A sensitive LC-MS/MS method for the study of exogenously administered 13 C-oleoylethanolamide in rat plasma and brain tissue.

Oleoylethanolamide is an endogenous molecule with neuroprotective effects. It has been reported that exogenous oleoylethanolamide can be administered therapeutically, but the confounding presence of the endogenous molecule has led to conflicting reports regarding the mechanisms of the effects and highlights a need for an adequate methodology to differentiate them. We have developed a liquid chromatography-tandem mass spectrometry method to study oleoylethanolamide in rat plasma and brain using a 13 C-labeled isotope, 13 C-oleoylethanolamide. 13 C-oleoylethanolamide was extracted using a liquid-liquid extraction employing acetonitrile and tert-butyl methyl ether (1:4). Analysis was performed using a gradient with a total run time of 12 min. 13 C-oleoylethanolamide, d4 -oleoylethanolamide (internal standard), and 12 C-oleoylethanolamide (endogenous background) eluted simultaneously at 1.64 min. The method was validated for specificity, sensitivity, accuracy, and precision and found to be capable of quantification within acceptable limits of ±15% over the calibration range of 0.39-25 ng/mL for the plasma and 1.17-75 ng/g for the brain. It was then applied to quantify 13 C-oleoylethanolamide over 90 min after intravenous administration of a solution (1 mg/kg) in rats. Results suggest that 13 C-oleoylethanolamide does not reach therapeutic concentrations in the brain, despite a relatively prolonged plasma circulation, suggesting that rapid degradation in the brain remains an obstacle to its clinical application to neurological disease.

Preclinical Development of a Nontoxic Oral Formulation of Monoethanolamine, a Lipid Precursor, for Prostate Cancer Treatment.

Purpose: Most currently available chemotherapeutic agents target rampant cell division in cancer cells, thereby affecting rapidly dividing normal cells resulting in toxic side-effects. This nonspecificity necessitates identification of novel cellular pathways that are reprogrammed selectively in cancer cells and can be exploited to develop pharmacologically superior and less toxic therapeutics. Despite growing awareness on dysregulation of lipid metabolism in cancer cells, targeting lipid biosynthesis is still largely uncharted territory. Herein, we report development of a novel nontoxic orally deliverable anticancer formulation of monoethanolamine (Etn) for prostate cancer by targeting the Kennedy pathway of phosphatidylethanolamine (PE) lipid biosynthesis.Experimental Design: We first evaluated gastrointestinal tract stability, drug-drug interaction liability, pharmacokinetic, and toxicokinetic properties of Etn to evaluate its suitability as a nontoxic orally deliverable agent. We next performed in vitro and in vivo experiments to investigate efficacy and mechanism of action.Results: Our data demonstrate that Etn exhibits excellent bioavailability, gastrointestinal tract stability, and no drug-drug interaction liability. Remarkably, orally fed Etn inhibited tumor growth in four weeks by approximately 67% in mice bearing human prostate cancer PC-3 xenografts without any apparent toxicity. Mechanistically, Etn exploits selective overexpression of choline kinase in cancer cells, resulting in accumulation of phosphoethanolamine (PhosE), accompanied by downregulation of HIF-1α that induces metabolic stress culminating into cell death.Conclusions: Our study provides first evidence for the superior anticancer activity of Etn, a simple lipid precursor formulation, whose nontoxicity conforms to FDA-approved standards, compelling its clinical development for prostate cancer management. Clin Cancer Res; 23(14); 3781-93. ©2017 AACR.

Discovery and Structure-Activity Relationship (SAR) of a Series of Ethanolamine-Based Direct-Acting Agonists of Sphingosine-1-phosphate (S1P1).

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates a multitude of physiological processes such as lymphocyte trafficking, cardiac function, vascular development, and inflammation. Because of the ability of S1P1 receptor agonists to suppress lymphocyte egress, they have great potential as therapeutic agents in a variety of autoimmune diseases. In this article, the discovery of selective, direct acting S1P1 agonists utilizing an ethanolamine scaffold containing a terminal carboxylic acid is described. Potent S1P1 agonists such as compounds 18a and 19a which have greater than 1000-fold selectivity over S1P3 are described. These compounds efficiently reduce blood lymphocyte counts in rats through 24 h after single doses of 1 and 0.3 mpk, respectively. Pharmacodynamic properties of both compounds are discussed. Compound 19a was further studied in two preclinical models of disease, exhibiting good efficacy in both the rat adjuvant arthritis model (AA) and the mouse experimental autoimmune encephalomyelitis model (EAE).

