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Ginkgo biloba extract attenuates warfarin-mediated anticoagulation through induction of hepatic cytochrome p450 enzymes by bilobalide in mice.

ABSTRACT

Ginkgo biloba extract (GBE) is a popular herbal ingredient used worldwide, but it is reported to induce bleeding as a serious adverse event. In this study we examined whether GBE induced spontaneous bleeding or accelerated warfarin anticoagulation via herb-drug interaction. Mice were given GBE or various active components of GBE orally for 5 days and blood coagulation parameters and hepatic cytochrome P450 enzymes (CYPs) were measured. Mice also received warfarin (racemate, (S)- or (R)-enantiomer) for the last 3 days of the 5-day regimen to examine GBE-warfarin interactions. Neither GBE (up to 1000 mg/kg) nor ginkgolide B (up to 140 mg/kg), a platelet-activating factor antagonist, influenced blood coagulation parameters. In contrast, GBE attenuated the anticoagulant action of warfarin. Bilobalide, a component of GBE that markedly induced hepatic CYPs including (S)-warfarin hydroxylase, showed similar effects. For (S)-warfarin, the anticoagulation action and the interaction with GBE was clear, while the influence on metabolism was greater for (R)-warfarin than for (S)-warfarin, which corresponded to the CYP types induced by GBE. These results suggest that GBE and ginkgolide B have no influence on blood coagulation in vivo, and that GBE attenuates the anticoagulation action of warfarin via induction of hepatic CYPs by bilobalide.

[c] 2011 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Article history:

Received 21 December 2010 Received in revised form 15 April 2011 Accepted 22 June 2011

Keywords: Ginkgo biloba Warfarin Bleeding Adverse event Bilobalide Cytochrome P450

Introduction

Herbal ingredients have received great attention in complementary and alternative medicine, and are used in dietary supplements or health products in many countries. With this increasing use, potential herb-drug interactions have been reported (Ulbricht et al. 2008). The most well-known example is St. John's Wort, which has been reported to induce cytochrome P450 enzymes (CYPs) and the key drug transporter P-glycoprotein, thereby attenuating the efficacy of various drugs (Zhao et al. 2006; Zhou and Lai 2008).

When analyzing adverse drug-herb interactions, it is generally hard to conclude whether the cause was a certain herbal ingredient itself or a combination of the many ingredients contained within a herbal product. Herbal products consist of many constituents, only some of which have been identified, and their natural origin means that these constituents vary. Moreover, idiosyncratic reactions or side effects of drugs taken simultaneously may be involved in the adverse events. Therefore, it is critical to examine the basic pharmacological effects of herbal ingredients and their interaction with drugs.

Ginkgo biloba extract (GBE) is one of the most popular herbal ingredients, being used for improvement of cognitive function and peripheral arterial disease (Sierpina et al. 2003); its effects in these conditions are thought to be related in part to the improvement of blood flow. GBE has also been shown to reduce blood viscosity and to improve ophthalmic artery blood flow (Lesk et al. 2008). Meanwhile, one of the most concerning adverse events associated with GBE is bleeding (Bent et al. 2005; Ulbricht et al. 2008), which has been reported in those simultaneously taking GBE and anticoagulant drugs such as aspirin and warfarin (Ulbricht et al. 2008).

One of the proposed mechanisms by which GBE could cause bleeding is via the action of ginkgolide B, a constituent of GBE that is reported to be a platelet-activating factor (PAF) antagonist (Smith et al. 1996). However, intake of GBE did not reduce PAF-mediated platelet aggregation (Kudolo et al. 2002), and did not reduce prothrombin times (Kohler et al. 2004) in clinical studies. Although GBE is also believed to increase the risk of bleeding by its interaction with anticoagulants such as warfarin (Matthews 1998; Ulbricht et al. 2008), some reports suggest that GBE does not increase the anticoagulant effect of warfarin in healthy people (Jiang et al. 2006) or patients that have a stable 1NR (Engelsen et al. 2003). An in vitro study (Gaudineau et al. 2004) reported that GBE inhibited major human cytochrome P450 enzymes (CYPs), particularly CYP2C9, an enzyme related to warfarin metabolism. The inhibition of CYP2C9 might result in an increase in warfarin levels and correspondingly greater anticoagulant action. In contrast to those studies, we have reported that GBE induced various types of hepatic CYP, including (S)-warfarin hydroxylase, and that it attenuated the efficacy of co-administered drugs in animal studies (Kubota et al. 2003, 2004; Sugiyama et al. 2004). We have also shown that components of GBE involved in the induction of hepatic CYPs existed in terpene trilac-tone fractions among which bilobalide was the active component (Taki et al. 2009; Umegaki et al. 2007). These findings contradict the reported adverse interaction of GBE and warfarin, because if GBE induced hepatic CYPs, the metabolism of warfarin might be enhanced and the drug's anticoagulant effect attenuated.

