ToxSci Advance Access originally published online on March 17, 2006
Toxicological Sciences 2006 91(2):356-364; doi:10.1093/toxsci/kfj164
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Ginsenoside Metabolites, Rather Than Naturally Occurring Ginsenosides, Lead to Inhibition of Human Cytochrome P450 Enzymes
* Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People's Republic of China; Graduate School of the Chinese Academy of Sciences, People's Republic of China; College of Life Sciences, Liaoning Normal University, Dalian 116029, People's Republic of China; and The Second Affiliated Hospital of Dalian Medical University, Dalian 116027, People's Republic of China
1 To whom correspondence should be addressed at 457, Zhong-shan Road, Dalian 116023, People's Republic of China. Fax: (86) 411-84676961. E-mail: yling{at}dicp.ac.cn.
Received November 25, 2005; accepted March 10, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is still an argument about ginseng–prescription drug interactions. To evaluate the influence on cytochrome P450 (P450) activities of ginseng in the present study, the influence on P450 activities of naturally occurring ginsenosides and their degradation products in human gut lumen was assayed by using human liver microsomes and cDNA-expressed CYP3A4. The results showed that the naturally occurring ginsenosides exhibited no inhibition or weak inhibition against human CYP3A4, CYP2D6, CYP2C9, CYP2A6, or CYP1A2 activities; however, their main intestinal metabolites demonstrated a wide range of inhibition of the P450-mediated metabolism. There was no mechanism-based inhibition found on these P450 isoforms. It is noteworthy that Compound K, protopanaxadiol (Ppd), and protopanaxatriol (Ppt) all exhibited moderate inhibition against CYP2C9 activity, and Ppd and Ppt also exhibited potent competitive inhibition against CYP3A4 activity. We suggest that after oral administration, naturally occurring ginsenosides might influence hepatic P450 activity in vivo via their intestinal metabolites.
Key Words: ginseng-drug interactions; ginsenosides; intestinal metabolites; cytochrome P450.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ginseng, one of the most commonly used herbal drugs, has been extensively used in traditional oriental medicine for over 2000 years and is also increasingly used as a general health tonic in many countries. There is some evidence that ginsenosides, the major active ingredients of ginseng, have a variety of biomedical efficacies such as antioxidation and anti-inflammatory activities (Attele et al., 1999
). According to the number and position of sugar moieties, ginsenosides are divided into two main categories, the 20(S)-protopanaxadiol and 20(S)-protopanaxatriol families (Fig. 1).
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-L-arabinopyranosyl; Araf: Numerous persons have taken ginseng or its derived products. However, they are not free from adverse effects, and there are a number of reports about ginseng–prescription drug interactions (Donovan et al., 2003
; Gurley et al., 2002, 2005; Janetzky and Morreale, 1997; Jiang et al., 2004; Smith et al., 2001). Metabolizing enzyme-based drug interactions constitute the major proportion of clinically important drug interactions. Cytochrome P450 isoforms (P450), a superfamily of heme-containing isoenzymes located primarily in hepatocytes, are responsible for the oxidative metabolism of the majority of drugs and xenobiotics (Estabrook, 1999). The inhibition of P450 activities may result in clinically significant drug-drug interactions (DDI) (Ekins et al., 2000).
There were several in vivo studies about the effects of ginseng, ginseng extracts, or naturally occurring ginsenosides on P450 activities. However, the reported effects of ginseng on P450 activities in the clinical trials are somewhat contradictory, as these studies have shown that ginseng may have no significant effects (Donovan et al., 2003; Gurley et al., 2002) or have statistically significant inhibition of some P450 activities (Gurley et al., 2005; Smith et al., 2001).
Because ginseng products are orally administered in most cases, the influence of ginsenosides on P450 activities in vivo includes the influence on both intestinal and hepatic P450 activities. The majority of naturally occurring ginsenosides including Rb1, Rb2, and Rg1 are poorly absorbed (Odani et al., 1983a,b), yet they have been found to be deglycosylated by colonic bacteria followed by transit to the systemic circulation (Hasegawa, 2004). Thus, each of the orally administered components and their intestinal bacterial metabolites in the gastrointestinal tract can possibly exert an influence on intestinal P450. However, the influence on hepatic P450 should be subject to the precondition that the ginsenosides can be absorbed from the gastrointestinal tract and enter systemic circulation. Therefore, to clarify whether or not ginseng can influence P450 activities in vivo, a systematic research is needed.
