Timothy H. Marczylo

Phase I clinical trial of oral curcumin : biomarkers of systemic activity and compliance

Curcumin, a polyphenolic antioxidant derived from a dietary spice, exhibits anticancer activity in rodents and in humans. Its efficacy appears to be related to induction of glutathione S-transferase enzymes, inhibition of prostaglandin E2 (PGE2) production, or suppression of oxidative DNA adduct (M1G) formation. We designed a dose-escalation study to explore the pharmacology of curcumin in humans. Fifteen patients with advanced colorectal cancer refractory to standard chemotherapies consumed capsules compatible with curcumin doses between 0.45 and 3.6 g daily for up to 4 months. Levels of curcumin and its metabolites in plasma, urine, and feces were analyzed by high-pressure liquid chromatography and mass spectrometry. Three biomarkers of the potential activity of curcumin were translated from preclinical models and measured in patient blood leukocytes: glutathione S-transferase activity, levels of M1G, and PGE2 production induced ex vivo. Dose-limiting toxicity was not observed. Curcumin and its glucuronide and sulfate metabolites were detected in plasma in the 10 nmol/L range and in urine. A daily dose of 3.6 g curcumin engendered 62% and 57% decreases in inducible PGE2 production in blood samples taken 1 hour after dose on days 1 and 29, respectively, of treatment compared with levels observed immediately predose (P < 0.05). A daily oral dose of 3.6 g of curcumin is advocated for Phase II evaluation in the prevention or treatment of cancers outside the gastrointestinal tract. PGE2 production in blood and target tissue may indicate biological activity. Levels of curcumin and its metabolites in the urine can be used to assess general compliance.

Dietary polyphenolic phytochemicals--promising cancer chemopreventive agents in humans? A review of their clinical properties.

Epidemiological and preclinical evidence suggests that polyphenolic phytochemicals exemplified by epigallocatechin gallate from tea, curcumin from curry and soya isoflavones possess cancer chemopreventive properties. Whilst such naturally occurring polyphenols have been the subject of numerous mechanistic studies in cells, information on their clinical properties, which might help assess their promise as human cancer chemopreventive agents, is scarce. Therefore, we present a review of pilot studies and trials with a cancer chemoprevention‐related rationale, in which either healthy individuals or patients with premalignant conditions or cancer received polyphenolic phytochemicals. The review identifies trial design elements specifically applicable to polyphenolic phytochemicals. The available evidence for tea polyphenols tentatively supports their advancement into phase III clinical intervention trials aimed at the prevention of progression of prostate intraepithelial neoplasia, leukoplakia or premalignant cervical disease. In the case of curcumin and soya isoflavones more studies in premalignacies seem appropriate to optimise the nature and design of suitable phase III trials. The abundance of flavonoids and related polyphenols in the plant kingdom makes it possible that several hitherto uncharacterised agents with chemopreventive efficacy are still to be identified, which may constitute attractive alternatives to currently used chemopreventive drugs.

Smoking induces differential miRNA expression in human spermatozoa: A potential transgenerational epigenetic concern?

Recent work has suggested that environmental chemicals, including those contained in cigarette smoke, can have adverse effects on the exposed individuals as well as their future progeny. The mechanisms underlying transmission of environmentally induced phenotypes through the germ line are not well understood. However, a predominant process appears to be the establishment of permanent heritable epigenetic alterations, and a number of studies have implicated microRNAs in such processes. Here, we show that cigarette smoke induces specific differences in the spermatozoal microRNA content of human smokers compared with non-smokers, and that these altered microRNAs appear to predominantly mediate pathways vital for healthy sperm and normal embryo development, particularly cell death and apoptosis. microRNA-mediated perturbation of such pathways may explain how harmful phenotypes can be induced in the progeny of smokers.

