European Neuropsychopharmacology (2008) 18, 157169

w w w. e l s e v i e r. c o m / l o c a t e / e u r o n e u r o

REVIEW

A review on the impact of P-glycoprotein on the penetration of drugs into the brain. Focus on psychotropic drugs
Kristian Linnet , Thomas Broeng Ejsing
The Department of Forensic Chemistry, Institute of Forensic Medicine, University of Copenhagen, Frederik Vs Vej 11, 2100 Copenhagen, Denmark Received 19 December 2006; received in revised form 15 May 2007; accepted 19 June 2007

KEYWORDS
P-glycoprotein; Psychotropic drugs; Bloodbrain barrier; Mdr1a/1b knock-out mice; Drugdrug interactions

Abstract In recent years there has been increasing focus on the role of the drug transporter P-glycoprotein (P-gp) with regard to drug penetration into the brain. Studies using mice devoid of functional P-gp have revealed that P-gp at the bloodbrain barrier (BBB) can exert a profound effect on the ability of some drugs to enter the brain, e.g. cardiovascular drugs (digoxin, quinidine), opioids (morphine, loperamide, methadone), HIV protease inhibitors, the new generation of antihistamines, and some antidepressants and antipsychotics. Among the latter group, risperidone is strongly influenced having about 10 times higher cerebral concentration in P-gp knock-out mice than in control mice. Taking into account that polytherapy is commonplace in psychiatry, theoretically there is a risk of drugdrug interactions with regard to P-gp at the BBB. Here we review the evidence for a role of P-gp with regard to psychoactive drugs from in vitro studies and experiments in knock-out mice devoid of functional P-gp. Moreover, the evidence for significant drugdrug interactions involving psychotropic drugs in rodents is considered. Clinical observations suggesting a role for P-gp in relation to drugdrug interactions at the BBB are sparse, and a definite conclusion awaits further studies. Also, the possible clinical relevance of P-gp genetic polymorphisms is questionable, and more investigations are needed on this subject. 2007 Elsevier B.V. and ECNP. All rights reserved.

1. Introduction
The bloodbrain barrier (BBB) is a major impediment to the entry of many therapeutic drugs into the brain, and during the last decade it has become clear that multispecific, xenobiotic transporters play an important role at the BBB. With the
Corresponding author. Tel.: +45 3532 6100; fax: +45 3532 6085. E-mail address: kristian.linnet@forensic.ku.dk (K. Linnet).

sequencing of the human genome, it has been estimated that approximately 5001200 genes code for transport proteins (Sakaeda et al., 2003). At present, messenger RNA (mRNA) from 15 of these drug transporters has been found at the BBB. They belong to the following subfamilies: the multidrug resistance protein (MDR), the multidrug resistance-associated protein (MRP), the organic anion transporter (OAT), the organic anion transporting polypeptide (OATP), the organic cation transporter (OCT), the concentrative nucleoside

0924-977X/$ see front matter 2007 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2007.06.003

158 transporter (CNT), and the equilibrative nucleoside transporter (ENT) (Bauer et al., 2005). P-glycoprotein (P-gp), which belongs to the MDR family, was one of the first of these proteins that was identified at the BBB, and thus the bulk of studies concerning drug efflux from the brain deal with P-gp. During the last decade it has been shown that P-gp exerts an important influence on the brain concentrations of some drugs (Lin and Yamazaki, 2003). Experiments in mice lacking functional P-gp at the BBB have revealed drastically enhanced brain levels (20 times or higher than those of the control animals) of e.g. the anthelmintic drug ivermectin, the cardiac glycoside digoxin and the HIV protease inhibitor nelfinavir (Schinkel et al., 1994; Choo et al., 2000; Mayer et al., 1996). Likewise, in a number of cases concerning Collie dogs devoid of functional P-gp severe signs of neurological symptoms were reported after treatment with the chemotherapeutic agent vincristine and the antidiarrheal agent loperamide (Mealey et al., 2003; Hugnet et al., 1996). P-gp at the BBB may in particular influence the effect of psychotropic drugs. Moreover, there is a possibility of drugdrug interactions with regard to P-gp. Here, we review the evidence of drugdrug interactions involving primarily psychotropic drugs and P-gp at the BBB on the basis of in vitro studies, animal experiments, and observations in humans. Additionally, pharmacogenetic aspects in relation to P-gp are considered.

K. Linnet, T.B. Ejsing The exact mechanism of drug transport has not been elucidated yet, but increasing amounts of evidence suggest that P-gp recognizes its substrates in the plasma membrane (Chen et al., 2001; Shapiro and Ling, 1998; Lugo and Sharom, 2005; Loo and Clarke, 2005). P-gp is able to recognize and transport an impressive array of substrates ranging in size from approximately 250 Da (cimetidine) to more than 1850 Da (gramicidin D) (Schinkel, 1999). These substrates include a wide variety of chemotherapeutic agents of natural origin such as anthracyclines (doxorubicin), vinca alkaloids (vinblastine), epipodophyllotoxins (etoposide), and taxanes (paclitaxel) (Kim, 2002). P-gp substrates also include drugs and pesticides such as the immunosuppressive agents cyclosporine A (CsA) and FK506 (Saeki et al., 1993), cardiac glycosides such as digoxin (Begley, 2004), antipsychotics and antidepressants like risperidone, nortriptyline, and citalopram (Uhr et al., 2000; Uhr and Grauer, 2003; Ejsing and Linnet, 2005; Ejsing et al., 2005), HIV protease inhibitors (Choo et al., 2000; Begley, 2004), and the anthelmintic pesticide ivermectin (Begley, 2004). P-gp substrates may act as competitive inhibitors of Pgp, e.g. the drugs cyclosporine A and verapamil (Saeki et al., 1993; Ford and Hait, 1990), which have been used as P-gp inhibitors since the early eighties. Many drugs are racemates and the question of stereoselectivity of transport mediated by P-gp thus is of relevance. A recent study by Miura et al. (2007) showed that the pharmacokinetics of fexofenadine, a probe substrate for P-gp, displays stereoselectivity. Apparently, P-gp has higher affinity for S(+)-fexofenadine than for R()-fenadine resulting in higher oral and renal clearance of the S(+)-form. Similarly, a study by Bertilsson et al. (1991) on the relationship between plasma and cerebrospinal fluid concentrations of the enantiomers of (E)-10OH-nortriptyline showed that the ()-enantiomer was more effectively transported out from the CNS than the (+)enantiomer. Studies on methadone pharmacokinetics also suggests stereoselectivity with regard to P-gp mediated transport (see later).

