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Ginkgo biloba extract modifies hypoglycemic action of tolbutamide via hepatic cytochrome P450 mediated mechanism in aged rats
Life Sciences 75 (2004) 1113 – 1122 www.elsevier.com/locate/lifescie
Ginkgo biloba extract modifies hypoglycemic action of tolbutamide via hepatic cytochrome P450 mediated mechanism in aged rats Tomomi Sugiyama a, Yoko Kubota b, Kazumasa Shinozuka b, Shizuo Yamada c, Jian Wu a, Keizo Umegaki a,* a National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku, Tokyo 162-8636, Japan Department of Pharmacology, School of Pharmaceutical Sciences, Mukogawa Women’s University, 11-68 Koshien Kyuban-cho, Nishinomiya 663-8179, Japan c Department of Biopharmacy and COE Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan b
Received 1 December 2003; accepted 24 February 2004
Abstract We examined hepatic cytochrome P450 (CYP)-mediated interactions between Ginkgo biloba extract (GBE) and tolbutamide, an oral anti-diabetic agent, in aged and young rats. Tolbutamide was orally given to rats with or without GBE treatment, and time-dependent changes in blood glucose were monitored. The basal activity of six CYP subtypes in liver was lower in the aged rats than in the young rats, while the inductions of these enzymes by 5 day pretreatment of 0.1% GBE diet were more in the aged rats. Further, the pretreatment of GBE significantly attenuated the hypoglycemic action of tolbutamide in the aged rats, corresponding well to the enhanced activity of (S)-warfarin 7-hydroxylase, which is responsible for CYP2C9 subtype, a major isoform metabolizing tolbutamide. In contrast, the simultaneous administration of GBE with tolbutamide potentiated the hypoglycemic action of this drug. The in vitro experiments revealed that GBE competitively inhibited the metabolism of tolbutamide by (S)-warfarin 7-hydroxylase in the rat liver microsomes. In the young rats, the 5 day pretreatment with GBE significantly attenuated the hypoglycemic action of tolbutamide, but a simultaneous treatment had little influence on the tolbutamide effect. In conclusion, the present study has shown that the simultaneous and continuous intake of GBE significantly affects the hypoglycemic action of tolbutamide, possibly via a hepatic CYP enzyme-mediated mechanism, particularly in the aged rats. Therefore, it is
* Corresponding author. Tel.: +81-3-3203-8063, fax: +81-3-3205-6549. E-mail address: [email protected] (K. Umegaki). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.02.020
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anticipated that the intake of GBE as a dietary supplement with therapeutic drugs should be cautious, particularly in elderly people. D 2004 Elsevier Inc. All rights reserved. Keywords: Ginkgo biloba extract; Cytochrome P450; Tolbutamide; Blood glucose; CYP2C9
Introduction Ginkgo biloba extract (GBE) is a popular herbal medicines, and the leaf extract of Ginkgo biloba contains some functional flavonoids and terpenoids (Kleijnen and Knipschild, 1992a; Gruenwald et al., 2000). In some European countries, standardized GBE has been clinically used mostly for cerebral insufficiency, dementia, memory impairment, tinnitus and intermittent claudication (Kleijnen and Knipschild, 1992b; Le Bars et al., 1997; Ernst, 1999; Pittler and Ernst, 2000), while it is marketed as a dietary supplement in the United States and Japan. GBE exerts many pharmacological effects, such as free radical-scavenging action and improvement of microcirculatory action (McKenna et al., 2001; De Smet, 2002; Ernst, 2002). On the other hand, GBE has been reported to have side effects such as headache, gastric symptoms, diarrhea, and allergic skin reactions, although the occasion of these side effects is rare (Cohen and Bartlik, 1998; De Smet, 2002; Ernst, 2002). Recently, adverse reactions of herbal remedies and potential herb-drug interactions have received a great deal of attention. As a well-known example, St John’s Wort has been reported to induce hepatic CYP3A4 activity, thereby leading to the attenuation of efficacy of therapeutic drugs such as cyclosporin, indinavir, and digoxin (Durr et al., 2000; Roby et al., 2000). In the case of GBE, the occurrence of bleeding has been reported in patients who took GBE and anticoagulant drugs such as aspirin, rofecoxib, or warfarin at the same time (Vaes and Chyka, 2000; Izzo and Ernst, 2001). It is proposed that the mechanism of this adverse interaction of GBE with these drugs is due to the inhibitory effect of platelet activating factors by ginkgolide B, which is one of the active constituents of GBE (Braquet and Hosford, 1991). However, other interactions between GBE and drugs have not been fully elucidated. In previous studies, we found that feeding GBE to rats markedly increased the concentration of hepatic cytochrome P450 (CYP), the expression of various CYP mRNA, and the enzyme activities in a dose- and time-dependent manner (Umegaki et al., 2000; Umegaki et al., 2002; Shinozuka et al., 2002). Moreover, we reported that pre-treatment with GBE decreased the hypotensive action by nicardipine, a calcium channel blocker, which is extensively metabolized by CYP3A type (Shinozuka et al., 2002). These findings indicate that similar interactions of GBE with other drugs through the mediation of CYPs might occur. Because of its reported beneficial effects, many elderly people take GBE (Cohen and Bartlik, 1998; De Smet, 2002; Ernst, 2002). Generally, elderly people tend to suffer from hypertension and diabetes and thereby take some other drugs simultaneously with GBE. In particular, GBE has been shown to improve peripheral blood flow (Iliff and Auer, 1983) and is expected to prevent periphery necrosis and retinopathy in severe diabetes (Lanthony and Cosson, 1988; Doly et al., 1992). It is also reported that GBE inhibits the increment of blood glucose concentration in glucose-loaded diabetic rats (Rapin et al., 1997). Thus, the simultaneous intake of GBE and anti-diabetic agents might occur, particularly in elderly people. The present study was undertaken to examine the hepatic CYP-mediated interactions of GBE with tolbutamide in aged and young rats. Tolbutamide is one of sulfonylureas applied for non-insulin-
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dependent, type 2 diabetes, and its metabolism is dependent mainly upon the CYP2C9 activity in the liver (Tal, 1993; Proks et al., 2002; Lee et al., 2003). The effect of GBE intake on the hypoglycemia of tolbutamide in rats was evaluated with a 5 day pretreatment and simultaneous treatment of GBE.
