Pharmacokinetics, Pharmacodynamics and Drug Metabolism
Enhancement of Zidovudine Transfer to Molt-4 Cells, a Human T-Cell Model, by Dehydroepiandrosterone Sulfate

https://doi.org/10.1002/jps.22624Get rights and content

Abstract

A possible approach to improve antiretroviral therapy with nucleoside reverse transcriptase inhibitors is to enhance inhibitor delivery to CD4-positive T cells. We previously showed that dehydroepiandrosterone sulfate (DHEAS) enhances zidovudine (AZT) transfer into syncytiotrophoblast. Here, we investigated whether DHEAS also enhances AZT transfer into a cellular model of human T lymphocytes, and whether AZT is taken up by a specific transport system. The effects of DHEAS and related compounds on the uptake of [3H]AZT and other nucleosides by Molt-4 cells (a model of human CD4-positive T cells) were measured. [3H]AZT uptake by Molt-4 cells was nitrobenzylthioinosine insensitive and pH dependent, and the uptake was significantly inhibited by 1 mm ethylisopropylamiloride. [3H]AZT uptake by Molt-4 cells was increased in the presence of DHEAS, whereas uptake of other nucleosides was reduced. Kinetic study revealed that the maximum uptake velocity (up to 30 min) was increased in the presence of DHEAS. The structural requirements for AZT uptake-enhancing activity were studied using structural analogues of DHEAS. Estrone-3-sulfate and 16α-hydroxy DHEAS also enhanced AZT uptake into Molt-4 cells. The use of uptake enhancers may be a good strategy to improve the efficacy of antiretroviral therapy. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3959–3967, 2011

Section snippets

INTRODUCTION

Zidovudine (AZT), a nucleoside reverse transcriptase inhibitor (NRTI), was one of the first drugs developed to treat human immunodeficiency virus type-1 (HIV-1) infection, and is still recommended for certain patients, especially in pregnancy. To improve the efficacy of antiretroviral therapy with NRTIs, strategies to enhance drug delivery to CD4-positive T cells, the primary target of HIV-1, are required. In particular, the enhancement of influx transporter activity into T cells seems to be a

Chemicals

[3H]AZT (0.47 TBq/mmol), 2′,3′-dideoxyinosine (ddI, [3H]ddI; 1.55 TBq/mmol), [3H]adenosine (0.96 TBq/mmol), [3H]uridine (1.67 TBq/mmol), and [3H] thymidine (2.53 TBq/mmol) were purchased from Moravek Biochemicals, Inc. (Brea, California). All other chemicals were commercial products of analytical grade.

Cell Culture

Molt-4 cells (Lot No. 3984740) were purchased from American Type Cell Culture (Manassas, Virginia). They were cultured in RPMI1640 medium (Sigma, St. Louis, Missouri) supplemented with 10% fetal

Characteristics of Nucleoside Transport in Molt-4 Cells

To characterize the nucleoside uptake mechanism, the effect of NBMPR, a specific inhibitor of ENT, on the uptake of nucleosides in Molt-4 cells was measured. In the untreated control cells, [3H]adenosine uptake was the highest, [3H]AZT and [3H]uridine uptakes were relatively high, and [3H]ddI and [3H]thymidine uptakes were moderate (Fig. 1). [3H]AZT uptake was not inhibited by NBMPR, but was almost completely saturated by an excess of unlabeled AZT (Fig. 1). [3H]ddI and [3H]thymidine uptakes

DISCUSSION

Involvement of the ENT family in uptake of nucleoside and nucleoside analogs by Molt-4 cells was investigated using NBMPR, a well-known ENT-specific inhibitor. Molt-4 cells have been shown to express equilibrative nucleoside transporter 1 (ENT1) and ENT2 mRNAs.17,18 ENT1 is very sensitive to NBMPR and is almost completely inhibited by 0.1 µM NBMPR. ENT2 is less sensitive to NBMPR but is inhibited by 100 µM NBMPR.19 Therefore, we can separate the functions of ENT1 and ENT2 by

Acknowledgements

This study was supported in part by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science and a grant for the adaptable and seamless technology transfer program through target-driven R&D from the Japan Science and Technology Agency, as well as grants from Suzuken Memorial Foundation, Uehara Memorial Foundation, and Nateglinide Memorial Toyoshima Research and Education Fund.

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