13.8 Uptake assay results
13.8.2 Experiments in GPNT cells treated with antiepileptic drugs
For the uptake assays in GPNT cells after the treatment with AEDs specific P gp substrate digoxin was chosen at concentration digoxin [1 μM]+3H-digoxin [10 kBq/ml].
Beginning of the treatment on the day of the confluence 20.06.08
Digoxin [1µM]+3H-Digoxin [10kBq/ml]
MED IUM
M DEX 1
M PB
300 M TQ
0.5 0
1000 2000 3000 4000
**
3 H-Digoxin uptake (DPM/mg protein)
Fig.29 Digoxin uptake assay on G PNT cells P35. Cells were tr eated with antiepiletic drugs Penobarbital (PB ), Pgp inducer De xamethasone (DE X), and Pgp inhibitor Tariquidar (TQ ) f or 3 days adtarting on the day of cell conf luence.
Drug concent rations are indicated under the graph. Accumulation of digoxin was investigated under the condition:
Digoxin [1µ M]+3H- Digoxin [10kB q/ml] and was normalized on DMP/mg protein. Data are shown as means ± SD of three repetitions. Data we re statistically compared by one-way analysis of variance (ANOVA) f ollowed by B onf errroni’s Multiple Comparison Test. Significant diff erences between compa red groups of data are indicated by two asterisks (P < 0.01).
Fig. 39 Digoxin uptake assay in GPNT cells P35. Cells were treated with antiepiletic drug phenobarbital (PB), Pgp inducer dexamethasone (DEX) for 3 days starting on the day of cell confluence and were stimulated with tariquidar (TQ) for 2 h on the day of the assay. Drug concentrations are indicated under the graph. Accumulation of digoxin was investigated under the condition: digoxin [1 µM]+3H-digoxin [10 kBq/ml] and was normalized on DMP/mg protein. Data are shown as means ± SD of three samples. Data were statistically compared by one -way analysis of variance (ANOVA) followed by Bonferrroni’s Multiple Comparison Test. Significant differences between compared groups of data are indicated by two asterisks (P<0.01).
GPNT cells were treated with AED phenobarbital at concentration 300 μM and with dexamethasone for three days starting on the day of confluence (Fig. 39). Statistical analysis showed significantly increased uptake in cells stimulated with tariquidar.
This experiment was performed when cells were treated with high concentration of phenobarbital (300 μM), in next trial lower concentration (100 μM) was tried which is in range of therapeutic concentrations. Treatment was prolonged up to seven days.
Beginning of the treatment on the day of the confluence Digoxin [1µM]+3H-Digoxin [10kBq/ml]
04.11.08
Treatment for 7 days
ME DIUM
M DE X
1 M DO
X
0.92
M PB
100 M T
Q
0.5 0
500 1000 1500 2000 2500 3000
3H-Digoxin uptake (DPM/mg protein)
Fig.30 Digoxin upta ke assay on GPNT cells P32. Cells were treated with antiepiletic drug Penobarbital (PB), Pgp inducers Dexam ethasone (DEX) and Doxorubicin (DOX), Pgp inhibitor Tariquidar (TQ) for 7 day s starting on the day of cell confluence. Drug concentrations are indicated under the graph. Accum ulation of digoxin was investigated under the condition: Digoxin [1µM]+3H-Digoxin [10kBq/m l] and was norm alized on DMP/m g protein. Data are shown as m eans ± SD of three repetitions. Data were statistically com pared by one-way analy sis of variance (ANOVA). There were no significant differences between sam ples.
Fig. 40 Digoxin uptake assay in GPNT cells P32. Cells were treated with antiepile ptic drug phenobarbital (PB), Pgp inducers dexamethasone (DEX) and doxorubicin (DOX) fo r 7 days starting on the day of cell confluence and were stimulated with tariquidar (TQ) fo r 2 h on the day o f the assay. Drug concentrations are indicated under the graph.
Accumulation of digoxin was investigated under the condition: digoxin [1 µM]+3H-digoxin [10 kBq/ml] and was normalized on DMP/mg protein. Data are shown as means ± SD o f three samples. Data were statistically compared by one -way analysis of variance (ANOVA). There were no significant differences between samples .
