Inhibition of P-Glycoprotein Function by Several Antidepressants may not Contribute to Clinical Efficacy

• Introduction
• Materials and Methods
• Materials
• Isolation of PBCEC's
• Cell culture
• Calcein-AM Assay
• ATP-Assay
• Statistics
• Results
• Discussion
• Acknowledgment
• References

Introduction: In many depressive patients the negative feedback mechanism of the HPA (hypothalamic-pituitary-adrenocortical) axis is impaired. It has been suggested that antidepressants inhibit membrane glucocorticoid transporters like P-Glycoprotein (Pgp) and hence enhance the intracellular glucocorticoid concentration, leading to an increased glucocorticoid-receptor mediated gene transcription and therefore to normalization of the function of the HPA axis. The aim of this study is to investigate inhibition of Pgp by several different antidepressants. Methods: We characterized the inhibitory potencies of the antidepressants in two in vitro assays by using calcein-AM as Pgp substrate. The two different cell-systems expressing Pgp were: 1. PBCEC (porcine brain capillary endothelial cells) as model for the blood-brain-barrier, and 2. A human lymphocytic leukaemia cell line CEM and the multi-drug-resistant (MDR) cell line VLB-100, expressing Pgp as model for the human protein. Results: All of the antidepressants tested inhibit the transport of calcein-AM by Pgp in the micromolecular range. Discussion: Because this inhibition is only seen at concentrations above therapeutically relevant plasma levels, their effect my not play a role for the mechanism of action of the antidepressants tested.

Introduction

In many depressed patients, the function of the HPA (hypothalamic-pituitary-adrenocortical) system is altered, probably because the corticosteroid-receptor (CR) signaling is impaired [14] [21]. This may lead to impaired feedback inhibition, which may result in increased production and secretion of CRH in various brain regions and in increased circulating levels of glucocorticoids [14]. Different studies from animals and in vitro data suggest that treatment with antidepressants leads to increased glucocorticoid-receptor (GR) expression and function, and thus normalizes the impaired negative feedback inhibition of the HPA axis.

Pariante et al. hypothesized that antidepressants enhance GR-mediated gene transcription by inhibiting membrane steroid transporters like P-Glycoprotein (Pgp) [22]. Inhibition of these transporters may lead to increased intracellular concentration of glucocorticoids and therefore enhanced gene transcription [22]. They showed evidence that clomipramine may normalize the function of the HPA axis by enhancing the function of the GR's. This effect could be blocked by co-incubation with verapamil, a known inhibitor of Pgp. Comparable effects were seen for several other antidepressants from different classes, for example fluoxetine [19]. They concluded that part of the efficacy of antidepressants could be explained by inhibition of Pgp and hence an increased GR-mediated gene transcription [20].

Since conflicting data have been reported concerning the inhibition of Pgp by antidepressants especially regarding specificity and potency [7] [8] [36]. We investigated several antidepressant drugs out of different classes in respect to their ability to inhibit Pgp function. We used two different systems: 1. CCRF-CEM cells, a human lymphocytic leucemia cell line, (not expressing Pgp) and its multidrug resistant variant CEM/VLB-100 cells (expressing Pgp) as a model for the human Pgp, and 2. Porcine brain capillary endothelial cells (PBCEC) expressing Pgp as a model for the blood brain barrier. To test the specificity of our findings, we additionally investigated several antipsychotics under similar conditions.

Materials and Methods

Materials

The CCRF-CEM (CEM) and CEM/VLB-100 (VLB) cells were kindly provided by Prof. Dr. William T. Beck, Department of Pharmaceutics and Pharmacodynamics, College of Pharmacy, Chicago, Illinois. These cells present a well-known model for modulation experiments on Pgp [4]. Porcine brains were obtained from the local slaughterhouse and immediately used to isolate PBCEC's. Citalopram was kindly provided by H. Lundbeck A/S (Copenhagen-DK), mirtazapine by Stada Arzneimittel AG and reboxetine by Pharmacia Upjohn. All other chemicals, culture media and substances were purchased in the highest grade commercially available.

