High Plasma Concentrations of Zidovudine (AZT) Do Not Parallel Intracellular Concentrations of AZT-Triphosphates in Infants During Prevention of Mother-to-Child HIV-1 Transmission.

OBJECTIVES: Zidovudine (AZT) is mainly used to prevent mother-to-child HIV-1 transmission (PMTCT). Despite serious concerns on AZT-associated toxicity, there is little information on pharmacokinetics of intracellular AZT metabolites in infants. METHODS: We conducted a prospective study in 31 HIV-uninfected infants who received AZT for PMTCT. Blood samples were obtained from 14 infants on postdelivery days (PDD) 1, 7, 14, and 28 and from 17 infants at 0 and 4 hours after dosing on PDD-1. Plasma AZT concentrations (pAZT) and intracellular concentrations of AZT-monophosphate (icAZT-MP), diphosphate (icAZT-DP), and triphosphate (icAZT-TP) were determined. RESULTS: Plasma AZT and icAZT-MP concentrations were 2713 nmol/L and 79 fmol/10 cells in PDD-1, but decreased to 1437 nmol/L and 31 fmol/10 cells by PDD-28 (P = 0.02 and P = 0.07 for all PDDs, respectively), whereas those of icAZT-DP and icAZT-TP remained low throughout the sampling period (P = 0.29 and P = 0.61 for all PDDs, respectively) There were no differences in icAZT-TP between infants of the 2 mg/kg 4 times a day dose and 4 mg/kg twice daily dose (P = 0.25), whereas pAZT and icAZT-MP levels were higher in the latter (P < 0.01 and <0.01, respectively). The pAZT and icAZT-MP significantly increased from 0 to 4 hours after dosing (P < 0.001 and <0.001, respectively), whereas icAZT-DP, icAZT-TP levels were not changed (P = 0.41 and 0.33, respectively). CONCLUSIONS: The level of icAZT-TP did not change with age, time, or a single dose despite the wide range of pAZT concentration. A safer dosage needs to be determined because high pAZT levels do not parallel those of icAZT-TP.

Pharmacokinetic Modeling of Lamivudine and Zidovudine Triphosphates Predicts Differential Pharmacokinetics in Seminal Mononuclear Cells and Peripheral Blood Mononuclear Cells.

The male genital tract is a potential site of viral persistence. Therefore, adequate concentrations of antiretrovirals are required to eliminate HIV replication in the genital tract. Despite higher zidovudine (ZDV) and lamivudine (3TC) concentrations in seminal plasma (SP) than in blood plasma (BP) (SP/BP drug concentration ratios of 2.3 and 6.7, respectively), we have previously reported lower relative intracellular concentrations of their active metabolites, zidovudine triphosphate (ZDV-TP) and lamivudine triphosphate (3TC-TP), in seminal mononuclear cells (SMCs) than in peripheral blood mononuclear cells (PBMCs) (SMC/PBMC drug concentration ratios of 0.36 and 1.0, respectively). Here, we use population pharmacokinetic (PK) modeling-based methods to simultaneously describe parent and intracellular metabolite PK in blood, semen, and PBMCs and SMCs. From this model, the time to steady state in each matrix was estimated, and the results indicate that the PK of 3TC-TP and ZDV-TP in PBMCs are different from the PK of the two in SMCs and different for the two triphosphates. We found that steady-state conditions in PBMCs were achieved within 2 days for ZDV-TP and 3 days for 3TC-TP. However, steady-state conditions in SMCs were achieved within 2 days for ZDV-TP and 2 weeks for 3TC-TP. Despite this, or perhaps because of it, ZDV-TP in SMCs does not achieve the surrogate 50% inhibitory concentration (IC50) (as established for PBMCs, assuming SMC IC50 = PBMC IC50) at the standard 300-mg twice-daily dosing. Mechanistic studies are needed to understand these differences and to explore intracellular metabolite behavior in SMCs for other nucleoside analogues used in HIV prevention, treatment, and cure.