Skin absorption of six performance amines used in metalworking fluids.

Every year, 10 million workers are exposed to metalworking fluids (MWFs) that may be toxic. There are four types of MWFs: neat oils and three water-based MWFs (soluble oil, semisynthetic and synthetic), which are diluted with water and whose composition varies according to the mineral oils ratio. MWFs also contain various additives. To determine the absorption of six amines used as corrosion inhibitors and biocides in MWFs, porcine skin flow-through diffusion cell experiments were conducted with hydrophilic ethanolamines (mono-, di- and triethanolamine, MEA, DEA and TEA respectively) and a mixture of lipophilic amines (dibutylethanolamine, dicyclohexylamine and diphenylamine). The six amines were dosed in four vehicles (water and three generic water-based MWF formulations) and analyzed using a scintillation counter or gas chromatography/mass spectrometry. These 24 h studies showed that dermal absorption significantly (P < 0.05) increased from water for the six amines (e.g. 1.15 ± 0.29% dose; DEA in water) compared to other formulations (e.g. 0.13 ± 0.01% dose; DEA in semisynthetic MWF) and absorption was greatest for dibutylethanolamine in all the formulations. The soluble oil formulation tended to increase the dermal absorption of the hydrophilic amines. The permeability coefficient was significantly higher (P < 0.05) with TEA relative to the other hydrophilic amines (e.g. 4.22 × 10(-4) ± 0.53 × 10(-4) cm h(-1) [TEA in synthetic MWF] vs. 1.23 × 10(-4) ± 0.10 × 10(-4) cm h(-1) [MEA in synthetic MWF]), except for MEA in soluble oil formulation. Future research will confirm these findings in an in vivo pig model along with dermatotoxicity studies. These results should help MWF industries choose safer additives for their formulations to protect the health of metalworkers.

The use of complexation with alkanolamines to facilitate skin permeation of mefenamic acid.

The preparation of mefenamic acid (MH)-alkanolamine [monoethanolamine, diethanolamine, triethanolamine and propanolamine] complexes was attempted to increase the transdermal flux of MH. A lipophilic enhancer system consisting of isopropyl myristate (IPM) and ethanol (9:1; EI system) produced a marked enhancement of MH flux from the alkanolamine complexes through hairless rat skin membrane. Among the alkanolamines examined, the propanolamine complex had the greatest enhancing effect on the permeation of MH. The observed permeation enhancement of MH-alkanolamine complexes by the EI system was explained by an analysis based on a two-layer diffusion model. The stratum corneum immersed in IPM forms a continuous phase of vehicle and stratum corneum and, from the phase, ethanol transport the MH-alkanolamine complexes to the epidermis and dermis, and the complexes, which are more water soluble than MH, exhibit increased partition into the epidermis and dermis, as the flux increases.

Aerobic biodegradability of methyldiethanolamine (MDEA) used in natural gas sweetening plants in batch tests and continuous flow experiments.

Mixtures of different amines including tertiary amines (methyldiethanolamine, MDEA) are commonly used for the removal of CO2 from gas mixtures or in gas sweetening processes for the extraction of CO2 and H2S. The absorber solutions used can be released into the industrial waste water due to continuous substitution of degraded MDEA, periodically cleaning processes or an accidental spill. In this study, the aerobic biodegradability of MDEA was investigated in a standardised batch test and a continuous flow experiment (40 l/d). The results of the batch test indicated that the MDEA-solution was non-biodegradable during the test period of 28 days, whereas the continuous flow experiments showed biodegradation of more than 96% based on TOC-measurements. This was probably due to the adaptation of the microorganisms to this particular waste water contamination during continuous flow experiment.

Anti-drug antibodies as drug carriers. I. For small molecules.

