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Peracetylation as a Means of Enhancing in Vitro Bioactivity and Bioavailability of Epigallocatechin-3-Gallate

Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, the State University, Piscataway, New Jersey (J.D.L, S.S., J.H., S.-J.K., M.-J.L., C.S.Y.); and Department of Food Science, Rutgers, the State University of New Jersey, New Brunswick, New Jersey (C.-T.H.)

(Received June 13, 2006; accepted September 20, 2006)

	Abstract

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(–)-Epigallocatechin-3-gallate (EGCG) is the widely studied catechin in green tea (Camellia sinensis). Previously, we have reported the low bioavailability of EGCG in rats and mice. As a means of improving the bioavailability of EGCG, we have prepared a peracetylated EGCG derivative (AcEGCG) and herein report its growth inhibitory activity and cellular uptake in vitro, as well as bioavailability in mice. AcEGCG exhibited enhanced growth inhibitory activity relative to EGCG in both KYSE150 human esophageal (IC₅₀ = 10 versus 20 µM) and HCT116 human colon cancer cells (IC₅₀ = 32 versus 45 µM). AcEGCG was rapidly converted to EGCG by HCT116 cells, and treatment of cells with AcEGCG resulted in a 2.8- to 30-fold greater intracellular concentration of EGCG as compared with treatment with EGCG. AcEGCG was also more potent than EGCG at inhibiting nitric oxide production (4.4-fold) and arachidonic acid release (2.0-fold) from lipopolysaccharide-stimulated RAW264.7 murine macrophages. Intragastric administration of AcEGCG to CF-1 mice resulted in higher bioavailability compared with administration of equimolar doses of EGCG. The plasma area under the curve from 0 to infinity (AUC₀) of total EGCG was 465.0 and 194.6 [(µg/ml) · min] from the administration of AcEGCG and EGCG, respectively. The t_1/2 of EGCG was also increased following administration of AcEGCG compared with EGCG (441.0 versus 200.3 min). The AUC₀ and t_1/2 were also increased in small intestinal (2.8- and 4.3-fold, respectively) and colonic tissues (2.4- and 6.0-fold, respectively). These data suggest that acetylation represents a means of increasing the biological potency in vitro, increasing the bioavailability of EGCG in vivo, and may improve cancer-preventive activity.

View larger version (21K): [in this window] [in a new window]   FIG. 1. HPLC analysis of EGCG and peracetylated EGCG.

Ester-based prodrugs are classical means to improve the bioavailability and reduce the toxicity of a compound (Liederer and Borchardt, 2006). For example, acetylation of salicylic acid to form aspirin accomplishes this, as does acetylation of morphine to produce heroin (Sollman, 1957; Klaassen, 1996). This improvement is accomplished by occlusion of polar side chains, increasing hydrophobicity, and making hydroxyl groups unavailable for phase II biotransformation or oxidative degradation. Previously Lam et al. (2004) have reported that peracetylated EGCG (AcEGCG, molecular formula C₃₈H₃₄O₁₉) (Fig. 1) represents a prodrug for inhibition of proteasome activity. The authors found that this compound had increased activity against proteasome activity in intact cells but not against pure enzymes.

In the present study, we have examined the in vitro growth inhibitory and anti-inflammatory activity of AcEGCG, the kinetics of AcEGCG de-esterification, and the oral bioavailability of this compound in mice.

	Materials and Methods

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Chemicals. EGCG (100% pure) was isolated from a crude green tea polyphenol extract and provided by Dr. Chi-Tang Ho (Department of Food Science, Rutgers University). AcEGCG was synthesized using a pyridine-catalyzed reaction of EGCG with acetic anhydride. ß-D-Glucuronidase (G-7896, EC 3.2.1.31 [EC] , from Escherichia coli with 9 x 10⁶ U/g solid) and sulfatase (S-9754, EC 3.1.6.1 [EC] , from Abalone entrails with 2.3 x 10 ⁵ U/g solid) were purchased from Sigma Chemical Co. (St. Louis, MO). All the other reagents were of the highest grade commercially available. Dosing solutions of EGCG or AcEGCG were prepared in 0.9% NaCl or polyethylene glycol 300/ethanol/polysorbate 80/water (2:1:0.98:0.02, v/v/v/v), respectively. For analytical purposes, a standard stock solution of EGCG, epigallocatechin, epicatechin, and epicatechin-3-gallate (10 mg/l each) was prepared in 11.4 mM ascorbic acid/0.13 mM EDTA (pH 3.8) and stored at –80°C.