Towards an improved anti-HIV activity of NRTI via metal-organic frameworks nanoparticles.

Nanoscale mesoporous iron carboxylates metal-organic frameworks (nanoMOFs) have recently emerged as promising platforms for drug delivery, showing biodegradability, biocompatibility and important loading capability of challenging highly water-soluble drugs such as azidothymidine tryphosphate (AZT-TP). In this study, nanoMOFs made of iron trimesate (MIL-100) were able to act as efficient molecular sponges, quickly adsorbing up to 24 wt% AZT-TP with entrapment efficiencies close to 100%, without perturbation of the supramolecular crystalline organization. These data are in agreement with molecular modelling predictions, indicating maximal loadings of 33 wt% and preferential location of the drug in the large cages. Spectrophotometry, isothermal titration calorimetry, and solid state NMR investigations enable to gain insight on the mechanism of interaction of AZT and AZT-TP with the nanoMOFs, pointing out the crucial role of phosphates strongly coordinating with the unsaturated iron(III) sites. Finally, contrarily to the free AZT-TP, the loaded nanoparticles efficiently penetrate and release their cargo of active triphosphorylated AZT inside major HIV target cells, efficiently protecting against HIV infection.

Biphasic elimination of tenofovir diphosphate and nonlinear pharmacokinetics of zidovudine triphosphate in a microdosing study.

OBJECTIVE: Phase 0 studies can provide initial pharmacokinetics (PKs) data in humans and help to facilitate early drug development, but their predictive value for standard dosing is controversial. To evaluate the prediction of microdosing for active intracellular drug metabolites, we compared the PK profile of 2 antiretroviral drugs, zidovudine (ZDV) and tenofovir (TFV), in microdose and standard dosing regimens. STUDY DESIGN: We administered a microdose (100 µg) of C-labeled drug (ZDV or tenofovir disoproxil fumarate) with or without a standard unlabelled dose (300 mg) to healthy volunteers. Both the parent drug in plasma and the active metabolite, ZDV-triphosphate (ZDV-TP) or TFV-diphosphate (TFV-DP) in peripheral blood mononuclear cells (PBMCs) and CD4 cells were measured by accelerator mass spectrometry. RESULTS: The intracellular ZDV-TP concentration increased less than proportionally over the dose range studied (100 µg-300 mg), whereas the intracellular TFV-DP PKs were linear over the same dose range. ZDV-TP concentrations were lower in CD4 cells versus total PBMCs, whereas TFV-DP concentrations were not different in CD4 cells and PBMCs. CONCLUSIONS: Our data were consistent with a rate-limiting step in the intracellular phosphorylation of ZDV but not TFV. Accelerator mass spectrometry shows promise for predicting the PK of active intracellular metabolites of nucleosides, but nonlinearity of PK may be seen with some drugs.

Topical delivery of DNA oligonucleotide to induce p53 generation in the skin via thymidine dinucleotide (pTT)-encapsulated liposomal carrier.

INTRODUCTION: Transcription factor p53 has a powerful tumor suppressing function that is associated with many cancers. Since the molecular weight of p53 is 53 kDa, it is difficult to transport across cell membranes. Thymidine dinucleotide (pTT) is an oligonucleotide that can activate the p53 transcription factor and trigger the signal transduction cascade. However, the negative charge and high water solubility of pTT limit its transport through cellular membranes, thereby preventing it from reaching its target in the nucleus. A suitable delivery carrier for pTT is currently not available. OBJECTIVE: The purpose of this study was to employ a nanoscale liposomal carrier to resolve the delivery problem, and increase the bioavailability and efficiency of pTT. METHODOLOGY: The approach was to employ liposomes to deliver pTT and then evaluate the particle size and zeta potential by laser light scattering (LLS), and permeation properties of pTT in vitro in a Franz diffusion assembly, and in vivo in a murine model using confocal laser scanning microscopy (CLSM). RESULTS: We found that dioleoylphosphatidylethanolamine (DOPE) combined with cholesterol 3 sulfate (C3S) were the best ingredients to achieve an average desired vehicle size of 133.6 ± 2.8 nm, a polydispersity index (PDI, representing the distribution of particle sizes) of 0.437, and a zeta potential of -93.3 ± 1.88. An in vitro penetration study showed that the liposomal carrier was superior to the free form of pTT at 2-24 hours. CLSM study observed that the penetration depth of pTT reached the upper epidermis and potential of penetration maintained up to 24 hours. CONCLUSION: These preliminary data demonstrate that nanosized DOPE/C3S liposomes can be exploited as a potential carrier of drugs for topical use in treating skin diseases.

