abcb1ab P-glycoprotein is involved in the uptake of citalopram and trimipramine into the brain of mice

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Abstract

The phenomenon of a heterogeneous response to the same drug in different patients is well-known. An important reason is that, even at equal concentrations, the bioavailability of a drug depends on the interaction of the drug with the blood–brain barrier (BBB). In part, this is due to the drug-transporting P-glycoprotein (P-gp), a product of the multiple drug resistance gene (ABCB1), which can transport drugs against a concentration gradient across the BBB back into the plasma and thereby reduce the bioavailability in the brain. In the present study, we have examined the uptake of the antidepressants citalopram and trimipramine into the brain of abcb1ab knockout mice compared with controls. One hour after s.c. injection of the drugs, concentrations of the two drugs and of the metabolite d-trimipramine in brain, spleen, kidney, liver and plasma were measured with HPLC. Significantly higher brain concentrations in knockout mice, showing that these drugs are substrates of P-gp and that the presence of P-gp reduces the effective bioavailability of these substances in the brain. The results of our study contradict an earlier report that citalopram is not actively transported from endothelial cells. These results were derived from an in vitro study, showing that due to the complexity of the BBB–drug interaction, it is difficult to transfer results from in vitro studies to the in vivo situation. We hypothesize that inter-individual differences in the activity of the ABCB1 gene can account in part for the great variation in clinical response to antidepressants in psychiatric patients, even at comparable plasma levels.

Introduction

The blood–brain barrier (BBB) regulates the uptake of substances from the plasma into the brain. It is formed by endothelial cells of the brain capillaries which are connected via tight junctions and the transport of substances across the BBB is regulated through endocytosis and/or transcytosis. The BBB has been studied intensively throughout the past few decades and the basic mechanisms are comparatively well-understood, although many details remain unclear. Any substance trying to cross into the brain has to interact with the BBB, and these interactions have been the subject of great scientific interest in the past, because they determine whether and to what extent a drug can enter the brain and subsequently exert a therapeutic effect. A complex interactive framework exists between the BBB and a drug, depending on many factors, among them molecular weight, hydrophobicity, degree of ionization, protein and tissue binding (Saunders et al., 1999). One cannot easily predict the ability of a certain drug to cross the BBB simply from the chemical structure. Strong hydrophobicity, for example, is considered a prerequisite for a substance to enter the brain (Habgood et al., 2000), however, various hydrophobic substances cannot easily penetrate the BBB.

One reason why it is difficult to anticipate the uptake of a certain drug into the brain is the existence of extrusion pumps, transporting substances against a concentration gradient from the brain back into the plasma. One of these extrusion pumps is P-glycoprotein (P-gp) which is expressed in the cells forming the BBB by the ABCB1 gene (Schinkel et al., 1996, Schinkel, 1999, von Moltke & Greenblatt, 2000). P-gp, a 170-kDa glycoprotein, is encoded by the ABCB1 gene in humans, shares many features with numerous bacterial and eucaryotic ATP-binding cassette (ABC) transport proteins and is a member of a phylogenetically highly conserved superfamily of transport proteins (Gottesman et al., 1995, Fromm, 2000, Kerb et al., 2001b). In mice, P-gp is encoded by the abcb1a (also called mdr1a and mdr3) and abcb1b (also called mdr1b and mdr1) gene (Devault and Gros, 1990) and although abcb1a and abcb1b are not always expressed in the same organs, the overall distribution in mice tissue overlaps well with the single ABCB1 gene in humans, suggesting that these two genes work in the same manner as the single ABCB1 (van de Vrie et al., 1998).

ABCB1 P-glycoprotein is a 1280 amino acid, glycosylated plasma membrane protein. It can actively transport its substrates against a concentration gradient, utilizing ATP hydrolysis as an energy source. The transported substrates for P-glycoprotein include anticancer drugs, such as anthracyclines, alkaloids, and immunosuppressive agent cyclosporin A, the glycoside digoxin, the synthetic glucocorticoid dexamethasone (Meijer et al., 1998), physiological steroids such as cortisol (Karssen et al., 2001), corticosterone, aldosterone and progesterone (Uhr et al., 2002), the antidepressant drug amitriptyline (Uhr et al., 2000), local anesthetics, and the anthelmintic drug ivermectin (Seelig, 1998).

ABCB1-type P-glycoprotein has been found in the apical membrane of intestinal epithelial cells (Mukhopadhyay et al., 1988), in the biliary canalicular membrane of hepatocytes, and in the lumenal membrane of proximal tubular epithelial cells in the kidney (Thiebaut et al., 1987, Thiebaut et al., 1989). High levels of ABCB1 P-glycoprotein have been found in the lumenal membrane of the endothelial cells that line small blood capillaries and form the blood–brain and blood–testis barrier (Cordon-Cardo et al., 1990, Tamai & Tsuji, 2000, Wijnholds et al., 2000).

P-glycoprotein is therefore an important component of the BBB and has an impact on the actual bioavailability of a substance in the brain. For this reason, interactions between central nervous system (CNS) drugs and P-gp are of clinical relevance. Any physician who administers antidepressants has experienced that it is impossible to predict the wide range of possible outcomes when therapy is initiated. The resistance rate of antidepressant therapy can vary between 15 and 30%. We hypothesize that P-gp can be an obstacle to the successful treatment of CNS diseases. Since the expression of the ABCB1 gene varies inter-individually, drug concentrations in the CNS can vary greatly, depending on P-glycoprotein levels. A high activity of the ABCB1 gene could lead to a considerably reduced bioavailability of a drug in the CNS and contribute to a reduced therapeutic effect or non-response. In these cases, administration of drugs that are not a substrate of P-gp would be warranted.

To test our hypothesis, we studied the brain uptake of two structurally different antidepressants, trimipramine and citalopram (Fig. 1) in knockout mice that lack the abcb1ab gene needed for P-glycoprotein production. Our goal was to determine whether the absence or presence of P-glycoprotein affects the concentrations of these substances in the brain.

Section snippets

Materials

Citalopram was used as a racemic mixture of s- and r-citalopram and obtained from Lundbeck (Copenhagen, Denmark), trimipramine was obtained from Rhone-Poulen-Rorer (Cologne, Germany).

Animals

Male abcb1ab(−/−) mice and FVB/N wildtype mice were housed individually and maintained on a 12:12 h light/dark cycle (lights on at 07:00), with food and water ad libitum. For age and body weight see Table 1. abcb1ab double knockout mice, originally created by A. Schinkel by sequential gene targeting in 129/Ola E14

Trimipramine

One hour after s.c. injection of 10 μg trimipramine/g bodyweight the trimipramine concentrations in the cerebrum were different between the abcb1ab(−/−) mutant and the wildtype controls. Analysis of variance revealed a significant group effect on the trimipramine concentrations [Wilks multivariate test of significance; effect of group: F(5, 14)=7.9; significance of F=0.001], to which reasonably only the concentration in the cerebrum contributed (univariate F-test; P-values <0.05). The cerebrum

Discussion

Our study shows considerable differences in the cerebral concentrations of citalopram between knock-out mice and controls. The differences were smaller for trimipramine and d-trimipramine, however they were still significant. This suggest that citalopram and possibly trimipramine and d-trimipramine are substrates of P-gp.

Any drug treatment requires sufficient bioavailability of the drug in the affected tissue. For cerebral disorders, this poses a particular problem: Substances that are

Acknowledgments

We would like to thank Ms. A. Rippl and Ms. M. Häusler for their very helpful technical assistance.

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