Interactions of attention-deficit/hyperactivity disorder therapeutic agents with the efflux transporter P-glycoprotein

Abstract

The objective of this study was to assess the potential interactions of the drug transporter P-glycoprotein with attention-deficit/hyperactivity disorder (ADHD) therapeutic agents atomoxetine--and the individual isomers of methylphenidate, amphetamine, and modafinil utilizing established in vitro assay. An initial ATPase assay indicated that both d- and l-methylphenidate have weak affinity for P-glycoprotein. The intracellular accumulation of P-glycoprotein substrates doxorubicin and rhodamine123 in the P-glycoprotein overexpressing cell line LLC-PK1/MDR1 was determined to evaluate potential inhibitory effects on P-glycoprotein. The results demonstrated that all compounds, except both modafinil isomers, significantly increased doxorubicin and rhodamine123 accumulation in LLC-PK1/MDR1 cells at higher concentrations. To investigate the P-glycoprotein substrate properties, the intracellular concentrations of the tested compounds in LLC-PK1/MDR1 and P-glycoprotein negative LLC-PK1 cells were measured in the presence and absence of the P-glycoprotein inhibitor PSC833. The results indicate that the accumulation of d-methylphenidate in LLC-PK1 cells was 32.0% higher than in LLC-PK1/MDR1 cells. Additionally, coadministration of PSC833 leads to 52.9% and 45.6% increases in d-modafinil and l-modafinil accumulation, respectively, in LLC-PK1/MDR1 cells. Further studies demonstrated that l-modafinil transport across LLC-PK1/MDR1 cell monolayers in the basolateral-to-apical (B-A) direction was significantly higher than in the apical-to-basolateral (A-B) direction. PSC833 treatment significantly decreased the transport of l-modafinil in B-A direction. In conclusion, our results suggest that all tested agents with the exception of modafinil isomers are relatively weak P-glycoprotein inhibitors. Furthermore, P-glycoprotein may play a minor role in the transport of d-methylphenidate, d-modafinil, and l-modafinil.

Fig. 1

Concentration-dependency of ortho-vanadate sensitive substrate-induced P-glycoprotein ATPase activity as measured by inorganic phosphate release. Data points are expressed as the means of duplicate incubations. Lines represent the nonlinear regression lines of best fit for the Michaelis–Menten equation for d- and l-methylphenidate and positive control verapamil.

Fig. 2

Effect of ADHD therapeutic agents on the intracellular accumulation of doxorubicin and rhodamine123 in LLC-PK1/MDR1 and LLC-PK1 cells. Inhibitory effects on P-glycoprotein function in LLC-PK1/MDR1 cells were assessed by measuring the intracellular concentrations of doxorubicin A, B and rhodamine123 D, E after incubation for 1 h in the presence or absence of specific concentrations of each tested drug. PSC833 (2 μM) and quinine (200 μM) were included as positive controls. The inhibitory efficiency of PSC833 was defined as 100%. LLC-PK1 cells were also incorporated as the negative controls C, F. Each data value is the mean with a bar representing S.D., n=3. *P<0.05, **P<0.01 versus control.

Fig. 2

Effect of ADHD therapeutic agents on the intracellular accumulation of doxorubicin and rhodamine123 in LLC-PK1/MDR1 and LLC-PK1 cells. Inhibitory effects on P-glycoprotein function in LLC-PK1/MDR1 cells were assessed by measuring the intracellular concentrations of doxorubicin A, B and rhodamine123 D, E after incubation for 1 h in the presence or absence of specific concentrations of each tested drug. PSC833 (2 μM) and quinine (200 μM) were included as positive controls. The inhibitory efficiency of PSC833 was defined as 100%. LLC-PK1 cells were also incorporated as the negative controls C, F. Each data value is the mean with a bar representing S.D., n=3. *P<0.05, **P<0.01 versus control.

Fig. 2

Effect of ADHD therapeutic agents on the intracellular accumulation of doxorubicin and rhodamine123 in LLC-PK1/MDR1 and LLC-PK1 cells. Inhibitory effects on P-glycoprotein function in LLC-PK1/MDR1 cells were assessed by measuring the intracellular concentrations of doxorubicin A, B and rhodamine123 D, E after incubation for 1 h in the presence or absence of specific concentrations of each tested drug. PSC833 (2 μM) and quinine (200 μM) were included as positive controls. The inhibitory efficiency of PSC833 was defined as 100%. LLC-PK1 cells were also incorporated as the negative controls C, F. Each data value is the mean with a bar representing S.D., n=3. *P<0.05, **P<0.01 versus control.

Fig. 3

P-glycoprotein substrate properties of ADHD therapeutic agents were assessed by measuring the intracellular accumulation of tested drugs in LLC-PK1 and LLC-PK1/MDR1 cells. Intracellular concentrations of tested agents were measured after incubation for 1 h in the presence or absence of the P-glycoprotein selective inhibitor PSC833 (2 μM) in LLC-PK1 (solid bars) and LLC-PK1/MDR1 (open bars). Data are presented as mean± S.D. (n=3). *P<0.05, **P<0.01 versus control.