Hybrid polymer nanocapsules enhance in vitro delivery of azidothymidine-triphosphate to macrophages.

One of the main limitations in the use of nucleoside reverse transcriptase inhibitors (NRTIs) such as azidothymidine (AZT) lies in their poor intracellular activation by cellular kinases into their active tri-phosphorylated form. Thus, the direct administration of triphosphate NRTIs like azidothymidine-triphosphate (AZT-TP), has been considered for bypassing this metabolic bottleneck, but these molecules do not diffuse intracellularly, due to their too hydrophilic character. Therefore, poly(iso-butylcyanoacrylate) (PIBCA) aqueous-cored nanocapsules have been tested as carriers to overcome the cellular delivery of AZT-TP. However, encapsulation of AZT-TP remained challenging because this molecule, due to its relatively low molecular weight, rapidly leaked out of the nanocapsules. In this study, we show that association of AZT-TP to a cationic polymer such as poly(ethyleneimine) (PEI) allowed to reach high entrapment efficiency of AZT-TP in PIBCA nanocapsules (up to 90%) as well as gradual in vitro release. The resulting hybrid PIBCA/PEI nanocapsules efficiently delivered AZT-TP in vitro to macrophages: the cellular uptake was increased by 30-fold compared to the free molecule, reaching relevant cellular concentrations for therapeutic purposes.

Cross-linked polymeric nanogel formulations of 5'-triphosphates of nucleoside analogues: role of the cellular membrane in drug release.

Activation of cytotoxic nucleoside analogues in vivo depends primarily on their cell-specific phosphorylation. Anticancer chemotherapy using nucleoside analogues may be significantly enhanced by intracellular administration of active phosphorylated drugs. However, the cellular transport of anionic compounds is very ineffective and restricted by many drug efflux transporters. Recently developed cationic nanogel carriers can encapsulate large amounts of nucleoside 5'-triphosphates that form polyionic complexes with protonated amino groups on the polyethylenimine backbone of the nanogels. In this paper, the 5'-triphosphate of an antiviral nucleoside analogue, 3'-azido-2',3'-dideoxythymidine (AZT), was efficiently synthesized and its complexes with nanogels were obtained and evaluated as potential cytotoxic drug formulations for treatment of human breast carcinoma cells. A selective phosphorylating reagent, tris-imidazolylphosphate, was used to convert AZT into the nucleoside analogue 5'-triphosphate using a one-pot procedure. The corresponding 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) was isolated with high yield (75%). Nanogels encapsulated up to 30% of AZTTP by weight by mixing solutions of the carrier and the drug. The AZTTP/nanogel formulation showed enhanced cytotoxicity in two breast cancer cell lines, MCF-7 and MDA-MB-231, demonstrating IC50 values 130-200 times lower than those values for AZT alone. The exact mechanism of drug release from nanogels remains unclear. One mechanism could involve interaction with negatively charged counterions. A high affinity of nanogels to isolated cellular membranes has been observed, especially for nanogels made of amphiphilic block copolymer, Pluronic P85. Cellular trafficking of nanogel particles, contrasted by polyethylenimine-coordinated copper(II) ions, was studied by transmission electron microscopy (TEM), which revealed membranotropic properties of nanogels. A substantial release of encapsulated drug was observed following interactions of drug-loaded nanogels with cellular membranes. A drug release mechanism triggered by interaction of the drug-loaded nanogels with phospholipid bilayer is proposed. The results illustrate therapeutic potential of the phosphorylated nucleoside analogues formulated in nanosized cross-linked polymeric carriers for cancer chemotherapy.