Isolation of PBCEC's

Porcine brain capillary endothelial cells were isolated as described before [1] [2] [9] [35]. Cortical gray matter from ten freshly obtained porcine brains was minced and incubated in Medium 199 containing 0.5 % Dispase II for 2 hours at 37 °C. Cerebral microvessels were obtained after centrifugation in Medium 199 containing 15 % dextran. Afterwards the microvessels were incubated in Medium 199 containing 1 mg/ml collagenase/dispase for 1.5 hours at 37 °C. The resulting cell suspension was filtered through a 150 μm nylon mesh. After centrifugation the cell pellet was resuspended in Medium 199 containing 10 % horse serum. This suspension was added to a discontinuous Percoll gradient consisting of Percoll 1.03 mg/ml and Percoll 1.07 mg/ml. The loaded Percoll gradient was centrifuged and the capillary endothelial cells were collected. The cells were again filtered through a nylon mesh. After another centrifugation the cells were resuspended in Medium containing 20 % horse serum and 10 % DMSO (dimethyl sulfoxide) and stored under liquid nitrogen until use.

Cell culture

CEM and VLB cells were cultivated in RPMI 1640 supplemented with 10 % FCS and penicillin/streptomycin (100 U/ml; 100 μg/ml). PBCEC's were cultivated in Medium 199 containing 10 % horse serum, penicillin/streptomycin (100 U/ml; 100 μg/ml), 0.8 mM l-glutamine, and 10 mM HEPES for seven days. On day three, the cells were passaged and seeded in 96 well plates coated with rat-tail collagen. The day prior to the experiment, the medium was changed to DMEM/Ham's F12 supplemented with 2 mM l-glutamine, penicillin/streptomycin (100 U/ml; 100 μg/ml) and 10 mM HEPES.

Calcein-AM Assay

Lipophilic non-fluorescent Calcein-AM can rapidly penetrate the plasma membrane. Inside the cell, endogenous unspecific esterases cleave the ester bonds quickly and irreversibly, producing the hydrophilic and fluorescent dye calcein, which cannot leave the cell via the plasma membrane. Calcein-AM is a highly specific substrate of Pgp as compared to calcein [27]. Cells expressing high levels of Pgp rapidly extrude non-fluorescent calcein-AM from the plasma membrane into the medium, thus preventing accumulation of fluorescent calcein in the cytosol. Inhibition of Pgp will lead to higher intracellular calcein concentration. Because the transport capacity of Pgp is inversely proportional to the inhibition of Pgp, the accumulation of intracellular calcein is a marker for the degree of inhibition [2].

For all tested compounds, stock solutions were prepared in either water or DMSO. Dilutions were made in KRB or in RPMI 1640 medium. The final concentration of DMSO in the assay did not exceed 1 %. At this concentration DMSO did not affect the assays.

The cells were washed at 37 °C with buffer and then incubated with the test compounds in different concentrations for 15 minutes at 37 °C. Subsequently the calcein-AM solution (final concentration 1 μM) was added and incubated for 30 minutes at 37 °C. The cells were washed three times with ice-cold buffer and lysed with 2 % Triton-X-100. The fluorescence was measured using a 1420 Victor² plate Reader with λ excitation = 485 nm and λ emission = 520 nm. Each tested compound was measured in triplicate.

None of the investigated compounds show any quenching effect on the fluorescence of calcein or any autofluorescence at the calcein excitation wavelength in the concentrations tested in the calcein-AM assay.

ATP-Assay

Pgp belongs to the superfamily of ABC-transporters, therefore the efflux of substances requires energy and depends on the intracellular ATP levels. The intracellular ATP levels were measured using the ViaLight HT kit (Cambrex). The kit allows a rapid and safe detection of ATP levels. It is based upon the bioluminescent measurement of ATP that is present in all metabolic active cells. The bioluminescent method utilizes an enzyme, luciferase, which catalyzes the formation of light from ATP and luciferin. The emitted light intensity is linearly related to the ATP concentration.

