Anti-retroviral strategies for AIDS and related diseases

The replication cycle of human immunodeficiency virus type 1 (HIV-1) and other retroviruses consists of four stages: attachment of the virus to specific receptors on the cell surface; uncoating of the viral nucleic acid and conversion to DNA; production of viral RNA and proteins; and assembly and liberation of progeny virus from the cell. Each of these steps represents a potential target for antiviral chemotherapy. Combinations of drugs which act against different steps in the viral replication cycle might be expected to have synergistic potential. Zidovudine (AZT) is the most widely used drug to date for impeding the replication of HIV-1. Although AZT therapy has been reasonably successful, it has not been free from toxicity. In addition, there have been several reports of isolation of AZT-resistant variants of HIV-1.     

Potent and selective inhibition of human immunodeficiency virus (HIV)-1 and HIV-2 replication by a class of bicyclams interacting with a viral uncoating event.

A series of bicyclams have been shown to be potent and selective inhibitors of human immunodeficiency virus (HIV). The compounds are inhibitory to the replication of various HIV-1 and HIV-2 strains in various human T-cell systems, including peripheral blood lymphocytes, at 0.14-1.4 microM, without being toxic to the host cells at 2.2 mM. The bicyclam JM2763 is active against 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant HIV-1 strains and acts additively with AZT. Mechanism of action studies revealed that the bicyclams (i.e., JM2763) interact with an early event of the retrovirus replicative cycle, which could be tentatively identified as a viral uncoating event.     

Synergistic anti-human immunodeficiency virus type 1 effect of hydroxamate compounds with 2'',3''-dideoxyinosine in infected resting human lymphocytes.

The cellular models generally used in the in vitro evaluation of anti-human immunodeficiency virus compounds are dividing cells. A model constituted by resting lymphocytes may more accurately reflect a drug's future efficacy in humans, since viral DNA synthesis is known to take place in quiescent cells, creating a reservoir of infected cells awaiting activation to complete their viral replication cycle and to produce infectious virions. We report here the activity of 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and two hydroxamates, D-aspartic acid beta-hydroxamate and hydroxycarbamate (hydroxyurea), alone and in various combinations, in an in vitro model based on resting lymphocytes. In our model, resting peripheral blood lymphocytes were infected with human immunodeficiency virus type 1 and treated with drugs for 7 days, at which time drugs were removed and the cells were activated by phytohemagglutinin. We show that under these conditions 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, and 2',3'-dideoxycytidine, alone or in combination, neither fully inhibit viral production nor protect lymphocytes from the cytopathic effect of viral replication, at concentrations corresponding to the peak plasma levels observed in a typical treatment schedule in humans. In contrast, we report the synergistic effect of treatment by each hydroxamate with 2',3'-dideoxyinosine of infected resting lymphocytes, resulting in the total suppression of viral production, total protection against the cytopathic effect induced by viral replication, and no effect on the ability of the cells to replicate in this cell culture system.     

Synergistic cytotoxic effect of azidothymidine and recombinant interferon alpha on normal human bone marrow progenitor cells

Azidothymidine (AZT) and interferon alpha (IFN-alpha) are among the drugs showing strong in vitro activity against the human immunodeficiency virus type-1 (HIV-1). Each drug, however, has significant toxicity against normal marrow progenitor cells that frequently proves dose-limiting in patients. In this study, AZT and recombinant IFN-alpha 2a (rIFN-alpha 2a) were tested as single agents and in combination against normal myeloid (CFU-GM) and erythroid (BFU- E) colony forming cells in a standard methylcellulose culture assay. The data were analyzed using a quantitative computerized analysis based on the median-effect principle and the isobologram equation as described by Chou and Talalay (Adv Enz Regul 22:27, 1984). The ED90 for BFU-E and CFU-GM inhibition was then compared with previously measured in vivo plasma levels of each drug and the ED90 for the anti-HIV-1 effect in vitro. We demonstrate that (a) the drugs are strongly synergistic in inhibiting marrow progenitor cell growth and that this synergism occurs at drug levels that are within the range of measured plasma levels in phase I clinical trials, (b) BFU-E are more sensitive than CFU-GM to the inhibiting effects of AZT, rIFN-alpha 2a or both drugs in combination, (c) the drug concentrations in combination that synergistically inhibit bone marrow progenitors are much higher than those required to inhibit HIV-1 replication in vitro, and (d) the anti- HIV-1 effect for the combination of AZT and rIFN-alpha 2a was clearly superior to the effect of AZT or rIFN-alpha 2a alone as indicated by the combination index and the dose-reduction index. These data suggest that substantially lower doses of AZT and rIFN-alpha than those currently being tested in clinical trials might not only maintain a strong synergistic anti-HIV-1 effect but might also avoid significant hematologic toxicity.     

