Initial structure-activity relationship of a novel class of nonpeptidyl GnRH receptor antagonists: 2-arylindoles.

A nonpeptidyl GnRH receptor antagonist (1), with a unique 2-arylindole core, was identified through the Merck in-house screening for binding affinity on the rat GnRH receptor. SAR studies directed toward the alkoxy-ethanolamine and 2-aryl groups resulted in a simpler lead structure with improved activity. This compound 50 exhibits a 60-fold improvement in binding activity over our initial lead 1.

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys.

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.

Potent, selective benzenesulfonamide agonists of the human beta 3 adrenergic receptor.

A cloned human beta 3 adrenergic receptor assay was used to identify phenoxypropanolamine agonist 1. SAR studies led to the identification of benzenesulfonamide derivative 20, a 6.3 nM beta 3 agonist which shows 30-fold selectivity for beta 3 agonist activity over beta 1 and beta 2 receptor binding. Further refinement of this lead provided 4-bromo derivative 39, a subnanomolar agonist with 660-fold and 230-fold selectivity over beta 1 and beta 2, respectively.

3-Pyridyloxypropanolamine agonists of the beta 3 adrenergic receptor with improved pharmacokinetic properties.

Pyridyloxypropanolamines L-749,372 (8, beta 3 EC50 = 3.6 nM) and L-750,355 (29, beta 3 EC50 = 13 nM) are selective partial agonists of the human receptor, with 33% and 49% activation, respectively. Both stimulate lipolysis in rhesus monkeys (ED50 = 2 and 0.8 mg/kg, respectively), with minimal effects on heart rate. Oral bioavailability in dogs, 41% for L-749,372 and 47% for L-750,355, is improved relative to phenol analogs.

Development of a highly quantitative, reproducible assay for determination of chicken T cell growth factor biological activity.

This report examines optimal culture conditions necessary for accurate and sensitive quantification of chicken T Cell Growth Factor (TCGF) activity. With this bioassay, TCGF is quantified by measuring its ability to cause proliferation of splenocytes prestimulated with mitogen. Proliferation is quantified by determining the optical density (OD) or "signal" of test samples in microtiter wells by measuring the incorporation of tetrazolium salt by live cells. To optimize assay conditions, systematic evaluation of the effects of cell culture variables was carried out with the constant aim of increasing signal to noise ratio in the assay. Higher signal to noise ratios were found when using Dulbecco's Modified Eagle's Medium (DMEM) rather than Roswell Park Memorial Institute Medium (RPMI) for basal tissue culture media containing the same supplements. The addition of lipid supplement to the assay system not only increased the proliferation signal, but also decreased the background OD. Incubation temperatures of 41 C rather than 37 C for both the mitogen prestimulation and proliferation phases of the assay also resulted in a higher signal to noise ratio. While incorporating the optimal experimental conditions, a finalized assay procedure employing test sample normalization with an internal assay standard was tested for accuracy. The assay can accurately detect 2 to 15 U/mL of TCGF activity. The within-assay variation ranged from 2 to 13% and the between-assay variation ranged from 11 to 22% depending upon the TCGF preparation being tested. The excellent reproducibility of this assay has facilitated investigations of TCGF production, processing, and purification.

High-performance liquid chromatographic method for the determination of spectinomycin in turkey plasma.

A selective high-performance liquid chromatographic (HPLC) method with ultraviolet-visible (UV-VIS) detection was developed to measure therapeutic concentrations of spectinomycin in turkey plasma. Treatment of plasma samples with 3% trifluoroacetic acid in acetonitrile facilitated spectinomycin extraction and protein precipitation. After centrifugation, the stable derivatization reagent, 2,4-dinitrophenyl-hydrazine, was added to an aliquot of the supernatant, and the mixture was incubated for 30 min at 70 degrees C. Excess reagent was quenched with acetone and additional heating. The resulting derivative, a proposed spectinomycin-hydrazone, was separated from other compounds by reversed-phase HPLC during a short gradient run. The absorbance of the effluent was monitored spectrophotometrically with the UV-VIS detector set at 205 nm. The detector response was linear through the range of interest, 2-100 micrograms/ml.