The VLB cells were washed with buffer at 37 °C and then incubated with the test compounds at different concentrations for 45 minutes at 37 °C. Subsequently the mixture of ATP-monitoring reagent and nucleotide releasing reagent was added. The luminescence was immediately measured using a 1420 Victor² plate reader. All tested concentrations were measured as triplicates.

Statistics

Data are given as mean ± SEM. Statistical analysis was performed using Student's t-test against the untreated control group. P value ≤ 0.05 was considered significant.

For calculation of the inhibitory effects, the EC50 values were determined. A nonlinear sigmoidal dose-response curve with variable slope was calculated for each substance using GraphPad Prism 4.0. The EC50 is the concentration leading to half-maximal effect in the calcein-AM assay. The determination of the EC50 of several antidepressants and antipsychotics are demonstrated graphically in Figs. [1] - [4]. The EC50 values of the other antidepressants are shown in Table [1] and of the antipsychotics in Table [2].

Results

We used two different cell-systems to characterize the inhibitory potencies of the drugs tested on Pgp function: 1. CCRF-CEM (CEM) cells, a human lymphocytic leucemia cell line, and its multidrug resistant variant CEM/VLB-100 (resistant to 100 ng/ml vinblastine) expressing Pgp and acting as a model for the human Pgp. 2. Porcine brain capillary endothelial cells (PBCEC) are well-known models for the blood-brain-barrier. For validation purposes, two typical Pgp inhibitors (verapamil and ivermectin) were tested in both cell systems in the calcein-AM assay. The EC50 values of both substances were comparable to others found in literature [verapamil: 3.2 μM (VLB); 3.1 μM (PBCEC)/ivermectin: 0.57 μM (VLB); 0.22 μM (PBCEC)] (data not shown) [36].

All psychotropic drugs tested inhibit the transport function of Pgp in a concentration dependent manner in both cell systems, but usually only at relatively high concentrations (Table [1], Table [2], Fig. [1] - [3]). VLB cells proved to be more sensitive than the PBCEC cells, since some antidepressants inhibit the transport in VLB cells at lower concentrations than in PBCEC cells.

EC50 values are also given in Table [1] (antidepressants) and Table [2] (antipsychotics), but could not be calculated for all compounds tested because saturation was not always reached.

Clomipramine inhibits the transport of calcein-AM in VLB cells above 3 μM and in PBCEC cells above 30 μM significantly [VLB: 3 μM*: 8.3 ± 4.4 %; 10 μM**: 27.3 ± 6.3 %; 30 μM**: 55.0 ± 9.5 %; 100 μM***: 88.8 ± 10.3 %/PBCEC: 30 μM**: 62.6 ± 11.0 %; 100 μM***: 142.6 ± 13.5 %] (Fig. [1]). In both cell systems it seems to be the most potent Pgp inhibitor of the tricyclics tested. Furthermore, clomipramine produces the highest accumulation of calcein-AM of all antidepressive drugs tested. Mirtazapine inhibits the transport activity of Pgp in VLB cells significantly but in PBCEC cells there is no significant effect shown [VLB: 10 μM: 19.6 ± 5.5 %; 30 μM: 31.2 ± 8.8 %; 100 μM: 43.2 ± 7.3 %].

Within the SSRI's analyzed, citalopram inhibits the transport activity in VLB cells more potent than fluoxetine [VLB-cells: 3 μM*: 6.0 ± 3.5 %; 10 μM*: 10.0 ± 4.9 %; 30 μM**: 15.3 ± 4.5 %; 100 μM**: 32.8 ±7.0 %/PBCEC-cells: 100 μM**: 25.6 ± 5.6 %] (Fig. [2]). Reboxetine, the only tested SNRI, also enhances the intracellular accumulation of calcein and thereby the intracellular fluorescence by inhibiting the transport activity of Pgp [VLB: 10 μM*: 15.4 ± 5.7 %; 30 μM***: 28.6 ± 3.2 %; 100 μM***: 44.9 ± 5.4 %/PBCEC: 100 μM*: 37.2 ± 14.3 %] (Fig. [3]).

