3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.

Attenuation of liver cancer development by oral glycerol supplementation in the rat.

PURPOSE: Glycerol usage is increasing in food industry for human and animal nutrition. This study analyzed the impact of glycerol metabolism when orally supplemented during the early stage of rat liver carcinogenesis. METHODS: Wistar rats were subjected to a 2-phase model of hepatocarcinogenesis (initiated-promoted, IP group). IP animals also received glycerol by gavage (200 mg/kg body weight, IPGly group). RESULTS: Glycerol treatment reduced the volume of preneoplastic lesions by decreasing the proliferative status of liver foci, increasing the expression of p53 and p21 proteins and reducing the expression of cyclin D1 and cyclin-dependent kinase 1. Besides, apoptosis was enhanced in IPGly animals, given by an increment of Bax/Bcl-2 ratio, Bad and PUMA mitochondrial expression, a concomitant increase in cytochrome c release and caspase-3 activation. Furthermore, hepatic levels of glycerol phosphate and markers of oxidative stress were increased in IPGly rats. Oxidative stress intermediates act as intracellular messengers, inducing p53 activation and changes in JNK and Erk signaling pathways, with JNK activation and Erk inhibition. CONCLUSION: The present work provides novel data concerning the preventive actions of glycerol during the development of liver cancer and represents an economically feasible intervention to treat high-risk individuals.

[Influence of cooking procedure on the bioavailability of lycopene in tomatoes]. / Influencia del procedimiento culinario sobre la biodisponibilidad del licopeno en el tomate.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the influence of raw and processed tomato consumption on plasma lycopene concentration in healthy volunteers. A cross-over dietary intervention study was employed. PATIENTS AND METHODS: Fifteen healthy subjects were included in the study. Plasma lycopene concentration was assayed by HPLC. RESULTS: Raw crushed tomato consumption did not significantly influence plasma lycopene concentration. Consumption of raw crushed tomato with olive oil and cooked tomatoes with olive oil, significantly increased blood lycopene levels.

Effectiveness of weekly percutaneous maxacalcitol injection therapy in patients with secondary hyperparathyroidism.

Percutaneous injection therapy with vitamin D has been applied in the treatment of hyperparathyroidism (HPT); however, the application of percutaneous injection therapy with vitamin D lacks established guidelines regarding the volume of injected solution and the frequency of injection. We have developed an outpatient treatment regimen using percutaneous maxacalcitol injection therapy (PMIT) on a weekly basis for 4-6 weeks following dialysis without major complications. Intact parathyroid hormone decreased from 797 +/- 178 pg/mL to 253 +/- 25 pg/mL, and the parathyroid gland volume initially increased during the first week, but thereafter, it gradually decreased with weekly PMIT (wPMIT). Finally, the parathyroid gland volume decreased from 1.27 +/- 1.06 cm(3) to 0.24 +/- 0.15 cm(3) after wPMIT. The benefits of our method were confirmed on weekly ultrasonographic examinations, which detailed the gradual reduction in gland size following an initial increase after the first injection. Therefore, we conclude that our carefully implemented PMIT method would be an effective treatment against refractory secondary HPT.

Phase I trial of daily oral Polyphenon E in patients with asymptomatic Rai stage 0 to II chronic lymphocytic leukemia.

PURPOSE: To define the optimal dose of Polyphenon E for chronic daily administration and tolerability in patients with chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: Previously untreated patients with asymptomatic Rai stage 0 to II CLL were eligible for participation. Polyphenon E with a standardized dose of epigallocatechin-3-gallate (EGCG) was administered using the standard phase I design with three to six patients per dose level (range, 400 to 2,000 mg by mouth twice a day). Trough plasma EGCG levels were measured 1 month after initiation of therapy. Response was classified using the National Cancer Institute (NCI) Working Group (WG) Criteria. RESULTS: Thirty-three eligible patients were accrued to dose levels 1 to 8. The maximum-tolerated dose was not reached. The most common adverse effects included transaminitis (33%, all grade 1), abdominal pain (30% grade 1, 0% grade 2, and 3% grade 3), and nausea (39% grade 1 and 9% grade 2). One patient experienced an NCI WG partial remission. Other signs of clinical activity were also observed, with 11 patients (33%) having a sustained > or = 20% reduction in absolute lymphocyte count (ALC) and 11 (92%) of 12 patients with palpable adenopathy experiencing at least a 50% reduction in the sum of the products of all nodal areas during treatment. Trough plasma EGCG levels after 1 month of treatment ranged from 2.9 to 3,974 ng/mL (median, 40.4 ng/mL). CONCLUSION: Daily oral EGCG in the Polyphenon E preparation was well tolerated by CLL patients in this phase I trial. Declines in ALC and/or lymphadenopathy were observed in the majority of patients. A phase II trial to evaluate efficacy using 2,000 mg twice a day began in November 2007.

