Design, synthesis and studies of triphosphate analogues for the production of anti AZT-TP antibodies.

Triphosphates anabolites are the active chemical species of nucleosidic reverse transcriptase inhibitors in HIV-therapy. Herein, we describe (i) the design of stable triphosphate analogues of AZT using molecular modelling, (ii) their synthesis and (iii) their use for producing anti AZT-TP antibodies in the aim of developing an immunoassay for therapeutic drug monitoring.

Adherence, virological outcome, and drug resistance in Chinese HIV patients receiving first-line antiretroviral therapy from 2011 to 2015.

Stavudine (D4T), zidovudine (AZT), and tenofovir (TDF) along with lamivudine (3TC) are the most widely used HIV treatment regimens in China. China's National Free Antiretroviral Treatment Programme (NFATP) has replaced D4T with AZT or TDF in the standard first-line regimens since 2010. Few studies have evaluated the adherence, virological outcome, and drug resistance in HIV patients receiving first-line antiretroviral therapy (ART) from 2011 to 2015 due to changes in ART regimen.From 2011 to 2015, 2787 HIV patients were examined, with 364, 1453, and 970 patients having initiated D4T-, AZT-, and TDF-based first-line ART regimens, respectively. The Cochran-Armitage test was used to examine the trends in clinical and virological outcomes during 2011 to 2015. Logistic regression was used to examine the effects of different regimens after 9 to 24 months of ART.From 2011 to 2014-2015, adverse drug reactions decreased from 18.9% to 6.7%, missed doses decreased from 9.9% to 4.6%, virological failure decreased from 16.2% to 6.4%, and drug resistance rates also significantly decreased from 5.4% to 1.1%. These successes were strongly associated with the standardized use of TDF- or AZT-based regimens in place of the D4T-based regimen. Poor adherence decreased from 11.3% in patients who initiated D4T-based regimens to 4.9% in those who initiated TDF-based regimens, adverse drug reactions decreased from 32.4% to 6.7%, virological failure reduced from 18.7% to 8.6%, and drug resistance reduced from 5.8% to 2.9%. Compared with patients who initiated AZT-based regimens, patients who initiated TDF-based regiments showed significant reductions in adherence issues, adverse drug reactions, virological outcomes, and drug resistance. Significant differences were also observed between those who initiated D4T- and AZT-based regimens.The good control of HIV replication and drug resistance was attributed to the success of China's NFATP from 2011 to 2015. This study provided real world evidence for further scaling up ART and minimizing the emergence of drug resistance in the "Three 90" era.

A killer mimotope with therapeutic activity against AIDS-related opportunistic micro-organisms inhibits ex-vivo HIV-1 replication.

OBJECTIVE: To verify whether a synthetic therapeutic killer decapeptide (KP), a functional mimotope of a yeast killer toxin with wide-spectrum microbicidal activity, inclusive of AIDS-related opportunistic micro-organisms, through interaction with beta-glucan receptors, which has been found to possess sequence homology with critical segments in gp160 V1/V2 and V3 loops, may also be inhibiting HIV-1 replication. METHODS: Primary peripheral blood mononuclear cells (PBMCs) cultures established from HIV-1-infected patients were treated with KP in comparison with zidovudine and supernatants and cells were harvested for analysis of HIV RNA and proviral contents, respectively. Virus production in exogenous in-vitro PBMCs infection with lymphocytotropic and monocytotropic HIV-1 strains was also assessed in presence of KP by enzyme-linked immunosorbent assay HIV p24 gag antigen detection. The binding affinity of KP to CD4, CCR5 and CXCR4 was evaluated on CD4-CCR5 or CD4-CXCR4 transfected astroglioma cell lines. RESULTS: KP was shown to be devoid of cytotoxicity on PBMCs and to inhibit HIV-1 replication in PBMCs of a patient in the acute phase of infection. The antiretroviral activity of KP, which proved to be more potent than zidovudine at micromolar concentrations, is abolished by beta 1,3-glucan but not by beta 1,6-glucan. Down-regulation of CCR5 co-receptor, and/or physical block of the gp120-receptor interaction are possible mechanisms of KP activity. CONCLUSION: KP appears to be the first antibody-derived short peptide displaying an inhibitory activity against HIV-1 and related opportunistic micro-organisms by different mechanisms of action.

Zidovudine-induced fever.