As a first AED, phenobarbital was used at concentration 100 µM (Fig. 39) and 300 µM (Fig. 40). As P gp inducer dexamethasone or doxorubicin were used. No P gp induction was observed either after dexamethasone, doxorubicin or phenobarbital. Even, when the treatment period was extended (from 3 day up to 7 days, Fig. 39 & 40) no Pgp alteration in P gp function was observed.However, increased accumulation of digoxin, after treatment with Pgp inhibitor tariquidar, indicates that Pgp was functionally active and inhibited by tariquidar.
Next AED that was tested in uptake assay in GPNT cells was phenytoin at concentration 50 μM (Fig. 41).
Beginning of the treatment on the day of the confluence
Digoxin [1µM]+3H-Digoxin [10kBq/ml]
22.08.08
ME DIUM
M DE X
1 M PH
T 50
M T Q
0.5 0
1000 2000 3000 3000 3500 4000
3 H-Digoxin uptake (DPM/mg protein)
Fig.31 Digoxin uptake ass ay on GPNT cells P3 1. Cells wer e treated with antiepiletic drugs Phenytoi n (PHT), Pgp induc er Dexamethasone (DEX) and Pgp inhibit or Tariquidar (TQ) f or 3 days starti ng on the day of c ell confluence. Drug conc entrations are indicated under the graph. Acc umulation of digoxin was investigated under the condition: Digoxin [ 1µM]+3H-Digoxin [10kBq/ml]
and was normalized on DMP/ mg protein. D ata are shown as means ±SD of three repetitions (except TQ n=1). Data were statisticall y compared by one- way anal ysis of varianc e (ANOVA). There were no significant dif ferences bet ween samples.
Fig. 41 Digoxin uptake assay in GPNT cells P31. Cells were treated with antiepileptic drug phenytoin (PHT), Pg p inducer dexamethasone (DEX) for 3 days starting on the day of cell confluence and were stimulated with tariquidar (TQ) for 2 h on the day of the assay. Drug concentrations are indicated under the graph. Accumulation of digoxin was investigated under the condition: digoxin [1 µM]+3H-digoxin [10 kBq/ml] and was normalized on DMP/mg protein. Data are shown as means ± SD of three samples (except TQ, n=1). Data were statistically compared by one -way analysis of variance (ANOVA). There were no significant differences between samples.
In this experiment no significant differences wer e observed. Anyhow, cells stimulated with tariquidar showed an increase in digoxin uptake assay but because of only one sample per treatment statistica l analysis could not be performed. Another used AED was carbamazepine at concentration 30 µM (Fig. 42).
MED
Fig.32 Digoxin uptake assay on G PNT cells P30, P32, P31 in three indenpendent e xperiments. . Cells were treated with antiepiletic drug Carbamazeine (CB Z), Pgp inducer De xamethasone (DEX) and Pgp inhibitor Tariquidar (TQ ) for 3 days starting on the day of cell confluence. Dr ug concentrations are indicated under the graph. A ccumulation of digoxin was investigated under t he condition: Digoxin [1µM]+3H- Digoxin [10kB q/ml] and was normalized on DMP/mg protein. Data are shown as means ± SD of three repetitions (except TQ n=1). Data were statistically compared by one-way analysis of variance (ANOVA). There were no signif icant diff erences between samples.
A B C
Fig. 42 Digoxin uptake assay in GPNT cells P30, P32, P31 in three independent experiments. Cells were treated with antiepileptic drug carbamazepine (CBZ), Pgp inducer dexamethasone (DEX) for 3 days starting on the day o f confluence and were stimulated with tariquidar (TQ, 0.5 µM) for 2 h on the day of the assay. Drug concentrations are indicated under the graph. Accumulation of digoxin was investigated under the condition: digoxin [1µM]+3 H-digoxin [10kBq/ml] and was normali zed on DMP/mg protein. Data are shown as means ± SD o f three samples (except TQ, n=1). Data were statistically compared by one -way analysis of variance (ANOVA). There were no significant differences between samples.
In previous experiments dexamethasone at concentration 1 µM did not significantly influence Pgp expression. One of the explanations could be the concentration. For that reason in experiment shown in Fig. 42C, concentration of dexamethasone was increased up to 50 µM. Nonetheless, no significant effect on Pgp functionality was observed.
