In vitro P-glycoprotein efflux inhibition by atypical antipsychotics is in vivo nicely reflected by pharmacodynamic but less by pharmacokinetic changes

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Abstract

Background

P-glycoprotein (P-gp), an efflux transporter of the blood–brain barrier, limits the access of multiple xenobiotics to the central nervous system (CNS). Thus drug-dependent inhibition, induction or genetic variation of P-gp impacts drug therapy.

Methods

We investigated atypical antipsychotics and their interaction with P-gp. Amisulpride, clozapine, N-desmethylclozapine, olanzapine, and quetiapine were assessed in vitro on their inhibitory potential and in vivo on their disposition in mouse serum and brain, and behaviourally on the RotaRod test. In vivo wildtype (WT) and mdr1a/1b double knockout mice (mdr1a/1b (−/−, −/−); KO) were investigated.

Results

In rhodamine 123 efflux assay drugs inhibitory potency to P-gp could be ranked quetiapine > N-desmethylclozapine > clozapine > olanzapine. When treating WT and KO mice i.p. and assessing brain and serum levels by HPLC analysis, P-gp expression has the highest but a rather short effect on the distribution of amisulpride, whereas the others ranked N-desmethylclozapine > olanzapine > quetiapine > clozapine; contrasted by in vivo behavioral changes at various time points. Here quetiapine > clozapine > olanzapine impacts behavior most when P-gp is lacking. Present results indicate the relevance of P-gp expression for CNS-drug therapy.

Conclusions

Combination of in vitro, and in vivo methods highlights that inhibitory potency did not reflect P-gp related drug disposition. But, when drugs were ranked for inhibitory potency, this order is reflected in pharmacodynamic changes or vice versa. Pharmacodynamic effects otherwise were at most correlated to drug brain levels, which however, were present only to a limited extent (by positron emission tomography) accessible in humans.

Highlights

► We investigated atypical antipsychotics and their interaction with P-gp at the BBB. ► Inhibitory potency, pharmacokinetics and pharmacodynamics were investigated. ► Amisulpride, N-desmethylclozapine, olanzapine and quetiapine are substrates of P-gp. ► Ranking of drugs for inhibitory potency is reflected in their pharmacodynamics. ► Pharmacodynamic effects were best correlated to brain levels not accessible in humans.

Introduction

Efflux transporters localized at several blood–tissue barriers in the body limit the access of different endogenous and exogenous substances to protected areas of the body and play an important role in disposition of drugs. Especially, the brain is guarded by several transporters localized in the blood–brain barrier. An important protein for the transport of drugs in this barrier is P-glycoprotein (P-gp) (Cordon-Cardo et al., 1989), which is also localized, for example in the hepatocyte canalicular membrane, renal proximal tubules, and the intestinal mucosa (Thiebaut et al., 1987). It is a member of the ATP-binding cassette (ABC) superfamily and is encoded by the multi-drug resistance gene (MDR1, ABCB1). Mice have two genes, mdr1a and mdr1b (Devault and Gros, 1990), which are supposed to have the same function as the sole MDR1 in humans. Single nucleotide polymorphism SNPs have been identified in the MDR1 gene, which lead to a changed P-gp expression and are associated with varying plasma levels of P-gp substrates, for example digoxine (Hoffmeyer et al., 2000). A wide spectrum of drugs, including central nervous system (CNS) acting substances, like antidepressants (amitriptyline, citalopram) (Uhr et al., 2000, Uhr and Grauer, 2003a, Uhr et al., 2003b) and antipsychotics (risperidone) (Doran et al., 2005, Wang et al., 2004a, Wang et al., 2004b) are transported by P-gp. In brains of mdr1a/1b (−/−, −/−) mice concentration differences were observed for several CNS acting P-gp substrates (Doran et al., 2005, Schinkel et al., 1996, Uhr and Grauer, 2003a, Uhr et al., 2003b, Wang et al., 2004a, Wang et al., 2004b). But not only variations in P-gp expression due to gene polymorphisms or deficiency can vary plasma and brain levels of substances, also inhibitors or inducers of P-gp, like verapamil (Nakagami et al., 2005) or St. John's wort (Pfrunder et al., 2003) lead to changes in the pharmacokinetics of concomitantly prescribed drugs.

In this study, affinities of the common atypical antipsychotics amisulpride, clozapine, its active metabolite N-desmethylclozapine, olanzapine, and quetiapine are investigated in terms of substrate and inhibitory properties of P-gp. All substances have antagonistic affinities to the dopamine D2 receptor in combination with properties mainly for the D3 receptor in case of amisulpride and for 5-HT2A, α1, H1, and several other receptors in the case of other antipsychotics. These atypical antipsychotics exhibit, because of their improved receptor profile, less incidence for extrapyramidal side effects compared to the typical antipsychotics, e.g. haloperidol, that are sole dopamine D2 antagonists.

To what extent variations in P-gp expression lead to functional consequences, like behavioral changes, of drugs transported by this protein is investigated in the second part of the study in mdr1a/1b (−/−, −/−) mice compared to wild type (WT) mice by measuring motor impairment typically induced by dopamine D2 receptor antagonism.

Section snippets

Drugs

Amisulpride pure drug substance was kindly supplied by Sanofi-Synthelabo (Berlin, Germany), clozapine was purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany), and its metabolite N-desmethylclozapine was kindly donated by Sandoz (Basel, Switzerland). Olanzapine was obtained from Mikromol GmbH (Luckenwalde, Germany) and quetiapine pure drug substance was kindly supplied by Astra Zeneca (London, United Kingdom). These drug substances were dissolved in methanol (HPLC-grade; Merck,

Inhibition of P-gp mediated R123 efflux in P388 cells

Inhibition of P-gp mediated R123 efflux was measured in a microtiter plate based uptake assay using mdr1 over-expressing mouse lymphocytes (P388/mdr1). The corresponding parental cell line P388/par was used to account for effects that are not related to P-gp. The antipsychotic compounds clozapine, N-desmethylclozapine, olanzapine, quetiapine and amisulpride were investigated up to a maximal concentration of 200 μM. In every experiment, 100 μM verapamil (a known P-gp inhibitor) was used as

Discussion

There is increasing evidence from in vitro assays that antipsychotic drugs may modulate P-gp function. However, so far, only little is known about the influence of newer atypical antipsychotic drugs on P-gp, and on its role in the distribution and elimination of antipsychotic compounds (Abou El Ela et al., 2004). Different antipsychotic compounds and a metabolite were investigated for their inhibitory properties in microtiter plate based efflux assays using mdr1 over-expressing P388/mdr1 cells,

Acknowledgments

The work was supported by the Deutsche Forschungsgemeinschaft (DFG Grant, Hi399/6-1).

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