The in vitro studies suggested that the standardized Panax ginseng (Asian ginseng) extract (G115) and the standardized Panax quinquefolius (North American ginseng) extract (NAGE) could influence some human P450 activities, but naturally occurring ginsenosides, including Rb1, Rb2, Rc, Rd, Re, Rf, or Rg1, were not likely to inhibit drug-metabolizing enzymes (Chang et al., 2002; Henderson et al., 1999). Rh1 and F1, the hydrolysis products of naturally occurring ginsenosides in the gastrointestinal tract, can enter systemic circulation (Tawab et al., 2003). Our recent studies showed that Rh1 and F1 had the potential to exert an influence on some human P450 activities in vitro, though their in vivo influence on the human hepatic P450 was likely to be very weak (Liu et al., 2006). We also found that naturally occurring ginsenosides including Rb1, Rb2, Rc, Re, Rg1, and one of their intestinal bacterial metabolites, Compound K (C-K), had no inhibitory effect, whereas another intestinal bacterial metabolite, protopanaxatriol (Ppt), exhibited potent inhibition against rat CYP3A activity (Liu et al., 2004). The intestinal bacterial metabolites, including C-K, protopanaxadiol (Ppd), and Ppt, were easily absorbed and appeared in the plasma of rats or humans after oral administration of Rb1, Re, or Rg1 (Akao et al., 1998; Bae et al., 2000; Hasegawa et al., 2002; Tawab et al., 2003). These results prompted us to consider whether the biotransformation of ginsenosides in the gastrointestinal tract plays an important role in ginseng-drug interactions via the influence of P450 and is responsible for the inconsistency of these clinical studies.
The aim of the present study was to explore the effects of naturally occurring ginsenosides and their intestinal metabolites, which could reach the liver and systemic circulation after oral administration of naturally occurring ginsenosides, on the activities of human P450. In the present study, using human liver microsomes and cDNA-expressed CYP3A4, we found that the intestinal metabolites of ginsenosides, including C-K, Ppd, and Ppt, demonstrated a wide range of inhibition of human P450-mediated metabolism, particularly the inhibition of CYP3A4 and CYP2C9. There was no mechanism-based inhibition found on CYP3A4, CYP2D6, CYP2C9, CYP2A6, or CYP1A2 in tested ginsenosides.
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MATERIALS AND METHODS |
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Chemicals.
D-Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, corticosterone, NADP+, phenacetin, acetaminophen, 7-hydroxycoumarin, 4'-hydroxydiclofenac, tolbutamide, 4'-hydroxytolbutamide, dextrorphan, furafylline, 8-methoxypsoralen, sulfaphenazole, quinidine, clomethiazole, and 6ß-hydroxytestosterone were purchased from Sigma-Aldrich (St. Louis, MO). Testosterone was obtained from Acros Organics (Morris Plains, NJ). Coumarin, diclofenac, dextromethorphan, and ketoconazole were purchased from ICN Biomedicals, Inc. (Aurora, OH). Ginsenoside Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, S-Rg3, Rh1, Rh2, F1, C-K, Ppd, and Ppt were generously provided by Dr. Hideo Hasegawa (Fermenta Herb Institute Inc., Tokyo, Japan). The purity of these compounds was >98% as assessed by HPLC. All other reagents were of the highest purity commercially available or HPLC grade.
Human liver microsomes and cDNA-expressed human CYP3A4.
The preparation of pooled human liver microsomes has been reported in our previous study (Liu et al., 2006). Protein concentrations of the microsomal fractions were determined by the Lowry method using bovine serum albumin as a standard (Lowry et al., 1951).
cDNA-expressed CYP3A4 in Escherichia coli coexpressing NADPH-P450 reductase was purchased from New England Biolabs (Beijing) Ltd. (Beijing, China). The P-450 contents were used as described in the data sheets provided by the manufacturers.
Enzymatic activity assay.