2. General properties of P-gp

2.1. Structure and function
P-gp is located mainly in the plasma membrane where it actively extrudes drugs from the cell. It was originally discovered in 1976 in drug-resistant ovary cells from Chinese hamsters (Juliano and Ling, 1976). Humans possess one gene (originally named MDR1 but today also denoted as ABCB1) encoding drug transporting Pgp whereas rodents have two (mdr1a and mdr1b). The combined tissue distribution of these two genes in rodents roughly coincides with that of the single MDR1 in humans, indicating that mdr1a and mdr1b together fulfil the same function as the human MDR1 (Bosch and Croop, 1998). Species variation with respect to P-gp exists. Cutler et al. (2006) found about similar behaviour of P-gp in mice and rats in relation to the potent P-gp inhibitor GF120918 (Elacridar) but not in guinea pigs. The latter species required about ten times higher concentration of GF120918 than rats and mice for a similar degree of inhibition. Murakami et al. (2000) compared BBB permeability in mice and rats for a range of compounds, including typical P-gp substrates such as quinidine, and found similar values. Generally, the functional consequences of species variation may vary from compound to compound (Yamazaki et al., 2001). Further studies, however, are needed on this aspect. The human P-gp consists of approximately 1280 amino acids and weighs around 170 kDa (Sharom, 1997; Schinkel, 1999). It contains two homologous, but not identical, parts joined together by a short linker region (Bosch and Croop, 1998). Each part comprises six transmembrane -helices and an ATP-binding site (Fig. 1). The 12 transmembrane segments fold together to form a barrel-like structure that traverses the plasma membrane. The two ATP-binding sites are located at the cytoplasmic site, and hydrolysis of ATP provides the energy necessary for drug transport (Schinkel, 1997).

2.2. Tissue distribution of P-gp

At the interface between the blood and the central nervous system P-gp is present in the microvessels and the choroid plexus. In the former, P-gp is found at the luminal membrane of the endothelial cells lining the capillaries (Tanaka et al., 1994; Beaulieu et al., 1997; Virginento et al., 2002) (Fig. 2A).

Figure 1 Two-dimensional representation of human P-gp. The 12 Transmembrane segments fold together to form a three dimensional barrel-structure in the membrane. N-linked glycosylation trees that are found in the first extracellular loop. The ATP binding domains are shown with circles. Modified from Schinkel et al. (1999).

A review on the impact of P-glycoprotein on the penetration of drugs into the brain. Focus on psychotropic drugs Here it excretes compounds into the blood, thus preventing e.g. drugs from gaining access to the brain. In the choroid plexus, P-gp is located at the apical surface of the epithelia cells that constitute the ventricular exposed surface of the plexus (Fig. 2B) (Rao et al., 1999; Warren et al., 2000). Furthermore, P-gp is expressed at the bloodspinal cord barrier (Sugawara et al., 1990). Apart from the central nervous system, P-gp is also found in the heart, lungs, pancreas, and other organs including the apical surface of the columnar epithelial cells of the intestines. This is the major uptake place for drugs into the body, and several studies have proved that P-gp actively secretes drugs into the intestinal lumen (Mayer et al., 1996; Sparreboom et al., 1997; Van Asperen et al., 2000). Likewise, P-gp is positioned at the major exit routes of the body, namely the biliary epithelial cells, and proximal renal tubules (Cordon-Cardo et al., 1990). P-gp is also located in the placenta (Young et al., 2003).

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3. In vitro studies on drug/P-gp relations

Several psychoactive drugs have been shown to be substrates and/or inhibitors of human P-gp in vitro. One principle of assessing interaction between a drug and P-gp is to measure P-gp mediated ATP-ase activity (Boulton et al., 2002). P-gp provides active transport by hydrolysis of ATP. The majority of drugs that stimulate the ATP-ase activity of P-gp are also transported by the protein. Thus, measurement of the ATPase activity in vitro can be used to identify P-gp substrates. In this way risperidone and quetiapine have been identified as good P-gp substrates comparable to the potent competitive inhibitor and model compound verapamil (Table 1) (Boulton et al., 2002; Ejsing et al., 2005). The main metabolite of risperidone, 9-OH-risperidone, and the antipsychotic drugs olanzapine and chlorpromazine are intermediate substrates (Boulton et al., 2002; Ejsing et al., 2005), whereas clozapine, haloperidol and nortriptyline were poor substrates with Km values that were 10-fold higher than that of verapamil (Boulton et al., 2002; Ejsing et al., 2006). In another in vitro model, El Ela et al. (2004) measured the P-gp mediated efflux of 14 psychoactive compounds across a human colon adenocarcinoma (Caco-2) cell monolayer. They classified six of these drugs as P-gp substrates: amisulpride, demethyl-clozapine, domperidone, flupentixol, fluphenazine, and fluvoxamine. The drugs not found to be P-gp substrates included quetiapine, olanzapine, clozapine, and haloperidol. This contrasts somewhat with the results of Boulton et al. (2002), who identified quetiapine and olanzapine as substrates. P-gp is thought to recognize its substrates in the plasma membrane, and one reason for the differences may therefore be the membrane composition, as the Caco-2 cells are of human origin, whereas the membrane fractions used in the ATP-ase assay were from insect cells. Several studies have shown that the membrane composition can influence the substrate specificity of P-gp (Germann et al., 1990; Romsicki and Sharom, 1999; Riou et al., 2003), and thus caution is needed when comparing results from different cell and membrane systems (Weiss et al., 2003). Permeation studies in primary porcine brain microvessel endothelial cells showed that P-gp influenced the penetration of amisulpride through the monolayer, whereas cloza-