Pgp inhibition is not a specific property of antidepressants, since some antipsychotics also inhibit the transport of calcein-AM by Pgp in a concentration dependent manner (Fig. [4]). The EC50 values in VLB cells for chlorpromazine (20.1 μM), clozapine (21.6 μM) and haloperidol (8.6 μM) are within the range of the value for antidepressants. In PBCEC cells the calculated EC50 values are comparable. Concentrations inhibiting Pgp significantly in both cells systems (VLB and PBCEC) for all antipsychotics tested are given in Table [2].

All substances did not enhance the calcein accumulation in CEM cells not expressing Pgp (data not shown), excluding non-specific effects on calcein-fluorescence.

To exclude possible involvement of the multidrug resistance associated protein MRP 1 and 2, which also transports calcein and calcein-AM, we tested probenecid (1 mM) in the calcein-AM assays. Probenecid is known to be a potent inhibitor of the transport of calcein and calcein-AM in MRP-overexpressing cell lines but not in Pgp overexpressing cell lines. In both cell systems used, PBCEC and VLB, probenecid has no influence on the calcein fluorescence (data not shown).

Because Pgp is an energy dependent efflux transporter, activity can be reduced by substances lowering the intracellular ATP-level. Therefore, we also measured possible effects of the antidepressants tested on intracelluar ATP-level in VLB-cells using the ViaLight plus Kit. H₂O₂ (100 μM), known to lower intracellular ATP levels, was used as control. It decreases the intracellular ATP concentration about 60 % in comparison to untreated cells. This effect, however, does not to influence the transport activity of Pgp (Fig. [5]). Some of the antidepressants and antipsychotics tested [clomipramine (30 % decrease at 100 μM**), haloperidol (20.6 % decrease at 100 μM*) and chlorpromazine (55.6 % decrease at 100 μM***)] reduce intracelluar ATP levels in VLB cells only weakly (data not shown). Thus, the weak effect on ATP can not explain the Pgp inhibiting properties of these drugs.

Discussion

In this study the well-known calcein-AM assay was used to characterize the Pgp inhibiting property of 9 antidepressants and 3 antipsychotics. The drugs were tested for their Pgp modulating property in two different cell lines, since plasma membrane composition of different cell lines may influence the activity and specificity of Pgp. This was confirmed in our studies showing that inhibiting effects for one drug can be different in the two cell systems.

Membrane steroid transporters like P-Glycoprotein (Pgp) regulate the function of the GR and the MR by modulating the intracellular concentration of glucocorticoids [6]. Some glucocorticoids like dexamethason are actively excreted out of cells by extrusion pumps like Pgp. But there is still conflicting data concerning the transport of glucocorticoids by Pgp. It seems quite possible that both corticosterone and cortisol are substrates of Pgp and therefore could be excreted out of cells by this transporter [18] [28] [31] [33]. P-Glycoprotein (Pgp, ABCB1), also known as an active efflux pump of antitumor agents in multidrug-resistant tumor cells, exists in several normal tissues like kidney, liver, colon, testes and capillary endothelium in brain. It can transport a wide range of chemically diverse compounds against a concentration gradient out of cells. The 170-180 kDa plasma-membrane associated protein, is a member of the ABC (ATP binding cassette) super family of transporters [25]. Therefore the transport activity of Pgp is energy-dependent.

Most of the analyzed antidepressants inhibit the transport of calcein-AM by Pgp at rather high concentrations in VLB cells as well as in PBCEC cells, however, sometimes to a different extent. In CEM cells (not expressing Pgp) the influence on calcein-fluorescence was absent indicating that the enhancement of the calcein fluorescence was based on inhibition of Pgp. Furthermore the MRP1 and 2 inhibitor probenecid did not increase the intracellular calcein fluorescence, this indicates that MRP1 and 2 are not involved. This was also seen by Weiss et al., although in different cell systems. In general, the antidepressants investigated are rather weak inhibitors of Pgp function, since mostly relatively high concentrations above therapeutical plasma levels (Table [1]) were needed to enhance calcein accumulation. This however does not exclude the possibility that the drugs are transported by Pgp at low concentrations in vivo. The data however are conflicting.