Methods Materials A standardized powder form of Ginkgo biloba extract (GBE) was supplied by Tama SeikagakuKogyo Co., Tokyo. The GBE contained 24.9% flavonoids and 10.6% total terpene, which consisted of 2.9% ginkgolide A, 1.4% ginkgolide B, 2.1% ginkgolide C, and 4.2% bilobalide, and less than 1 ppm of ginkgolic acid. Tolbutamide, resorufin, ethoxyresorufin, methoxyresorufin, pentoxyresorufin, testosterone, 6h-hydroxytestosterone, corticosterone, p-nitrophenol, 4-nitrocatechol, and 7-ethoxycoumarin were purchased from Sigma (St. Louis, MO, USA). (S)-Warfarin and 7-hydroxywarfarin were obtained from Ultrafine (Manchester, England). NADPH was obtained from Oriental Yeast (Tokyo, Japan). All other reagents were obtained from Wako Pure Chemical Ltd. (Osaka, Japan). Animal experiment Male Wistar aged (19 months old) and young (7 weeks old) rats obtained from Japan SLC (Shizuoka, Japan) were housed individually in stainless steel, wire-bottomed cages in a constant temperature room (23 F 1jC) under 12 hr light-dark cycle. Rats were divided into several groups with or without GBE treatment. GBE pretreated group was fed commercial rodent diet CE2 (Japan Clea, Tokyo) containing 0.1% GBE for 5 days. After the treatment, rats were orally administered tolbutamide (40 mg/kg). The simultaneous GBE treated group was given GBE (100 mg/kg body weight, p.o.) simultaneously with tolbutamide. After the tolbutamide administration, the blood was collected at predetermined time points and immediately blood glucose concentration was determined using a blood glucose detector Dexter Z II (Bayer Corp. Mishawaka, IN). After the treatment with or without GBE, the rat liver was removed and weighed, and a part of the liver was subjected to the enzyme assay. All procedures were in accordance with National Institute of Health and Nutrition guidelines for the Care and Use of Laboratory Animals.
Analytical methods Preparation of microsome and cytosolic fractions from the liver The liver was rinsed with 0.9% NaCl solution, weighed and homogenized in 50 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose. The homogenate was centrifuged at 10,000 g at 4jC for 30 min. The supernatant was further centrifuged at 105,000 g at 4jC for 60 min. The supernatant was used as the cytosolic fraction for the assay of glutathione S-transferase, the activity of which was determined by the method of Habig and Jakoby using 1-chloro-2,4-dinitrobenzene as a substrate (Habig and Jakoby, 1981). The pellet was washed once with 50 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose by
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centrifugation at 105,000 g at 4jC for 60 min, and the concentration and activities of CYP were analyzed. Protein concentrations of microsome and cytosolic fractions were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Analysis of CYP enzyme activities Cytochrome P450 content was quantified by the method of Omura and Sato (Omura and Sato, 1964). The activities of various CYP enzymes were determined by HPLC methods as reported previously (Umegaki et al., 2002). The subtypes of CYP enzymes examined and the corresponding CYPs were methoxyresorufin O-demethylase, CYP1A2; ethoxyresorufin O-deethylase, CYP1A1; pentoxyresorufin O-dealkylase, CYP2B; p-nitrophenol hydroxylase, CYP2E1; testosterone 6h-hydroxylase, CYP3A; and (S)-warfarin 7-hydroxylase, CYP2C9. The interaction between GBE and tolbutamide toward (S)-warfarin hydroxylase activity in vitro were evaluated by adding these substances at various concentrations (GBE; 1.0–30 Ag/mL, tolbutamide; 0.1– 30 Ag/mL) to the enzyme assay system ((S)-warfarin concentration; 2–16 AM). Statistical analysis Statistical analysis of the data was carried out using ANOVA followed by a post hoc test of Fisher’s PLSD. These statistical analyses were performed using a computer program (Stat View 5.0, ASA Institute Inc, Cary, NC, USA).
Results Induction of hepatic drug-metabolizing enzymes by GBE Effects of 5 days of feeding of 0.1% GBE diet on the liver weight and hepatic drug metabolizing enzymes are shown in Table 1. The calculated intake of GBE was about 14 mg per day in both groups and
Table 1 Effects of 5 day pretreatment with GBE on the liver weight, cytochrome P450 content, and glutathione S-transferase activity in aged and young rats Aged rats Body weight (g) Liver weight (% /Body weight) Cytochrome P450 content (nmol/mg protein) Glutathione S-transferase (nmol/mg protein/min)
Young rats
Control
0.1% GBE
Control
0.1% GBE
433 F 26 2.76 F 0.09 0.731 F 0.08 565 F 197
450 F 18 3.28 F 0.06a 1.692 F 0.33a 1054 F 217a
189 F 7 4.14 F 0.19 0.921 F 0.05 667 F 104
190 F 6 4.12 F 0.29 1.749 F 0.22b 934 F 236b
Male Wistar aged (19 months old) and young (7 weeks old) rats received commercial rodent diet CE2 with or without 0.1% GBE for 5 days. Each value is the mean F SD from six rats. Significant differences from each control level are indicated by a P < 0.01 and bP < 0.05.
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was 32 mg (per kg body weight in a day) in the aged rats and 72 mg in the young rats. The liver weight in the aged rats but not in the young rats was significantly increased to 1.2-fold by the GBE treatment. Significant increases in the cytochrome P450 content and glutathione S-transferase activity were also detected by the GBE treatment, both in the aged rats (2.3- and 1.9-fold, respectively) and young rats (1.9- and 1.4-fold, respectively). Activities of various CYP are shown in Table 2. The basal levels of the activities of six CYP subtypes were clearly lower in the aged rats than in the young rats. The activity of (s)-warfarin 7-hydroxylase, which is responsible for the metabolism of tolbutamide, in the aged rats was about 41% of that in the young rats. Both in the aged and young rats, the GBE pretreatment significantly increased all CYP enzymes, especially in pentoxyresorufin O-dealkylase and (s)-warfarin 7-hydroxylase activity. The induction ratios in both groups were apparently higher in the aged rats; the ratios in the young and aged rats were 13- and 42-fold for pentoxyresorufin O-dealkylase, respectively, and 2.1- and 4.7-fold for (s)-warfarin 7-hydroxylase activity, respectively. Effects of GBE on the efficacy of tolbutamide The time-dependent changes in the blood glucose concentration by tolbutamide administration in the aged rats are shown in Fig. 1. The baseline concentration of blood glucose was approximately 100 mg/ dl. In the control group, the blood glucose concentration decreased to the minimum level (the reduction: 32.2 F 2.4 mg/dl, n = 6) at 3 hr after oral administration of tolbutamide (40 mg/kg) and remained at the lower level until for least 7 hr. In the 0.1% GBE diet (5 days) pretreated group, the hypoglycemic action of tolbutamide was considerably attenuated when compared with that in the control group without GBE, and the statistical significance was detected at 2–5 and 7 hr. This result indicated that the pretreatment of GBE reduced the pharmacological action of tolbutamide. On the other hand, a simultaneous treatment of tolbutamide with GBE at a single dose (100 mg/kg body weight) significantly lowered (at 4–6 hr) the blood glucose concentrations in the aged rats, compared with the treatment of tolbutamide alone in the control group (Fig. 1). This result indicated that the simultaneous treatment of tolbutamide and GBE potentiated the hypoglycemic efficacy of tolbutamide. In the young rats, the pretreatment (5 days) of 0.1% GBE diet significantly attenuated the hypoglycemic action of tolbutamide as in the aged rats,
Table 2 Inductions of various hepatic CYP activities by 5 day pretreatment with GBE in aged and young rats Activity (pmol/mg protein/min) Aged rats Control Ethoxyresorufin O-deethylase (CYP1A1) Methoxyresorufin O-demethylase (CYP1A2) Pentoxyresorufin O-dealkylase (CYP2B) (s)-Warfarin 7-hydroxylase (CYP2C9) p-Nitrophenol hydroxylase (CYP2E1) Testosterone 6h-hydroxylase (CYP3A)
13.5 15.5 8.2 2.6 1453 218
F F F F F F
Young rats 0.1% GBE
0.9 1.1 0.3 0.4 99 15
37.9 33.2 348.4 12.1 3580 564
F F F F F F
Control a
1.4 2.0a 39.6a 0.8a 215a 42a
24.7 27.6 35.5 6.3 1717 295
F F F F F F
0.1% GBE b
1.5 2.4b 6.4 0.4b 76 30
44.2 41.7 476.7 13.5 4064 739
F F F F F F
1.6a 1.9a 23.6a 1.2a 170a 20a
Male Wistar aged (19 months old) and young (7 weeks old) rats received commercial rodent diet CE2 with or without 0.1% GBE for 5 days. Each value is the mean F SD from six rats. Significant difference from each control level is indicated by a P < 0.001. Significant difference from control of aged rats is indicated by bP < 0.01.
