Azido-3'-deoxythymidine Treatment Research Articles

Emodin and AZT synergistically inhibit the proliferation and induce the apoptosis of leukemia K562 cells through the EGR1 and the Wnt/β‑catenin pathway.

The aim of the present study was to investigate the synergistic antitumor effects of emodin and 3'‑azido‑3'‑deoxythymidine (AZT) on human chronic myeloid leukemia cells and to explore the possible underlying mechanisms. The K562 cells were treated with emodin and AZT, and the rates of cell inhibition and apoptosis were determined by MTT assay and flow cytometry, respectively. The mRNA expression of EGR1 was detected by reverse transcription‑polymerase chain reaction (RT‑PCR) analysis. The expression of EGR1 was silenced using siRNA, and then protein expression of β‑catenin was detected by western blotting. The results demonstrated that AZT enhanced the inhibitory effect of emodin in K562 cells. The IC50 of the emodin/AZT combination at 24, 48 or 72h was 23.6/235.6, 10.2/101.6 or 5.9/58.5µmol/l, respectively, which was significantly lower compared with the IC50 of emodin (all >32µmol/l) or AZT (all >320µmol/l) alone. There was a dose‑dependent response to the combined emodin and AZT treatment, and the calculation of the combination index yielded values <1, demonstrating the synergistic effect of the combined treatment compared with the control (P<0.05). Furthermore, the combination of emodin and AZT increased apoptosis in K562 cells (P<0.05). Apoptosis was higher in the combination group compared with that of either treatment alone or control groups. The expression of early growth response‑1 (EGR1) in K562 cells was upregulated in a time‑dependent manner. The expression of EGR1 was higher in the combination group compared with that in the emodin or AZT alone groups. The expression of the Wnt/β‑catenin signaling pathway in the combination group was lower compared with that in the emodin or AZT alone groups. The expression of the Wnt/β‑catenin signaling pathway was significantly increased following EGR1 siRNA transfection. These data suggest that treating K562 cells with a combination of emodin and AZT exhibits reduced toxicity and improves therapeutic efficacy, and that the growth, inhibition, apoptosis and regulation of the Wnt/β‑catenin signaling pathway in human chronic myeloid leukemia cells by emodin and AZT may be associated with the expression of EGR1.

Compartmentalized Accumulation of cAMP near Complexes of Multidrug Resistance Protein 4 (MRP4) and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Contributes to Drug-induced Diarrhea

Diarrhea is one of the most common adverse side effects observed in ∼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3'-azido-3'-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea.

Palm Fruit Juice phenolics reduce the number of mutations caused by 3′‐Azido‐3′‐deoxythymidine treatment in a cultured human cell line

3′‐Azido‐3′‐deoxythymidine (AZT), used to treat HIV/AIDS, has been shown to cause mitochondrial dysfunction over time. How this dysfunction develops and its relation to long‐term adverse side effects of treatment, that include diabetes and Alzheimer's disease, is not known. One hypothesis is that AZT causes mutations in mitochondrial DNA (mtDNA) that build up over time, and that these mutations lead to mitochondrial dysfunction and disease. A broadly applicable approach to detect mutations without sequencing was developed using a LATE‐PCR Lights‐On/Lights‐ Off multiplex assay. The study objective tested whether AZT increases mitochondrial DNA mutations over time and whether palm fruit juice (PFJ) would reduce this damage. PFJ contains phenolics that might act to reduce the amount of reactive oxygen species, which are mutagenic to mtDNA. Three gene targets, HV2, CO2, and ND1, from the mitochondria of HepG2 cells were studied to assess AZT‐induced mutations in cell culture. Initial results revealed that AZT treatment for 30 days induced up to a 9‐ fold increase in mutations in all three target genes compared to normal culture media. When palm fruit juice was added with AZT, mutations decreased by 35% versus AZT alone. These results provide insight into how AZT damages mtDNA and suggest a possible way to ameliorate AZT effects in patients.This work was supported by a grant from the Malaysian Palm Oil Board.

