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Zidovudine (ZDV) is sequentially phosphorylated by host intracellular enzymes to its active form, ZDV triphosphate (ZDVTP) (4). ZDVTP competes with endogenous dTTP for incorporation into viral DNA, thus inhibiting viral DNA synthesis. Following incorporation of ZDVTP, the azido (N₃) group results in chain termination (9). As the effect of ZDV is dependent on the rate and extent of intracellular activation, concentrations in plasma are of limited value in predicting efficacy or toxicity (1, 10). Intracellular phosphorylation studies performed with human immunodeficiency virus (HIV)-infected patients and healthy volunteers have been performed mostly with Caucasian patients (1, 7, 10-12).

ZDV has a major role in resource-poor countries in the prevention of vertical transmission of HIV. A recent report from Thailand showed that ZDV administered orally during late pregnancy and delivery reduced HIV transmission from infected mothers to infants by 50% compared with a placebo group (16). However, no studies to date have investigated the metabolism of ZDV in an Asian population. There are known ethnic differences in drug metabolism (6, 17), and thus any differences in expression of enzymes involved in ZDV metabolism (glucuronyltransferase, CYP 3A, or, more importantly, cellular kinases [14]) would alter plasma and intracellular pharmacokinetics. In this study, we have examined ZDV pharmacokinetics with ZDV-naive, HIV-infected Thai patients and healthy volunteers.

Twenty antiretroviral drug-naive HIV-positive patients, 3 females and 17 males, aged 21 to 54 years (median age, 26 years), and 7 male volunteers, aged 24 to 30 years (median age, 28 years), participated in this study. The patients had a median body weight of 53 kg (range, 42.5 to 73.0 kg). HIV-positive patients were at different disease stages (A1, n = 2; A2, n = 4; B1, n = 1; B2, n = 4; B3, n = 5; and C3, n = 4) (Centers for Disease Control and Prevention 1993 classification system), but all were stable at the time of sampling. Median CD4 cell counts were 246 cells/mm³ (range, 26 to 810 cells/mm³). All patients and volunteers had normal renal function and a hemoglobin value of more than 10 g/dl at the time of the study. Two patients were taking co-trimoxazole, one patient was receiving 600 mg of rifampin and 300 mg of isoniazid daily for tuberculosis, and another patient was receiving 300 mg of phenytoin (Dilantin) therapy daily for epilepsy. The serum alanine and aspartate transaminases were within the normal limit except in one patient, whose values were three times higher than normal. Written informed consent was obtained from the subjects, and the study was approved by the ethics committee of Mahidol University, Thailand.

After overnight fasting by the study subjects, blood was sampled for baseline drug concentrations and CD4 cell count. Further samples (20 ml) were collected by venipuncture at 1, 2, 4, and 6 h after supervised ingestion of a single 300-mg dose of ZDV.

After separation of plasma, peripheral blood mononuclear cells (PBMCs) were isolated by density cushion centrifugation, washed, and quantified using a hemocytometer. PBMCs (5 × 10⁶ cells) were extracted with 60% methanol prior to separation of ZDV and its phosphate metabolites by high-performance liquid chromatography, as described previously (1). In brief, samples were eluted on a Partisil 10-SAX anion-exchange column (4.6 by 250 mm) using a mobile phase of ammonium dihydrogen phosphate buffer-methanol run as a gradient over 40 min. Fractions eluted from the column corresponding to ZDV, ZDV monophosphate (ZDVMP), ZDV diphosphate (ZDVDP), and ZDVTP were collected. Collection periods were determined from the retention times of authentic phosphorylated anabolites of ZDV (13, 15). Phosphorylated fractions were hydrolyzed by overnight incubation with acid phosphatase (40 U/ml). Samples were cleaned using C₁₈ Sep-Pak cartridges, and ZDV concentrations were quantified by a commercially available radioimmunoassay kit (2).

ZDV concentrations (nanograms per milliliter) obtained from the radioimmunoassay were converted to intracellular concentrations (picomoles per 10⁶ cells) by correcting for sample volume and cell number. The lower limit of detection of this assay was 0.2 ng/ml, or 0.01 pmol/10⁶ cells. Validation studies using this assay have been described previously (2, 11).

Concentrations in plasma were determined directly from the data. The area under the ZDV concentration time curve from 0 to 6 h (AUC_0-6) was determined by the log-linear trapezoidal rule using the TOPFIT computer program (Gustav Fischer Verlag, Stuttgart, Germany). Correlations between levels of total ZDV phosphates and plasma ZDV or CD4 cell count (in HIV-positive individuals) were assessed by simple linear regression. The Mann-Whitney U test was used to assess differences in intracellular ZDV phosphate metabolites between patients and volunteers.

