WO2007033356A2

WO2007033356A2 - Compositions and methods for green tea catechins for modification of detoxification enzymes

Info

Publication number: WO2007033356A2
Application number: PCT/US2006/035972
Authority: WO
Inventors: Hsiao-Hui Sherry Chow
Original assignee: Mitsui Norin Co., Ltd; University Of Arizona
Priority date: 2005-09-14
Filing date: 2006-09-13
Publication date: 2007-03-22
Also published as: WO2007033356B1; WO2007033356A3

Abstract

Compositions and methods useful for increasing GST-pi expression and/or GST-pi activity in human penpheral blood cells by administering daily in an oral dosage a green tea polyphenol composition consisting of epigallocatechin gallate, epigallocathechin, epicatechin, epicatechin gallate, gallocatechin gallate and/or polyphenon E.

Description

COMPOSITIONS AND METHODS FOR GREEN TEA CATECHINS FOR MODIFICATION OF DETOXIFICATION ENZYMES

This application claims priority to our copending U.S. provisional patent application with the serial number 60/717,561, which was filed September 14, 2005.

This invention was made with government support under NCI Grant Number NOl-

CN-25119. The government has certain rights in the invention. Field of The Invention

The field of the invention is modulation of expression of detoxifying enzymes, and particularly as it relates to boosting of glutathione-S-transferase (GST) isotype π expression in lymphocytes of human having low expression of GST-π.

Background of The Invention

Glutathione S-transferases (EC 2.5.1.18) are multifunctional and multigene products that catalyze nucleophilic attack of sulphur on electrophilic centers of various endogenous and xenobiotic compounds. The superfamily of GSTs is typically organized into groups of isozymes (including alpha, mu, pi, sigma and theta), which exhibit functional polymorphic variations with respect to substrates, kinetic parameters, and/or locus of expression.

Over the last decades, data from cancer studies have linked aberrant expression of GST isotypes with the development and expression of resistance to a variety of chemicals, including cancer drugs. For example, Mcllwain et al. recently discussed how differences in certain human GST isozyme expression patterns influence cancer susceptibility, prognosis and treatment, and pointed out that in addition to the well-characterized catalytic activity, selected GST isozymes also regulate mitogen-activated protein kinases and/or facilitate the addition of glutathione to cysteine residues in target proteins (Oncogene 2006 Mar 13;25(11): 1639-48).

One of the GST isotypes, GST-pi (also known as GST-π or GST-P), has gained particular attention with respect to its involvement in the etiology of various diseases, and particularly cancer. For example, as GST-pi is often overexpressed in neoplastic diseases (e.g., chemically induced hepatocarinoma), GST-pi was proposed as a marker for diagnosis of selected cancers and/or for detection of chemical toxicity and carcinogenesis (MoI Cell Biochem. 2003 Nov;253(l-2):319-27). Other groups have investigated the role of GST-pi in a mouse line that lacks this enzyme to determine its effects on tumorigenesis in selected organs, drug metabolism, and toxicity (Methods Enzymol. 2005;401 : 116-35).

Recently, overexpression of GST-pi in cancers resistant to drugs has also been shown to provide an attractive target for prodrug activation. Moreover, GST-pi was found to possess noncatalytic ligand-binding properties important in the direct regulation of kinase pathways. This has led to the development and testing of agents that bind to GST-pi and interfere with protein-protein interactions (Methods Enzymol. 2005;401:287-307). While such diagnostic and prodrug-related approaches have shown at least some promise, effective use as a curative strategy for neoplastic diseases is generally not considered realistic.

Alternative approaches have focused on the reduction of toxins and mutagens using compounds that stimulate selected GST isoforms in certain tissues. For example, Chou et al reported that glutathione S-transferase GSTM2 subunit expression is promoted in rat liver by epigallocatechin gallate in a dose- and time-dependent manner (Biochem Pharmacol. 2000 Sep l;60(5):643-50). Remarkably, while three GST isoforms (GSTA1/2, GSTMl, and GSTM2) were tested for changes in protein level affected by EGCG, only GSTM2 revealed a significant time-dependent increase, with a maximal induction of approximately 2.0-fold. This differential effect of EGCG on GST subunit expression was verified by immunocyto- chemical examination and showed strong induction of GSTM2 but not GSTA1/2 and GSTMl level in liver. Based on these results, the authors propose a hepatic chemopreventive effect of EGCG on tumorigenesis. While such finding is consistent with anecdotal beneficial effects of green tea on chemoprevention of cancer, several uncertainties remain. Among other things, the observed level of overexpression is likely not sufficient to conclusively argue that hepatic overexpression of GSTM2 has significant chemopreventive effect.

Similarly, Schilter et al reported placental glutathione S-transferase (GST-P) induction in liver as a potential mechanism for the anti-carcinogenic effect of the coffee- specific components cafestol and kahweol (Carcinogenesis. 1996 Nov;17(l l):2377-84). Remarkably, in this study isoforms mu and alpha were substantially unaffected while hepatic GST-P expression was increased in a relatively slow and time-dependent manner. While these known studies provide at least some support for a mechanism of chemoprevention via increase of GST isoforms, the results are somewhat inconclusive and generally limited to hepatic expression of selected isoforms in response to selected chemicals. Moreover, and especially where carcinogenesis occurs in a tissue other than the liver, or where a carcinogenic drug is not or only to a relatively small degree exposed to the liver, chemopreventive effects are unknown.

