Miguel Muzzio

Subchronic oral toxicity and cardiovascular safety pharmacology studies of resveratrol, a naturally occurring polyphenol with cancer preventive activity.

To characterize the subchronic oral toxicity of resveratrol, CD rats received daily gavage doses of 0, 200, 400, or 1000 mg resveratrol/kg/day, and beagle dogs received daily capsule doses of 0, 200, 600, or 1200 mg resveratrol/kg/day for 90 days. Resveratrol induced only minimal toxicity, consisting of dose-related reductions in body weight gain in female rats and both sexes of dogs, and a statistically significant increase in bilirubin levels in rats at the 1000 mg/kg/day dose. Clinical observations, hematology, ophthalmology, neurotoxicity evaluations (functional observational batteries), organ weights, and gross pathology provided no biologically significant evidence of resveratrol toxicity in either species. In rats, the high dose of resveratrol reduced the incidence of cardiomyopathy; no other microscopic changes were seen. Histopathologic changes in dogs were limited to minimal inflammatory infiltrates in the kidney and urinary bladder, which were not considered toxicologically significant. A cardiovascular safety pharmacology (telemetry) study in dogs revealed no evidence of resveratrol toxicity. Based on body weight effects, the No Observed Adverse Effect Level (NOAEL) for resveratrol was 200mg/kg/day in rats and 600 mg/kg/day in dogs. The apparent cardioprotective activity of resveratrol in rats demonstrates that its potentially beneficial activities may extend beyond efficacy in cancer prevention.

A Randomized Phase II Presurgical Trial of Transdermal 4-Hydroxytamoxifen Gel versus Oral Tamoxifen in Women with Ductal Carcinoma In Situ of the Breast

Purpose: Local transdermal therapy to the breast may achieve effective target-organ drug delivery, while diminishing systemic effects. We conducted a randomized, double-blind, placebo-controlled phase II trial comparing transdermal 4-hydroxytamoxifen gel (4-OHT) to oral tamoxifen (oral-T) in women with ductal carcinoma in situ (DCIS). Methods: Twenty-seven pre- and postmenopausal women were randomized to 4-OHT (4 mg/day) or oral-T (20 mg/day) for 6 to 10 weeks before surgery. Plasma, nipple aspirate fluid, and breast adipose tissue concentrations of tamoxifen and its major metabolites were determined by liquid chromatography/tandem mass spectrometry. The primary endpoint was Ki67 labeling in DCIS lesions, measured by immunohistochemistry. In plasma, insulin-like growth factor-1 (IGFI), sex hormone–binding globulin (SHBG), and coagulation protein concentrations were determined. Results: Posttherapy Ki67 decreased by 3.4% in the 4-OHT and 5.1% in the oral-T group (P ≤ 0.03 in both, between-group P = 0. 99). Mean plasma 4-OHT was 0.2 and 1.1 ng/mL in 4-OHT and oral groups, respectively (P = 0.0003), whereas mean breast adipose tissue concentrations of 4-OHT were 5.8 ng/g in the 4-OHT group and 5.4 ng/g in the oral group (P = 0.88). There were significant increases in plasma SHBG, factor VIII, and von Willebrand factor and a significant decrease in plasma IGFI with oral-T, but not with 4-OHT. The incidence of hot flashes was similar in both groups. Conclusions: The antiproliferative effect of 4-OHT gel applied to breast skin was similar to that of oral-T, but effects on endocrine and coagulation parameters were reduced. These findings support the further evaluation of local transdermal therapy for DCIS and breast cancer prevention. Clin Cancer Res; 20(14); 3672–82. ©2014 AACR.

Deguelin action involves c-Met and EGFR signaling pathways in triple negative breast cancer cells.