Hence, from the available evidence so far, it remains unclear whether GBE and ginkgolide B increase bleeding, and whether GBE enhances the anticoagulant effect of warfarin in vivo. Thus, in the present study, we examined whether GBE and ginkgolide B itself influenced blood coagulation parameters in vivo in mice. We also examined the effect of GBE and bilobalide on warfarin-induced blood anticoagulation, and its possible mechanism of interaction, in particular through induction of hepatic CYPs.

Materials and methods

Materials

Powdered GBE was supplied by Tama Seikagaku-Kogyo Co. (Tokyo, Japan).This preparation was created by extraction of Ginkgo biloba leaves with hydrous ethanol, followed by elimination of ginkgolic acid at less than 1 ppm, and drying. Previous HPLC analysis showed that it contained 24.9% flavonoids and 10.6% total terpene trilactones, which consisted of 2.9% ginkgolide A, 1.4% ginkgolide B, 2.1% ginkgolide C, and 4.2% bilobalide (Umegaki et al. 2007). The content of bilobalide in this GBE preparation was higher than that of EGb461, (1) a well known standardized GBE preparation (4.2% versus 2.9%). NADPH was obtained from Oriental Yeast (Tokyo, Japan). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Reagents for blood coagulation tests were obtained from Sysmex Co. (Hyogo, Japan).

Animal experiments

Male ICR mice (5 weeks old; CLEA Japan Inc., Tokyo, Japan) were kept in polypropylene cages. They had free access to laboratory feed (CE2; CLEA Japan Inc., Tokyo, Japan) and tap water, and were kept at a constant temperature (23 [+ or -] 1 [degree] C) with a 12-h light-dark cycle. In blood coagulation and warfarin treatment studies, mice were orally given the test samples (GBE, ginkgolide B, or bilobalide), suspended in 0.5% carboxymethylcellulose (CMC) for 5 days, with or without warfarin co-treatment (1.5 mg/kg for racemate or 0.75 mg/kg for (S)-form and (R)-form) dissolved in 0.5% CMC for the last 3 days (days 3-5). Two hours after the last administration of test sample and warfarin, animals were anaesthetized with pentobarbital and blood was collected from the descending aorta into tubes containing 3.8% sodium citrate (1:10 dilution).

In the study of induction of hepatic CYPs by various terpene trilactones, mice were orally given the test samples (ginkgolide A, B, C, or bilobalide; l0 mg/kg body weight) suspended in 0.5% CMC or vehicle for 5 days. One day after the last administration, they were anaesthetized with pentobarbital and exsanguinated from the descending aorta. The liver was then removed and rinsed with 0.9% (w/v) NaCl, and stored at -80 [degree] C until analysis.

All procedures were in accordance with the National Institute of Health and Nutrition guidelines for the Care and Use of Laboratory Animals and were approved by the ethics committee of our institution.

Analysis of hepatic CYPs

The liver was rinsed with 0.9% (w/v) NaCl and homogenized in 50 mmol/1 Tris-HCl buffer (pH 7.4) containing 0.25 mol/1 sucrose. The homogenate was centrifuged at 10,000 x g at 4 C for 30min and the resulting supernatant was centrifuged at 105,000 x g at 4 [degree] C for 60min. The pellet was washed once with 50 mmol/1 Tris-HCl buffer (pH 7.4) containing 0.25 mol/1 sucrose by centrifugation at 105,000 x g at 4 [degree] C for 60min, and the concentrations and activities of CYPs were analyzed. The overall concentration of CYPs and the enzyme activities were determined as previously described (Umegaki et al. 2002). The subtypes of CYP enzymes tested for were: ethoxyresorufin O-de-ethylase, CYPIA1; methoxyresorufin O-demethylase, CYPIA2; pentoxyresorufin O-dealkylase, CYP2B; (S)-warfarin 7-hydroxyIase, CYP2C; p-nitrophenol hydroxylase, CYP2E1; and testosterone 6[beta]-hydroxylase, CYP3A. Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA).