Thus, while numerous compositions and methods for GST induction are known in the art, all or almost all of them, suffer from one or more disadvantages. Therefore, there is still a need for improved nutrition and/or pharmaceutical agents for treatment and chemoprevention of neoplastic diseases.

Summary of the Invention

The present invention is directed to compositions, methods, and uses of selected green tea polyphenol compositions to increase GST-π and/or GST-π activity in human peripheral blood lymphocytes, especially in persons having low baseline GST-π activity in lymphocytes. Among other advantages, a population with low baseline GST-π activity may be expect to receive an increased chemopreventive effect from the green tea compositions contemplated herein, which may reduce the cancer risk for such population.

In one aspect of the inventive subject matter, a method of increasing at least one of a GST-π expression and a GST-π activity in peripheral blood lymphocytes of a person determined to have low baseline GST-π activity will include a step of administering a green tea polyphenol composition to the person at a dosage effective to increase the at least one of the GST-π expression and the GST-π activity in peripheral blood lymphocytes.

In another aspect of the inventive subject matter, use of a green tea polyphenol composition is contemplated in the manufacture of a medicament or nutraceutical preparation for increasing GST-π activity in peripheral blood lymphocytes of a person having low baseline GST-π activity, wherein such use includes a step of ascertaining that the person has a low baseline GST-π activity in peripheral blood lymphocytes, and a further step of administering to the person with the low baseline GST-π activity the green tea polyphenol composition at a dosage effective to increase GST-π expression in peripheral blood lymphocytes.

Viewed from a different perspective, a method of increasing the potential efficacy of treatment of a person with a green tea polyphenol composition to thereby reduce cancer risk in a person will include a step of ascertaining that the person has a low baseline GST-π activity in peripheral blood lymphocytes, and a further step of administering to the person with the low baseline GST-π activity the green tea polyphenol composition at a dosage effective to increase GST-π expression in peripheral blood lymphocytes.

Low baseline GST-π activity is preferably characterized by a GST-π activity that is within the lower tertile of GST-π activities measured in a group of at least 40 individuals, and it is further preferred that the low baseline GST-π activity is determined by measuring GST-π quantity from a blood sample and/or measuring conversion of a marker substrate. Preferred green tea polyphenol compositions comprise polyphenon E, or components thereof (and particularly epigallocatechin gallate, epigallocatechin, epicatechin, epicatechin gallate, and/or gallocatechin gallate), and it is particularly preferred that the polyphenol composition is orally and daily administered in an amount corresponding to an amount of total polyphenols in 8-16 cups of green tea. Where desired, information may be given to the person that administration of the green tea polyphenol composition increases at least one of the GST-π expression and the GST-π activity (and thereby reduces risk of cancer or diseases associated free radical damage, low antioxidant status, etc.).

Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention.

Brief Description of the Drawings

Figure 1 is a graph depicting relationship between post-treatment changes in GST activity and in GST-π enzyme level in human blood lymphocytes.

Detailed Description

The inventors have unexpectedly discovered that GST-π activity in peripheral blood lymphocytes of individuals having low baseline GST-π activity can be significantly increased to normal, and even above normal values when a green tea polyphenol composition is given to the individually, preferably in oral form at a daily dosage equivalent to the total polyphenol amount contained in about 8-16 cups of tea. Such increase in GST-π activity is expected to substantially reduce cancer risk in individuals having low baseline GST-π activity.

As used herein, the term "increasing potential efficacy of a treatment to reduce cancer risk" refers to an action that results in a desirable physiological change in a person, wherein that change is associated with an expected reduction in cancer incidence, multiplicity, and/or progression (which may or may not include an increase in apoptosis of precancerous and/or cancerous cells). Therefore, the term "reduce cancer risk" refers to a reduction in incidence, multiplicity, and/or progression of a cancer of precancerous condition.

As also used herein, the term "low baseline GST-π activity" refers to a GST-π activity in a person (particularly in peripheral blood lymphocytes) that is measured under conditions where the person was not exposed to daily doses of greater than 20 mg of green tea catechins for at least one week, and where the measured activity (in peripheral blood lymphocytes) was determined to belong to the lower tertile of measured activities in a random population of at least 40 individuals. Therefore, individuals with low baseline GST-π activity will typically exhibit activities of equal or less than 25 nmol/min/mg as measured in the examples below (see table 1). Alternatively, GST-π protein concentration may also be used as a surrogate marker for activity. Under such scenario, levels of equal or less than 1900 ng/mg protein as measured in the examples below (see table 2) are deemed to reflect low baseline GST-π activity.