Considering citalopram, cyclosporin A, a well-known Pgp inhibitor, does not modify its transport in bovine brain microvessel endothelial cells [24] indicating that citalopram is not a substrate of Pgp. Conversely, Uhr et al. reported, that Pgp is involved in the uptake of citalopram into the brain of mice. They examined the uptake of citalopram and trimipramine into the brain of abcb1ab knock out mice and showed that presence of Pgp in control mice reduces the bioavailability of these substances in the brain [29]. Our data, indicating that citalopram inhibits Pgp in a concentration dependent manner are in line with the former data. The second SSRI tested, fluoxetine, also inhibits the transport of calcein-AM in both cell systems but only at even higher concentrations. In contrast, Peer et al. investigated the inhibitory potency of fluoxetine in eight different MDR cell systems (drug resistant cells) and found that fluoxetine is highly effective in doses that are well below its human safety limits [23]. Another group reported that the penetration of fluoxetine into brain is not enhanced in Pgp knock out mice [32]. The Pgp inhibition therefore might be allosteric, if fluoxetine is not a substrate of Pgp. Data generated by Juurlink et al. indicate that the inhibition of Pgp activity by SSRIs has no clinical relevance. They analyzed 245 305 older patients treated with digoxin and found no major discrepancy in the risk of digoxin toxicity after initiation of various SSRIs [16]. These data confirm our results showing that Pgp inhibition is seen only at high concentrations. In summary the concentrations needed to inhibit the transport function of Pgp by SSRI's are much higher than average therapeutical plasma levels (Table [1]). This suggests that inhibition of Pgp probably does not play a role for the antidepressive action of the SSRI's.

The only selective noradrenaline-reuptake-inhibitor tested, reboxetine, inhibits the transport activity of Pgp above 10 μM in VLB cells and only at 100 μM in PBCEC cells. It seems to be a moderate inhibitor. Our data are in line with other findings using L-MDR cells and also PBCEC cells [36]. The inhibiting concentrations for reboxetine are much higher than average therapeutical plasma levels which range from 30-320 nM (Table [1]).

All the tricyclic antidepressants tested also inhibit Pgp in a concentration dependent manner. Clomipramine seems to be the most potent. This supports the results of Pariante et al. However, the concentrations needed to inhibit the transport of calcein-AM significantly are again much higher than therapeutical plasma levels (0.56-1.4 μM) (Table [1]). Amitriptyline has been shown to be a potent inhibitor of the transport activity of Pgp by reversing multidrug resistance of human colon cancer cells and mouse lymphoma cells in vitro [34]. Its penetration into brain is enhanced after single administration in mice with BBB deficiency due to mdr1 Pgp gene disruption [32]. The same group showed again that Pgp also reduces the brain-bioavailability of amitriptyline metabolites like nortriptyline after a 10 day administration [12]. Similarly inhibition of Pgp by cyclosporin A increases the accumulation of nortriptyline in the brain [7]. In our hands, both amitriptyline and its metabolite nortriptyline, are moderate inhibitors of the transport activity of Pgp in the calcein-AM assay in VLB cells and PBCEC cells, however only at concentrations substantially above therapeutical plasma levels [3] [10].

The same is the case of mirtazapine where inhibition of transport activity of Pgp is only seen at concentrations higher than the recommended plasma concentration ranges (150-300 nM, Table [1]) [3] [11]. Moreover, Uhr et al. showed that mirtazapine may not be a substrate of this transporter [30].

