title: Decrease in intracellular concentration causes the shift in Km value of efflux pump substrates.
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 P-glycoprotein 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.
authors.affiliation: Department of Pharmaceutics, University of Kuopio, Yliopistonranta 1C, FI-70210, Kuopio, Finland. Timo.Korjamo@uku.fi
authors.forename: Timo
authors.initials: T
authors.lastname: Korjamo
authors.forename: Heidi
authors.initials: H
authors.lastname: Kemiläinen
authors.forename: Aki T
authors.initials: AT
authors.lastname: Heikkinen
authors.forename: Jukka
authors.initials: J
authors.lastname: Mönkkönen
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000378:metabolism; D000962:Antimalarials / Q000378:metabolism; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003198:Computer Simulation; D004077:Digoxin / Q000378:metabolism; D004285:Dogs; D006801:Humans; D007700:Kinetics; D015233:Models, Statistical; D010539:Permeability; D011802:Quinidine / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000889:Anti-Arrhythmia Agents; D000962:Antimalarials; D002352:Carrier Proteins; D004077:Digoxin; D011802:Quinidine