Determination of a Degradation Constant for @entity5322 by Direct Suppression of mRNA in a Novel XXXX Hepatocyte Model, HepatoPac    .

@entity1 @entity11670 @entity650 @entity5322 @entity5396
UNASSIGNED: Accurate determination of rates of de novo synthesis and degradation of cytochromes @entity11670 ( @entity11670 ) has been challenging. There is a high degree of variability in the multiple published values of turnover for specific P450s which is likely exacerbated by differences in methodologies. For @entity5322 , half-life values range from 10 to 140h (Yang et al., 2008). An accurate value for kdeg has been identified as a major limitation for prediction of drug interactions involving mechanism based inhibition and/or induction. Estimation of @entity11670 half-life from in vitro test systems, such as @entity1 hepatocytes, is complicated by differential decreased enzyme function over culture time, attenuation of the impact of enzyme loss through inclusion of glucocorticoids in media, and viability limitations over long-term culture times. HepatoPac overcomes some of these challenges providing an extended stability of enzymes (2.5 weeks in our hands). As such it is a unique tool for studying rates of enzyme degradation achieved through modulation of enzyme levels. @entity5322 mRNA levels were rapidly depleted by >90% using either siRNA or addition of @entity650 which allowed an estimation of the degradation rate constant for @entity5322 protein over an incubation time of 96 hours. The degradation rate constant of 0.0240    0.005 h(-1) was reproducible in hepatocytes from five different @entity1 donors. These donors also reflected the overall population with respect to @entity5396 genotype. This methodology can be applied to additional enzymes and may provide a more accurate in vitro derived kdeg value for predicting clinical DDI outcomes.
