Thiol Reduction of 3′-Azidothymidine to 3′-Aminothymidine: Kinetics and Biomedical Implications

The ability of thiols to reduce 3-azidothymidine (AZT) to 3-aminothymidine has been investigated. Incubation with glutathione, dithiothreitol (DTT), or mercaptoethanol at pH 7.2 and 37°C leads to quantitative reduction of the azido moiety to an amine. The reaction is first order in AZT and first order in reducing agent (mono- or dithiol). The second-order rate constants are 2.77 × 10^–3, 6.55 × 10^–5, and 6.35 × 10^–6 M ^–l sec^–1 for the dithiothreitol, glutathione, and mercaptoethanol reductions, respectively. The thiol reduction of alkyl azide to amine under mild conditions is a synthetic method particularly suitable for water-soluble azido compounds that are sensitive to catalytic hydrogenation. The potential for the mono- or dithiol-mediated reduction of alkyl azides under biological conditions must be considered when conducting studies of azido drugs.     

Genetic damage detected in CD-1 mouse pups exposed perinatally to 3'-azido-3'-deoxythymidine or dideoxyinosine via maternal dosing, nursing, and direct gavage: II. Effects of the individual agents compared to combination treatment

We previously reported extraordinary increases in micronucleated erythrocytes in CD-1 mouse pups exposed to 3'-azido-3'-deoxythymidine (AZT) and dideoxyinosine (ddI; 50/250, 75/375, 150/750 mg/kg/day AZT/ddI) by gavage throughout gestation and lactation, followed by direct pup dosing beginning postnatal day (PND) 4 (Bishop et al. [2004]: Environ Mol Mutagen 43: 3-9). That study was conducted to explore the potential for genetic damage in newborns exposed perinatally to antiretrovirals in order to reduce maternal-infant transmission of HIV-1. Because dramatic increases in frequencies of micronucleated erythrocytes were seen in exposed pups, additional studies were conducted to clarify the relative contribution of each drug to the observed damage. Pregnant CD-1 mice were administered AZT (50, 75, 150 mg/kg/day) or ddI (250, 375, 750 mg/kg/day) by gavage twice daily in equal fractions beginning prior to mating and continuing throughout gestation and lactation. Direct pup dosing (same regimens) began on PND 4. Peripheral blood erythrocytes of male pups were screened for micronuclei on PNDs 1, 4, 8, and 21. Significant increases in micronucleated erythrocytes were observed in pups and dams exposed to AZT at all doses and sampling times. The highest micronucleus levels were observed in pups on PND 8 after the initiation of direct dosing. In contrast, effects seen in pups and dams treated with ddI were minimal. These results demonstrate that AZT, a component of many anti-HIV combination therapies, induces chromosomal damage in perinatally exposed neonatal mice. Comparison of micronucleated cell frequencies induced by AZT alone or in combination with ddI suggests that ddI potentiates AZT-induced chromosomal damage following direct exposure.     

Toward hyperpolarized molecular imaging of HIV: synthesis and longitudinal relaxation properties of 15N‐Azidothymidine

Previously unreported ¹⁵N labeled Azidothymidine (AZT) was prepared as an equimolar mixture of two isotopomers: 1‐¹⁵N‐AZT and 3‐¹⁵N‐AZT. Polarization decay of ¹⁵N NMR signal was studied in high (9.4 T) and low (~50 mT) magnetic fields. ¹⁵N T₁ values were 45 ± 5 s (1‐¹⁵N‐AZT) and 37 ± 2 s (3‐¹⁵N‐AZT) at 9.4 T, and 140 ± 16 s (3‐¹⁵N‐AZT) at 50 mT. ¹⁵N‐AZT can be potentially ¹⁵N hyperpolarized by several methods. These sufficiently long ¹⁵N‐AZT T₁ values potentially enable hyperpolarized in vivo imaging of ¹⁵N‐AZT, because of the known favorable efficient (i.e., of the time scale shorter than the longest reported here ¹⁵N T₁) kinetics of uptake of injected AZT. Therefore, 3‐¹⁵N‐AZT can be potentially used for HIV molecular imaging using hyperpolarized magnetic resonance imaging.     