In the experiments presented above, GPNT cells were treated with various AEDs. Treatment was started on the day of the confluence and lasted for 3 days. As a control P gp inducers doxorubicin and dexamethasone were used. However, no increase in P gp expression was found in uptake assays with digoxin. Thus, in next trial digoxin was replaced by vinblastine.
Vinblastine is not so specific like digoxin and could be a better substrate for P gp in presented experiments. As it is shown in Fig. 39 vinblastine as a substrate for Pgp in uptake assay worked well and provided significant results after the treatment with Pgp inducers. In next trials with vinblastin e applied concentration was vinblastine [1 μM]+3H-vinblastine [4.2 kBq/ml].
Next experiments were performed either on the day of confluence (Fig. 43) or 6 days after confluence (Fig. 44). GPNT cells were treated with several AEDs: phenobarbital, carbamazepine and phenytoin.
As positive control dexamethasone was used. Functionality of assays was confirmed by tariquidar.
ME DIUM
M DE X 1
M PB
100 M CB Z 30
M PH T 50
M TQ
0.5 0
2000 4000 6000 8000 10000 12000 14000
**
***
3 H-Vinblastine uptake (DPM/mg protein)
Fig. 43 Vinblastine uptake assay in GPNT cells P40. Cells were treated with antiepileptic drugs phenobarbital (PB), carbamazepine (CBZ), phenytoin (PHT) and Pgp inducer dexamethasone (DEX) fo r 3 days starting on the day o f confluence and were stimulated with tariquidar (TQ) for 2 h on the day o f the assay. Drug concentrations are indicated under the graph. Accumulation of vinblastine was investigated under the condition: vinblastine [1 µM]+3H-vinblastine [4.2 kBq/ml] and was normalized on DMP/mg protein. Data are shown as means ± SD of three samples (except MEDIUM, n=6). Data were statistically compared by one -way analysis of variance (ANOVA) followed by Bonferrroni’s Multiple Comparison Test. Significant differences between compared groups of data are indicated by two asterisks (P<0.01) and three asterisks (P < 0.001). This uptake assay was performed by Carlos Luna Tórtos.
Beginning of the treatment 6 days after confluence
16.12.08 Vinblastine [1µM]+
3H-Vinblastine [4.2kBq/ml]
Carlos
Fig.34 Vinbla stine upta ke assay on G PNT cells P40. Cells were treated with antiepileptic drugs pheno barbital (PB), carbamazepine (CBZ), phenytoin (PHT) and Pgp inducer dexamethasone (DEX) and Pg p i nhi bitor tariquidar (TQ ) for 3 days starting 6 days after confluence. Drug concentrations are indicated under the graph. A ccumulatio n of vi nbla stine was i nvestigated under the condition:
Vinblasi ne [1µM]+3HVinblasine [4.2kBq/ ml] and was norma lized o n DMP/mg protein. Data were statistically compared by one -way analysis of variance (ANOVA) followed by Bonferrroni’s Multiple Comparison Test. Significant differences between compared groups of data are indicated by one asteris k (P<0.05), two asterisks (P<0.01) and three asterisks (P < 0.001). Thi s upta ke assay was performed by Carlos Luna Tórtos.
Fig. 44 Vinblastine uptake assay in GPNT cells P40. Cells were treated with antiepileptic drugs phenobarbital (PB), carbamazepine (CBZ), phenytoin (PHT) and Pgp inducer dexamethasone (DEX) for 3 days starting 6 days after confluence and were stimulated with tariquidar (TQ) for 2 h on the day o f the assay. Drug concentrations are indicated under the graph. Accumulation o f vinblastine was investigated under the condition: vinblastine [1 µM]+3H-vinblastine [4.2 kBq/ml] and was normalized on DMP/mg protein. Data are shown as means ± SD of three samples (except MEDIUM, n=6). Data were statistically co mpared by one -way analysis of variance (ANOVA) followed by Bonferrroni’s Multiple Comparison Test. Significant differences between compared groups of data are indicated by one asterisk (P<0.05), two asterisks (P<0.01) and three asterisks (P < 0.001). This uptake assay was performed by Carlos Luna Tórtos.
