Aryl Phosphate Derivatives of Bromo-Methoxy-Azidothymidine: MATERIALS AND METHODS(2)
HIV-1 Replication Assay
To evaluate anti-HIV-1 activities of AZT, WHI compounds, and N-9, peripheral blood mononuclear cells (PBMC) from HIV-1-negative donors were cultured for 72 h in RPMI 1640 medium (Gibco-BRL, Grand Island, NY) supplemented with 20% (v:v) heat-inactivated fetal calf serum, 5% interleukin-2, 2 mM glutamine, 25 mM Hepes, 2 g/L NaHCO3, 50 ^g/ml gentamicin, and 4 ^g/ml phytohemagglutinin prior to exposure to HIV-1 at a multiplicity of infection of 0.1 during a 1-h adsorption period. Subsequently, cells were cultured in 96-well microtiter plates (100 ^l/well; 2 X 106/ml, triplicate wells) in the presence and absence of various concentrations (0.001-100 ^M) of drugs for 7 days. asthma inhalers
Aliquots of culture supernatants were removed from the wells on the 7th day after infection for p24 antigen enzyme immunoassay as an indication of viral replication. Percentage inhibition of virus replication was calculated by comparing the p24 antigen values from the test drug-treated infected cells with p24 antigen values from untreated infected cells. The anti-HIV activity of tested compounds was expressed as the IC50 values (the final concentration of the compound in culture medium that decreases the replication of HIV-1 by 50%). Cell viability was quantified by a colorimetric assay monitoring the ability of viable cells to reduce 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide.