Abacavir pharmacokinetics during chronic therapy in HIV-1-infected adolescents and young adults.

The pharmacokinetics of abacavir and its metabolites were investigated in 30 human immunodeficiency virus (HIV)-infected adolescents and young adults 13-25 years of age, equally divided into two groups: <18 years of age and >or=18 years of age. All the subjects received the recommended adult dose of 300 mg twice daily. The area under the plasma concentration-time curve (AUC) and half-life of abacavir did not differ significantly between the age groups or by gender or race, and there were only modest associations of age with apparent abacavir clearance and with volume of distribution. There were no significant correlations of carboxylate or glucuronide metabolite levels with age or gender, although glucuronide AUC was higher in Hispanic subjects than in African-American subjects. Zidovudine and lamivudine concentration profiles were also similar in the two age groups. A novel aspect of the study included an assessment of intracellular carbovir, zidovudine, and lamivudine triphosphate levels, and these were found to be similar in the two age-based groups. Overall, these findings suggest that current recommendations relating to adult dosages are appropriate for adolescents and young adults.

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.

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.

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.

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.

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.

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.

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.

Metabolism and in vitro antiretroviral activities of bis(pivaloyloxymethyl) prodrugs of acyclic nucleoside phosphonates.

Bis(pivaloyloxymethyl) [bis(pom)] derivatives of various acyclic nucleoside phosphonates--9-(2-phosphonylmethoxyethyl)adenine (PMEA), 9-(2-phosphonylmethoxypropyl)adenine (PMPA), and 9-(2-phosphonylmethoxypropyl)diaminopurine (PMPDAP)--were found to exhibit 9- to 23-fold greater antiviral activity than their corresponding unmodified compounds. The cytotoxicity of the bis(pom) analogs was also increased by various degrees, thus altering the therapeutic indexes of these compounds. Metabolic studies using [3H]bis(pom)PMEA and [3H]PMEA as model compounds suggested a > 100-fold increase in the cellular uptake of the bis(pom) derivative and formation of active diphosphorylated metabolite. However, the bis(pom) derivatives were chemically unstable and highly susceptible to serum-mediated hydrolysis, factors which limit their potential utility for intracellular drug delivery.

Mechanism of uptake of the phosphonate analog (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) in Vero cells.

The cellular uptake of phosphonylmethoxypropyl cytosine (HPMPC) was characterized to gain insight into the molecular properties that allow this anticytomegalovirus drug to permeate cell membranes. The time course of uptake of HPMPC into Vero cells was linear between 10 and 75 min and proportional to the concentration in the medium from 10(-6) to 10(-2) M. HPMPC uptake was temperature sensitive and the rate of uptake was considerably lower at 27 degrees than at 37 degrees and almost totally inhibited at 4 degrees. In competition studies with naturally occurring nucleosides, nucleotides or the phosphonylmethoxyethyl derivatives, none affected the uptake of HPMPC at concentrations up to 2000-fold molar excess. The uptake of [3H]HPMPC into Vero cells was compared with that of [14C]sucrose, a probe for fluid-phase endocytosis. Kinetics for both compounds were very similar, as were the effects of the microtubule antagonist colchicine and the tumor promoting agent phorbol myristate acetate. Colchicine and the phorbol ester are known to, respectively, inhibit and stimulate endocytosis. It is concluded from these data that HPMPC enters Vero cells by fluid-phase endocytosis and that once internalized it may accumulate in the lysosome. Protonation of the negative charge on the phosphonyl group in HPMPC may allow its diffusion across the lysosome membrane and eventual activation to its putative active diphosphorylated form in the cell cytoplasm.

Metabolic intermediates as potential regulators of glucose-6-phosphatase.

Twenty-five metabolites of glucose, gluconeogenic substrates, and related compounds were examined as potential inhibitors of glucose-6-phosphatase (EC 3.1.3.9) catalytic unit and substrate transport function, using disrupted and intact rat liver microsomes. Inhibitions (competitive) were noted with six. Calculated per cent inhibitions with presumed near-physiologic concentrations of inhibitor and substrate were small. However, when hepatic fructose-1-P concentration is elevated in response to a fructose load, inhibition of glucose-6-phosphatase by fructose-1-P may play a regulatory role, along with fructose-1-P-associated deinhibition of glucokinase, by directing glucose-6-P away from glucose formation and towards glycogen synthesis and glycolysis.