Pharmacokinetics of oral zidovudine administered during labour: a preliminary study.

OBJECTIVES: The aim of this study was to determine whether oral zidovudine (ZDV) given during labour would provide a similar systemic exposure to the established intravenous regimen used to prevent mother-to-child transmission in HIV-infected pregnant women. METHODS: ZDV pharmacokinetic parameters following oral administration during labour were determined in 10 HIV-infected pregnant women in active labour. All subjects were converted to intravenous ZDV prior to delivery. RESULTS: In cohort 1 (n=6), subjects received 300 mg oral ZDV every 3 h for three doses. Oral therapy was well tolerated but plasma ZDV concentrations were substantially lower than previously reported with continuous intravenous therapy. Based on the pharmacokinetic results from cohort 1, women in cohort 2 (n=4) received an initial 600 mg dose followed by two 400 mg doses every 3 h. ZDV area under the curve and concentrations in cohort 2 increased approximately in proportion to the increase in dose but varied 6-7-fold. In both cohorts, ZDV pharmacokinetic parameters suggested erratic absorption. CONCLUSIONS: While ZDV exposure improved with the increased dosing regimen, our sample size was small and larger studies are needed to establish whether oral ZDV administration during labour can consistently provide equivalent exposure to intravenous administration.

Cellular factors for resistance against antiretroviral agents.

Substantial advancements have been made in our understanding of the complex replication cycle of, and immunopathology associated with HIV infection as well as the drugs used to treat the disease. The nucleoside reverse transcriptase inhibitors remain the cornerstones of current antiviral treatment modalities. Unfortunately, their longterm use often leads to adverse reactions and the emergence of virus mutants with decreased susceptibility to therapeutic agents. In addition to viral resistance, prolonged antiviral treatment may affect metabolic changes in the host cells that can diminish the efficacy of the treatment. Thus, both viral and cellular resistance mechanisms must be considered in the context of failing antiviral chemotherapy. This review article concerns the intracellular pharmacology of antiviral nucleoside analogues in human lymphoid cells and the possible impact of a newly identified nucleotide transporter on drug resistance.

Cellular phosphorylation of 2',3'-dideoxyadenosine-5'-monophosphate, a key intermediate in the activation of the antiviral agent DDI, in human peripheral blood mononuclear cells.

2',3'-dideoxyadenosine 5-monophosphate (ddAMP), is a key intermediate in the metabolism of the antiviral agent 2',3'-dideoxyinosine (ddI) to its active triphosphate derivative, 2',3'-dideoxyadenosine-5'-triphosphate (ddATP). The potential role of adenylate kinase in the phosphorylation of ddAMP was studied in human peripheral blood mononuclear cells (PBMC) and a human T cell line, CEMss. Subcellular distribution, sulfhydryl inhibitor, and substrate specificity studies support the hypothesis that the mitochondrial adenylate kinase (AK2) is a major route of cellular activation of these compounds in human lymphocytes.

Single-dose pharmacokinetics and safety of the oral antiviral compound adefovir dipivoxil in children infected with human immunodeficiency virus type 1. The Pediatrics AIDS Clinical Trials Group.

The acyclic phosphonate analog adefovir is a potent inhibitor of retroviruses, including human immunodeficiency virus (HIV) type 1, and, unlike some antiviral nucleosides, does not require the initial phosphorylation step for its activity. Two oral dosages of the adefovir prodrug adefovir dipivoxil were evaluated in a phase I study with children with HIV infection. A total of 14 patients were stratified into age groups ranging from 6 months to 18 years of age. Eight patients received 1.5 mg of adefovir dipivoxil per kg of body weight, and six patients received 3.0 mg of adefovir dipivoxil per kg. Serum samples were obtained at intervals during the 8 h postdosing and were analyzed for adefovir concentrations. Patients were monitored for adverse effects. All samples collected resulted in quantifiable levels of adefovir (lower limit of quantitation, 25 ng/ml) from each patient. The areas under the concentration-versus-time curves (AUCs) were similar (P = 0.85) for the 1.5- and 3.0-mg/kg doses, while the apparent oral clearance (CL/F) was significantly higher (P = 0.05) for the 3-mg/kg dose. Pharmacokinetic parameters differed by patient age. In comparing those children older and younger than the median age of 5.1 years, AUC (P = 0.03), maximum concentration of drug in serum (P = 0.004), and the concentration at 8 h postdosing (P = 0.02) were significantly lower for the younger children. There were no significant differences for apparent volume of distribution and CL/F normalized to body surface area, but there was a suggestive difference in half-life (P = 0.07) among the subjects in the older and younger age groups. No significant adverse events were encountered. These data provide the basis for a multidose phase II study of adefovir dipivoxil in HIV-infected infants and children.

