中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/25128

行政院國家科學委員會專題研究計畫 期中精簡報告

中藥食品與西藥之交互作用(1/2)

中 華 民 國 92 年 5 月 5 日

MARKED DECREASE OF CYCLOSPORINE ABSORPTION CAUSED BY COADMINISTRATION OF GINKGO AND ONION IN RATS

Chao P.D.L. 3Yang C.Y.1 , Yeh Y. 1 , Hou Y.C.2 , Hsiu S.L. 3 ,

1

Institute of Chinese Pharmaceutical Science, Taiwan, R.O.C. 2Graduate Institute of Natural

Products, Chang-Gung University, Taiwan, R.O.C. 3

Department of Pharmacy, China Medical College, Taichung, Taiwan, R.O.C

Introduction: Cyclosporine is an immunosuppressive agent with narrow

therapeutic range and was found to be substrate of CYP3A4 and P-glycoprotein (Pgp). Ginkgo and onion contain quercetin and its glycosides which were reported to alter

the activit ies of CYP3A4 and Pgp. For organ transplant patients treated with cyclosporine, ginkgo and onion might affect the therapeutic outcome.

M ethods: Rats were given Neoral® (cyclosporine) alone or coadministered with quercetin, ginkgo and onion respectively. The blood samples were withdrawn via cardiopuncture. The blood concentration of cyclosporine was determined by a specific monoclonal FPIA method. The in vitro everted intestine sac study was conducted to explore the mechanism of interaction.

Results: C yc lo spo r ine abso r pt io n wa s s ig nif ica nt ly de creased by t he

coadministrations of quercetin, ginkgo and onion, respectively, by 43 %, 51 % and 82 %, respectively. The mechanism for this interaction was not explainable by their in vitro effects on Pgp.

Conclusion: Herb-food-drug interaction is a hidden risk for patients. When

patients are prescribed Neoral®, concurrent use of quercetin glycoside-containing dietary supplements and herbs should be taken into consideration by clinicians.

Flavonoids have attracted increasing attention in recent years because of their

various beneficial bioactivities including antioxidation (1), free radical scavenging (2),

anticancer (3), antivirus (4) etc. and furthermore, their additional abilities to modulate

both CYP 3A4 and P-glycoprotein (Pgp), the product of mdr (multidrug - resistance)

genes (5-8). CYP3A4 is mainly present in intestine and liver. The significant role of

CYP 3A4 for drug – drug interactions was well recognized. Pgp is expressed in

various normal human tissues such as small intestine, kidney, liver and capillary

e nd o t he lia l c e l ls o f br a in a nd t e st e s ( 9-11), and it s s ig nif ic a nt ro les fo r

chemoprevention of organisms and drug - drug interaction had been proposed (12-14).

Quercetin was shown to be a potent inhibitor of CYP3A4 in in vitro studies (15). In

our laboratory, an in vitro everted intestinal sac study indicated that quercetin

inhibited the function of P-gp in a dose – dependent manner as shown in Fig. 1.

Quercetin, the most popular flavonoid in food and herbs, is present mostly as

glycoside in nature. Ginkgo, an herbal product of the leaves of Ginkgo biloba widely

used for treating neurodegeneration and cardiovascular disease as well as a dietary

supplement worldwide, contains quercetin glycoside which was considered as one of

the active components for standardization. Onion, a common food and also a dietary

supplement in Europe, contain 0.03% of quercetin glycoside. The fate of flavonoid

hydrolyzed by enterobacteria in gastrointestinal tract and transformed into quercetin

which was absorbable into circulation.

Cyclosporin is a widely used immunosuppressant with a narrow therapeutic

range. Cyclosporin is a substrate of both CYP 3A4 (16) and Pgp (17). In a recent

report, inhibition of P-glycoprotein was proposed to be a more important mechanism for

enhanced cyclosporin absorption than inhibition of CYP 3A4 (18). In this study, we

attempted to investigate the in vivo effect of quercetin, ginkgo and onion on the

pharmacokinatics of cyclosporin in rats

Materials and Methods

Chemicals

Cyclosporin (Sandimmune Neoral®, 100 mg/ml) was kindly offered by

Novartis (Taiwan) Co. Ltd.. Rhodamine 123 was purchased from Aldrich (Milw. WI,

U. S. A.). Glycofurol and medium 199 were supplied by Sigma (St. Louis, MI, U. S.

A.) Milli-Q plus Water (Millipore, Bedford, MA, U. S. A.) was used for all

preparation.