Site-Specific Reduction of Oxidative and Lipid Metabolism in Adipose Tissue of 3′-Azido-3′-Deoxythymidine-Treated Rats

Although it is well accepted that treatment with some nucleoside reverse transcriptase inhibitors modifies both fat metabolism and fat distribution in humans, the mechanisms underlying these modifications are not yet known. The present investigation examined whether a decrease in oxidative capacity, induced by a chronic oral administration of 3'-azido-3'-deoxythymidine (AZT) in rats, could be associated with an alteration of the lipogenic capacity of white adipose tissues. The impact of obesity as a factor was then evaluated. Results showed that AZT treatment induced differential effects depending on anatomical localization. Indeed, in the inguinal adipose tissue, the specific activities of cytochrome c oxidase and fatty acid synthase, two rate-controlling enzymes in energy and lipogenic metabolisms, respectively, both decreased under AZT treatment, thus leading to a lowered cell lipid accumulation. Moreover, the AMP-activated protein kinase phosphorylation level tended to increase, thus implying that AZT causes an energy imbalance. Furthermore, the inguinal tissue of obese rats presented a sensitivity to AZT treatment that was higher than that of lean rats. In contrast, for epididymal tissue, no significant change in all these parameters could be detected under AZT treatment, regardless of the nutritional status of the animals. Taken together, these data demonstrate differential effects of AZT on subcutaneous adipose tissue and visceral white adipose tissue. It could be considered that the chronic decreases in energy and lipogenic metabolism of inguinal adipocyte, consecutive to AZT treatment, may lead, in the long term, to adipose tissue atrophy.

Site specific alterations of adipose tissue mitochondria in 3′-azido-3′-deoxythymidine (AZT)-treated rats: An early stage in lipodystrophy?

Although it is well accepted that treatment with nucleoside reverse transcriptase inhibitors (NRTIs) modifies fat metabolism and fat distribution in humans, the mechanisms underlying these modifications are not yet known. The present investigation examines the effects of chronic oral administration of 3′-azido-3′-deoxythymidine (AZT) on the mitochondrial metabolism and the redox status management of rat white adipose tissues originating from two anatomical sites, as well as of the rat liver. Results showed that AZT treatment induced differential effects on the mitochondrial functions depending on the anatomical localisation. Indeed, in inguinal adipose tissue, a significant decrease in the cytochrome c oxidase activity and in the mitochondrial DNA (mtDNA) content was observed, whereas the activity of citrate synthase, a mitochondrial protein exclusively encoded by the nucleus, was not affected. In contrast, no significant change in these parameters could be detected for epididymal tissue and for liver. In parallel, no oxidative stress could be detected after treatment, for both white adipose tissues and for liver, even though treated liver exhibited several modifications in redox management. Taken together, these data demonstrate differential mitochondrial effects of AZT on subcutaneous versus visceral white adipose tissue. Moreover, the decrease in mitochondrial oxidative capacity of inguinal adipocyte consecutive to AZT treatment is not primarily due to an oxidative stress per se, but rather to a depletion of the mtDNA content per cell.

Effect of nucleoside reverse transcriptase inhibitors on mitochondrial DNA synthesis in rats and humans.

Nucleoside reverse transcriptase inhibitors (NRTIs) have been hypothesized to inhibit mitochondrial DNA polymerase gamma, resulting in decreased mtDNA synthesis and mitochondrial insufficiency in HIV-1-infected patients. mtDNA synthesis was measured directly using a stable isotope mass spectrometric method following NRTI treatment in rodents. 3'-Azido-3'-deoxythymidine (AZT) was added to water (1 mg/mL) and administered ad libitum to female Sprague-Dawley rats for 1-8 weeks (n = 4 or 5 animals/timepoint). Neither body weight nor food intake was affected by AZT intake. Untreated controls and AZT-treated rats were given 4% H2O as drinking water for 2 weeks. AZT (approximately 100 mg/kg/d) produced a significant (P < 0.05) decrease in cardiac and hindlimb muscle mtDNA fractional synthesis compared with control groups (from 13.8 +/- 4.2% to 7.0 +/- 4.8% and from 7.6 +/- 1.8% to 4.5 +/- 0.4%, respectively) after 4 weeks. Cytochrome c oxidase content in hindlimb muscle was also decreased by 50% compared with controls after 4 weeks of AZT treatment (P < 0.07) and a calculated index of absolute mitochondrial biogenesis rate was significantly reduced by week 2 of AZT (P < 0.05) in hindlimb muscle. In preliminary studies, platelet mtDNA enrichments were compared to monocyte nDNA enrichments (used as a marker of a fully turned over tissue) in healthy human subjects. Fractional synthesis of mtDNA in platelets reached 98 +/- 3% after 5 weeks of H2O labeling. It is concluded that NRTIs decrease mtDNA synthesis and oxidative enzyme content and thus mitochondrial biogenesis in rodents and that the effects of NRTIs on mitochondrial biogenesis in tissues of HIV-1- infected humans can in principle be measured using this approach.

