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Cancer Prevention |
Authors' Affiliations: 1 Arizona Cancer Center, The University of Arizona, Tucson, Arizona; 2 Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland; and 3 Mitsui Norin Co., Ltd., Shizuoka, Japan
Requests for reprints: H-H. Sherry Chow, Arizona Cancer Center, The University of Arizona, Tucson, AZ 85724. Phone: 520-626-3358; Fax: 520-626-5348; E-mail: schow{at}azcc.arizona.edu.
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Abstract |
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Experimental Designs: Thirty healthy volunteers were randomly assigned to one of the following doses of Polyphenon E (a decaffeinated and defined green tea catechin mixture): 400, 800, or 1,200 mg, based on the epigallocatechin gallate content (10 subjects per dose group). After an overnight fast, study participants took a single dose of Polyphenon E with or without a light breakfast, which consisted of one or two 4-oz muffins and a glass of water. Following a 1-week wash-out period, subjects were crossed over to take the same dose of Polyphenon E under the opposite fasting/fed condition. Tea catechin concentrations in plasma and urine samples collected after dosing were determined by high-pressure liquid chromatography analysis.
Results: Consistent with previous reports, epigallocatechin gallate and epicatechin gallate were present in plasma mostly as the free form, whereas epicatechin and epigallocatechin were mostly present as the glucuronide and sulfate conjugates. There was >3.5-fold increase in the average maximum plasma concentration of free epigallocatechin gallate when Polyphenon E was taken in the fasting condition than when taken with food. The dosing condition led to a similar change in plasma-free epigallocatechin and epicatechin gallate levels. Taking Polyphenon E in the fasting state did not have a significant effect on the plasma levels of total (free and conjugated) epigallocatechin, but resulted in lower plasma levels of total epicatechin. Urinary epigallocatechin gallate and epicatechin gallate levels were very low or undetectable following Polyphenon E administration with either dosing condition. Taking Polyphenon E under the fasting state resulted in a significant decrease in the urinary recovery of total epigallocatechin and epicatechin. Polyphenon E administered as a single dose over the dose range studied was generally well-tolerated by the study participants. Mild and transient nausea was noted in some of the study participants and was seen most often at the highest study agent dose (1,200 mg epigallocatechin gallate) and in the fasting condition.
Conclusions: We conclude that greater oral bioavailability of free catechins can be achieved by taking the Polyphenon E capsules on an empty stomach after an overnight fast. Polyphenon E up to a dose that contains 800 mg epigallocatechin gallate is well-tolerated when taken under the fasting condition. This dosing condition is also expected to optimize the biological effects of tea catechins.
Key Words: Oral bioavailability • Green tea catechins • Food effect • Doing condition
The epidemiologic evidence on the protective effect of green tea consumption against the development of human cancers is not conclusive. Some studies suggested that green tea consumption could reduce the risk of certain cancers (7–9); such a protective effect has not been observed in other studies (10, 11). The inconsistent epidemiologic findings may be attributed to confounding variables such as individualized differences in tea preparation and consumption patterns, variability associated with tea production, variability in the bioavailability of the active green tea constituents, concomitant use of tobacco and alcohol, and individualized differences in lifestyle. Controlled prospective human intervention trials are clearly necessary to evaluate the chemopreventive activity of green tea or green tea constituents.
We have recently determined the clinical pharmacokinetics of green tea catechins following single and multiple dose administration of a defined green tea catechin extract (Polyphenon E) and epigallocatechin gallate (12, 13). The oral bioavailability of tea catechins was found to be low in humans, resulting in plasma concentrations 5 to 50 times less than concentrations shown to exert biological activities in in vitro systems (14–16). A number of factors may affect the oral bioavailability of green tea catechins and subsequently their biological responses. In a small pilot study, free epigallocatechin gallate plasma concentrations determined at a single time point (90 minutes post-dose) after ingestion of 3, 5, or 7 capsules of Sunphenon DCF-1 (corresponding to 225, 375, and 525 mg epigallocatechin gallate) was 300, 1,970, and 2,020 ng/mL, respectively (17). These levels were significantly higher than those reported by Yang et al. (18) and by us (12, 13). Upon further comparison of the design of these studies, we hypothesized that the dosing condition could have a significant impact on the systemic availability of green tea catechins. We conducted this clinical study to test the hypothesis that the oral bioavailability of green tea catechins can be enhanced when consumed in the absence of food. The information generated from this study is important for the design of future intervention trials, interpretation of epidemiologic findings, and extrapolation of animal data to human situations.