Evaluation of silica cartridge purification and hemiacetal formation for liquid chromatographic determination of aflatoxins in corn.

Rapid silica cartridge cleanup, acid-catalyzed conversion of aflatoxins B1 and G1 to hemiacetals, and reverse-phase liquid chromatography with fluorescence detection were evaluated for effectiveness in determining aflatoxins B1, B2, G1, and G2 in corn at concentrations ranging from 2 ng/g to 100 micrograms/g. In testing the method, aflatoxins applied to silica cartridges were recovered at greater than 97.1% overall. Conversion of aflatoxins B1 and G1 to their hemiacetals was shown to be complete (150 micrograms of toxin per extract). Application of these techniques to spiked corn extracts yielded data indicating excellent repeatability of derivatization relative to paired standards; coefficients of variation ranged from 1.08% to 5.81%. The repeatability of the method with naturally contaminated corn was also excellent; coefficients of variation ranged from 1.15% to 3.97%. Liquid chromatographic determination of aflatoxins in corn using fluorescence detection was sensitive, accurate, and precise resulting in applicability from less than 1 ng/g of aflatoxin B1 to greater than 100,000 ng/g.

Purification of adenovirus hexon by high performance liquid chromatography.

Hexon is the major structural protein of adenovirus, and has significance in studies of virus structure and function, vaccine development, and immunodiagnosis. We describe a simple, single-step, anion-exchange high performance liquid chromatography (HPLC) method for the high yield purification of hexon. Purity of the isolated hexon was assessed by SDS-PAGE and HPLC methods. The isolated hexon was immunologically reactive with anti-hexon monoclonal antibody in a dot-blot assay. It also retained immunogenicity, as polyclonal antisera from rabbits immunized with hexon showed the desired antigen specificity. The enhanced speed of this purification method allows for the efficient isolation of hexon from various serotypes, and thus may facilitate comparative studies of hexon immunobiology.

Liquid chromatographic determination of aflatoxicol in porcine liver.

A liquid chromatographic (LC) method for determination of aflatoxicol in porcine liver was developed. Liver sample is homogenized with water, diluted with saturated Na2SO4 solution, and extracted with acetone. After filtration, less polar interferences are removed by partition with isooctane. Aflatoxicol in the aqueous fraction is partitioned into CHCl3. The extract is dried over anhydrous Na2SO4 and evaporated nearly to dryness at 35 degrees C under a gentle flow of dry filtered air or nitrogen. Residue is dissolved in CHCl3-hexane and applied to a hexane-activated silica cartridge. The cartridge is washed with hexane-CHCl3, then aflatoxicol is eluted with CHCl3-acetone. Purified extract is evaporated to dryness, dissolved in methanol, and analyzed by C18 reverse phase liquid chromatography using a water-CH3CN-acetic acid mobile phase and fluorescence detection. Recovery of aflatoxicol from spiked liver samples at levels ranging from 0.25 to 4.0 ng aflatoxicol/g wet tissue averaged 92% with a limit of detection of about 0.1 ng aflatoxicol/g liver.

Determination of ampicillin in serum by using simple ultrafiltration technique and liquid chromatographic analysis.

A new liquid chromatographic method for determination of ampicillin in canine and equine serum has been developed. The serum sample (500 microL) is vortex-mixed with 20% ethanol (500 microL) and filtered using a 30,000 molecular weight cutoff microseparation tube to separate high molecular weight solutes following low-speed centrifugation. Ampicillin is then separated from other serum components by reverse phase ion-pair liquid chromatography (LC). The ultraviolet (UV) absorbance of the column effluent is monitored at 230 nm. Recoveries of ampicillin from canine serum spiked at concentrations of 10, 40, and 60 micrograms/mL were 93.1, 100.9, and 87.8%, respectively, with coefficients of variation (CVs) of 2.91, 3.08, and 4.08%, respectively. Recoveries of ampicillin from equine serum spiked at the same concentrations were 91.6, 90.1, and 88.7%, respectively, with CVs of 3.03, 2.61, and 3.35%, respectively. The limit of detection for ampicillin by this method is less than 0.5 micrograms/mL serum.