Based on the below observations and unexpected findings, the inventors unexpectedly discovered that green tea polyphenol compositions, and especially Polyphenon E (defined green tea polyphenol composition commercially available from Mitsui Norin, Hibiya Central Building, 1-2-9 Nishi-Shinbashi, Minato-Ku - Tokyo 105-8427, Japan) has only insignificant effects on the four major CYP isozymes (1A2, 2D6, 2C9, and 3A4), but significantly induces GST enzyme activity, and especially GST-π activity in peripheral blood lymphocytes. The inventors further surprisingly discovered that the quantitative increase of GST-π activity in peripheral blood lymphocytes by Polyphenon E was a function of the baseline GST-π activity in the tested individuals. Remarkably, individuals with low baseline GST-π activity in peripheral blood lymphocytes were found to respond strongest (i.e., had the quantitatively largest increase of GST-π activity) to Polyphenon E administration, while the response was less pronounced (but still significant) in individuals with about average baseline GST-π activity. On the other hand, Polyphenon E administration to individuals with above average baseline GST-π activity failed to increase GST-π activity. On the contrary, and some cases, Polyphenon E administration to individuals with above average baseline GST-π activity reduced GST-π activity to about average values, thus acting as a GST-π activity normalizing treatment. These results suggest that Polyphenon E administration is not likely to affect the pharmacokinetics of commonly used pharmaceutical drugs but could enhance the detoxification of carcinogens in individuals with low baseline GST-π activity.

With respect to compositions suitable for use herein, it is contemplated that various polyphenol, and especially catechin-containing compositions are appropriate, most typically, such compositions will be derived from one or more plants, and most preferably from the tea plant {Camilla sinensis), grapes (Vitis vinifera) olives (Olea europaea), etc. For example, preferred polyphenol containing compositions include green tea polyphenol compositions obtained from drying and/or extraction of green tea (e.g., polyphenon E). Such compositions may be fortified with one or more isolated polyphenols. Alternatively, the green tea polyphenol composition may also be formulated to comprise one or more natural or synthetic green tea polyphenol. Particularly suitable green tea polyphenols include epigallocatechin gallate, epigallocatechin, epicatechin, epicatechin gallate, and gallocatechin gallate. Thus, suitable green tea polyphenol compositions may be solid, liquids, or suspensions and may be prepared using methods well known in the art.

Contemplated compositions comprising green tea polyphenol compositions may be formulated in numerous manners. However, particularly preferred formulations include those in which the polyphenol composition is incorporated into a food product (e.g., nutritional supplements [snack bar, pill, powder, etc.], beverages [soda, green tea, etc.], pet food, etc), or a pharmaceutical product (e.g., antineoplastic formulations) that may include a second active ingredient (antineoplastic drug, chemopreventive drug,), etc. Therefore, contemplated routes and schedules for administration of contemplated compositions will vary substantially. However, it is generally preferred that the polyphenol composition is orally administered over a period of at least several days, more preferably at least several weeks, and most preferably, at least several months. Furthermore, while once or twice daily administration is typically preferred to sustain a desired minimum plasma concentration over a predetermined time, alternative schedules are also deemed suitable and include 1-6 administrations per week, or even less. Such schedules will preferably, but not necessarily, employ sustained release formulations.

With respect to preferred dosages per administration, it is contemplated that the green tea polyphenol composition is administered at a daily amount corresponding to the amount of total polyphenols present in 8-16 cups of green tea. Therefore, especially preferred amounts will range between 50 mg and 1500 mg of total polyphenols, more preferably between 200 mg and 1000 mg of total polyphenols, and most preferably between 400 mg and 800 mg of total polyphenols. However, larger dosages (e.g., between 1500 mg and 3000 mg, and even higher) are also deemed suitable, especially where such dosages are administered over a relatively short period or in a sustained release formulation.

There are numerous manners known in the art to determine GST-π activity and thus to ascertaining low baseline GST-π activity. For example, suitable methods are described in the experimental section below. Thus, preferred test samples will be whole blood and/or isolated peripheral lymphocytes. It is further generally contemplated that GST-π activity may be either directly or indirectly determined. Direct determination includes measurement of GST activity using a colorimetric or isotopically labeled substrate, while indirect determination is preferably based on quantification of the protein (e.g., via ELISA) or the RNA encoding for GST-π (e.g., via quantitative rtPCR). Such quantification may then be correlated with the GST-π activity the known using specific activity of the enzyme. Alternatively, indirect determination may also be based on quantification of metabolites associated with GST-π metabolism. In still further contemplated manners, ascertaining of GST-π activity may also include a presumptive assertion without validation by a laboratory test.

Once the GST-π activity is ascertained, low baseline GST-π activity is determined by either statistical methods ore absolute measurements. For example, statistical determination may define an individual as having low baseline activity if the GST-π activity is in the lower tertile, qύartile, or quintile of activities of an average population after washout of polyphenols On the other hand, quantitative criteria may also be employed to determine low baseline GST-π activity using quantitative methods as described in the experimental section below. In such case, individuals with low baseline GST-π activity will be defined as having GST-π activities of equal or less than 25 nmol/min/mg as measured in the examples below (see table 1). Alternatively, individuals with low baseline GST-π activity will be defined as having levels of equal or less than 1900 ng/mg protein as measured in the examples below (see table 2). In yet further alternative methods, low baseline activity may be genetically determined by a test for the presence of low-activity allele or mutant. Where desired, the person may be informed about the outcome of the test result and/or the possibility that administration of the green tea polyphenol composition increases at least one of the GST-π expression and the GST-π activity in a person in which low baseline GST-π activity was previously ascertained. Therefore, the inventors contemplate a method of increasing GST-π expression and/or GST-π activity in peripheral blood lymphocytes of an individual that is determined to have low baseline GST-π activity. In such methods, a green tea polyphenol composition is orally (or otherwise) administered to the individual at a dosage effective to increase the GST-π expression and/or GST-π activity in peripheral blood lymphocytes. Consequently, and viewed from a different perspective, the inventors also contemplate use of a green tea polyphenol composition in the manufacture of a medicament or nutraceutical preparation for increasing GST-π activity in peripheral blood lymphocytes of a person having low baseline GST-π activity. In such use, the person has previously been ascertained as having a low baseline GST-π activity in peripheral blood lymphocytes, and the medicament or nutraceutical preparation is formulated to allow administration of the green tea polyphenol composition to the person with the low baseline GST-π activity at a dosage effective to increase GST-π expression in peripheral blood lymphocytes.