The P450 activities were characterized based on their probe reactions: CYP1A2 (phenacetin O-deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (diclofenac 4'-hydroxylation), CYP2D6 (dextromethorphan O-demethylation), and CYP3A4 (testosterone 6ß-hydroxylation). Preliminary experiments were performed to determine incubation conditions specific to each isoform that was linear with time and protein concentrations. The percentage of conversion of metabolites never exceeded 20% of substrate added. Incubation and analytical conditions varied depending on the characteristics of the probe drug, which had been reported in our previous study (Liu et al., 2006). In brief, each incubation was performed in a 100mM phosphate buffer at pH 7.4 containing human microsomal protein (final protein concentrations were 0.2 mg/ml for CYP1A2, 0.1 mg/ml for CYP2A6, 0.3 mg/ml for CYP2C9, 0.2 mg/ml for CYP2D6, or 0.5 mg/ml for CYP3A4), NADPH-generating system (10mM glucose 6-phosphate, 1mM NADP+, 4mM magnesium chloride, 1 unit/ml of glucose 6-phosphate dehydrogenase), and various probe substrates of P450 (dextromethorphan previously dissolved in water, the others previously dissolved in methanol, whose final concentration was 1%, v/v) with a range of concentrations in a total volume of 400 µl (microsomes) or 200 µl (cDNA-expressed CYP3A4). There was a 3-min preincubation step at 37°C before the reaction was started by the addition of NADP+. The reactions were continued for 10 min (30 min for CYP1A2) at 37°C, and the reactions were quenched by adding the same volume of acetonitrile or 10% trichloroacetic acid, and an internal standard. The incubation mixtures were then centrifuged for 10 min at 20,000 x g. An aliquot of the supernatant was analyzed by HPLC. The HPLC system (SHIMADZU, Kyoto, Japan) consisted of an SCL-10A system controller, two LC-10AT pumps, an SIL-10A autoinjector, and an SPD-10AVP UV detector or an RF-10AXL fluorescence detector. The chromatographic separation was achieved using a C18 column (4.6 x 150 mm internal diameter, 5-µm particle size) at a flow rate of 1 ml/min.
Inhibition of P450 activity assay.
A typical incubation mixture contained human microsomal protein (0.2 mg/ml for CYP1A2, 0.1 mg/ml for CYP2A6, 0.3 mg/ml for CYP2C9, 0.2 mg/ml for CYP2D6, or 0.5 mg/ml for CYP3A4) or 40 pmol/ml cDNA-expressed CYP3A4, NADPH-generating system, various probe substrates of P450, and different concentrations (0–100µM) of individual ginsenosides, in a 100mM phosphate buffer (pH 7.4). The selective inhibitors of each P450 isoform (furafylline for CYP1A2, 8-methoxypsoralen for CYP2A6, sulfaphenazole for CYP2C9, quinidine for CYP2D6, ketoconazole for CYP3A4) were used as positive controls. There was a 3-min preincubation step at 37°C before the reaction was started by the addition of NADP+. These ginsenosides and inhibitors were previously dissolved in methanol or water. The final concentration of methanol in the incubation system was 1% (v/v). The reactions were continued for 10 min (30 min for CYP1A2) at 37°C. The reactions were quenched by adding the same volume of acetonitrile or 10% trichloroacetic acid, and internal standard. The incubation mixtures were then centrifuged for 10 min at 20,000 x g. Finally, an aliquot of supernatant was used for HPLC analysis. All experiments were separately performed in duplicate three times.
Enzyme kinetics analysis.
The apparent Km and Vmax values were determined in a range of concentrations of probe substrates. The substrate concentrations were as follows: phenacetin 10–100µM, coumarin 0.3–3µM, diclofenac 3–30µM, dextromethorphan 0.5–30µM, and testosterone 10–100µM. The inhibition constant Ki values were determined in a range of concentrations of testosterone (30, 50, 80, and 100µM) and different concentrations of Ppd (0–50µM), Ppt (0–20µM), or ketoconazole (0–1000nM). All experiments were separately performed in duplicate three times.
Time- and NADPH-dependent inactivation assay.
