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Antimicrobial Agents and Chemotherapy, February 2007, p. 405-411, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.00730-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Quantitative Immunoassay To Measure Plasma and Intracellular Atazanavir Levels: Analysis of Drug Accumulation in Cultured T Cells

Laboratoire de Chimie des Molécules Bioactives et Arômes UMR 6001 CNRS-Université de Nice-Sophia Antipolis, Nice, France,¹ CEA, Service de Pharmacologie et d'Immunologie, DRM, CEA Saclay, 91191 Gif-sur-Yvette cedex, France,² Service de Pharmacologie, Hôpital Pasteur, Nice, France³

Received 14 June 2006/ Returned for modification 8 August 2006/ Accepted 7 November 2006

We have developed an enzyme immunoassay to measure atazanavir (ATV) levels in plasma and cells. Anti-ATV polyclonal antibodies were raised in rabbits by using a synthetic ATV derivative coupled to bovine serum albumin as the immunogen, and the enzyme tracer was prepared by chemically coupling the ATV derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates, and the lowest limit of quantification was 150 pg/ml, which is about 10 times more sensitive than previously published techniques. The plasma assay was performed, after a simple methanol extraction, with a minimum of 30 µl of plasma. This assay showed good precision and efficiency, since the rates of recovery from human plasma and cell extracts spiked with ATV ranged form 93 to 113%, with coefficients of variation of less than 10%. ATV concentrations were measured in peripheral blood mononuclear cells incubated with various ATV concentrations and in CEM cells in the absence or presence of antiretroviral drugs and drug transporter inhibitors. The results indicated a dose-dependent uptake (intracellular concentration/extracellular concentration ratio range, 0.04 to 19). A significant increase in the accumulation of ATV was noticed in the presence of P-glycoprotein and MRP1 inhibitors (dipyridamole, inter alia). Interestingly, efavirenz significantly increased the baseline accumulation of ATV, whereas nevirapine induced a marked reduction. This new enzyme immunoassay for measuring plasma and intracellular ATV levels was fully validated and provides an inexpensive and useful tool for routine therapeutic drug monitoring. Moreover, in vitro results suggested the implication of drug transporters and interactions with other antiviral drugs that should be further explored in human immunodeficiency virus-infected patients.

Published ahead of print on 20 November 2006.