In Silico and in Vitro Modeling of Hepatocyte Drug Transport Processes: Importance of ABCC2 Expression Levels in the Disposition of Carboxydichlorofluroscein

Abstract

ABSTRACT:The impact of transport proteins in the disposition of chemicals is becoming increasingly evident. Alteration in disposition can cause altered pharmacokinetic and pharmacodynamic parameters, potentially leading to reduced efficacy or overt toxicity. We have developed a quantitative in silico model, based upon literature and experimentally derived data, to model the disposition of carboxydichlorofluroscein (CDF), a substrate for the SLCO1A/B and ABCC subfamilies of transporters. Kinetic parameters generated by the in silico model closely match both literature and experimentally derived kinetic values, allowing this model to be used for the examination of transporter action in primary rat hepatocytes. In particular, we show that the in silico model is suited to the rapid, accurate determination of K i values, using 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571) as a prototypical pan-ABCC inhibitor. In vitro-derived data are often used to predict in vivo response, and we have examined how differences in protein expression levels between these systems may affect chemical disposition. We show that ABCC2 and ABCC3 are overexpressed in sandwich culture hepatocytes by 3.5-and 2.3-fold, respectively, at the protein level. Correction for this in markedly different disposition of CDF, with the area under the concentration versus time curve and C max of intracellular CDF increasing by 365 and 160%, respectively. Finally, using kinetic simulations we show that ABCC2 represents a fragile node within this pathway, with alterations in ABCC2 having the most prominent effects on both the K m and V max through the pathway. This is the first demonstration of the utility of modeling approaches to estimate the impact of drug transport processes on chemical disposition.

An introduction to systems toxicology

“…This allows other users to both use published models for their individual research projects, but also fosters an environment of collaborative development, where several groups may produce iterative improvements of a single biological model. The models available from both these resources simulate many aspects of biology, from regulatory gene and signalling networks such as MAPK 44,45 and nuclear receptors, 46,47 through drug transport 48 and nuclear transport 49,50 and, to more complex behaviours such as circadian rhythmicity. 51,52 A good example of how small-scale modelling can be used to understand the design principles of an adverse outcome is demonstrated through the study of the glutathione antioxidant defence network in mammals.…”

Can a systems approach produce a better understanding of mood disorders?

“…This would include the absolute concentration of every mRNA or protein (accurately), plus the kinetic parameters for all enzymatic reactions, the rate of transcription, translation etc. This level of detail is seldom available for all components of a biological system, meaning that fully quantitative models are usually limited to small-scale 'bottom-up' approaches [24][25][26]. One potential work-around for this problem can be seen in physiologically based pharmacokinetic models; these predict the movement of chemicals around the entire body in a quantitative manner.…”

Molecular biology II: protein function

“…Regardless of the specificity of the antibody used, one important limitation of Western blotting is that it can only really be seen as semi quantitative, looking for the relative expression of a target in different samples, for example (5). For true quantification of protein species it is necessary to turn to methods that combine chromatography with mass spectrometry of NMR, which are both more sensitive/specific and able to provide absolute expression levels for a protein (6).…”