Direct measurement of nucleoside monophosphate delivery from a phosphoramidate pronucleotide by stable isotope labeling and LC-ESI(-)-MS/MS.

Amino acid phosphoramidates of nucleosides have been shown to be potent antiviral and anticancer agents with the potential to act as nucleoside monophosphate prodrugs. To access their ability to deliver 3'-azido-3'-deoxythymidine (AZT) 5'-monophosphate to cells, the decomposition pathway of an 18O-labeled AZT amino acid phosphoramidate was investigated by capillary reverse-phase high-performance liquid chromatography interfaced with negative ion electrospray ionization mass spectrometry (LC-ESI(-)-MS/MS). 18O-labeled L-AZT tryptophan phosphoramidate methyl ester ([18O]2) was synthesized with an 18O/16O relative ratio of 1.22 +/- 0.18. For CEM cells, a human T-lymphoblast leukemia cell line, incubated with [18O]2, values of 1.55 +/- 0.37, 0.34, and 0.13 were found for the 18O/16O relative ratio of intracellular AZT-MP for time intervals of 0.5, 4, and 20 h, respectively. The decrease in the level of labeled AZT-MP in CEM cells corresponded to a rapid increase in the amount of intracellular AZT presumably by dephosphorylation of AZT-MP. In contrast, for peripheral blood mononuclear cells (PBMCs), the 18O/16O relative ratio values of intracellular AZT-MP were 1.43, 1.06, and 0.61 for time intervals of 0.5, 4, and 20 h, respectively. Intracellular AZT in PBMCs was nearly undetectable for each time interval. Taken together, these results are consistent with the detection of direct P-N bond cleavage by CEM cells and PBMCs. However, AZT phosphoramidates are able to more effectively deliver AZT-MP to PBMCs than to CEM cells. Differential expression of 5'-nucleotidase in CEM cells relative to PBMCs is likely the reason for this discrepancy. Although applied to a phosphoramidate pronucleotide, the judicious use of 18O labeling and LC-MS is a general approach that could be applied to the investigation of the intracellular fate of other pronucleotides.

Novel nucleotide analogues as potential substrates for TMPK, a key enzyme in the metabolism of AZT.

Novel cyclic and acyclic analogues of dTMP and AZTMP were synthesized from the corresponding cycloSal-phosphotriesters. This method yielded the nucleotides in good yields with a simple work-up. Investigation of the substrate properties of the modified nucleotides towards TmpK showed, that they are very poor substrates for this key enzyme in the bioactivation of AZT.

SATE (aryl) phosphotriester series. I. Synthesis and biological evaluation.

Synthesis and biological activities of several phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and aryl residues derived from L-tyrosine are reported. All compounds showed marked anti-HIV activity in thymidine kinase-deficient CEM cells demonstrating their ability to deliver intracellularly the parent 5'-mononucleotide.

SATE (aryl) phosphotriester series. II. Stability studies and physicochemical parameters.

The stability of phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and various aryl residues derived from L-tyrosine was evaluated in biological media. The results demonstrate that such compounds give rise to intracellular delivery of the parent mononucleotide through esterase and phosphodiesterase hydrolytic steps, successively.

Development of a pharmacodynamic model for HIV treatment with nucleoside reverse transcriptase and protease inhibitors.

