Hydroxyurea in the treatment of HIV-1

OBJECTIVE: To describe the role of hydroxyurea in the treatment of HIV-1. DATA SOURCES: Published clinical studies using hydroxyurea in HIV treatment were accessed through MEDLINE (January 1994-March 1999) and conference abstracts. All relevant studies were evaluated. DATA SYNTHESIS: Adherence, expense, and resistance limit the pharmacotherapeutic options in the management of patients with HIV. Hydroxyurea may be an alternative to conventional HIV treatments. CONCLUSIONS: Several potential advantages of adding hydroxyurea to antiretroviral treatment regimens include the drug's well-documented toxicity, convenient dosing, good tolerability and low cost, and its unique mechanism of action. Hydroxyurea may have synergistic effects that prove promising in initial and salvage therapy antiretroviral regimens. Larger, well-controlled clinical studies are needed to adequately define the role of hydroxyurea in the treatment of HIV.     

Reduction of HIV-1 antigen production by phosphatidylcholine containing formulations via growth inhibition of HIV-1-infected cells

Phosphatidylcholine (PC) and licensed formulations containing PC were tested for their influence on the proliferation and viability of cells permanently infected with HIV-1 (human immunodeficiency virus type 1). PC alone, as well as pharmaceutical formulations containing PC, selectively inhibited the growth of productively infected lymphoid cells. The strongest growth inhibition was observed with formulations containing PC, glycerol and triglyceride together. The growth inhibition was dose-dependent for HIV-1-infected cells. Additionally, PC-containing formulations dramatically reduced antigen production from peripheral blood mononuclear cells (PBMCs) infected in vitro with HIV-1. In vivo experiments with Rauscher-MuLV-infected mice showed that PC administered either intraperitoneally or orally was able to inhibit Rauschervirus-induced splenomegaly. PC-containing formulations are currently used in man for supportive therapy at doses, which in vitro induced 50% growth inhibition of HIV-1-infected cells in vitro. Such doses have been used in man without side effects for many years. Thus, PC-containing formulations may be valuable for the treatment of HIV-1-infected individuals.     

Long-term RNase P-mediated inhibition of HIV-1 replication and pathogenesis

Advances in genetic analysis and a greater understanding of human immunodeficiency virus (HIV) molecular pathogenesis have identified critical viral targets for gene interference strategies. RNase P molecules have been proposed as a novel approach for gene targeting based upon their potent catalytic activity, as well as versatile external guide sequence (EGS) which can be modified to specifically recognize almost any target mRNA. We designed a truncated EGS to specifically recognize the highly conserved U5 region of HIV-1 mRNA and mediate subsequent cleavage of hybridized mRNA by the RNase P enzyme component. The active U5-EGS (560), as well as a disabled U5 EGS (560D) control, were cloned into plasmids containing proviral constructs and transfected into a CD4(+) T cell line that was thereafter infected with HIV-1 MN. CD4(+) T cells treated with the active U5 EGS (560) were observed to maintain CD4(+) expression and did not produce HIV p24 gag antigen, form syncytia or undergo apoptosis up to 30 days after infection. Identical cells expressing the inactivated form of the U5 RNase P EGS completely down-regulated CD4 expression, produced elevated levels of HIV-1, formed large syncytia and underwent apoptosis similar to untreated cells. HIV-1 replication and related cytopathology can be effectively inhibited in CD4(+) T cells expressing a protective U5 EGS (560).     

Novel phorbol esters exert dichotomous effects on inhibition of HIV-1 infection and activation of latent HIV-1 expression

Two new phorbol esters, NPB-11 (12-O-methoxymethylphorbol-13-decanoate) and NPB-15 (12-O-benzyloxymethylphorbol-13-decanoate) were synthesized. The compounds exhibited potent anti-HIV-1 activity and low cytotoxicity in MT-4 cells by MTT assay even at a high concentration [50% cytotoxic concentrations (CC50) were 8.32 and 4.39 microg/ml, respectively]. Two inhibitors strongly suppressed HIV-1 (IIIB strain) replication in MT-4 cells with a 50% effective concentration (EC50) of 1.3 and 0.27 ng/ml, respectively. NPB-11 efficiently blocked replication of both X4 and R5 HIV-1 in PHA-activated peripheral blood mononuclear cells and MT-4 cells as revealed by p24 assay. The antiviral activity appeared to be mediated, at least partially, by the down-regulation of the expression of CD4 and the HIV-1 co-receptors, CXCR4 and CCR5. The compounds were also capable of selectively up-regulating HIV-1 expression in a variety of latently infected cell lines and inducing cell death in HIV-1 infected cells. The effect of NPBs on the induction of HIV-1 was specifically blocked by nontoxic doses of a protein kinase C blocker, staurosporine. NPB-11 blocked the spread of HIV-1 released from latently infected ACH-2 cells to MT-4 cells in a co-culture system. When combined with AZT, NPB-11 synergistically inhibited HIV-1 replication in MTT assay using MT-4 cells. These data suggest that these agents might be useful in reducing persistent viral reservoirs in patients and as adjuvant therapy in patients treated with HAART.     