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Materials and Methods |
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Participants. Thirty nonsmoking healthy men and women 
18 years of age participated in the study. The participants had normal liver and renal function. Participants were excluded if they were pregnant, had cancers of any type within the past 5 years, had severe metabolic disorders or other serious acute or chronic diseases, had consumed tea regularly, or had participated in other clinical research studies within the past 3 months. The study was approved by the University of Arizona Human Subjects Committee. Written informed consent was obtained from all participants.
Study design. During the initial clinic visit, study participants completed a medical history form and underwent a brief physical examination. A fasting blood sample was collected and subjected to a complete blood count with differential leukocyte count and a comprehensive blood chemistry analysis. Eligible subjects were randomly assigned to one of the Polyphenon E doses (400, 800, and 1,200 mg, based on epigallocatechin gallate content) and were required to refrain from the ingestion of tea, apples, chocolate, and their products 2 weeks prior to the first pharmacokinetic study day and until the end of the second pharmacokinetic study day. The night prior to the first pharmacokinetic study day, subjects fasted after midnight except for water. On the morning of the first pharmacokinetic study day, each subject was randomly assigned to receive Polyphenon E with or without breakfast. Breakfast consisted of one or two 4-oz muffins and a glass of water. Study participants had the option of selecting from three varieties of Otis Spunkmeyer brand muffins: cheese streusel, wild blueberry, and banana nut flavors. The muffins contain 420 to 480 calories each, with 48 to 60 g of carbohydrates, 20 to 24 g of fat, and 6 g of protein. Blood samples were collected prior to and at 0.5, 1, 2, 4, 6, 8, 10, and 24 hours after Polyphenon E administration. A sandwich lunch was provided to all study subjects following the 4-hour blood collection. Urine samples were collected prior to and up to 24 hours (divided into two intervals: 0-8 and 8-24 hours post-dose) after dosing. Study subjects left the clinic after the 10-hour blood collection and continued their urine collection at home. Study subjects brought back the overnight urine collection the next morning and had a 24-hour blood collection. There was no restriction on the dinner with the exception of items to be refrained from the entire study period. Following a 1-week wash-out period, study subjects fasted overnight and were crossed-over to receive the same dose of Polyphenon E under the other fasting/fed condition. A complete blood count and comprehensive blood chemistry analysis was repeated after the subject completed the cross-over portion of the study.
Sample collection and processing. Blood samples were collected into Vacutainer tubes containing sodium heparin. Within 30 minutes of collection, tubes were centrifuged for 10 minutes at 2,000 rpm. After centrifugation, plasma was mixed with ascorbate-EDTA solution [0.4 mol/L NaH2PO4 buffer containing 20% ascorbic acid and 0.1% EDTA (pH 3.6)] in a fixed volume ratio and stored at –80°C until sample analysis. Urine specimen container was pre-added with 1.38 g NaH2PO4, 1 g ascorbic acid, and 5 mg EDTA to prevent degradation of tea catechins. Urine specimen was kept cold by storing the specimen container in a cooler chest with freezer packs during each collection period. The total volume of each urine sample was measured. An aliquot of urine sample was mixed with the ascorbate-EDTA solution and stored at –80°C until sample analysis.
Tea polyphenol concentration measurements. Epigallocatechin gallate, epicatechin, epigallocatechin, epicatechin gallate concentrations in plasma and urine samples were determined using a published method (18) with minor modifications. In brief, for determination of free green tea catechins, plasma or urine samples were extracted with ethyl acetate. The ethyl acetate layer was mixed with a small aliquot of 0.1% ascorbic acid before drying by vacuum centrifugation. The dried residue was redissolved in 15% acetonitrile and injected onto high-pressure liquid chromatography. For determination of the total of free and glucuronic acid/sulfate conjugates of tea catechins, plasma or urine samples were mixed with an aliquot of ß-glucuronidase and sulfatase in the presence of ascorbate-EDTA solution. Following pretreatment, the samples were extracted as described above for the free catechins.