Zidovudine (3'-azido-3'deoxythymidine, AZT, Retrovir) is the first antiretroviral drug to demonstrate clinical efficacy for symptomatic human immunodeficiency virus infection. In a large, placebo-controlled trial, nausea and hematologic toxicity, but not fever, occurred more frequently in zidovudine- than in placebo-treated patients. However, in an open label study, fever severe enough to halt zidovudine administration occurred in 10% of 70 acquired immune deficiency syndrome (AIDS) patients receiving the drug. We now describe three AIDS patients with severe zidovudine-induced fever in whom other causes of fever were excluded. Zidovudine-induced fever was confirmed in each case by drug rechallenge. Using an enzyme immunoassay, we detected IgM antibodies directed against a zidovudine-serum protein conformational determinant in one of these three patients. Neither IgG nor IgM anti-zidovudine antibodies were present in sera from the other two patients with zidovudine fever, from four AIDS patients who discontinued zidovudine for reasons other than fever, or from five AIDS patients who never received zidovudine. Zidovudine may cause fever as a severe adverse effect in patients with AIDS. Either type III or type IV hypersensitivity may mediate this reaction.

Monitoring of intracellular levels of 5'-monophosphate-AZT using an enzyme immunoassay.

We have developed a competitive enzyme immunoassay suitable for routine monitoring of intracellular levels of 5'-monophosphate-AZT (AZT-MP). This assay is performed in 96-well microtiter plates coated with anti-rabbit immunoglobulin antibodies and is based on the use of rabbit polyclonal antibodies raised against an AZT-MP analog and of an AZT-MP/acetylcholinesterase conjugate as tracer. It is very sensitive, with a detection limit close to 0.1 ng/ml (0.2 pmol/ml), and precise (CV < 20% from 20 to 0.3 ng/ml). Very low cross-reactivities were observed with AZT and the corresponding di- and triphosphate derivatives as well as with other related nucleotides and nucleosides. The validity of the assay was demonstrated by measuring intracellular concentrations of AZT-MP in peripheral blood mononuclear cells (PBMCs) and in monocyte-derived macrophages (MDMs) cultured in the presence of various concentrations of AZT (from 0.01 microM to 10 microM). We observed very high levels of AZT-MP in stimulated (PHA + IL2) PBMCs (> 100 pmol/10(6) cells) while, as expected, much lower concentrations were measured in resting PBMCs or MDMs (0.1 to 2 pmol/10(6) cells). The assay constitutes a very convenient tool permitting easy, precise studies of the first step of the intracellular metabolism of AZT leading to the formation of AZT-TP in cultured cells.

The production and evaluation of antibodies for enzyme immunoassay of AZTTP.

We describe the development of the first enzyme immunoassay for quantifying AZTTP that does not use of radioactive labeling. Anti-AZTTP antibodies were raised in rabbits by immunizing with an AZTTP-kelhoyle limpet hemocyanin (KLH) conjugate. Competitive immunoassays indicated a nanomolar sensitivity to AZTTP. One of the antisera produced was specific for AZTTP.

Maternal immune responses and risk of infant infection with HIV-1 after a short course Zidovudine in a cohort of HIV-1 infected pregnant women in rural Kenya.

OBJECTIVE: To investigate the effects of short-course nucleoside reverse transcriptase inhibitor (Zidovudine, ZDW/AZT) on maternal immune responses and risk of infant infection with HIV-1 among rural-based mothers in western Kenya. DESIGN: A prospective cohort study involving HIV-1 seropositive pregnant mothers and their infants. SUBJECTS: One hundred and seven HIV-1 seropositive asymptomatic pregnant women and their infants. METHODS: After informed consent, the women were enrolled at gestation age between 16-24 weeks. For cultural and economic reasons, all mothers were allowed to breast feed their infants. Short-course antepartum regime of AZT was administered to all mothers starting at 36 weeks gestation until start of labour. Maternal absolute CD4+ T cell subset assays were performed before 3rd trimester (about 36 weeks gestation) and after a 4-week therapy of AZT (at least one month post-nuptially). Infant HIV-1 status was determined by HIV-1 DNA polymerase chain reaction (PCR) on samples sequentially taken at 1, 2, 3, 4, 6 and 9 months and confirmed by serology at 18 months of age. INTERVENTIONS: Antepartum short-course orally administered AZT: 300mg twice-daily starting at 36 weeks gestation until start of labour, 300mg at labour onset and 300mg every three hours during labour until delivery. MAIN OUTCOME MEASURES: Maternal CD4+ T cell counts before and after AZT treatment. Determination of infant HIV-1 infection status. RESULTS: Among 107 women sampled, only 59 received full dose of AZT and thus qualified for present analysis. Of these, 12 infected their children with HIV, while 47 did not. Comparison of CD4+ T cells before and after AZT treatment scored a significant rise in all mothers (P = 0.01). This increase in CD4+ T cells was not significant among mothers who infected their infants with HIV-1 (P = 0.474). However, a significant rise in CD4+ T cells following AZT therapy was observed only in mothers who did not transmit HIV-1 to their infants (P=0.014). CONCLUSION: These data suggest that a rise in the CD4+ T cell counts following short AZT regimen, now widely in use in resource-weak countries, may be evidence of the active suppression of the replication of HIV. However, further studies to examine the multi-factorial effect of CD4+ lymphocytes and pregnancy on MTCT of HIV need to be carried out to help fully explain the effect of AZT on immune response and whether the CD4+T cell count can be used as a true test of immunological normalisation during antiretroviral therapy.