Effect of cyanidin-3-glucoside and an anthocyanin mixture from bilberry on adenoma development in the ApcMin mouse model of intestinal carcinogenesis : Relationship with tissue anthocyanin levels

Anthocyanins are dietary flavonoids, which can prevent carcinogen‐induced colorectal cancer in rats. Here, the hypotheses were tested that Mirtoselect, an anthocyanin mixture from bilberry, or isolated cyanidin‐3‐glucoside (C3G), the most abundant anthocyanin in diet, interfere with intestinal adenoma formation in the ApcMin mouse, a genetic model of human familial adenomatous polyposis, and that consumption of C3G or Mirtoselect generates measurable levels of anthocyanins in the murine biophase. ApcMin mice ingested C3G or Mirtoselect at 0.03, 0.1 or 0.3% in the diet for 12 weeks, and intestinal adenomas were counted. Plasma, urine and intestinal mucosa were analyzed for presence of anthocyanins by high‐pressure liquid chromatography with detection by UV spectrophotometry (520 nm) or tandem mass spectrometry (multiple reaction monitoring). Ingestion of either C3G or Mirtoselect reduced adenoma load dose‐dependently. At the highest doses of C3G and Mirtoselect adenoma numbers were decreased by 45% (p < 0.001) or 30% (p < 0.05), respectively, compared to controls. Anthocyanins were found at the analytical detection limit in the plasma and at quantifiable levels in the intestinal mucosa and urine. Anthocyanin glucuronide and methyl metabolites were identified in intestine and urine. Total anthocyanin levels in mice on C3G or Mirtoselect were 43 ng and 8.1 μg/g tissue, respectively, in the intestinal mucosa, and 7.2 and 12.3 μg/ml in the urine. The efficacy of C3G and Mirtoselect in the ApcMin mouse renders the further development of anthocyanins as potential human colorectal cancer chemopreventive agents worthwhile.

Effect of cyanidin-3-glucoside and an anthocyanin mixture from bilberry on adenoma development in the ApcMin mouse model of intestinal carcinogenesis--relationship with tissue anthocyanin levels.

Anthocyanins are dietary flavonoids, which can prevent carcinogen‐induced colorectal cancer in rats. Here, the hypotheses were tested that Mirtoselect, an anthocyanin mixture from bilberry, or isolated cyanidin‐3‐glucoside (C3G), the most abundant anthocyanin in diet, interfere with intestinal adenoma formation in the ApcMin mouse, a genetic model of human familial adenomatous polyposis, and that consumption of C3G or Mirtoselect generates measurable levels of anthocyanins in the murine biophase. ApcMin mice ingested C3G or Mirtoselect at 0.03, 0.1 or 0.3% in the diet for 12 weeks, and intestinal adenomas were counted. Plasma, urine and intestinal mucosa were analyzed for presence of anthocyanins by high‐pressure liquid chromatography with detection by UV spectrophotometry (520 nm) or tandem mass spectrometry (multiple reaction monitoring). Ingestion of either C3G or Mirtoselect reduced adenoma load dose‐dependently. At the highest doses of C3G and Mirtoselect adenoma numbers were decreased by 45% (p < 0.001) or 30% (p < 0.05), respectively, compared to controls. Anthocyanins were found at the analytical detection limit in the plasma and at quantifiable levels in the intestinal mucosa and urine. Anthocyanin glucuronide and methyl metabolites were identified in intestine and urine. Total anthocyanin levels in mice on C3G or Mirtoselect were 43 ng and 8.1 μg/g tissue, respectively, in the intestinal mucosa, and 7.2 and 12.3 μg/ml in the urine. The efficacy of C3G and Mirtoselect in the ApcMin mouse renders the further development of anthocyanins as potential human colorectal cancer chemopreventive agents worthwhile.

Ultra performance liquid chromatography tandem mass spectrometry method for the measurement of anandamide in human plasma.

