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Antimicrobial Agents and Chemotherapy, October 1998, p. 2656-2660, Vol. 42, No. 10
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Development of a New Cartridge Radioimmunoassay for Determination of Intracellular Levels of Lamivudine Triphosphate in the Peripheral Blood Mononuclear Cells of Human Immunodeficiency Virus-Infected Patients

Brian L. Robbins,1 Thu T. Tran,1 Frank H. Pinkerton Jr.,1 Fatima Akeb,2 Roger Guedj,2 Jacques Grassi,3 Danny Lancaster,4 and Arnold Fridland1,5,*

Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 381051; Laboratoire de Chimie Bioorganique, UPRES-A 6001 CNRS, Université de Nice-Sophia Antipolis, 06108 Nice, Cedex 22, and Commissariat à l'Energie Atomique, Service de Pharmacologie et d'Immunologie, C.E.-Saclay, F-91191 Gif sur Yvette Cedex,3 France; Methodist Hospital, Memphis, Tennessee 381044; and Department of Pharmacology, University of Tennessee, Memphis, Tennessee 381015

Received 2 February 1998/Returned for modification 19 May 1998/Accepted 25 July 1998

A new sensitive method for the measurement of lamivudine triphosphate (3TC-TP), the active intracellular metabolite of lamivudine in human cells in vivo, has been established. The procedure involves rapid separation of 3TC-TP by using Sep-Pak cartridges, dephosphorylation to 3TC by using acid phosphatase, and measurement by radioimmunoassay using a newly developed anti-3TC serum. The radioimmunoassay had errors of less than 21% and a cross-reactivity of less than 0.016% with a wide variety of other nucleoside analogs. The limit of quantitation of the assay for intracellular 3TC-TP was 0.195 ng/ml (0.212 pmol/106 cells), and a cell sample of only 4 million cells was ample for the assay. This procedure, combined with our previously developed method for measuring zidovudine (ZDV) metabolite levels, proved capable of measuring 3TC-TP, ZDV monophosphate (ZDV-MP) and ZDV triphosphate (ZDV-TP) in human immunodeficiency virus (HIV)-infected subjects treated with combination 3TC and ZDV therapy. In seven subjects, intracellular 3TC-TP levels ranged from 2.21 to 7.29 pmol/106 cells, while intracellular ZDV-MP and ZDV-TP levels ranged from <0.01 to 1.76 and 0.01 to 0.07 pmol/106 cells, respectively. Concentrations of 3TC in plasma determined in these subjects ranged from 0.34 to 9.40 µM, which was about fivefold higher than ZDV levels in plasma of 0.04 to 1.4 µM. This is the first study to determine the intracellular levels of the active metabolites in HIV-infected subjects treated with this combination. These methods should prove very useful for in vivo pharmacodynamic studies of combination therapy.

* Corresponding author. Mailing address: Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 N. Lauderdale Blvd., Memphis, TN 38105. Phone: (901) 495-3486. Fax: (901) 495-3099. E-mail: arnold.fridland{at}stjude.org.

Antimicrobial Agents and Chemotherapy, October 1998, p. 2656-2660, Vol. 42, No. 10
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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