Duration and degree of cyclosporin induced P-glycoprotein inhibition in the rat blood–brain barrier can be studied with PET

doi:10.1016/j.neuroimage.2006.05.047

NeuroImage

Volume 32, Issue 3, September 2006, Pages 1134-1141

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Abstract

Active efflux transporters in the blood–brain barrier lower the brain concentrations of many drug molecules and endogenous substances and thus affect their central action. The objective of this investigation was to study the dynamics of the entire inhibition process of the efflux transporter P-glycoprotein (P-gp), using positron emission tomography (PET). The P-gp marker [¹¹C]verapamil was administered to anesthetized rats as an i.v. bolus dose followed by graded infusions via a computerized pump system to obtain a steady-state concentration of [¹¹C]verapamil in brain. The P-gp modulator cyclosporin A (CsA) (3, 10 and 25 mg/kg) was administered as a short bolus injection 30 min after the start of the [¹¹C]verapamil infusion. The CsA pharmacokinetics was studied in whole blood in a parallel group of rats. The CsA blood concentrations were used as input to model P-gp inhibition. The inhibition of P-gp was observed as a rapid increase in brain concentrations of [¹¹C]verapamil, with a maximum after 5, 7.5 and 17.5 min for the respective doses. The respective increases in maximal [¹¹C]verapamil concentrations were 1.5, 2.5 and 4 times the baseline concentration. A model in which CsA inhibited P-gp by decreasing the transport of [¹¹C]verapamil out from the brain resulted in the best fit. Our data suggest that it is not the CsA concentration in blood, but rather the CsA concentration in an effect compartment, probably the endothelial cells of the blood–brain barrier that is responsible for the inhibition of P-gp.

Introduction

The uptake of drugs by tissues such as the brain is decreased by biological membranes containing active efflux transporters. The blood–brain barrier consists of endothelial cells connected by “tight junctions”. The tight junctions prevent drug molecules from diffusing between the endothelial cells. Hence, drug molecules have to pass the two membranes of the blood–brain barrier in order to penetrate into the brain tissue. These two membranes are the luminal membrane, facing capillary blood, and the abluminal membrane, facing brain interstitial fluid. P-glycoprotein (P-gp), the most well known and, according to current understanding, the most important efflux transporter for exogenous substances, is present at the luminal membrane of the blood–brain barrier (Cordon-Cardo et al., 1989, Tsuji et al., 1992).

Positron emission tomography (PET) is a unique in vivo method of assessing drug interactions with biochemical target systems such as cerebral receptors, enzyme systems and transporters in the brain (Bergstrom et al., 2000, Lammertsma, 2002). PET has gained an important role in drug development, especially in the development of neuropsychiatric drugs (Andree et al., 2000, Bergstrom et al., 2003). Because of the broad substrate specificity of P-gp, it is likely that many drug candidates will be modulators of the P-gp system and therefore not be suitable for further development as drugs. Verapamil, a calcium channel blocker, interacts with P-gp in the blood–brain barrier and is excluded from the brain because of P-gp action (Chikhale et al., 1995). ¹¹C-labeled verapamil has been used in PET studies of P-gp-mediated efflux in rats and mice (Bart et al., 2003, Hendrikse et al., 1999) and recently in humans (Sasongko et al., 2005). These studies have shown that Cyclosporin A (CsA), a potent P-gp modulator, increases the brain uptake of [¹¹C]verapamil.

The aim of this project was to develop new methodology involving PET for kinetic studies of P-gp modulation in vivo and to investigate possible mechanisms for the inhibition process. In earlier studies, CsA was administered as a bolus or an infusion prior to a bolus injection of [¹¹C]verapamil (Bart et al., 2003, Doze et al., 2000, Hendrikse et al., 1999, Sasongko et al., 2005). In the present study, [¹¹C]verapamil was administered as a bolus and graded infusions by a computerized pump system to obtain a steady-state condition in the animal. The inhibitor drug, CsA, was administered as a single bolus injection when steady-state [¹¹C]verapamil concentrations were reached. The magnitude and duration of P-gp inhibition were investigated dynamically in a sequence of PET scans, thus providing an opportunity to study the influence of time on the process. This enabled the investigation of the whole P-gp inhibition process, something that has not been focused on in earlier studies.

Section snippets

Materials and methods

CsA (50 mg/ml) in cremophore EL (650 mg/ml) (Sandimmune) was obtained from Novartis (Basel, Switzerland), normethylverapamil hydrochloride was obtained from RBI/Sigma-Aldrich (Stockholm, Sweden), and isoflurane was obtained from Baxter Medical (Stockholm, Sweden). All other chemicals were obtained commercially and were of reagent grade.

The uptake of [¹¹C]verapamil in the brain was studied in male Sprague-Dawley rats weighing on average (±SD) 425 ± 100 g. The CsA concentration in rat whole blood

Results

Two PET images of [¹¹C]verapamil in the brain are shown in Fig. 2. The left image is a summation over the first 30 min of the emission scan, i.e., before CsA administration. The same rat is shown in the right image which is a summation over the next 30 min after administration of 25 mg/kg CsA. The uptake of [¹¹C]verapamil is clearly increased after CsA administration. The [¹¹C]verapamil concentration–time profiles in the brain before and after the three CsA doses of 3 (n = 3), 10 (n = 3) and 25

Discussion

In this paper, we developed a new method for studying P-gp modulation in vivo, using PET. We administered [¹¹C]verapamil as a series of short infusions utilizing a computerized pump infusion system to rapidly obtain steady-state concentrations of the tracer prior to the administration of the P-gp inhibitor CsA, thus allowing us to investigate the dynamics of the whole inhibition process. This differs from earlier methods in which the inhibitor has been given prior to a bolus injection of the

Conclusions

We have shown that the inhibition of P-gp by CsA is a rapid process, both in the sense that the transporters rapidly become blocked when CsA is administered but also in the sense that this inhibition is rapidly reversible due to the rapid brain pharmacokinetics of [¹¹C]verapamil. Our experiments indicate that CsA increases [¹¹C]verapamil uptake in the brain by reducing its transport out of the brain rather than by increasing its transport into the brain. Our data also suggest that it is not the

Acknowledgments

We wish to thank the staff at Uppsala Imanet for excellent research conditions and the staff at the Department of Clinical Immunology, Autoimmunity and Allergy (University Hospital Uppsala, Sweden) for the CsA analyses.

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Citation Excerpt :
Positron emission tomography (PET) with [11C]verapamil, either in racemic form or in form of the (R)-enantiomer ((R)-[11C]verapamil), has been frequently used to non-invasively study the functional activity of the adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) at the blood–brain barrier (BBB) of animals and humans (see, for instance, Refs. [1–7]).
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Duration and degree of cyclosporin induced P-glycoprotein inhibition in the rat blood–brain barrier can be studied with PET

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Conclusions

Acknowledgments

Nucl. Med. Biol.

NeuroImage

Neuropharmacology

Eur. Neuropsychopharmacol.

Nucl. Med. Biol.

Nucl. Med. Biol.

Trends Pharmacol. Sci.

Neuroscience

Biochem. Pharmacol.

Life Sci

Biochem. Pharmacol.

Appl. Radiat. Isot.

Nucl. Med. Biol.

Information theory and extension of maximum likelihood principle