Systemic pharmacokinetics and cellular pharmacology of zidovudine in human immunodeficiency virus type 1-infected women and newborn infants.

Systemic and intracellular pharmacokinetics of zidovudine were determined for 28 human immunodeficiency virus type 1-infected pregnant women and their newborn infants. Plasma zidovudine and intracellular zidovudine monophosphate and triphosphate concentrations were determined in serial maternal samples and cord blood at delivery. Higher levels of cord blood zidovudine were associated with lower maternal zidovudine clearance and longer infusion times. Median levels of zidovudine monophosphate and triphosphate in maternal (1556 and 67 fmol/106 cells) and cord (1464 and 70 fmol/106 cells) blood were similar but highly variable. Intersubject pharmacokinetic variability for zidovudine is substantial, but intravenous therapy provides plasma concentrations and intracellular zidovudine triphosphate levels consistent with high antiviral activity. The substantial amount of intracellular zidovudine triphosphate in cord blood provides an explanation for the clinical success of zidovudine in reducing vertical transmission. Studies of simpler oral regimens of zidovudine can now be evaluated regarding the ability to achieve these pharmacologic end points associated with highly effective parenteral therapy.

MRP4: A previously unidentified factor in resistance to nucleoside-based antiviral drugs.

Dideoxynucleosides, which are potent inhibitors of HIV reverse transcriptase and other viral DNA polymerases, are a common component of highly active anti-retroviral therapy (HAART) (ref. 1). Six reverse transcriptase inhibitors have been approved for human use: azidothymidine; 2'3'-dideoxycytidine; 2'3'-dideoxyinosine; 2', 3'-didehydro-3'deoxythymidine; 2',3'-dideoxy-3'-thiacytidine; and 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-++ +metha nol. Although drug-resistant HIV strains resulting from genetic mutation have emerged in patients treated with HAART (ref. 1), some patients show signs of drug resistance in the absence of drug-resistant viruses. In our study of alternative or additional mechanisms of resistance operating during antiviral therapy, overexpression and amplification of the MRP4 gene correlated with ATP-dependent efflux of PMEA (9-(2-phosphonylmethoxyethyl)adenine) and azidothymidine monophosphate from cells and, thus, with resistance to these drugs. Overexpression of MRP4 mRNA and MRP4 protein severely impaired the antiviral efficacy of PMEA, azidothymidine and other nucleoside analogs. Increased resistance to PMEA and amplification of the MRP4 gene correlated with enhanced drug efflux; transfer of chromosome 13 containing the amplified MRP4 gene conferred resistance to PMEA. MRP4 is the first transporter, to our knowledge, directly linked to the efflux of nucleoside monophosphate analogs from mammalian cells.

Human immunodeficiency virus protease inhibitors serve as substrates for multidrug transporter proteins MDR1 and MRP1 but retain antiviral efficacy in cell lines expressing these transporters.

The human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs)-saquinavir, ritonavir, nelfinavir, and indinavir-interact with the ABC-type multidrug transporter proteins MDR1 and MRP1 in CEM T-lymphocytic cell lines. Calcein fluorescence was significantly enhanced in MDR1(+) CEM/VBL100 and MRP1(+) CEM/VM-1-5 cells incubated in the presence of various HIV PIs and calcein acetoxymethyl ester. HIV PIs also enhanced the cytotoxic activity of doxorubicin, a known substrate for MDR1 and MRP1, in both VBL100 and VM-1-5 CEM lines. Saquinavir, ritonavir, and nelfinavir enhanced doxorubicin toxicity in CEM/VBL100 cells by approximately three- to sevenfold. Saquinavir and ritonavir also enhanced doxorubicin toxicity in CEM/VM-1-5 cells. HIV-1 replication was effectively inhibited by the various PIs in all of the cell lines, and the 90% inhibitory concentration for a given compound was comparable between the different cell types. Therefore, overexpression of MDR1 or MRP1 by T lymphocytes is not likely to limit the antiviral efficacy of HIV PI therapy.

