Extrapolation of Elementary Rate Constants of P-glycoprotein–Mediated Transport from MDCKII-hMDR1-NKI to Caco-2 Cells

Abstract

The best parameters for incorporation into mechanistic physiologically based pharmacokinetic models for transporters are system-independent kinetic parameters and active (not total) transporter levels. Previously, we determined the elementary rate constants for P-glycoprotein (P-gp)-mediated transport (on- and off-rate constants from membrane to P-gp binding pocket and efflux rate constant into the apical chamber) using the structural mass action kinetic model in confluent MDCKII-hMDR1-NKI cell monolayers. In the present work, we extended the kinetic analysis to Caco-2 cells for the first time and showed that the elementary rate constants are very similar compared with MDCKII-hMDR1-NKI cells, suggesting they primarily depend on the interaction of the compound with P-gp and are therefore mostly independent of the in vitro system used. The level of efflux active (not total) P-gp is also fitted by our model. The estimated level of efflux active P-gp was 5.0 ± 1.4-fold lower in Caco-2 cells than in MDCKII-hMDR1-NKI cells. We also kinetically identified the involvement of a basolateral uptake transporter for both digoxin and loperamide in Caco-2 cells, as found previously in MDCKII-hMDR1-NKI cells, due to their low passive permeability. This demonstrates the value of our P-gp structural model as a diagnostic tool in detecting the importance of other transporters, which cannot be unambiguously done by the Michaelis-Menten approach. The system-independent elementary rate constants for P-gp obtained in vitro are more fundamental parameters than those obtained using Michaelis-Menten steady-state equations. This suggests they will be more robust mechanistic parameters for incorporation into physiologically based pharmacokinetic models for transporters.

Derivation of a System-IndependentKifor P-glycoprotein Mediated Digoxin Transport from System-Dependent IC50Data

“…The k 1 for association of substrates to P-gp is essentially the same for all compounds used for model validation (Lumen et al, 2013;Meng et al, 2017a), consistent with a large open binding site on P-gp (Li et al, 2014). The inhibitor dissociation constant K i = k rQ /k 1 , with respect to the inhibitor concentration in the membrane, so the system independence of k 1 allows K i to be calculated for each inhibitor from k rQ alone.…”

“…We can calculate the inhibitor dissociation constant, K i , from P-gp into the membrane from the inhibitor k rQ and k 1 (see Materials and Methods). We have found previously that k 1 is essentially the same for all of the drugs we have studied in both MDCKII-hMDR1-NKI and Caco-2 cells (Agnani et al, 2011;Lumen et al, 2013;Meng et al, 2017a). %CV is the coefficient of variation as a percentage between the data and the fitted data points.…”

“…The same value has been assumed for the LLC-PK1-hMDR1-NKI cells. However, for Caco-2 cells, Meng et al (2017a) found that k 1Q was about 1.7fold larger. This means that the K i for an inhibitor with the Caco-2 cells would be 1.7-fold smaller than with MDCKII-hMDR1-NKI cells.…”

Modeling Substrate Entry into the P-Glycoprotein Efflux Pump at the Blood–Brain Barrier

“…Therefore, a rhodamine molecule needs to be within ∼2.8 nm of a P-gp to allow direct entry into the central binding cavity prior to jumping out of the second energy minimum. Assuming a P-gp density on the cell surface of 100 μm −2 , 30 the average protein− protein distance is 100 nm, and hence the maximum solute− protein distance of 50 nm. Therefore, the probability of a rhodamine diffusing directly from the energy minimum in the bilayer into the central binding cavity is about 5.6%.…”

A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics

“…In their study, Shirasaka et al found that estimated K m and V max values for quinidine, verapamil, and vinblastine increased with increasing P-gp expression levels. If other transport systems, such as a basolateral uptake transporter 62 or another efflux transporter, is involved in the observed transport of a substrate, then the calculated values for K m and V max becomes convolutions of all transport processes involved, considering the dependency on P-gp expression levels and the potential contribution from other transport systems coexpressed in the cell model. Estimations of K m and V max values will, therefore, be dependent on the used model system.…”