Figure 2 P-gp at the bloodcentral nervous system barrier. A: The BBB barrier (modified from Schinkel et al. (1999)). B: The choroid plexus.

pine and N-desmethylclozapine were not transported (Hrtter et al., 2003). The results were supported by Caco2 cell transport studies. Using monolayers of bovine brain microvessel endothelial cells, Rochat et al. (1999) did not find any interaction between P-gp and citalopram. However, in vivo studies in mice have later verified that citalopram is a P-gp substrate (see later). As P-gp is one of the important proteins involved in multidrug resistance of tumours, extensive research has been undertaken to find drugs that can reverse the resistance. In this process numerous psychotropic drugs have been screened, and some exhibited a high inhibitory potential. Among these are the antidepressants sertraline and paroxetine, both of which inhibited the uptake of calceinacetoxymethylester (calcein-AM) in a porcine cell line transfected with human P-gp (Weiss et al., 2003). The IC50 (the concentration leading to half maximum inhibition of the calcein AM transport) was 29.8 M for paroxetine and 31.8 for sertraline, which is comparable to the value of the efficient P-gp inhibitor quinidine (33.8 M) but still higher than that of verapamil (18.9 M). Szab et al. (1999) tested the effect of psychotropic drugs on cellular uptake of the model compound rhodamine 123 in cell lines transfected with human P-gp and uptake of daunorubicin in tumour cell lines selected for P-gp mediated resistance. They found that the tricyclic antidepressant amitriptyline as well as fluphenazine and haloperidol were good inhibitors comparable to the powerful P-gp inhibitor cyclosporine A. Moreover, they found that the remaining drugs (maprotiline, trimipramine, desipramine, imipramine and doxepin) all to a greater or lesser extent

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Table 1 Psychotropic drugs identified in vitro as substrates/inhibitors for P-gp In vitro interaction principle Amisulpride Microvessel permeation Caco-2 cell transport

K. Linnet, T.B. Ejsing

References Hrtter et al. (2003) Hrtter et al. (2003) El Ela et al. (2004) Schmitt et al. (2006) Ibrahim et al. (2000) Boulton et al. (2002) Ibrahim et al. (2000) Boulton et al. (2002) Boulton et al. (2002) Ibrahim et al. (2000) Szab et al. (1999) El Ela et al. (2004) Szab et al. (1999) Szab et al. (1999) El Ela et al. (2004) El Ela et al. (2004) El Ela et al. (2004) Szab et al. (1999) Ibrahim et al. (2000) Boulton et al. (2002) Szab et al. (1999) Szab et al. (1999) Ejsing et al. (2005) Boulton et al. (2002) Weiss et al. (2003) Ibrahim et al. (2000) Ibrahim et al. (2000) Boulton et al. (2002) Boulton et al. (2002) Ejsing et al. (2005) Ejsing et al. (2005) Weiss et al. (2003) Ibrahim et al. (2000) Szab et al. (1999)

Chlorpromazine Clozapine Demethyl-clozapine Desipramine Domperidone Doxepin Fluphenazine Flupentixol Fluvoxamine Haloperidol

Imipramine Maprotiline Nortriptyline Olanzapine Paroxetine Pimozide Protriptyline Quetiapine Risperidone 9-HO-Risperidone Sertraline Trimipramine

Rhodamine 123 uptake in human Caco-2 cells ATP-ase Rhodamine 123 uptake in human Caco-2 cells ATP-ase Caco-2 cell transport Rhodamine 123 uptake in human Caco-2 cells Rhodamine123/daunorubicin cellular uptake Caco-2 cell transport Rhodamine123/daunorubicin cellular uptake Rhodamine123/daunorubicin cellular uptake Caco-2 cell transport Caco-2 cell transport Caco-2 cell transport Rhodamine123/daunorubicin cellular uptake Rhodamine 123 uptake in human Caco-2 cells ATP-ase Rhodamine123/daunorubicin cellular uptake Rhodamine123/daunorubicin cellular uptake ATP-ase ATP-ase Calcein AM transport Rhodamine 123 uptake in human Caco-2 cells Rhodamine 123 uptake in human Caco-2 cells ATP-ase ATP-ase ATP-ase Calcein AM transport Rhodamine 123 uptake in human Caco-2 cells Rhodamine123/daunorubicin cellular uptake

increased the cellular uptake of daunorubicin. Finally, Ibrahim et al. (2000) examined the effect of 33 drugs and metabolites on the cellular uptake of the P-gp substrate rhodamine 123 in human Caco-2 cells. Among the drugs were several psychoactive drugs, and especially amitriptyline, chlorpromazine, pimozide and protriptyline displayed a high degree of P-gp inhibition, reaching at least 80% inhibition. They also found haloperidol, trimipramine, clozapine, and desipramine to be intermediate inhibitors displaying 2060% inhibition. Some drugs, such as nortriptyline, chlorazepate, triazolam, and estazolam displayed no inhibitory effect at all. Interaction between P-gp and nutritional components is also a subject of interest. The active components of St. Johns wort hypericin and hyperforin are able to inhibit P-gp in vitro (Wang et al., 2004a). More important, however, is the induction effect of St. Johns wort (see later). Another nutritional component of interest is grape fruit juice. Generally, effects of grapefruit juice have been ascribed to inhibition of the CYP3A4 enzyme. However, grapefruit juice components are also able to inhibit P-gp as demonstrated by in vitro studies (Wang et al., 2001).