Background Treatment of breast cancer patients with antiestrogens and aromatase inhibitor(s) or Herceptin have shown significant success in steroid receptor positive or Her-2+ breast cancers respectively. However, choice of treatments for breast cancer patients with negative status for estrogen, progesterone receptors and HER2/neu is limited. As a result, search for appropriate therapy regimen for these triple negative breast cancers (TNBC) has become a major focus of investigations for many laboratories. Recently, Deguelin, a natural product isolated from African plant Mundulea sericea (Leguminossae) has shown both antiproliferative actions in various cancers including breast as well as chemoprenventive activity against carcinogen induced experimental cancers. In this report we evaluated efficacy and mechanism of action of Deguelin in triple negative breast cancer cell lines. Methods/Findings In vitro, Deguelin in a dose and time dependent manner inhibited the growth of MDA-MB-231, MDA-MB-468, BT-549 and BT-20 cells. Deguelin (2 or 4 mg/kg body weight), when injected intraperitoneally, reduced the in vivo tumor growth of MDA-MB-231 cells transplanted subcutaneously in athymic mice. Moreover it was nontoxic as evident from daily observations on mobility, food and water consumption and comparison of bodyweight and other visceral organ weights with those in control animals at the termination of the study. The western blot analyses and immunostaining studies indicated that the deguelin effects may be mediated through EGFR-PAKT/c-Met p-ERK and NF-κB by down regulating their downstream targets such as p-STAT3, c-Myc, Survivin. Conclusion/Significance These results suggest that Deguelin may have a significant therapeutic value for the treatment of TNBC patients.

Effect of filtration by activated charcoal on the toxicological activity of cigarette mainstream smoke from experimental cigarettes

Activated charcoal (AC) filtration reportedly decreases the yields of smoke vapor phase constituents including some identified as human carcinogens and respiratory irritants. Non-clinical studies including chemical smoke analysis, in vitro cytotoxicity and mutagenicity (bacterial and mammalian cells), and in vivo subchronic rat inhalation studies were carried out using machine smoking at ISO conditions with lit-end research cigarettes containing AC filters. The objective was to assess whether AC filter technology would alter the established toxicity profile of mainstream smoke by increasing or decreasing any known toxicological properties, or elicit new ones. The reduced yield of vapor phase irritants from AC filter cigarettes correlated with markedly decreased in vitro cytotoxicity and in vivo morphology of the nose and lower respiratory tract. Increased yields of particulate phase constituents (e.g. polycyclic aromatic hydrocarbons) in AC filtered smoke were noted in comparison to controls in some studies. The in vitro bacterial mutagenicity of AC filtered smoke particulate preparations was occasionally increased over control levels. Laryngeal epithelial thickness was increased in some rats inhaling AC filtered smoke in comparison to controls, an effect perhaps related to higher inspiratory flow. When tested under more intense Massachusetts Department of Public Health smoking conditions, AC filter associated reductions in vapor phase constituent yields were smaller than those seen with ISO conditions, but the effect on in vitro cytotoxicity remained.

Direct quantitation of glucoraphanin in dog and rat plasma by LC-MS/MS

A rapid method to quantify levels of the beta-thioglycoside N-hydroxyl sulfate, glucoraphanin, in dog and rat plasma to support pre-clinical toxicological and pharmacological studies has been developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Glucoraphanin was extracted from plasma by protein precipitation with acetonitrile and separated via hydrophilic interaction liquid chromatography (HILIC) using a Luna 5microm Silica (2) 100A column (50mmx2.0mm) at a flow rate of 0.3mL/min. Solvent A consisted of 200mM ammonium acetate and formic acid (99:1, v/v) and Solvent B was acetonitrile. Initial conditions (90% Solvent B) were held for 0.01min after injection, decreased to 40% in 0.5min and held constant for 2.5min, returning to initial conditions for 3min (reequilibration time). Glucoraphanin was detected by MS/MS using a turbo ion spray interface as the ion source operating in negative ion mode. Acquisition was performed in multiple reaction monitoring mode at m/z 435.8-->96.7. The method was validated for the calibration range 10-2000ng/mL. Within- and between-run precision for the low, mid and high QC levels was 8% R.S.D. or less and accuracy ranged from 100 to 113%. The lower limit of quantification was 10ng/mL; calibration curves encompassed the range of plasma concentrations expected to be found in bioavailability and pharmacokinetics studies with glucoraphanin. The method has successfully been applied to the determination of glucoraphanin in dog and rat plasma and should be extendable to other species as well.