Cell Culture. KYSE150 human esophageal squamous cell carcinoma cells were maintained in log-phase growth in Ham's F-12/RPMI-1640 media (1:1) supplemented 5% fetal bovine serum. HCT116 human colon adenocarcinoma cells and RAW264.7 murine macrophages were maintained in log-phase growth in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. All the media were supplemented with 100 U/ml penicillin and 0.1 mg/ml streptomycin.

Animals. Male CF-1 mice (6–8 weeks) were purchased from Charles River Laboratories (Wilmington, MA) and allowed to acclimate for at least 1 week before the start of the experiment. The mice were housed 10 per cage and maintained in air-conditioned quarters with a room temperature of 20 ± 2°C, relative humidity of 50 ± 10%, and an alternating 12-h light/dark cycle. Mice were given Purina Rodent Chow 5001 (Research Diets, New Brunswick, NJ) and water ad libitum. For bioavailability studies, animals were deprived of food for 12 h before administration of test compounds.

Growth Inhibition and Intracellular Concentration. To compare the growth inhibitory activity of EGCG and AcEGCG, KYSE150 human esophageal cancer cells and HCT116 human colon adenocarcinoma cells were plated in 96-well plates (2–5 x 10³ cells/well) and allowed to attach for 24 h. The medium was replaced with fresh, serum-free medium containing 0 to 40 µM EGCG or AcEGCG. Cells were incubated for 24 h at 37°C. The medium was then replaced with fresh serum-complete medium, and the cells were incubated for an additional 24 h at 37°C. Growth inhibition was determined using the 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (Bold et al., 2001).

Cellular uptake and cytosolic levels of EGCG and AcEGCG following treatment of HCT116 cells with either AcEGCG or EGCG were determined as previously reported (Hong et al., 2003). In brief, cells were allowed to grow to 70 to 90% confluence in 12-well plates. The medium was replaced with fresh medium containing 20 µM EGCG or AcEGCG and 5 U/ml superoxide dismutase (to stabilize the EGCG). The cells were incubated for 0.25 to 24 h at 37°C, after which the medium was removed, the cells were washed two times with cold phosphate-buffered saline, 2% ascorbic acid was added to each well, and the cells were scraped and sonicated. The resulting solution was centrifuged at 10,000g for 20 min. The supernatant was combined with an equal volume of ice-cold methanol and centrifuged for 20 min at 10,000g to precipitate the protein. The resulting supernatant was analyzed by high-performance liquid chromatography (HPLC). Cytosolic EGCG was normalized to cytosolic protein concentration.

Inhibition of Arachidonic Acid Release and NO Production. The ability of EGCG and AcEGCG to inhibit the release of arachidonic acid and production of NO by lipopolysaccharide (LPS)-stimulated murine macrophages was determined using previously described methods (Sang et al., 2004). In brief, to determine arachidonic acid release, RAW264.7 cells were incubated overnight with 0.1 µCi/ml [5,6,8,9,11,12,14,15-³H](N)-arachidonic acid to allow membrane incorporation. Cells were then washed with phosphate-buffered saline containing 0.1% bovine serum albumin. Cells were then stimulated with 2 µg/ml LPS for 1 h; the cells were washed; and fresh medium containing AcEGCG or EGCG (0–40 µM) was added. Following 18 h of incubation, radioactivity in the medium was determined by scintillation counting. To determine inhibition of NO formation, cells were stimulated and treated with test compounds as above. NO levels were determined by measuring nitrite production spectrophotometrically using the Greiss reagent (Ryu et al., 2003).

Esterase-Mediated Deacetylation. To determine the capability of plasma esterases to convert AcEGCG to EGCG, freshly isolated plasma from CF-1 mice was combined with AcEGCG (final concentration, 5 µM) and incubated at 37°C for 0 to 60 min. The reaction was stopped by addition of CH₂Cl₂, and EGCG was extracted and analyzed as described below. Time-dependent deacetylation by plasma or hepatic microsomal esterases was determined by incubating 10 µM AcEGCG with mouse plasma or hepatic microsomes (20 µg of protein) in Tris-HCl (20 mM, pH 6.8 containing 0.02% ascorbic acid and 1 mM CaCl₂). Samples were preincubated for 3 min at 37°C, and the reaction was started by addition of AcEGCG. Reactions were carried out for 0 to 30 min at 37°C and terminated by addition of an equal volume of acetonitrile. Following centrifugation at 10,000g for 10 min, 100-µl aliquots of supernatant were analyzed by HPLC-UV to determine the disappearance of AcEGCG. To determine the K_m and V_max for plasma-mediated de-esterification of AcEGCG, the reactions were prepared as above with the modification that the concentration of AcEGCG was changed from 2 to 200 µM. The reactions were preincubated and started as above. The reaction time was held constant at 15 min. Samples were then prepared and analyzed as above.