Inhibition of hematopoietic progenitor cell proliferation by ethanol in human immunodeficiency virus type 1 tat-expressing transgenic mice

BACKGROUND: A number of hematological abnormalities are associated with both human immunodeficiency virus type 1 (HIV-1) infection and alcohol abuse. There is little information on how alcohol abuse might further influence the survival and growth of hematopoietic progenitors in HIV-infected individuals in the presence of immune system abnormalities and anti-HIV drugs. Because there is evidence that viral transactivator Tat itself can induce hematopoietic suppression, in this study we examined the role of ethanol as a cofactor in transgenic mice that expressed HIV-1 Tat protein. METHODS: Tat transgenic mice and nontransgenic littermates were given ethanol (20% v/v) and the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT; 1 mg/ml) in drinking water. Immunosuppression in mice was induced by weekly intraperitoneal injections of anti-CD4 antibody. Hematopoiesis was examined by erythroid colony forming unit (CFU-E) and granulocyte/macrophage colony-forming unit (CFU-GM) assays of the bone marrow progenitor cells. RESULTS: Administration of ethanol for 7 weeks resulted in a 50% decrease in the proliferative capacity of CFU-E- and CFU-GM-derived progenitors from transgenic mice compared with that of ethanol-treated nontransgenic controls. Similar decreases also were observed in transgenic mice treated with AZT or a combination of AZT and ethanol. Furthermore, ethanol and AZT were significantly more toxic to the granulopoietic progenitors (40-50% inhibition) than to the erythropoietic progenitors (10-20% inhibition) in Tat transgenic mice. Although a 10 day exposure of Tat transgenic and nontransgenic mice to a combination of ethanol and AZT had no suppressive effect on the erythropoietic and granulopoietic progenitor cells, there was a marked decrease (40-60%) in CFU-GM in mice made immunodeficient by CD4+ T-lymphocyte depletion. The ethanol-treated Tat transgenic mice but not the nontransgenic litter-mates also showed a significant decrease (25%) in CFU-GM. CONCLUSION: Our in vivo study strongly suggests that ethanol ingestion in HIV-1-infected individuals, particularly those on antiretroviral drugs, might increase bone marrow toxicity and contribute to HIV-1-associated hematopoietic impairment.     

Contraceptive activity of a spermicidal aryl phosphate derivative of bromo-methoxy-zidovudine (compound WHI-07) in rabbits

OBJECTIVE: To determine the vaginal contraceptive activity of WHI-07 in the rabbit model. DESIGN: Prospective, controlled study. SETTING: Center for advanced preclinical sciences. ANIMAL(S): Subgroups of 15, 16, or 24 New Zealand White does and 24 bucks per experiment. INTERVENTION(S): Ex vivo (Experiment 1) and in vivo (Experiments 2 and 3) treatment of semen with WHI-07 or Nonoxynol-9 (N-9). In Experiment I, ovulated does in subgroups of 15 were artificially inseminated with semen mixed with WHI-07 or vehicle. In Experiment 2, ovulated does in subgroups of 24 were artificially inseminated within 2 min after intravaginal administration of 2% WHI-07 gel-microemulsion or 2% N-9 gel and allowed to complete term pregnancy. In Experiment 3, ovulated does in subgroups of 16 were artificially inseminated at 15, 30, or 60 minutes. MAIN OUTCOME MEASURE(S): The numbers of implanted embryos on postinsemination day 8 or the proportion of does that became pregnant and delivered newborn rabbits; the litter size, weight, growth, and viability of pups until lactation day 5. RESULT(S): Exposure of semen to WHI-07 at the time of artificial insemination completely inhibited pregnancy rates (WHI-07-pretreated, 0%, vs. control, 60%) and embryo implantation (WHI-07-pretreated, 0/175 vs. control, 68/170). Intravaginal administration of a 2% WHI-07 gel-microemulsion or 2% N-9 gel before artificial insemination significantly inhibited pregnancy rates (81% and 85% inhibition, respectively) when compared with control. Furthermore, the 2% WHI-07 gel-microemulsion provided >90% inhibition of fertility even when insemination was delayed until 60 minutes after intravaginal application. Rabbits that delivered litters despite intravaginal application of 2% WHI-07 gel-microemulsion had healthy offsprings with no perinatal or postnatal repercussions. CONCLUSION(S): WHI-07 is a potent contraceptive agent in vivo. Intravaginal use of WHI-07 gel-microemulsion has clinical potential as a safe prophylactic contraceptive, in addition to its microbicide activity to curb the sexual transmission of HIV.     

