Lack of genotoxicity potential and safety assessment of 4-(2-hydroxyethyl) morpholine present as an impurity in pharmaceuticals and nutritional supplements.

4-(2-Hydroxyethyl) morpholine (HEM) is widely used as a building block of macromolecules in the manufacture of pharmaceuticals and dietary supplements and could remain as an impurity in the finished products. An evaluation of HEM was conducted to identify endpoints that could be used to determine the point-of-departure (POD) for use in assessing the potential risk from exposure to HEM. No oral repeated dose toxicological studies of appropriate duration were found for HEM. Therefore, suitable analogue(s) were identified. Although oral repeated dose studies were available for the analogues, the studies were not of sufficient duration for use in the assignment of a POD for risk evaluation. Accordingly, the Threshold of Toxicological Concern (TTC) approach, which proposes that a de minimis value can be derived to qualitatively assess risk, was considered for HEM. To determine the appropriate TTC approach (genotoxic or non-genotoxic), the genotoxicity of HEM and its analogues were evaluated. The weight of the evidence indicated that HEM, and the appropriate analogues, are not genotoxic. Considering the chemical structure of HEM, the non-genotoxic Cramer class III TTC value of 1.5 µg/kg bw/day was determined to be appropriate for use in safety assessment of HEM as an impurity in products intended for human consumption.

Assessment of consumer exposure to boron in cleaning products: a case study of Canada.

Boron is an essential mineral for plants, and as such, is a normal dietary constituent for humans. Humans may be naturally exposed to boron through food and drinking water, or via anthropogenic sources such as consumer products. The World Health Organisation established an acceptable safe range of population mean intakes for boron of 1-13 mg/day. Most studies of dietary boron intake show a range of 1-2 mg/day. Consumer products have been estimated to contribute a geometric mean daily intake of 0.1 mg to total boron exposure; however, there are few published surveys of consumer exposure to boron from use of cleaning products. The Government of Canada published a draft screening assessment report of boric acid, its salts and precursors that included estimates of consumer exposure to boron found as ingredients in consumer products. The manufacturers of consumer cleaning products conducted a survey of boron content of current products and estimated exposure using the publicly available exposure tool ConsExpo Web. Dermal exposures to boron during cleaning product use were estimated to result in annual internal exposures ranging from ≪0.001 to 0.36 µg/kg bw/day based on dermal absorption of 0.5%. Using a conservative point of departure for hazard assessment (2,900 µg boron/kg bw/day), estimated margins of exposure for dermal exposures to boron from cleaning product use range from 8,056 to >1,000,000. This work demonstrates that exposure to boron from cleaning product use is very low and essentially insignificant when compared to other (e.g. dietary) sources of boron intake by Canadian consumers.

Tracking of In-111-labeled human umbilical tissue-derived cells (hUTC) in a rat model of cerebral ischemia using SPECT imaging.

BACKGROUND: In order to increase understanding of how infused cells work, it becomes important to track their initial movement, localization, and engraftment efficiency following transplantation. However, the available in vivo cell tracking techniques are suboptimal. The study objective was to determine the biodistribution of intravenously administered Indium-111 (In-111) oxine labeled human umbilical tissue-derived cells (hUTC) in a rat model of transient middle cerebral occlusion (tMCAo) using single photon emission computed tomography (SPECT). METHODS: Rats received 3 million In-111 labeled hUTC (i.v.) 48 hrs after tMCAo. Following the administration of either hUTC or equivalent dose of In-111-oxine (18.5 MBq), animals underwent SPECT imaging on days 0, 1, and 3. Radioactivity in various organs as well as in the stroke area and contralateral hemisphere was determined, decay corrected and normalized to the total (whole body including head) radioactivity on day 0. Immunohistochemical analysis was also performed to confirm the beneficial effects of hUTC on vascular and synaptic density, and apoptosis. RESULTS: Most of the radioactivity (43.36±23.07% on day 0) trafficked to the lungs immediately following IV administration of In-111 labeled hUTC (day 0) and decreased drastically to 8.81±7.75 and 4.01±4.52% on days 1 and 3 post-injection, respectively. In contrast, radioactivity measured in the lung of animals that received In-111-oxine alone remained relatively unchanged from day 0 to day 1 (18.38±5.45% at day 0 to 12.59±5.94%) and decreased to 8.34±4.25% on day 3. Significantly higher radioactivity was observed in stroke areas of animals that received In-111 labeled hUTC indicating the presence of cells at the site of injury representing approximately 1% of total administered dose. In addition, there was significant increase in vascular and synaptophysin immunoreactivity in stroke areas of rats that received In-111 labeled hUTC. CONCLUSIONS: The present studies showed the tracking of In-111 labeled hUTC to the sites of stroke in a rat model of tMCAo using SPECT. Animals treated with In-111 labeled hUTC showed histological improvements, with higher vascular and synaptic densities observed in the ischemic boundary zone (IBZ).

