Case Study 8: Status of the Structural Mass Action Kinetic Model of P-gp-Mediated Transport Through Confluent Cell...

In the first edition of this book, we presented the basics of explicitly incorporating the lipid biochemistry into a confluent cell monolayer transport model and the novel findings of this model up to 2013, including the use of global optimization to fit the elementary rate constants and the efflux active P-glycoprotein (P-gp) membrane concentrations for the transport of four P-gp substrates across MDCKII-hMDR1-NKI confluent cell monolayers. This chapter is an update on that model, which has been focused primarily on discovering how microvilli morphology regulates the efflux active P-gp and the existence of, as yet, unidentified uptake transporters of P-gp substrates in all of the commonly used P-gp expressing cell lines used in the pharmaceutical industry, thereby adding new players to DDI predictions and IVIVE. The structural mass action kinetic model uses the general mass action reactions for P-gp binding and efflux, with the membrane structural parameters for the confluent cell monolayer to predict drug transport over time. Binding of drug to P-gp occurs within the cytosolic monolayer of the apical membrane, according to (a) the molar partition coefficient of the drug to the cytosolic monolayer and (b) the association rate constant, k₁ (M^-1 s^-1), of the drug from the basolateral or apical outer monolayers into the P-gp binding site. Release of substrate from P-gp back into the cytosolic monolayer occurs with a dissociation rate constant k_r (s^-1) or, much less frequently, into the apical aqueous chamber with an efflux rate constant k₂ (s^-1). The model fits the efflux active P-gp concentration, T(0), i.e., the P-gp whose effluxed drug actually reaches the apical aqueous chamber, as opposed to the majority of P-gp whose effluxed drug is reabsorbed back into the same or neighboring microvilli prior to reaching the apical aqueous chamber. Efflux active P-gp largely resides near the tips of the microvilli. We have shown using kinetics and structured illumination microscopy that: (a) efflux active P-gp is controlled by microvilli morphology; (b) there are apical (AT) and basolateral (BT) uptake transporters for P-gp substrates in most, if not all, P-gp expressing cell lines used in the pharmaceutical industry, which exist, but which remain unidentified; (c) the lab-to-lab variability in P-gp IC₅₀ values observed in the P-gp IC₅₀ initiative was due to the conflated inhibition of P-gp and the basolateral digoxin uptake transporters by all 15 P-gp substrates tested in that study; (d) even the IC₅₀ values for P-gp inhibition alone do not obey the Cheng-Prusoff relationship; (e) the fitted elementary rate constants and the molecular dissociation constant Ki for this kinetic model are system independent; and (f) the time dependence of product formation for these confluent cell monolayers is correlated with the P-gp V_max/K_m, when defined by its fitted elementary rate constants and uptake transporter clearances, without any steady-state assumptions.