Cytosolic and mitochondrial deoxyribonucleotidases: activity with substrate analogs,inhibitors and implications for therapy

Nucleoside analogs act as prodrugs that must be converted to 5'-phosphates by intracellular kinases to become active in the treatment of viral and oncological diseases. Activation may be reversed by dephosphorylation if the 5'-phosphates are substrates for 5'-nucleotidases. Dephosphorylation by cytosolic enzymes decreases the efficacy of the analogs, whereas dephosphorylation by mitochondrial enzymes may decrease mitochondrial toxicity. Both effects may influence the outcome of therapy. We investigated the dephosphorylation of the 5'-phosphates of commonly used nucleoside analogs by two cytosolic (cN-II and dNT-1) and one mitochondrial (dNT-2) nucleotidase. Most uracil/thymine nucleotide analogs were dephosphorylated by all three human enzymes but cytosine-containing nucleotide analogs were inactive. Only cN-II showed some activity with the monophosphates of the two purine analogs 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosylguanine. We conclude that overproduction of any of the three 5'-nucleotidases cannot explain development of resistance against cytosine analogs but that overproduction of cN-II could lead to resistance against purine analogs. Of the tested analogs, only (E)-5-(2-bromovinyl)-2'-deoxyuridine was preferentially dephosphorylated by mitochondrial dNT-2. We propose that in future developments of analogs this aspect be considered in order to reduce mitochondrial toxicity. We tested inhibition of dNT-1 and dNT-2 by a large variety of synthetic metabolically stable nucleoside phosphonate analogs and found one (PMcP-U) that inhibited dNT-1 and dNT-2 competitively and a second (DPB-T) that inhibited dNT-2 by mixed inhibition. Both inhibitors are useful for specific 5'-nucleotidase assays and structural studies and may open up possibilities for therapy.     

Synergy Between 3′Azido-3′deoxythymidine and Paclitaxel in Human Pharynx FaDu Cells

Purpose. We recently demonstrated simultaneous targeting of telomere and telomerase as a novel cancer therapeutic approach, and that telomerase inhibitors such as 3azido-3deoxythymidine (AZT) significantly enhanced the antitumor activity of paclitaxel, which causes telomere erosion, in telomerase-positive human pharynx FaDu tumors in vitro and in vivo (1). The present study evaluated the synergy between AZT and paclitaxel to identify optimal combinations for future clinical evaluation. Methods. FaDu cells were incubated with or without AZT for 24 h and then treated with AZT with or without paclitaxel for an additional 48 h. Under these conditions, single agent paclitaxel produced a 60% maximum reduction of cell number (IC₅₀ was 7.3 nM), and single agent AZT produced a 97% reduction (IC₅₀ was 5.6 M). Synergy was evaluated using fixed-concentration and fixed-ratio methods, and data were analyzed by the combination index method. Results. The results indicate a concentration-dependent synergy between the two drugs; the synergy was higher for combinations containing greater paclitaxel-to-AZT concentration ratios and increased with the level of drug effect. For example, in combinations containing 1 M AZT, synergy was 1.3-fold at the 20% effect level and 3.1-fold at the 60% effect level. Because the major antitumor activity, determined by comparing the posttreatment cell number to the pretreatment cell number, was antiproliferation at the 20% effect level and cell kill at the 60% effect level, our results suggest that AZT mainly enhances the cell kill effect of paclitaxel. Conclusion. In summary, the present study demonstrates a synergistic interaction between paclitaxel and AZT and supports a combination using a low and nontoxic AZT dose in combination with a therapeutically active dose of paclitaxel.