Determination of blood coagulation

Plasma samples were immediately centrifuged at 4320 x g at 4 [degree] C for l0 min. The coagulation parameters (prothrombin time, PT; activated partial thromboplastin time, APTT; fibrinogen, Fbg; and thrombotest (Owren), TTO) were measured with an automated blood coagulation analyzer (CA-50; Sysmex Co., Hyogo, Japan) according to the manufacturer's protocol. Blood loss in vivo was determined by a method reported elsewhere (Sato et al. 1998). Briefly, mice given test samples with and without warfarin were anesthetized with pentobarbital, and the tail was amputated 5 mm from the distal end with a surgical blade. The proximal tail stump was immersed in a tube containing 10 ml of saline at 37 [degree] C for 20 min. After adding 1 ml of 20% Triton X-100 into the tube, the volume of blood was measured photometrically at 546 nm using a standard curve prepared with pooled blood samples.

Determination of the concentration of warfarin and its metabolites

Preparation of plasma samples and column-switching chromatographic conditions were according to the method reported by Uno et al. (2007), with some modification. In particular, 20 [micro]l of diclofenac sodium solution (50[micro]g/ml) as an internal standard (I.S.) and 400 [micro]l of 0.01 M [Na.sub.2][HPO.sub.4] (pH 2.0) were added to 400 [micro]l of plasma. The tubes were well vortexed and 2 ml of diethyl ether-chloroform (80:20, v/v) was added as extract solvent. After 10 min of shaking, the sample was centrifuged at 2500 x g at 4 [degree] C for 10 min, and 1.5 ml of the organic phase was dried under nitrogen at 50 [degree] C. The residue was dissolved with 100 [micro]l of acetonitrile in water (25:75, v/v) and 30 [micro]l of the aliquot was applied to HPLC. The column-switching HPLC system consisted of two pumps (Model 582 Solvent Delivery System; ESA Biosciences. Inc., Chelmsford, MA, USA), a valve unit (FLO 401; ESA Biosciences. Inc., Chelmsford, MA, USA), an auto sampler (Model 542; ESA Biosciences. Inc., Chelmsford, MA. USA), and a UV detector (L-4200; Hitachi Ltd., Tokyo, Japan). The columns and separating conditions were the same as those reported by Uno et al. (2007).

Statistical analysis

Data are presented as the mean and standard error (SE) for the individual group. Statistical analyses were carried out by one-way ANOVA with Dunnett's multiple comparison post hoc test. P values < 0.05 were considered significant. These statistical analyses were performed with Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA).

Results

Interaction of GBE and warfarin in blood coagulation

Administration of GBE for 5 days at a dose of 10, 100, or l000 mg/kg did not influence blood coagulation parameters (PT, APTT, Fbg, and TTO). Similarly, administration of ginkgolide B, a GBE constituent considered to be a PAF antagonist, for 5 days did not influence these blood coagulation parameters, even at doses of 1.4, 14, and 140 mg/kg, which are equivalent to 100,1000, and 10,000 mg/kg of GBE (data not shown). Warfarin racemate at a dose of 1.5 mg/kg for the last 3 days of the regimen impeded blood coagulation in terms of all parameters except fibrinogen levels. The anticoagulation effects of warfarin were significantly attenuated by co-treatment with GBE at 100 mg/kg (Fig. la). The same interaction was detected when bilobalide was administered at 4.2 mg/kg (equivalent to GBE 100 mg/kg) (Fig. lb). The in vivo blood loss test was performed to corroborate the effect of this GBE-warfarin interaction on blood coagulation. Bleeding volume ([micro]l) in the 20min after tail amputation (mean [+ or-] SE [5 mice]) was 40 [+ or-] 23 for mice treated with vehicle, 48 [+ or-] 24 for those given GBE, 426 [+ or-] 68 for those administered warfarin, and 191 [+ or-] 80 when warfarin and GBE co-treatment was used. Because of fluctuation of the data, no significant effect of GBE was detected, but the blood loss data suggested that GBE influenced the anticoagulation effects of warfarin, as shown in Fig. 1.