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Fig. 1. Effects of simultaneous treatment and 5 day pretreatment with Ginkgo biloba extract (GBE) on the hypoglycemic effect of tolbutamide in aged rats. Aged rats (19 months old) were administered tolbutamide (40 mg/kg, p.o.) with or without GBE treatment. The GBE pretreated group was given feed containing 0.1% GBE for 5 days, and a simultaneous GBE treated group was given a single dose of GBE (100 mg/kg, p.o.) with tolbutamide. After the tolbutamide administration, blood was collected for the analysis of blood glucose concentrations. Each point represents the mean F SD from six rats. , control group; E, GBE pretreated group; and 5, a simultaneous GBE treated group. Significant difference from the control group is indicated by *P < 0.05.
.
whereas the simultaneous GBE treatment at a single dose exhibited little significant interaction with tolbutamide (Fig. 2). Competitive inhibition of GBE and tolbutamide on CYP2C9 activity in vitro The direct interaction of tolbutamide and GBE toward (S)-warfarin 7-hydroxylase (CYP2C9) activity was examined using rat liver microsome in vitro. As shown in a Dixon plot, tolbutamide competitively
Fig. 2. Effects of simultaneous treatment and 5 day pretreatment with Ginkgo biloba extract (GBE) on the hypoglycemic effect of tolbutamide in young rats. Young rats (7 weeks old) were administered tolbutamide (40 mg/kg, p.o.) with or without GBE treatment. The GBE pretreated group was given feed containing 0.1% GBE for 5 days, and a simultaneous GBE treated group was given a single dose of GBE (100 mg/kg, p.o.) with tolbutamide. After tolbutamide administration, blood was collected for analysis of blood glucose concentrations. Each point represents the mean F SD from six rats. , control group; E, GBE pretreated group; and 5, a simultaneous GBE treated group. Significant difference from the control group is indicated by *P < 0.05.
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Fig. 3. Effects of tolbutamide (A) and Ginkgo biloba extract (B) on the (S)-warfarin 7-hydroxylase (CYP2C9) activity in rat liver microsome in vitro (Dixon plot). The (S)-warfarin 7-hydroxylase (CYP2C9) activities were measured in the presence of various concentrations of tolbutamide (0.1 – 30 Ag/mL) or GBE (1.0 – 30 Ag/mL) in rat liver microsome in vitro. Loaded (S)warfarin concentrations were , 2 AM; D, 4 AM; n, 8 AM and w , 16 AM. Each point represents the mean from five experiments.
.
inhibited the metabolism of (S)-warfarin by the (S)-warfarin 7-hydroxylase (Fig. 3A). Similarly, GBE decreased the (S)-warfarin 7-hydroxylase activity in vitro in a concentration-dependent manner, and showed competitive inhibition toward the enzyme activity (Fig. 3B). These results indicated that the hepatic metabolism of tolbutamide was competitively inhibited by GBE. The calculated Ki values were 19 Ag/mL for GBE and 75 Ag/mL for tolbutamide, respectively.
Discussion Elderly people often suffer from functional impairments in their bodies and diabetes in these days. Considering these situations and the efficacy of GBE reported so far (Iliff and Auer, 1983; Lanthony and Cosson, 1988; Doly et al., 1992; Rapin et al., 1997), the simultaneous intake of GBE and anti-diabetic drug is most likely to occur in the elderly. For the safe use of both GBE and drugs, it is important to elucidate how GBE taken as a dietary supplement modifies the efficacy of therapeutic drugs. In the present study, we investigated the influence of GBE intake on the hypoglycemic efficacy of tolbutamide. We focused particularly on the elderly of advanced age and on the hepatic CYP-mediated interactions. The pharmacodynamic interaction was evaluated by inducing hypoglycemia with an excess administration of tolbutamide in aged (19-month-old) and young (7-week-old) rats. As reported previously (Szabo et al., 1995; Umegaki et al., 2000), the intake of GBE did not change the basal blood glucose level in the aged and young rats with the pretreatment of GBE with diet (data not shown). However, the pretreatment and simultaneous treatment of GBE markedly influenced the hypoglycemic action of tolbutamide in the aged rats (Fig. 1). In the case of 5 days pretreatment of GBE, the hypoglycemic action of tolbutamide was attenuated both in the aged and young rats. As shown
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in Table 2, the 5 day pretreatment of GBE markedly induced hepatic drug metabolizing enzymes, especially (S)-warfarin 7-hydroxylase, an enzyme corresponding to CYP2C9 which mainly metabolizes tolbutamide in the liver (Lee et al., 2003). Therefore, the induction by GBE pretreatment of hepatic CYP2C9 may reduce the plasma concentration of tolbutamide after oral administration, thereby leading to the attenuation of the hypoglycemic action of tolbutamide. In the present study, the plasma concentration of tolbutamide was not determined, due to the limited blood sample from the GBEpretreated rats. However, the change in the blood glucose concentration was taken as the final outcome of interaction between GBE and tolbutamide, and the observed changes of CYP2C9 responsible for tolbutamide metabolism following the GBE pretreatment could well explain the mechanism of the pharmacodynamic interaction. Kudolo (Kudolo, 2001) also reported that the ingestion of GBE by non-insulin-dependent diabetes mellitus subjects induced the hepatic metabolic clearance rate of insulin and hypoglycemic agents, suggesting an increase of blood glucose. In the previous paper (Shinozuka et al., 2002), we also showed that pretreatment of GBE significantly reduced the hypotensive action of nicardipine, which is metabolized by CYP3A2 isoform in rats. The mechanism of these interactions is similar to that in St John’s Wort (Durr et al., 2000; Roby et al., 2000), a well-known inducer of CYP, indicating that the induction of CYP by pretreatment with GBE attenuates the efficacy of various drugs. Inasmuch as the beneficial effects of GBE in humans are expected after the continuous intake for more than 4 weeks (Gruenwald et al., 2000; Blumenthal, 1998), the induction of hepatic CYP would be taken into account for the interaction with drugs seen after a long-term intake of GBE as a dietary supplement. In contrast to the GBE pretreatment, simultaneous treatment of tolbutamide with GBE as a single dose potentiated hypoglycemic action of tolbutamide in the aged rats (Fig. 1). In order to clarify the underlying mechanism of this interaction, a direct effect of GBE on the hepatic (S)-warfarin 7hydroxylase (subtype of CYP2C9) activity was examined in vitro. As tolbutamide is mainly metabolized by (S)-warfarin 7-hydroxylase (Lee et al., 2003), tolbutamide competitively inhibited the hepatic metabolism of (S)-warfarin as shown in the Dixon plot analysis (Fig. 3A). Interestingly, GBE also showed the