In both trials (Fig. 43 & 44) dexamethasone affected P gp function and resulted in significant decrease in vinblastine uptake. Additionally, treatment with phenytoin also gave significant effect on Pgp functionality (Fig. 34). The correctness of the assays was proved by Pgp inhibitor tariquidar.
Stimulation with Pgp inducer dexamethasone caused lower accumulation of radioactive vinblastine compared to controls. In cells, where tariquidar was used, transport of vinblastine was inhibited and resulted in higher accumulation of substrate in the cells.
Apart from GPNT cells, MDCK-WT cells were also used in uptake assay. Uptake assays were performed with both Pgp substrates digoxin (Fig. 46) and vinblastine (Fig. 45). Vinblastine is not as specific as digoxin and can be transported also by multidrug resistance-associated proteins (MRPs).
Thus, to confirm that vinblastine uptake was due to P gp induction, specific inhibitor of MRPs called MK571 was used (Fig. 45).
In both experiments treatment was initated on the day of the cell confluence and was performed for three days. Uptake assays were made on 4th day of confluence.
Beginning of the treatment on the day of the confluence 27.01.09
Carlos Vinblastine [1µM]+3H-Vinblastine [4.2kBq/ml]
MEDIUM M DEX 1
M PB
100 M CBZ 30
M PHT 50
M TQ
0.5 M MK571
50 0
2500 5000 7500 10000 12500 60000 70000 80000
***
***
3H-Vinblastine uptake (DPM/mg protein)
Fig.35 Vinblastine uptake assay on MDCK-W T cells P9. C ell s wer e treated with antiepi leptic drug s phenobarbital (PB), carbam azepin e (CBZ), phen ytoin (PHT) and Pgp inducer d examethason e (DEX), Pgp inhibitor tariqu idar (TQ) and MRPs inhibitor MK571 for 3 d ays starting on the day of conflu ence. Drug concentration s are indicated under the graph.
Accumulation of vinblastine was investigated under the condition: Vinblasine [1µM]+3H-Vinblasine [4. 2kBq/ml] and was normalized on DMP/mg protein. Data wer e statisti cal l y compar ed b y one- way an al ysis of vari ance (ANOVA) follo wed b y Bonferrroni’s Multiple Comparison Test. Significant differences b etween compared groups of data are indicated b y three asterisks (P < 0. 001). This uptake assay was p erformed b y Carlos Luna Tórtos.
Fig. 45 Vinblastine uptake assay in MDCK-WT cells P9. Cells were treated with antiepileptic drugs phenobarbital (PB), carbamazepine (CBZ), phenytoin (PHT) and Pgp inducer dexamethasone (DEX) for 3 days starting on the day of confluence and were stimulated with Pgp inhibito r tariquidar (TQ) and MRPs inhibito r MK571 for 2 h on the day o f the assay. Drug concentrations are indicated under the graph. Accumulation o f vinblastine was investigated under the condition: vinblastine [1 µM]+3H-vinblastine [4.2 kBq/ml] and was normalized on DMP/mg protein. Data are shown as means ± SD o f three samples (except MEDIUM, n=6). Data were statistically compared by o ne -way analysis of variance (ANOVA) follo wed by Bonferrroni’s Multiple Comparison Test. Significant differences between compared groups of data are indicated by three asterisks (P<0.001). This uptake assay was performed by Carlos Luna Tórtos.
Beginning of the treatment on the day of the confluence Carlos 20.02.09 Digoxin [1µM]+3H-Digoxin [10kBq/ml
MEDIUM M DEX 1
M PB
100 M CBZ 30
M PHT 50
M TQ
0.5 0
5000 10000 15000
3 H-Digoxin uptake (DPM/mg protein)
***
Fig.36 Digoxin uptake assay on MDCK-WT cell s P16. Cel ls wer e treated with antiepi leptic drugs ph enobarbital (PB), carbam azepin e (CBZ), phen ytoin (PHT) and Pgp inducer dexamethasone (DEX), Pgp inhib itor tariquidar (TQ) and MRPs inhibitor MK571 for 3 days starting on the day of confluence. Drug concentrations ar e indicated under the graph.