Mechanism of interaction between GBE and warfarin

In our previous study using mice, GBE induced hepatic CYPs at a dose comparable to that of bilobalide in the GBE used here (Umegaki et al. 2007). However, CYPs inducing ability among ter-pene trilactones in GBE was not clear. Thus, in this study mice were orally given one of four terpene trilactones (ginkgolide A, B, C and bilobalide at 10 mg/kg) for 5 days, and the ability to induce CYPs was compared among them. As Table 1 shows, all terpene trilactones significantly increased overall CYP concentration, and bilobalide was the most potent. In addition, bilobalide significantly induced all but one of the CYP subtypes including (S)-warfarin 7-hydroxylase, while the three ginkgolides did not. The GBE used in our study consisted of 2.9% ginkgolide A, 1.4% ginkgolide B, 2.1% ginkgolide C, and 4.2% bilobalide; thus, the contribution of bilobalide to CYP induction by GBE was higher than that of the other terpene trilactones. These data on hepatic CYP induction corroborated our previous findings in rats and mice (Taki et al. 2009; Umegaki et al. 2007).

Table 1
Concentrations of hepatic CYPs and their subtypes in mice
given various terpene trilactones.
                            Control             Ginkgolide A
Overall
concentration
(nmol/mg protein)
Cytochrome P450       0.40 [+ or -] 0.03  0.53 [+ or -] 0.01*
                                   [100]                [133]

Activities (nmol/mg
protein/min)
Ethoxyresorufin O-de  9.42 [+ or -] 1.80  10.05 [+ or -] 2.76
ethylase (CYPIA1)
                                   [100]                [107]

Methoxyresorufin      22.76[+ or -] 1.73  24.31 [+ or -] 2.55
O-demethylase
(CYPIA2)
                                   [100]                [107]

Pentoxyresorfin       6.54 [+ or -] 0.44  10.75 [+ or -] 1.95
O-dealkylase
(CYP2B)
                                   [100]                [164]

(S)-Warfarin          1.25 [+ or -] 0.15   1.77 [+ or -] 0.20
7-hydroxylase
(CYP2C)
                                   [100]                [142]

p-Nitrophenol             682.7 [+ or -]  773.3 [+ or -] 31.5
hydroxylase                         48.7
(CYP2E1)
                                   [100]                [113]

Testosterone           5001 [+ or -] 618    7084 [+ or -] 792
6[beta]-hydroxylase
(CYP3A)
                                   [100]                [142]
                          Ginkgolide B           Ginkgolide C

Overall
concentration
(nmol/mg protein)
Cytochrome P450       0.54 [+ or -] 0.03*  0.57 [+ or -] 0.04**
                                    [135]                 [143]

Activities (nmol/mg
protein/min)
Ethoxyresorufin O-de  11.15 [+ or -] 2.93   11.87 [+ or -] 3.33
ethylase (CYPIA1)
                                    [118]                 [126]

Methoxyresorufin      22.57 [+ or -] 4.37   33.14 [+ or -] 3.15
O-demethylase
(CYPIA2)
                                     [99]                 [146]

Pentoxyresorfin       12.97 [+ or -] 1.83    3.66 [+ or -] 0.58
O-dealkylase
(CYP2B)
                                    [198]                  [56]

(S)-Warfarin           2.06 [+ or -] 0.14    1.54 [+ or -] 0.16
7-hydroxylase
(CYP2C)
                                    [165]                 [123]

p-Nitrophenol         793.3 [+ or -] 47.1   810.0 [+ or -] 26.0
hydroxylase
(CYP2E1)
                                    [116]                 [119]

Testosterone            8231 [+ or -] 566     6135 [+ or -] 639
6[beta]-hydroxylase
(CYP3A)
                                    [165]                 [123]
                            Bilobdlide

Overall
concentration
(nmol/mg protein)

Cytochrome P450                0.74 [+ or -]
                                     0.05***
                                       [1851

Activities (nmol/mg
protein/min)
Ethoxyresorufin O-de  46.67 [+ or -] 8.51***
ethylase (CYPIA1)
                                       [495]