Toxicologic evaluation of tungsten: 28-day inhalation study of tungsten blue oxide in rats

The toxicity and toxicokinetics of tungsten blue oxide (TBO) were examined. TBO is an intermediate in the production of tungsten powder, and has shown the potential to cause cellular damage in in vitro studies. However, in vivo evidence seems to indicate a lack of adverse effects. The present study was undertaken to address the dearth of longer-term inhalation toxicity studies of tungsten oxides by investigating the biological responses induced by TBO when administered via nose-only inhalation to rats at levels of 0.08, 0.325, and 0.65 mg TBO/L of air for 6 h/day for 28 consecutive days, followed by a 14-day recovery period. Inhaled TBO was absorbed systemically and blood levels of tungsten increased as inhaled concentration increased. Among the tissues analyzed for tungsten levels, lung, femur and kidney showed increased levels, with lung at least an order of magnitude greater than kidney or femur. By exposure day 14, tungsten concentration in tissues had reached steady-state. Increased lung weight was noted for both terminal and recovery animals and was attributed to deposition of TBO in the lungs, inducing a macrophage influx. Microscopic evaluation of tissues revealed a dose-related increase in alveolar pigmented macrophages, alveolar foreign material and individual alveolar foamy macrophages in lung. After a recovery period there was a slight reduction in the incidence and severity of histopathological findings. Based on the absence of other adverse effects, the increased lung weights and the microscopic findings were interpreted as nonadverse response to exposure and were not considered a specific reaction to TBO.

Oral Toxicity and Bacterial Mutagenicity Studies with a Spunbond Polyethylene and Polyethylene Terephthalate Polymer Fabric

Spunbond, nonwoven fabrics consisting of polyethylene and polyethylene terephthalate, which meet food contact requirements, may be used as pouch materials for products containing food and/or flavor ingredients that are held in the mouth. In these situations ingestion may occur, resulting in exposure to the fabric and potentially antimony, a catalyst used in polyethylene terephthalate. To assess potential adverse effects when such a material is ingested, a 13week dietary study in Sprague-Dawley CD rats and a Salmonella reverse mutation assay were conducted. Ground fabric was dosed at target concentrations of 0.5%, 2.5%, and 5% in the dietary study. Antimony trioxide in the polyethylene terephthalate was used to determine the test material concentration in the diet and was also assessed for bioavailability. Detectable levels of antimony were found in 2/20 blood samples of control rats, and in 20/20 high-dose group rats. No toxicologically relevant treatment related effects were observed in any of end points evaluated in the feeding study. In the mutagenicity assay, ground fabric was extracted in phosphate buffered saline or dimethysulfoxide and tested in Salmonella strains TA98, TA100, TA102, TA1535, and TA1537 with and without S9 activation. No mutagenic response was observed at any dose level tested. These results demonstrate that repeated daily ingestion of a spunbond, nonwoven polymer fabric consisting of polyethylene and polyethylene terephthalate for up to 13 weeks is well tolerated in rats, with no apparent target-organ toxicity at dietary levels up to 5%, and that fabric extracts are not mutagenic in a bacterial reverse mutation assay.

Methodological considerations in estrogen assays of breast fluid and breast tissue

Estradiol (E2) in nipple aspirate fluid (NAF), ductal lavage fluid (DLF), and random fine needle aspirates (rFNA) are compared. Quantification was by immunoassay or tandem MS. The percent of women yielding NAF varied between 24% and 48% and for DLF was 86.3%. Variation between ducts within a breast was not less than variation between breasts within women but variation between breasts and within women over time was significantly less than variation between women. Serum E2 was highly significantly different among phases of the menstrual cycle but NAF E2 was not different. The correlation between serum and breast fluid E2 concentrations in premenopausal women had coefficients of determination of less than 15%. The correlation between serum and NAF in studies of postmenopausal women varied greatly and may depend on patient selection. The difference between NAF E2 between pre- and postmenopausal women was only 22%; for rFNA it was non-significantly 44% lower in a similar group of postmenopausal women. Progesterone was 96% and 98% lower in postmenopausal NAF and rFNA samples, respectively. Measurements of E2 in breast fluid or breast tissue appears to provide similar estimates of E2 exposure. E2 levels in breast fluid do not reflect the rapid changes that occur in serum and, thus, serum availability of E2 is only one factor determining its levels in the breast. The similarity of levels between breasts and between ducts suggests that estimates of estrogen exposure does not require multiple samples, however, unavailability of fluid may require rFNA in some cases.

