Several 4- or 5-monosubsituted and 4,5-disubstituted 1,2,3-triazole analogues of the anti-HIV-1 lead compound [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D- ribofuranosyl]thymine]-3'-spiro-5"-(4"-amino-1",2"-oxathiole 2",2"-dioxide) (TSAO-T) have been prepared and evaluated as inhibitors of HIV-1-induced cytopathicity. These analogues have been prepared by 1,3-diplar cycloaddition of [2,5-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]- 3-spiro-5'-(4'-amino- and 4'-(N-acetylamino)-1',2'-oxathiole 2',2'-dioxide) (TSAO) azides to various substituted acetylenes. Several 4- and 5-substituted 1,2,3-triazole-TSAO analogues proved superior to the unsubstituted derivative by 1-2 orders of magnitude. In particular the 5-substituted amido-, (methylamido)-, and (dimethylamido)-1,2,3-triazole derivatives of TSAO were endowed with potent anti-HIV-1 activity (50% effective concentration: 0.056-0.52 microM). They show a similar resistance spectrum as previously noted for TSAO-T and related derivatives.

The plant lectins derived from Galanthus nivalis (Snowdrop) (GNA) and Hippeastrum hybrid (Amaryllis) (HHA) selectively inhibited a wide variety of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains and clinical (CXCR4-and CCR5-using) isolates in different cell types. They also efficiently inhibited infection of T lymphocytes by a variety of mutant virus strains. GNA and HHA markedly prevented syncytium formation between persistently infected HUT-78/HIV cells and uninfected T lymphocytes. The plant lectins did not measurably affect the antiviral activity of other clinically approved anti-HIV drugs used in the clinic when combined with these drugs. Short exposure of the lectins to cell-free virus particles or persistently HIV-infected HUT-78 cells markedly decreased HIV infectivity and increased the protective (microbicidal) activity of the plant lectins. Flow cytometric analysis and monoclonal antibody binding studies and a PCR-based assay revealed that GNA and HHA do not interfere with CD4, CXCR4, CCR5, and DC-SIGN and do not specifically bind with the membrane of uninfected cells. Instead, GNA and HHA likely interrupt the virus entry process by interfering with the virus envelope glycoprotein. HHA and GNA are odorless, colorless, and tasteless, and they are not cytotoxic, antimetabolically active, or mitogenic to human primary T lymphocytes at concentrations that exceed their antivirally active concentrations by 2 to 3 orders of magnitude. GNA and HHA proved stable at high temperature (50°C) and low pH (5.0) for prolonged time periods and can be easily formulated in gel preparations for microbicidal use; they did not agglutinate human erythrocytes and were not toxic to mice when administered intravenously.

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The activity of three human immunodeficiency virus (HIV) protease inhibitors was investigated in human primary monocytes/macrophages (M/M) chronically infected by HIV-1. Saquinavir, KNI-272, and ritonavir inhibited the replication of HIV-1 in vitro, with EC50s of approximately 0.5-3.3 microM. However, only partial inhibition was achievable, even at the highest concentrations tested. Also, the activity of these drugs in chronically infected M/M was approximately 7- to 26-fold lower than in acutely infected M/M and approximately 2- to 10-fold lower than in chronically infected H9 lymphocytes. When protease inhibitors were removed from cultures of chronically infected M/M, production of virus rapidly returned to the levels found in untreated M/M. Therefore, relatively high concentrations of protease inhibitors are required to suppress HIV-1 production in chronically infected macrophages, and such cells may be a vulnerable point for the escape of virus in patients taking these drugs.