Development of a new cartridge radioimmunoassay for determination of intracellular levels of lamivudine triphosphate in the peripheral blood mononuclear cells of human immunodeficiency virus-infected patients.

A new sensitive method for the measurement of lamivudine triphosphate (3TC-TP), the active intracellular metabolite of lamivudine in human cells in vivo, has been established. The procedure involves rapid separation of 3TC-TP by using Sep-Pak cartridges, dephosphorylation to 3TC by using acid phosphatase, and measurement by radioimmunoassay using a newly developed anti-3TC serum. The radioimmunoassay had errors of less than 21% and a cross-reactivity of less than 0.016% with a wide variety of other nucleoside analogs. The limit of quantitation of the assay for intracellular 3TC-TP was 0.195 ng/ml (0.212 pmol/10(6) cells), and a cell sample of only 4 million cells was ample for the assay. This procedure, combined with our previously developed method for measuring zidovudine (ZDV) metabolite levels, proved capable of measuring 3TC-TP, ZDV monophosphate (ZDV-MP) and ZDV triphosphate (ZDV-TP) in human immunodeficiency virus (HIV)-infected subjects treated with combination 3TC and ZDV therapy. In seven subjects, intracellular 3TC-TP levels ranged from 2.21 to 7.29 pmol/10(6) cells, while intracellular ZDV-MP and ZDV-TP levels ranged from <0. 01 to 1.76 and 0.01 to 0.07 pmol/10(6) cells, respectively. Concentrations of 3TC in plasma determined in these subjects ranged from 0.34 to 9.40 microM, which was about fivefold higher than ZDV levels in plasma of 0.04 to 1.4 microM. This is the first study to determine the intracellular levels of the active metabolites in HIV-infected subjects treated with this combination. These methods should prove very useful for in vivo pharmacodynamic studies of combination therapy.

Antiviral activities of 9-R-2-phosphonomethoxypropyl adenine (PMPA) and bis(isopropyloxymethylcarbonyl)PMPA against various drug-resistant human immunodeficiency virus strains.

9-R-2-Phosphonomethoxypropyl adenine (PMPA) is an acyclic nucleoside phosphonate analog that has demonstrated efficacy against human immunodeficiency virus (HIV). We recently described the synthesis, metabolism, and biological activities of bis(isopropyloxymethylcarbonyl)PMPA [bis(poc)PMPA] as an orally bioavailable prodrug for PMPA. Among a large panel of drug-resistant HIV type 1 variants, only the K65R virus was resistant to PMPA. K65R virus also showed reduced susceptibility to bis(poc)PMPA, although the prodrug could still inhibit these viruses at submicromolar, nontoxic concentrations. Among a panel of seven primary clinical isolates from patients with diverse treatment histories, only one isolate showed reduced susceptibility to PMPA and was found to carry three mutations (M41L, T69N, R73K) in its reverse transcriptase catalytic domain.

Anti-human immunodeficiency virus activity and cellular metabolism of a potential prodrug of the acyclic nucleoside phosphonate 9-R-(2-phosphonomethoxypropyl)adenine (PMPA), Bis(isopropyloxymethylcarbonyl)PMPA.

Bis(isopropyloxymethylcarbonyl) 9-R-(2-phosphonomethoxypropyl)adenine [bis(POC)PMPA] has been identified as a novel prodrug of PMPA. The anti-human immunodeficiency virus activity of bis(POC)PMPA was >100-fold greater than that of PMPA in both an established T-cell line and primary peripheral blood lymphocytes. This improved efficacy was shown to be due to a rapid intracellular uptake of the prodrug resulting in an increased intracellular accumulation of PMPA diphosphate (PMPApp), the pharmacologically active metabolite. PMPApp levels in bis(POC)PMPA-treated cells exceeded by >1,000-fold the levels seen in cells treated with unmodified PMPA in both resting and activated peripheral blood lymphocytes. Significant differences in the intracellular catabolism of PMPA metabolites were noted between the resting and activated lymphocytes. The half-life for the disappearance of PMPApp, derived from either bis(POC)PMPA or PMPA, was 12 to 15 h in the activated lymphocytes and 33 to 50 h in the resting lymphocytes. This long persistence of PMPApp, particularly in resting lymphocytes, may be unique to the nucleoside phosphonate analogs and indicates that effective levels of the active metabolite can be achieved and maintained with relatively infrequent administration of the parent drug.

Intracellular metabolism and action of acyclic nucleoside phosphonates on DNA replication.