Incorporation of zidovudine into cord blood DNA of infants and peripheral blood DNA of their HIV-1-positive mothers.

The nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) is a weak carcinogen in adult female mice and a moderately strong carcinogen in the offspring of female mice given the drug during gestation. In addition, incorporation of AZT into DNA was observed in multiple organs of transplacentally exposed newborn mice. Here we investigate the incorporation of AZT into peripheral leukocyte DNA of HIV-1-positive adult pregnant women given AZT for variable times during gestation and cord blood of infants exposed to AZT in utero. The length of treatment varied between 10 days and 9 months. High molecular weight DNA was extracted from maternal peripheral blood mononuclear cells (PBMC) and infant cord blood. A specific AZT-DNA radioimmunoassay was used to determine the amount of AZT incorporated into leukocyte DNA. Incorporation of AZT into DNA ranged up to 183.3 and 344.5 molecules of AZT/10(6) nucleotides in the mothers and infants, respectively, and was detected in about 70% of samples. Therefore, AZT-induced mutagenic events are possible in the majority of adults and infants. No correlation was found between level of incorporation and length of AZT treatment, suggesting that the differences observed among the individuals arise from variability in AZT metabolism. These data support previous observations that a high degree of inter-individual variability in AZT phosphorylation occurs in primates.

3′-Azidothymidine Potently Inhibits the Biosynthesis of Highly Branched N-Linked Oligosaccharides and Poly-N-acetyllactosamine Chains in Cells

3′-Azidothymidine Potently Inhibits the Biosynthesis of Highly Branched N-Linked Oligosaccharides and Poly-N-acetyllactosamine Chains in Cells

3'-Azidothymidine potently inhibits the biosynthesis of highly branched N-linked oligosaccharides and poly-N-acetyllactosamine chains in cells.

Previous studies in our laboratory have characterized 3'-azido-3'-deoxythymidine (AZT) as a potent inhibitor of glycosphingolipid biosynthesis in cultured cells (Steet, R., Alizadeh, M., Melançon, P., and Kuchta, R. D. (1999) Glycoconj. J. 16, 237-245; Yan, J.-P., Ilsley, D. D., Frohlick, C., Steet, R., Hall, E. T., Kuchta, R. D., and Melançon, P. (1995) J. Biol. Chem. 270, 22836-22841). Here, we report that AZT treatment of K562 cells results in significant alterations in the profile of N-linked oligosaccharides. Fractionation of [(3)H]mannose-labeled oligosaccharides from AZT-treated K562 cells using lectin affinity chromatography revealed striking changes in the branching and processing of N-linked glycoconjugates. AZT treatment resulted in the production of fewer highly branched complex glycans (60% of control at 20 micrometer AZT) and a significant accumulation of core-fucosylated biantennary oligosaccharides. In addition, extension of branched oligosaccharides with multiple poly-N-acetyllactosamine repeats is nearly abolished by AZT concentrations as low as 2 micrometer. A shift from multiantennary to moderately branched oligosaccharides was also apparent in the melanoma cell line SK-MEL-30 upon AZT treatment. N-Linked glycans from both cell lines exhibited increased affinity for the beta-galactoside-binding lectin RCA-I in the presence of AZT, suggesting that the addition of terminal sialic acid is sensitive to the drug. These results demonstrate the ability of AZT to modulate strongly the processing of asparagine-linked glycoconjugates in whole cells and reveal a novel mechanism by which AZT treatment may cause anemia.

Long-term effects of prenatal 3'-azido-3'-deoxythymidine (AZT) exposure on intermale aggressive behaviour of mice.

AZT treatment of seropositive pregnant women and their neonates has been widely used due to its effectiveness in reducing vertical transmission of HIV, but medium- and long-term effects of AZT on neurobehavioural development and adult responding are still poorly described. The aim of the present study was to evaluate the long-term effects of prenatal AZT treatment on aggressive behaviour of adult male mice. Pregnant CD-1 mice were given saline vehicle, 0.4, or 0.8 mg/ml AZT in their drinking water from gestation day 10 to delivery. Social-aggressive types of interaction were assessed in their male offspring following a 4-week isolation period. Two groups of subjects were used, each undergoing a different type of test: test 1 consisted of a single 20-min encounter with an isolated same-strain opponent on postnatal day (PND) 90, while in test 2 (PND 150) subjects were paired for 10 min for 5 consecutive days with a non-isolated opponent. Slight changes in both aggressive and defensive components of the male-specific agonistic pattern were evident only in test 1, AZT mice displaying a limited increase of aggressive behaviour compared to their controls. Although the long-term effects of prenatal AZT on social behaviour are limited, they may be of some relevance for paediatricians in order to plan a follow-up of infants, children and adolescents exposed in utero to antiretroviral drugs.

