Multiorgan transplacental and neonatal carcinogenicity of 3'-azido-3'-deoxythymidine in mice

The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is used successfully for reduction of perinatal viral transmission. However toxic side effects including carcinogenesis are possible. To test this, pregnant CD-1 Swiss mice were given 25.0 or 12.5 mg AZT on gestation days 12-18. Previously we reported an increase in lung, liver, and female reproductive system tumors in offspring euthanized at 1 year (Olivero et al., J. Natl. Cancer Inst. 89, 1602-1608, 1997). Findings for all remaining offspring up to 2 years old are reported here. AZT effects were most prominent in female offspring, with a significant threefold increase in lung tumors, a reduction in lymphoblastic and follicle center cell lymphomas, and a significant increase in histiocytic sarcomas (0 in controls, 3% after low-dose AZT, and 8% after high-dose AZT, p = 0.022). Dose-dependent incidences of mammary gland, ovarian, and seminal vesicle tumors were low but significant: 0/106 controls, 3/105 low-dose, and 8/105 high-dose mice presented one of these neoplasms (p = 0.0025). Incidences of females showing any clearly AZT-related neoplasm, in lung, liver, ovary, or mammary gland or histiocytic sarcoma, in the second year, were 12/32 after the low dose and 14/27 after the high dose vs 3/23 controls (p = 0.0045). Also, the sensitivity of neonatal mice was assessed by administration of 25, 50, 100, or 200 mg/kg AZT on postnatal days 1 through 8. The effects at 2 years were similar to those seen after transplacental exposure, with significant increases in lung, liver, and mammary tumors in females. The results confirm that AZT is a moderately effective perinatal carcinogen in mice, targeting several tissue types.     

Synthesis and antiviral activity of several 2,5'-anhydro analogues of 3'-azido-3'-deoxythymidine, 3'-azido-2',3'-dideoxyuridine, 3'-azido-2',3'-dideoxy-5-halouridines, and 3'-deoxythymidine against human immunodeficiency virus and Rauscher-murine leukemia virus

Several 2,5'-anhydro analogues of 3'-azido-3'-deoxythymidine (AZT), 3'-azido-2'3'-dideoxyuridine (AZU), 3'-azido-2'3'-dideoxy-5-bromouridine, 3'-azido-2',3'-dideoxy-5-iodouridine, and 3'-deoxythymidine and the 3'-azido derivative of 5-methyl-2'-deoxyisocytidine have been synthesized for evaluation as potential anti-HIV (human immunodeficiency virus) agents. These 2,5'-anhydro derivatives, compounds 13-17, demonstrated significant anti-HIV-1 activity with IC50 values of 0.56, 4.95, 26.5, 27.1, and 48 microM, respectively. Compared to that of the parent compounds AZT and AZU, the respective 2,5'-anhydro analogues, compounds 13 and 14, were somewhat less active. Whereas AZT was cytotoxic with a TCID50 of 29 microM, the toxicity of the 2,5'-anhydro derivative of AZT, compound 13, was reduced considerably to a TCID50 value of greater than 100 microM. The 2,5'-anhydro analogue of 5-methyl-2'-deoxyisocytidine also demonstrated anti-HIV-1 activity with an IC50 value of 12 microM. These compounds were also evaluated against Rauscher-Murine leukemia virus (R-MuLV) in cell culture. Among them, AZT, 3'-azido-2',3'-dideoxy-5-iodouridine, 3'-azido-2',3'-dideoxy-5-bromouridine, and 2,5'-anhydro-3'-azido-3'-deoxythymidine (13) were found to be most active, with IC50 values of 0.023, 0.21, 0.23, and 0.27 microM, respectively.     