competitive inhibition toward the metabolism of (S)-warfarin (Fig. 3B), suggesting that GBE competitively inhibited the metabolism of tolbutamide by the CYP2C9 enzyme in the liver of rats. Thus, the pharmacodynamic interaction between GBE and tolbutamide after their single administration could be explained by the following mechanism. Following the simultaneous intake of GBE and tolbutamide, the hepatic metabolism of tolbutamide would be attenuated via the significant occupancy by GBE of the same active site on the CYP2C9 enzymes where this drug binds, resulting in an enhancement of hypoglycemic action of tolbutamide. The induction of hepatic drug metabolizing enzymes and attenuation of hypoglycemic efficacy of tolbutamide were observed by the pretreatment of GBE both in the young rats and aged rats. However, the extent of these effects by GBE pretreatment was greater in the aged rats than in the young rats. This distinction may be explained by the difference between the two groups of rats in the induced level of CYP enzymes. In fact, the activities of pentoxyresorufin O-dealkylase and (S)-warfarin 7-hydroxylase were induced more (2 to 3-fold) by the GBE treatment in the aged rats than in the young rats (Table 2). This greater induction of CYP2B and CYP2C9 in the aged rats may be somehow related to the lower basal activities of these enzymes, which displayed about 23 and 41%, respectively, of the enzyme activity in the young rats. Interestingly, in the case of simultaneous treatment of GBE and tolbutamide, the potentiation of hypoglycemic action of tolbutamide was not detected in the young rats (Fig. 2). A clear explanation for the absence of enhancement of the pharmacological effect by tolbutamide in the young rats is lacking. Further
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detailed experiments will be necessary to clarify pharmacokinetic and pharmacodynamic difference of tolbutamide between young and aged rats observed by the GBE treatment. In any event, it is evident that the aged rats may be more susceptible to the CYP-mediated interactions of GBE and tolbutamide when compared to the young rats. This finding may be applied for humans. As a dietary supplement, 120 to 240 mg of GBE are generally taken in a day (Blumenthal, 1998; Gruenwald et al., 2000; Ernst, 2002), and this dose can be estimated as 2 to 4 mg/kg body weight. In this animal study, the intake of GBE was 30 f 100 mg/kg body weight per day both in dietary pretreatment and in a single dose treatment. In our previous study, a significant induction of (s)-warfarin 7hydroxylase was detected even at the lower dose (10 mg/kg body weight) of GBE (Umegaki et al., 2002). Owing to the species difference in the susceptibility to GBE, it is still not clear whether the interaction of tolbutamide and GBE also occurs in humans. Although the extrapolation of pharmacokinetic and pharmacodynamic interaction of GBE and tolbutamide in the aged rats to the clinical situation should be interpreted with caution, it is anticipated, from our recent preliminary study with healthy volunteers, that the repeated oral intake of GBE may influence the pharmacokinetics and pharmacodynamics of tolbutamide in humans (unpublished observation). In conclusion, the present study has shown that the intake of GBE significantly affects the efficacy of tolbutamide in the aged rats. Therefore, it is anticipated that the simultaneous and continuous intake of GBE as a dietary supplement with therapeutic drugs should be cautious, particularly in elderly people.
Acknowledgements This work was financially supported in part by a research grant of Ministry of Health Labor and Welfare in Japan. Authors are grateful to Ms. Keiko Saito for her technical assistance.
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Izzo, A.A., Ernst, E., 2001. Interactions between herbal medicines and prescribed drugs: a systematic review. Drugs 61, 2163 – 2175. Kleijnen, J., Knipschild, P., 1992a. Ginkgo biloba. Lancet 340, 1136 – 1139. Kleijnen, J., Knipschild, P., 1992b. Ginkgo biloba for cerebral insufficiency. British Journal of Clinical Pharmacology 34, 352 – 358. Kudolo, G.B., 2001. The effect of 3-month ingestion of Ginkgo biloba extract (EGb 761) on pancreatic beta-cell function in response to glucose loading in individuals with non-insulin-dependent diabetes mellitus. Journal of Clinical Pharmacology 41, 600 – 611. Lanthony, P., Cosson, J.P., 1988. The course of color vision in early diabetic retinopathy treated with Ginkgo biloba extract. A preliminary double-blind versus placebo study. Journal Francais d’Ophtalmologie 11, 671 – 674. Le Bars, P.L., Katz, M.M., Berman, N., Itil, T.M., Freedman, A.M., Schatzberg, A.F., 1997. A placebo-controlled, double-blind, randomized trial of an extract of Ginkgo biloba for dementia. North American EGb Study Group. JAMA 278, 1327 – 1332. Lee, C.R., Pieper, J.A., Frye, R.F., Hinderliter, A.L., Blaisdell, J.A., Goldstein, J.A., 2003. Tolbutamide, flurbiprofen, and losartan as probes of CYP2C9 activity in humans. Journal of Clinical Pharmacology 43, 84 – 91. McKenna, D.J., Jones, K., Hughes, K., 2001. Efficacy, safety, and use of ginkgo biloba in clinical and preclinical applications. Alternative Therapies Health Medicine 7, 70 – 86, 88 – 90. Omura, T., Sato, R., 1964. The carbon monoxide-binding pigment of liver microsomes. Journal of Biological Chemistry 239, 2370 – 2378. Pittler, M.H., Ernst, E., 2000. Ginkgo biloba extract for the treatment of intermittent claudication: a meta-analysis of randomized trials. American Journal of Medicine 108, 276 – 281. Proks, P., Reimann, F., Green, N., Gribble, F., Ashcroft, F., 2002. Sulfonylurea stimulation of insulin secretion. Diabetes 51 (Suppl. 3), S368 – S376. Rapin, J.R., Yoa, R., Bouvier, C., Drieu, K., 1997. Effect of repeated treatments with an extract of Ginkgo biloba (EGb761) and bilobalide on liver and muscle glycogen contents in the non-insulin-dependent diabetic rat. Drug Development Research 40, 68 – 74. Roby, C.A., Anderson, G.D., Kantor, E., Dryer, D.A., Burstein, A.H., 2000. St John’s Wort: effect on CYP3A4 activity. Clinical Pharmacology and Therapeutics 67, 451 – 457. Shinozuka, K., Umegaki, K., Kubota, Y., Tanaka, N., Mizuno, H., Nakamura, K., Kunitomo, M., 2002. Feeding of Ginkgo biloba extract (GBE) enhances gene expression of hepatic cytochrome P-450 and attenuates the hypotensive effect of nicardipine in rats. Life Sciences 70, 2783 – 2792. Szabo, M.E., Droy-Lefaix, M.T., Doly, M., 1995. EGb 761 and the recovery of ion imbalance in ischemic reperfused diabetic rat retina. Ophthalmic Research 27, 102 – 109. Tal, A., 1993. Oral hypoglycemic agents in the treatment of type II diabetes. American Fam Physician 48, 1089 – 1095. Umegaki, K., Yoshimura, M., Higuchi, M., Esashi, T., Shinozuka, K., 2000. Influence of Ginko biloba Extract feeding on blood pressure, heart rate, blood glucose, and various hepatic parameters in spontaneously hypertensive rats. Shokuhin Eiseigaku Zasshi 41, 171 – 177. Umegaki, K., Saito, K., Kubota, Y., Sanada, H., Yamada, K., Shinozuka, K., 2002. Ginkgo biloba extract markedly induces pentoxyresorufin O-dealkylase activity in rats. Japanese Journal of Pharmacology 90, 345 – 351. Vaes, L.P., Chyka, P.A., 2000. Interactions of warfarin with garlic, ginger, ginkgo, or ginseng: nature of the evidence. Annals of Pharmacotherapy 34, 1478 – 1482.