Evasion mechanisms to Igf1r inhibition in rhabdomyosarcoma

Inhibition of the insulin-like growth factor 1 receptor (Igf1r) is an approach being taken in clinical trials to overcome the dismal outcome for metastatic alveolar rhabdomyosarcoma (ARMS), an aggressive muscle cancer of children and young adults. In our study, we address the potential mechanism(s) of Igf1r inhibitor resistance that might be anticipated for patients. Using a genetically engineered mouse model of ARMS, validated for active Igf1r signaling, we show that the prototypic Igf1r inhibitor NVP-AEW541 can inhibit cell growth and induce apoptosis in vitro in association with decreased Akt and Mapk phosphorylation. However, drug resistance in vivo is more common and is accompanied by Igf1r overexpression, Mapk reactivation, and Her2 overexpression. Her2 is found to form heterodimers with Igf1r in resistant primary tumor cell cultures, and stimulation with Igf2 leads to Her2 phosphorylation. The Her2 inhibitor lapatinib cooperates with NVP-AEW541 to reduce Igf1r phosphorylation and to inhibit cell growth even though lapatinib alone has little effect on growth. These results point to the potential therapeutic importance of simultaneous targeting of Igf1r and Her2 to abrogate resistance.     

Differential inhibition of HIV-1 cell binding and HIV-1-induced syncytium formation by low molecular weight sulphated polysaccharides.

Dextran sulphate (MW 5000 and 8000) and a polysulphated glycosaminoglycan (MW 10,000), at concentrations that provided complete protection in a homologous infection assay, failed to block syncytium formation and the resulting cytopathic effect when MT-2 cells were mixed with H9/HIV-1 cells. These substances also had no antiviral activity when added to cells, after virus challenge, at a time when binding and entry were complete. However, a high molecular weight (500,000) dextran sulphate blocked HIV-1 infection at both stages. Thus, the gp120-CD4 interactions mediating HIV-1 binding and HIV-1-induced syncytium formation are differentially affected by this class of polyanionic substances. Furthermore, size may be a determining factor in their potential application as anti-HIV treatment.     

Growth inhibition of HIV-1-infected cells and membrane alterations induced by phosphatidylcholine

Cultured T-lymphoma cell lines H9 and KE37-1 permanently infected with human immunodeficiency virus (HIV-1, strain HTLV-III B) were exposed to phosphatidylcholine (PC) and a PC-containing formulation “Essentiale” (PC-E). PC and PC-E, but not triglyceride, were found to inhibit growth of virus-infected cells. Additionally, the membrane lipid composition of infected and uninfected H9 cells was investigated upon exposure to PC. The HIV-1-infected cells showed a 25% increase in membrane triglyceride content and a 15% increase in membrane phospholipid saturated fatty acids. In the presence of PC, there is a further increase in triglyceride content up to 180% compared with uninfected control cells, suggsting a possible cause for the selective growth inhibition of HIV-1-infected cells by PC. The PC-E dose range effective in vitro for inhibition of HIV-1-infected cell growth falls within the range that can be reached invivo. Formulations containing PC are well tolerated by humans and might be applicable at an early stage of HIV-1 infection to reduce the number of virus-producing cells.     

Early therapy in HIV-1-infected children: effect on HIV-1 dynamics and HIV-1-specific immune response

BACKGROUND: Perinatal HIV-1 infection is acquired in the milieu of a developing immune system, leading to high levels of uncontrolled viral replication. Few data have been reported that address the viral dynamics and immunological response in infants who initiated aggressive antiretroviral therapy (ART) shortly after birth. METHODS: Six HIV-1-infected infants who started ART within 3 months of age were studied. The median followup was 61 months. Plasma HIV-1 RNA, cell-associated HIV-1 DNA, unspliced and multiply spliced HIV-1 mRNAs, HIV-1 antibodies, and CD4+ and CD8+ T-cell subsets were assessed in sequential peripheral blood samples. HIV-1 cellular immune response was measured by EliSpot assay. RESULTS: All children showed a decline in plasma viraemia to undetectable levels. HIV-1 DNA persisted in four children, but only two of these had detectable HIV-1 mRNA. All viral parameters remained persistently negative in two children. Only two children produced HIV-1 antibodies, while the others, after having lost maternal antibodies, remained seronegative. No HIV-1 cellular immune response was observed in any child. Therapy interruption was performed in two children: one HIV-1-seropositive and one HIV-1-seronegative with persistently undetectable levels of all viral parameters. Rebound of HIV-1 plasma viraemia in the seronegative child was more rapid and higher than that observed in the seropositive child. CONCLUSIONS: Early ART treatment in infants modifies the natural course of infection by controlling HIV-1 replication and reducing viral load to below the threshold levels required for onset of HIV-1 immune response, but does not prevent the establishment of a reservoir of latently infected cells that precludes virus eradication.     

In vitro inhibition of HIV-1 by Met-SDF-1beta alone or in combination with antiretroviral drugs

Compounds that can block the CXCR4 chemokine receptor are a promising new class of antiretroviral agents. In these experiments we studied the effect of a modified form of the native stromal cell-derived factor-1 (SDF-1), Met-SDF-1beta. The in vitro susceptibility of two different CXCR4-tropic HIV-1 strains was determined. Antiviral effect was assessed by the reduction of p24 antigen production in PHA-stimulated peripheral blood mononuclear cells with exposure to the modified SDF-1 molecule. The 50% inhibitory concentrations (IC50) were derived from six separate experiments. The IC50 against the two HIV-1 isolates was in 1.0-2.8 microg/ml range for Met-SDF-1beta. Met-SDF-1beta showed synergy to additivity with either zidovudine or nelfinavir at IC75 IC90 and IC95. Additivity was seen when Met-SDF-1beta was combined with efavirenz. No cellular toxicity was observed at the highest concentrations when these agents were used either singly or in combination. This compound is a promising new candidate in a receptor-based approach to HIV-1 infection in conjunction with currently available combination antiretroviral drug therapies.     

Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components

New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1–inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2_ROD, SIV_AGMtan, FIV_PET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti–HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5α, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary CD4⁺ T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, Rag2^–/–γc^–/– mice were engrafted with human CD4⁺ T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human CD4⁺ T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti–HIV-1 gene therapy candidate.