Glucuronidation of 3'-azido-3'-deoxythymidine: human and rat enzyme specificity

Since preclinical studies indicated that 3'-azido-3'-deoxythymidine (AZT, zidovudine, Retrovir, BW A509U), a potent anti-HIV agent, is not metabolized extensively in rats, rabbits, mice, guinea pigs, cats, or dogs, the extensive biotransformation of AZT observed in humans was not expected. On average, approximately 75% of an oral AZT dose is recovered in human urine as a single metabolite while only 14-18% of the dose is recovered unchanged. Ultraviolet, infrared, nuclear magnetic resonance, and mass spectra and enzymatic degradation characterized the isolated major metabolite as a 5'-O-glucuronide (3'-azido-3'-deoxy-5'-beta-D-glucopyranuronosylthymidine, GAZT), a very unique nucleoside metabolite. These observations suggest that UDP-glucuronosyltransferase (UDPGT), EC2.4.1.17, mediates the in vivo biotransformation of AZT to GAZT. Since glucuronidation is one of the major conjugation reactions involved in the metabolic conversion of xenobiotics to more polar, water-soluble metabolites, it is an important detoxification pathway in humans. Therefore, it is important to understand the enzymatic basis for the discrepancy between metabolism of AZT in laboratory mammals and humans. This is especially relevant in light of the use of laboratory mammals to predict the metabolism of novel pharmaceutical agents in humans. The study presented herein confirms that liver UDPGT does catalyze the glucuronidation of AZT and that the higher substrate efficiency of AZT with human enzyme compared to rodent enzyme may account for metabolic differences observed in vivo.     

Glucuronidation of 3'-azido-3'-deoxythymidine by rat and human liver microsomes

The glucuronidation of 3'-azido-3'-deoxythymidine (AZT) by rat and human liver microsomes has been studied in vitro. The AZT-glucuronide was preliminarily identified through specific hydrolysis by beta-glucuronidase and rigorous product identification was performed by high-field proton nuclear magnetic resonance and fast-atom-bombardment mass spectrometry. A beta-linked 5'-O-glucuronide was the exclusive product formed in liver microsomes. Rat and human liver microsomal uridine 5'-diphosphoglucuronyltransferase activities toward AZT were investigated. These studies revealed that AZT had a lower Km and a 5-6-fold higher relative catalytic efficiency for uridine 5'-diphosphoglucuronyltransferase in human as compared to rat liver microsomes which may play a role in the quantitative differences observed in the degree of AZT glucuronidation between rat and human.     

Cellular pharmacology of 3'-azido-3'-deoxythymidine with evidence of incorporation into DNA of human bone marrow cells

We previously demonstrated that 3'-azido-3'-deoxythymidine (AZT) inhibits growth proliferation of human bone marrow progenitor cells in vitro [Antimicrob. Agents Chemother. 31:452-454 (1987)]. The present study evaluates the effect of toxic concentrations of AZT on possible sites of toxicity in human bone marrow cells. Exposure of cells over a 6-hr period to AZT concentrations between 0.5 and 50 microM resulted in a decreased incorporation of tritiated deoxyguanosine into DNA. Unchanged AZT and its phosphorylated metabolites accumulated within cells after exposure to 10 microM [3H]AZT. 3'-Azido-3'-deoxythymidine-5'-monophosphate was the predominant metabolite, reaching a concentration of 49.2 +/- 14.1 pmol/10(6) cells after 48 hr, and a continuous increase was observed in all phosphorylated derivative levels between 2 and 48 hr of incubation. Using a highly sensitive and specific DNA polymerase assay, endogenous deoxyribonucleotide pool size(s) were analyzed for 48 hr after incubation of cells with a pharmacologically relevant concentration of 10 microM AZT. After a 6-hr exposure, 2'-deoxycytidine-5'-triphosphate and 2'-deoxythymidine-5'-triphosphate pools represented approximately 86 and 70% of the control values; levels returned to normal after 24 hr and remained subsequently unchanged. Nucleic acids of human bone marrow cells exposed for 24 hr to 10 microM [3H]AZT were purified and analyzed by cesium sulfate density gradient. No radioactivity was detected in the RNA region, whereas a significant amount was associated with the DNA region. Hydrolysis of radiolabeled DNA and subsequent analysis by high performance liquid chromatography demonstrated specific incorporation of AZT into DNA. In additional studies, the amount of AZT incorporated into DNA was correlated with the initial extracellular AZT concentration. In particular, a significant relationship (p less than 0.0001) between the level of AZT incorporated into DNA and the inhibition of clonal growth was observed at concentrations of AZT between 1 and 25 microM (IC50 and IC85 for human bone marrow cells). In summary, these studies demonstrate that AZT is incorporated into DNA of human bone marrow cells and suggest that incorporation of AZT into DNA may be one mechanism responsible for AZT-induced bone marrow toxicity. In contrast, imbalance of deoxyribonucleotide pools by AZT appears unlikely to be associated with inhibition of DNA synthesis and toxicity in human bone marrow cells.     