Contemplated methods and uses are deemed especially significant as numerous GST levels, mutations, and expression patterns have been reported to be associated with disease susceptibility. Thus, various GSTs appear to play a major role in determining the sensitivity of cells to toxic and carcinogenic agents (see e.g., Crit Rev Biochem MoI Biol. 1995;30:445- 600; Basic Life Sci. 1993;61: 127-136). For example, low GST-alpha expression was associated with reduced detoxification of various environmental pollutants and tobacco smoke, and increased susceptibility towards colorectal cancer (see e.g., Methods Enzymol. 2005 ;401:9-42) and prostate cancer (see e.g., Pharmacogenetics. 2004 Jan;14(l):35-44). Also, various mutations in several GST isoforms were associated with increased breast cancer risk (Afr J Reprod Health. 2003 Dec;7(3): 17-28).

Lack in GST-π was associated with various cancers, including skin and lung cancer (Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5275-80), and low-activity of GST-π was reported as risk factor for oral cancer (Pharmacogenetics. 1999 Apr;9(2): 165-9) and bladder cancer (Arch Toxicol. 2001 Oct;75(8):459-64). Without wishing to be bound by any specific theory or hypothesis, the inventors contemplate that as GST-π has a relatively broad substrate specificity (e.g., with respect to its capacity to detoxify carcinogenic products of polycyclic aromatic hydrocarbon metabolism (Int J Cancer. 1997;73:897-902)), stimulation of GST-π may confer reduced risk for various cancers. Such reduction should be particularly desirable where an individual has a low base line activity of GST-π. Therefore, in further aspects of the inventive subject matter, a method of increasing potential efficacy of a treatment to reduce cancer risk in a person is contemplated in which it is first ascertained that the person has a low baseline GST-π activity in peripheral blood lymphocytes. In another step a green tea polyphenol composition is administered to the person with the low baseline GST-π activity at a dosage effective to increase GST-π activity and/or expression in peripheral blood lymphocytes, wherein administration increases potential efficacy of the treatment relative to a person having normal or high baseline GST-π activity in peripheral blood lymphocytes. Among other cancer risks, especially contemplated cancer risks include lung cancer risk, bladder cancer risk, oral cancer risk, colon cancer risk, prostate cancer risk, and breast cancer risk. With respect to the compositions, dosages, routes, and administration schedules for such methods, the same considerations as provided above apply.

Experiments

The following experiments were performed to investigate whether or not green tea polyphenol compositions, and especially Polyphenon E would be effective to provide cancer preventive activities (as evidenced in increased GST-π activity), especially in individuals that have a relatively low GST-π baseline activity. Earlier work by the inventors (data not shown) suggested that green tea or green tea catechins can modulate the activities of drug and carcinogen metabolizing enzymes. Here, the inventors conducted a clinical study to test the hypothesis that Polyphenon E administration would selectively and advantageously modulate drug/carcinogen metabolizing enzymes in individuals, and particularly individuals with low GST-π baseline activity in peripheral blood lymphocytes.

More specifically, forty two healthy volunteers underwent a 4-week washout period by refraining from tea or tea related products. At the end of the washout period, study participants received a cocktail of cytochrome P450 (CYP) metabolic probe drugs (caffeine, dextromethorphan, losartan, and buspirone for assessing the activity of CYP 1A2, 2D6, 2C9, and 3A4, respectively). Blood and urine samples before and for 8 hours after probe drag administration were collected for measurements of baseline CYP enzyme activities. Baseline glutathione-S-transferase (GST) activity was determined in blood lymphocytes collected prior to probe drug administration. Following the baseline evaluation, study participants underwent 4 weeks of Polyphenon E intervention at a dose that contains 800 mg epigallocatechin gallate once a day. Upon completion of the Polyphenol! E intervention, the post-intervention CYP and GST enzyme activities were evaluated as described above.

Remarkably, the inventors discovered that there are large between-subject variations in CYP and GST enzyme activities in healthy individuals, and that four weeks of Polyphenon E administration at a dosing schedule currently implemented in cancer prevention trials had no significant effects on CYP enzyme activities. However, Polyphenon E intervention significantly increased GST-π activity in blood lymphocytes in individuals with baseline GST activity in the lowest and intermediate tertiles and did not significantly change the GST activity in individuals with high baseline GST-π activity.

Methods and Materials

Study Drugs: Polyphenon E is a green tea catechin enriched and defined product produced by Mitsui Norin, Ltd. It contains 80-98% total catechins by weight with epigallocatechin gallate (EGCG) as the main component accounting for 50-75% of the material. Other catechins, including epicatechin, epigallocatechin, epicatechin gallate, and gallocatechin gallate, are present in levels ranging from approximately 2 to 12% each.