Oral Bioavailability. Male, CF-1 mice (20–25 g, six per group) were given a single dose of EGCG or AcEGCG (163.8 µmol/kg i.g.) and sacrificed at 20, 50, 90, 180, and 300 min. Plasma, small intestine, and colon were collected and processed by previously reported methods (Lee et al., 2002; Lambert et al., 2003). In brief, 100 µl of plasma was hydrolyzed with 1 U of sulfatase and 250 U of ß-glucuronidase at 37°C for 45 min and extracted with methylene chloride and ethyl acetate. The ethyl acetate fractions were pooled and dried under vacuum. Samples were reconstituted in 10% aqueous acetonitrile and analyzed by HPLC.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Biological activities and cellular uptake of AcEGCG. A, growth inhibition of KYSE150 human esophageal cancer cells and HCT116 human colon adenocarcinoma cells following treatment with either EGCG or AcEGCG for 24 h. Inhibition of NO production (B) or arachidonic acid release (C) by LPS-stimulated RAW264.7 murine macrophages by AcEGCG or EGCG (0–40 µM, 18-h treatment). D, cytosolic levels of EGCG in HCT116 human colon cancer cells treated with EGCG or AcEGCG (10 µM) for 0.25 to 24 h. n = 4 ± S.D. *, p < 0.05; **, p < 0.01.

HPLC Analysis of AcEGCG and EGCG. HPLC analysis of EGCG in cell culture medium and biological samples was performed using our previously reported methods (Lee et al., 2002; Hong et al., 2003). AcEGCG concentrations in enzyme reactions were analyzed by HPLC-UV with _max = 280 nm. The mobile phase was a binary gradient of solvent A (4% sodium monophosphate buffer containing 5% acetonitrile, pH 3.2) and solvent B (1% sodium monophosphate buffer containing 70% acetonitrile, pH 3.2). The initial phase was a 12-min gradient from 57 to 86% B, followed by a 6-min isocratic period at 86% B. The mobile phase was then re-equilibrated at 57% B for 7 min. The flow rate was 1.5 ml/min. The limit of detection for the method was determined as the minimum quantifiable peak at a signal-to-noise ratio of 3 to 1.

Statistical Analyses. Pharmacokinetic parameters were calculated using Microsoft (Redmond, WA) Excel functions developed by Usansky and colleagues (http://www.boomer.org/pkin/soft.html). The area under the curve from 0 to infinity (AUC₀) was calculated by the linear trapezoidal rule; elimination rate constant (k_elim) was calculated from least-squares curve fit of the plot of ln concentration as a function of time; and the half-life (t_1/2) was determined as ln(2)/k_elim. K_m and V_max values for the de-esterification of AcEGCG were determined using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA). Differences in pharmacokinetic parameters, inhibition values, and cytosolic levels were assessed using the paired Student's t test, and p < 0.05 was regarded as significant.

