Anti-human immunodeficiency virus type 1 activity of an anti-CD4 immunoconjugate containing pokeweed antiviral protein.

The ability of an alpha CD4-pokeweed antiviral protein (PAP) immunoconjugate to inhibit replication of human immunodeficiency virus type 1 (HIV-1) was evaluated in vitro with 22 clinical HIV-1 strains obtained from four seropositive asymptomatic individuals, three patients with AIDS-related complex, and four patients with AIDS. Fifteen isolates were from zidovudine-untreated individuals, whereas seven isolates were obtained after 24 to 104 weeks of therapy with zidovudine, alone or alternating with zalcitabine. Mean zidovudine 50% inhibitory concentrations (IC50s) were 126 nM (range, 1 to 607 nM) for isolates from zidovudine-untreated individuals and 2,498 nM (range, 14 to 6,497 nM) for strains from patients treated with antiretroviral agents. Mean alpha CD4-PAP IC50s were 48 x 10(-3) nM (range, 0.02 x 10(-3) to 212 x 10(-3) nM) for isolates from zidovudine-untreated individuals, and 16 x 10(-3) nM (range, 2 x 10(-3) to 28 x 10(-3) nM) for isolates from treated patients. Overall, higher concentrations of alpha CD4-PAP were necessary to inhibit HIV-1 strains from untreated individuals at more advanced stages of disease. Seventeen isolates were susceptible to zidovudine (mean IC50, 117 nM), and five were resistant to zidovudine (mean IC50, 3,724 nM). Mean alpha CD4-PAP IC50s were 43 x 10(-3) nM for zidovudine-susceptible isolates and 19 x 10(-3) nM for isolates resistant to zidovudine. All HIV-1 strains had IC50s greater than 0.5 nM for unconjugated PAP, the alpha CD19-PAP immunoconjugate, and monoclonal antibody alpha CD4. At concentrations as high as 5,000 nM, alphaCD4-PAP did not inhibit colony formation by normal bone marrow progenitor cells(BFU-E, CFU-GM , and CFU-GEMM) or myeloid cell lines (KG-1 and HL-60) and did not decrease cell viabilities of T-cell (Jurkat) or B-cell (FL-112 and Raji) precursor lines. Overall, alphaCD4-PAP demonstrated more potent anti-HIV-1 activity than zidovudine and inhibited replication of zidovudine-susceptible and zidovudine-resistant viruses at concentrations that were not toxic to lymphohematopoietic cell populations.     

(-)-2''-deoxy-3''-thiacytidine is a potent, highly selective inhibitor of human immunodeficiency virus type 1 and type 2 replication in vitro.

The (-)-enantiomer of 2'-deoxy-3'-thiacytidine (3TC) was found to be a potent and selective inhibitor of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) in vitro. We determined its antiviral activity against a number of laboratory strains of HIV-1 and HIV-2 in a range of CD4-bearing lymphocyte cell lines (mean 50% inhibitory concentration [IC50] range, 4 nM to 0.67 microM). 3TC was also active against a range of HIV-1 strains in peripheral blood lymphocytes (mean IC50 range, 2.5 to 90 nM). The IC50 for cytotoxicity in seven lymphocyte cell cultures, including human peripheral blood lymphocytes, ranged from 0.5 to 6 mM. 3TC had no detectable antiviral activity against a range of other viruses or in cells chronically infected with HIV-1 or HIV-2. The effects of time of addition of the compound and varying the multiplicity of infection on the antiviral activity of 3TC were determined. The results showed that 3TC is a potent and selective inhibitor of HIV-1 and HIV-2 replication in vitro.     

Genotypic and phenotypic resistance patterns in early-stage HIV-1-infected patients failing initial therapy with stavudine,didanosine and nevirapine

The objectives of this study were to determine the genotypic and phenotypic patterns of resistance in a group of early-stage antiretroviral-naive patients failing initial therapy with didanosine, stavudine and nevirapine. These patterns of resistance were determined at baseline and at time of virological failure in 89 antiretroviral-naive patients with CD4 cells >500 cells/ml and viral load >5000 copies/ml who received initial antiretroviral therapy with didanosine plus stavudine and nevirapine as part of the SCAN study, and who failed after having reached undetectable plasma levels (<200 copies/ml). Of the 89 patients recruited in the SCAN study, 14 (16%) developed a virological failure after reaching a viral load below 200 copies/ml after a median of 20 months of follow-up. At baseline, none of these 14 patients had genotypic resistance. At time of failure, six out of 14 (43%) failing patients had wild-type genotype and no phenotypic resistance. Suboptimal compliance could be documented in four of these six patients. Seven patients (50%) had nevirapine resistance mutations (mainly K103N [4/7], Y181C/I [2/7], G190A/S [2/7] and V108I [1/7]) associated with phenotypic high-level resistance to nevirapine, delavirdine and efavirenz (nevirapine >47.4- to 58.1-fold, delavirdine >74.4- to 168.9-fold and efavirenz >56.0- to 347.2-fold). Four of these seven patients also had thymidine analogue-associated mutations (TAM) (T215Y/F [2/4], M41L [1/4], D67N [2/4] and K70R [1/4]). Finally, one patient (7%) had exclusively TAM mutations (M41L). None of the patients developed mutations associated with didanosine resistance or phenotypic resistance to didanosine or stavudine. Suboptimal compliance or selection of nevirapine resistance often with TAM mutations was frequently associated with virological failure in a cohort of early-stage chronic HIV-1-infected patients treated with a protease inhibitor-sparing regimen.     