Polyphenon E contains small quantities of caffeine (around 0.5% w/w) and can be considered a decaffeinated product. Polyphenon E, formulated as oral capsules (200 mg EGCG per capsule), was supplied by the Chemoprevention Agent Development Research Group, National Cancer Institute (Bethesda, MD). The study capsules were stored at room temperature and protected from environmental extremes.

Study Participants: Forty-two non-smoking 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 or breast feeding, had invasive cancers within the past 5 years, had uncontrolled severe metabolic disorders or other serious acute or chronic diseases, consumed more than 3 drinks of alcohol per week on average, consumed tea regularly, had known hypersensitivity to green tea or CYP metabolic probe drugs, were taking medications and/or supplements that are known P450 enzyme inducers or inhibitors, or had participated in other clinical research studies within the past 3 months.

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 underwent a 4- week washout period in which they were required to refrain from tea or related products and herbal/botanical supplements, and to minimize the consumption of cruciferous vegetables. At the end of the washout period, a fasting blood sample was collected into heparinized collection tubes. Plasma was separated and lymphocytes were isolated using Ficoll-Hypaque medium (Amersham Biosciences, Piscataway, NJ) for assessment of glutathione S- transferase activities and levels. Following the fasting blood sample collection, study subjects underwent baseline CYP enzyme activity assessment. Clinical procedures for CYP enzyme activity assessment are detailed elsewhere and are well known in the art.

Following completion of the washout period and after baseline enzyme activity determination, study participants were provided with a 4- week supply of Polyphenon E. They continued to complete a daily diary of any adverse reactions, and an intake calendar for recording the daily intake of the study agent and any non-routine medications. Study participants were instructed to take 4 Polyphenon E capsules every day in the morning, on an empty stomach. Breakfast could be consumed one hour after Polyphenon E dosing. Participants continued to refrain from tea or its related products and herbal/botanical supplements, and to minimize the consumption of cruciferous vegetables.

A fasting blood sample was collected the day after completing the 4- week Polyphenon E treatment period. Plasma and lymphocytes were separated for measurement of post- intervention glutathione S-transferase activities and levels. A fasting blood sample was also collected for a complete blood count and comprehensive blood chemistry. Following the fasting blood collection, study participants underwent post-intervention CYP enzyme activity assessment.

Measurements of GST Activities and Levels: Cell lysates were prepared by sonicating the lymphocyte pellets twice at 15-s intervals. The lysed cells were centrifuged at 10,000 x g at 4°C for 30 min. The supernatant was collected and stored at -7O⁰C prior to the analysis. Total GST activity in lymphocyte lysates was determined using a GST assay kit (Cayman Chemical, Ann Arbor, MI) by measuring the conjugation of l-chloro-2,4- dinitrobenzene (CDNB) with reduced glutathione. This GST mediated conjugation reaction is accompanied by an increase in absorbance at 340 nm. GST activity was expressed as nmol/min/mg protein, with protein concentrations assayed by the Bio-Rad protein assay kit (Bio-Rad Lab, Hercules, CA). GST-π concentrations in lymphocyte lysates were measured using an enzyme-linked immunoassay kit (Human pi GST EIA assay, Biotrin International, Dublin, Ireland). The assay procedure is based on sequential addition of samples or standards, anti-GST-π IgG conjugated with horseradish peroxidase, and substrate to microassay wells coated with anti-GST-π IgG. The assay range is 3.12 to 100 ng/ml. Standards of known concentrations were included in every run, and the enzyme levels were calculated from a standard curve. GST-π concentrations were normalized to protein concentrations and expressed as pg/mg protein. Plasma GST-α concentrations were measured using an enzyme-linked immunoassay kit (High sensitivity alpha GST EIA assay, Biotrin International, Dublin, Ireland). The assay procedure is based on sequential addition of samples or standards, anti-GST-α IgG conjugated with horseradish peroxidase, and substrate to microassay wells coated with anti-GST-α IgG. The assay range is 0.0625 - 2 ng/ml. Standards of known concentrations were included in every run, and the enzyme levels were calculated from a standard curve and expressed as ng/ml plasma.

Measurements of Bilirubin Levels: Total serum bilirubin levels were determined in a certified commercial laboratory (LabCorp, Phoenix, AZ) with a modified Jendrassik-Grof method [7] as part of the comprehensive blood chemistry analysis.

Data Analysis: The effects of tea catechin intervention on the enzyme activity/level were analyzed using a conditional change model, in which baseline activity/level was included as a covariate. The model is written as

y\ - yo = βo + βij>o + e

whereto and ^₁ are baseline and post-treatment values for the outcome and e is the error term. The primary effect measure was assessed after centering the data by testing the statistical significance of β₀. For an intervention effect that was found to be significantly affected by the baseline activity/level, the data was further divided into tertiles based on baseline activity/level. The significance level of the intervention effect within each tertile was determined by a paired t-test. Kp < 0.05 was considered statistically significant. Results

Table 1 summarizes the GST activity determined in peripheral blood lymphocytes before and after 4 weeks of daily Polyphenon E administration. Polyphenon E intervention was found to have a significant effect on the GST activity (p < 0.001) and the effect was dependent on baseline GST activity (p < 0.001). GST activity data was further divided into tertiles based on baseline GST activity. Polyphenon E intervention significantly induced the GST activity in individuals in the lowest tertile (p = 0.003), but did not change the GST activity in individuals in the middle tertile. GST activity was decreased in individuals in the highest tertile (p = 0.036).