Synthesis and antiretrovirus properties of 5'-isocyano-5'-deoxythymidine, 5'-isocyano-2',5'-dideoxyuridine, 3'-azido-5'-isocyano-3',5'-dideoxythymidine, and 3'-azido-5'-isocyano-2',3',5'-trideoxyuridine

The 5'-azidonucleosides 3 and 4 were obtained by treating thymidine and 2'-deoxyuridine with TPP/DEAD/HN3. The 3'-O-silylated 5'-azido-5'-deoxythymidine 5 and the corresponding 2'-deoxyuridine derivative 6 were transformed to the formamides (7 and 8, respectively) and dehydrated to the protected 5'-isocyano derivatives 9 and 10; deblocking gave 5'-isocyano-5'-deoxythymidine (11) and 5'-isocyano-2',5'-dideoxyuridine (12). 2,3'-Anhydro-5'-formamido derivatives of thymidine and 2'-deoxyuridine (19 and 20, respectively) were prepared by three different ways. In the most direct synthesis 3 and 4 were transformed to the 2,3'-anhydro-5'- azidonucleosides 17 and 18 by using TPP/DEAD; following the reaction with TPP/HCO2COCH3 gave 19 and 20. Nucleophilic opening reaction with LiN3 yielded the 3'-azido-5'-formylamino derivatives 21 and 22. Dehydration to 3'-azido-5'-isocyano-3',5'-dideoxythymidine (23) and 3'-azido-5'-isocyano-2',3',5'-trideoxyuridine (24) was achieved with tosyl chloride/pyridine. In contrast with 3'-azido-3'-deoxythymidine, compounds 11, 12, 23, and 24 were devoid of any marked inhibitory effect against DNA and RNA viruses including human immunodeficiency virus type I (HIV).     

Synergic effect of 3'-azido-3'-deoxythymidine and arsenic trioxide in suppressing hepatoma cells

The aim of this study was to investigate the synergic antitumor effects of arsenic trioxide (As2O3) and 3'-azido-3'-deoxythymidine (AZT) on hepatoma cells and explore the possible molecular basis of these effects. These results showed that AZT enhanced the inhibitory effect of As2O3 on HepG2 and SMMC-7721 cell growth. The IC50 of As2O3 in combination with AZT was lower than that of As2O3 alone. A concentration-dependent synergic effect of As2O3 and AZT (CI < 1) was observed in all the tested combinations of these compounds. These results also showed that the combination of As2O3 and AZT dramatically and significantly increased the number of apoptotic cells in HepG2 and SMMC-7721 cells. Studies in vivo showed that the combination of As2O3 and AZT was statistically superior to either As2O3 or AZT alone in the treatment of tumor-bearing mice. As2O3 (1 mg/kg) containing AZT (50 mg/kg) inhibits proliferation of implanted hepatoma 22 by 56.35%. These results suggest that treating hepatoma with a combination of As2O3 and AZT offers the advantages of reduced toxic side effects and improved therapeutic efficacy. To understand the mechanism through which As2O3 and AZT suppress tumors, we studied the effects of these compounds, both separately, and in combination, on telomerase and caspase-3 activity. The results showed that the growth inhibitory and apoptotic effects of As2O3 and AZT on human hepatoma cells could be related to the inhibition of telomerase and the activation of caspase 3.     