Incorporation of 12-methoxydodecanoate into the human immunodeficiency virus 1 gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line.

Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55gag) is necessary for its proteolytic processing and viral assembly. We have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) despite their reduced hydrophobicity. Some inhibit HIV-1 replication in acutely infected CD4+H9 cells without accompanying cellular toxicity. To examine the mechanism of their antiviral effects, we performed labeling studies with two analogs, 12-methoxydodecanoate (13-oxamyristate; 13-OxaMyr) and 5-octyloxypentanoate (6-oxamyristate; 6-OxaMyr), the former being much more effective than the latter in blocking virus production. [3H]Myristate and [3H]13-OxaMyr were incorporated into Pr55gag with comparable efficiency when it was coexpressed with S. cerevisiae NMT in Escherichia coli. [3H]6-OxaMyr was not incorporated, even though its substrate properties in vitro were similar to those of 13-OxaMyr and myristate. [3H]13-OxaMyr, but not [3H]6-OxaMyr, was also efficiently incorporated into HIV-1 Pr55gag and nef (negative factor) in chronically infected H9 cells. Analog incorporation produced a redistribution of Pr55gag from membrane to cytosolic fractions and markedly decreased its proteolytic processing by viral protease. 13-OxaMyr and 3'-azido-3'-deoxythymidine (AZT) act synergistically to reduce virus production in acutely infected H9 cells. Unlike AZT, the analog is able to inhibit virus production (up to 70%) in chronically infected H9 cells. Moreover, the inhibitory effect lasts 6-8 days. These results suggest that (i) its mechanism of action is distinct from that of AZT and involves a late step in virus assembly; (ii) the analog may allow reduction in the dose of AZT required to affect viral replication; and (iii) combinations of analog and HIV-1 protease inhibitors may have synergistic effects on the processing of Pr55gag.     

Three-drug synergistic inhibition of HIV-1 replication in vitro by zidovudine, recombinant soluble CD4, and recombinant interferon-alpha A

Optimal management of human immunodeficiency virus type 1 (HIV-1) infections may require combinations of agents that attack different targets in the viral replicative cycle. Zidovudine (AZT), recombinant soluble CD4 (rsCD4), and recombinant interferon-alpha A (rIFN-alpha A) were evaluated in 2- and 3-drug regimens against HIV-1 replication in vitro. Peripheral blood mononuclear cells and a CD4+ T cell line (H9) were studied using multiple HIV-1 replicative end points. Drug interactions were evaluated by the median-effect principle and the isobologram technique. AZT, rsCD4, and rIFN-alpha A inhibited HIV-1 synergistically in 2- and 3-drug combinations. The 3-drug regimen provided more complete virus suppression than the 2-drug regimens. In H9 cells, single-drug regimens lost effectiveness at 10-14 days and 2-drug regimens lost effectiveness at 14-18 days. In contrast, the 3-drug regimen showed nearly complete suppression over 28 days in culture without toxicity. Clinical trials of these 3 drugs in combination should be considered.     

Effect of zidovudine and granulocyte-macrophage colony-stimulating factor on human immunodeficiency virus replication in alveolar macrophages

The alveolar macrophage (AM), as a representative human tissue macrophage, was used in an in vitro system to examine the anti-human immunodeficiency virus type-1 (HIV-1) activity of zidovudine (AZT) and granulocyte-macrophage colony-stimulating factor (GM-CSF). AMs were infected with the IIIB strain of HIV-1 and exposed to AZT (1 mumol/L), GM-CSF (30 U/mL), a combination of AZT (1 mumol/L)/GM-CSF (30 U/mL), or medium control. At 10 or 20 days post-infection, phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear leukocytes (PBMLs) were added to the AM cultures as stimulated target cells. AZT effectively suppressed HIV replication and prevented transfer/amplification in target PBMLs as long as the drug was maintained in the medium. GM-CSF neither suppressed nor augmented HIV replication. The combination of AZT/GM-CSF was comparable with AZT alone in suppressing both the initial infection of AMs and the transfer to target PBMLs as long as the agents were maintained in the cultures. However, when the drugs were removed at the same time that PHA-stimulated PBMLs were added to the culture, the combination of AZT/GM-CSF was found to be more effective than AZT alone in preventing the transfer/amplification of HIV in the target lymphocytes. These results suggest that (1) AZT is effective in inhibiting HIV-1 infection in mononuclear phagocytes; (2) GM-CSF neither inhibits nor augments the replication of the IIIB strain of HIV in human AMs; and (3) the combination of AZT and GM-CSF may have an enhanced anti-HIV-1 activity compared with AZT alone. Clinical trials with the two agents in combination appear warranted.     

In vitro inhibition of a variety of human immunodeficiency virus isolates by a broadly reactive, V3-directed heteroconjugate antibody.