Anandamide (N-arachidonoylethanolamine, AEA) is an endocannabinoid present in human plasma that is associated with several physiological functions and disease states. Significant variability in AEA plasma concentrations has been reported between studies, because quantification of AEA is fraught with methodological difficulties. A rapid, highly sensitive, robust, specific, and highly reproducible ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method is described here for the analysis of AEA in human plasma. This fully validated method using octa-deuterated AEA (AEA-d8) as an internal standard represents an improvement over previous analyses in terms of run time (4 min), limit of detection (0.055 fmol on column, 18.75 fmol/ml plasma), precision (relative standard deviations of 3.7, 3.9, and 4.8% for 1.66, 6.65, and 133 fmol on column), and accuracy (97.5-104.5%). AEA analysis was linear over the range 0.23 to 19 nM (1.66 to 133 fmol on column). To demonstrate the usefulness of this method for the measurement of AEA levels in clinical samples, plasma samples obtained from female volunteers at different stages of the menstrual cycle and pregnant women were assayed. Plasma AEA concentrations were significantly (P=0.0078) lower in the luteal phase of the menstrual cycle compared to the follicular phase. In pregnancy, the concentrations were lowest in the first and second trimesters with levels comparable to those observed in the luteal phase of the menstrual cycle and modestly increased in the third trimester. The highest plasma AEA levels were observed in women in active labour, and these were significantly (P=0.0147) higher than those observed in women at term but not in active labour. Postmenopausal women had AEA concentrations comparable to levels observed during the luteal phase of premenopausal women and were significantly (P=0.0389) lower than AEA plasma concentrations obtained during the follicular phase. The sensitivity and precision of the validated method described here suggests that this method is suitable for the analysis of AEA in clinical samples and may be utilised for the investigation of biomatrices containing limited amounts of AEA.

The role of sex steroid hormones, cytokines and the endocannabinoid system in female fertility

BACKGROUND Marijuana, the most used recreational drug, has been shown to have adverse effects on human reproduction. Endogenous cannabinoids (also called endocannabinoids) bind to the same receptors as those of Δ(9)-tetrahydrocannabinol (THC), the psychoactive component of Cannabis sativa. The most extensively studied endocannabinoids are anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol. The endocannabinoids, their congeners and the cannabinoid receptors, together with the metabolic enzymes and putative transporters form the endocannabinoid system (ECS). In this review, we summarize current knowledge about the relationships of ECS, sex steroid hormones and cytokines in female fertility, and underline the importance of this endocannabinoid-hormone-cytokine network. METHODS Pubmed and the Web of Science databases were searched for studies published since 1985, looking into the ECS, sex hormones, type-1/2 T-helper (Th1/Th2) cytokines, leukaemia inhibitory factor, leptin and reproduction. RESULTS The ECS plays a pivotal role in human reproduction. The enzymes involved in the synthesis and degradation of endocannabinoids normalize levels of AEA for successful implantation. The AEA degrading enzyme (fatty acid amide hydrolase) activity as well as AEA content in blood may potentially be used for the monitoring of early pregnancies. Progesterone and oestrogen are involved in the maintenance of endocannabinoid levels. The ECS plays an important role in the immune regulation of human fertility. CONCLUSIONS The available studies suggest that tight control of the endocannabinoid-hormone-cytokine network is required for successful implantation and early pregnancy maintenance. This hormone-cytokine network is a key element at the maternal-foetal interface, and any defect in such a network may result in foetal loss.

Rapid Analysis of Curcumin and Curcumin Metabolites in Rat Biomatrices Using a Novel Ultraperformance Liquid Chromatography (UPLC) Method

The bioavailability of the putative cancer chemopreventive agent curcumin is limited, making measurement either in target tissues or in biofluids difficult and variable between studies. The purposes of these investigations were to develop validated methods of extraction of curcumin from biomatrices and of detection of curcumin and its conjugated metabolites using ultraperformance liquid chromatography (UPLC) and to identify metabolites of curcumin using online tandem mass spectrometry (MS/MS). The limit of detection for curcumin after solid-phase extraction from plasma or urine was 2.5 ng/mL. Extraction efficiencies were 62 and 64% for urine and plasma. Intra- and interday variabilities (RSD) for extraction of curcumin from biofluids were less than 10 and 15%, respectively, and accuracies were 92 +/- 10% for plasma and 95 +/- 6% for urine. Curcumin was extracted from tissues using protein precipitation with quercetin as internal standard. Curcumin extraction from intestinal mucosa spiked with 0.2, 1, and 5 mug/g curcumin was validated. Extraction efficiency was 65-84%, accuracy was 94-106%, limit of detection was 12.5 ng/g, and intra- and interday variabilitiies (RSD) were 0.7-4.9 and 4.9-5.5%, respectively. The methods were applied to measure curcumin in tissues from rats that had received oral curcumin (340 mg/kg). Curcumin was found in plasma (16.1 ng/mL), urine (2.0 ng/mL), intestinal mucosa (1.4 mg/g), liver (3671.8 ng/g), and, for the first time, kidney (206.8 ng/g) and heart (807.6 ng/g). Curcumin metabolites identified by UPLC-MS/MS in plasma and urine were phenolic glucuronides and, probably, alcoholic glucuronides. Products of reduction of curcumin and their metabolites were found in the liver. The methods described here represent improvements on existing analytical methods for curcuminoids and metabolites in terms of sensitivity, speed, and separation.