The in vitro study results suggest generally that some psychotropic drugs can exert an important effect with regard to drug uptake in the brain. However, two factors ought to be kept in mind. First, the inhibitory effect of most of the drugs are only manifest when the concentration is in the micromolar range, which is larger than the serum levels typically observed during therapeutic conditions (Ibrahim et al., 2000). Moreover, studies in cell lines easily lead to exaggerated expectations regarding the effects in vivo, as cell lines, chosen for their resistance to P-gp substrates, often express amounts of P-gp that by far exceeds those observed in vivo (Litman et al., 2001), thereby overshadowing other factors that may influence drug distribution. Polli et al. (2001) concluded that some in vitro techniques were reliable for high affinity- and others for low-affinity drugs with respect to P-gp. Thus, when several in vitro techniques are applied on the same drugs, discrepant results are obtained in some cases. Accordingly, in vitro studies may provide an indication on the relationship to P-gp, but in vivo techniques as described in the following more clearly reveal the functional consequences (with reservations for possible species variation).

A review on the impact of P-glycoprotein on the penetration of drugs into the brain. Focus on psychotropic drugs

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4. Distribution of drugs over the bloodbrain barrier in knock-out mice versus control mice
A simple method to estimate the influence of P-gp on the distribution of drugs over the BBB is to compare brain concentrations in P-gp knock-out (KO) and wild-type (WT) mice. At present three targeted KO mice strains are available: the mdr1a (abcb1a) (/) KO mice (Schinkel et al., 1994), the mdr1b (abcb1b) (/) KO mice (Schinkel et al., 1997) and the mdr1a/mdr1b (/) double KO mice (Schinkel et al., 1997). It is now well established that only mdr1a P-gp is expressed at the BBB and not mdr1b P-gp (Barrand et al., 1995; Regina et al., 1998; Demeule et al., 2001). Thus the mdr1a and the mdr1a/1b (/) KO mice should be equally good models for the absence of P-gp at the BBB. P-gp KO mice are phenotypically normal, but are more sensitive towards the toxicity of P-gp substrates (Schinkel, 1997). Numerous drugs have been screened in KO mice, and quite dramatic differences between KO and WT mice have been observed for some drugs. These include nelfinavir, digoxin, ivermectin, the antiarrhythmic drugs amiodarone, quinidine and verapamil (Kusuhara et al., 1997; Dagenais et al., 2001; Doran et al., 2005) as well as methadone (Wang et al., 2004b) and the chemotherapeutic agent vinblastine (Van Asperen et al., 1996). All these drugs had brain concentrations in the KO-mice that were at least 10 times larger than those observed in WT mice. Several psychotropic drugs have been screened in KO mice, and they generally showed smaller differences between KO and WT mice (Tables 2 and 3). Table 2 gives the brainserum and in some instances the brainkidney ratios for a number of psychotropic drugs after acute administration (for amitriptyline also after repeated dosing). The KO/WT ratios of brainserum ratios show that the absence of P-gp has a rather moderate effect on most drugs with ratios below 3. The only exceptions are risperidone, 9-OH-risperidone, the hydroxylated amitriptyline metabolites and E-10-OH-nortriptyline. As the brain concentrations have been normalized against the serum or kidney concentration, elevated serum concentrations following decreased P-gp efflux in the intestines, liver and kidneys can be ignored. In a few cases, however, the brain serum ratios were not available. Instead the brain(KO mice) brain(WT mice) concentration ratios are given along with the plasmaplasma ratios (Table 3). Again, the effect was quite moderate. When considering the displayed results of cerebral drug concentrations in KO-mice, it should be noted that acute drug administration experiments form the basis. In the clinical situation, chronic administration is of primary relevance. Grauer and Uhr (2004) studied the distribution of amitriptyline and its metabolites in KO- and control mice after administration for 10 days. Somewhat surprisingly, only the metabolites and not amitriptyline itself had significantly higher cerebral concentrations in KO-mice than in controls (Table 2). Partial saturation of P-gp may play a role for this unexpected result, but further studies on the conditions during chronic dosing would be desirable. With regard to antiepileptic drugs (not included in Tables 2 and 3), the absence of P-gp had a limited effect. Drugs such as phenytoin, phenobarbital, lamotrigine, topiramate and carbamazepine had ratios between 1 and 1.8 (Doran et al.,

2005; Sills et al., 2002). Finally, P-gp is of importance for several opioids. Methadone has more than 10 times higher brain concentration in KO-mice than in controls (R-methadone 15- and S-methadone 23-fold) (Wang et al., 2004b). For morphine, the influence is smaller, amounting to a factor 1.7 (Doran et al., 2005). The peripherally acting opioid, loperamide, on the other hand, has a ratio of 65 (Kalvass et al., 2004). The in vitro studies above suggested that psychotropic drugs were good P-gp substrates and inhibitors. The data in Tables 2 and 3, however, show that although P-gp influences the brain penetration of many psychotropic drugs and metabolites in KO mice, the effect is rather limited in most cases. One factor that may affect the ratios is other transport proteins at the BBB. It has previously been shown that at the BBB of mdr1a KO mice the mRNA level of Breast Cancer Resistance Protein (BCRP), which is an ABC transporter that has overlapping substrate specificities with P-gp (Litman et al., 2001), is threefold higher than that of the WT mice. The substrate specificity of BCRP with regard to psychotropic drugs has not been examined, and thus it is difficult estimate the significance of the increased expression of BCRP. Additionally, when interpreting the mice results, the possibility of species variation should be kept in mind. Although not in focus here, it should be briefly mentioned that also endogenous compounds are transported by P-gp. The steroids corticosterone, cortisol and aldosterone have been shown to attain higher cerebral concentrations in KOmice than in controls (Uhr et al., 2002). Further, a role for Pgp concerning transport of -amyloid out of the CNS has been suggested (Kandimalla et al., 2005, Thuerauf and Fromm, 2006). Thus, the level of P-gp expression might hypothetically play a role for development of Alzheimers disease.