The cytotoxic efficacy of a heteroconjugate antibody composed of OKT3 cross-linked to a broadly reactive antibody directed against the GPGRAF sequence of the gp120 V3 region has been characterized. The heteroconjugate antibody could completely inhibit viral replication of both the IIIB and MN isolates of human immunodeficiency virus (HIV) at concentrations as low as 0.5 ng/ml. At an antibody concentration of 1 micrograms/ml, heteroconjugate-mediated cytotoxicity occurred at effector-to-target ratios as low as 0.006:1. Eight different HIV isolates were tested for in vitro inhibition by the anti-V3-OKT3 conjugate, and all but one were completely inhibited for at least 7 days. These results indicate that heteroconjugate antibodies are a potent, effective means by which HIV-infected cells can be killed and viral replication suppressed.     

Zidovudine (AZT) causes an oxidation of mitochondrial DNA in mouse liver

Zidovudine (3'-azido-2',3'-dideoxythymidine [AZT]) inhibits human immunodeficiency virus replication and delays progression of acquired immune deficiency syndrome. We have recently found that, in muscle, AZT causes oxidative damage to mitochondrial DNA (mtDNA) and other signs of mitochondrial oxidative damage. The aim of this work was to test if AZT causes oxidative damage to liver mtDNA. In our study, an experimental mouse model was used in which mice were administered AZT (10 mg/kg body weight/d) in drinking water. Liver mtDNA of mice treated with AZT had 40% more of the oxidized, mutagenic nucleoside, 8-oxo-7,8-dihydroxy-2'deoxyguanosine (8-oxo-dG) than untreated controls. This oxidative damage to mtDNA is caused by a significant increase (of over 240%) in peroxide production by liver mitochondria from AZT-treated mice, which was prevented by dietary administration with vitamins C and E.     

Effect of duration of hepatitis B virus infection on the association between human immunodeficiency virus type-1 and hepatitis B viral replication.

This study examined the effect of duration of hepatitis B virus infection on the association between human immunodeficiency virus type-1 infection and hepatitis B viral replication. Twenty-five chronic HBsAg carriers were studied. Presence of hepatitis B virus DNA and expression of HBeAg were more frequent among 20 chronic HBsAg carriers positive for human immunodeficiency virus type-1 antibody compared with five chronic HBsAg carriers negative for human immunodeficiency virus type-1 antibody, but the associations were not statistically significant. Hepatitis B virus DNA and HBeAg were inversely related to duration of hepatitis B virus infection (p less than 0.001). Stratifying for duration of hepatitis B virus infection, the presence of viral replication was similar among patients negative and positive for antibody to human immunodeficiency virus type-1. Hepatitis B virus DNA levels did not increase with the decline of cellular immunity over time. In conclusion, hepatitis B virus replication among chronic carriers may be a function of duration of hepatitis B virus infection rather than of an effect of human immunodeficiency virus type-1.     

Human immunodeficiency virus-associated progressive multifocal leukoencephalopathy: apparent response to 3'-azido-3'-deoxythymidine

We present the case of a 26-year-old human immunodeficiency virus-seropositive man who developed progressive multifocal leukoencephalopathy as the initial manifestation of AIDS. He appears to have responded dramatically to therapy with 3'-azido-3'-deoxythymidine (AZT). His neurologic status deteriorated shortly after an AZT dose reduction. He has stabilized since resuming his previous AZT dose. Although it remains unclear whether AZT is useful in the treatment of JC virus infection, we think that all AIDS patients with progressive multifocal leukoencephalopathy should be offered treatment with AZT, especially in light of recent reports describing a possible potentiation of human immunodeficiency virus infection of the central nervous system in this setting.     

Use of self-sustained sequence replication amplification reaction to analyze and detect mutations in zidovudine-resistant human immunodeficiency virus

Mutations at amino acid positions 67, 70, 215, and 219 in the human immunodeficiency virus type 1 (HIV-1) pol gene correlate with the emergence of resistance to zidovudine (AZT). These four positions were monitored in viral RNA extracted from infected peripheral blood mononuclear cells (PBMC) and viral stocks obtained after coculture with uninfected lymphocytes. Genotype determinations were made using the self-sustained sequence replication (3SR) and differential bead-based sandwich hybridization (BBSH) assay. The hybridization results obtained by 3SR and BBSH analyses were verified by dideoxynucleotide sequencing of the 3SR products. Correlation of 3SR and BBSH with polymerase chain reaction and Southern hybridization analyses of the PBMC and corresponding viral isolates indicated that PBMC and corresponding HIV-1 isolates may differ in their genotypes at the monitored amino acid positions, variations from the wild-type nucleotide sequence may occur proximal to the codons being monitored, and viral isolates possessing the same genotypes at the four monitored amino acid positions showed a threefold variation in their ID50 measurements.