A solid-phase method for the extraction and measurement of anandamide from multiple human biomatrices

N-Arachidonoylethanolamine (AEA, anandamide) was the first endocannabinoid to be identified and has since become associated with the mediation of several physiological functions and disease states. AEA has been isolated from numerous tissues and biofluids, in the low nanomolar range, using lipid extraction techniques with organic solvents. These techniques require the drying down of relatively large volumes of solvents, making them unsuitable for high-throughput analysis. Here we describe a solid-phase extraction (SPE) method for the investigation of AEA concentrations in human plasma, serum, milk, urine, amniotic fluid, peritoneal fluid, saliva, follicular fluid, and fluid from an ovarian cyst. AEA was detected in serum and plasma from blood isolated from 20 adult women (means+/-standard deviations: 0.68+/-0.29 and 0.64+/-0.28 nM, respectively), from pregnant women at term (1.37+/-0.42 nM), and from umbilical vein (1.26+/-0.33 nM) and umbilical artery (1.14+/-0.35nM), in milk (0.12+/-0.05 nM) and from amniotic (0.03+/-0.02 nM), peritoneal (0.93+/-0.27 nM), follicular (1.17+/-0.51 nM), and ovarian cyst (0.32+/-0.01 nM) fluids. AEA was undetectable in saliva and urine. The 60% AEA extraction efficiency achieved with SPE from plasma was superior to the 19% efficiency achieved using the existing organic solvent extraction method. Limits of quantification and detection for AEA were also improved dramatically using SPE (8 and 4 fmol/ml) compared with organic extraction (25 and 18.75 fmol/ml plasma). These improvements allow the use of smaller plasma samples with SPE. Intra- and interday variability were comparable, and the mean AEA concentration of pooled plasma samples (1.18 nM, n=15) was identical with the two techniques. Similarly, when 56 plasma samples from laboring and nonlaboring women were analyzed using both techniques, no extraction method-dependent differences were observed. Consequently, we provide evidence for a robust SPE technique for the extraction of AEA from biomatrices to replace the existing liquid extraction methods, with the SPE technique being superior in terms of speed, extraction efficiency, and sample size required.

N-Acylethanolamine Levels and Expression of Their Metabolizing Enzymes during Pregnancy

Decidualization is essential for a successful pregnancy and is a tightly regulated process influenced by the local microenvironment. Lipid-based mediators, such as the endocannabinoid anandamide, and other compounds that have cannabimimetic actions may act on the decidua during early pregnancy. In this study, the levels of N-arachidonoylethanolamine (anandamide) and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in rat plasma and maternal tissues between d 8 and 19 of pregnancy by ultraperformance liquid chromatography tandem mass spectrometry. The spatiotemporal expression of N-acylethanolamine metabolizing enzymes in implantation units were also determined by quantitative PCR, Western blot, and immunohistochemistry and shown to vary with gestation being mainly localized in decidual cells. The data also indicated that plasma and tissues levels of all three N-acylethanolamines fluctuate throughout pregnancy. Tissue levels of endocannabinoids did not correlate with plasma, suggesting that during pregnancy, maternal tissue levels of endocannabinoids are primarily regulated by in situ production and degradation to create endocannabinoid gradients conducive to successful pregnancy.