There is a need for models useful for predicting the efficacy of agents developed for treating human immunodeficiency virus (HIV) based on information obtained during the drug development process. A pharmacodynamic model that superimposes the pharmacokinetics of anti-HIV nucleoside reverse transcription (RT) and protease inhibitors over a previously published predator-prey model of HIV and CD4 dynamics was developed to address this need. This model was applied to in vitro measurements and patient-derived pharmacokinetics of the unbound antiviral drugs to simulate HIV-1 and CD4 counts versus time and dose. The primary mechanism for nucleoside RT inhibitors was assumed to be competitive inhibition of HIV-1-RT by the active nucleoside triphosphates (NTP). Cellular accumulation and breakdown rates of the NTP were estimated from previous in vivo pharmacokinetic studies. Median inhibition concentrations for the HIV-1 RT enzyme were estimated from previously published cell-free binding studies. The concentration of active protease inhibitor available for binding with HIV-1 protease was assumed equal to the unbound fraction in the plasma. The resulting simulations for mono- and dual nucleoside therapy with zidovudine and lamivudine single dose regimen with the protease inhibitor indinavir, produced similar HIV and CD4 response profiles to those reported in large Phase II and III clinical trials. Based on these findings this pharmacodynamic model can be applied to predict starting doses for a new agent based on simulated biological responses as a function of time for dosage regimens comprising one or two agents. However, the model overestimated the efficacy of highly effective drug combinations where all three agents are combined as in highly active anti-retroviral therapy.

The characteristics of nucleobase transport and metabolism by the perfused sheep choroid plexus.

The uptake of nucleobases was investigated across the basolateral membrane of the sheep choroid plexus perfused in situ. The maximal uptake (U(max)) for hypoxanthine and adenine, was 35.51+/-1.50% and 30.71+/-0.49% and for guanine, thymine and uracil was 12.00+/-0.53%, 13.07+/-0.48% and 12.30+/-0.55%, respectively with a negligible backflux, except for that of thymine (35.11+/-5.37% of the U(max)). HPLC analysis revealed that the purine nucleobase hypoxanthine and the pyrimidine nucleobase thymine can pass intact through the choroid plexus and enter the cerebrospinal fluid CSF so the lack of backflux for hypoxanthine was not a result of metabolic trapping in the cell. Competition studies revealed that hypoxanthine, adenine and thymine shared the same transport system, while guanine and uracil were transported by a separate mechanism and that nucleosides can partially share the same transporter. HPLC analysis of sheep CSF collected in vivo revealed only two nucleobases were present adenine and hypoxanthine; with an R(CSF/Plasma) 0.19+/-0.02 and 3.43+/-0.20, respectively. Xanthine and urate, the final products of purine catabolism, could not be detected in the CSF even in trace amounts. These results suggest that the activity of xanthine oxidase in the brain of the sheep is very low so the metabolic degradation of purines is carried out only as far as hypoxanthine which then accumulates in the CSF. In conclusion, the presence of saturable transport systems for nucleobases at the basolateral membrane of the choroidal epithelium was demonstrated, which could be important for the distribution of the salvageable nucleobases, adenine and hypoxanthine in the central nervous system.

Zidovudine triphosphate and lamivudine triphosphate concentration-response relationships in HIV-infected persons.