9-(2-phosphonylmethoxyethyl)guanine (PMEG) is an acyclic nucleoside phosphonate derivative that has demonstrated significant anticancer activity in a number of in vitro and in vivo animal model systems. In this study, we compared the cellular metabolism of PMEG and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a clinically active anti-HIV and antihepatitis agent, and the inhibitory activities of their putative active diphosphate derivatives, PMEGpp and PMEApp, respectively, toward human cellular DNA polymerases. PMEG was significantly more cytotoxic than PMEA against a panel of human leukemic cells. The diphosphate derivatives were the major metabolites formed in cells on both these agents, with PMEGpp reaching cellular concentration approximately 4-fold higher than that achieved for PMEApp. These differences in cellular accumulation of the diphosphate derivatives were not, however, sufficient to account for the 30-fold difference in cytotoxicity between the two analogs. PMEGpp was also at least a 7-fold more effective inhibitor of in vitro simian vacuolating virus 40 DNA replication system than that of PMEApp (IC50 = 4.6 microM). Studies with a defined primed DNA template showed that PMEGpp was a potent inhibitor of both human polymerases alpha and delta, two key enzymes involved in cellular DNA replication, whereas PMEApp inhibited these enzymes relatively poorly. From these studies, we can conclude that the factors that contribute to the enhanced antileukemic activity of PMEG derives both from its increased anabolic phosphorylation and the increased potency of the diphosphate derivative to target the cellular replicative DNA polymerases.

In vitro induction of human immunodeficiency virus type 1 variants resistant to 2'-beta-Fluoro-2',3'-dideoxyadenosine.

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA) is an acid-stable purine dideoxynucleoside analog active against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains in vitro. F-ddA is presently undergoing a phase I clinical trial at the National Cancer Institute. We induced HIV-1 variants resistant to F-ddA by exposing wild-type HIV-1 (HIV-1LAI) to increasing concentrations of F-ddA in vitro. After 18 passages, the virus was fourfold less sensitive to F-ddA than HIV-1LAI. Sequence analyses of the passage 18 virus revealed changes in three amino acids in the reverse transcriptase (RT)-encoding region of the pol gene: P to S at codon 119 (P119S; present in 3 of 13 and 28 of 28 molecular clones before and after F-ddA exposure, respectively), V179D (0 of 13 and 9 of 28, respectively), and L214F (9 of 13 and 28 of 28, respectively). Drug sensitivity assays using recombinant infectious clones confirmed that P119S was directly responsible for the reduced sensitivity of HIV-1 to F-ddA. Various infectious clones with single or multiple amino acid substitutions conferring viral resistance against nucleoside RT inhibitors, including HIV-1 variants with multi-dideoxynucleoside resistance, were generally sensitive to F-ddA. The moderate level of resistance of HIV-1 to F-ddA, together with the lack of conferment of significant cross-resistance by the F-ddA-associated amino acid substitutions, warrants further investigation of F-ddA as a potential antiviral agent for use in treatment of HIV-1 infection.

(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) inhibits HIV-1 replication in epithelial cells, but not T-lymphocytes.

PMEA [9-(2-phosphonylmethoxyethyl)adenine] inhibited both HSV-1 and HIV-1 replication in MT-2 and HeLa-CD4 cells. (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) inhibited both these viruses in the epithelioid HeLa-CD4 cells, but did not inhibit either virus in the T-lymphocytic MT-2 cells. PMEA and HPMPC are metabolized to their diphosphorylated forms within cells, which then inhibit viral polymerases. We therefore compared the metabolism of PMEA and HPMPC in MT-2 and HeLa CD4 cells. PMEApp formation was efficient in both the cell types, whereas HPMPCpp levels were approximately 3-10 fold lower in MT-2 cells, compared to HeLa-CD4 cells. These results indicate that HPMPC can inhibt HIV replication in the appropriate cell types, and show that differences in their metabolism cannot account entirely for the lack of antiviral efficacy of HPMPC in MT-2 cells.

Quantitation of intracellular zidovudine phosphates by use of combined cartridge-radioimmunoassay methodology.

This report describes the development of a potentially clinical method to measure the cellular metabolites of zidovudine (ZDV) in patients receiving the drug. This new method combines the use of Sep-Pak cartridges to separate ZDV phosphates with radioimmunoassaying to quantitate ZDV. The detection limit is 0.02 pmol/10(6) cells, and this assay can measure a wide range of intracellular drug concentrations. The use of the cartridge-radioimmunoassay methodology should prove very useful for in vivo cellular pharmacokinetic studies of ZDV.