Ginkgo biloba extract modifies hypoglycemic action of tolbutamide via hepatic cytochrome P450 mediated mechanism in aged rats Tomomi Sugiyama a, Yoko Kubota b, Kazumasa Shinozuka b, Shizuo Yamada c, Jian Wu a, Keizo Umegaki a,* a National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku, Tokyo 162-8636, Japan Department of Pharmacology, School of Pharmaceutical Sciences, Mukogawa Women’s University, 11-68 Koshien Kyuban-cho, Nishinomiya 663-8179, Japan c Department of Biopharmacy and COE Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan b
Received 1 December 2003; accepted 24 February 2004
Abstract We examined hepatic cytochrome P450 (CYP)-mediated interactions between Ginkgo biloba extract (GBE) and tolbutamide, an oral anti-diabetic agent, in aged and young rats. Tolbutamide was orally given to rats with or without GBE treatment, and time-dependent changes in blood glucose were monitored. The basal activity of six CYP subtypes in liver was lower in the aged rats than in the young rats, while the inductions of these enzymes by 5 day pretreatment of 0.1% GBE diet were more in the aged rats. Further, the pretreatment of GBE significantly attenuated the hypoglycemic action of tolbutamide in the aged rats, corresponding well to the enhanced activity of (S)-warfarin 7-hydroxylase, which is responsible for CYP2C9 subtype, a major isoform metabolizing tolbutamide. In contrast, the simultaneous administration of GBE with tolbutamide potentiated the hypoglycemic action of this drug. The in vitro experiments revealed that GBE competitively inhibited the metabolism of tolbutamide by (S)-warfarin 7-hydroxylase in the rat liver microsomes. In the young rats, the 5 day pretreatment with GBE significantly attenuated the hypoglycemic action of tolbutamide, but a simultaneous treatment had little influence on the tolbutamide effect. In conclusion, the present study has shown that the simultaneous and continuous intake of GBE significantly affects the hypoglycemic action of tolbutamide, possibly via a hepatic CYP enzyme-mediated mechanism, particularly in the aged rats. Therefore, it is
* Corresponding author. Tel.: +81-3-3203-8063, fax: +81-3-3205-6549. E-mail address: [email protected] (K. Umegaki). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.02.020
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anticipated that the intake of GBE as a dietary supplement with therapeutic drugs should be cautious, particularly in elderly people. D 2004 Elsevier Inc. All rights reserved. Keywords: Ginkgo biloba extract; Cytochrome P450; Tolbutamide; Blood glucose; CYP2C9
Introduction Ginkgo biloba extract (GBE) is a popular herbal medicines, and the leaf extract of Ginkgo biloba contains some functional flavonoids and terpenoids (Kleijnen and Knipschild, 1992a; Gruenwald et al., 2000). In some European countries, standardized GBE has been clinically used mostly for cerebral insufficiency, dementia, memory impairment, tinnitus and intermittent claudication (Kleijnen and Knipschild, 1992b; Le Bars et al., 1997; Ernst, 1999; Pittler and Ernst, 2000), while it is marketed as a dietary supplement in the United States and Japan. GBE exerts many pharmacological effects, such as free radical-scavenging action and improvement of microcirculatory action (McKenna et al., 2001; De Smet, 2002; Ernst, 2002). On the other hand, GBE has been reported to have side effects such as headache, gastric symptoms, diarrhea, and allergic skin reactions, although the occasion of these side effects is rare (Cohen and Bartlik, 1998; De Smet, 2002; Ernst, 2002). Recently, adverse reactions of herbal remedies and potential herb-drug interactions have received a great deal of attention. As a well-known example, St John’s Wort has been reported to induce hepatic CYP3A4 activity, thereby leading to the attenuation of efficacy of therapeutic drugs such as cyclosporin, indinavir, and digoxin (Durr et al., 2000; Roby et al., 2000). In the case of GBE, the occurrence of bleeding has been reported in patients who took GBE and anticoagulant drugs such as aspirin, rofecoxib, or warfarin at the same time (Vaes and Chyka, 2000; Izzo and Ernst, 2001). It is proposed that the mechanism of this adverse interaction of GBE with these drugs is due to the inhibitory effect of platelet activating factors by ginkgolide B, which is one of the active constituents of GBE (Braquet and Hosford, 1991). However, other interactions between GBE and drugs have not been fully elucidated. In previous studies, we found that feeding GBE to rats markedly increased the concentration of hepatic cytochrome P450 (CYP), the expression of various CYP mRNA, and the enzyme activities in a dose- and time-dependent manner (Umegaki et al., 2000; Umegaki et al., 2002; Shinozuka et al., 2002). Moreover, we reported that pre-treatment with GBE decreased the hypotensive action by nicardipine, a calcium channel blocker, which is extensively metabolized by CYP3A type (Shinozuka et al., 2002). These findings indicate that similar interactions of GBE with other drugs through the mediation of CYPs might occur. Because of its reported beneficial effects, many elderly people take GBE (Cohen and Bartlik, 1998; De Smet, 2002; Ernst, 2002). Generally, elderly people tend to suffer from hypertension and diabetes and thereby take some other drugs simultaneously with GBE. In particular, GBE has been shown to improve peripheral blood flow (Iliff and Auer, 1983) and is expected to prevent periphery necrosis and retinopathy in severe diabetes (Lanthony and Cosson, 1988; Doly et al., 1992). It is also reported that GBE inhibits the increment of blood glucose concentration in glucose-loaded diabetic rats (Rapin et al., 1997). Thus, the simultaneous intake of GBE and anti-diabetic agents might occur, particularly in elderly people. The present study was undertaken to examine the hepatic CYP-mediated interactions of GBE with tolbutamide in aged and young rats. Tolbutamide is one of sulfonylureas applied for non-insulin-
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dependent, type 2 diabetes, and its metabolism is dependent mainly upon the CYP2C9 activity in the liver (Tal, 1993; Proks et al., 2002; Lee et al., 2003). The effect of GBE intake on the hypoglycemia of tolbutamide in rats was evaluated with a 5 day pretreatment and simultaneous treatment of GBE.
Methods Materials A standardized powder form of Ginkgo biloba extract (GBE) was supplied by Tama SeikagakuKogyo Co., Tokyo. The GBE contained 24.9% flavonoids and 10.6% total terpene, which consisted of 2.9% ginkgolide A, 1.4% ginkgolide B, 2.1% ginkgolide C, and 4.2% bilobalide, and less than 1 ppm of ginkgolic acid. Tolbutamide, resorufin, ethoxyresorufin, methoxyresorufin, pentoxyresorufin, testosterone, 6h-hydroxytestosterone, corticosterone, p-nitrophenol, 4-nitrocatechol, and 7-ethoxycoumarin were purchased from Sigma (St. Louis, MO, USA). (S)-Warfarin and 7-hydroxywarfarin were obtained from Ultrafine (Manchester, England). NADPH was obtained from Oriental Yeast (Tokyo, Japan). All other reagents were obtained from Wako Pure Chemical Ltd. (Osaka, Japan). Animal experiment Male Wistar aged (19 months old) and young (7 weeks old) rats obtained from Japan SLC (Shizuoka, Japan) were housed individually in stainless steel, wire-bottomed cages in a constant temperature room (23 F 1jC) under 12 hr light-dark cycle. Rats were divided into several groups with or without GBE treatment. GBE pretreated group was fed commercial rodent diet CE2 (Japan Clea, Tokyo) containing 0.1% GBE for 5 days. After the treatment, rats were orally administered tolbutamide (40 mg/kg). The simultaneous GBE treated group was given GBE (100 mg/kg body weight, p.o.) simultaneously with tolbutamide. After the tolbutamide administration, the blood was collected at predetermined time points and immediately blood glucose concentration was determined using a blood glucose detector Dexter Z II (Bayer Corp. Mishawaka, IN). After the treatment with or without GBE, the rat liver was removed and weighed, and a part of the liver was subjected to the enzyme assay. All procedures were in accordance with National Institute of Health and Nutrition guidelines for the Care and Use of Laboratory Animals.
Analytical methods Preparation of microsome and cytosolic fractions from the liver The liver was rinsed with 0.9% NaCl solution, weighed and homogenized in 50 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose. The homogenate was centrifuged at 10,000 g at 4jC for 30 min. The supernatant was further centrifuged at 105,000 g at 4jC for 60 min. The supernatant was used as the cytosolic fraction for the assay of glutathione S-transferase, the activity of which was determined by the method of Habig and Jakoby using 1-chloro-2,4-dinitrobenzene as a substrate (Habig and Jakoby, 1981). The pellet was washed once with 50 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose by
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centrifugation at 105,000 g at 4jC for 60 min, and the concentration and activities of CYP were analyzed. Protein concentrations of microsome and cytosolic fractions were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Analysis of CYP enzyme activities Cytochrome P450 content was quantified by the method of Omura and Sato (Omura and Sato, 1964). The activities of various CYP enzymes were determined by HPLC methods as reported previously (Umegaki et al., 2002). The subtypes of CYP enzymes examined and the corresponding CYPs were methoxyresorufin O-demethylase, CYP1A2; ethoxyresorufin O-deethylase, CYP1A1; pentoxyresorufin O-dealkylase, CYP2B; p-nitrophenol hydroxylase, CYP2E1; testosterone 6h-hydroxylase, CYP3A; and (S)-warfarin 7-hydroxylase, CYP2C9. The interaction between GBE and tolbutamide toward (S)-warfarin hydroxylase activity in vitro were evaluated by adding these substances at various concentrations (GBE; 1.0–30 Ag/mL, tolbutamide; 0.1– 30 Ag/mL) to the enzyme assay system ((S)-warfarin concentration; 2–16 AM). Statistical analysis Statistical analysis of the data was carried out using ANOVA followed by a post hoc test of Fisher’s PLSD. These statistical analyses were performed using a computer program (Stat View 5.0, ASA Institute Inc, Cary, NC, USA).
Results Induction of hepatic drug-metabolizing enzymes by GBE Effects of 5 days of feeding of 0.1% GBE diet on the liver weight and hepatic drug metabolizing enzymes are shown in Table 1. The calculated intake of GBE was about 14 mg per day in both groups and
Table 1 Effects of 5 day pretreatment with GBE on the liver weight, cytochrome P450 content, and glutathione S-transferase activity in aged and young rats Aged rats Body weight (g) Liver weight (% /Body weight) Cytochrome P450 content (nmol/mg protein) Glutathione S-transferase (nmol/mg protein/min)
Young rats
Control
0.1% GBE
Control
0.1% GBE
433 F 26 2.76 F 0.09 0.731 F 0.08 565 F 197
450 F 18 3.28 F 0.06a 1.692 F 0.33a 1054 F 217a
189 F 7 4.14 F 0.19 0.921 F 0.05 667 F 104
190 F 6 4.12 F 0.29 1.749 F 0.22b 934 F 236b
Male Wistar aged (19 months old) and young (7 weeks old) rats received commercial rodent diet CE2 with or without 0.1% GBE for 5 days. Each value is the mean F SD from six rats. Significant differences from each control level are indicated by a P < 0.01 and bP < 0.05.