Preoperative levels of plasma micronutrients are related to endometrial cancer risk.

OBJECTIVE: To examine the relation between the plasma concentration of antioxidant micronutrients and endometrial cancer risk in Korean women. DESIGN: Hospital-based case-control study. SETTING: Seven tertiary medical institutes in Korea. POPULATION: Incidence of 28 endometrial cancer cases were identified and 140 age-matched controls selected for the same period. METHODS: Preoperative plasma concentrations of beta-carotene, lycopene, zeaxanthin plus lutein, retinol, alpha-tocopherol, and gamma-tocopherol were measured by reverse-phase, gradient high-pressure liquid chromatography. Conditional logistic regression was used to evaluate micronutrient effect after adjustment for body mass index (BMI), menopause, parity, oral contraceptive use, smoking status, and alcohol consumption status. MAIN OUTCOME MEASURES: Effect of micronutrients on endometrial cancer risk. RESULTS: The mean concentration of plasma beta-carotene (p=0.001), lycopene (p=0.008), zeaxanthin plus lutein (p=0.031), retinol (p=0.048), and gamma-tocopherol (p=0.046) were significantly lower in endometrial cancer patients than in controls. Plasma levels of beta-carotene (p for trend=0.0007) and lycopene (p for trend=0.007) were inversely associated with endometrial cancer risk across tertiles. Women in the highest tertile of plasma beta-carotene and lycopene had a 0.12-fold (95% confidence intervals (CIs) 0.03-0.48) and 0.15-fold (95% CIs 0.04-0.61) decreased risk of endometrial cancer compared to women in the lowest tertile, respectively. Other micronutrients such as zeaxanthin plus lutein (p for trend=0.142), retinol (p for trend=0.108), alpha-tocopherol (p for trend=0.322), and gamma-tocopherol (p for trend=0.087) showed no association with endometrial cancer risk. CONCLUSIONS: Plasma levels of beta-carotene and lycopene are inversely associated with the risk of endometrial cancer in Korean women.

Distribution of anthocyanins delivered from a bioadhesive black raspberry gel following topical intraoral application in normal healthy volunteers.

PURPOSE: Results from our oral cavity chemoprevention trial demonstrated appreciable interpatient variations regarding chemopreventive efficacy of a freeze dried black raspberry (FBR) gel. We speculated these data reflected individual patient-related differences in absorption, target tissue uptake and local compound metabolism of key FBR compounds (anthocyanins). Accordingly, this study assessed the distribution of anthocyanins from the 10% (w/w) FBR gel in saliva, oral tissues and plasma. METHODS: Human subject participation entailed collection of: (1) saliva, tissue and plasma (5 min following gel application, keratinized tissues), (2) saliva and plasma (5 min after sublingual gel application), (3) saliva and plasma at 1, 2, and 4 h post gel application (keratinized tissues), and (4) saliva (cyanidin 3-rutinoside incubations). Levels of FBR anthocyanins in the respective samples were analyzed by LC/MS/MS. RESULTS: Our data show: significantly higher anthocyanin levels in saliva and oral tissues relative to matched plasma samples, marked donor-specific variations in anthocyanin uptake, sustainability of anthocyanins at the target site, pH affects anthocyanin penetration and intraoral anthocyanin decomposition and/or metabolism. CONCLUSIONS: No previous oral cavity chemoprevention trials evaluated compound distribution at the treatment site. Our data, which demonstrate a local delivery-derived pharmacologic advantage, provide insights which could advance oral cavity chemoprevention strategies.

Development of a high performance liquid chromatography method for quantification of PAC-1 in rat plasma.

A sensitive and specific high performance liquid chromatography method with UV detection was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on a Diamonsil C(18) column (150mmx4.6mm i.d., 5microm particle size, Zhonghuida) protected by a ODS guard column (10mmx4.6mm i.d., 5microm particle size), using acetonitrile-methanol-phosphate buffer (pH 3.0, 30mM) (31:3:66, v/v/v) as mobile phase at a flow rate of 1.0mL/min, and wavelength of the UV detector was set at 281nm. No interference from any endogenous substances was observed during the elution of PAC-1 and internal standard (IS, indapamide). The calibration curve was linear over the range of 0.05-20microg/mL (r>0.99). The lower limit of quantification was evaluated to be 50ng/mL. The method was successfully applied to the pharmacokinetic study of PAC-1 after intravenous and oral administration in rats, respectively.