Diamond paste based immunosensor for the determination of azidothymidine.

An amperometric immunosensor, based on diamond paste (diamond powder and paraffin oil), has been constructed for the assay of azidothymidine (AZT). The diamond paste is impregnated with anti-AZT. The immunosensor can be used reliably for the assay of azidothymidine in its pharmaceutical formulations. The potential used for azidothymidine assay was + 240 mV vs. Ag/AgCl electrode. The surface of the immunosensor can be regenerated by simply polishing, thereby obtaining fresh immunocomposite ready to be used in a new assay. The new amperometric immunosensor, based on diamond paste, gives reliable results for the assay of AZT as raw material and from its pharmaceutical formulation.

Use of a regenerable immobilized second antibody to determine azidothymidine in a flow system.

A regenerable immobilized second-antibody reactor was used to measure azidothymidine (AZT) by competitive enzyme immunoassay in a flow system. The immobilized antibody was regenerated 4 times with little loss of immunospecificity. A residual enzymatic activity of about 7% of the total response was obtained with horseradish peroxidase as label. AZT was measured below the nanomolar level by allowing competition to proceed for about 2 min. A limit of detection of 6.57 x 10(-11) +/- 1.56 x 10(-11) M AZT was obtained when the concentration of AZT-peroxidase conjugate was 0.125 ng/mL. This system was used to determined azidothymidine in Retrovir capsule with good results. In replicate measurements, RSDs of 5.51% and 2.44%, n = 6, were obtained at 7.48 x 10(-11) and 3.74 x 10(-9) M AZT, respectively.

Hyaline globules reacting positively with zidovudine antibody in brain and spinal cord of AIDS patients.

Histology of the central nervous system in nine AIDS showed extracellular hyaline globules in the white matter of the brain and the spinal cord. In immunohistochemical studies with a battery of antibodies, the only positive reaction of these globules was with an antibody to zidovudine. High-performance liquid chromatography showed the presence of a zidovudine isomer in eluates of brain tissue from these patients.

In vitro immunotoxicity of +/- 2'-deoxy-3'-thiacytidine, a new anti-HIV agent.

The present study compares the in vitro effect of (+/-)-2'-deoxy-3'-thiacytidine (BCH 189) a new synthetic anti-HIV-1 dideoxynucleoside, with 3'-azido-3'-deoxythymidine (AZT) on the immune function of lymphocytes from 10 normal and 12 HIV-1+ patients (CDC II and III). The effect of different doses of BCH 189 and AZT was analysed in vitro on: (i) T cell proliferation after stimulation with concanavalin A (Con A) or anti-CD3 MoAb; (ii) B cell proliferation and immunoglobulin production after stimulation with pokeweed mitogen (PWM); (iii) cytokine production (IL-2, IL-6, GM-CSF, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) from lymphocytes stimulated with anti-CD3 MoAb or phytohaemagglutinin (PHA). BCH 189 inhibited the proliferation of B and T lymphocytes from normal and HIV+ subjects less than AZT; even if lymphocytes from HIV+ (CDC III) subjects produced higher levels of IL-6 and TNF-alpha, neither BCH 189 nor AZT molecule interfered with cytokine release. Immunoglobulin production from B lymphocytes was inhibited only by a high concentration (50 microM) of BCH 189 or AZT. These results show that BCH 189 affects lymphocyte proliferation in vitro less than AZT, and support its use in clinical trials in HIV-infected patients.

Enzymatic assay for measurement of zidovudine triphosphate in peripheral blood mononuclear cells.