NCI Comparative Oncology Program Testing of Non-Camptothecin Indenoisoquinoline Topoisomerase I Inhibitors in Naturally Occurring Canine Lymphoma

Purpose: Only one chemical class of topoisomerase I (TOP1) inhibitors is FDA approved, the camptothecins with irinotecan and topotecan widely used. Because of their limitations (chemical instability, drug efflux-mediated resistance, and diarrhea), novel TOP1 inhibitors are warranted. Indenoisoquinoline non-camptothecin topoisomerase I (TOP1) inhibitors overcome chemical instability and drug resistance that limit camptothecin use. Three indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan), and LMP744, were examined in a phase I study for lymphoma-bearing dogs to evaluate differential efficacy, pharmacodynamics, toxicology, and pharmacokinetics. Experimental Design: Eighty-four client-owned dogs with lymphomas were enrolled in dose-escalation cohorts for each indenoisoquinoline, with an expansion phase for LMP744. Efficacy, tolerability, pharmacokinetics, and target engagement were determined. Results: The MTDs were 17.5 mg/m2 for LMP 776 and 100 mg/m2 for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m2 LMP400 was well-tolerated and MTD was not reached. None of the drugs induced notable diarrhea. Sustained tumor accumulation was observed for LMP744; γH2AX induction was demonstrated in tumors 2 and 6 hours after treatment; a decrease in TOP1 protein was observed in most lymphoma samples across all compounds and dose levels, which is consistent with the fact that tumor response was also observed at low doses LMP744. Objective responses were documented for all indenoisoquinolines; efficacy (13/19 dogs) was greatest for LMP744. Conclusions: These results demonstrate proof-of-mechanism for indenoisoquinoline TOP1 inhibitors supporting their further clinical development. They also highlight the value of the NCI Comparative Oncology Program (https://ccr.cancer.gov/Comparative-Oncology-Program) for evaluating novel therapies in immunocompetent pets with cancers.

Breast Hormone Concentrations in Random Fine-Needle Aspirates of Healthy Women Associate with Cytological Atypia and Gene Methylation

Sex steroid hormones contribute to breast cancer development, but data on concentrations of these within breast tissue are limited. We performed simultaneous multiparameter measurement of breast sex steroids, breast epithelial cytology, and DNA methylation in 119 healthy women (54 pre- and 65 postmenopausal) without a history of breast cancer. Random fine-needle aspiration (rFNA) of the breast was performed simultaneously with blood collection. Breast samples were analyzed by LC/MS-MS for estrone, estradiol, progesterone, androstenedione, and testosterone. Blood samples were assayed for estradiol and progesterone by immunoassay. Cytomorphology was classified using the Masood Score, and DNA methylation of eight genes was analyzed using quantitative multiplexed methylation-specific PCR, and expressed as the cumulative methylation index (CMI). Serum and breast concentrations of estradiol and progesterone showed significant correlation (Spearman r = 0.34, Padj = 0.001 and r = 0.69, Padj < 0.0006, respectively). Progesterone concentration was significantly higher in the premenopausal breast (Padj < 0.0008), and showed a luteal surge. Breast estrone and estradiol concentrations did not differ significantly by menopause, but androstenedione concentration was higher in the breasts of postmenopausal women (P = 0.026 and Padj = 0.208). Breast androgens were significantly correlated with breast density (Spearman r = 0.27, Padj = 0.02 for testosterone) and CMI (Spearman r = 0.3, Padj = 0.038 for androstenedione). Our data indicate that future larger studies of breast steroid hormones along with other parameters are feasible. Significant associations of breast androgen concentrations with breast density and gene methylation warrant future study. Cancer Prev Res; 11(9); 557–68. ©2018 AACR.