MRI detects brain reorganization after human umbilical tissue-derived cells (hUTC) treatment of stroke in rat.

Human umbilical tissue-derived cells (hUTC) represent an attractive cell source and a potential technology for neurorestoration and improvement of functional outcomes following stroke. Male Wistar rats were subjected to a transient middle cerebral artery occlusion (tMCAo) and were intravenously administered hUTC (N = 11) or vehicle (N = 10) 48 hrs after stroke. White matter and vascular reorganization was monitored over a 12-week period using MRI and histopathology. MRI results were correlated with neurological functional and histology outcomes to demonstrate that MRI can be a useful tool to measure structural recovery after stroke. MRI revealed a significant reduction in the ventricular volume expansion and improvement in cerebral blood flow (CBF) in the hUTC treated group compared to vehicle treated group. Treatment with hUTC resulted in histological and functional improvements as evidenced by enhanced expression of vWF and synaptophysin, and improved outcomes on behavioral tests. Significant correlations were detected between MRI ventricular volumes and histological lesion volume as well as number of apoptotic cells. A positive correlation was also observed between MRI CBF or cerebral blood volume (CBV) and histological synaptic density. Neurological functional tests were also significantly correlated with MRI ventricular volume and CBV. Our data demonstrated that MRI measurements can detect the effect of hUTC therapy on the brain reorganization and exhibited positive correlation with histological measurements of brain structural changes and functional behavioral tests after stroke. MRI ventricular volumes provided the most sensitive index in monitoring brain remodeling and treatment effects and highly correlated with histological and functional measurements.

The alkaloid conessine and analogues as potent histamine H3 receptor antagonists.

The naturally occurring alkaloid, conessine (6), was discovered to bind to histamine H3 receptors in a radioligand-based high-throughput screen. Conessine displayed high affinity at both rat and human H3 receptors (pKi = 7.61 and 8.27) and generally high selectivity against other sites, including histamine receptors H1, H2, and H4. Conessine was found to efficiently penetrate the CNS and reach very high brain concentrations. Although the very slow CNS clearance and strong binding to adrenergic receptors discouraged focus on conessine itself for further development, its potency and novel steroid-based skeleton motivated further chemical investigation. Modification based on introducing diversity at the 3-nitrogen position generated a new series of H3 antagonists with higher in vitro potency, improved target selectivity, and more favorable drug-like properties. One optimized analogue (13c) was examined in detail and was found to be efficacious in animal behavioral model of cognition.

Synthesis, potency, and in vivo profiles of quinoline containing histamine H3 receptor inverse agonists.

A new structural series of histamine H3 receptor antagonist was developed. The new compounds are based on a quinoline core, appended with a required basic aminoethyl moiety, and with potency- and property-modulating heterocyclic substituents. The analogs have nanomolar and subnanomolar potency for the rat and human H3R in various in vitro assays, including radioligand competition binding as well as functional tests of H3 receptor-mediated calcium mobilization and GTPgammaS binding. The compounds possessed favorable drug-like properties, such as good PK, CNS penetration, and moderate protein binding across species. Several compounds were found to be efficacious in animal behavioral models of cognition and attention. Further studies on the pharmaceutic properties of this series of quinolines discovered a potential problem with photochemical instability, an issue which contributed to the discontinuation of this series from further development.

