TY - JOUR
T1 - Intracellular zidovudine (ZDV) and ZDV phosphates as measured by a validated combined high-pressure liquid chromatography-radioimmunoassay procedure
AU - Slusher, J. T.
AU - Kuwahara, S. K.
AU - Hamzeh, F. M.
AU - Lewis, L. D.
AU - Kornhauser, D. M.
AU - Lietman, P. S.
PY - 1992
Y1 - 1992
N2 - In vitro studies of zidovudine (ZDV) phosphorylation may not accurately reflect the in vivo dose-response relationship, which is crucial to determining the relationship between ZDV exposure, efficacy, and toxicity. However, measurement of ZDV phosphorylated anabolites in peripheral blood mononuclear cells (PBMCs) from ZDV-treated human immunodeficiency virus (HIV)-infected patients would be extremely useful in the more appropriate utilization of ZDV in the treatment of HIV infection. We developed a specific and sensitive combined high-pressure liquid chromatography (HPLC)- radioimmunoassay (RIA) procedure for the determination of ZDV, ZDV- monophosphate, ZDV-diphosphate, and ZDV-triphosphate in PBMCs taken from ZDV- treated HIV-infected patients. ZDV and its anabolites were extracted from washed, Ficoll-Paque-isolated PBMCs and then separated by HPLC using a strong anion-exchange column. The anabolites were then hydrolyzed to ZDV with acid phosphatase. ZDV was then measured by using a modified commercially available RIA protocol. Our method was validated by measuring [3H]ZDV anabolites generated in Molt-4 cells radioisotopically and simultaneously by the combined HPLC-RIA procedure. The ZDV determinations correlated well (r2 = 0.97) over the range of 0.037 to 5.2 pmol (10 to 1,400 pg) per assay tube. Furthermore, we defined the stability of ZDV anabolites during ficoll isolation and the recovery after extraction and cleanup. We then measured intracellular parent ZDV and its phosphorylated anabolites in PBMCs from six ZDV-treated HIV-infected patients (PBMCs were taken 2 h after a 300-mg oral dose). The mean concentrations (± standard deviations) of parent and of mono-, di-, and triphosphates were 0.15 ± 0.08, 1.4 ± 1, 0.082 ± 0.02, and 0.081 ± 0.03 pmol/106 PBMC, respectively (one pmol/106 PBMC represents a concentration of approximately 1 μM). Concurrent serum ZDV concentrations were between 1.3 and 7.1 μM. This method should provide a useful tool for evaluating in vivo pharmacokinetics of ZDV anabolites in PBMCs and possibly other cell types, even at the low doses of ZDV currently administered therapeutically.
AB - In vitro studies of zidovudine (ZDV) phosphorylation may not accurately reflect the in vivo dose-response relationship, which is crucial to determining the relationship between ZDV exposure, efficacy, and toxicity. However, measurement of ZDV phosphorylated anabolites in peripheral blood mononuclear cells (PBMCs) from ZDV-treated human immunodeficiency virus (HIV)-infected patients would be extremely useful in the more appropriate utilization of ZDV in the treatment of HIV infection. We developed a specific and sensitive combined high-pressure liquid chromatography (HPLC)- radioimmunoassay (RIA) procedure for the determination of ZDV, ZDV- monophosphate, ZDV-diphosphate, and ZDV-triphosphate in PBMCs taken from ZDV- treated HIV-infected patients. ZDV and its anabolites were extracted from washed, Ficoll-Paque-isolated PBMCs and then separated by HPLC using a strong anion-exchange column. The anabolites were then hydrolyzed to ZDV with acid phosphatase. ZDV was then measured by using a modified commercially available RIA protocol. Our method was validated by measuring [3H]ZDV anabolites generated in Molt-4 cells radioisotopically and simultaneously by the combined HPLC-RIA procedure. The ZDV determinations correlated well (r2 = 0.97) over the range of 0.037 to 5.2 pmol (10 to 1,400 pg) per assay tube. Furthermore, we defined the stability of ZDV anabolites during ficoll isolation and the recovery after extraction and cleanup. We then measured intracellular parent ZDV and its phosphorylated anabolites in PBMCs from six ZDV-treated HIV-infected patients (PBMCs were taken 2 h after a 300-mg oral dose). The mean concentrations (± standard deviations) of parent and of mono-, di-, and triphosphates were 0.15 ± 0.08, 1.4 ± 1, 0.082 ± 0.02, and 0.081 ± 0.03 pmol/106 PBMC, respectively (one pmol/106 PBMC represents a concentration of approximately 1 μM). Concurrent serum ZDV concentrations were between 1.3 and 7.1 μM. This method should provide a useful tool for evaluating in vivo pharmacokinetics of ZDV anabolites in PBMCs and possibly other cell types, even at the low doses of ZDV currently administered therapeutically.
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U2 - 10.1128/AAC.36.11.2473
DO - 10.1128/AAC.36.11.2473
M3 - Article
C2 - 1489190
AN - SCOPUS:0026446188
SN - 0066-4804
VL - 36
SP - 2473
EP - 2477
JO - Antimicrobial agents and chemotherapy
JF - Antimicrobial agents and chemotherapy
IS - 11
ER -