Evaluation of zebrafish embryos as a model for assessing inhibition of hERG.

INTRODUCTION: It has been proposed that the analysis of heart rate in zebrafish embryos can be used to assess the potential effects of compounds on hERG. The purpose of this study was to investigate the effect of compounds on the heart rate and atrioventricular dissociation in zebrafish. The compounds investigated were chosen based on the association or lack of association with QTc prolongation in the clinic. METHODS: Three-day-old embryos were incubated in buffered embryo medium. On the day of the study, fish were placed in a petri dish containing 5.0 mL of embryo medium and 125 mg/L MS-222 anesthetic. Drugs to be tested were added to the medium from a stock solution to achieve the desired target concentration. Ten fish were incubated in each concentration of drug for 80 min. Beat rates of the atrium and ventricle were recorded after the incubation by counting beats of the respective chambers using standard brightfield stereomicroscopy. RESULTS: All of the compounds associated with QT prolongation induced dissociation between the atrium and ventricular rates except D,L-sotalol and procainamide. The concentrations that induced dissociation tended to be higher than the hERG IC50. None of the negative control compounds caused atrioventricular dissociation at clinically efficacious concentrations. DISCUSSION: In conclusion, the present data demonstrate that zebrafish can be utilized to assess the effects of chemicals on hERG. However, the practical use of this assay may be difficult because of the high concentrations that must be reached to see those pharmacological effects.

Effects of fasting on evaluation of gastrointestinal transit with charcoal meal.

INTRODUCTION: At the present time, most studies investigating gastrointestinal transit time with charcoal are conducted in fasted rats. It seems reasonable to hypothesize that the fasting state of rats could influence the effect a compound had on gastrointestinal transit time. The purpose of this study was to investigate the effects of food on the pharmacological effects on gastrointestinal transit. METHODS: For each drug investigated, two sets of 32 male Sprague-Dawley rats were used. One set was studied after being fasted for approximately 6 h, the second set was studied after free access to food. Each set had 4 groups of animals (n=8/group) that were administered different doses, allowing the assessment of the drug effect after fasting and after free access to food. Animals were administered 0, 10, 25, and 75 mg/kg of morphine; 0, 10, 20, and 40 mg/kg loperamide, or 0, 0.05, 0.5, and 3.0 mg/kg clonidine. At predetermined times, an activated charcoal suspension was administered by oral gavage. Thirty minutes after receiving the charcoal meal, rats were euthanized and the small intestine was removed. The length of the small intestine and the distance traveled by the charcoal were recorded. For each animal, gastrointestinal transit was calculated as the percentage of the distance traveled relative to the total length of the small intestine. RESULTS: Baseline (vehicle dosed animals) gastrointestinal transit was significantly greater in fasted versus fed rats. In fasted rats, morphine did not have a significant effect on transit. In fed rats, 25 and 75 mg/kg morphine caused a significant decrease in transit. In fed and fasted rats, 0.5 and 3 mg/kg clonidine caused a significant decrease in transit. Loperamide did not affect gastrointestinal transit in fed or fasted rats at doses up to 40 mg/kg. DISCUSSION: These data demonstrate that food does not reduce the sensitivity of the gastrointestinal transit time.

Soluble, template-dependent extracts from Nicotiana benthamiana plants infected with potato virus X transcribe both plus- and minus-strand RNA templates.

We have developed a method to convert membrane-bound replication complexes isolated from Nicotiana benthamiana plants infected with potato virus X (PVX) to a soluble, template-dependent system for analysis of RNA synthesis. Analysis of RNA-dependent RNA polymerase activity in the membrane-bound, endogenous template extracts indicated three major products, which corresponded to double-stranded versions of PVX genomic RNA and the two predominant subgenomic RNAs. The endogenous templates were removed from the membrane-bound complex by treatment with BAL 31 nuclease in the presence of Nonidet P-40 (NP-40). Upon the addition of full-length plus- or minus- strand PVX transcripts, the corresponding-size products were detected. Synthesis was not observed when red clover necrotic mosaic dianthovirus (RCNMV) RNA 2 templates were added, indicating template specificity for PVX transcripts. Plus-strand PVX templates lacking the 3' terminal region were not copied, suggesting that elements in the 3' region were required for initiation of RNA synthesis. Extracts that supported RNA synthesis from endogenous templates could also be solublized using sodium taurodeoxycholate and then rendered template-dependent by BAL 31 nuclease/NP-40 treatment. The solubilized preparations copied both plus- and minus-strand PVX transcripts, but did not support synthesis from RCNMV RNA 2. These membrane-bound and soluble template-dependent systems will facilitate analyses of viral and host components required for PVX RNA synthesis.