Decrease in Intracellular Concentration Causes the Shift in K_m Value of Efflux Pump Substrates

Abstract

ABSTRACT:Passive permeability and active efflux are parallel processes in transcellular flux. Therefore, the observed kinetics of a transporter substrate depends on both of these factors. The transporter expression has been shown to affect both the apparent K m and V max values. Kinetic parameters can be obtained from various experimental settings, but these do not necessarily reflect the situation in transcellular flux. Kinetic absorption models need reliable estimates of saturable kinetics when accurate in silico predictions are to be made. The effect of increasing P-glycoprotein expression on apparent transport kinetics was studied using quinidine and digoxin as model compounds. The intracellular concentrations of drugs during the transport process were also measured. A dynamic simulation model was constructed to study the observed data. The apparent K m and V max values increased as the P-glycoprotein expression increased. Simulations reproduced the shift in both kinetic parameters as a function of efflux pump expression. In addition, the apparent K m value showed a strong inverse relationship to the passive permeability. In contrast, the apparent V max value reached a maximum at intermediate passive permeability and declined above and below this passive permeability. The true V max and K m values were never reached. The shift in K m was assigned to a decrease in intracellular concentration at the Pglycoprotein interaction site with both experimental and simulation data. In conclusion, the apparent kinetic parameters in transcellular permeability assays depend on passive permeability and efflux pump activity. Therefore, parameters that are obtained from in vitro assays should be cautiously applied to in vivo predictions.

Intestinal transporters for endogenic and pharmaceutical organic anions: the challenges of deriving in-vitro kinetic parameters for the prediction of clinically relevant drug–drug interactions

“…It is interesting to notice that in the study from Balimane et al [187] , the A-B transport data gave more stringent IC50 values than the B-A transport data, when the opposite was the case for the deduction of Km by others. [188,189] A more stringent IC50 value from the A-B data may be explained by a lower apical permeability of the probe substrate thus limiting its intracellular concentration. Consequently, a lower concentration of the drug substance (acting as an inhibitor) is required for the inhibition of the efflux transporter.…”

“…In addition, it has been shown that Km values deducted with basis in extracellular drug concentrations are dependent on the expression levels of the efflux transporter, since an increased efflux capacity due to a high expression level will result in a lower intracellular drug concentration. [189,194] Hence, it is not surprising, that kinetic constants differ greatly between in-vitro systems for the human efflux transporter P-gp even though the same calculation method was applied, as the results are dependent on both influx permeability and expression level of the transporter. [30,189,194,195] In addition to the above presented drawbacks in using Michaelis-Menten kinetics for estimating drug affinity for efflux transports, the interpretation of transcellular transport data across a double-membrane as if they were from a singlemembrane system, has been criticized also.…”

“…[189,194] Hence, it is not surprising, that kinetic constants differ greatly between in-vitro systems for the human efflux transporter P-gp even though the same calculation method was applied, as the results are dependent on both influx permeability and expression level of the transporter. [30,189,194,195] In addition to the above presented drawbacks in using Michaelis-Menten kinetics for estimating drug affinity for efflux transports, the interpretation of transcellular transport data across a double-membrane as if they were from a singlemembrane system, has been criticized also. [196] The ongoing transgression of Michaelis-Menten kinetics for determining kinetic constants for efflux transporters and the difficulties in estimating the enterocytic concentration has been recognized by several groups that have attempted to derive Ki and Km values from more or less complex three-compartment models and simulation models.…”

“…[196] The ongoing transgression of Michaelis-Menten kinetics for determining kinetic constants for efflux transporters and the difficulties in estimating the enterocytic concentration has been recognized by several groups that have attempted to derive Ki and Km values from more or less complex three-compartment models and simulation models. [30,189,190,[196][197][198][199] However, a thorough validation of these models is still lacking and it is outside the scope of this review to further address these models. As emphasized by Tachibana et al [193] , there is a need of standardizing both the in-vitro methods and the method used for the Ki/IC50 calculations.…”

“…This has been shown in both Caco-2 cells and single transfected MDCK cells. [188,189] The discrepancy has been explained by differences in the passive influx permeability across the apical and basolateral membrane that is suggested to arise from different compositions or surface areas of these membranes. [188,190] Alternatively, it may reflect that the binding site of the transporter is located within the apical membrane and not in the cytosol.…”