Evaluation of predictive CYP2C19 genotyping assays relative to measured phenotype in a South African cohort.

AIM: To align predicted and measured CYP2C19 phenotype in a South African cohort. MATERIALS & METHODS: Genotyping of CYP2C19*2, *3, *9, *15, *17, *27 and *28 was performed using PCR-RFLP, and an activity score (AS) system was used to predict phenotype. True phenotype was measured using plasma concentrations of omeprazole and its metabolite 5'-hydroxyomperazole. RESULTS: Partial genotype-phenotype discrepancies were reported, and an adapted AS system was developed, which showed a marked improvement in phenotype prediction. Results highlight the need for a more comprehensive CYP2C19 genotyping approach to improve prediction of omeprazole metabolism. CONCLUSION: Evidence for the utility of a CYP2C19 AS system is provided, for which the accuracy can be further improved by means of comprehensive genotyping and substrate-specific modification.

Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays.

BACKGROUND: The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration assays; the neutral red uptake (NRU), resazurin reduction (RES) and sulforhodamine B (SRB) assays, in comparison to the MTT assay. Interference between the MTT assay and three glycolysis inhibitors, 2-deoxyglucose, 3-bromopyruvate and lonidamine, was investigated. RESULTS: Data indicate that the NRU, RES and SRB assays showed the smallest variability across the linear range, while the largest variation was observed for the MTT assay. This implies that these assays would more accurately detect small changes in cell number than the MTT assay. The SRB assay provided the most reproducible results as indicated by the coefficient of determination after a limited number of experiments. The SRB assay also produced the lowest variance in the derived 50% inhibitory concentration (IC50), while IC50 concentrations of 3-bromopyruvate could not be detected using either the MTT or RES assays after 24 hours incubation. Interference in the MTT assay was observed for all three tested glycolysis inhibitors in a cell-free environment. No interferences were observed for the NRU, SRB or RES assays. CONCLUSIONS: This study demonstrated that the MTT assay was not the best assay in a number of parameters that must be considered when a cell enumeration assay is selected: the MTT assay was less accurate in detecting changes in cell number as indicated by the variation observed in the linear range, had the highest variation when the IC50 concentrations of the glycolysis inhibitors were determined, and interference between the MTT assay and all the glycolysis inhibitors tested were observed. The SRB assay performed best overall considering all of the parameters, suggesting that it is the most suitable assay for use in preclinical screening of novel therapeutic compounds with oxido-reductive potential.

Quantitative plasma analysis using automated online solid-phase extraction with column switching LC-MS/MS for characterising cytochrome P450 2D6 and 2C19 metabolism.

In this study an easy and efficient assay for simultaneous quantitation of plasma concentrations of probe drugs and their metabolically relevant metabolites for the phenotypic analysis of cytochrome P450 2D6 and 2C19, respectively, has been established. This sensitive method makes use of a simple initial sample preparation, followed by a 6-min automated analysis that includes online solid-phase extraction (SPE), column switching and tandem mass spectrometry. Validation over a concentration range of 1.3-2500 ng/mL for dextromethorphan, omeprazole, dextrorphan and 5'-hydroxyomeprazole was performed with LOQ between 215 and 1145 pg/mL. Intra- and inter-day precision and accuracy over the calibration ranges were within 15% for all analytes with recoveries of greater than 85%. Advantages are small sample volumes required, a robust, sensitive and highly selective method suitable for pre-prescription metabolic screening. This method could compliment or offer an alternative to DNA mutation analysis for determining appropriate dosage regimens for personalised medicine.

The antioxidant potential of Sutherlandia frutescens.

One of the best-known multi-purpose medicinal plants in Southern Africa, Sutherlandia frutescens subsp. microphylla (family: Fabaceae/Leguminosa), is used for a wide range of conditions, including cancer, viral diseases and inflammatory conditions. Little scientific data has been documented on the mechanism by which Sutherlandia frutescens acts on the immune system. Phagocyte derived reactive oxygen species, such as hydrogen peroxide and superoxide radicals, are responsible for the pathogenesis of various inflammatory conditions. Anti-inflammatory properties of various medicinal-plant extracts have been explained, at least in part, by their antioxidant activities. We investigated the effects of a hot water extract of Sutherlandia frutescens on both luminol and lucigenin enhanced chemiluminescence of neutrophils stimulated with L-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) as well as its superoxide and hydrogen peroxide scavenging properties in a cell free system. The results indicate that Sutherlandia frutescens extract possesses superoxide as well as hydrogen peroxide scavenging activities at concentrations as low as 10 microg/ml, which could account for some of the anti-inflammatory properties that have been described.