Fifth International Congress Drug Therapy in HIV Infection 22-26 October, 2000 Glasgow, UK

MEASUREMENT OF NUCLEOSIDE ANALOGUE TRIPHOSPHATES BY ENZYMATIC ASSAY IN HIV PATIENTS IN A CLINICAL SETTING

P.G. Hoggard¹, S. Kewn¹, S.D. Sales¹, J. Lloyd¹, B. Maher¹, S.H. Khoo¹, E.Wilkins², T. Jones³, D. Pillay⁴, C. Sabin⁵ and D.J. Back^1
1Dept. Pharmacology and Therapeutics, University of Liverpool, Liverpool, UK; ²North Manchester General Hospital, Manchester, UK; ³Glaxo Wellcome, UK; ⁴University of Birmingham Medical School, Birmingham, UK; ⁵Royal Free and University College Medical School, London, UK

Int Cong Drug Therapy HIV 2000 Oct 22-26;5:Abstract No. PL8-4
AIDS 2000, Oct 22-26;14(Suppl. 4); S10

Intracellular phosphorylation of the antiretroviral nucleoside analogues (NAs) has been studied in patients attending three adherence clinics. Intracellular ZDVTP, d4TTP, 3TCTP and CBVTP were determined enzymatically, based on the competition for HIV reverse transcriptase by radiolabelled deoxynucleoside triphosphate (dNTP) and drug triphosphate.

The study has involved both longitudinal (n = 122) and cross-sectional (n = 65) measurement of NA triphosphates. Patients with viral loads > 1000 copies/ml entered the cross-sectional study (at failure). Patients with viral loads < 1000 copies/ml were recruited in the longitudinal study to be followed until failure. Isolated PBMCs were extracted (60% methanol: perchloric acid) and neutralized before determination. Initially endogenous dNTPs were measured by a DNA polymerase assay. For measurement of drug triphosphate, the reaction mixtures included ³H-dNTP, template primer, reverse transcriptase and the appropriate amount of dNTP in a total volume of 50 µl. Radioactivity bound to the primer was separated by spotting onto filter paper (DE81), washing and scintillation counting.

There was considerable variability in endogenous dNTP levels (eg dTTP < 0.01 to 1.40 pmol/10⁶ cells, dGTP < 0.01 to 0.28 pmol/10⁶ cells). Drug triphosphate levels were also variable with CBVTP values from < 0.01 to 0.54 pmol/10⁶ cells. Data analysis of all patients in both studies showed a significant difference in 3TCTP/dCTP ratio between failures () and non-failures (); Mann–Whitney (see Figure PL8.4).

These data illustrate the importance of determining drug triphosphate/ dNTP ratios in HIV-positive patients.

Presenting author: P.G. Hoggard

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2000-10-22
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Originally published in AIDS Volume 14, Supplement 4 and hosted with permission of the publisher Lippincott Williams & Wilkins, 250 Waterloo Road, London, SE1 8RD, UK. Tel: +44 (0)20 7981 0700 Fax: +44 (0) 7981 0701

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