The benzamide derivative N-[1-(7-tert-Butyl-1H-indol-3-ylmethyl)-2-(4-cyclopropanecarbonyl-3-methyl-piperazin-1-yl)-2-oxo-ethyl]-4-nitro-benzamide (SP-10) reduces HIV-1 infectivity in vitro by modifying actin dynamics

Current treatments for patients infected with HIV are suboptimal. There is a need for new HIV therapies that act through different mechanisms than current treatments. We investigated the in vitro efficacy, safety and mechanism of action of the benzamide derivative N-[1-(7-tert-Butyl-1H-indol-3-ylmethyl)-2-(4-cyclopropanecarbonyl-3-methyl-piperazin-1-yl)-2-oxo-ethyl]-4-nitro-benzamide (SP-10), a potential new HIV treatment. When HIV-1-responsive engineered HeLa cells were pre-incubated for 48 h with either SP-10 or zidovudine (AZT), SP-10 was able to inhibit viral replication at much lower concentrations (IC50 = 0.036 nM) than AZT (IC50 = 27.4 nM). In contrast to AZT, SP-10 also inhibited replication of the multidrug-resistant HIV-1 strain MDR-769 in the HeLa cell model. In co-incubation experiments, SP-10 also inhibited the CCR5-sensitive HIV-1 BaL virus replication in human peripheral blood mononuclear cells. SP-10 displayed very low toxicity compared with current antiviral treatments. Confocal laser scanning microscopy and immunoprecipitation studies showed that SP-10 reduced the expression of CD4 and CCR5 on the surface of the host cell. SP-10 also reduced the level of gp120 binding to the cell surface. Confocal laser scanning microscopy studies showed that SP-10 blocked the formation of actin filaments (F-actin) and altered actin accumulation near the cell surface. These promising results suggest that SP-10 has a novel mechanism of action that enables effective inhibition of HIV-1 binding and cell entry. Further development of SP-10 as a new HIV treatment appears warranted.     

Drug resistance and drug combination features of the human immunodeficiency virus inhibitor, BCH-10652 [(+/-)-2'-deoxy-3'-oxa-4'-thiocytidine, dOTC

The heterosubstituted nucleoside analogue dOTC [( )-2'-deoxy-3'-oxa-4'-thiocytidine, BCH-10652] is a racemic compound structurally related to 3TC (lamivudine), but has the oxygen and sulphur in the furanosyl ring transposed. Both the enantiomers (-)dOTC (BCH-10618) and (+)dOTC (BCH-10619) had equivalent activity against wild-type strains of HIV-1 in C8166 T-cells (EC50 1.0-10.0 microM) and in PBMCs (EC50 0.1-3.0 microM). Investigation of the activity of dOTC and its enantiomers against laboratory strains of HIV-1 with defined resistance to 3TC, AZT (zidovudine), ddl (didanosine), PMEA (adefovir), nevirapine and saquinavir indicated that sensitivity was maintained (<3-fold change in EC50) in all cases, with the exception of HIV-1RF 3TC-resistant viruses. The degree of resistance recorded for dOTC (four- to sevenfold), (-)dOTC (five- to eightfold) and (+)dOTC (five- to >18-fold) against these M1841 or M184V mutants, was significantly less than that recorded for 3TC (>100-fold). In addition, the inhibitory effect of the compounds against clinical isolates of HIV-1 recovered from patients with suspected resistance to 3TC and AZT was investigated. Clinical isolates were genotyped using the Murex Line Probe Assay (LiPA) and subgrouped into wild-type, 3TC-resistant and dual 3TC/AZT-resistant, as well as undefined or mixed genotype populations. Compared with the mean EC50 values obtained with genotypically and phenotypically wild-type clinical isolates, the mean EC50 values calculated for isolates phenotypically resistant to 3TC or 3TC and AZT were only 2.6-, 1.6- and 8.2-fold higher for dOTC, (-)dOTC and (+)dOTC, respectively. When the rate of emergence of virus resistant to dOTC and its enantiomers in vitro was investigated, virus resistant to (+)dOTC was readily selected for (<10 passages), and a methionine (ATG) to isoleucine (ATA) amino acid change at codon 184 was identified. In contrast, virus resistant to dOTC and (-)dOTC took longer to appear (15-20 passages), with a methionine (ATG) to valine (GTG) amino acid change at position 184 identified in both cases. In addition, virus passaged 20 times in the presence of dOTC also had a partial lysine (AAA) to arginine (AGA) exchange at position 65. These viruses showed only low-level resistance to dOTC and its enantiomers, but were highly resistant to 3TC. The antiviral effects of dOTC in combination with the nucleoside RT inhibitors AZT, 3TC, d4T (stavudine) and ddl, the non-nucleoside RT inhibitor nevirapine and the protease inhibitors saquinavir, ritonavir and indinavir was investigated. Two-way drug combination assays were carried out in peripheral blood mononuclear cell (PBMC) cultures by measuring the reduction in p24 viral antigen levels, and data was analysed using the MacSynergy II program. dOTC in combination with 3TC or d4T showed a moderate synergistic effect while all other combinations had an additive interaction.