Adaptation of an in vitro phospholipidosis assay to an automated image analysis system.

ABSTRACT Phospholipidosis is the excessive intralysosomal accumulation of phospholipids and is induced in humans and animals by the chronic administration of cationic amphiphilic drugs. To identify compounds that may induce phospholipidosis early in the discovery process, we have developed a predictive fluorescent cell-based assay amenable to automated high content screening using the 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (ss-BODIPY C(12)-HPC) dye and primary rat hepatocytes. ss-BODIPY C(12)-HPC localized to lysosomes that accumulate phospholipids and not to lipid droplets, indicating the selectivity for phospholipid-containing granules. Accumulation of ss-BODIPY C(12)-HPC was monitored in primary rat hepatocytes plated onto 96-well plates and 24 h after exposure to increasing concentrations of 13 drugs known to induce phospholipidosis and four negative compounds. Fluorescent images were captured and analyzed using the Discovery-1 automated cellular imaging system. Eleven out of the 12 selected positive compounds and all negative compounds were properly assigned as positive and negative inducers of phospholipidosis, respectively, indicating the high degree of sensitivity and specificity of this assay. The ability of ss-BODIPY C(12)-HPC to detect and quantify phospholipidosis is similar to that of the well-established probe, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidylethanolamine (NBD-PE).

Synthesis and SAR of 5-amino- and 5-(aminomethyl)benzofuran histamine H3 receptor antagonists with improved potency.

A new series of H3 receptor antagonists was discovered with nanomolar and subnanomolar affinities at human and rat H3 receptors. Starting from an earlier, more structurally limited series of benzofurans, the present series of compounds demonstrated increased structural variety and flexibility with greater in vitro potency. One compound in particular, [2-[2-(2-(R)-methylpyrrolidin-1-yl)ethyl]benzofuran-5-yl](5-nitropyridin-2-yl)amine (7h), gave the best binding potency (human K(i) of 0.05 nM, rat K(i) of 0.11 nM), which represented a 9-fold (in human) and an 11-fold (in rat) improvement over ABT-239 (compound 5), a compound previously reported to have excellent in vitro potency and in vivo efficacy. The synthesis, SAR of the H3 binding affinities, in vitro assay for phospholipidosis, and pharmacokinetic properties of the new compounds are described.

Cyclical mitochondrial deltapsiM fluctuations linked to electron transport, F0F1 ATP-synthase and mitochondrial Na+/Ca+2 exchange are reduced in Alzheimer's disease cybrids.

Reduced complex IV, increased oxidative stress and beta amyloid peptide secretion in Alzheimer's disease (AD) can be replicated in cybrid models. We characterized cyclical mitochondrial deltapsiM fluctuations ('flickering') in neuroblastoma cells and AD/CTL cybrids. Flickering was blocked by ATP-synthase inhibition, was not observed in rho0 cells and was not blocked by antioxidant treatment. Flickering was not affected by the Ca(+2) uniporter antagonist Ru360 but was eliminated by BAPTA or CGP37137 blockade of the mitochondrial Na(+)/Ca(+2) exchanger. AD cybrid mitochondria showed reduced flickering. Flickering seems to represent coupling of deltapsiM to F0F1 ATP-synthase; reduction of flickering in AD cybrids suggests dysfunction of this coupling.

Cerebrospinal fluid 3,4-dihydroxyphenylacetic acid level after tolcapone administration as an indicator of nigrostriatal degeneration.