Methoxyresorufin      55.33 [+ or -] 6.74***
O-demethylase
(CYPIA2)
                                       [243]

Pentoxyresorfin       37.94 [+ or -] 2.84***
O-dealkylase
(CYP2B)
                                       [580]

(S)-Warfarin            2.51 [+ or -] 0.35**
7-hydroxylase
(CYP2C)
                                       [201]

p-Nitrophenol            807.6 [+ or -] 59.3
hydroxylase
(CYP2E1)
                                       [118]

Testosterone          10,014 [+ or -] 1405**
6[beta]-hydroxylase
(CYP3A)
                                       [200]

Mice were orally given various terpene trilactones that are constituents
of Ginkgo biloba extract (CBE) at a dose of l0 mg/kg for 5 days.
Mean [+ or -] SE for 5 mice. Numbers in parenthesis
indicate % of control.
* p < 0.05.
** p < 0.01.
*** p < 0.001.


The metabolites of warfarin were evaluated in plasma samples, which were obtained in mice given 1.5 mg/kg racemic warfarin for the last 3 days of the 5-day regimen (with or without 100 mg/kg GBE or 4.2 mg/kg bilobalide). As Table 2 shows, concentrations of (S)-warfarin were markedly higher than those of (R)-warfarin. Both GBE and bilobalide significantly decreased the concentration of (R)-warfarin and tended to decrease the concentration of (S)-warfarin. The concentration of the warfarin metabolites (S)-7-hydroxywarfarin and (R)-7-hydroxywarfarin were unaffected by treatment with GBE or bilobalide. The effects of the interactions of warfarin and GBE on blood coagulation were also examined after giving (S)-warfarin or (R)-warfarin at a dose of 0.75 mg/kg. As Fig. 2 shows, only (S)-warfarin acted as an anticoagulant and this action was significantly prevented by GBE co-treatment. The plasma concentration of (S)-warfarin was higher than that of (R)-warfarin even though the same dose of each enantiomer was given; the mean [+ or -] SE (vehicle vs. GBE, ng/ml) was 38 [+ or -] 21 vs. 13 [+ or -] 7 in the (R)-warfarin treated group, and 837 [+ or -] 80 vs. 405 [+ or -] 95 in the (S)-warfarin treated group. These data were similar to the results shown in Table 2.

Table 2
Effects of GBE and bilohalide on plasma concentration of warfarin and
7-hydroxywarfarin enantiomers in mice given warfarin racemate.
                Plasma
             concentration
                (ng/ml)
              (S)-Warfarin       (R)-Warfarin         (S)-7-OH
                                                      warfarin

 Warfarin   730 [+ or -] 77  132 [+ or -] 14  4.4 [+ or -] 0.4

  GBE +     635 [+ or -] 66   67 [+ or -] 5"  4.5 [+ or -] 0.3
 warfarin

Bilobafide  661 [+ or -] 21     77 [+ or -]7  4.2 [+ or -] 0.3
+ warfarin
                (R)-7-OH
                warfarin

 Warfarin   4.4 [+ or -] 0.4

  GBE +     4.5 [+ or -] 0.3
 warfarin

Bilobafide  4.2 [+ or -] 0.3
+ warfarin

Mice were administered Ginkgo biloba extract (GBE: l00 mg/kg)
or bilobalide (4.2 mg/kg) for 5 days and warfarin (racemate,
1.5 mg/kg) for the last 3 days of this 5-day regimen. Concentrations
of warfarin and its metabolites in plasma were measured 2 h after the
last administration. Each value
indicates the mean [+ or -] SE for 4-5 mice.
P < 0.05 vs. warfarin.


Discussion

GBE is a frequently prescribed herbal medicine in Germany and is also used an ingredient of dietary supplements and health products worldwide. To ensure its safe use, it is important to clarify any adverse events. These include gastrointestinal upset, headaches, and dizziness, as well as the potentially serious adverse event of bleeding (Sierpina et al. 2003; Ulbricht et al. 2008). The present mouse study demonstrated that GBE and its constituent ginkgolide B, a PAF antagonist, did not affect coagulation parameters in vivo. Further, GBE did not enhance, but rather reduced the anticoagulation action of warfarin as evaluated by coagulation parameters and an in vivo blood loss test. These results do not support the greatest concern about the safety of GBE, i.e., that it contributes to bleeding episodes (Bent et al. 2005; Ulbricht et al. 2008).