5. Drugdrug interaction experiments in relation to P-gp in rats and mice

The large number of psychoactive drugs that are substrates of P-gp could potentially be involved in a significant number of drugdrug interactions regarding P-gp. Because of overlapping substrate specificities between CYP3A4 and P-glycoprotein, many drug interactions may involve both CYP3A4 and Pglycoprotein. Therefore, it is important to distinguish CYP3A4mediated from P-glycoprotein-mediated inhibition in order to make appropriate interpretation of drug interaction data. The following studies all use brainserum or dialysateserum ratios, and thus there should be accounted for potential metabolic effects influencing the serum levels. Numerous interaction studies with chemosensitizers and chemotherapeutic agents in rats and mice have been made, and these show that significant increases of drug brain levels can occur (Choo et al., 2000; Cisternino et al., 2004). One of the early chemosensitizers, cyclosporine A (Ford and Hait, 1990), has also been used in studies including psychotropic drugs. Cotreatment of rats with nortriptyline in different doses and cyclosporine A (200 mg/kg, i.p.) increased the nortriptyline brainserum ratios with roughly 25% for nortriptyline and 30% for the major metabolite, E-10-OH-nortriptyline (Table 4) (Ejsing and Linnet, 2005). Taking into account that the therapeutic interval in humans covers plasma concentrations from 190 to 570 nM (Task Force on the Use of Laboratory Tests

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Table 2 Drug Amitriptyline Amitriptyline (chronic dosing) E-10-OH-amitriptyline E-10-OH-amitriptyline Z-10-OH-amitriptyline Z-10-OH-amitriptyline Buspirone Chlorpromazine Citalopram Clozapine Diazepam Fluoxetine Fluoxetine Norfluoxetine Fluvoxamine Haloperidol Meprobamate Midazolam Nortriptyline

K. Linnet, T.B. Ejsing

Brainserum ratios of psychotropic drugs and metabolites in either knock-out (KO) or wild-type (WT) mice KO 13.3 2.2 7.4 2.0 29 9.7 6.6 2.3 18 14 18 0.70 0.24 20 30 3.1 13 2.0 7.1 8.0 5.8 5.6 1.0 2.9 3.2 27 0.15 0.54 7.7 0.40 WT 10.3 1.6 1.5 1.6 23 5.1 4.1 2.0 12 6.1 13 0.42 0.23 11 18 0.48 8.5 0.9 3.3 0.78 0.4 0.4 0.060 0.1 0.26 24 0.078 0.61 4.2 0.29 KO/WT 1.9 1.3 3.2 1.4 4.5 4.9 1.3 1.3 1.9 1.6 1.2 1.1 1.5 1.1 2.3 1.4 1.7 1.0 1.8 2.5 1.6 2.9 6.4 2.4 1.5 2.6 2.2 10 12 14 17 29 12 1.1 1.9 0.9 1.8 1.4 Mouse type Mdr1a Mdr1a/1b Mdr1a Mdr1a/1b Mdr1a Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a Mdr1a/1b Mdr1a Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a Mdr1a Mdr1a Mdr1a Mdr1a Mdr1a Mdr1a Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a Mdr1a/1b Mdr1a/1b Mdr1a Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a/1b Reference Uhr et al. (2000) Grauer and Uhr (2004) Uhr et al. (2000) Grauer and Uhr (2004) Uhr et al. (2000) Grauer and Uhr (2004) Doran et al. (2005) Doran et al. (2005) Doran et al. (2005) Doran et al. (2005) Doran et al. (2005) Uhr et al. (2000) Doran et al. (2005) Uhr et al. (2000) Doran et al. (2005) Doran et al. (2005) Doran et al. (2005) Doran et al. (2005) Doran et al. (2005) Uhr et al. (2000) Ejsing et al. (2006) Uhr et al. (2000) Ejsing et al. (2006) Uhr et al. (2000) Ejsing et al. (2006) Wang et al. (2004c) Doran et al. (2005) Doran et al. (2005) Wang et al. (2004d) Ejsing et al. (2005) Doran et al. (2005) Wang et al. (2004d) Ejsing et al. (2005) Doran et al. (2005) Doran et al. (2005) Doran et al. (2005) Doran et al. (2005) Doran et al. (2005)

E-10-OH-nortriptyline Z-10-OH-nortriptyline Demethyl-nortriptyline Olanzapine Paroxetine Risperidone

9-OH-risperidone

Sertraline Sulpiride Trazodone Venlafaxine Zolpidem

Significance of deviation from 1 (p b 0.05) is marked with an asterisk whereas absence of an asterisk denotes no significance. Results based on brainkidney ratios are shown in italics. -: no brainserum values were available. : In the article the ratio was given graphically.

in Psychiatry, 1985) (i.e. a variation corresponding to a factor of three), an increase of about 1.25 for the brainserum ratio is not dramatic. Substitution of cyclosporine A with verapamil
Table 3 Drug Citalopram Doxepin Mirtazapine Paroxetine Trimipramine Desmethyl-trimipramine Venlafaxine Brainbrain (cbrain,
KO mice/cbrain, WT mice)

(50 mg/kg) led to a 60% increase in brainserum ratios of nortriptyline (Table 4) (Ejsing et al., 2006). Likewise, the abovementioned KO/WT nortriptyline ratios of 1.6, 1.8 and 2.6

ratio of psychotropic drugs and metabolites screened in KO mice Ratioplasma 0.9 1.1 1.3 1.4 0.9 1.2 1.3 Mice type Mdr1a Mdr1a/1b Mdr1a/1b Mdr1a/1b Mdr1a Mdr1a Mdr1a/1b Reference Uhr and Grauer (2003) Uhr et al. (2003) Uhr et al. (2003) Uhr et al. (2003) Uhr and Grauer (2003) Uhr and Grauer (2003) Uhr and Grauer (2003)

Ratiobrain 3.0 1.2 1.3 2.1 1.2 1.5 2.3

: p b 0.05. No asterisk denotes no significant difference.