In this report, we describe a new method to measure intracellular zidovudine triphosphate (ZDV-TP) levels in peripheral blood mononuclear cells (PBMCs) from patients treated with ZDV by utilizing inhibition of human immunodeficiency virus type 1 reverse transcriptase activity by ZDV-TP. Intracellular levels of ZDV-TP were determined with our enzymatic assay in PBMCs isolated from the blood of healthy individuals incubated with different concentrations of labeled ZDV and were validated by high-performance liquid chromatography separation and liquid scintillation counting of the radioactive ZDV-TP. These methods gave virtually identical results over a range of ZDV-TP concentrations from 150 to 900 fmol. ZDV-TP recoveries were over 90%, and the limit of quantitation of ZDV-TP by this method was 20 to 50 fmol. To demonstrate the utility of the method, plasma ZDV and intracellular ZDV-TP concentrations were measured at serial time points over 6 h in 12 human immunodeficiency virus-infected volunteers following a single 100- or 500-mg oral dose of ZDV. Systemic oral clearance rates were similar to those in previous studies with adults but were highly variable (range, 0.86 to 2.75 liters/h/kg of body weight). The area under the plasma concentration versus time curve increased significantly (P < 0.0005) with the dose from a median value of 1.2 mg.h/liter at the lower dose to 4.2 mg.h/liter at the higher dose. Median intracellular ZDV-TP levels ranged from 5 to 57 and 42 to 92 fmol/10(6) cells in volunteers administered 100 and 500 mg of ZDV, respectively. Intracellular ZDV-TP levels rose to a plateau value by 2 h and remained consistent to 6 h. Although the higher dose and higher areas under the curve yielded consistently higher intracellular ZDV-TP levels, systemic pharmacokinetics explains only a modest proportion of the variability in cellular pharmacokinetic. The ZDV-TP bioassay should prove useful in further studies of ZDV metabolism in patient-derived PBMCs at the doses of ZDV currently administered.

Inhibitory effect of 3'-azido-3'-deoxythymidine on proliferative responsiveness of CD8+ lymphocytes to interleukin-2.

Although several studies have shown that 3'-azido-3'-deoxythymidine (AZT) is not toxic for CD4+ lymphocytes, its effect on CD8+ cells has never been studied in a systematic way. We purified CD8+ cells from the peripheral blood mononuclear cells of both human immunodeficiency virus (HIV)-seronegative and HIV-infected individuals by means of magnetic beads that had been coated with monoclonal antibodies. We report that AZT, but not two other nucleosides tested, inhibited the interleukin-2-dependent proliferation of CD8+ lymphocytes in a dose-dependent manner. No such effect was observed with regard to CD4(+)-enriched populations. The AZT-mediated antiproliferative effect did not appear to be related to either the CD4+ count or to prior treatment with this drug in the case of HIV-seropositive subjects.

Resistencia del virus de inmunodeficiencia humano (VIH) a zidovudina (AZT): análisis genotípico en cepas aisladas de pacientes chilenos / Resistance of human immunodeficiency virus (VIH) to zidovudine: genotypic analysis in strains isolated from chilean patients

Background: Resistance of HIV to AZT is the result of mutations in the pol gene that codifies the enzyme reverse transcriptase. Aim: To asses the resistance to antiretroviral drugs in Chhilean patients infected with HIV. Material and methods: The presence of mutations was searched in 22 patients infected with HIV. The emergence or persistence of these mutations was studiend in sequential samples of 19 patients. The presence of the mutation that confers resistance to didanosine (ddi) was studied in those subjects exposed to the drug. Polymerase chain reaction techniques were used to analyze mutations in codons 41, 70 and 215 of the pol gene (resistance to AZT) and the mutation in codon 71 (resistance to DDI). Results: On admission, none of the patients without previous exposure to AZT had drug resistance mutations. Seven of 12 patients (58.3 percent) that had received AZT had mutations in codon 215. In two, they were associated to a mutation in codon 41 and in two, to a mutation in codon 70. After a mean follow up of 14 months, 13 of 15 patients (86 percent) that received AZT had viral strains genotypically resistant to the drug. In nine of these, the resistance was associated with disease progression. None of the 10 patients that received DDI had the mutation in codon 74 that confers resistance to the drug. However, in one of these patients, that never receided AZT, virus with a mutation in codon 215 was detected. Conclusions: A high percentage of patients that have received monotheraphy with AZT have genotypic resistance to the drug. This resistance is associated with clinical and immunological derangement in 70 percent of these subjects