The high-pressure liquid chromatography system consisted of an ESA Model 465 refrigerated autosampler, an ESA Model 580 two-pump solvent delivery system, an ESA 5500 Coulochem electrode array system, and a Supelcosil C18 reversed-phase column (150 x 4.6 mm; particle size, 5 µm; Supelco, Inc., Bellefonte, PA). The autosampler and column temperatures were maintained at 6°C and 35°C, respectively. This assay employed a gradient of two mobile phases. Buffer A consisted of 30 mmol/L NaH2PO4 buffer, acetonitrile, and tetrahydrofuran in the volume ratio of 98.13:1.75:0.12 (pH 3.35). Buffer B consisted of 15 mmol/L NaH2PO4 buffer, acetonitrile, and tetrahydrofuran in the volume ratio of 41.5:58.5:12.5 (pH 3.45). The flow rate was maintained at 1 mL/minute. The column was eluted with 96% buffer A and 4% buffer B from 0 to 7 minutes. Then the linear gradient was changed progressively to 17% buffer B at 25 minutes, 28% at 31 minutes, 33% at 37 minutes, and 98% at 38 minutes. It was maintained at 98% from 38 to 43 minutes and finally changed back to 4% buffer B at 44 minutes for the analysis of the next sample. The eluent was monitored by the Coulochem electrode array system with potential settings at –10, 150, 300, and 500 mV.
Data analysis. The following pharmacokinetic variables of free epigallocatechin gallate were estimated using the WINNONLIN program (version 4.0.1) with the noncompartment approach: time to reach the maximum plasma drug concentration (Tmax), maximum plasma drug concentration (Cmax), area under the plasma drug concentration-time profile (AUC), systemic clearance/bioavailability (CL/F), apparent volume of distribution/bioavailability (Vd/F), and elimination half-life (t1/2). The Cmax of free epigallocatechin, epicatechin, and epicatechin gallate, and of total catechins (free and conjugated) was obtained by visual inspection of the concentration-time data.
The amount of total epigallocatechin and epicatechin excreted in the urine for each collection interval was calculated by the product of urinary catechin concentration and urine volume. The total amount excreted over 24 hours after dosing was obtained by adding the amount excreted over 0 to 8 and 8 to 24 hours after dosing.
The distribution of pharmacokinetic variable data was normalized by logarithmic transformation prior to statistical analyses. The primary analyses are to determine whether the pharmacokinetics of tea catechins are different between fasting versus fed states. Pharmacokinetic variables such as AUC, Cmax, Tmax, half-life, CL/F, Vd/F, and total amount excreted over 24 hours were compared between the fasting and fed conditions by standard cross-over statistical analyses. The pkcross routine in Stata 8.0 (StataCorp 2003) was used. In this software, the default parameterization estimates overall mean, period effects, treatment effects, and sequence effects, assuming no carryover effects. A P < 0.05 was considered statistically significant. In secondary analysis, epigallocatechin gallate pharmacokinetic variables such as CL/F, Vd/F, dose-normalized AUC, and dose-normalized Cmax were compared among different dose levels using one-way ANOVA followed by post hoc t tests corrected for multiple comparisons. For the post hoc t test, a P < 0.0167 was considered statistically significant.
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Results |
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Discussion |
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Because a significant fraction of the orally administered green tea catechins is not absorbed or is eliminated presystemically, small changes in factors limiting the systemic availability of green tea catechins could have a significant impact on their oral bioavailability. We have shown in this study that taking Polyphenon E on an empty stomach after an overnight fast resulted in a dramatic increase in the blood levels of free epigallocatechin gallate, epigallocatechin, and epicatechin gallate. Nevertheless, taking Polyphenon E under fasting conditions resulted in a decrease in the blood and urine levels of total epigallocatechin and epicatechin. Because epigallocatechin and epicatechin are mostly present as the conjugated form, a decrease in the total catechin concentration and an increase in the free catechin concentration suggests that less epigallocatechin and epicatechin conjugates are formed and bioavailable in the fasting condition. Formation of conjugated metabolites of soy polyphenols, daidzein and genistein, has been shown to be modulated in rodents after an acute fast (21). It has been postulated that an acute fast depletes precursors for the glucuronidation reaction (22, 23). It is plausible that less epigallocatechin and epicatechin undergo presystemic glucuronidation and/or sulfation reactions in the fasting condition, resulting in more free catechins escaping the presystemic loss and being available in the systemic blood. Unlike epigallocatechin and epicatechin, epigallocatechin gallate and epicatechin gallate were mostly present as the free form in plasma and in low quantities in urine. It is not known whether an overnight fast will have a similar effect on the conjugation of epigallocatechin gallate and epicatechin gallate.