Effects of granulocyte-macrophage colony-stimulating factor on 3'-azido-3'-deoxythymidine uptake, phosphorylation and nucleotide retention in human U-937 cells

Previous studies have demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) both increases and decreases levels of 3'-azido-3'-deoxythymidine (AZT) nucleotides in certain human myeloid cells. The present studies have examined the effects of GM-CSF on AZT metabolism in U-937 cells. The results demonstrate that GM-CSF stimulated AZT nucleotide formation in these cells. This stimulation was detectable during concurrent exposure to GM-CSF and AZT or as a result of pretreatment with GM-CSF. The GM-CSF-induced enhancement in AZT nucleotide formation was associated with a 4-fold increase in AZT uptake. The finding that uptake of AZT into U-937 cells was only partially sensitive to 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) suggested a process primarily involving nonfacilitated diffusion. The results also demonstrate that treatment of U-937 cells with GM-CSF was associated with nearly a 2-fold increase in thymidine kinase activity. Moreover, the findings indicate that retention of AZT-MP and AZP-TP was prolonged significantly (P less than 0.05 and P less than 0.01 respectively) in association with GM-CSF treatment. Taken together, these results suggest that GM-CSF enhances the formation of AZT nucleotides by increasing AZT uptake and phosphorylation, as well as increasing retention of phosphorylated derivatives.     

Comparative pharmacokinetics of 3'-azido-3'-deoxythymidine (AZT) and 3'-azido-2',3'-dideoxyuridine (AZddU) in mice

The pharmacokinetics of 3'-azido-3'-deoxythymidine (AZT) and 3'-azido-2',3'-dideoxyuridine (AZddU, CS-87), active anti-HIV compounds, were characterized in uninfected mice. Sensitive and specific HPLC techniques were used to quantitate AZT and AZddU concentrations in serum and brain homogenates following iv doses of 50 mg/kg and 250 mg/kg. The pharmacokinetic parameters of t1/2, CIt, and Vss were similar for both compounds at each dose; however, CIt and Vss decreased at the higher dose, indicating a dose dependency. At the 50 mg/kg doses, the CIt of AZddU and AZT was 1.27 liters/hr/kg and 1.38 liters/hr/kg, respectively, which is analogous to the clearance value of AZT observed in humans. Brain/serum concentration ratios for AZddU tended to be greater than those obtained for AZT and were significantly different at the 50 mg/kg dose, being 0.234 +/- 0.282 for AZddU and 0.064 +/- 0.025 for AZT.     

The effect of malaria infection on 3'-azido-3'-deoxythymidine and paracetamol glucuronidation in rat liver microsomes.

The effect of malaria infection on UDP-glucuronosyltransferase (UDPGT) activity was investigated in rat liver microsomes using 3'-azido-3'-deoxythymidine and paracetamol. The Michaelis-Menten parameters, Km and Vmax were calculated and intrinsic clearance values were estimated for normal and infected livers. The results show that malaria infection alters the activity of UDPGT.     

Effect of dipyridamole on transport and phosphorylation of thymidine and 3'-azido-3'-deoxythymidine in human monocyte/macrophages

Dipyridamole (DPM), a commonly used coronary vasodilator and antithrombotic drug, was shown recently to potentiate the antiviral effect of 3'-azido-3'-deoxythymidine (AZT) in HIV-1 infected human monocyte-derived macrophages (M/M) in vitro. We report in the present study that in uninfected M/M, DPM markedly inhibited cellular uptake of [3H]thymidine (dThd) and its incorporation into the nucleotide pools, particularly the dThd-triphosphate pool. In contrast, DPM did not affect cellular uptake and phosphorylation of [3H]AZT. Since dThd counteracts the phosphorylation and antiviral action of AZT, these findings support the hypothesis that the potentiation of the anti-HIV effect of AZT is due, at least in part, to differential inhibition of nucleoside salvage.     