The PAF antagonist action of ginkgolide B, which has been confirmed in vitro (Chung et al. 1987; Lamant et al. 1987), is an attractive explanation of bleeding episodes associated with GBE. However, in the present in vivo studies ginkgolide B did not affect coagulation parameters, even at extremely high doses (up to 140 mg/kg). These data are consistent with clinical studies that showed lack of influence of GBE intake on PAF-mediated platelet aggregation (Kudolo et al. 2002) and prothrombin times (Kohler et al. 2004). The half-maximal inhibitory concentration (1C50) of ginkgolide B for PAF-mediated human platelet aggregation was reported to be about 100 times those attained after oral intake of standardized GBE (EGb 761) at the recommended doses (120-240 mg) (Koch 2005). It is therefore unlikely that GBE and ginkgolide B, when used at the doses present in GBE products, induce spontaneous bleeding in vivo via antagonism of PAF.

In the present study the anticoagulation effect of warfarin was attenuated by co-treatment with GBE. This warfarin-GBE interaction in vivo is in line with our previous findings. We previously showed that GBE induced hepatic CYPs and attenuated the hypoglycemic action of tolbutamide (Sugiyama et al. 2004), the hypotensive action of nicardipine (Kubota et al. 2003), and the sedative effects of phenobarbital (Kubota et al. 2004). In our previous study, we also showed that the terpene trilactone fraction of GBE was responsible for induction of hepatic CYPs, and that bilobalide is one of the substances within this fraction. However, we could not clearly show which of the terpene trilactones was the most potent in terms of CYP induction. Thus, in the present study, we compared the induction of CYPs by four terpene trilactones and confirmed that bilobalicle produced marked increases in overall CYP concentration and activities of various CYP enzymes. The three ginkgolides tested (A, B, and C) enhanced hepatic CYP concentration slightly, but did not significantly increase CYP enzyme activities. The proportion of terpene trilactones in the GBE used in the present study was 4.2% for bilobalide, 2.9% for ginkgolide A, 1.4% for ginkgolide B, and 2.1% for ginkgolide C. Based on its high content in the present GBE and its potent CYP induction ability, it is reasonable to conclude that bilobalide is a major contributor to warfarin-GBE interactions. In fact, GBE and bilobalide (at the amount equivalent to that present in GBE) showed quite similar inhibitory effects on warfarin-induced anticoagulation and had a similar influence on warfarin metabolism, as shown in Fig. 1 and Table 2.

In blood, warfarin binds exclusively to the protein albumin, and an increase in unbound warfarin enhances the anticoagulant action (Palareti and Legnani 1996). In a clinical study, Jiang et al. (2005) reported that GBE did not influence the protein binding of warfarin. In our preliminary study, GBE did not influence warfarin-protein binding as assessed in mouse serum by the equilibrium dialysis method (data not shown). Therefore, we may rule out the possibility that warfarin-protein binding is involved in GBE-warfarin interactions in vivo.