An additional factor that could affect the oral bioavailability of green tea catechins is luminal catechin degradation. Green tea catechins have been shown to be stable in acidic conditions, but degrade more rapidly at pH levels above 6.5 (24, 25). Thirty-eight percent of epigallocatechin gallate was found to remain intact when incubated at 37°C (pH 7.4) for 5 minutes (24) and minimal amounts of epigallocatechin gallate remained when incubated for 3 hours (pH 7.4; ref. 25). Following a meal, the gastric pH increases to a peak range (pH 5.8-6.7) from an acidic range (pH 1.1-1.6; ref. 26). The presence of food also delays the gastric emptying rate. Based on these physiologic changes and the in vitro stability data, it is possible that green tea catechins may be more stable in a fasted stomach than a fed stomach, which could contribute to the enhanced oral bioavailability observed in the fasting condition.
Food has also been shown to decrease the intestinal absorption of pharmaceutical drugs through irreversible interactions between drugs and dietary components or reversible interactions but exhibiting an absorption window in the proximal small intestine (27), a decrease in drug dissolution rate as a result of elevation of the luminal viscosity (28), and interactions between drugs and bile acids secreted following food ingestion (29). These factors could also contribute to the dramatic increase in the oral bioavailability of green tea catechins observed in the fasting condition.
As shown in Tables 2-4, there is a large subject-to-subject variability in the pharmacokinetics of green tea catechins. This variability has been previously observed by us (12, 13) and others (18) and is consistent with compounds that undergo extensive presystemic elimination. The appropriate sample size for this study was calculated during the clinical protocol development process with the consideration of this variability. A sample size of 30 was found to result in 80% power to observe a 20% difference in epigallocatechin gallate AUC between dosing conditions based on a 5% level of significance and a cross-over design. The higher percentage of difference observed in this study suggests a higher statistical power.
All doses of Polyphenon E used in this trial were generally well-tolerated after single-dose administration. No significant differences were observed in the adverse events reported for the 400 and 800 mg dose levels between the dosing conditions. Mild and transient nausea was noted in some of the study participants and was seen most often at the highest study agent dose (1,200 mg epigallocatechin gallate) and in the fasting condition. Because of this adverse event, taking Polyphenon E at the 1,200 mg dose in the fasting condition is not considered feasible for chronic use.
It is not known whether taking Polyphenon E with a meal with a composition different from that used in our study would allow for better gastrointestinal tolerance at higher doses but not impair the oral bioavailability of green tea catechins. A recent study has assessed the impact of different macronutrients on flavanol (epicatechin + catechin) absorption from sugar-free, flavanol-rich cocoa (30). It was found that flavanol absorption is increased significantly by concurrent consumption of carbohydrate-rich meals, including sugar, bread, and grapefruit juice (
140% increase in AUC values). Lipid and protein-rich meals such as butter, milk, and steak had minimal effects on flavanol absorption. The muffins provided in our study are rich in carbohydrate and fat content. Because carbohydrate-rich meals already provided facilitated flavanol absorption when compared with lipid and protein rich meals (30), it is expected that lipid- and protein-rich meals would also reduce the oral bioavailability of green tea catechins in comparison with the fasting condition.
We conclude that greater oral bioavailability of free catechins can be achieved by taking Polyphenon E on an empty stomach after an overnight fast. Polyphenon E up to a dose that contains 800 mg epigallocatechin gallate is well-tolerated when taken under the fasting condition. This dosing condition is also expected to optimize the biological effects of tea catechins.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12/10/04; revised 2/ 3/05; accepted 3/17/05.
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