Membrane permeation characteristics of abacavir in human erythrocytes and human T-lymphoblastoid CD4+ CEM cells: comparison with (−)-carbovir
Introduction
Abacavir, (−)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol (Fig. 1), is a novel purine carbocyclic nucleoside analogue that exhibits potent and selective activity against HIV-1 and HIV-2 [1]. The hemisulfate salt of this compound has been evaluated clinically as an anti-HIV agent [2], [3], [4], [5], [6], [7] and has received approval by the FDA for the treatment of HIV (as Ziagen™ [abacavir sulfate] and, more recently, as part of the anti-HIV triple combination tablet, Trizivir™). Abacavir shares structural similarity with CBV, the first carbocyclic nucleoside reported to have anti-HIV activity [8], differing at the 6-position of the purine ring, where abacavir contains a cyclopropylamino moiety instead of the 6-lactam functionality of CBV (Fig. 1). Like CBV, abacavir is metabolized intracellularly to carbovir triphosphate [9], [10]; CBV triphosphate can then potently and selectively inhibit HIV reverse transcriptase, without significant inhibition of human DNA polymerases or DNA primase [1], [11], [12]. In anabolic studies using human T-lymphoblastoid CD4+ CEM cells, Faletto et al. [10] showed that the formation of CBV triphosphate from abacavir did not involve the intermediate formation of CBV but rather proceeded through the direct phosphorylation of abacavir, followed by a dealkylamination step at the monophosphate level.
Abacavir has been found to be superior to CBV both in its pharmacokinetics and in its ability to penetrate the CNS [4], [13], [14], [15]. We have previously shown that the membrane permeation of CBV in human erythrocytes (at 37 °C) is very slow and is dependent upon facilitated diffusion, primarily via the nucleobase carrier but also to a small extent via the es nucleoside transporter [16]. Because membrane permeation is an important determinant of drug pharmacology, we have compared the membrane permeation characteristics of abacavir and CBV in human erythrocytes and in human T-lymphoblastoid CD4+ CEM cells.
In this report, we describe dramatic differences in both the kinetics and the mechanism of influx of these carbocyclic nucleoside analogues in the two types of human cells. Also, we demonstrate that both abacavir and CBV are concentrated in these cells and provide evidence suggesting that this effect is attributable to cytosolic protein binding of these agents following cellular uptake. We also describe the development of a novel preparation of “sucrose-dense” erythrocyte ghosts which sediment through an oil phase.
Section snippets
Materials
Abacavir [1] and CBV [16] were synthesized in these laboratories; purity levels were >99% as determined by reversed-phase high-performance liquid chromatography [17]. Adenine, hypoxanthine, NBMPR, papaverine hydrochloride, sucrose, mannitol, and 1-bromododecane were purchased from Sigma. Dow Corning 550 silicone fluid was obtained from Dow Corning Corp. Dilazep was kindly provided from Hoffmann-La Roche. Hepes and RPMI-1640 were obtained from GIBCO. [3H]Abacavir (1.6 Ci/mmol) and [3H]CBV (2
Uptake equilibrium studies of permeants with human erythrocytes, erythrocyte ghosts, and CD4+ CEM cells
The time-dependent influx of 5 μM concentrations of [3H]abacavir and [3H]CBV at 20 °C is shown in human erythrocytes (Fig. 2A), in erythrocyte ghosts (Fig. 2B), and in CD4+ CEM cells (Fig. 2C). The uptake of both abacavir and CBV into erythrocytes reached equilibration within 3 min; at equilibrium, intracellular permeant concentrations were substantially higher than extracellular concentrations: by 3.2- to 3.8-fold for abacavir and by 1.8- to 2.2-fold for CBV. In parallel incubations with CD4+ CEM
Discussion
The present investigation demonstrates, in two different types of human cells, the superior membrane permeation characteristics of abacavir relative to those of CBV and provides a detailed biochemical understanding of the differing mechanisms by which these two structurally related carbocyclic nucleosides enter cells.
Abacavir was found to enter both human erythrocytes and CD4+ CEM cells exclusively by nonfacilitated diffusion. This conclusion is based upon the linear relationship of influx rate
Acknowledgements
The authors thank Barbara J. Rutledge, Ph.D., for editing assistance.
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