A systemic and cellular model for zidovudine plasma concentrations and intracellular phosphorylation in patients.

In a pharmacokinetic model for the systemic and cellular disposition of zidovudine in patients, serial measurements of plasma zidovudine and intracellular metabolites were used to simultaneously characterize systemic pharmacokinetics and intracellular phosphorylation in 6 human immunodeficiency virus-infected patients. First-order processes are sufficient to describe zidovudine monophosphate kinetics in peripheral blood mononuclear cells (PBMC), and the pharmacokinetic model provided reliable parameter estimates for each subject. The amount of zidovudine monophosphate in PBMC was inversely correlated with plasma zidovudine concentrations, and patients with higher systemic clearance had less intracellular zidovudine monophosphate. Zidovudine triphosphate values were measurable but not different at each time point. Lower lymphocyte counts were associated with higher intracellular zidovudine monophosphate but lower zidovudine triphosphate. The pharmacokinetic model proposed provides a quantitative link between systemic zidovudine concentrations and zidovudine monophosphate in PBMC from patients that will be useful in evaluating alternative therapeutic strategies.

Metabolic pathways for activation of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine in human lymphoid cells.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA), the acyclic phosphonate analog of adenine monophosphate, is a promising antiviral drug with activity against herpesviruses, Epstein-Barr virus, and retroviruses, including the human immunodeficiency virus. In order to be active, it must be converted to the diphosphate derivative, the putative inhibitor of viral DNA polymerases. The metabolic pathway responsible for activation of PMEA is unclear. The metabolism of PMEA was investigated in human T-lymphoid cells (CEMss) and a PMEA-resistant subline (CEMss(r-1)) with a partial deficiency in adenylate kinase activity. Experiments with [3H]PMEA showed that extracts of CEMss phosphorylated PMEA to its mono- and diphosphate in the presence of ATP as the phosphate donor. No other nucleotides or 5-phosphoribosyl pyrophosphate displayed appreciable activity as a phosphate donor. Subcellular fractionation experiments showed that CEMss cells contained two nucleotide kinase activities, one in mitochondria and one in the cytosol, which phosphorylated PMEA. The PMEA-resistant CEMss mutant proved to have a deficiency in the mitochondrial adenylate kinase activity, indicating that this enzyme was important in the phosphorylation of PMEA. Other effective antiviral purine phosphonate derivatives of PMEA showed a profile of phosphorylating activity similar to that of PMEA. By comparison, phosphorylation of the pyrimidine analog (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine proceeded by an enzyme present in the cytosol. We conclude from these studies that adenylate kinase which has been localized in the intermembrane space of mitochondria is the major route for PMEA phosphorylation in CEMss cells but that another hitherto unidentified enzyme(s) present in the cytosol may contribute to the anabolism of the phosphonates.

Metabolic diversity and antiviral activities of acyclic nucleoside phosphonates.

The acyclic nucleoside phosphonates (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) inhibited herpes simplex virus-1 replication in Vero cells, and the IC50 values ranged from 4 microM (for HPMPC and HPMPA) to 40 microM (for PMEA). Pretreatment of cells with HPMPC for 12-24 hr induced an effective antiviral state, and the cells maintained this antiviral state for > 7 days. In contrast, much larger amounts (approximately 2.5-5 x IC50 doses) of PMEA or HPMPA were required to establish an antiviral state, which lasted for only approximately 24 or 72 hr, respectively. A 12-hr treatment of the cells with the phosphonates was required for the establishment of optimal antiviral activity; surprisingly, longer durations of exposure to PMEA (but not HPMPA or HPMPC) resulted in diminished antiviral effect. We investigated the metabolism of PMEA and HPMPC to determine the cellular basis for these differences. The cellular uptake of HPMPC was approximately 8-fold greater than that of PMEA. The levels of the PMEA metabolites PMEA monophosphate and PMEA diphosphate increased for approximately 12 hr and plateaued thereafter. PMEA and its metabolites were cleared from the cells with a half-life of 4.9 hr. In contrast, the HPMPC metabolites HPMPC monophosphate (HPMPCp) and HPMPC diphosphate (HPMPCpp) accumulated throughout the 24-hr study period and, at equimolar drug concentrations (25 microM), reached intracellular levels approximately 2-3-fold greater than those of the PMEA metabolites. HPMPC also differed from PMEA in its capacity to generate a phosphodiester metabolite (HMPCp-choline), which was a predominant metabolite in HPMPC-treated cells. In addition, the rates of disappearance of intracellular metabolites of the two drugs were significantly different. Thus, the decay of HPMPCpp was quite slow and biphasic (t1/2 = 24 and 65 hr) and that of HMPCp-choline was monophasic (t1/2 = 87 hr). Together, these factors can explain the differing antiviral potencies seen with PMEA and HPMPC. The possible role of the choline adduct in the expression of antiviral activity of the drug remains to be elucidated, but the adduct may serve as an intracellular store for the long term maintenance of active HPMPCpp in cells. The results also highlight the extent of diversity in the cellular pharmacology and antiviral activities of the acyclic nucleoside phosphonates.