A review on the impact of P-glycoprotein on the penetration of drugs into the brain. Focus on psychotropic drugs
Table 4 Drug Amisulpride + Cyclosporine A(6h) Amisulpride + Cyclosporine A Fluphenazine + Cyclosporin A (6h) Nortriptyline + Cyclosporine A E-10-OH-nortriptyline + Cyclosporine A Nortriptyline + Cyclosporine A Nortriptyline + Verapamil Nortriptyline + Methadone E-10-OH-nortriptyline + Cyclosporine A E-10-OH-nortriptyline + Verapamil E-10-OH-nortriptyline + Methadone Risperidone + Cyclosporine A Risperidone + Nortriptyline Risperidone + Sertraline 9-OH-risperidone + Cyclosporine A 9-OH-risperidone + Nortriptyline 9-OH-risperidone + Sertraline Brainserum ratios in psychotropic drugdrug interaction experiments in rats or mice Brain/serum ratio 0.22 0.13 0.16 0.10 12.2 27 20 25 1.3 1.6 22.5 28.9 15.5 24.8 16 16 1.6 2.6 1.6 0.7 1.9 1.9 0.60 0.77 0.65 0.49 1.6 2.3 0.15 0.17 0.16 0.19 0.9 2.5 Rel. change after inhibition 0.6 Rat 0.6 Rat 2.2 Rat 1.3 Rat 1.2 Rat 1.3 Rat 1.6 Rat 1.0 Rat 1.6 Rat 0.5 Rat 1.0 Rat 1.3 Rat 0.8 Mice 1.4 Rat 1.1 Rat 1.3 Mice 2.8 Wang et al. (2006) Ejsing et al. (2005) Ejsing et al. (2005) Wang et al. (2006) Ejsing et al. (2005) Ejsing et al. (2005) Ejsing et al. (2006) Ejsing et al. (2006) Ejsing et al. (2006) Ejsing et al. (2006) Ejsing et al. (2006) Ejsing et al. (2006) El Ela et al. (2004) Schmitt et al. (2006) Species Rat Reference El Ela et al. (2004)

163

Ejsing and Linnet (2005) Ejsing and Linnet (2005)

Significance of deviation from 1 (p b 0.05) is marked with an asterisk whereas absence of an asterisk denotes no significance.

(Table 2) suggest that even complete inhibition of P-gp is unlikely to yield serious toxicity. We here assume similar functionality of mice and rat P-gp with respect to nortriptyline. In order to study possible interactions between psychotropic drugs at realistic concentration levels risperidone ( mg/kg) or methadone (1 mg/kg) were co-administered with nortriptyline (5 mg/kg) (Ejsing and Linnet, 2005; Ejsing et al., 2006). No significant alterations in the nortriptyline brainserum ratios were observed. Likewise, there was no effect with regard to E-10-OH-nortriptyline (Table 4). As the KO/WT brainserum ratio for risperidone is higher than 10 (Table 2), even a 50% inhibition would give rise to a 5fold higher brain concentration. Interaction studies with cyclosporine A (200 mg/kg), nevertheless, demonstrated a rather limited effect of the inhibitor, with no more than a 1.3-fold increase in the brainserum ratio. For the main metabolite, 9-OH-risperidone, only a 1.1-fold increase in the brainserum ratio was observed (Table 4) (Ejsing et al., 2005). Furthermore, substitution of cyclosporine A with a

dose of nortriptyline yielding serum concentrations close to the therapeutic interval gave identical brainserum ratios for control and nortriptyline treated rats (Ejsing et al., 2005). In a recent study, Wang et al. (2006) showed that coadministration of sertraline and risperidone gave 1.4- and 2.8-fold higher brainserum AUC ratios of risperidone and 9OH-risperidone in mice, respectively (Table 4). Interestingly, the plasma concentrations of sertraline were comparable to those reported in humans after normal therapeutic doses of sertraline. In another study, cyclosporine A was used to inhibit the brain uptake of fluphenazine or amisulpride (El Ela et al., 2004). Cyclosporine A did not affect the brainserum ratio of amisulpride, whereas it enhanced the fluphenazine brain serum ratio with roughly a factor of 2. Schmitt et al. (2006) also studied the possible interaction between amisulpride and cyclosporine A in the rat. They found that both the plasma and the brain concentration of amisulpride were increased resulting in an increased pharmacodynamic effect (the ratios shown in Table 4 have been derived from AUCvalues).

