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The effects of 3'-azido-3'-deoxythymidine (AZT), phosphonoformate (PFA), and 2',3'-dideoxythymidine (ddT) and their combinations on human immunodeficiency type 1 (HIV-1) replication were studied by measuring the HIV-1 p24 antigen expression and reverse transcriptase (RT) release in HIV-1-infected MT4 cells in vitro. RT activity was also measured in a cell-free system by using poly(rA)-oligo(dT) as the primer-template, and cell growth inhibition was measured in noninfected MT4 cells. The interactions of these two-and three-drug combinations were evaluated by the combination index (CI) method and isobologram techniques. The 50% effective concentrations (EC50s) of AZT, PFA, and ddT were 0.014 to 0.005, 9.4 to 8.8, and 8.4 to 2.5 ,uM, respectively, for p24 enzyme-linked immunosorbent assays (ELISAs) and 0.0055 to 0.0034, 1.43 to 1.37, and 2.87 to 2.83 ,uM, respectively, for RT activity in vitro; for RT activity in the cell-free system, the EC50s were 0.00019 to 0.00024, 0.012 to 0.02, and 0.00074 to 0.0005 ,uM, for AZT-5'-triphosphate, PFA, and ddT-5'-triphosphate, respectively. AZT in combination with PFA (1:200) or ddT (1:5) as well as the combination of these three drugs (1:200:5) synergistically inhibited HIV-1 replication and RT activity in the cell-free system over a wide range of drug concentrations, with the CIs ranging from 0.5 to 0.9, in which CIs of <1, 1, and >1 indicate synergism, additive effect, and antagonism, respectively. Three-and two-drug combinations of AZT, PFA, and ddT showed similar degrees of synergism against HIV-1 replication in p24 assays and RT release assays, whereas the combination of AZT and ddT was found to be the most selective in terms of its anti-HIV-1 effect versus cytotoxicity. Dose reduction indices calculated from both HIV-1 replication inhibition, as measured by p24 ELISA and by RT activity in the cell-free system, indicated that two-and three-drug combinations at high effect levels and the selected combination ratios allow 2-to 240-fold dose reduction over the single drug alone in terms of their anti-HIV-1 effects. The three-drug combination showed the highest dose reduction index. These findings suggest that increased efficacy and reduced toxicity may be achieved in AIDS therapy by using AZT, PFA, and ddT in two-or three-drug combinations.

Epithelial Na(+) channels (ENaCs) are ion channels that play important roles in physiology as well as pathophysiology. Inhibiting ENaCs using amiloride and its derivatives has been suggested in treatment of cardiovascular disease and hypertension. By immunoblotting, we demonstrated the presence of ENaC-alpha protein in the flagellar midpiece of both rat and human sperm. Immunohistochemistry analyses in rat testis localized ENaC-alpha expressed in the Leydig cell, Sertoli cell, Ap spermatogonium, spermatocyte, spermatid and residual body. Importantly, using computer-assisted sperm motility analysis, we first observed that EIPA [5-(N-ethyl-N-isopropyl)-amiloride hydrochloride] inhibition of ENaCs, possibly including ENaC-alpha and ENaC-delta, significantly improved the sperm motility in healthy donors by 14.23% (mean +/- SEM, 68.75 +/- 9.76% vs. 78.53 +/- 6.20%, p < 0.001) and in asthenospermia patients by 115.89% from 9.50 +/- 6.11% to 20.51 +/- 12.13% (mean +/- SEM, p < 0.001). The improved sperm motility by EIPA has important clinical implications in the treatment of asthenospermia and certainly warrants further investigation.

2'-Fluoro-5-ethyl-1-4-D-arabinofuranosyluracil (FEAU) was synthesized, and its biological activities were compared with those of 2'-fluoro-5-methyl-1-oi-D-arabinofuranosyluracil (FMAU). Earlier studies indicated that both compounds showed potent anti-herpes simplex virus activity, with a 50% effective dose (EDSO) of <0.25 ,uM. In the present study the cell growth inhibitory activity of FEAU (ED50, 200 to 2,060 ,uM) was found to be about 100-fold less than that of FMAU. With an ED50 ranging from 630 to 3,700 ,uM, FEAU only weakly inhibited thymidine incorporation into DNA, as compared with FMAU with an ED50 of 9 to 28 ,uM A series of studies on the structure-activity relationships of pyrimidine nucleoside analogs indicated that the antiherpes simplex virus effect was greatly increased by 2'-fluoro-arabinosyl group substitution and by 5-iodo or 5-methyl group substitution (7,11,13). 2'-Fluoro-5-methyl-1-p-D-arabinofuranosyluracil (FMAU) emerged as the most potent anti-herpes simplex virus agent in a mouse model (13). However, subsequent studies indicated that FMAU caused neurotoxicity in humans (6) and was incorporated into mammalian cell DNA as well as into viral DNA (8).Earlier studies of FMAU showed that the 50% effective doses (ED50s) for inhibiting herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) replication in vitro are 0.006 and 0.023 ,uM, respectively, whereas the ED50 for inhibiting the growth of Vero cells is 2.8 ,uM (1). Preliminary studies of 2'-fluoro-5-ethyl-1-,-D-arabinofuranosyluracil (FEAU) indicated that the ED50s for the inhibition of HSV-1 and HSV-2 replication are 0.024 and 0.24 ,uM, respectively. However, the ED50 for the inhibition of Vero cell growth by FEAU is >200 FiM (7,10,13). FMAU, therefore, is 4-and 10-fold more potent than FEAU against HSV-1 and HSV-2 replication, respectively, but 71-fold more toxic than FEAU toward host cells.In this study, FEAU is compared with FMAU for cytotoxicity in a variety of mammalian cells, kinetic constants for host and viral thymidine kinases, metabolic properties which may be modulated to reduce toxicity toward host cells, and toxicity in several animal species. The detailed synthesis procedure for FEAU is also reported. * Corresponding author. MATERIALS AND METHODSSynthesis of FEAU. The chemical synthesis of FEAU was performed by two different procedures (11, 13): (i) catalytic reduction of 5-vinyl-1-(2'-deoxy-2'-fluoro-,-D-arabinofuranosyl) uracil and (ii) condensation of 5-ethyluracil with 3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranosyl bromide. The latter method is more practical than the former. It should be noted that condensation of the silylated 5-ethyluracil with the 2-fluoro-arabinosyl bromide without the Lewis acid catalyst in methylene chloride at room temperature yielded and desired P-nucleoside as the major product, and very little formation of the ax-isomer was observed. Saponification of the condensation product with methanolic ammonia yielded FEAU, which was directly crystallized from a mixture of chloroform and aceton...

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