To test whether there was mechanism-based inhibition of these ginsenosides on P450, the time- and NADPH-dependent inhibition was assayed. In brief, the preincubation reactions were conducted at 37°C. Ginsenoside Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, S-Rg3, Rh1, Rh2, F1, C-K, Ppd, or Ppt of a certain concentration (40 or 100µM) was preincubated with phosphate buffer (100mM, pH 7.4) and human liver microsomes (the final protein concentrations were 2 mg/ml for CYP1A2, 1 mg/ml for CYP2A6, 3 mg/ml for CYP2C9, 2 mg/ml for CYP2D6, and 5 mg/ml for CYP3A4, respectively) in the presence of the NADPH-generating system (as described above). The preincubation reactions were initiated by the addition of an NADPH-generating system for a final incubation volume of 1 ml. At various times (0, 5, 10, 15, and 20 min), preincubation mixtures (40 µl) were removed from the preincubation reaction tubes and were diluted 10-fold into small tubes already containing substrates and NADPH-generating system (as described above). After transfer, the total incubation volume in each small tube was 400 µl, and the final concentrations of substrates in each small tube were 80µM for phenacetin, 2µM for coumarin, 50µM for dextromethorphan, 100µM for testosterone, or 20µM for diclofenac. The incubation was continued for 10 min (30 min for CYP1A2) at 37°C. The reaction was terminated by the addition of the same volume of acetonitrile or 10% trichloroacetic acid containing respective internal standards. The mixtures were then centrifuged for 10 min at 20,000 x g. Finally, an aliquot of supernatant was used for HPLC analysis. P450 activity was assayed. Activities were normalized to the activity at 0 min so that the percent decrease in P450 activity reflected activity loss due only to inactivation and not reversible inhibition. All experiments were performed in duplicate.
Kinetics data analysis.
The apparent kinetic parameters for each probe reaction (i.e., Vmax and Km) were estimated from nonlinear regression analysis of experimental data that were conducted using multiple compound concentrations to the Michaelis-Menten equation. Inhibition data from experiments that were conducted using multiple compound concentrations were graphically represented by Dixon plots, and inhibition constant (Ki) values were calculated with the use of nonlinear regression according to the equations for competitive inhibition (Equation 1) and noncompetitive inhibition (Equation 2) (Copeland, 2000),
 | (1) |
 | (2) |
The calculation of possible concentrations of ginsenosides in blood and gut lumen.
Based on the hypothesis that the possible maximum concentrations of naturally occurring ginsenosides in gut lumen were the ratio of orally administered dose excluding the fraction absorbed into blood to the volume of that in the gut lumen, the possible maximum concentrations of Rb1, Rb2, and Rg1 in human blood and gut lumen after a single oral administration of G115 were estimated according to the equations for gut lumen (Equation 3) and blood (Equation 4),
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 | (4) |
The Fc values of Rb1, Rb2, and Rg1 in G115 were about 1.07, 0.66, and 0.44%, respectively (Chang et al., 2002), and the common average recommended dosage for G115 was about 700 mg/day as a single oral dose (Tawab et al., 2003). The reported VL was 1650 ml/70 kg (Davies and Morris, 1993). The reported oral bioavailabilities of Rb1, Rb2, and Rg1 were 4.35, 3.7, and 18.40%, respectively (Hasegawa, 2004; Xu et al., 2003).
The intestinal metabolites of naturally occurring ginsenosides C-K and Ppd are likely to be derived from the hydrolysis of Rb1 or Rb2, and another intestinal metabolite Ppt is likely to be derived from hydrolysis of Rg1 by intestinal bacteria in the gut lumen (Hasegawa, 2004; Hasegawa et al., 1996; Tawab et al., 2003). Their amounts in the gut lumen cannot be directly estimated because of the absence of quantitative data of biotransformation and absorption. However, their estimated maximal amounts in the gut lumen should be less than the sum of amounts of parental ginsenosides after a single oral dose. In addition, after the oral administration of ginseng total saponin (1 g/kg), the ratio of C-K to Ppd concentration in rat blood is 6.3 (Hasegawa et al., 1996). We arbitrarily assumed that the ratio in rat equaled to that in human blood.
The calculation of ratio of Cmax/Ki in humans and rats.