3'-Azidothymidine significantly alters glycosphingolipid synthesis in melanoma cells and decreases the shedding of gangliosides.

In this report, we establish that 3'-azido-3'-deoxythymidine (AZT) treatment of melanoma cells greatly alters the pattern of glycosphingolipid biosynthesis. In SK-MEL-30 cells, synthesis of the gangliosides GM3 and GD3 was significantly inhibited (60% and 50% of control, respectively) and the production of their precursor, lactosylceramide, was stimulated by 2.5-fold. Control experiments established that phospholipid synthesis was not affected by AZT treatment, consistent with AZT treatment only affecting lipid biosynthetic reactions that involve glycosylation. Likely as a consequence of decreased rates of ganglioside synthesis, AZT treatment of SK-MEL-30 cells also significantly suppressed the amount of gangliosides shed from the membranes of these cells. Since shedding of gangliosides has been proposed to allow melanoma cells to avoid destruction by the immune system and alterations of glycosphingolipid levels are likely important for the malignant cell phenotype, these results may have important implications regarding the potential use of AZT or related glycosylation inhibitors as cancer chemotherapeutics.

Deoxyribonucleoside triphosphate pool imbalances in vivo are associated with an increased retroviral mutation rate.

Deoxyribonucleoside triphosphate (dNTP) pool imbalances are associated with an increase in the rate of misincorporation and hypermutation during in vitro reverse transcription reactions. However, the effects of in vivo dNTP pool imbalances on the accuracy of reverse transcription are unknown. We sought to determine the effects of in vivo dNTP pool imbalances on retroviral mutation rates and to test our hypothesis that 3'-azido-3'-deoxythymidine (AZT) increases the retroviral mutation rates through induction of dNTP pool imbalances. D17 cells were treated with thymidine, hydroxyurea (HU), or AZT, and the effects on in vivo dNTP pools were measured. Thymidine and HU treatments induced significant dNTP pool imbalances. In contrast, AZT treatment had very little effect on the dNTP pools. The effects of in vivo dNTP pool imbalances induced by thymidine and HU treatments on the retroviral mutation rates were also determined. Spleen necrosis virus (SNV)-based and murine leukemia virus (MLV)-based retroviral vectors that expressed the lacZ mutant reporter gene were used. The frequencies of inactivating mutations introduced in the lacZ gene in a single replication cycle provided a measure of the retroviral mutation rates. Treatment of D17 target cells with 500 microM thymidine increased the SNV and MLV mutant frequencies 4.7- and 4-fold, respectively. Treatment of D17 target cells with 2 mM HU increased the SNV and MLV mutant frequencies 2.1- and 2.7-fold, respectively. These results demonstrate that dNTP pool imbalances are associated with an increase in the in vivo retroviral mutation rates, but AZT treatment results in an increase in the retroviral mutation rates by a mechanism not involving alterations in dNTP pools.

The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis.

AZT-induced hypermethylation of human thymidine kinase gene in the absence of total DNA hypermethylation

Genome-wide DNA hypermethylation induced by 3′-azido-3′-deoxythymidine (AZT) has been suggested to be involved in the development of AZT resistance. We used a CD4 T-lymphoblastoid CEM line and its AZT-resistant MT500 variant with reduced thymidine kinase activity. Evaluation of total DNA methylation, after AZT treatment, failed to show an increase in the 5-methylcytosine level in both parental and AZT-resistant cells. The effect was instead observed at a more specific gene level, on the three HpaII sites present in exon 1 of the human thymidine kinase gene. These results suggest that AZT treatment can induce site-specific hypermethylation, even in the absence of a more general DNA hypermethylating effect.

Skeletal muscle mitochondria from AZT-treated rats have a diminished response to chronic electrical stimulation.