OBJECTIVE: To quantitate intracellular concentrations of zidovudine and lamivudine triphosphate and explore relationships with virologic and immunologic responses to antiretroviral therapy. DESIGN: Eight antiretroviral-naive, HIV-infected persons with CD4 T cell counts > 100 x 10(6) cells/l, and HIV RNA in plasma > 5000 copies/ml participating in a prospective, randomized, open-label study of standard dose versus concentration-controlled therapy with zidovudine, lamivudine, and indinavir. METHODS: Peripheral blood mononuclear cells and plasma were collected frequently throughout the study for quantitation of intracellular zidovudine triphosphate and lamivudine triphosphate concentrations, and zidovudine and lamivudine concentrations in plasma. CD4 T cells and HIV RNA in plasma (Roche Amplicor Ultrasensitive Assay) were measured at baseline and every 4 weeks throughout the study. Relationships among intracellular and plasma concentrations, and CD4 T cells and HIV RNA in plasma were investigated with regression analyses. RESULTS: Significant relationships were observed between the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate and the baseline level of CD4 cells. Lamivudine triphosphate concentrations were related in a linear manner to the apparent oral clearance of lamivudine from plasma. A direct linear relationship was found between the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate. The percent change in CD4 cells during therapy and the rate of decline in HIV RNA in plasma were related to the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate. CONCLUSION: These studies into the intracellular clinical pharmacology of nucleoside reverse transcriptase inhibitors illustrate potential clinical implications as determinants of therapeutic success. Moreover, these findings provide several leads and a strong impetus for future investigations with nucleoside reverse transcriptase inhibitors particularly when given in combination and sequentially.

Modifying human thymidylate kinase to potentiate azidothymidine activation.

Based on the knowledge of the crystal structures of yeast and Escherichia coli thymidylate kinases (TmpKs) and the observation that TmpK from E. coli can phosphorylate azidothymidine monophosphate (AZT-MP) much more efficiently than either the yeast or the highly homologous human enzyme, we have engineered yeast and human TmpKs to obtain enzymes that have dramatically improved AZT-MP phosphorylation properties. These modified enzymes have properties that make them attractive candidates for gene therapeutic approaches to potentiating the action of AZT as an inhibitor of human immunodeficiency virus (HIV) replication. In particular, insertion of the lid domain of the bacterial TmpK into the human enzyme results in a pronounced change of the acceptance of AZT-MP such that it is now phosphorylated even faster than TMP.

Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3'-azido-3'-deoxythymidine.

The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase.

Phosphorylated zidovudine concentrations in mononuclear cells in pediatric patients with human immunodeficiency virus infections.

PURPOSE: The efficacy of zidovudine (ZDV) in patients with HIV-1 infection may decrease over time due to its decreased activation. The objectives of this study were to determine ZDV concentrations in plasma, active phosphorylated zidovudine (pZDV) concentrations in mononuclear cells, and assess the markers of immune function and drug toxicity during extended therapy. METHODS: Pediatric patients (aged 3 months to 18 years) with HIV-1-infection were enrolled in the study. For each patient, one blood sample was collected at each of eight routine visits to measure plasma ZDV and ZDV concentrations by a radioimmunoassay. Data including demographic information, immunological markers (CD2+, CD3+, CD4+, CD5+/19+, CD8+, CD16+, CD19+, CD38+/8+ lymphocytes), hematologic function (absolute neutrophil count, white blood cell with differential, hemoglobin, and red blood cell count), concurrent medications, and dosage regimens were obtained. RESULTS: The data from 13 patients were as follows: age: 2-18 years; range of ZDV dose: 76-238 mg/m2, total ZDV daily dosage: 264-720 mg/m2; duration of ZDV therapy prior to study: 1 to 37 months; time in study: 180-394 days; plasma ZDV concentration range: 5-1021 ng/ml; and pZDV concentration range: 0-5.382 pmol/10(6) cells. Both plasma ZDV and intracellular pZDV concentrations had a marked inter- and intrapatient variability. The pZDV concentrations decreased significantly over time in one pediatric patient (p < 0.05), tended to decrease but not significantly in three patients, and no decrease was detected in nine patients due to high variability. In our population, neither immunological nor drug toxicity markers changed over time. CONCLUSIONS: Marked inter- and intrapatient variability in pZDV concentrations was observed. The ability to phosphorylate ZDV, however, did not appear to change significantly in 12 of 13 pediatric patients with HIV-1 infection during the study period of 6-13 months.

Pharmacokinetics and biodistribution of oligonucleotide adsorbed onto poly(isobutylcyanoacrylate) nanoparticles after intravenous administration in mice.