The ratios of Cmax/Ki of Ppd and Ppt were estimated according to the equations for humans (Equation 5) and rats (Equation 6) (Bjornsson et al., 2003),
 | (5) |
 | (6) |
), and Ki,r is the reported Ki value in rat liver microsomes (3.2µM for Ppt inhibiting rat CYP3A activity, and no data obtained for Ppd) (Liu et al., 2004). |
RESULTS |
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Enzymatic Kinetic Parameters for P450 in Human Liver Microsomes and cDNA-Expressed CYP3A4
The apparent Km values for phenacetin O-deethylation, coumarin 7-hydroxylation, diclofenac 4'-hydroxylation, dextromethorphan O-demethylation, and testosterone 6ß-hydroxylation in human liver microsomes were 43.4, 1.1, 13.7, 5.1, or 52.1µM, respectively. The Vmax values for phenacetin O-deethylation, coumarin 7-hydroxylation, diclofenac 4'-hydroxylation, dextromethorphan O-demethylation, and testosterone 6ß-hydroxylation in human liver microsomes were 0.28, 0.40, 0.47, 0.56, or 2.59 nmol/min/mg protein, respectively. The apparent Km and Vmax values for testosterone 6ß-hydroxylation in cDNA-expressed CYP3A4 were 54.2µM and 104.3 nmol/min/nmol P450, respectively.
Inhibition of P450 Activities by the Intestinal Metabolites of Ginsenosides in Human Liver Microsomes
To investigate whether these ginsenosides affect the catalytic activity of P450, the probe reaction assays were conducted with various concentrations of ginsenosides. The specific inhibitors of each P450 isoform were used as positive controls.
Results from our study (Table 1) showed that naturally occurring ginsenosides, including Rb1, Rb2, Rc, Re, and Rg1, exhibited no inhibition on CYP3A4, CYP2D6, CYP2C9, CYP2A6, or CYP1A2 activities, and another naturally occurring ginsenoside Rd exhibited inhibition on some P450 activities with an IC50 value of 78.4 ± 5.3µM for CYP1A2, 85.1 ± 9.1µM for CYP2C9, 58.0 ± 4.5µM for CYP2C9, or 81.7 ± 2.6µM for CYP3A4. Their degradation products Rg2, S-Rg3, and Rh2 also exhibited no inhibition on tested P450 activities, except the inhibition of S-Rg3 on CYP2D6 with an IC50 value of 81.0 ± 6.5µM and of Rh2 on CYP3A4 with an IC50 of 94.1 ± 7.9µM.
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C-K, one of the intestinal metabolites of 20(S)-protopanaxadiol derivatives, exhibited an inhibition against the activity of CYP2C9 in human liver microsomes with an IC50 value of 32.0 ± 3.6µM, a weak inhibition against the activity of CYP2A6 in human liver microsomes with an IC50 value of 63.6 ± 4.2µM, and an even weaker inhibition against the activity of CYP2D6 in human liver microsomes with an IC50 value of more than 100µM. However, at 100µM, C-K did not exhibit inhibitory activity against the activities of CYP1A2 and CYP3A4 in human liver microsomes but exhibited weak stimulation with an increase of 14.8% in the activity of CYP1A2 at 100µM.
Different from C-K, Ppd, the other intestinal metabolite of 20(S)-protopanaxadiol derivatives, was found to strongly inhibit CYP3A4-mediated testosterone 6ß-hydroxylation activity in human liver microsomes with an IC50 value of 14.1 ± 2.3µM. Ppd also exhibited an inhibition against the activity of CYP2C9 in human liver microsomes with an IC50 value of 42.7 ± 2.2µM and weak inhibition against the activities of CYP1A2, CYP2A6, and CYP2D6 in human liver microsomes with an IC50 value of more than 100µM.
Ppt, the intestinal metabolite of 20(S)-protopanaxatriol derivatives, exhibited stronger inhibition against CYP3A4 activity in human liver microsomes with an IC50 value of 7.1 ± 0.9µM. Ppt also exhibited an inhibition against CYP2C9 activity in human liver microsomes with an IC50 value of 33.7 ± 2.7µM and exhibited weaker inhibition against the activities of CYP1A2, CYP2A6, and CYP2D6 in human liver microsomes with an IC50 value of more than 100µM.