164 A number of antiepileptic drugs have been studied by microdialysis experiments in rats by Potschka and co-workers. They found that local administration of verapamil led to a 1.25-fold increase in the dialysateplasma ratio of carbamazepine (Potschka et al., 2001) and a 1.8-fold increase for phenytoin (Potschka and Lscher, 2001). Subsequent studies showed that verapamil gave between 1.5- and 2-fold increases in the dialysateplasma ratios of lamotrigine, felbamate and phenobarbital (Potschka et al., 2002), whereas no effect was observed for levitiracetam (Potschka et al., 2004). Under normal conditions P-gp is not expressed in neurons (Volk et al., 2004), but recently some studies have shown that neurons in the rat hippocampus begin to express P-gp after a chemically induced status epilepticus (Lazarowski et al., 2004b; Volk et al., 2004). Neuronal P-gp expression in epileptic patients has also been reported (Lazarowski et al., 2004a; Volk et al., 2004). Taking the above-mentioned results into account, P-gp could be involved in drug-refractory epilepsy (Sisodiya et al., 2002). Interestingly, a recent study has shown that local administration (microdialysis) of verapamil and i.p. administration of oxcarbazepine can decrease the number of pilocarpine induced limbic seizures in rats (Clinckers et al., 2005). Inhibition of P-gp has also been tested with regard to analgesic drugs. Methadone is a good P-gp substrate in mice, as mentioned above. In a study, where rats were pretreated with the powerful P-gp inhibitor valspodar 30 min prior to methadone administration, a threefold increase in the antinociceptive effect was observed. After 20 min the methadone brainplasma ratios were five times higher in the valspodar treated group than in the control group (Rodriguez et al., 2004). Likewise, the P-gp inhibitor elacridar (GF120918) gave a threefold increase in the brainserum ratios of morphine, and, as it also was the case for methadone, a twofold increase in the antinociceptive effect was observed (Letrent et al., 1999). In contrast, valspodar had no effect on the brain penetration of the opioid oxycodone (Bostrm et al., 2005).

K. Linnet, T.B. Ejsing methadone given orally was increased by quinidine, it was concluded that quinidine enhanced the absorption of methadone by inhibition of intestinal P-gp, but that quinidine in the administered dose did not inhibit P-gp at the BBB. An additional contributing factor might be inhibition of CYP2D6 by quinidine, since CYP2D6 is involved in the metabolism of methadone (Eap et al., 2001). Recently, an interaction between colchicine and verapamil with regard to P-gp at the BBB was proposed, based on a case report (Trger et al., 2005). In this case the drug combination resulted in enhanced neurotoxicity of colchicine in the form of tetraparesis. As previously mentioned, P-gp and CYP3A4 have a striking substrate overlap (Fromm, 2004). Since the brain concentrations are not readily available in humans, CNS effects will often be ascribed solely to metabolic interactions involving the CYP enzymes, even though CNS adverse effects could arise from the combination of drugdrug interactions in relation to CYP enzymes and with regard to P-gp at the BBB. An example is the interaction between risperidone and the HIV protease inhibitor ritonavir. Following concomitant administration of the drugs, extrapyramidal symptoms (Kelly et al., 2002) and reversible coma (Jover et al., 2002) have been reported. These effects were ascribed to metabolic interactions, but as ritonavir is a good P-gp inhibitor in vitro (Drewe et al., 1999; Van der Sandt et al., 2001) and can inhibit P-gp at the renal tubules in humans (Ding et al., 2004), inhibition of P-gp at the BBB might also be involved. Although the reported cases point to drugdrug interactions with regard to P-gp at the BBB, it should be kept in mind that few reports exist for these relatively widely used drugs. Thus, further systematic research is needed to delineate the interaction potential in a clinical context. As mentioned previously, interaction between P-gp and nutritional components takes place. Ingestion of a single dose of St. Johns wort increased the maximum plasma concentration of the P-gp model compound fexofenadine by 45%, whereas long-term treatment caused a 35% decrease due to induction (Wang et al., 2002). Specific interactions between Pgp and St. Johns wort components with regard to psychotropic drugs at the BBB have apparently not been assessed. However, the well-known inducing effect of St. Johns wort with regard to CYP3A4 will influence the effect of many psychotropic drugs via a decrease of the plasma concentration.

6. Relevance in humans of P-gp in relation to CNS effects of drugs

6.1. Interactions
The experimental findings of the interaction studies described above clearly support the notion that P-gp plays an important role in brain uptake of drugs. The results also point to the potential risk of neurotoxicity when potent P-gp inhibitors are co-administered. A good example is the study by Sadeque et al. (2000). They treated healthy volunteers with the antidiarrhoeal agent loperamide (16 mg, oral) with or without co-administration of quinidine (600 mg, oral). When loperamide was administered alone, no adverse effects were observed. Contrary to this, serious respiratory depression occurred when the drug was given with quinidine, which was ascribed to inhibition of P-gp. In another study, however, also performed on healthy volunteers, quinidine (800 mg/kg, oral) did not enhance the central nervous effects of morphine (7.5 mg, infusion) (Skarke et al., 2003). Kharasch et al. (2004) found that quinidine enhanced the effects of orally administered methadone but not of methadone given by the intravenous route. Since the plasma concentration of

6.2. Pharmacogenomics
Recently, attention has been directed towards the pharmacogenomics of P-gp. As many central nervous system-active drugs are P-gp substrates, differences in P-gp expression at the BBB could, at least in part, be of importance for inter-individual variation in response and the occurrence of side effects at identical plasma concentrations. More than 50 single nucleotide polymorphisms (SNPs) have been detected in the human MDR1 gene (Marzolini et al., 2004, Kimchi-Sarfaty et al., 2007). SNPs in exon 21 and 26 have been associated with differences in P-gp expression and function in humans (Sakaeda et al., 2003). The majority of SNP related reports focus on the silent C3435T SNP of exon 26. This SNP has been associated with both increased and decreased expression of Pgp in the intestines and with changes in serum levels of digoxin and fexofenadine (Hoffmeyer et al., 2000; Kurata et al., 2002;