PURPOSE: To better understand the pharmacokinetics of drugs compounds that bind endogenous antibodies METHODS: Three groups of mice with differing anti-fluorescein (FL) titers were established by empirically developed immunization protocols. These with two control groups were given intravenously [3H]-ethanolamine conjugate of FL (FL-EA). The latter was synthesized using isothiocyanate chemistry. Radioactivity in the circulation, and occasionally in peritoneal ascites, was monitored for 7 days. A group of mice was immunized with eosin Y and given FL-EA. Conversely, eosin Y conjugate of radiolabeled EA (EY-EA) was given to mice immunized with FL. These two groups represented animals of low affinity to probe haptens. The affinity was assessed by a precipitation procedure, while titer was determined by a standard ELISA. Dose of FL-EA varied over a 100-fold. RESULTS: On average, the three immunized groups showed a 1:13:85 ratio of anti-FL titer, with remarkably consistent levels within each group. Elimination rates of FL-EA from the serum of very high-titer mice and high-titer mice were similar, however, were substantially lower than that found in low-titer mice. The latter was in turn lower than that found in non- or mock-immunized mice. Serum of mice immunized with FL showed approximately 200-fold lower affinity towards EY-EA than FL-EA. In these mice and in mice immunized with eosin Y and given FL-EA, the elimination of the probe haptens was again fast, reminiscent of low-titer mice. Mice of either low titer or low affinity showed more rapid redistribution of the conjugate between serum and peritoneal fluid. In a group of mice with comparable anti-FL titer, elimination from serum was independent of dose over a 100-fold difference. The bi-phasic concentration-time profile observed was accommodated by a physiologically meaningful pharmacokinetic model incorporating two compartments in which antibody binding can occur. CONCLUSIONS: Monovalent antigenic substance cannot trigger immune clearance. As such, endogenous antibodies that recognize the molecule can serve as a carrier to result in a substantial decrease in clearance.

Antibodies as drug carriers. II. For proteins.

PURPOSE: To evaluate the potential use of antibodies as a carrier for monovalent protein haptens. METHODS: A single -SH functionality present in the human IgG light chain was fluoresceinated. This conjugate, FL-LC, was treated with pepsin to obtain FL conjugate of half light chain, FL-(LC)1/2, of MW 11 kDa. These two were radiolabeled using [3H]-propionic acid N-hydroxysuccinimide ester, and administered via tail vein to FL-immunized or mock-immunized mice. The blood radioactivity was measured over a 72-h period. Attempts were made to measure the affinity constant for the interaction between the conjugates and anti-FL antibodies by fluorescence quenching, surface plasmon resonance spectroscopy, and competitive ELISA. RESULTS: All of the three methods used produced supportive, if not conclusive, evidence of decreased binding affinity with increased conjugate size. Subsequent to tail-vein injection to FL-immunized mice, FL-LC showed approximately 4-fold smaller volume of distribution than mock-immunized mice: 0.041 +/- 0.005 vs. 0.16 +/- 0.02 mL/g. Corresponding values for FL-(LC)1/2, were significantly larger: 0.070 +/-0.013 and 0.30 +/- 0.02 mL/g, respectively. Compared with a small FL conjugate of ethanolamine, FL-EA, we studied earlier, the dose-normalized concentrations of the protein conjugates started at a higher level but declined more rapidly with time. In mock-immunized mice, the radioactivity disappeared very rapidly after administration, followed by an extremely slow decline with half-life close to 60 h. Evidence is provided to support that the radiolabel dissociated in the kidney, however, binding to anti-FL antibodies greatly stabilized the conjugate. CONCLUSIONS: Based on an entropic principle alone the affinity of monovalent hapten-antibody interaction is expected to diminish with increase in hapten size. As such, the size of a hapten should be an important determinant of its pharmacokinetics in animals harboring antibodies that recognize the hapten. Relative to what was observed with small MW FL-EA, the protein conjugates showed substantially sustained circulation as a result of antibody binding, but this effect was diminished at later time points. Both affinity and pharmacokinetic data are consistent with the hypothesis of reduced affinity with increasing MW for monovalent hapten conjugates, but neither offered overwhelming proof.

Effect of n-3 fatty acid deficiency on fatty acid composition and metabolism of aminophospholipids in rat brain synaptosomes.

Docosahexaenoic acid (DHA, 22:6n-3) is one of the major polyunsaturated fatty acids esterified predominantly in aminophospholipids such as ethanolamine glycerophospholipid (EtnGpl) and serine glycerophospholipid (SerGpl) in the brain. Synaptosomes prepared from rats fed an n-3 fatty acid-deficient safflower oil (Saf) diet had significantly decreased 22:6n-3 content with a compensatory increased 22:5n-6 content when compared with rats fed an n-3 fatty acid-sufficient perilla oil (Per) diet. When the Saf group was shifted to a diet supplemented with safflower oil plus 22:6n-3 (Saf + DHA) after weaning, 22:6n-3 content was found to be restored to the level of the Per group. The uptake of [3H]ethanolamine and its conversion to [3H]EtnGpl did not differ significantly among the three dietary groups, whereas the formation of [3H]lysoEtnGpl from [3H]ethanolamine was significantly lower in the Saf group than in the other groups. The uptake of [3H]serine, its incorporation into [3H]SerGpl, and the conversion into [3H]EtnGpl by decarboxylation of [3H]SerGpl did not differ among the three dietary groups. The observed decrease in lysoEtnGpl formation associated with a reduction of 22:6n-3 content in rat brain synaptosomes by n-3 fatty acid deprivation may provide a clue to reveal biochemical bases for the dietary fatty acids-behavior link.

