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PLoS One
2013 May 07;85:e64854. doi: 10.1371/journal.pone.0064854.
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A multi-system approach assessing the interaction of anticonvulsants with P-gp.
Dickens D
,
Yusof SR
,
Abbott NJ
,
Weksler B
,
Romero IA
,
Couraud PO
,
Alfirevic A
,
Pirmohamed M
,
Owen A
.
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30% of epilepsy patients receiving antiepileptic drugs (AEDs) are not fully controlled by therapy. The drug transporter hypothesis for refractory epilepsy proposes that P-gp is over expressed at the epileptic focus with a role of P-gp in extruding AEDs from the brain. However, there is controversy regarding whether all AEDs are substrates for this transporter. Our aim was to investigate transport of phenytoin, lamotrigine and carbamazepine by using seven in-vitro transport models. Uptake assays in CEM/VBL cell lines, oocytes expressing human P-gp and an immortalised human brainendothelial cell line (hCMEC/D3) were carried out. Concentration equilibrium transport assays were performed in Caco-2, MDCKII ±P-gp and LLC-PK1±P-gp in the absence or presence of tariquidar, an inhibitor of P-gp. Finally, primary porcine brain endothelial cells were used to determine the apparent permeability (Papp) of the three AEDs in the absence or presence of P-gp inhibitors. We detected weak transport of phenytoin in two of the transport systems (MDCK and LLC-PK1 cells transfected with human P-gp) but not in the remaining five. No P-gp interaction was observed for lamotrigine or carbamazepine in any of the seven validated in-vitro transport models. Neither lamotrigine nor carbamazepine was a substrate for P-gp in any of the model systems tested. Our data suggest that P-gp is unlikely to contribute to the pathogenesis of refractory epilepsy through transport of carbamazepine or lamotrigine.
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23741405
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Figure 2. Uptake of AEDs into CEM and VBL100 cell lines. Cells were incubated for 30 minutes in transport buffer with (a) 5µM 3H-digoxin or (b) 5µM 14C-phenytoin or (c) 5µM 14C-lamotrigine or (d) 5µM 14C-carbamazepine in the absence or presence of 300nM tariquidar (TQR). Uptake into cell lines is shown as pmoles per million cells and the data is expressed as mean ±SD (n = 3). ** Significantly different from the appropriate control sample as indicated (P<0.01).
Figure 3. Concentration equilibrium approach in LLC-PK1 transfected with human P-gp for the transport of AEDs. Transport of a) 5µM 3H-digoxin or (b) 5µM 14C-phenytoin or (c) 5µM 14C-lamotrigine or (d) 5µM 14C-carbamazepine in LLC-PK1±P-gp in the absence or presence of 300nM tariquidar. Samples were taken at each indicated time point over a 6 hour time course with the percentage concentration of drug determined in the apical and basal compartments. Data are expressed as mean ±SD (n = 3). * significantly different compared to wild type cells (* P<0.05, ** P<0.01). # significantly different compared to LLC-PK1+P-gp cells in the absence of tariquidar (# P<0.05, ## P<0.01, ### P<0.001).
Figure 4. Transport of AEDs in oocytes expressing human P-gp.a) Percentage efflux of digoxin from cRNA injected oocytes compared to water injected negative control oocytes. Intra-oocyte concentration of 1µM 3H-digoxin ±40µM PSC833 with data expressed as mean ± SD (n≥3, 8–10 oocytes per experiment). The significance values are * (P<0.05) compared to water injected oocytes and # (P<0.05) compared to P-gp injected oocytes. b) Percentage efflux of 14C-phenytoin from cRNA injected oocytes compared to water injected negative control oocytes. Intra-oocyte concentration of 5µM 14C-phenytoin ±40µM PSC833 with data expressed as mean ± SD (n≥3, 8–10 oocytes per experiment). c) Accumulation of 14C-phenytoin, 14C-lamotrigine or 14C-carbamazepine, in oocytes expressing human P-gp. The accumulation of drug into oocytes with 20µM drug ±40µM PSC833 in transport buffer was determined as pmoles per oocyte, from oocytes expressing human wild-type P-gp, triple SNP variant or ATPase dead mutant (AD) compared to water injected negative control oocytes. Data are expressed as mean ± SD (n≥3, 8–10 oocytes per experiment).
Figure 5. Uptake of AEDs into a human brain endothelial cell line (hCMEC/D3). Cells were incubated for 30 minutes in transport buffer with (a) 5µM 3H-digoxin or (b) 5µM 14C-phenytoin or (c) 5µM 14C-lamotrigine or (d) 5µM 14C-carbamazepine in the absence or presence of 300nM tariquidar (TQR). Uptake into cell lines shown as pmoles per million cells and the data is expressed as mean ±SD (n = 3). ** significantly different compared to cells without inhibitor (** P<0.01).
Figure 6. Apparent permeability of AEDs in apical to basal direction in a primary porcine brain endothelial monolayer. Cells were grown on transwells and drug added to apical compartment in transport buffer with (a) 6µM 3H-digoxin or (b) 6µM 14C-phenytoin or (c) 6µM 14C-lamotrigine or (d) 6µM 14C-carbamazepine in the absence or presence of 300nM tariquidar, 100µM verapamil and 100µM prazosin. Data are expressed as mean ±SD (n = 3) with * indicating significant difference compared to control Papps (* P<0.05, ** P<0.01, *** P<0.001).
Figure 1. Relative membrane expression of P-gp in cell lines.Expression of P-gp determined using an extracellular epitope-specific antibody with flow cytometry performed. P-gp protein is expressed as relative fluorescence units with values relative to the CEM cell line and expressed as mean ±SD (n = 3).
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