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So324 is a 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4T-MP) prodrug containing at the phosphate moiety a phenyl group and the methylester ofalanine linked to the phosphate through a phosphoramidate linkage. So324 has anti-HIV activity in human CEM, MT4, and monocyte/macrophage cells that is superior to that of d4T. In contrast to d4T, So324 is also able to inhibit HIV replication in thymidine kinase-deficient CEM cells. After uptake of So324 by intact human lymphocytes, d4T-MP is released and subsequently converted intracellularly to d4T-TP. In addition, accumulation of substantial amounts of a novel d4T derivative has been found. This d4T metabolite has been characterized as alaninyl d4T-MP. The latter metabolite accumulates at "13-to 200-fold higher levels than d4T-TP depending the experimental conditions. Alaninyl d4T-MP should be considered as an intra-and/or extracellular depot form of d4T and/or d4T-MP. These findings may explain the superior anti-retroviral activity of So324 over d4T in cell culture.2',3'-Dideoxynucleoside (ddN) analogues are potent inhibitors of HIV replication in cell culture. To date, five ddN analogues have been formally licensed for treatment of HIVinfected individuals [(i) zidovudine (azidothymidine; AZT), (ii) zalcitabine (dideoxycytidine; ddC), (iii) didanosine (dideoxyinosine; ddI), (iv) stavudine (didehydrodideoxythymidine; d4T), and (v) lamivudine (3'-thia-2',3'-dideoxycytidine; 3TC)]. The ddN analogues have to be converted to their corresponding 5'-triphosphate derivatives to act as inhibitors of the retroviral reverse transcriptase. However, the rate and extent of phosphorylation of the ddN analogues to their active 5'-triphosphate metabolite may be insufficient. In particular, the first activation (phosphorylation) step may be rate-limiting in that most ddN analogues show poor affinity for nucleoside kinases [i.e., thymidine kinase (TK), 2'-deoxycytidine kinase, adenosine kinase] or other cellular enzymes (i.e., 5'-nucleotidase) that play a crucial role in the conversion of the ddN analogues to their 5'-monophosphates (1-6). In efforts to circumvent the dependence of ddN analogues on activation by the nucleoside kinases, prodrugs of the 5'-monophosphate forms of several nucleoside analogues have been prepared. The phosphoramidate derivatives of azidothymidine and fluorothymidine (FLT), two good substrates for cytosol TK, have been found to exhibit marked antiviral activity in cell culture (7,8). We have recently focused on masked nucleoside phosphoramidate derivatives of nucleoside analogues [i.e., d4T and dideoxyuridine (ddU)] that have relatively poor affinity for cytosol TK (2,(9)(10)(11). In this study, we revealed that the phosphoramidate derivative of d4T, designated So324, not only proved able to deliver intracellularly the free 5'-monophosphate of d4T (d4T-MP) [leading to pronounced levels of the corresponding active d4T 5'-triphosphate (d4T-TP)], but also released a phosphoramidate derivative of d4T-MP. This latter metabolite markedly accumul...

The role of macrophages in the pathogenesis and progression of human immunodeficiency virus (HIV)-related infection is substantiated by in vitro and in vivo evidence. The unique ability to survive HIV infection and produce viral particles for long periods is postulated. Detailed studies of this phenomenon are lacking. The dynamics of HIV-1 replication and cumulative virus production was studied in long-term cultures of macrophages in the presence or in the absence of antiviral drugs. Multiply spliced and unspliced HIV-RNA production was assessed by quantitative PCR, and the number of infected cells was monitored by FACS analysis. Cumulative HIV-1 production was determined by a trapezoidal equation, including such parameters as times of collection and experimental values of genomic-RNA and p24 gag antigen. Unspliced and multiply spliced HIV-RNA increased linearly after macrophage infection; reached levels of 1.5 x 10(8) and 2.8 x 10(5) copies/10(5) cells, respectively, at day 10; and then remained stable throughout the course of the experiment. Cumulative production of genomic-RNA and p24 gag antigen was 10(10) copies/10(6) cells and 10(7) pg/10(6) cells, respectively, with an average of >200 virus particles produced daily by each macrophage. AZT decreased the cumulative production of both genomic-RNA and p24 gag antigen down to 2.5 x 10(9) copies and 1.1 x 10(6) pg/10(6) cells (73.8% and 88.9% inhibition, respectively) up to day 50 without virus breakthrough. Ritonavir had a limited, but consistent, efficacy on the release of mature virus proteins (about 40% inhibition), but not on HIV-RNA production. In conclusion, the long-term dynamics and the high cumulative virus production that characterize HIV-1 infection of macrophages underscore the peculiar role of these cells as a persistently infected reservoir of HIV.

Nerve growth factor (NGF) is a neurotrophin with the ability to exert specific effects on cells of the immune system. Human monocytes͞macrophages (M͞M) infected in vitro with HIV type 1 (HIV-1) are able to produce substantial levels of NGF that are associated with enhanced expression of the high-affinity NGF receptor (p140 trkA) on the M͞M surface. Treatment of HIVinfected human M͞M with anti-NGF Ab blocking the biological activity of NGF leads to a marked decrease of the expression of p140 trkA high-affinity receptor, a concomitant increased expression of p75 NTR low-affinity receptor for NGF, and the occurrence of apoptotic death of M͞M. Taken together, these findings suggest a role for NGF as an autocrine survival factor that rescues human M͞M from the cytopathic effect caused by HIV infection.