Inhibition Kinetic Analysis
To further characterize the inhibition of CYP3A4 activity by Ppd and Ppt, enzyme inhibition kinetic experiments were performed. We calculated IC50 and Ki values of ketoconazole to compare with previous studies. As positive control, ketoconazole inhibited testosterone 6ß-hydroxylation with an IC50 value of 52.1 ± 5.4nM, which was comparable to published data (40–100nM) in human liver microsomes (Eagling et al., 1998; McKillop et al., 1998). From the relevant data, the Ki value of ketoconazole calculated was 30.8 ± 9.5nM.
Ppd and Ppt were found to strongly inhibit testosterone 6ß-hydroxylation in both human liver microsomes and cDNA-expressed CYP3A4. The representative Lineweaver-Burk plots for the inhibition of testosterone 6ß-hydroxylation by Ppd and Ppt (Figs. 2A–5A) and analysis of the parameters of the enzyme inhibition model suggested that the inhibition types of Ppd and Ppt were competitive. Based on analysis of nonlinear regression of inhibition data and Dixon plots presented in Figs. 2B and 3B, Ppd showed a competitive inhibition with Ki of 9.7 ± 2.0µM in human liver microsomes and 2.7 ± 0.5µM in cDNA-expressed CYP3A4, respectively. Ppt was found to have strongly competitive inhibitory activities against testosterone 6ß-hydroxylation in human liver microsomes and cDNA-expressed CYP3A4 with Ki of 2.3 ± 0.2µM and 3.2 ± 0.4µM, respectively (Figs. 4B and 5B). These results showed that intestinal metabolites of ginsenosides Ppd and Ppt had the potential to exert an influence on the CYP3A4 activity.
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Lack of Time- and NADPH-Dependent Inactivation
To test whether there was mechanism-based inhibition of ginsenosides on P450 activities, the time- and NADPH-dependent inhibition was assayed.
There was no time- and NADPH-dependent inactivation of CYP3A4 found in both naturally occurring ginsenosides and their degradation products and also no time- and NADPH-dependent inactivation of CYP2D6, CYP2C9, CYP2A6, or CYP1A2 found in Rb1, Rh1, F1, C-K, Ppd, or Ppt (data not shown).
The Calculated Concentrations of Ginsenosides in Blood and Gut Lumen
The single oral dose of G115, the respective contents of these ginsenosides, the oral bioavailability of Rb1, Rb2, and Rg1, and the average human gut volume were taken from published literature data. The possible maximum concentrations of Rb1, Rb2, and Rg1 in human gut lumen after a single oral dose were calculated to be about 3.9, 2.5, and 1.9 µM, respectively.
C-K and Ppd are likely to be derived from the hydrolysis of Rb1 or Rb2, and Ppt is likely to be derived from the hydrolysis of Rg1 by intestinal bacteria in the large intestine (Hasegawa, 2004; Hasegawa et al., 1996; Tawab et al., 2003). It is to be supposed that these naturally occurring ginsenosides totally transform into metabolites, and the ratio of C-K to Ppd concentration in human blood is equal to the reported ratio in rat blood. Thus, the possible maximum concentration of C-K in the gut lumen of humans after a single oral dose was calculated to be about 5.5 µM and that in blood was likely to be less than 5.5µM, the possible maximum concentration of Ppd in the gut lumen of humans after a single oral dose was calculated to be about 0.9µM and that in blood was likely to be less than 0.9µM, and the possible maximum concentration of Ppt in the gut lumen of humans after a single oral dose was calculated to be about 1.9µM and that in blood was likely to be less than 1.9µM.
The Calculated Values of Cmax/Ki
Rh determined from the data of human liver microsomes was 0.83 for Ppt and 0.09 for Ppd, Rh determined from those of cDNA-expressed CYP3A4 was 0.59 for Ppt and 0.33 for Ppd, and Rr determined from reported data of rat liver microsomes was 0.47 for Ppt.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, using human liver microsomes and cDNA-expressed CYP3A4, characteristic effects of naturally occurring ginsenosides and their intestinal metabolites on human P450 activity were achieved by examining the activities of marker reactions, and mechanism-based inhibition was also assayed.
