Comparison of alamar blue and MTT assays for high through-put screening.

The performance of alamar blue and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell viability assays in a high through-put format were compared. A total of 117 drugs chosen for their wide range of therapeutic areas were screened at 10 microM using both assays in human hepatoma cell line HepG2. Except for terfenadine and astemizole, which performed consistently in both assays, the alamar blue assay was slightly more sensitive than the MTT assay for most compounds. The MTT assay was less sensitive detecting an effect for daunorubicin and trifluoperazine. Seven drugs, astemizole, daunorubicin, ellipticine, fluphenazine, terfenadine, thioridazine and trifluoperazine, had percent viability results of 55% or less in the alamar blue assay at the single point screen. These were re-tested in both assays for reconfirmation of cytotoxicity and determination of the EC50 values. Except for daunorubicin, the EC50 values were comparable in both assays. Based on these results and the Z'-factor assessment of assay quality, both assays provided useful information to identify in vitro cytotoxic drugs at early stages of drug candidate selection. However, careful interpretation of data is warranted due to the possibility of false positive or negative results caused by inducers and/or inhibitors of metabolic enzymes that are responsible for transformation of cell toxicity end points, as we demonstrated using dicumarol.

Application of modified in vitro screening procedure for identifying herbals possessing sulfonylurea-like activity.

We describe here the application of a modified in vitro procedure for identifying herbs potentially possessing sulfonylurea-like activity. The procedure consists of the combination of an SUR1 receptor binding assay and an insulin secretion assay in cultures of HIT-T15 cells. This procedure could be used as an initial step in identifying new safe and efficacious agents for the management of Type II diabetes. The application of this screening procedure to a set of selected herbs produced results that were consistent with the previously reported properties of those herbs. The collected data suggest that the hypoglycemic properties of bitter melon (Momordica charantia, Linn. Family, Cucurbitacea), cerasse (Momordica charantia, Linn. wild variety, Family, Cucurbitacea) and American ginseng (Panax quinquefolius, Linn., Family Araliacea) are at least partially due to their sulfonylurea-like activity.

A highly sensitive assay for the simultaneous determination of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma by high-performance liquid chromatography with electrochemical and fluorescence detection.

A novel, highly sensitive and specific bioanalytical method has been developed for the simultaneous determination of morphine and its major metabolites, morphine-3-glucuronide and morphine-6-glucuronide, in human plasma, using noroxymorphone as the internal standard. The analytes are isolated from human plasma using a nonpolar/polar C2 solid-phase extraction cartridge and analyzed by high-performance liquid chromatography with serial detection using electrochemical detection for morphine, morphine-6-glucuronide (M6G), and noroxymorphone and fluorescence detection for morphine-3-glucuronide (M3G). The limit of quantitation (sensitivity) using a 0.5-ml sample of plasma is 1 ng/ml, 10 ng/ml, and 5 ng/ml for morphine, M3G, and M6G, respectively. Standard curves were linear (correlation coefficients > 0.999) over the ranges 1-30 ng/ml, 10-500 ng/ml, and 5-100 ng/ml for morphine, M3G, and M6G, respectively. The overall interday accuracy of the method was -1.58% for morphine, 2.27% for M3G, and -5.34% for M6G. The assay is routinely used for the study of morphine, M3G, and M6G pharmacokinetics after oral administration of morphine.