp 11
inhibitor), quercetin and rutin significantly decreased the intracellular accumulation of 12
rhodamine 123, indicating activation of P-gp, which was in agreement with the findings 13
of two previous studies (13, 31) and could in part explain the decreased blood levels of 14
CSP in rats. On the contrary, quercetin has been reported as an inhibitor of P-gp in 15
numerous studies using breast and pancreatic cell lines (12, 13, 32-34), which was 16
apparently not consistent with our in vivo evidences. In regard to these discrepant 17
effects of quercetin on P-gp among in vitro studies, either inhibition or stimulation, we 18
contemplate that it might be arisen from the differences of cell models in use. We 19
14
suspect that different metabolic capability among cell lines may result in differential 1
amount of quercetin metabolites after incubation with quercetin for certain duration, 2
which may lead to discrepant effects on P-gp activity. Therefore, cellular metabolism of 3
quercetin in various cell lines requires more future studies. 4
Pharmacokinetic studies of quercetin and rutin have identified 5
glucuronides/sulfates of quercetin being the major molecules in the circulation (25, 27). 6
We proposed that the serum metabolite of rutin could mimic the molecules interacting 7
with enteric or hepatic CYP3A, which located in the microsome of cells, after intake of 8
either rutin or quercetin. Therefore, we had prepared and characterized the serum 9
metabolite of rutin from rats to evaluate the in vivo effects of rutin and quercetin on 10
CYP 3A activity. Our results showing that the serum metabolite of rutin, containing 11
mainly quercetin glucuronides/sulfates, increased CYP 3A activity clearly implied that 12
CYP3A–mediated mechanism can explain in part the decreased bioavailability of CSP 13
caused by rutin or quercetin in rats. This novel approach was different from most in 14
vitro studies reporting the effects of herbal extract or natural compounds on CYP 3A4
15
by using their parent forms, which may not represent the true molecules interacting 16
with CYP 3A4 in vivo. Herein our finding is opposite to a previous in vitro study 17
reporting that quercetin was an inhibitor of CYP 3A (12), which apparently had not 18
taken the metabolism of quercetin by gut into consideration and could not explain our 19
15
in vivo evidence. Therefore, we suggest that in order to mimic the biological system,
1
understanding of presystemic metabolism of natural polyphenols is very important 2
before in vitro studies. 3
In recent decade, many cases of subtherapeutic blood CSP concentration caused by 4
SJW has brought about increasing interests in herb - drug interactions, because many of 5
which were life-threatening (28, 29, 35, 36). In regard to the mechanism of interaction, 6
SJW has been shown to increase the metabolism of various drugs, such as CSP, oral 7
contraceptives and indinavir, through induction of CYP3A4 activity (37, 38). We 8
suspect that the antioxidant supplements rutin and quercetin may bring about risks of 9
critical interactions with western medicines as SJW did. 10
In conclusion, quercetin and rutin significantly reduced the oral bioavailability of 11
CSP through activating P-gp and CYP 3A4. We suggest that transplant patients treated 12
with CSP should avoid concomitant intake of dietary supplements containing rich 13
quercetin and rutin to minimize the risk of allograft rejection. 14
16
ABBREVIATIONS USED
1
P-gp, p-glycoprotein; CYP3A4, cytochrome P-450 3A4; SJW, St. John's Wort. 2
ACKNOWLEDGEMENT
3This work was in part supported by the National Science Council, ROC. (NSC
4
99-2320-B-039-017-MY3, NSC99-2628-B-039-005-MY3), Taiwan Department of 5
Health Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004) and 6
China Medical University, Taichung, Taiwan, ROC. (CMU98-S-34, CMU98-S-32). 7
17
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Figure captions
Figure 1. Chemical structures of quercetin and rutin.
Figure 2. Mean (±S.D.) blood concentration-time profiles of cyclosporine (CSP) after oral administration of CSP alone (1.25 mg kg-1) (
z) and coadministration
with quercetin (50 mg kg-1) (
c) and rutin (110 mg kg-1) (▼) to six rats in
each group.
Figure 3. Effects of quercetin (Q, μM), rutin (R, μM) and verapamil (V, 100 μM) on the accumulation of rhodamine 123 in LS 180 cells.
*p < 0.05, *** p < 0.001
Figure 4. Effects of serum metabolite of rutin (R, 1/4- and 1/8 -fold serum concentration) and ketoconazole (Keto, 10 μM) on CYP3A4 activity.
24
Table 1. Pharmacokinetic parameters of cyclosporine (CSP) after oral administration of CSP alone (1.25 mg kg-1) and coadministration with quercetin (50 mg kg-1) and rutin (110 mg kg-1) to six rats in each group.
Treatments Parameters
CSP alone CSP + quercetin CSP + rutin
Cmax (ng mL-1) 261.5 ± 114.0 a 84.1 ± 6.9 b (-67.8%) 96.3 ± 45.1 b (-63.2%) AUC0-540 (μg‧min mL-1) 65.5 ± 25.8 a 37.2 ± 2.2b (-43.3%) 28.0 ± 11.1b (-57.2%) MRT (min) 225.5 ± 17.7 267.3 ± 5.2 224.4 ± 21.1
Data expressed as mean ± S.D. Means in a row without a common superscript differ.
P <0.05. Cmax: peak blood concentration. AUC0-540: area under the blood
25 O OH HO O OH OH OH O OH HO O O OH OH CH2 OH OH O O OH CH3 HO HO HO Quercetin Rutin Figure 1.
26 Time (min) 20 40 60 180 300 540 Bl ood concent rat ion of cy cl os po ri ne (ng/ m L ) 100 200 300 400 cyclosporine alone cyclosporine + quercetin cyclosporine + rutin Figure 2.
27 Con trol V-10 0 Q-1 0 Q-5 0 R-10 R-50 Relative intensit y (%) 0 20 40 60 80 100 120 140 160 180 *** *** *** *** * Figure 3.
28 control keto R-1/4 R-1/8 CYP3A4 activ ity (%) 0 50 100 150 200 250 * *** *** Figure 4.