Everted intestine sac study

Fe ma le Spr ague-Dawley rats were sacrificed. The jejunum (30 cm long from

stomach) and ileum (30 cm long from the ileocecum) were isolated. After flushing

prepare a 25 cm long everted sac. Then the sac was immersed into 50 ml medium 199

prewarmed at 37 0C and preoxygenated with 95% O2/5% CO2.. After incubating for

20 min, 3 ml rhodamine 123 solution (20 µg/ml in medium 199) was introduced into

the everted sac (serosal side). Under bubbling with the 95% O2/5% CO2 mixture gas,

the transport of rhodamine 123 solution from serosal to mucosal surfaces across the

intestine was measured by sampling the mucosal medium every 20 min until 100 min.

On the other hand, quercetin or rutin was dissolved with glycofurol and added to the

mucosal medium in order to give designated final concentrations of 200 and 400 µM.

The transport of rhodamine 123 in the absence (control) or presence of the modulator

was measured fluorometrically using Luminescence Spectrometer LS-50B (Perkin

Elmer, U. S. A.).

Drug administration and blood collection

Female Sprague-Dawley rats (n = 6-12) weighing 200 ~ 300 g were fasted for

12 hr before drug administration. Half of them were given 1.25 mg/kg cyclosporin

which had been prepared by diluting Neoral® with deionized water, whereas the other

half rats were administered a single dose of 50 mg/kg quercetin, 2 g/kg ginkgo leaf (as

water decoction) and 2 ml/rat onion juice 30 sec before cyclosporin via gastric gavage.

The rats were randomly assigned to receive monotherapy and combined therapy in a

treatment. Blood samples (0.3 ml) were withdrawn via cardiopuncture at 0, 20, 40

min, 1, 3, 5, 7 and 9 hr after drug administration. The blood was collected into small

plastic vials containing EDTA and assayed within one week.

Quantitation of blood cyclosporin concentrations

Cyclosporin concentration in blo o d was measured by using a specific

monoclonal fluorescence polarization immunoassay (Abbott, Abbott Park, Ill, USA).

The assay was calibrated for concentrations from 25.0 to 1500.0 ng/ml.

Data analysis

Pharmacokinet ic parameters of cyclo sporin were calculated by using

noncompartment model of WINNONLIN (version 1.1, SCI software, Statistical

Consulting, Inc., Apex, NC). Unpaired and paired Student’s t-tests were used for quercetin treatment and ginkgo or onion treatment, respectively, taking p < 0.05 as

significan

t.

Results and Discussion

Fig. 1 shows the effect of quercetin on the efflux transport of rhodamin 123

fro m the serosal side to the mucosal side. The result indicated that quercetin

significantly inhibited the function of intestinal Pgp in a dose - dependant manner for

ginkgo and onion on the absorption and disposition of cyclosporin. Fig. 2, 3 and 4

depict the blood profiles of cyclosporin after administration of cyclosporin alone and

coadministration with a single dose of quercetin, ginkgo and onion, respectively. The

pharmacokinetic parameters of cyclosporin for monotherapy and combined therapy

are given in Table 1, 2 and 3. Our results showed that quercetin, ginkgo and onion

significantly decreased the AUC of cyclosporin by 43 %, 51 % and 82 %, respectively,

indicating that the bioavailability of cyclosporin was markedly decreased. These

results could not be explainable by the inhibition of intestinal Pgp by quercetin as

observed from the in vitro studies. Modulat ion of Pgp and/or CYP 3A4 is an

important mechanism of drug interaction. However, the fate of a drug and metabolites

frequently is not only determined by Pgp and/or CYP 3A4, but also by other

metabolic enzymes and possibly by other transporters e.g. MRP (multidrug resistance

protein) (14).

Several phamacokinetic studies of quercetin glycosides reported that it is

indispensable to be hydrolyzed into quercetin before absorption. Quercetin was then

rapidly metabolized into its glucuronides/sulfates by the enterocytes and hepatocytes

and circulating in the bloodstream as these conjugated metabolites. Therefore, the

glucuronides/sulfates of quercetin derived in vivo from glycosides might play a more

significant role of the glucuronidated/sulfated metabolites of flavonoids for drug -

d r u g i n t e r a c t io n i s w o r t h y o f i n v e s t ig a t io n . T h e d ir e c t a c t io n o f t h e

glucuronides/sulfates on Pgp and CYP 3A4 need to be further studied.

The pharmacokinetic interaction study was carried out in a crossover design for

ginkgo and onion, whereas for quercetin a parallel design was conducted because

glycofurol, the solvent used to dissolve quercetin, would even lower the blood

cyclosporin level at the second treatment after one week wash – out. The data analysis

of quercetin study was thus calculated based on the comparisons between two parallel

groups. It is suggested that when using organic solvent to dissolve the precipitant drug

in a drug - cyclosporin (Sandimmun Neoral®) study, a parallel design is preferable to

crossover design.