Due to a problem with high-performance liquid chromatography separation, samples from one healthy volunteer were not available for further analysis. Considerable variability was associated with the measurement of intracellular phosphates (Table 1). Occasionally an intracellular anabolite was below the limit of quantification (0.01 pmol/10⁶ cells). The number of ZDVMP, ZDVDP, and ZDVTP AUC_0-6 values that were below this limit was 7 out of a total of 78. The plasma ZDV levels at each time point for HIV-positive patients (n = 20) are shown in Fig. 1a. The highest concentration in plasma was found at the 1-h time point. The AUC_0-6 was 11.09 µmol/liter · h (range, 3.45 to 38.95 µmol/liter · h), which was not significantly different from that in healthy volunteers (17.98 µmol/liter · h; range, 7.15 to 26.18 µmol/liter · h).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   (a) Concentration of ZDV in plasma; (b) ZDVMP, ZDVDP, and ZDVTP in PBMCs from HIV-infected Thai patients after oral administration of 300 mg of ZDV. Values are expressed as the means ± the standard errors of the means.

The intracellular concentrations of ZDVMP, ZDVDP, and ZDVTP in HIV-positive patients (n = 20) over time are shown in Fig. 1b. Most of the intracellular phosphate was present as ZDVMP, with its highest concentration at the 1-h time point. Concentrations of ZDVDP and ZDVTP were similar between 1 and 6 h (Fig. 1b). Although the intracellular AUC_0-6 of ZDVDP and ZDVTP was similar for the two groups (Table 1), the ZDVMP AUC_0-6 was significantly greater in HIV patients than in healthy volunteers. There was a weak but significant correlation between ZDV plasma AUC_0-6 and ZDVMP AUC_0-6 (r² = 0.243; P = 0.027) but no relationship between ZDV plasma AUC_0-6 and active anabolite ZDVTP AUC_0-6 (r² = 0.080; P = 0.228).

Previous pharmacokinetic studies with the nucleoside analogues have been performed mostly in developed countries for Caucasian patients. However, as these drugs are being used for some patients in emerging or developing countries with non-Caucasian populations, it is important to know if the intracellular activation is similar in such populations.

The major metabolite in PBMCs from HIV-infected Thais was ZDVMP, with smaller amounts of ZDVDP and ZDVTP (Table 1). This indicates that the enzyme thymidylate kinase (responsible for conversion of ZDVMP to ZDVTP) is the rate-limiting step of ZDV activation. This also explains the lack of a relationship between ZDV plasma AUC_0-6 and ZDVTP AUC_0-6 (r² = 0.080; P = 0.228). In healthy volunteers, no anabolite predominated. The increase in ZDVMP may represent an increase in the activity of thymidine kinase and/or a reduction in the degradation of intracellular phosphates by phosphatases in HIV patients. This is in contrast to in vitro studies (5) in which PBMCs stimulated in culture with the mitogen phytohemagglutinin had less thymidine kinase activity. A possible explanation for this difference is that although PBMCs from HIV patients have greater thymidine kinase activity in vivo, following culture their responsiveness to mitogens such as phytohemagglutinin is less (3).

Interpatient variability was marked, and in both groups there were some subjects for whom, at individual time points, intracellular concentrations of ZDV phosphates could not be detected. To reduce the limit of quantification of intracellular nucleoside triphosphates, a more sensitive assay is required, and such an assay has now been developed (8).

Overall, the intracellular pharmacokinetics for HIV-positive Thai patients were comparable to those described previously for Caucasians (1, 10-12). Higher intracellular levels of ZDVMP in HIV-positive Thais than in seronegative Thai subjects are in agreement with the results of similar studies with Caucasians. Secondly, there were no significant differences in ZDVDP and ZDVTP levels in seronegative and HIV-seropositive Thai subjects, in accordance with an earlier study with Caucasians (1).

The levels of ZDVMP in Thai HIV-positive patients were lower than those reported by Barry et al. (1), who also showed significantly higher ZDVMP concentrations in Caucasian patients than volunteers. It is possible that the higher levels of phosphates seen in the study of Barry et al. may be related to the lower CD4 cell counts in that study (mean, 155 versus 298 cells/mm³). It has been previously demonstrated that there are differences in phosphorylation in patients with widely different CD4 cell counts and disease states (1, 10, 11).

In conclusion, in agreement with previously published data for Caucasians, there was a significant difference in the levels of intracellular phosphates in Thai healthy volunteers and HIV-positive patients, which is due solely to a difference in ZDVMP. Overall there were no marked differences between Thai and Caucasian subjects.