Effects of phytoestrogen genistein on cytogenetic biomarkers in postmenopausal women: 1 year randomized, placebo-controlled study.

To evaluate in a twelve-month, randomized placebo-controlled study whether pure administration of phytoestrogen genistein (54 mg/day) might reduce cytogenetic biomarkers in peripheral lymphocytes of postmenopausal women. A total of 57 postmenopausal women met the criteria and were randomly assigned to receive phytoestrogen genistein (n = 30) or placebo (n = 27). There was no significant difference in age, length of time since menopause or body mass index between the two groups. After one year, plasma genistein level was 0.14 +/- 0.01 micromol/L in the control group and 0.72 +/- 0.08 micromol/L in the genistein group (P < 0.0001). At baseline, sister chromatid exchange rate was 4.97 +/- 2.17 in the control group and 4.96 +/- 1.83 in the genistein group (P = 0.89). After one year, sister chromatid exchange rate was 4.96 +/- 2.16 in the control group and 3.98 +/- 1.14 in the genistein group (P < 0.05). High frequency cells count was 3% in the genistein group and 5% in the control group (P < 0.05) at the end of the study. Chromosomal aberration frequency was 5.55% in the control group at time 0 and 5.75% in the genistein group; after one year, the figures were 5.86% in the control group and 4.5% in the genistein group (P < 0.05). After one year, there was a negative relationship between sister chromatid exchange rate and plasma levels (r = - 0.43; P < 0.05) in the genistein group. Phytoestrogen genistein has been shown in postmenopausal women to be effective in the reduction of cytogenetic biomarkers. The protective effect on genomic damage appears to be a particularly promising tool in reducing the risk of cancer.

Evaluation of the cancer chemopreventive efficacy of silibinin in genetic mouse models of prostate and intestinal carcinogenesis: relationship with silibinin levels.

Silibinin, a flavonolignan from milk thistle seeds, possesses cancer chemopreventive properties in rodent models of carcinogenesis. We tested the hypotheses that silibinin or silipide, silibinin formulated with phospholipids, delays tumour development in TRAMP or Apc(Min) mice, genetic models of prostate or intestinal malignancies, respectively. Mice received silibinin or silipide with their diet (0.2% silibinin equivalents) from weaning. Intervention with silipide reduced the size of well differentiated TRAMP adenocarcinomas by 31%. Silipide and silibinin decreased the incidence of poorly differentiated carcinomas by 61% compared to mice on control diet. Silipide decreased plasma levels of insulin-like growth factor (IGF)-1 by 36%. Levels of circulating IGF binding protein (IGFBP)-3 in mice on silipide or silibinin were 3.9- or 5.9-fold, respectively, elevated over those in control TRAMP mice. In Apc(Min) mice silibinin, but not silipide, had only a marginal adenoma number-reducing effect. The results cautiously support the advancement of silipide to the stage of clinical investigation in prostate cancer.

A randomized trial of lycopene supplementation in Tobago men with high prostate cancer risk.

This unblinded, randomized, Phase I clinical trial was conducted to determine whether lycopene supplementation lowered serum prostate specific antigen (PSA), surrogate endpoint for prostate cancer initiation or progression, in men with elevated prostate cancer risk. Afro-Caribbean men (n=81) with high-grade prostatic intraepithelial neoplasia, atypical foci or repeated non-cancerous biopsies, ascertained in a population-based screening program, were randomized to four months intervention with 30 mg/day lycopene (Lyc-O-Mato) plus a multivitamin, or to multivitamin, only. Serum PSA and lycopene were compared at randomization, 1, and 4 mo using two-sided chi2 and t-tests for independent samples. Treatment groups were similar at baseline. Serum lycopene levels approximately doubled in the lycopene intervention group. Serum PSA declined during the first month of treatment, but returned to randomization level by month 4. The PSA response was nearly identical in both treatment groups. No adverse effects attributed to lycopene supplementation were documented. We conclude that the PSA lowering response to antioxidant supplementation observed in previous 3-wk studies in men awaiting prostatectomy may have been a transient response, perhaps not specific to lycopene. Lowering of serum PSA may not be an appropriate endpoint for the long-term studies needed to evaluate lycopene supplementation for reducing prostate cancer initiation or progression.