	Results

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Synthesis and HPLC Analysis of AcEGCG. AcEGCG was obtained in 95% yield following peracetylation by reaction with acetic anhydride in a pyridine-catalyzed reaction. The structure was confirmed by one- (¹H and ¹³C) and two-dimensional (HMBC and HMQC) NMR. ¹H NMR [(CD₃)₂CO, 600 MHz]: 5.53 (1H, s, H-2), 5.77 (1H, m, H-3), 3.22 (1H, dd, J = 4.8, 18.0 Hz, H-4a), 3.11 (1H, dd, J = 1.8, 18.0 Hz, H-4b), 6.61 (1H, d, J = 2.4 Hz, H-6), 6.74 (1H, d, J = 2.4 Hz, H-8), 7.42 (2H, s, H-2' and H-6'), 7.63 (2H, s, H-2'' and H-6''), 2.29 (3H, CH₃CO), 2.28 (6H, CH₃CO), 2.28 (3H, CH₃CO), 2.252 (3H, CH₃CO), 2.251 (3H, CH₃CO), and 2.23 (6H, CH₃CO); ¹³C NMR [(CD₃)₂CO, 150 MHz]: 79.0 (d, C-2), 71.1 (d, C-3), 28.1 (t, C-4), 152.7 (s, C-5), 111.8 (d, C-6), 152.6 (s, C-7), 110.3 (d, C-8), 157.5 (s, C-9), 112.6 (s, C-10), 130.1 (s, C-1'), 121.6 (d, C-2' and C-6'), 146.4 (s, C-3' and C-5'), 137.3 (s, C-4'), 141.9 (s, C-1''), 124.5 (d, C-2'' and C-6''), 146.3 (s, C-3'' and C-5''), 138.4 (s, C-4''), 166.0 (s, C-7''), 22.6 (q, CH₃CO), 22.3 (q, CH₃CO), 22.2 (q, 2 x CH₃CO), 22.1 (q, 2 x CH₃CO), 21.7 (q, CH₃CO), 21.6 (q, CH₃CO), 171.1 (s, CH₃CO), 170.6 (s, CH₃CO), 170.0 (s, 2 x CH₃CO), 169.9 (s, 2 x CH₃CO), 169.1 (s, CH₃CO), and 168.8 (s, CH₃CO).

AcEGCG was not detectable using our standard electrochemical method for EGCG (Lee et al., 2002; Hong et al., 2003). Therefore, we developed an HPLC-UV method to analyze AcEGCG in cell culture medium and enzyme incubations. Analysis of standard solutions of AcEGCG revealed a single peak with a retention time of 12 min with a _max = 280 nm. The apparent limit of detection was 400 ng/ml (Fig. 1). Although this limit of detection was not sensitive enough to detect AcEGCG levels in plasma and other biological samples, it was sufficient to monitor enzyme assays and cell uptake studies.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Enzyme-mediated deacetylation of AcEGCG. Conversion of AcEGCG (5 µM) to EGCG by mouse plasma (A). Time-dependent deacetylation (10 µM) by mouse plasma (B) and hepatic microsomes (C). Concentration-dependent deacetylation by mouse plasma (D). Time- and concentration-dependent kinetics were based on the disappearance of AcEGCG. Each point represents the average of three experiments. Error bars represent the S.D.

The ability of AcEGCG and EGCG to inhibit NO production and arachidonic acid release by LPS-stimulated RAW 264.7 murine macrophages was compared. At 40 µM, AcEGCG inhibited NO production by 81%, whereas EGCG treatment reduced NO by only 15% (Fig. 2B). Similarly, AcEGCG reduced LPS-induced arachidonic acid release dose-dependently and was approximately 2-fold more potent than EGCG (Fig. 2C).

To determine whether the increased biological potency of AcEGCG was caused by increased cytosolic levels of EGCG in treated cells, we determined the cytosolic levels of EGCG in AcEGCG-treated HCT116 cells. Cells treated with AcEGCG had a maximal cytosolic concentration of 2088.0 ng/mg EGCG 2 h after treatment, whereas those treated with EGCG had a maximal cytosolic level of 65.0 ng/mg 6 h after treatment (Fig. 2D). After 24 h, the cytosolic levels of EGCG were 2.3-fold higher in cells treated with AcEGCG than in those treated with EGCG.

Enzyme-Mediated Deacetylation of AcEGCG. EGCG could be detected within 5 min following incubation of AcEGCG in mouse plasma at 37°C (Fig. 3A). Time-dependent incubation with lower concentrations of mouse plasma and with hepatic microsomes resulted in decreases in the levels of AcEGCG and the formation of at least two putative metabolites (Fig. 3, B and C). We are currently trying to identify the structures of these compounds. Incubations in the absence of plasma or hepatic microsomes did not result in a significant decrease in the concentration of AcEGCG, nor the formation of M4.8 and M7.01 (data not shown). The kinetics of de-esterification of AcEGCG were determined in mouse plasma (Fig. 3D). Following a 10-min incubation, the V_max and K_m of de-esterification were determined as 10,615 pmol/min/mg and 16.7 µM, respectively.