Accumulation of vinblastine was investigat ed under the condition: Digoxin [1µM]+3H-Digoxin [4.2kBq/ml] and was normalized on DMP/mg protein . Data wer e statisticall y comp ared b y on e- way an al ysis of varian ce ( ANOVA) follo wed b y Bonferrroni’s Multiple Comparison Test. Significant differen ces b et ween compared groups of data are ind icated b y three asteri sks (P < 0.001). Thi s uptake assay was p erformed b y Carlo s Luna Tórtos.
Fig. 46 Digoxin uptake assay in MDCK-WT cells P16. Cells were treated with antiepileptic drugs phenobarbital (PB), carbamazepine (CBZ), phenytoin (PHT) and Pgp inducer dexamethasone (DEX) for 3 days starting on the day of confluence and were stimulated with tariquidar (TQ) for 2 h on the day o f the assay. Drug concentrations are indicated under the graph. Accumulation of digoxin was investigated under the condition: digoxin [1 µM]+3 H-digoxin [10 kBq/ml] and was normalized on DMP/mg protein. Data are shown as means ± SD o f three samples (except MEDIUM, n=6). Data were statistically compared by one -way analysis of variance (ANOVA) follo wed by Bonferrroni’s Multiple Comparison Test. Significant differences between compared groups of data are indicated by three asterisks (P < 0.001). This uptake assay was performed by Carlos Luna Tórtos.
Uptake assays in MDCK cells have revealed no alternations in Pgp expression after dexamethasone and AEDs treatment. There was significantly increased vinblastine and digoxin uptake after Pgp inhibitor tariquidar and MRP inhibitor MK571.
ME DIUM
M DE X 1 M DO
X
0.92
M PB 300 M CB
Z 30
M PH T 50
M T Q
0.5 0
50 100 150
**
***
200 250 300
3 H-Digoxin uptake (DPM/mg protein)
Fig. 47 Digoxin uptake assay in GPNT cells within P30 & P35 in seven indenpendent experiments. Cells were treated with antiepiletic drugs phenobarbital (PHT), carbamazepine (CBZ), phenytoin (PHT) and Pgp inducer dexamethasone (DEX) and doxorubicin (DOX) for 3 days starting on the day of confluence and were stimulated with tariquidar (TQ, 0.5 µM) for 2 h on the day of the assay. Drug concentrations are indicated under the graph.
Accumulation of digoxin was investigated under the condition: digoxin [1µM]+3H-digoxin [10kBq/ml] and was normalized on DMP/mg protein. Data are shown as means ± SD of samples (MEDIUM, n=28; DEX, n=19; DOX, n=3; PB, n=3; CBZ, n=11; PHT, n=5; TQ, n=11). Data were statistically compared by one -way analysis of variance (ANOVA) and post test t-test (MEDIUM vs DEX & MEDIUM vs TQ). Significant differences between compared groups of data are indicated by two asterisks (P<0.01) and three asterisks (P < 0.001).
In Fig. 47, data from 7 independent xperiements in GPNT cells are summarized. Overall, there was significantly decreased uptake with Pgp substrate digoxin after dexamethasone and no changes in Pgp functionality after AED treatments. Functionality of Pgp was proven by using specific Pgp inhibitor tariquidar, which significantly inhibited accumulation of digoxin (Fig. 47).
14 Discussion
The main question addressed in presented thesis dealt with possible P gp induction after AEDs.
First suggestion that AEDs can be substrates for P gp came from experiments by Tishler (Tishler et al., 1995). It has been reported that several major AEDs (carbamazepine, phenobarbital, phenytoin) are transported either by Pgp or MRPs or both (Löscher and Potschka, 2002). This would explain lower concentration of AEDs in the brain, which would be a result of active transport of drugs by multidru g transporters. However, which AEDs are substrates for P gp is still very controversial issue because of contradictory data about multidrug transporter substrates (Owen et al., 2001; Crowe and Teoh, 2006;
Luna-Tórtos et al., 2008).
The purpose of presented PhD thesis was to check whether AEDs can induce Pgp expression in different cells lines. However, in order to start answering this question, funtiona l protocols for methods used in thesis had to be established. Western blotting method, used for Pgp expression and uptake assay, applied for Pgp functionality, had to be specified.