GST Activity (nmol/min/mg protein) P value

Baseline Post-Treatment

All participants (n = 42) 30.7 ± 12.2' 35 .1 ± 14.3 < 0.001

Baseline activity in the lowest 19.6 ± 3.7 35 .6 ± 16.2 0.003 tertile (n = 14)

Baseline activity in the middle 29.2 ± 3.0 34 .8 ± 15.1 0.22 tertile (n = 14)

Baseline activity in the highest 43.5 ± 11.8 34 .8 ± 12.3 0.036 tertile (n = 14)

Table 1. GST activity in blood lymphocytes before and after 4 weeks of daily Polyphenon E administration; ' mean ± ISD

Because GST-π is the major GST isozyme present in blood lymphocytes (Cancer Epidemiology, Biomarkers & Prevention, 7: p 891-9, 1998), the inventors have determined whether Polyphenon E intervention affected GST-π enzyme levels in blood lymphocytes and the data is summarized in Table 2. Polyphenon E intervention was found to have a significant effect on the GST-π enzyme level in blood lymphocytes (p = 0.001) and the effect was dependent on the baseline GST-π enzyme level (p = 0.005). GST-π data was further divided into tertiles based on the baseline GST-π enzyme level. Polyphenon E intervention significantly increased GST-π protein levels in individuals in the lowest tertile (p = 0.003), but did not change GST-π levels in individuals in the middle and highest tertiles. Figure 1 illustrates the relationship between post-treatment changes in GST activity and GST-π enzyme levels in blood lymphocytes. Post-treatment changes in GST activity correlated significantly with changes in GST-π enzyme level (r^{2 =} 0.35, p < 0.0001).

GST-πLevel (ng/mg protein)

Baseline Post-Treatment P value

All participants (n = 42) 2252.9 ± 734.2¹ 2634.4 ± 1138.3 0.001

Baseline level in the lowest 1505.7 ± 434.9 2754.5 ± 1456.8 0.003 tertile (n = 14)

Baseline level in the middle 2254.1 ± 144.3 2561.8 ± 762.3 0.179 tertile (n = 14)

Baseline level in the highest 2998.8 ± 538.2 2587.0 ± 1166.1 0.157 tertile (n = 14)

Table 2. GST-π enzyme level in blood lymphocytes before and after 4 weeks of daily Polyphenon E administration; ' mean ± ISD

Table 3 summarizes plasma GST-α levels obtained before and after Polyphenon E intervention. GSTs are cytosolic or membrane bound enzymes. GST protein detected in serum/plasma is due to release of GSTs from normal cell turnover. The average baseline plasma GST-α concentration was 7.46 ng/ml with values ranging from 2.51 to 40.13 ng/ml. Following 4 weeks of daily Polyphenon E administration, the average plasma GST-α concentration was 7.59 ng/ml with values ranging from 2.49 to 42.99 ng/ml.

Results

Baseline Post-Treatment P value

GST-α concentration (ng/ml) 7.46 ± 6.54¹ 7.59 ± 6.91 P = 0.508

Table 3. Plasma GST-α concentration before and after 4 weeks of daily Polyphenon E administration; ' mean ± ISD

Table 4 summarizes the changes in total serum bilirubin concentrations. The averaged serum total bilirubin concentrations decreased significantly from a mean value of 0.63 to 0.55 mg/dL (p - 0.005) following 4 weeks of Polyphenon E intervention and the effect was dependent on the baseline bilirubin level (p < 0.001). Since bilirubin is cleared from the body by the liver through conjugation reactions mediated by UDP-glucuronosyl- transferase IAl (Annual Review of Pharmacolgy and Toxicology, 40: p 581-616, 2000), reduction in serum bilirubin concentrations after Polyphenon E intervention suggests an induction of this isozyme. Individuals with baseline bilirubin levels in the highest tertile showed the most significant reduction (from a mean of 0.96 to 0.72 mg/dL,p = 0.003). Individuals with baseline bilirubin levels in the middle tertile showed a moderate change (from a mean of 0.65 to 0.56 mg/dL,/? = 0.04). Polyphenon E intervention did not change serum bilirubin levels in individuals with baseline levels in the lowest tertile.

Bilirubin Levels

(mg/ dL) P value

Baseline Post-Treatment

All participants (n = 42) 0.63 ± 0.24¹ 0.56 ± 0.21 0.005

Baseline level in the lowest 0.41 ± 0.20 0.43 ± 0.21 0.56 tertile (n = 14)

Baseline level in the middle 0.65 ± 0.05 0.56 ± 0.17 0.04 tertile (n = 14)

Baseline level in the highest 0.96 ± 0.08 0.72 ± 0.16 0.003 tertile (n = 14)

Table 4. Serum bilirubin levels before and after 4 weeks of daily Polyphenon E administration; ^λ mean ± ISD

The present data show that four weeks of green tea catechin intervention significantly enhanced the human GST activity and GST-π levels in peripheral blood lymphocytes in individuals with low baseline activity or level. Furthermore, repeated green tea catechin intervention significantly lowered serum bilirubin levels, implicating an induction of human UGTlAl activity, in individuals with low baseline activity.