Inhibition of DNA polymerase gamma-function mediated by 3'-azido-3'-deoxythymidine (AZT) has been proposed to cause a myopathy by reducing mitochondrial DNA (mtDNA) replication. Repeated bouts of exercise stimulate an increase in mtDNA replication, mitochondrial content, and mitochondrial volume fraction. Therefore, adaptation of rat skeletal muscle [tibialis anterior (TA)] mitochondria exposed to AZT (1 mg/ml for 35 days) and then to electrical stimulation for 8 h/day (7, 14, 21 days) with continued AZT treatment was examined. Fourteen and 21 days of stimulation increased TA cytochrome oxidase (CO) activity, mtDNA, and CO subunit III and VIc mRNA levels in both groups. The TA CO activity and CO III mRNA increases after 14 and 21 days of stimulation were diminished in AZT-treated rats. TA glyceraldehyde-3-phosphate dehydrogenase was reduced in normal rats after chronic stimulation but was unchanged in AZT-treated rats. Chronic stimulation increased the mitochondrial volume fraction by 80 and 40% in normal and AZT-treated rats, respectively. These results indicate diminution, but not complete inhibition, of mitochondrial adaptation by AZT-treated skeletal muscle in response to stimulation.

3'-Azido-3'-deoxythymidine inhibits erythroid-specific transcription factors in human erythroid K562 leukemia cells.

The present study examines genetic mechanism(s) possibly involved in the observed 3'-azido-3'-deoxythymidine (AZT)-induced inhibition of globin gene transcription by evaluating the direct phenotypic erythroid effects of AZT on erythroid-specific transcription factors which regulate globin gene promoters. In vitro binding of GATA-1 or NFE-2 to its consensus sequence was decreased in the presence of AZT reaching a maximum inhibition as early as 24 h after AZT treatment. Nuclear extracts from butyric acid-induced K562 cells treated with an IC50 concentration of AZT exhibited a decrease in GATA-1 and NFE-2 binding by approximately 30% and 35%. In contrast, 2',3'-dideoxycytidine which inhibits cell growth without affecting hemoglobin synthesis, had no effect on binding of GATA-1 and NFE-2 factors. Northern blot analysis revealed a 25% decrease by AZT in GATA-1 mRNA steady-state levels at 24 h and this inhibitory effect was maintained until 72 h after drug addition. A similar decrease in NFE-2 mRNA steady-state levels was observed at 72 h after AZT treatment. This study suggests that AZT inhibition of erythroid differentiation is subsequent to a decrease of nuclear factors gene expression which affect their DNA binding.

Predominance of codon 215 mutation in reverse transcriptase-coding region of 3'-azido-3'-deoxythymidine (AZT)-resistant HIV-1 isolates after long-term AZT therapy.

Among human immunodeficiency type 1 viruses (HIV-1) isolated during long-term 3'-azido-3'-deoxythymidine (AZT) therapy, 4 condoms (Asp67, Lys70, Thr215, and Lys219) in the HIV-1-reverse transcriptase (RT)-coding region have been considered to be related to the AZT resistance of HIV-1. Therefore we determined these mutation patterns in HIV-1 isolates from patients undergoing long-term AZT treatment. In 41 clones of HIV-1 from 7 patients, the Thr215 mutation was the most predominant (97.6%), and more frequent than Asp67 (48.8%), Lys70 (31.7%) and Lys219 (9.8%) mutations. All 22 clones from isolates cultured in the presence of 1 microM AZT Thr215 mutation; such a high frequency was not found for the other 3 codon mutations. In a clinical follow-up study, Thr215 mutation appeared in the late stage of AZT treatment parallel with the emergence of AZT insusceptibility. It is worth noting that this Thr215 mutation to Tyr or Phe is a 2-nucleotide mutation in contrast to the 1-nucleotide mutations seen in the other 3 codons. Isolates with the single amino acid change of Thr215 of the RT-coding region obtained after long-term AZT treatment grew in the presence of 1 microM AZT. This single amino acid mutation in the Thr215 codon is the most important factor in AZT resistance.

