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

A new sensitive method for the measurement of lamivudine triphosphate (3TC-TP), the active intracellular metabolite of lamivudine in human c ells in vivo, has been established. The procedure involves rapid separ ation 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 le ss than 21% and a cross-reactivity of less than 0.016% with a wide var iety of other nucleoside analogs. The limit of quantitation of the ass ay for intracellular 3TC-TP was 0.195 ng/ml (0.212 pmol/10(6) cells), and a cell sample of only 4 million cells was ample for the assay, Thi s procedure, combined with our previously developed method for measuri ng 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 combinati on 3TC and ZDV therapy. In seven subjects, intracellular 3TC-TP levels ranged from 2.21 to 7.29 pmol/10(6) cells, while intracellular ZDV-MP and ZDV-TP levels ranged from <0.01 to 1.76 and 0.01 to 0.07 pmol/10( 6) cells, respectively. Concentrations of 3TC in plasma determined in these subjects ranged from 0.34 to 9.40 mu M, which was about fivefold higher than ZDV levels in plasma of 0.04 to 1.4 mu M This is the firs t study to determine the intracellular levels of the active metabolite s in HIV-infected subjects treated with this combination. These method s should Drove very useful for in vivo pharmacodynamic studies of comb ination therapy.