The family of GSTs represents a major group of detoxification enzymes. GSTs catalyze the reaction of glutathione with electrophiles, resulting in elimination of potentially carcinogenic chemicals (Critical Reviews in Biochemistry and Molecular Biology, 30: p 445- 600, 1995). GSTs also exhibit glutathione peroxidase activity and catalyze the reduction of organic hydroperoxides to their corresponding alcohols (Critical Reviews in Biochemistry and Molecular Biology, 30: p 445-600, 1995). The family of UGTs catalyzes the detoxification and elimination of a large number of endogenous (e.g., bilirubin, bile acids, steroid hormone) and exogenous compounds (drugs, pesticides, components of tobacco smoke) in the liver and extrahepatic tissues. Both the glutathione conjugation and glucuronidation reactions are considered important Phase II detoxification reactions for elimination of carcinogens or for reducing the interactions of carcinogens with cellular macromolecules. Induction of Phase II enzymes, such as GSTs or UGTs, has been suggested as a potential mechanism for the cancer chemopreventive activities observed with a number of nonnutrient phytochemicals (Mutation Research, 480-481: p 285-297, 2001; Experimental Biology & Medicine, 216: p 192-200, 1997). Our data provided the first clinical evidence of human Phase II enzyme induction with green tea catechin intervention at a daily EGCG dose level equivalent to that in 8-16 cups of green tea.

Modulation of GST activity with green tea or green tea catechins has been observed in some preclinical studies, however, have been inconclusive. For example, Maliakal et al. (Journal of Pharmacy & Pharmacology, 53: p 569-77, 2001) showed an induction of hepatic GST activity in rats fed with green tea preparation, while Sohn et al. (Xenobiotica, 24: p 119- 27, 1994) demonstrated that hepatic GST activity in rats was not affected by chronic green tea feeding. Green tea catechin feeding to mice has been shown to induce GST activity in the small bowel and the liver (Cancer Research, 52: p 4050-2, 1992). Here, the inventors demonstrated that repeated green tea catechin administration significantly induced human GST activity in blood lymphocytes in individuals with low baseline activity. GST activity measured using CDNB, a substrate conjugated by all the GSTs, represents total GST activity. Total GST activities measured in blood lymphocytes have been found to correlate well with those determined in colon mucosa in humans (Clinical Cancer Research, 3: p 25-30, 1997; Cancer Research, 55: p 2789-93, 1995).

The most abundant cytosolic GST enzymes in humans are encoded by three distinctly related groups; α, μ, and π. These isozymes are differentially expressed in hepatic and extrahepatic tissues. GST-π is the major GST isozyme present in human blood lymphocytes. GST-π is also abundant in human intestine and colon. The inventors showed that green tea catechin intervention significantly increased GST-π protein levels in blood lymphocytes in individuals with low baseline level. Post-treatment changes in blood lymphocyte GST activity correlated significantly with changes in GST-π protein level. Expression of GST-μ is also evident in human blood lymphocytes. The inventors have measured changes in GST-μ levels in lymphocyte lysates semi-quantitatively using western blotting. No consistent changes were observed in the expression of this isozyme (data not shown). However, the sample size of the current study is limited for such an evaluation since ~50% of individuals have a null mutation for the expression of GST-μ. GST-α is the major GST isozyme found in human liver. Plasma GST-α has been shown to reflect induction of hepatic GST-α levels in mice when fed with Phase II enzyme inducers, and has been used to assess the effect of intervention with vegetable diets in the clinical setting. The inventors showed that repeated green tea catechin administration had minimal effects on plasma GST-α levels. This suggests that human GST isozymes may be differentially induced by green tea catechins. Also, tissue specific induction of human GST activity may be observed with green tea catechin intervention since GST isozymes are differentially expressed in various tissues.

Moreover, green tea feeding has been consistently shown to induce hepatic UGT activity towards multiple phenolic substrates in rats. Embola et al. showed that induction of UGT activity by green tea may involve the UGTlA gene complex of the UGT multigene family. UGTlAl is the major UGT isozyme responsible for glucuronidation of bilirubin. Inheritable benign unconjugated hyperbilirubinemia (Gilbert syndrome) is associated with reduced hepatic UGTlAl expression. Using serum bilirubin levels as a surrogate biomarker, our data suggested that UGTlAl activity was induced with repeated green tea catechin administration in individuals with low baseline activity/level. Intervention trials have been conducted to evaluate the effect of supplementation of cruciferous vegetables on UGT activity. Using acetaminophen as a metabolic probe drug, Pantuck et al. showed that supplementation of cruciferous vegetables increased acetaminophen glucuronidation. Hecht et al. demonstrated that three days of cruciferous vegetable supplementation increased glucuronidation of nicotine in current smokers. Further studies are needed to determine whether other human UGT isozymes are induced by green tea catechin intervention.