The development of reliable biological markers of nigrostriatal degeneration has important implications from both experimental and clinical viewpoints, since such biomarkers could be used for diagnostic and monitoring purposes in models of parkinsonism as well as in Parkinson's disease patients. In this study, levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in the cerebrospinal fluid (CSF) of normal and parkinsonian squirrel monkeys in order to assess their reliability as indicators of nigrostriatal injury. In particular, we tested the hypothesis that these measurements may become more accurate by inhibiting catecholamine-O-methyltransferase (COMT) activity and therefore blocking the conversion of DOPAC to homovanillic acid. Oral administration of the COMT inhibitor tolcapone (2 doses of 15 mg/kg each with a 4-h interval) significantly reduced enzyme activity in the monkey brain. Tolcapone treatment enhanced CSF DOPAC concentrations in unlesioned animals (by approximately four times) as well as monkeys rendered parkinsonian after severe nigrostriatal dopaminergic injury caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Importantly, however, COMT inhibition greatly magnified the differences in CSF DOPAC levels between control and parkinsonian monkeys, since MPTP-induced DOPAC depletion was 35% in the absence vs >60% in the presence of tolcapone. Thus, tolcapone administration enhances the detection of DOPAC in the CSF and, by doing so, improves the reliability of CSF DOPAC as a marker of nigrostriatal degeneration.

Neurotoxic nitric oxide rapidly depolarizes and permeabilizes mitochondria by dynamically opening the mitochondrial transition pore.

Exposure of SH-SY5Y neuroblastoma or rat cortical neurons to diethylenetriamine-NO (DETA-NO) rapidly depolarized mitochondria. In SH-SY5Y DETA-NO activated caspase 3 and produced cell death. Mitochondrial depolarization in SH-SY5Y was visualized both with JC-1 accumulation and as dequenching of calcein fluorescence in mitochondria initially loaded with calcein-AM and tetramethylrhodamine methyl ester (TMRM). Calcein/TMRM-visualized mitochondrial depolarization was prevented by cyclosporin A (CsA) or approximately two-fold increased levels of BclXL protein. Dynamic imaging of mitochondrial potential (Deltapsi M) with TMRM showed that DETA-NO induced cycles of mitochondrial depolarization/repolarization ("flickering"). Fifteen-30 min of DETA-NO exposure caused high-frequency flickering with small peak size; 2 h of DETA-NO produced large peaks with prolonged depolarization. NO-induced flickering but not that from Bax was blocked by the calcium uniporter antagonist Ru360. Our findings show rapid-onset, dynamic regulation of Deltapsi M by NO, implying that neuroprotective therapies for brain ischemia target cell death processes downstream of effects of NO on mitochondria.

Environmental risk factors and Parkinson's disease: selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat.

Environmental toxicants and, in particular, pesticides have been implicated as risk factors in Parkinson's disease (PD). The purpose of this study was to determine if selective nigrostriatal degeneration could be reproduced by systemic exposure of mice to the widely used herbicide paraquat. Repeated intraperitoneal paraquat injections killed dopaminergic neurons in the substantia nigra (SN) pars compacta, as assessed by stereological counting of tyrosine hydroxylase (TH)-immunoreactive and Nissl-stained neurons. This cell loss was dose- and age-dependent. Several lines of evidence indicated selective vulnerability of dopaminergic neurons to paraquat. The number of GABAergic cells was not decreased in the SN pars reticulata, and counting of Nissl-stained neurons in the hippocampus did not reveal any change in paraquat-treated mice. Degenerating cell bodies were observed by silver staining, but only in the SN pars compacta, and glial response was present in the ventral mesencephalon but not in the frontal cortex and cerebellum. No significant depletion of striatal dopamine followed paraquat administration. On the other hand, enhanced dopamine synthesis was suggested by an increase in TH activity. These findings unequivocally show that selective dopaminergic degeneration, one of the pathological hallmarks of PD, is also a characteristic of paraquat neurotoxicity. The apparent discrepancy between pathological (i.e., neurodegeneration) and neurochemical (i.e., lack of significant dopamine loss) effects represents another important feature of this paraquat model and is probably a reflection of compensatory mechanisms by which neurons that survive damage are capable of restoring neurotransmitter tissue levels.