Liquid chromatographic determination of aflatoxin M1 in milk.

The official AOAC method for aflatoxin M1 in milk was modified by replacing cellulose column chromatography with cartridge chromatographic cleanup and replacing thin layer chromatographic (TLC) determination with liquid chromatographic (LC) quantitation to yield a new method for bovine and porcine milk. An acetone extract of milk is treated with lead acetate and defatted with hexane, and M1 is partitioned into chloroform as in the AOAC method. Chloroform is removed by evaporation under a stream of nitrogen at 50 degrees C. The residue is dissolved in chloroform, the vessel is rinsed with hexane, and the 2 solutions are applied in sequence to a hexane-activated silica Sep-Pak cartridge. Less polar impurities are removed with hexane-ethyl ether, and M1 is eluted with chloroform-methanol, and determined by C18 reverse phase LC using fluorescence detection. Recoveries of M1 added to bovine milk at 0.25, 0.50, and 1.0 ng/mL were 90.8, 93.4, and 94.1%, respectively. The limit of detection was less than 0.1 ng M1/mL for both bovine and porcine milk.

Models of feed refusal syndrome in poultry.

Research on feed refusal syndrome(s), an important problem in the poultry industry, has been hindered by a lack of sensitive and quantitative laboratory models. Suitable models were developed using five groups of 30-week-old male chickens per treatment. Feed or water, depending on which was to be measured, was withdrawn overnight. Then consumption of treated water or feed was measured over a 6-hr period of rehydration or refeeding. In aqueous solutions NaCl, H2SO4, and Na2CO3 reduced liquid consumption in a dose-related manner. Ammonia caused a similar refusal when added to feed. Consumption of feed and water was not influenced by pH over the ranges likely to occur as a result of fungal activity. Consumption of feed on a wet weight, but not dry weight, basis was influenced by the moisture content of feed. A culture filtrate of Fusarium roseum NRRL 1181 containing diacetoxyscirpenol, a trichothecene mycotoxin, reduced consumption of feed by about 77% when added to feed and reduced liquid consumption by about 92% when substituted for drinking water. Thus, the models for consumption of liquids and solids appear to have the attributes necessary for quantitative investigation into the relationships of mycotoxins to feed refusal syndromes.

Rapid liquid chromatographic determination of aflatoxins in heavily contaminated corn.

A procedure is described for rapid, quantitative determination of aflatoxins B1, B2, G1, and G2 in heavily contaminated corn (greater than 500 micrograms B1/kg) from field, greenhouse, and growth chamber experiments employing artificial inoculation of corn with Aspergillus flavus. Whole kernel corn is ground to pass a 1 mm screen and mixed before extraction of a water-wetted (25 mL) 50 g subsample with 250 mL chloroform. The filtered extract is diluted 1:1 with hexane and applied to a hexane-wetted (10 mL) disposable silica cartridge. Interferences are removed with chloroform-hexane (1 + 3), and aflatoxins are quantitatively eluted with hexane-acetone (1 + 1). Aflatoxins B1 and G1 are converted to the more intensely fluorescent hemiacetals, B2a and G2a, by treatment with trifluoroacetic acid-water. Derivatized aflatoxins are separated by reverse phase liquid chromatography (LC) and quantitated fluorometrically. Compared with AOAC method I (CB) for corn, using samples containing approximately 50 and 10 000 micrograms B1/kg, agreement between methods was good at the lower level while the rapid method yielded a considerably larger mean at the higher level. A precision study of 30 replicate samples produced a coefficient of variation of 8.46% at a mean value of 1066 micrograms B1/kg. The cartridge method was developed for LC analysis of samples that contain greater than 500 micrograms aflatoxin B1/kg corn, but it may be used to quantitate as little as 10 micrograms B1/kg with no modification.