A review on the impact of P-glycoprotein on the penetration of drugs into the brain. Focus on psychotropic drugs Nakamura et al., 2002). With regard to the effect of the C3435T SNP on P-gp at the BBB few studies are available. Roberts et al. (2002) genotyped a number of depressed patients for the C3435T SNP and randomized them to either nortriptyline or fluoxetine treatment. Fifty-four of the nortriptyline treated patients completed the 6-week trial, and among these patients no difference in nortriptyline serum levels between C/C, C/T, and TT genotypes were found. They did, however, observe that there was an increased frequency of postural hypotension for the patients homozygous for T. No association between any of the genotypes and postural hypotension was apparent for the 72 fluoxetine treated patients that completed the trial. In a similar study Laika et al. (2006) found no correlation between the G2677T/A SNPs and the therapeutic outcome and occurrence of side effects in depressed inpatients treated with amitriptyline. De Luca et al. (2003) investigated the involvement of the C3435T SNP in antidepressant-induced mania in depressed patients. The study included 55 patients treated with fluoxetine, fluvoxamine, sertraline, imipramine, moclobemide, venlafaxine, paroxetine, nefazodone or combination therapy with fluoxetine/fluvoxamine. They found no association, but the large number of different drugs included in the study is a problem. Thus, further studies are needed to clarify the possible influence of the C3435T SNP at the BBB on the effects of psychotropic drugs. Apart from antidepressants and antipsychotics a number of studies have focused on the possible association between the C3435T SNP and drug-resistant epilepsy. Siddiqui et al. (2003) genotyped 200 patients with drug-resistant epilepsy as well as 115 patients with drug-sensitive epilepsy. They reported that patients with drug-resistant epilepsy were more likely to have the CC genotype than the TT genotype. Using identical inclusion criteria, Tan et al. (2004) studied 401 cases of drug-resistant epilepsy and 208 drug responsive epileptics. In contrast to Siddique and co-workers, they found no association between the C3435T SNP and lack of response to antiepileptic treatment. Likewise, Sills et al. (2005) and Kim et al. (2006) did not find any correlation between C3435T and multidrug resistance in patients with epilepsy. Finally, Zimprich et al. (2004) studied patients that were homozygous for either of two haplotypes. One haplotype (called TTT) included a thymine at positions 1236, 2677, and 3435, whereas the other haplotype (CGC) had cytosine at positions 1236 and 3435 and guanine at position 2677. They reported a correlation between the CGC haplotype and treatment failure. A discussion of possible confounders and problems with data analysis in these studies has been presented by Ott (2004). He states that due to the low sample size and low prior probability of true association, the simplest and most plausible explanation is that the significant results are false positive findings. Finally, no correlation between C3435T and central nervous system effects of loperamide treatment in healthy volunteers was found (Pauli-Magnus et al., 2003). In another study, Brunner et al. (2005) measured the brain uptake of 11Cverapamil by PET (positron emission tomography) in 20 volunteers. Ten of these had the abovementioned TTT haplotype whereas the remainders had the CGC haplotype. In this study no difference between the two groups was observed. The importance of SNPs has also been investigated in relation to P-gps role in the placenta. Hitzl et al. (2004) found

165

that the polymorphisms C3435T and G2677T were associated with lower P-gp expression in the placenta. Rahi et al. (in press) studied placental transport of the antipsychotic quetiapine, and observed that C3435T was associated with a high placental transfer value. However, there was no correlation between P-gp expression levels and quetiapine transfer suggesting that further studies are needed on this issue. Generally, the majority of studies concerning the effect of P-gp polymorphisms on adverse effects or brain penetration of drugs do not show any significant effects. A loss of function mutation could, however, have quite drastic effects, as several examples from Collie dogs devoid in functional P-gp have shown. When these Collie dogs are treated with standard doses of loperamide they are subject to severe neurotoxic effects, including mydriasis, ataxia, prostration and disorientation (Hugnet et al., 1996; Sartor et al., 2004). Likewise, the antiparasitic drug ivermectin gives rise to neurotoxicity when dogs are treated for e.g. mite infections (Mealey et al., 2004). No loss-of-function mutations have been described for MDR1 in humans (Eichelbaum et al., 2004). However, recently, Kimchi-Sarfaty et al. (2007) have suggested that silent SNPs in P-gp may be associated with changed folding patterns of the protein resulting in changed functionality. Further studies are needed on the significance of this finding (Komar, 2007). Currently, it seems that genetic polymorphisms in the MDR1 gene are not of importance in the context of mono- or polypharmacy with psychoactive drugs.

7. Conclusion
The various in vitro studies give an indication of which psychoactive drugs are substrates of P-gp. An impression of the functional significance can be gained by KO mice studies, although some reservation should be taken with regard to possible species variation. For antidepressants that are P-gp substrates the brain concentrations in KO mice are up to about 2.5 times higher than in control mice. With regard to antipsychotics P-gp exerts a moderate influence in most cases, except for risperidone and its active metabolite, which both have a more than 10-fold higher brain concentration in KO mice than in control mice. However, in humans, no loss-of-function mutation in P-gp has hitherto been discovered. Animal experiments show that drugdrug interactions of psychotropic drugs in relation to P-gp may occur, but when considering the relatively low impact of P-gp absence in KO mice in relation to most psychotropic drugs, the possible clinical effects are probably limited in most cases. Yet, for a drug like risperidone important effects cannot be excluded.

Role of the funding source

There was no specific funding for this work.

Contributors
Kristian Linnet planned the structure of the review. Thomas B. Ejsing made the first draft of the ms. including one table. Kristian

166
Linnet produced the rest of the tables and re-wrote the ms. Both authors contributed to the literature search. Both authors approved the final ms.

K. Linnet, T.B. Ejsing

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Conflicts of interest
Kristian Linnet has not had any financial, personal or other relationships that have influenced the work. Thomas B. Ejsing has not had any financial, personal or other relationships that have influenced the work.

Acknowledgements
We thank the reviewers for helpful suggestions that have improved the present work.

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