PURPOSE: The goal of this study was to evaluate the ability of nanoparticles to be used as a targeted delivery system for oligonucleotides. METHODS: Pharmacokinetic and tissue distribution were carried out in mice by measuring radioactivity associated to the model oligothymidylate 33P-pdT16 loaded to poly(isobutylcyanoacryate) (PIBCA) nanoparticles. In addition, we have used a TLC linear analyzer to measure quantitatively on a polyacrylamide gel electrophoresis, the amount of non degraded pdT16. RESULTS: Organ distribution study has shown that nanoparticles deliver 33P-pdT16 specifically to the liver reducing its distribution in the kidney and in the bone marrow. Nanoparticles could partially protect pdT16 against degradation in the plasma and in the liver 5 min after administration, whereas free oligonucleotide was totally degraded at the same time. CONCLUSIONS: Nanoparticles protect oligonucleotides in vivo against degradation and deliver them to the liver.

Mononucleoside phosphotriester derivatives with S-acyl-2-thioethyl bioreversible phosphate-protecting groups: intracellular delivery of 3'-azido-2',3'-dideoxythymidine 5'-monophosphate.

The synthesis, in vitro anti-HIV-1 activity, and decomposition pathways of several mononucleoside phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) incorporating a new kind of carboxylate esterase-labile transient phosphate-protecting group, namely, S-acyl-2-thioethyl, are reported. All the described compounds showed marked antiviral activity in thymidine kinase-deficient CEM cells in which AZT was virtually inactive. The results strongly support the hypothesis that such pronucleotides exert their biological effects via intracellular delivery of the 5'-mononucleotide of AZT. This point was corroborated by decomposition studies in cell extracts and culture medium.

Cytotoxicity of 3'-azido-3'-deoxythymidine correlates with 3'-azidothymidine-5'-monophosphate (AZTMP) levels, whereas anti-human immunodeficiency virus (HIV) activity correlates with 3'-azidothymidine-5'-triphosphate (AZTTP) levels in cultured CEM T-lymphoblastoid cells.

Activation of the anti-human immunodeficiency virus (HIV) compound 3'-azido-3'-deoxythymidine (AZT) is dependent on its 5'-phosphorylation by cellular nucleoside and nucleotide kinases. Azidothymidine 5'-triphosphate (AZTTP) is considered to be the metabolite responsible for both the anti-HIV effect of AZT, via inhibition of reverse transcriptase, and cytoxicity by interference with cellular DNA polymerases. During the characterization of AZT metabolism in cultured human T-lymphoblastoid CEM cells, a spontaneously occurring variant cell line, CEM/Ag-1, was found that showed approximately 10-fold resistance to AZT growth inhibition as compared to wild type (wt) cells (EC50 = 2 mM as compared to 350 microM for wt cells). CEM/Ag-1 cells had a 3-fold reduced capacity to accumulate azidothymidine monophosphate (AZTMP) compared to wt cells whereas similar levels of AZTTP were found in both cell lines. The intracellular half-life of AZTMP was approximately 70 min in both wt and CEM/Ag-1 cells. A 3-fold lower specific activity of cytoplasmic thymidine kinase was observed in CEM/Ag-1 extracts as compared to wt. The reduced thymidine kinase activity was not correlated to a decreased level of thymidine kinase mRNA. Syncytium formation of CEM/Ag-1 cells infected with HIV-2 as well as HIV-1 antigen production was inhibited at the same concentrations of AZT (approx. 0.01 microM) as were HIV-1 and HIV-2 infected wt cells. Thus, minor decreases in cellular thymidine kinase levels may markedly affect the cytoxicity of AZT but have no major effect on the antiviral activity of AZT. Our results strongly suggest that AZTMP is responsible for a major part of the growth inhibitor effects, while AZTTP mainly mediates the antiviral activity of AZT.