Oral Bioavailability of AcEGCG and EGCG. The oral bioavailability of EGCG in CF-1 mice was compared following administration of equimolar doses of AcEGCG and EGCG. The peak concentration (C_max) of total EGCG in the plasma, small intestine, and colon was not significantly different between the two treatment groups (Fig. 4). By contrast, the AUC₀ of total EGCG increased by 2.4-, 2.8-, and 2.4-fold in the plasma, small intestine, and colon, respectively, of mice treated with AcEGCG compared with those treated with EGCG (Table 1). Likewise, the t_1/2 of total EGCG was increased by 2.2-, 2.4-, and 6.0-fold in the plasma, small intestine, and colon, respectively, in mice treated with AcEGCG compared with those treated with EGCG (Table 1). There was no significant difference in the lung levels of EGCG of the two treatment groups (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 4. Plasma and tissue levels of EGCG in male CF-1 mice treated with equimolar doses of EGCG (75 mg/kg i.g.) and AcEGCG (130 mg/kg i.g.). n = 6, error bars represent the S.E.M.

	Discussion

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EGCG is the most abundant catechin in green tea (C. sinensis, Theaceae). This compound has been reported to have cancer-preventive activity in vivo and associated activities in vitro; however, it is extensively biotransformed and has relatively poor bioavailability (Lambert and Yang, 2003a,b). In an effort to improve the bioavailability of EGCG, we prepared AcEGCG and investigated the effect of peracetylation on the in vitro biological activities of EGCG related to cancer prevention and the bioavailability of AcEGCG in vivo.

Acetylation improved the cell uptake and growth inhibitory activity of EGCG against both human esophageal squamous cell carcinoma cells (KYSE150) and human colon adenocarcinoma cells (HCT116). Incubation of AcEGCG with KYSE150 cells resulted in rapid conversion to EGCG and in the formation of at least two unknown compounds. These unknown compounds may represent partially deacetylated AcEGCG products. We are attempting to identify these putative metabolites. It appears that AcEGCG can increase the intracellular concentrations as a result of increased cell uptake over the short term (1 h), but once the compound is converted back to EGCG, it is then subject to biotransformation and efflux at a rate similar to EGCG over the long term (24 h). This may explain why the increase in growth inhibitory activity, which is measured after 24 h of treatment, was only a 2-fold. Based on these data, it would appear that AcEGCG in combination with inhibitors of phase II metabolism and active efflux would result in an even greater increase in biological potency.

AcEGCG increased the oral bioavailability of EGCG, as measured by AUC₀. Increases in the AUC₀ and t_1/2 of plasma (240 and 220%), small intestine (280 and 240%), and colon (240 and 600%) were observed following oral gavage with AcEGCG compared with treatment with equimolar doses of EGCG. Interestingly, there was little difference in the C_max of EGCG between the AcEGCG- and EGCG-treated groups. This could be because of incomplete conversion of AcEGCG to EGCG following p.o. administration to mice. It is also possible that although acetylation is expected to increase absorption of the test compound, it is not expected to affect biotransformation and elimination of the deacetylated product, EGCG.

Because of limitations in the sensitivity of our current analytical method, we were unable to measure AcEGCG or partially deacetylated AcEGCG in the tissues of treated mice. Although we showed that AcEGCG is readily deacetylated by mouse plasma and hepatic microsomes, it is possible that a pool of partially deacetylated products is present in the tissue and plasma and that these products may have biological activity. We are currently developing a more sensitive method to study these partially deacetylated AcEGCG products.

In summary, we have shown that AcEGCG has improved biological activity in vitro and improved bioavailability in vivo. This compound is apparently readily converted back to EGCG both in vitro and in vivo, and the improved biological activity in vitro appears to be caused by increased intracellular levels of EGCG. These data suggest that AcEGCG may have improved cancer preventive activity in vivo. Future studies in animal models of carcinogenesis are needed to test this hypothesis.

	Footnotes

 
Supported by National Institutes of Health Grant CA88961 (C.S.Y.) and American Institute for Cancer Research Grant AICR 05A047 (J.D.L.).

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.011460.

ABBREVIATIONS: EGCG, (–)-epigallocatechin-3-gallate; AcEGCG, peracetylated (–)-epigallocatechin-3-gallate; HPLC, high-performance liquid chromatography; LPS, lipopolysaccharide; AUC₀, area under the curve from 0 to infinity.

Address correspondence to: Chung S. Yang, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854. E-mail: csyang{at}rci.rutgers.edu

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