Formulation and in-vitro and in-vivo evaluation of a mucoadhesive gel containing freeze dried black raspberries: implications for oral cancer chemoprevention.

PURPOSE: The purpose of these studies was to formulate mucoadhesive gels containing freeze dried black raspberries (FBR) and to determine optimum parameters for a subset of FBR bioactive compounds including anthocyanin stability, absorption and penetration in-vitro and in-vivo. MATERIALS AND METHODS: Berry gels were prepared having FBR at 5% and 10% w/w and final pHs ranging from 3.5 to 7.5. A HPLC assay was developed to quantify and determine the stability of the anthocyanins in the gels. A single time-point study was performed to determine anthocyanin uptake when the gels were applied to oral mucosa. Penetration of anthocyanins into human oral tissue explants was determined as a function of gel pH and FBR content. A HPLC-mass spectroscopy assay was utilized to quantify the anthocyanin levels in human oral tissue explants, saliva, and blood. RESULTS: The stability of anthocyanins in the gel was directly related to gel pH and storage temperature. Maximum stability of anthocyanins was found at lower pH (pH 3.5) and storage temperature (4 degrees C). Anthocyanins contained in mucoadhesive berry gel formulations were readily absorbed into human oral mucosa tissue as evidenced by detectable blood levels within 5 min after gel application. There was a trend for greater penetration of anthocyanins into tissue explants for berry gels with a final pH of 6.5 versus pH 3.5. CONCLUSIONS: Formulation and characterization of a novel gel formulation for local delivery of chemopreventive compounds to human oral mucosal tissues has been described. The results show anthocyanin stability was dependent upon gel pH and storage temperature and also demonstrate that the gel composition is well-suited for absorption and penetration into the target oral mucosal tissue site.

Pilot study of oral silibinin, a putative chemopreventive agent, in colorectal cancer patients: silibinin levels in plasma, colorectum, and liver and their pharmacodynamic consequences.

Silibinin, a flavonolignan from milk thistle, has intestinal cancer chemopreventive efficacy in rodents. It is a strong antioxidant and modulates the insulin-like growth factor (IGF) system by increasing circulating levels of IGF-binding protein 3 (IGFBP-3) and decreasing levels of IGF-I. Here, the hypothesis was tested that administration of oral silibinin generates agent levels in human blood and colorectal and hepatic tissues consistent with pharmacologic activity. Patients with confirmed colorectal adenocarcinoma received silibinin formulated with phosphatidylcholine (silipide) at dosages of 360, 720, or 1,440 mg silibinin daily for 7 days. Blood and biopsy samples of normal and malignant colorectum or liver were obtained before dosing, and blood and colorectal or hepatic tissues were collected at resection surgery after the final silipide dose. Levels of silibinin were quantified by high-pressure liquid chromatography-UV, and plasma metabolites were identified by liquid chromatography-mass spectrometry. Blood levels of IGFBP-3, IGF-I, and the oxidative DNA damage pyrimidopurinone adduct of deoxyguanosine (M1dG) were determined. Repeated administration of silipide was safe and achieved levels of silibinin of 0.3 to 4 micromol/L in the plasma, 0.3 to 2.5 nmol/g tissue in the liver, and 20 to 141 nmol/g tissue in colorectal tissue. Silibinin monoglucuronide, silibinin diglucuronide, silibinin monosulfate, and silibinin glucuronide sulfate were identified in the plasma. Intervention with silipide did not affect circulating levels of IGFBP-3, IGF-I, or M1dG. The high silibinin levels achieved in the human colorectal mucosa after consumption of safe silibinin doses support its further exploration as a potential human colorectal cancer chemopreventive agent.

Effects of ingestion of tomatoes, tomato juice and tomato purée on contents of lycopene isomers, tocopherols and ascorbic acid in human plasma as well as on lycopene isomer pattern.