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was 32 mg (per kg body weight in a day) in the aged rats and 72 mg in the young rats. The liver weight in the aged rats but not in the young rats was significantly increased to 1.2-fold by the GBE treatment. Significant increases in the cytochrome P450 content and glutathione S-transferase activity were also detected by the GBE treatment, both in the aged rats (2.3- and 1.9-fold, respectively) and young rats (1.9- and 1.4-fold, respectively). Activities of various CYP are shown in Table 2. The basal levels of the activities of six CYP subtypes were clearly lower in the aged rats than in the young rats. The activity of (s)-warfarin 7-hydroxylase, which is responsible for the metabolism of tolbutamide, in the aged rats was about 41% of that in the young rats. Both in the aged and young rats, the GBE pretreatment significantly increased all CYP enzymes, especially in pentoxyresorufin O-dealkylase and (s)-warfarin 7-hydroxylase activity. The induction ratios in both groups were apparently higher in the aged rats; the ratios in the young and aged rats were 13- and 42-fold for pentoxyresorufin O-dealkylase, respectively, and 2.1- and 4.7-fold for (s)-warfarin 7-hydroxylase activity, respectively. Effects of GBE on the efficacy of tolbutamide The time-dependent changes in the blood glucose concentration by tolbutamide administration in the aged rats are shown in Fig. 1. The baseline concentration of blood glucose was approximately 100 mg/ dl. In the control group, the blood glucose concentration decreased to the minimum level (the reduction: 32.2 F 2.4 mg/dl, n = 6) at 3 hr after oral administration of tolbutamide (40 mg/kg) and remained at the lower level until for least 7 hr. In the 0.1% GBE diet (5 days) pretreated group, the hypoglycemic action of tolbutamide was considerably attenuated when compared with that in the control group without GBE, and the statistical significance was detected at 2–5 and 7 hr. This result indicated that the pretreatment of GBE reduced the pharmacological action of tolbutamide. On the other hand, a simultaneous treatment of tolbutamide with GBE at a single dose (100 mg/kg body weight) significantly lowered (at 4–6 hr) the blood glucose concentrations in the aged rats, compared with the treatment of tolbutamide alone in the control group (Fig. 1). This result indicated that the simultaneous treatment of tolbutamide and GBE potentiated the hypoglycemic efficacy of tolbutamide. In the young rats, the pretreatment (5 days) of 0.1% GBE diet significantly attenuated the hypoglycemic action of tolbutamide as in the aged rats,
Table 2 Inductions of various hepatic CYP activities by 5 day pretreatment with GBE in aged and young rats Activity (pmol/mg protein/min) Aged rats Control Ethoxyresorufin O-deethylase (CYP1A1) Methoxyresorufin O-demethylase (CYP1A2) Pentoxyresorufin O-dealkylase (CYP2B) (s)-Warfarin 7-hydroxylase (CYP2C9) p-Nitrophenol hydroxylase (CYP2E1) Testosterone 6h-hydroxylase (CYP3A)
13.5 15.5 8.2 2.6 1453 218
F F F F F F
Young rats 0.1% GBE
0.9 1.1 0.3 0.4 99 15
37.9 33.2 348.4 12.1 3580 564
F F F F F F
Control a
1.4 2.0a 39.6a 0.8a 215a 42a
24.7 27.6 35.5 6.3 1717 295
F F F F F F
0.1% GBE b
1.5 2.4b 6.4 0.4b 76 30
44.2 41.7 476.7 13.5 4064 739
F F F F F F
1.6a 1.9a 23.6a 1.2a 170a 20a
Male Wistar aged (19 months old) and young (7 weeks old) rats received commercial rodent diet CE2 with or without 0.1% GBE for 5 days. Each value is the mean F SD from six rats. Significant difference from each control level is indicated by a P < 0.001. Significant difference from control of aged rats is indicated by bP < 0.01.
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Fig. 1. Effects of simultaneous treatment and 5 day pretreatment with Ginkgo biloba extract (GBE) on the hypoglycemic effect of tolbutamide in aged rats. Aged rats (19 months old) were administered tolbutamide (40 mg/kg, p.o.) with or without GBE treatment. The GBE pretreated group was given feed containing 0.1% GBE for 5 days, and a simultaneous GBE treated group was given a single dose of GBE (100 mg/kg, p.o.) with tolbutamide. After the tolbutamide administration, blood was collected for the analysis of blood glucose concentrations. Each point represents the mean F SD from six rats. , control group; E, GBE pretreated group; and 5, a simultaneous GBE treated group. Significant difference from the control group is indicated by *P < 0.05.
.
whereas the simultaneous GBE treatment at a single dose exhibited little significant interaction with tolbutamide (Fig. 2). Competitive inhibition of GBE and tolbutamide on CYP2C9 activity in vitro The direct interaction of tolbutamide and GBE toward (S)-warfarin 7-hydroxylase (CYP2C9) activity was examined using rat liver microsome in vitro. As shown in a Dixon plot, tolbutamide competitively
Fig. 2. Effects of simultaneous treatment and 5 day pretreatment with Ginkgo biloba extract (GBE) on the hypoglycemic effect of tolbutamide in young rats. Young rats (7 weeks old) were administered tolbutamide (40 mg/kg, p.o.) with or without GBE treatment. The GBE pretreated group was given feed containing 0.1% GBE for 5 days, and a simultaneous GBE treated group was given a single dose of GBE (100 mg/kg, p.o.) with tolbutamide. After tolbutamide administration, blood was collected for analysis of blood glucose concentrations. Each point represents the mean F SD from six rats. , control group; E, GBE pretreated group; and 5, a simultaneous GBE treated group. Significant difference from the control group is indicated by *P < 0.05.
.
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Fig. 3. Effects of tolbutamide (A) and Ginkgo biloba extract (B) on the (S)-warfarin 7-hydroxylase (CYP2C9) activity in rat liver microsome in vitro (Dixon plot). The (S)-warfarin 7-hydroxylase (CYP2C9) activities were measured in the presence of various concentrations of tolbutamide (0.1 – 30 Ag/mL) or GBE (1.0 – 30 Ag/mL) in rat liver microsome in vitro. Loaded (S)warfarin concentrations were , 2 AM; D, 4 AM; n, 8 AM and w , 16 AM. Each point represents the mean from five experiments.
.
inhibited the metabolism of (S)-warfarin by the (S)-warfarin 7-hydroxylase (Fig. 3A). Similarly, GBE decreased the (S)-warfarin 7-hydroxylase activity in vitro in a concentration-dependent manner, and showed competitive inhibition toward the enzyme activity (Fig. 3B). These results indicated that the hepatic metabolism of tolbutamide was competitively inhibited by GBE. The calculated Ki values were 19 Ag/mL for GBE and 75 Ag/mL for tolbutamide, respectively.
