Dean E. Brenner

Dose escalation of a curcuminoid formulation

BackgroundCurcumin is the major yellow pigment extracted from turmeric, a commonly-used spice in India and Southeast Asia that has broad anticarcinogenic and cancer chemopreventive potential. However, few systematic studies of curcumins pharmacology and toxicology in humans have been performed.MethodsA dose escalation study was conducted to determine the maximum tolerated dose and safety of a single dose of standardized powder extract, uniformly milled curcumin (C3Complex™, Sabinsa Corporation). Healthy volunteers were administered escalating doses from 500 to 12,000 mg.ResultsSeven of twenty-four subjects (30%) experienced only minimal toxicity that did not appear to be dose-related. No curcumin was detected in the serum of subjects administered 500, 1,000, 2,000, 4,000, 6,000 or 8,000 mg. Low levels of curcumin were detected in two subjects administered 10,000 or 12,000 mg.ConclusionThe tolerance of curcumin in high single oral doses appears to be excellent. Given that achieving systemic bioavailability of curcumin or its metabolites may not be essential for colorectal cancer chemoprevention, these findings warrant further investigation for its utility as a long-term chemopreventive agent.

Phase I Dose Escalation Pharmacokinetic Study in Healthy Volunteers of Resveratrol, a Potential Cancer Chemopreventive Agent

The red grape constituent resveratrol possesses cancer chemopreventive properties in rodents. The hypothesis was tested that, in healthy humans, p.o. administration of resveratrol is safe and results in measurable plasma levels of resveratrol. A phase I study of oral resveratrol (single doses of 0.5, 1, 2.5, or 5 g) was conducted in 10 healthy volunteers per dose level. Resveratrol and its metabolites were identified in plasma and urine by high-performance liquid chromatography-tandem mass spectrometry and quantitated by high-performance liquid chromatography-UV. Consumption of resveratrol did not cause serious adverse events. Resveratrol and six metabolites were recovered from plasma and urine. Peak plasma levels of resveratrol at the highest dose were 539 ± 384 ng/mL (2.4 μmol/L, mean ± SD; n = 10), which occurred 1.5 h post-dose. Peak levels of two monoglucuronides and resveratrol-3-sulfate were 3- to 8-fold higher. The area under the plasma concentration curve (AUC) values for resveratrol-3-sulfate and resveratrol monoglucuronides were up to 23 times greater than those of resveratrol. Urinary excretion of resveratrol and its metabolites was rapid, with 77% of all urinary agent-derived species excreted within 4 h after the lowest dose. Cancer chemopreventive effects of resveratrol in cells in vitro require levels of at least 5 μmol/L. The results presented here intimate that consumption of high-dose resveratrol might be insufficient to elicit systemic levels commensurate with cancer chemopreventive efficacy. However, the high systemic levels of resveratrol conjugate metabolites suggest that their cancer chemopreventive properties warrant investigation. (Cancer Epidemiol Biomarkers Prev 2007;16(6):1246–52)

Adherence to oral tamoxifen: a comparison of patient self-report, pill counts, and microelectronic monitoring.

PURPOSE Recent innovations allow the integration of microelectronics into drug packaging, providing a continuous record of the interactions of the patient with the drug package. We hypothesized that adherence to oral tamoxifen, as measured by a pressure-activated microelectronic monitoring device, would be significantly discrepant from traditional measures of patient adherence, ie, patient self-report (SR) and pill counts (PCs). PATIENTS AND METHODS Twenty-six patients receiving oral tamoxifen therapy were assessed by patient SR, PCs, and Medication Event Monitoring System (MEMS; Aprex Corp, Fremont, CA) microelectronic monitoring. A microprocessor in the MEMS cap recorded each opening as a presumptive dose, listing the date, time, and duration of opening for later retrieval on a microcomputer. Patients were not informed that their adherence was to be monitored electronically or that PCs would be performed. RESULTS A total of 2,102 days (70.1 months) of tamoxifen therapy were monitored; patients were monitored for a mean of 2.92 months of tamoxifen therapy. SR adherence to oral tamoxifen was significantly higher than that suggested by either PCs (SR missed doses only v PC, P = .008) or MEMS adherence monitoring (SR missed doses only v MEMS missed doses only, P = .005; SR dosing-interval errors only v MEMS dosing-interval errors only, P < .0001; SR all dosing errors v MEMS all dosing errors, P < .0005). PC data also suggested significantly higher adherence rates than MEMS monitoring. CONCLUSION Microelectronic adherence monitoring provides both confirmatory and complimentary data regarding adherence behavior, while also allowing for the evaluation of patterns of nonadherence. Patient SRs and PCs likely overestimate the degree to which patients adhere to their tamoxifen regimen.

