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Antimicrobial Agents and Chemotherapy, January 2003, p. 255-261, Vol. 47, No. 1
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.1.255-261.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Enzymatic Assay for Measurement of Intracellular DXG Triphosphate Concentrations in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus Type 1-Infected Patients

Stephen Kewn,1* Laurene H. Wang,2 Patrick G. Hoggard,1 Franck Rousseau,2 Robert Hart,2 John P. MacNeela,2 Saye H. Khoo,1 and David J. Back1

Department of Pharmacology & Therapeutics, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, United Kingdom,1 Triangle Pharmaceuticals, Inc., Durham, North Carolina 275142

Received 21 June 2002/ Returned for modification 16 September 2002/ Accepted 17 October 2002

DXG {[2R-cis]-2-amino-1,9-dihydro-9-[2-[hydroxymethyl]-1,3-dioxolan-4-yl]-6H-purin-6-one} and its prodrug DAPD ([2R-cis]-4-[2,6-diamino-9H-purin-9-yl]-1,3-dioxolane-2-methanol; amdoxovir) are novel 2',3'-dideoxynucleosides (ddNs) displaying activity against human immunodeficiency virus type 1 (HIV-1). In this paper, we describe the development of an enzymatic assay for determining the intracellular active metabolite of DXG and DAPD, DXG triphosphate (DXGTP), in peripheral blood mononuclear cells (PBMCs) from HIV-infected patients. The assay involves inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates (dNTPs) into a synthetic template primer. DXGTP (0.6 pmol) inhibited control product formation with or without a preincubation step. Inhibition was greatest when the template primer was most diluted. DAPDTP inhibited control product formation only at very high levels (50 pmol) and when a preincubation procedure was used. However, reduced template primer stability in assays using preincubation steps, coupled with potential interference by DAPDTP, led to the current assay method for DXGTP being performed without preincubation. Standard DXGTP inhibition curves were constructed. The presence of PBMC extracts or endogenous dGTP did not interfere with the DXGTP assay. Intracellular DXGTP and dGTP concentrations were determined in PBMCs from HIV-infected patients receiving oral DAPD (500 mg b.i.d.). Peak concentrations of DXGTP were obtained 8 h after dosing and were measurable through 48 h postdose. Levels of endogenous dGTP were also determined over 48 h. No direct relationship was observed between concentrations of DXGTP and dGTP. Quantification of DXGTP concentrations in PBMCs from patients receiving a clinically relevant dose of DAPD is possible with this enzymatic assay.

* Corresponding author. Mailing address: Department of Pharmacology & Therapeutics, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, United Kingdom. Phone: (0151) 794-5919. Fax: (0151) 794-5656. E-mail: Kewny{at}liverpool.ac.uk.

Antimicrobial Agents and Chemotherapy, January 2003, p. 255-261, Vol. 47, No. 1
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.1.255-261.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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