Tomatoes are an important part of the diet. Lycopene, the predominant carotenoid in tomatoes, is hypothesised to mainly mediate the health benefits of tomato products. Anticancer activity of tomato products and lycopene has been suggested by numerous studies. The aim of the present study was to investigate the effect of ingestion of three different tomato-based foodstuffs on plasma contents of lycopene, tocopherols and ascorbic acid. Because isomers of lycopene may have different biological activities, a special interest was to look how the lycopene isomer pattern is changed depending on the matrix of tomato products. Following a 2-week depletion phase volunteers ingested 12.5 mg lycopene/d for 4 weeks comprising tomatoes, tomato juice or tomato purée. The basal levels of lycopene in plasma were comparable for all groups and decreased significantly during the 2 weeks of depletion to approximately half of the basal values. Following intervention, plasma lycopene concentration increased significantly. Conversely, supplementation did not significantly affect levels of tocopherols and ascorbic acid in plasma. Regarding isomers of lycopene, the (Z)-lycopene:(all-E)-lycopene plasma isomer ratio was significantly changed during the study for all groups. A remarkable enrichment of the relative contents of (5Z)-lycopene was observed during the depletion period, which supports the hypothesis that lycopene (Z)-isomers are formed within the human body after ingestion of (all-E)-lycopene. After dietary intervention with lycopene-rich products the isomer ratios returned to those observed at the start of the study. Further investigations will clarify the process of isomerisation in more detail.

Antioxidant status and risk of cancer in the SU.VI.MAX study: is the effect of supplementation dependent on baseline levels?

The SUpplementation en VItamines et Mineraux AntioXydants (SU.VI.MAX) study, a randomised double-blind, primary-prevention trial showed that after 7.5 years, low-dose antioxidant supplementation lowered the total cancer incidence in men, but not in women. To explain this difference in the impact of antioxidant supplementation in men and women, we hypothesised that the effect of supplementation is dependent on initial antioxidant status; 12 741 French adults (7713 females aged 35--60 years; 5028 males aged 45--60 years) received daily antioxidant supplementation (120 mg vitamin C, 30 mg vitamin E, 6 mg beta-carotene, 100 microg Se, 20 mg Zn daily) or a matching placebo. Cut-off limits for baseline serum concentrations of the different antioxidant vitamins and minerals were defined as follows for both men and women: 0.3 micromol/l for beta-carotene, 11.4 micromol/l for vitamin C, 15 micromol/l for vitamin E, 0.75 micromol/l for Se and 10.7 micromol/l for Zn. The percentage of men with serum concentrations under cut-off limits was higher for vitamins C and E and beta-carotene in those who developed a cancer than in those who did not. The risk of cancer was higher in men with baseline concentrations of serum vitamin C or vitamin E under cut-off limits, but not in women. The effect of supplementation was greater in men with baseline serum concentrations of vitamin C, vitamin E and beta-carotene below the cut-off limits compared with those above it. This effect was maintained only for vitamin E after adjustment for age, tobacco, and alcohol consumption and BMI. No effect of supplementation could be seen in women. Baseline antioxidant status is related to the risk of cancer in men but not in women and therefore does not entirely explain the differences observed in the effect of antioxidant supplementation on cancer risk between sexes in the SU.VI.MAX study.

The epidemiology of vitamin D and cancer incidence and mortality: a review (United States).

In vitro and animal studies indicate that vitamin D may have anti-cancer benefits, including against progression and metastasis, against a wide spectrum of cancers. Supporting an anti-cancer effect of vitamin D is the ability of many cells to convert 25(OH)D, the primary circulating form of vitamin D, into 1,25(OH)2D, the most active form of this vitamin. No epidemiologic studies have directly measured vitamin D concentrations or intakes on risk of total cancer incidence or mortality. However, higher rates of total cancer mortality in regions with less UV-B radiation, and among African-Americans and overweight and obese people, each associated with lower circulating vitamin D, are compatible with a benefit of vitamin D on mortality. In addition, poorer survival from cancer in individuals diagnosed in the months when vitamin D levels are lowest suggests a benefit of vitamin D against late stages of carcinogenesis. The only individual cancer sites that have been examined directly in relation to vitamin D status are colorectal, prostate and breast cancers. For breast cancer, some data are promising for a benefit from vitamin D but are far too sparse to support a conclusion. The evidence that higher 25(OH)D levels through increased sunlight exposure or dietary or supplement intake inhibit colorectal carcinogenesis is substantial. The biologic evidence for an anti-cancer role of 25(OH)D is also strong for prostate cancer, but the epidemiologic data have not been supportive. Although not entirely consistent, some studies suggest that higher circulating 1,25(OH)2D may be more important than 25(OH)D for protection against aggressive, poorly-differentiated prostate cancer. A possible explanation for these divergent results is that unlike colorectal tumors, prostate cancers lose the ability to hydroxylate 25(OH)D to 1,25(OH)2D, and thus may rely on the circulation as the main source of 1,25(OH)2D. The suppression of circulating 1,25(OH)2D levels by calcium intake could explain why higher calcium and milk intakes appear to increase risk of advanced prostate cancer. Given the potential benefits from vitamin D, further research should be a priority.