Our results showed that the naturally occurring ginsenosides exhibited no inhibition or weak inhibition against human CYP3A4, CYP2D6, CYP2C9, CYP2A6, or CYP1A2 activities, which was consistent with the previous studies (Chang et al., 2002; Henderson et al., 1999), however, the intestinal metabolites of ginsenosides including C-K, Ppd, and Ppt demonstrated a wide range of inhibition of P450-mediated metabolism (Table 1). There was no mechanism-based inhibition found in these P450 induced by naturally occurring ginsenosides or their degradation products.
C-K, Ppd, and Ppt exhibited inhibition against CYP2C9 activity in human liver microsomes with an IC50 value of 32.0 ± 3.6µM, 42.7 ± 2.2µM, or 33.7 ± 2.7µM, respectively. CYP2C9 is important in the metabolism of therapeutically used drugs including the anticoagulant drug warfarin and a number of nonsteroidal anti-inflammatory drugs (Goldstein and de Morais, 1994). The influence on this enzyme is particularly important if a drug has a narrow therapeutic index, such as warfarin. A recent clinical trial in healthy subjects showed that ginseng diminished the urine excretion rate of S-7-hydroxywarfarin, the metabolite from S-warfarin by CYP2C9 (Jiang et al., 2004). Our results offer new in vitro evidence that ginsenosides might inhibit the activity of CYP2C9 after oral administration.
Unfortunately, there are no quantitative data for human blood and gut concentrations of C-K, Ppd, and Ppt. Their in vivo influence on P450 is difficult to be predicted quantitatively. The reasons might include the difference of biotransformation potential in the gastrointestinal tract, the rapid disappearance from the circulation and appearance in the liver, as well as succedent accumulation in the form of fatty acid esterified C-K in the liver for C-K or the rapid fatty acid esterification of Ppt in mesenteric lymph (Hasegawa, 2004). Based on the published literature data, and some assumptions, we calculated the possible maximum concentrations of C-K, Ppd, and Ppt. Considering their low concentrations estimated in the blood and their in vitro inhibitory potential for CYP2C9, the in vivo influence of C-K, Ppd, and Ppt on the hepatic CYP2C9 is likely to be weak, except in the case that their levels in the liver markedly rise.
C-K also exhibited inhibition against the activity of CYP2A6 in human liver microsomes with an IC50 value of 63.6 ± 4.2µM and inhibition against the activity of CYP2D6 in human liver microsomes with an IC50 value of more than 100µM. Ppd and Ppt also exhibited inhibition against the activity of CYP1A2, CYP2A6, and CYP2D6 in human liver microsomes with an IC50 value of more than 100µM. However, clinically significant inhibition is uncommon for these interactions because sufficiently high plasma levels of these compounds are not clinically achieved.
It is particularly noteworthy that Ppd and Ppt inhibited the activity of CYP3A4-mediated testosterone 6ß-hydroxylation in human liver microsomes with an IC50 value of 14.1 ± 2.3µM or 7.1 ± 0.9µM, respectively. The further inhibition kinetics studies using human liver microsomes and cDNA-expressed CYP3A4 suggested that the inhibition types of both Ppd and Ppt were competitive. The Ki values of Ppd and Ppt in human liver microsomes were 9.7 ± 2.0µM and 2.3 ± 0.2µM, respectively, and the Ki values in cDNA-expressed CYP3A4 were 2.7 ± 0.5µM and 3.2 ± 0.4µM, respectively.