High-performance liquid chromatographic analysis of 3'-azido-3'-deoxythymidine monophosphate diglyceride, an anti-HIV glycerophospholipid

A reversed-phase chromatographic method is described for the analysis of an experimental anti-AIDS drug 3'-azido-3'-deoxythymidine monophosphate diglyceride (AZT-MP-DG) [J.M. Steim et al., Biochem. Biophys. Res. Commun. 171, 458-464 (1990)] [1], a phosphatidic acid derivative of AZT. Analytical conditions were based upon conventional separations of glycerophospholipid species. Where AZT-MP-DG was monitored by UV absorption, there were two wavelength maxima. The response was linear in the concentration range used in this study. The peak was characterized by absorbance ratios with a rapid scanning UV detector.     

Synthesis and biological evaluation of dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine and 2', 3'-dideoxycytidine

The 5'----5' dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC) were prepared and evaluated for their inhibitory properties against different viruses, including human immunodeficiency virus (HIV). The synthesis of the compounds was achieved by reaction of AZT or N4-(4-monomethoxytrityl)-2',3'-dideoxycytidine with in situ prepared methylphosphonic bis (triazolide), followed in the latter case by an acidic treatment. The two title compounds showed in vitro anti-HIV activity, that was 200- to 450-fold less pronounced that that shown by the corresponding monomeric nucleosides AZT and DDC. The decreased antiviral activity may be ascribed to nuclease resistance of the methylphosphonate linkage.     

Modulation of 3'-azido-3'-deoxythymidine catabolism by probenecid and acetaminophen in freshly isolated rat hepatocytes

Metabolic studies of 3'-azido-3'-deoxythymidine (AZT) in humans have demonstrated that this compound is primarily eliminated as a 5'-O-glucuronide, 3'-azido-3'-deoxy-5'-beta-D-glucopyranuronosylthymidine (GAZT), accounting for approximately 80% of the administered dose. Recently, we characterized the complete catabolic pathway of AZT in freshly isolated rat hepatocytes in suspension, demonstrating extensive formation of three catabolites, including GAZT, 3'-amino-3'-deoxythymidine (AMT), and 3'-amino-3'-deoxy-5'-beta-D-glucopyranuronosylthymidine (GAMT). The present study evaluated the effects of probenecid (PROB) and acetaminophen (ACET), two agents which are also metabolized by UDP-glucuronyltransferase, on the metabolism and transmembrane distribution of AZT in rat hepatocytes. Pre-exposure of cells to 350 microM PROB 30 min prior to the addition of 10 microM [3H]AZT decreased intracellular GAZT levels by approximately 10-fold. Interestingly, AMT formation was enhanced approximately 1.5-fold in the presence of PROB, probably resulting from increased AZT availability. In contract, pre-exposure to 50 microM ACET 30 min prior to addition of 10 microM [3H]AZT did not substantially alter AZT glucuronidation. Additionally, decreased AZT catabolism by PROB did not contribute to the formation of 5'-phosphorylated derivatives of AZT. Agents which undergo glucuronidation may thus not necessarily affect AZT conversion to GAZT, and their potential interactions should be investigated using in vitro systems prior to co-administration with AZT.     

Doses and time-dependent effects of 3'-azido-3'-deoxythymidine on T47D human breast cancer cells in vitro

The objective of the present work was to study the effects of 3'-azido-3'-deoxythymidine (azidothymidine, Zidovudine) on human breast cancer cells by using, as a model, the T47D cell line (typified as oestrogen-dependent and p53-mutated). Low azidothymidine doses (3.125 microM) increase the percentage of cells in S-phase, with the effect reversing after 24 hr of incubation; as azidothymidine doses increase, the magnitude and duration of its effect increase proportionally, although, even with the highest concentrations (50-100 microM) the effects decline after 48 hr of incubation. If media (containing azidothymidine or vehicle) are daily renewed, the azidothymidine effects (accumulation of cells in S-phase) are higher and decline later than when media and drug are not changed during the whole culture period, thereby suggesting that the reversion of azidothymidine effects could be related with a degradation of the drug or accumulation in media of substances which counteract its effects. Azidothymidine inhibits T47D cell proliferation at concentrations higher than 50 microM. The exposure to 50 or 100 microM azidothymidine induced cell apoptosis after 48 hr or more of incubation. We conclude that: a) azidothymidine, with appropriate doses and duration of treatment, synchronizes cells in S-phase, inhibits proliferation, and induces apoptosis, b) the discontinuous application of the drug rather than continuous exposure to it increases its efficiency to synchronize the T47D cell cycle. This in vitro anti-breast cancer activity suggests that a possible clinical usefulness of azidothymidine, either alone or associated with other drugs with cycle-specific antitumoural activity circumscribed to the S-phase of cell cycle, is worthy of investigation.     

