Correlation between intracellular pharmacological activation of nucleosideanalogues and HIV suppression in vitro

Following intracellular activation of HIV nucleoside analogue reverse trans criptase inhibitors, their triphosphates (ddNTPs) compete with endogenous n ucleoside triphosphates (dNTPs) for incorporation into proviral DNA. In thi s study we have examined the effect of combinations of two thymidine analog ues, stavudine (d4T) and zidovudine (ZDV), and two cytidine analogues, lami vudine (3TC) and zalcitabine (ddC) on intracellular drug activation and on the relevant competing dNTP in uninfected and persistently HIV-infected cel ls. Endogenous triphosphates of deoxycytidine (dCTP) and deoxythymidine (dT TP) were measured using a template primer assay and the ratio of ddNTP:dNTP was calculated. Antiviral activity of two-drug combinations was also assay ed by p24 ELISA. A significant reduction in d4T triphosphate (d4TTP) [0.11/-0.09 pmol/10(6) cells to undetectable (<0.01); P=0.039] in the presence o f equimolar concentrations of ZDV and d4T, resulted in a decrease in the d4 TTP/dTTP ratio of 90%. ZDVTP/dTTP was not significantly altered in the pres ence of d4T. 3TC (10 <mu>M) reduced total ddC phosphates by 57% and ddCTP/d CTP by 27%. 3TC phosphorylation was comparatively unaffected by ddC, up to a concentration of 10 muM ddC (>100 times the plasma concentration achieved following standard dosing). 3TC plus ddC resulted in greater p24 inhibitio n than 3TC or ddC alone (P<0.001). Combining one thymidine analogue (ZDV or d4T) with one cytidine analogue (3TC or ddC) resulted in greater inhibitio n of p24 inhibition than with any single agent. From a pharmacological view point, the combination of ZDV plus d4T should be avoided, but in vitro the combination of 3TC plus ddC confers modest benefit over either drug alone. This in vitro study illustrates that decreases in ddNTP/dNTP are consistent with a reduction in antiviral effect.