Toxicity and bioavailability of copper herbicides (Clearigate, Cutrine-Plus, and copper sulfate) to freshwater animals.

In designing aquatic herbicides containing copper, an important goal is to maximize efficacy for target species while minimizing risks for nontarget species. To have a margin of safety for nontarget species, the concentration, duration of exposure (i.e., uptake), and form (i.e., species) of copper used for herbicidal properties should not elicit adverse effects on populations of nontarget species. To determine the potential for risk or adverse effects (conversely the margin of safety), data regarding the comparative toxicity of copper-containing herbicides are crucial. A series of comparative toxicity experiments was conducted, including baseline estimates of toxicity (LC50s, LOECs), sensitive species relationships (thresholds and exposure-response slopes), and bioavailability of toxic concentrations and forms of copper 7 days after initial herbicide application. Aqueous 48-h toxicity experiments were performed to contrast responses of Daphnia magna Strauss, Hyalella azteca Saussure, Chironomus tentans Fabricius, and Pimephales promelas Rafinesque to copper herbicides: Clearigate(R), Cutrine(R)-Plus, and copper sulfate. D. magna was the most sensitive aquatic animal tested for all three herbicides; 48-h LC50s for organisms exposed to Clearigate, Cutrine-Plus, and copper sulfate were 29.4, 11.3, and 18. 9 microg Cu/L, respectively. In terms of potency (calculated from the linearized portion of the exposure-response curves, which included 50% mortality), D. magna was the most sensitive animal tested. Organisms exposed to Clearigate, Cutrine-Plus, and copper sulfate had exposure-response slopes of 2.55, 8.61, and 5.07% mortality/microg Cu/L, respectively. Bioavailability of Clearigate and Cutrine-Plus was determined by comparing survival data (LC50s) of test organisms exposed to herbicide concentrations during the first and last 48-h of a 7-day exposure period. Even in these relatively simplified water-only exposures, a transformation of copper to less bioavailable species over time was observed with a 100-200% decrease in toxicity (i.e., an increase in 48-h LC50s) for all four test animals. This series of laboratory experiments provides a worst-case scenario for determining the risk associated with the manufacturer's recommended application rates of Clearigate (100-1,000 microg Cu/L), Cutrine-Plus (200-1,000 microg Cu/L), and copper sulfate (100-500 microg Cu/L) in natural waters for four nontarget freshwater animals.

Lateral iontophoretic solute transport in skin.

PURPOSE: The lateral iontophoretic transport of three solutes (sodium, ethanolamine, lidocaine) from an active electrode through skin and other tissues to an indifferent electrodes was investigated. METHODS: Anodal epidermal iontophoresis was carried out on an in vivo rat model using constant direct current of 0.38 mA/cm2. Cells were fixed on the epidermis of anesthetized rats at distances of adjacent, 3 cm and 7 cm apart. After iontophoresis, tissues were dissected at I cm intervals between the electrodes. Concentrations of the radiolabelled solutes in tissues were determined by liquid scintillation counting or gamma counting. RESULTS: The concentration of each solutes in the epidermis, dermis and other tissues was found to decrease in an exponential manner with lateral distance from the active electrode to the indifferent electrode. The detectable lateral distance for ethanolamine and lidocaine was less than 2 cm from the donor sites, at which distance the concentrations were not significantly different to those found in the corresponding contralateral site. The lateral drift velocities for all solutes in the epidermis and dermis were consistent with diffusivities of the order of 10(-6) cm2/s. The drift velocity of sodium was greater than either lidocaine or ethanolamine. CONCLUSIONS: The decline in solute concentration with lateral distance is mainly due to clearance from the site of application by the skin's microcirculation and decreases with distance from the active electrode until a baseline concentration, similar to the contralateral tissue concentration is reached.