TAKE HOME MESSAGES FORM THE ITC PAPERS part 5
Ultimately the goal is to translate in vitro and preclinical data into clinically meaningful parameters. This requires derivation of accurate measurements from in vitro and in vivo preclinical assays that can be used to populate predictive modeling programs. Zamek-Glyszczynski et al. provides an overview of the different kinetic models that can be used and the considerations when evaluating the utility of the data produced.
Uptake: For uptake substrate kinetics, often the conventional 2 step method is used, based on data generated mostly from transfected cell lines. This model, however, has its limitations in that it does not take into account ’the bidirectional nature of passive diffusion, intracellular binding, metabolism or active efflux.’ Additional experiments for mechanistic compartmental modeling are required to address parameters beyond active uptake. If not only uptake but also metabolism and active efflux are modeled, which will affect the ability to study specific interactions, the use of more complex models such as suspension hepatocytes or sandwich cultures is required. Regardless of the assay format chosen, care must be taken in assay design to limit the contribution of confounding processes.
Efflux: For polar, hydrophilic substrates, inside out vesicles are a good tool to study enzyme kinetics, as the transporter can interact directly with the substrate binding site. For more lipophilic substrates, however, polarized cell monolayers are a preferred assay system. However, monolayers do not follow the principles of simple enzyme kinetics, due to their complexity. The authors state that the traditional model, using Jmax (maximum flux) and EC50 (half maximum flux), where Jmax is often interpreted as the Vmax and EC50 as the Km, is not appropriate since C0 in monolayers does not represent the actual concentration at the transporter since the transporter binding site is inside the cells. Three different compartmental models are described: a three compartment model (basolateral, intracellular and apical), a five compartment model (basolateral, basolateral membrane, intracellular, apical membrane and apical) and a structural model, which considers binding of substrate from within the inner leaflet of the apical membrane. The use of these models is recommended in order to estimate correct kinetic parameters when using monolayer assays.
In vivo knock-out models can be a useful tool to understand the impact of transporters on ADME properties of a compound. Three points need to be considered, though:
1) compound should have similar PK in rodents as in human
2) species differences in expression, affinity and inhibitory potency should be assessed in vitro
3) compensatory mechanisms should be understood (up- or downregulation of other transporters or enzymes in the knock-out)
Zamek-Gliszczynski, M.J., et al., ITC Recommendations for Transporter Kinetic Parameter Estimation and Translational Modeling of Transport-Mediated PK and DDIs in Humans. Clin Pharmacol Ther, 2013. 94(1): p. 64-79.