A human T lymphoid cell variant resistant to the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine shows a unique combination of a phosphorylation defect and increased efflux of the agent.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral agent with activity against herpes viruses and retroviruses, including human immunodeficiency virus, but its metabolism and mechanism of action remain unclear. We have isolated a human T lymphoid cell line (CEMr-1) that is resistant to the antiproliferative effects of PMEA. The antiviral effects of PMEA against human immunodeficiency virus-1 infection were also greatly reduced in CEMr-1 cells, compared with the parental cells. This mutant showed cross-resistance to the related acyclic nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)diaminopurine and 9-(2-phosphonylmethoxyethyl)guanine and the lipophilic prodrug bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine-( bispome-PMEA), as well as partial resistance to the purine nucleosides 2-chlorodeoxyadenosine, 2-fluro-9-beta-D-arabinosylfuranosyladenine, and adenosine, but did not show resistance to 2'-deoxyadenosine or 9-beta-D-arabinosylfuranosyladenine. We compared the uptake and metabolism of [3H]PMEA and [3H]-bispom-PMEA in the mutant and parental cells. The analysis of radioactive products by high pressure liquid chromatography revealed marked alterations in the ability of the mutant cell line to accumulate PMEA and its anabolites, compared with the parental cells. Accumulation of PMEA, PMEA monophosphate, and PMEA bisphosphate (major metabolites formed with either PMEA or bispom-PMEA) decreased by 50, 95, and 97%, respectively. Compared with the parental cells, the variant cells showed a approximately 7-fold increase in the rate of efflux of PMEA and a 2-fold decrease in the activity of adenylate kinase. In contrast, other enzymes of nucleotide metabolism, such as adenosine kinase, deoxycytidine kinase, and 5-phosphoribosyl-1-pyrophosphate synthetase, showed no significant change in the two cell lines. Overall, these results suggest that the mutation in this resistant cell line is of a novel type, involving an alteration in the cellular efflux of PMEA as the major basis for the resistant phenotype.

Susceptibilities of zidovudine-resistant variants of human immunodeficiency virus type 1 to inhibition by acyclic nucleoside phosphonates.

The acyclic purine nucleoside phosphonates, a newly described class of broad-spectrum antiviral agents, effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro and in animal AIDS models. 9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is currently being evaluated in clinical trials in patients with AIDS. In this study, we investigated the efficacy of PMEA and a related analog, 9-(2-phosphonylmethoxypropyl)diaminopurine (PMPDAP), against HIV-1 isolates exhibiting various degrees of resistance to zidovudine (azidothymidine [AZT]). HIV isolates highly (approximately 50 to 200-fold) resistant to AZT were found to be about two- to eightfold less susceptible to PMEA. A comparable degree of cross-resistance to PMPDAP, a structurally related analog of PMEA, was also observed. However, the 50% effective dose values of PMEA or PMPDAP against a panel of HIV isolates showing intermediate levels (approximately 8 to 25-fold) of AZT resistance was indistinguishable from the 50% effective dose values of PMEA (0.7 to 1.7 versus 2 microM) or PMPDAP (0.4 to 1.4 versus 0.8 to 1 microM) against HIV isolates from patients who had not previously used AZT. In addition, we were unable to generate PMEA- (or PMPDAP)-resistant HIV-1 variants by > 30 serial passages of the virus in the presence of increasing concentrations of PMEA. Careful analysis of HIV-1 isolates from patients previously treated with AZT for cross-resistance to PMEA are needed to evaluate the significance of these observations.

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

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