Phase IIA Clinical Trial of Curcumin for the Prevention of Colorectal Neoplasia

Curcumin is derived from the spice tumeric and has antiinflammatory and antineoplastic effects in vitro and in animal models, including preventing aberrant crypt foci (ACF) and adenomas in murine models of colorectal carcinogenesis. Inhibiting the production of the procarcinogenic eicosanoids prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5-HETE) can suppress carcinogenesis in rodents. Curcumin reduces mucosal concentrations of PGE2 (via inhibition of cyclooxygenases 1 and 2) and 5-HETE (via inhibition of 5-lipoxygenase) in rats. Although preclinical data support curcumin activity in many sites, the poor bioavailability reported for this agent supports its use in the colorectum. We assessed the effects of oral curcumin (2 g or 4 g per day for 30 days) on PGE2 within ACF (primary endpoint), 5-HETE, ACF number, and proliferation in a nonrandomized, open-label clinical trial in 44 eligible smokers with eight or more ACF on screening colonoscopy. We assessed pre- and posttreatment concentrations of PGE2 and 5-HETE by liquid chromatography tandem mass spectroscopy in ACF and normal-tissue biopsies; ACF number via rectal endoscopy; proliferation by Ki-67 immunohistochemistry; and curcumin concentrations by high-performance liquid chromatography in serum and rectal mucosal samples. Forty-one subjects completed the study. Neither dose of curcumin reduced PGE2 or 5-HETE within ACF or normal mucosa or reduced Ki-67 in normal mucosa. A significant 40% reduction in ACF number occurred with the 4-g dose (P < 0.005), whereas ACF were not reduced in the 2-g group. The ACF reduction in the 4-g group was associated with a significant, five-fold increase in posttreatment plasma curcumin/conjugate levels (versus pretreatment; P = 0.009). Curcumin was well tolerated at both 2 g and 4 g. Our data suggest that curcumin can decrease ACF number, and this is potentially mediated by curcumin conjugates delivered systemically. Cancer Prev Res; 4(3); 354–64. ©2011 AACR.

Pharmacokinetics of Curcumin Conjugate Metabolites in Healthy Human Subjects

Background: Curcumin is a polyphenol, found in the spice turmeric, that has promising anticancer properties, but previous studies suggest that absorption of curcumin may be limited. Methods: This study examined the pharmacokinetics of a curcumin preparation in healthy human volunteers 0.25 to 72 h after a single oral dose. Curcumin was administered at doses of 10 g (n = 6) and 12 g (n = 6). Subjects were randomly allocated to dose level for a total of six subjects at each dose level. Serum samples were assayed for free curcumin, for its glucuronide, and for its sulfate conjugate. The data were fit to a one-compartment absorption and elimination model. Results: Using a high-performance liquid chromatography assay with a limit of detection of 50 ng/mL, only one subject had detectable free curcumin at any of the 14 time points assayed, but curcumin glucuronides and sulfates were detected in all subjects. Based on the pharmacokinetic model, the area under the curve for the 10 and 12 g doses was estimated (mean ± SE) to be 35.33 ± 3.78 and 26.57 ± 2.97 μg/mL × h, respectively, whereas Cmax was 2.30 ± 0.26 and 1.73 ± 0.19 μg/mL. The Tmax and t1/2 were estimated to be 3.29 ± 0.43 and 6.77 ± 0.83 h. The ratio of glucuronide to sulfate was 1.92:1. The curcumin conjugates were present as either glucuronide or sulfate, not mixed conjugates. Conclusion: Curcumin is absorbed after oral dosing in humans and can be detected as glucuronide and sulfate conjugates in plasma. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1411–7)

A Mouse to Human Search for Plasma Proteome Changes Associated with Pancreatic Tumor Development

Background The complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer. Methods and Findings Plasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer. Conclusions Our findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection.

Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays.

Carbohydrate post-translational modifications on proteins are important determinants of protein function in both normal and disease biology. We have developed a method to allow the efficient, multiplexed study of glycans on individual proteins from complex mixtures, using antibody microarray capture of multiple proteins followed by detection with lectins or glycan-binding antibodies. Chemical derivatization of the glycans on the spotted antibodies prevented lectin binding to those glycans. Multiple lectins could be used as detection probes, each targeting different glycan groups, to build up lectin binding profiles of captured proteins. By profiling both protein and glycan variation in multiple samples using parallel sandwich and glycan-detection assays, we found cancer-associated glycan alteration on the proteins MUC1 and CEA in the serum of pancreatic cancer patients. Antibody arrays for glycan detection are highly effective for profiling variation in specific glycans on multiple proteins and should be useful in diverse areas of glycobiology research.

Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

BackgroundCancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response.MethodsEighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance.ResultsSeven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified.ConclusionOur results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.

Occurrence of Autoantibodies to Annexin I, 14-3-3 Theta and LAMR1 in Prediagnostic Lung Cancer Sera

PURPOSE We have implemented a high throughput platform for quantitative analysis of serum autoantibodies, which we have applied to lung cancer for discovery of novel antigens and for validation in prediagnostic sera of autoantibodies to antigens previously defined based on analysis of sera collected at the time of diagnosis. MATERIALS AND METHODS Proteins from human lung adenocarcinoma cell line A549 lysates were subjected to extensive fractionation. The resulting 1,824 fractions were spotted in duplicate on nitrocellulose-coated slides. The microarrays produced were used in a blinded validation study to determine whether annexin I, PGP9.5, and 14-3-3 theta antigens previously found to be targets of autoantibodies in newly diagnosed patients with lung cancer are associated with autoantibodies in sera collected at the presymptomatic stage and to determine whether additional antigens may be identified in prediagnostic sera. Individual sera collected from 85 patients within 1 year before a diagnosis of lung cancer and 85 matched controls from the Carotene and Retinol Efficacy Trial (CARET) cohort were hybridized to individual microarrays. RESULTS We present evidence for the occurrence in lung cancer sera of autoantibodies to annexin I, 14-3-3 theta, and a novel lung cancer antigen, LAMR1, which precede onset of symptoms and diagnosis. CONCLUSION Our findings suggest potential utility of an approach to diagnosis of lung cancer before onset of symptoms that includes screening for autoantibodies to defined antigens.

Human Urine Contains Small, 150 to 250 Nucleotide-Sized, Soluble DNA Derived from the Circulation and May Be Useful in the Detection of Colorectal Cancer

Human urine has been shown to possess submicrogram per milliliter amounts of DNA. We show here that DNA isolated from human urine resolves into two size categories: the large species, greater than 1 kb, being predominantly cell associated and heterogeneous in size, and the smaller, between 150 to 250 bp, being mostly non-cell associated. We showed that the low molecular weight class of urine DNA is derived from the circulation, by comparing the mutated K-ras sequences present in DNA isolated from tumor, blood, and urine derived from an individual with a colorectal carcinoma (CRC) containing a mutation in codon 12 of the K-ras proto-oncogene. In the urine, mutated K-ras sequences were abundant in the low molecular weight species, but far less abundant in the large molecular weight-derived DNA. Finally, the possibility that detection of mutant K-ras sequences in DNA derived from the urine correlates with the occurrence of a diagnosis of CRC and polyps that contain mutant K-ras was explored in a blinded study. There was an 83% concurrence of mutated DNA detected in urine and its corresponding disease tissue from the same individuals, when paired urine and tissue sections from 20 subjects with either CRC or adenomatous polyps were analyzed for K-ras mutation. The possibility that the source of the trans renal DNA is apoptotic cells, and the potential use of this finding for cancer detection and monitoring is discussed.