In recent years, many transplant recipients were reported to show subtherapeutic

cyclosporin concentrations after they started self-medicating with St. John’s Wort (Hypericum perforatum) (19, 20). St. John’s Wort, containing quercetin and its glycosides, was likely to act as potent inducer of hepatic enzymes. Most in vitro

studies have agreed that St. John’s Wort doubles the metabolic activity of CYP 3A4.

Other cytochrome P-450 isoenzymes as well as Pgp, may be affected by St. John’s Wort (21). It is proposed that herbs represent a potential and possibly an overlooked

In summary, ginkgo and onion markedly decreased the oral bioavailability of

cyclosporin. Because cyclosporin is a substrate of both Pgp and CYP 3A4, we suggest

that the coadministration of quercetin or quercetin – containing herb or food with cyclosporin or other medications whose absorption and metabolism are mediated by

Pgp and/or CYP 3A4 should require close monitoring. Healthcare providers should be

cautious of the hidden risk of these herb – drug interactions.

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( a )

( b )

Fig.. 1. Mean (



quercetin ; (n=3, * p < 0.05). T i m e ( m i n ) 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 Co n ce n tr a ti o n o f rh o d a m in e 1 2 3 ( g /m l) 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 c o n t r o l q u e r c e t i n ( 2 0 0  M ) q u e r c e t i n ( 4 0 0  M ) Tim e (m in) 0 20 4 0 6 0 80 1 0 0 1 2 0 C o n ce n tr a ti o n o f rh o d a m in e 1 2 3 ( g /m l) 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 control quercetin (200 M ) quercetin (400 M ) * * * *

Time (min) 0 2040 60 180 300 540 Cy c lo s p o ri n e b lo o d c o n c e n tr a ti o n ( n g /mL ) 0 100 200 300

cyclosporine with tetraglycol cyclosporine with quercetin

Fig. 2 Mean (±S.E.) blood concentration-time profiles of cyclosporine after oral administration of cyclosporine with tetraglycol in 6 rats (●) and coadministration with quercetin (50 mg/kg) in another 6 rats (△),

Fig. 3. Mean ( s. e.) blood concentration – time profiles of cyclosporin after administation of cyclosporin alone (●) and coadministration with ginkgo (o) in

6 rats. T im e (m in) 0 20 40 60 1 8 0 3 0 0 N e o ra l (n g /m L ) 0 50 100 150 200 250 C o n tro l G in kg o b iloba

T im e ( m in ) 0 1 0 0 20 0 3 0 0 4 0 0 5 0 0 6 0 0 cyclos porine conc. ( ng/ml ) 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 _{o n io n} c o n tr o l

Fig. 4. Mean ( s. e.) blood concentration – time profiles of cyclosporin after

administation of cyclosporin alone (o) and coadministration with onion (●) in 7 rats.

Table 1. Comparison of pharmacokinetic parameters of cyclosporine in rats between receiving 1.25mg/ kg cyclosporine alone and coadministration with 50 mg/kg quercetin.

With quercetin Cyclosporin alone

P a r a met e r s

Mean  S.E. Mean  S.E.

Difference (%)

AUC0-5h(ngminmL-1) 3.72 (± 0.09)E4 6.55 (± 1.05)E4 -43.3

* Tmax(min) 30.0 ± 6.8 43.3 ± 3.3 -30.7 Cmax(ngmL-1) 84.1 ± 2.8 261.5 ± 46.6 -67.8** MRT(min) 267.3 ± 2.1 225.5 ± 7.2 18.5*** ＊ p＜0.05, ＊＊p＜0.01, ＊＊＊p＜0.001

Table 2. Comparison of pharmacokinetic parameters of cyclosporine in 6 rats between receiving cyclosporine alone and coadministration with decoction of Gnkgo biloba.

Cyclosporin Alone With Ginkgo

P a r a met e r s

Mean  S.E. Mean  S.E.

Difference (%) AUC0-5h(ngminmL-1) 26024.4  4011.3 12737.0  1306.4 -51.1* Tmax(min) 30.0  4.5 36.7  3.3 22.2 Cmax(ngmL-1) 169.4  35.6 65.2  7.9 -61.5* MRT(min) 11 4 . 6  4.6 134.1  3.5 17.0* *p < 0.05

Table 3. Comparision of pharmacokinetic parameters of cyclosporine in 7 rats etween receiving cyclosporin alone and coadministration with onion juice

Par a met er Cyclosporin Alone M e a nS.E With Onion Mea nS.E Difference (%) AUC0-9h（ng.min.mL-1） 89594.219343.9 -82.1** Tmax 88. 9 Cmax（ng.mL-1） 2 5 . 73.7 331.169.0 16068.23758.7 4 8 . 64.0 9 8 . 416.5 -70.3**