Considering the present findings, GBE interacted with warfarin through the induction of hepatic CYPs by bilobalide, which resulted in increased warfarin metabolism, thereby decreasing the concentration of warfarin and its pharmacological effect. As shown in the results, GBE had a clearer influence on the anticoagulant action of warfarin than on the concentration of warfarin and its metabolites in plasma. This may be related to the time lag after administration between the maximum plasma concentration of warfarin and its anticoagulant action. Warfarin is known to produce an anticoagulant effect by inhibiting the cyclic interconversion of vitamin K and its epoxide, which is needed for the carboxylation of several blood coagulation proteins (Hirsh et al. 2003). Moreover, the approximate duration of action of warfarin after administration is a few days. This mode of action of warfarin on anticoagulation may explain the difference in the influence of GBE on warfarin concentration in plasma and anticoagulation action. Generally warfarin consists of a racemic mixture of two active enantiomers, (R)-form and (S)-form, and the metabolism and anticoagulant potency of each enantiomer differs, with the anticoagulant action of (S)-warfarin being about 3-5 times higher than that of (R)-warfarin (Breckenridge et al. 1974; Palareti and Legnani 1996). In humans, (S)-warfarin is metabolized almost exclusively by CYP 2C9 to its inactive form, (S)-7-hydroxywarfarin, while (R)-warfarin is metabolized by multiple CYPs such as CYP1A1 and CYP3A4 (Kaminsky and Zhang 1997). In this mouse study, bilobalide induced multiple CYPs, with an increase in activity of 5-fold for CYP1A1, and 2-fold for CYP2C (Table 1). The effect of bilobalide on the decrease in plasma concentration of warfarin enantiomers was clearer for the (R)-isomer than for the (S)-isomer. These data are consistent with those of a rat study reported by Yacobi and Levy (1977), suggesting that the (R)-isomer is more metabolizable than the (S)-isomer in rats and mice. The induction pattern of CYPs by bilobalide and the nature of warfarin metabolism may explain the quicker disappearance of (R)-warfarin and the clearer influence of bilobalide on plasma (R)-warfarin concentration in this study. We administered warfarin for 3 consecutive days, so evaluation of the metabolism and excretion of (S)-warfarin is likely to be complicated. This may also have contributed to the unclear influence of bilobalide on (S)-warfarin concentration. Species differences exist in warfarin metabolism; (S)-warfarin is mainly metabolized to (S)-7-hydroxywarfarin in humans and to (S)-4-hydroxywarfarin with some (S)-7-hydroxywarfarin in mice (Inoue et al. 2009). In the present study, it is difficult to extrapolate the findings to how GBE and bilobalide influence the pharmacokinetics of warfarin in humans. Further detailed pharmacokinetic studies in humans or in chimeric mice with humanized liver will be needed to confirm the influence of GBE and bilobalide on the metabolism of warfarin.

In general, when patients take a standardized formula such as EGb761, which generally contains 2.9% of bilobalide, the intake of GBE is less than 240 mg/day. In our previous dose-evaluation study in rats, a significant increase in hepatic CYP enzyme activity was detected after administering a GBE dose higher than l0 mg/kg for 5 days (Umegaki et al. 2002). The concentration of bilobalide in the GBE used in our study was about twice that in EGb761 (4.2% versus 2.9%). If these GBE doses are applied to humans, more than 600 mg/day are likely to be needed for significant induction of CYPs and ensuing GBE-drug interactions. Although the susceptibility to GBE-drug interaction may vary from person to person because of individual differences in sensitivity, our data suggest that GBE-drug interaction via bilobalide-mediated induction of CYPs should not generally be expected.

In conclusion, we have shown that GBE and ginkgolide B did not cause bleeding in vivo, and that GBE attenuated rather than promoted the anticoagulant action of warfarin through the induction of hepatic CYPs by bilobalide. Nonetheless, we advise careful observation for adverse events of bleeding and hemorrhage related to GBE-containing products in clinical practice.

Conflict of interest

No conflict to disclose.

Acknowledgement

This study was financially supported in part by a Grant-in-Aid from the Food Safety Commission, Japan (No. 0807).

* Corresponding author at: Information Center, National Institute of Health and Nutrition, 1-23-1 Toy a ma, Shinjuku-ku, Tokyo 162-8636, Japan. Tel.: +81 3 3203 5721; fax: +81 3 3202 3278.

E-mail address: umegaki@nih.go.jp (K. Umegaki).

0944-7113/$ - see front matter [C] 2011 Elsevier GmbH. All rights reserved, doi: 10.1016/j.phymed.2011.06.020

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Yuko Taki (a), (b), Kaori Yokotani (a), Shizuo Yamada (b), Kazumasa Shinozuka (c), Yoko Kubota (d), Yasuo Watanabe (d), Keizo Umegaki (a), (*)

(a) National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan

(b) Department of Pharmacokinetics and Pharmacodynamics and Global COE Program, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan

(c) Department of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya 663-8179, Japan

(d) Department of Pharmacology, School of Medical Pharmaceutical Sciences, Nihon Pharmaceutical University, Saitama 362-0806, Japan
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Author:Taki, Yuko; Yokotani, Kaori; Yamada, Shizuo; Shinozuka, Kazumasa; Kubota, Yoko; Watanabe, Yasuo; Ume
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9JAPA
Date:Feb 15, 2012
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