It was estimated that for reversible inhibition, an in vivo interaction via the inhibition of CYP3A4 would likely occur if the ratio of inhibitor Cmax/Ki was greater than 1 and possible if that was between 1 and 0.1 (Cmax is the inhibitor concentration at steady state and at the highest clinical dose, and Ki is the inhibition constant determined from in vitro data) (Bjornsson et al., 2003). Based on their possible maximum concentrations calculated in blood, for Ppt, the values of the ratio of Cmax/Ki after a single dose of G115 in humans were more than 0.5 from both the data of human liver microsomes and those of cDNA-expressed CYP3A4. For Ppd, the value was more than 0.3 from the data of cDNA-expressed CYP3A4 and close to 0.1 from those of human liver microsomes. Considering the rate of biotransformation and absorption, the actual values of Cmax/Ki in humans may be less than those presented. However, the calculated value of the ratio of Cmax/Ki in rat was also more than 0.4 for Ppt. In addition, among the naturally occurring ginsenosides in G115, the total contents of Rc, Rd, Re, and Rf are close to those of Rb1, Rb2, and Rg1 (Chang et al., 2002), and there is also some evidence that Rc, Rd, and Re are transformed into C-K, Ppd, or Ppt by intestinal bacteria, though there are still no quantitative data (Hasegawa, 2004). Moreover, ginseng or ginseng-derived products are usually long-term administered. Thus, it is possible that naturally occurring ginsenosides could result in an in vivo drug interaction via the inhibitory effect on hepatic CYP3A4 activity of their intestinal metabolites, Ppd and Ppt.
Although the liver is the main site of P450-catalyzed oxidative metabolism for orally administered drugs, P450 have also been found in extrahepatic tissues. In the human intestine, CYP3A4 is the predominant P450, while only a very limited number of other P450 including CYP2C9, CYP2C19, and CYP1A1 are expressed (Obach et al., 2001; Zhang et al., 1999). The inhibition of CYP3A4 in the small intestine by two prominent furanocoumarins, 6',7'-dihydroxybergamottin and bergamottin, in grapefruit juice is, presumably, the main mechanism for enhanced bioavailability of coadministered drugs with grapefruit juice (Dahan and Altman, 2004; Paine et al., 2004, 2005), resulting in a significant reduction of drug presystemic metabolism. In the present study, testosterone 6ß-hydroxylation (the commonly used specific marker reaction for CYP3A4) was selected as an indication for the activity of CYP3A4. In addition, although hepatic and intestinal CYP3A4 are different in tissue sources, they are the same in quality. The inhibition experiments with liver microsomes and cDNA-expressed CYP3A4 should be representative. However, in view of the inhibition potential of these tested ginsenosides on certain P450, their estimated concentrations, together with the fact that there is no mechanism-based inhibition against CYP3A4, the influence of ginsenosides on the bioavailability of coadministered drugs or compounds is likely to be not comparable to that of grapefruit juice.
Both the content and activity of P450 exhibit a high degree of inter- and intraindividual variability (Shimada et al., 1994; Shu et al., 2001). The genetic polymorphism of P450 is extensive, and the rate of metabolism for a certain drug can even differ 1000-fold between phenotypes (Ingelman-Sundberg, 2004). In addition, human intestinal bacteria exhibit a high degree of intraindividual variability, as that which are very changeable in dependence of host conditions, including diet, health, and even stress. The bacterial ginsenoside-hydrolyzing potentials also exhibit a high degree of interindividual variability in humans and experimental mice (Hasegawa, 2004). Moreover, substantial variability in ginsenoside content has been reported among commercial ginseng preparations (Harkey et al., 2001). These reports imply that the clinically significant effects of ginseng on P450 activity might be individual dependent and product dependent. Combined with our present results, the biotransformation of naturally occurring ginsenosides in the gastrointestinal tract could play a key role in the ginseng-associated DDI. The high-degree variability of the products, P450, and intestinal bacteria, as well as the biotransformation potential in the gastrointestinal tract, might be responsible for the contradictory of the current clinical studies.
In summary, naturally occurring ginsenosides might influence the in vivo hepatic P450 activities via their intestinal metabolites producing after oral administration; however, the effect might be product and individual dependent. This study might also be important for the prediction of the effects on in vivo P450 activity of the other orally administered herbal medicines from their in vitro data.
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ACKNOWLEDGMENTS |
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This work was supported by the 973 Program (2003CB716005) of the Ministry of Science and Technology of China and the Leading Program of (KGCXZ-SW-213-04) the Chinese Academy of Sciences. The authors thank Dr. Hideo Hasegawa of Fermenta Herb Institute Inc., Tokyo, Japan, for the gifts of ginsenosides. The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
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