All of the tested antipsychotics also inhibit the transport of calcein-AM by Pgp in both cell systems. Again inhibition is only seen at substantially higher concentrations than the therapeutical plasma levels. Various and contradictory effects of antipsychotics concerning their ability to inhibit the transport activity of Pgp have been reported. For example El Ela et al. tested clozapine and haloperidol in a Pgp inhibition assay using talinolol as Pgp substrate. The EC50 values are comparable to those created in our group (clozapine 92.78 μM ± 1.13 and haloperidol 12.43 μM ± 0.03) [8]. Another group tested the ATPase modulatory potency of some atypical (clozapine) and conventional (haloperidol and chlorpromazine) antipsychotics. All of the tested substrates stimulated Pgp ATPase activity to some degree in a time- and dose-dependent fashion. They classified the tested antipsychotics as class I compounds (stimulation of ATPase activity at low concentration and inhibition at higher concentration). Furthermore, chlorpromazine had a moderate affinity for Pgp and clozapine and haloperdidol may be transported by Pgp, but only to a small extent [5]. On the contrary, Henning et al. concluded that clozpine is not transported by Pgp [13]. They detected, that the intracellular accumulation of clozapine is not altered by incubation with cyclosporine A, vinblastine and verapamil, all of them known to be potent Pgp inhibitors. These data are confirmed by Lane et al. They pointed out that clozapine is unlikely a Pgp substrate [17]. Ibrahim et al. pointed out that chlorpromazine blocks Pgp at concentrations used to achieve pharmacological effects in patients in three cell lines. Haloperidol and clozapine also inhibit Pgp but only at higher concentrations [15]. Another group tested several antipsychotic drugs in the rhodamine 123 efflux assay concerning their ability to inhibit the transport activity of Pgp. Haloperdiol and Fluphenazine were the most active substances and increased the relative fluorescence intensity [26]. In conclusion, the data concerning the modulatory potencies of antipsychotics on Pgp activity are very inconsistent. The results obtained in our group suggest that the inhibition of Pgp by the tested antipsychotics is not clinically relevant. Antipsychotic drugs are used at plasma levels of 10 to 500 ng/ml, depending on the drug used and on the sensitivity of the patient [26]. The EC50 values of the antipsychotics determined in our assays are above the reached blood-levels.

In summary, we have shown that antidepressants of different groups (tricyclics, tetracyclics, SSRI's and SNRI's) inhibit the transport activity of Pgp at high concentrations. Because this inhibition is only pronounced at concentrations above plasma therapeutical levels it might not play a role for the antidepressant activity. Therefore, we can not confirm the hypothesis by Pariante et al. that a part of the antidepressive effect is the normalization of the function of the HPA axis by inhibiting Pgp and enhancing intracellular concentrations of glucocorticoids. Moreover, if this inhibition is specific for antidepressant activity, other substance groups which act in the CNS like antipsychotics should not be active under similar conditions.

Acknowledgment

The cell lines CCRF-CEM and CEM/VLB-100 were kindly provided by Prof. Dr. William T. Beck, Department of Pharmaceutics and Pharmacodynamics, College of Pharmacy, Chicago (Illinois).

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• 36 Weiss J, Dormann S M, Martin-Facklam M, Kerpen C J, Ketabi-Kiyanvash N, Haefeli W E. Inhibition of P-glycoprotein by newer antidepressants.  J Pharmacol Exp Ther. 2003;  305 197-204
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. W. E. Müller

Department of Pharmacology

Biocenter

Marie-Curie-Str. 9

60439 Frankfurt

Germany

Phone: +69 79829373

Fax: +69 79829374

Email: PharmacolNat@em.uni-frankfurt.de

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• 36 Weiss J, Dormann S M, Martin-Facklam M, Kerpen C J, Ketabi-Kiyanvash N, Haefeli W E. Inhibition of P-glycoprotein by newer antidepressants.  J Pharmacol Exp Ther. 2003;  305 197-204
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. W. E. Müller

Department of Pharmacology

Biocenter

Marie-Curie-Str. 9

60439 Frankfurt

Germany

Phone: +69 79829373

Fax: +69 79829374

Email: PharmacolNat@em.uni-frankfurt.de