The inventors have shown that induction of GSTs or UGTlAl with repeated tea catechin intervention is affected by baseline enzyme level or activity, with significant effects observed in individuals with low baseline level or activity. Recent studies have shown that modulation of GSTs or UGTs with dietary constituents can be affected by genotypes or phenotypes of these enzymes. Lampe et al. (Cancer Epidemiology, Biomarkers & Prevention, 9: p 787-93, 2000) illustrated that botanically defined vegetable diets can modulate GST isozyme level or activity and the modulation effects are affected by GST genotypes. Consistent with this, Pool-Zobel (Cancer Epidemiology, Biomarkers & Prevention, 7: p 891-9, 1998) demonstrated that induction of lymphocyte GST-π levels by vegetable consumption is affected by GST genotype and baseline GST-π levels. In an observational study, Peterson et al. found that increased Cruciferae intake correlated with lower serum bilirubin levels only in individuals with the UGT genotype that is associated with decreased transcription and activity of UGTl Al isozyme. It is plausible that there may be a threshold in Phase II enzyme expression, thus induction of these detoxification enzymes is more pronounced in individuals with low baseline activity or level. It is also likely that the baseline Phase II enzyme activity or level affects the systemic bioavailability of green tea catechins since these phytochemicals are extensively glucuronidated or sulfated. Individuals with low baseline Phase II enzyme activity or level may have higher systemic bioavailability of unchanged green tea catechins, resulting in greater magnitude of enzyme induction. The observations of phenotype/genotype dependent induction of Phase II enzymes suggest the potential of selecting individuals with low baseline enzyme activity for cancer prevention trials of chemopreventive agents or dietary interventions that affect the Phase II enzymes.

The inventors therefore conclude that green tea catechin administration significantly induced GST and UGTlAl activities in individuals with low baseline enzyme activity. Thus, the results also suggest that green tea catechin intervention could enhance the detoxification of carcinogens in individuals with low baseline detoxification capacity.

Thus, specific embodiments and applications of green tea catechins for modification of detoxification enzymes have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

Claims

CLAIMSWhat is claimed is:

1. A method of increasing at least one of a GST-π expression and a GST-π activity in peripheral blood lymphocytes of a person determined to have low baseline GST-π activity comprising administering a green tea polyphenol composition to the person at a dosage effective to increase the at least one of the GST-π expression and the GST-π activity in peripheral blood lymphocytes.

2. The method of claim 1 wherein the low baseline GST-π activity is characterized by a GST-π activity that is within a lower textile of GST-π activities measured across a group of at least forty individuals.

3. The method of claim 1 wherein the green tea polyphenol composition comprises polyphenon E.

4. The method of claim 1 wherein the green tea polyphenol composition comprises a polyphenol selected from the group consisting of epigallocatechin gallate, epigallocatechin, epicatechin, epicatechin gallate, and gallocatechin gallate.

5. The method of claim 1 wherein the green tea polyphenol composition is orally daily administered in an amount corresponding to an amount of total polyphenols in 8-16 cups of green tea.

6. The method of claim 1 wherein the low baseline GST-π activity is determined by at least one of measuring GST-π quantity from a blood sample and measuring conversion of a marker substrate.

7. The method of claim 1 further comprising a step of providing information to the person that administration of the green tea polyphenol composition increases at least one of the GST-π expression and the GST-π activity.

8. Use of a green tea polyphenol composition in the manufacture of a medicament or nutraceutical preparation for increasing GST-π activity in peripheral blood lymphocytes of a person having low baseline GST-π activity, wherein the person is a person previously ascertained as having a low baseline GST-π activity in peripheral blood lymphocytes, and wherein the medicament or nutraceutical preparation is formulated to allow administration of the green tea polyphenol composition to the person with the low baseline GST-π activity at a dosage effective to increase GST-π expression in peripheral blood lymphocytes.

9. Use of claim 8 wherein the medicament or nutraceutical preparation is formulated for oral administration.

10. Use of claim 8 wherein the step of administering comprises daily oral administration of the polyphenol composition in an amount corresponding to an amount of total polyphenols in 8-16 cups of green tea.

11. Use of claim 8 wherein green tea polyphenol composition comprises polyphenon E.

12. Use of claim 8 wherein the green tea polyphenol composition comprises a polyphenol selected from the group consisting of epigallocatechin gallate, epigallocatechin, epicatechin, epicatechin gallate, and gallocatecliin gallate.

13. A method of increasing potential efficacy of a treatment to reduce cancer risk in a person, comprising:

ascertaining that the person has a low baseline GST-π activity in peripheral blood lymphocytes; and administering to the person with the low baseline GST-π activity the green tea polyphenol composition at a dosage effective to increase GST-π expression in peripheral blood lymphocytes; wherein administration increases potential efficacy of the treatment relative to a person having normal or high baseline GST-π activity in peripheral blood lymphocytes.

14. The method of claim 13 wherein the green tea polyphenol composition comprises polyphenon E.

15. The method of claim 13 wherein the green tea polyphenol composition comprises at least one of epigallocatechin gallate, epigallocatechin, epicatechin, epicatechin gallate, and gallocatechin gallate.

16. The method of claim 13 wherein the cancer risk is selected from the group of lung cancer risk, oral cancer risk, colon cancer risk, prostate cancer risk, and breast cancer risk.

17. The method of claim 13 wherein the step of ascertaining is performed by measuring GST-π quantity from a blood sample.

18. The method of claim 13 wherein the step of ascertaining is performed by measuring conversion of a marker substrate.

19. The method of claim 13 wherein the step of administering comprises oral administration.

20. The method of claim 19 wherein the step of administering comprises daily oral administration of the polyphenol composition in an amount corresponding to an amount of total polyphenols in 8-16 cups of green tea.