Discussion Elderly people often suffer from functional impairments in their bodies and diabetes in these days. Considering these situations and the efficacy of GBE reported so far (Iliff and Auer, 1983; Lanthony and Cosson, 1988; Doly et al., 1992; Rapin et al., 1997), the simultaneous intake of GBE and anti-diabetic drug is most likely to occur in the elderly. For the safe use of both GBE and drugs, it is important to elucidate how GBE taken as a dietary supplement modifies the efficacy of therapeutic drugs. In the present study, we investigated the influence of GBE intake on the hypoglycemic efficacy of tolbutamide. We focused particularly on the elderly of advanced age and on the hepatic CYP-mediated interactions. The pharmacodynamic interaction was evaluated by inducing hypoglycemia with an excess administration of tolbutamide in aged (19-month-old) and young (7-week-old) rats. As reported previously (Szabo et al., 1995; Umegaki et al., 2000), the intake of GBE did not change the basal blood glucose level in the aged and young rats with the pretreatment of GBE with diet (data not shown). However, the pretreatment and simultaneous treatment of GBE markedly influenced the hypoglycemic action of tolbutamide in the aged rats (Fig. 1). In the case of 5 days pretreatment of GBE, the hypoglycemic action of tolbutamide was attenuated both in the aged and young rats. As shown
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in Table 2, the 5 day pretreatment of GBE markedly induced hepatic drug metabolizing enzymes, especially (S)-warfarin 7-hydroxylase, an enzyme corresponding to CYP2C9 which mainly metabolizes tolbutamide in the liver (Lee et al., 2003). Therefore, the induction by GBE pretreatment of hepatic CYP2C9 may reduce the plasma concentration of tolbutamide after oral administration, thereby leading to the attenuation of the hypoglycemic action of tolbutamide. In the present study, the plasma concentration of tolbutamide was not determined, due to the limited blood sample from the GBEpretreated rats. However, the change in the blood glucose concentration was taken as the final outcome of interaction between GBE and tolbutamide, and the observed changes of CYP2C9 responsible for tolbutamide metabolism following the GBE pretreatment could well explain the mechanism of the pharmacodynamic interaction. Kudolo (Kudolo, 2001) also reported that the ingestion of GBE by non-insulin-dependent diabetes mellitus subjects induced the hepatic metabolic clearance rate of insulin and hypoglycemic agents, suggesting an increase of blood glucose. In the previous paper (Shinozuka et al., 2002), we also showed that pretreatment of GBE significantly reduced the hypotensive action of nicardipine, which is metabolized by CYP3A2 isoform in rats. The mechanism of these interactions is similar to that in St John’s Wort (Durr et al., 2000; Roby et al., 2000), a well-known inducer of CYP, indicating that the induction of CYP by pretreatment with GBE attenuates the efficacy of various drugs. Inasmuch as the beneficial effects of GBE in humans are expected after the continuous intake for more than 4 weeks (Gruenwald et al., 2000; Blumenthal, 1998), the induction of hepatic CYP would be taken into account for the interaction with drugs seen after a long-term intake of GBE as a dietary supplement. In contrast to the GBE pretreatment, simultaneous treatment of tolbutamide with GBE as a single dose potentiated hypoglycemic action of tolbutamide in the aged rats (Fig. 1). In order to clarify the underlying mechanism of this interaction, a direct effect of GBE on the hepatic (S)-warfarin 7hydroxylase (subtype of CYP2C9) activity was examined in vitro. As tolbutamide is mainly metabolized by (S)-warfarin 7-hydroxylase (Lee et al., 2003), tolbutamide competitively inhibited the hepatic metabolism of (S)-warfarin as shown in the Dixon plot analysis (Fig. 3A). Interestingly, GBE also showed the competitive inhibition toward the metabolism of (S)-warfarin (Fig. 3B), suggesting that GBE competitively inhibited the metabolism of tolbutamide by the CYP2C9 enzyme in the liver of rats. Thus, the pharmacodynamic interaction between GBE and tolbutamide after their single administration could be explained by the following mechanism. Following the simultaneous intake of GBE and tolbutamide, the hepatic metabolism of tolbutamide would be attenuated via the significant occupancy by GBE of the same active site on the CYP2C9 enzymes where this drug binds, resulting in an enhancement of hypoglycemic action of tolbutamide. The induction of hepatic drug metabolizing enzymes and attenuation of hypoglycemic efficacy of tolbutamide were observed by the pretreatment of GBE both in the young rats and aged rats. However, the extent of these effects by GBE pretreatment was greater in the aged rats than in the young rats. This distinction may be explained by the difference between the two groups of rats in the induced level of CYP enzymes. In fact, the activities of pentoxyresorufin O-dealkylase and (S)-warfarin 7-hydroxylase were induced more (2 to 3-fold) by the GBE treatment in the aged rats than in the young rats (Table 2). This greater induction of CYP2B and CYP2C9 in the aged rats may be somehow related to the lower basal activities of these enzymes, which displayed about 23 and 41%, respectively, of the enzyme activity in the young rats. Interestingly, in the case of simultaneous treatment of GBE and tolbutamide, the potentiation of hypoglycemic action of tolbutamide was not detected in the young rats (Fig. 2). A clear explanation for the absence of enhancement of the pharmacological effect by tolbutamide in the young rats is lacking. Further
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detailed experiments will be necessary to clarify pharmacokinetic and pharmacodynamic difference of tolbutamide between young and aged rats observed by the GBE treatment. In any event, it is evident that the aged rats may be more susceptible to the CYP-mediated interactions of GBE and tolbutamide when compared to the young rats. This finding may be applied for humans. As a dietary supplement, 120 to 240 mg of GBE are generally taken in a day (Blumenthal, 1998; Gruenwald et al., 2000; Ernst, 2002), and this dose can be estimated as 2 to 4 mg/kg body weight. In this animal study, the intake of GBE was 30 f 100 mg/kg body weight per day both in dietary pretreatment and in a single dose treatment. In our previous study, a significant induction of (s)-warfarin 7hydroxylase was detected even at the lower dose (10 mg/kg body weight) of GBE (Umegaki et al., 2002). Owing to the species difference in the susceptibility to GBE, it is still not clear whether the interaction of tolbutamide and GBE also occurs in humans. Although the extrapolation of pharmacokinetic and pharmacodynamic interaction of GBE and tolbutamide in the aged rats to the clinical situation should be interpreted with caution, it is anticipated, from our recent preliminary study with healthy volunteers, that the repeated oral intake of GBE may influence the pharmacokinetics and pharmacodynamics of tolbutamide in humans (unpublished observation). In conclusion, the present study has shown that the intake of GBE significantly affects the efficacy of tolbutamide in the aged rats. Therefore, it is anticipated that the simultaneous and continuous intake of GBE as a dietary supplement with therapeutic drugs should be cautious, particularly in elderly people.
Acknowledgements This work was financially supported in part by a research grant of Ministry of Health Labor and Welfare in Japan. Authors are grateful to Ms. Keiko Saito for her technical assistance.
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