Simian immunodeficiency virus (SIV) infection of infant rhesus macaques as a model to test antiretroviral drug prophylaxis and therapy: oral 3'-azido-3'-deoxythymidine prevents SIV infection

The prophylactic and therapeutic properties of 3'-azido-3'-deoxythymidine (AZT) against simian immunodeficiency virus (SIV) infection were tested in four 3-month-old rhesus macaques. The infant monkeys were inoculated intravenously with a low dose (1 to 10 100% animal infectious doses) of uncloned SIVmac. The monkeys were treated orally with 50 mg of AZT per kg of body weight every 8 h; two animals were started on treatment 2 h prior to virus inoculation, and two animals were started on treatment 6 weeks later. All four animals were treated for a period of 6 to 10 weeks. Outward signs of AZT toxicity were absent, but a mild macrocytic anemia occurred soon after therapy was started and resolved shortly after it was discontinued. The two infants that were begun on AZT treatment 2 h prior to virus inoculation never became infected, as demonstrated by the inability to detect cell-free or cell-associated virus in the blood, proviral DNA in peripheral blood mononuclear cells, or anti-SIV antibodies. AZT administration over a 10-week period had no detectable effect on the course of disease in the two animals that were begun on treatment after the infection had been established. In addition to demonstrating the prophylactic effect of AZT against low-dose SIV exposure, the study demonstrated the ease with which infant rhesus macaques can be used for antiretroviral drug testing.

Anti-HIV-1 activity of 3′-azido-3′-deoxythymidine (AZT) in primary mononuclear phagocytes

Conflicting data have been reported on ability of 3′-azido-3′-deoxythymidine (AZT) to protect mononuclear phagocytes from HIV-1 infection. We compared the antiviral potency of AZT in three types of primary human mononuclear phagocytes: peripheral blood monocytes, monocyte-derived macrophages (in vitro differentiated) and alveolar macrophages (in vivo differentiated). To establish highly-productive virus infection, purified cells (> 99%) from healthy donors were challenged with the macrophage-tropic HTLV-III Ba-L strain at input multiplicities ranging from 0.05 to 20 TCID 50 per cell. AZT (0.1 nM–10 μM) was added immediately after infection and either continued for the duration of the experiment or stopped 1–7 days after infection. The kinetics of HIV-1 Ba-L replication were assessed by measuring p24 antigen production on days 4–28 post-infection. Continuous treatment with AZT reproducibly inhibited viral replication in a concentration-dependent manner in all three cell types. The IC 90 of AZT was 0.04 μM in blood monocytes, 0.009 μM in monocyte-derived macrophages, and 0.0001 μM in alveolar macrophages (mean of 3–4 donors for each cell type). AZT was not cytotoxic at < 10 μM as assessed by cell viability, cell protein, and interferon-γ-activated H 2O 2-release. In experiments in which AZT treatment was stopped after infection, viral replication resumed after a lag of 7–14 days and increased exponentially toward control levels. This occurred despite initial inhibition of virus production to below the limit of p24 detection (∼ 50 pg/ml). These results indicate that AZT is a potent inhibitor of HIV-1 replication in primary mononuclear phagocytes regardless of the stage of cell differentiation, and that AZT is most active in tissue (alveolar) macrophages. AZT does not irreversibly block infection of mononuclear phagocytes, however, as viral replication resumes after removal of AZT.

Pharmacokinetics of 3'-azido-3'-deoxythymidine and its catabolites and interactions with probenecid in rhesus monkeys

The pharmacokinetics and metabolism of 3'-azido-3'-deoxythymidine (AZT) were investigated in rhesus monkeys after subcutaneous administration of 33.3 mg of AZT per kg of body weight alone or in the presence of 100 mg of probenecid per kg. In addition to unchanged drug, two catabolites, 5'-O-glucuronide (GAZT) and 3'-amino-3'-deoxythymidine (AMT), were detected in plasma within 30 min. GAZT exhibited a kinetic profile similar to that of AZT, with an elimination half-life of approximately 1 h, while AMT was more variable, with an apparent half-life of 1.6 +/- 1.5 h. Approximately 90% of the total administered dose was recovered in urine within 24 h as AZT, GAZT, AMT, and the 5'-O-glucuronide of AMT. AZT and AMT demonstrated similar cerebrospinal fluid (CSF) penetration 1 h after AZT treatment, while GAZT poorly crossed the blood-brain barrier. Concomitant administration of probenecid greatly altered the pharmacokinetics of AZT, GAZT, and AMT, resulting in prolongation of their apparent elimination half-lives, increased concentrations in plasma, and marked reduction in renal clearances. In addition, the CSF/plasma concentration ratios for AZT and its catabolites were greatly increased, suggesting that probenecid inhibits efflux of AZT and its catabolites from CSF to plasma. The substantial levels of AMT in plasma suggest that this catabolite affects the pharmacodynamic properties of AZT in relation to its activity against human immunodeficiency virus replication and cytotoxicity to host cells. Enhanced AMT levels in plasma in the presence of probenecid may decrease the therapeutic efficacy of the AZT-probenecid combination.