Amino acid phosphoramidate monoesters of 3'-azido-3'-deoxythymidine: relationship between antiviral potency and intracellular metabolism

A series of phosphoramidate monoesters of 3'-azido-3'-deoxythymidine (AZT) bearing aliphatic amino acid methyl esters (3a, 3c, 4a, 4c, 5-7) and methyl amides (3b, 3d, 4b, 4d) was prepared and evaluated for anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs). These compounds, which showed no cytotoxicity at concentrations of 100 microM, were effective at inhibiting HIV-1 replication at concentrations of 0.08-30 microM. Since the D-phenylalanine and D-tryptophan derivatives exhibited equivalent or enhanced antiviral activity compared to their L-counterparts, there appears to be no specific stereochemical requirement for the amino acid side chain. In addition, except for the D-phenylalanine derivatives, the methyl amides had greater antiviral activity than the corresponding methyl esters. On the basis of the observed antiviral activity of AZT phosphoramidate monoesters 3a and 4a in PBMCs and CEM cells, the mechanism of action of these two compounds was investigated. AZT-MP and substantial amounts of either phosphoramidate were detected in PBMCs and CEM cells treated with either 3a or 4a. Biological mechanistic studies demonstrated that 3a and 4a affect viral replication at a stage after virus entry and preceding viral DNA integration. Quantitation of the intracellular levels of AZT-TP in PBMCs and CEM cells treated with 3a and 4a in the presence and absence of exogenous thymidine correlated the intracellular levels of AZT-TP to the antiviral activity and suggested that AZT-TP was responsible for the activity observed. In addition, the reduced toxicity of 3a and 4a toward CEM cells relative to AZT correlated with reduced levels of total phosphorylated AZT and not AZT-TP. Stable carbamate analogues of 3a and 4a were prepared and shown to inhibit the production of AZT-MP from cell-free extracts of CEM cells, further suggesting that a phosphoramidate hydrolase may be responsible for intracellular P-N bond cleavage. Taken together, these results suggest that the biological activity and intracellular metabolism of nucleoside phosphoramidate monoesters are distinct from that of phosphoramidate diesters.     

3'-azido-3'-deoxythymidine (AZT) induces apoptosis and alters metabolic enzyme activity in human placenta

The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is the drug of choice for preventing maternal-fetal HIV transmission during pregnancy. Our aim was to assess the cytotoxic effects of AZT on human placenta in vitro. The mechanisms of AZT-induced effects were investigated using JEG-3 choriocarcinoma cells and primary explant cultures from term and first-trimester human placentas. Cytotoxicity measures included trypan blue exclusion, MTT, and reactive oxygen species (ROS) assays. Apoptosis was measured with an antibody specific to cleaved caspase-3 and by rescue of cells by the general caspase inhibitor Boc-D-FMK. The effect of AZT on the activities of glutathione-S-transferase, beta-glucuronidase, UDP-glucuronosyl transferase, cytochrome P450 (CYP) 1A, and CYP reductase (CYPR) in the placenta was assessed using biochemical assays and immunoblotting. AZT increased ROS levels, decreased cellular proliferation rates, was toxic to mitochondria, and initiated cell death by a caspase-dependent mechanism in the human placenta in vitro. In the absence of serum, the effects of AZT were amplified in all the models used. AZT also increased the amounts of activity of GST, beta-glucuronidase, and CYP1A, whereas UGT and CYPR were decreased. We conclude that AZT causes apoptosis in the placenta and alters metabolizing enzymes in human placental cells. These findings have implications for the safe administration of AZT in pregnancy with respect to the maintenance of integrity of the maternal-fetal barrier.