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Antimicrobial Agents and Chemotherapy, July 2008, p. 2555-2563, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.01130-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

High Levels of Zidovudine (AZT) and Its Intracellular Phosphate Metabolites in AZT- and AZT-Lamivudine-Treated Newborns of Human Immunodeficiency Virus-Infected Mothers{triangledown}

Lucie Durand-Gasselin,1 Alain Pruvost,1 Axelle Dehée,2 Genevieve Vaudre,3 Marie-Dominique Tabone,3 Jacques Grassi,4 Guy Leverger,3 Antoine Garbarg-Chenon,2 Henri Bénech,1* and Catherine Dollfus3

CEA, iBiTecS, Service de Pharmacologie et d'Immunoanalyse, Gif sur Yvette F-91191, France,1 AP-HP, Hôpital Armand Trousseau, Service de Virologie, Paris F-75012, France, and Université Pierre et Marie Curie-Paris 6, EA 3500, Paris F-75012, France,2 AP-HP, Hôpital Armand Trousseau, Service d'Hématologie et d'Oncologie Pédiatrique, Paris F-75012, France,3 CEA, Direction des Sciences du Vivant, CEA/Saclay, 91191 Gif-sur-Yvette Cedex, France4

Received 27 August 2007/ Returned for modification 5 October 2007/ Accepted 11 April 2008


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ABSTRACT
 
Newborns from human immunodeficiency virus-infected mothers are given antiretroviral prophylaxis against mother-to-child transmission, including predominantly nucleoside reverse transcriptase inhibitors. Pharmacological monitoring of these drugs in newborns has so far been limited to plasma and cord blood. In this study, samples from newborns (up to 45 days old) treated with zidovudine (AZT) alone (n = 29) or in combination with lamivudine (3TC) (n = 20) were analyzed for both intracellular concentrations of phosphate metabolites in peripheral blood mononuclear cells and levels of parent drugs in plasma. Plasma AZT and intracellular AZT-monophosphate and AZT-triphosphate (TP) concentrations were significantly higher during the first 15 days of life (199 versus 52.7 ng/ml [P < 0.0001], 732 versus 282 fmol/106 cells [P < 0.0001], and 170 versus 65.1 fmol/106 cells [P < 0.0001], respectively) and then became comparable to those of adults. No difference in intracellular AZT metabolite concentrations was found when AZT- and AZT-3TC-treated groups were compared. Plasma 3TC levels (lower limit of quantification [LLOQ], 1,157 ng/ml; median, 412.5 ng/ml) were not associated with the newborn's age, gender, or weight. Intracellular 3TC-TP concentrations (LLOQ, 40.4 pmol/106 cells; median, 18.9 pmol/106 cells) determined for newborns receiving the AZT-3TC combination were associated with neither the age nor weight of the newborns. Concentrations in females were significantly higher (1.8-fold [P = 0.0415]) than those in males. Unexpectedly, newborns on AZT monotherapy whose mothers' treatment included 3TC displayed residual plasma 3TC and intracellular 3TC-TP levels up to 1 week after birth.


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INTRODUCTION
 
Zidovudine (AZT), a nucleoside reverse transcriptase inhibitor (NRTI), is the first antiretroviral drug that has proven effective in preventing vertical transmission of human immunodeficiency virus (HIV), thus reducing mother-to-child HIV transmission (MTCT) from 25% to 8% in the absence of breastfeeding (16). Today, AZT is recommended for newborns of HIV-infected mothers as a three-part regimen (antenatal, intrapartum, and postpartum). The use of combined therapy, mostly including AZT, further contributed to decreases in MTCT rates to less than 1% (17, 24, 37, 50). In newborns, AZT alone or in association with lamivudine (3TC) is the most commonly used regimen in Western countries. Short-term toxicity related to MTCT prophylaxis in newborns appears to be limited mostly to mild hematological disturbances. Indeed, biological abnormalities on blood cell counts and asymptomatic hyperlactatemia are frequent, affecting one-third to one-half of uninfected infants at various degrees of severity (30, 49). However, long-lasting effects on hematopoiesis up to 18 months after birth (36) and neutrophil cell counts over the first 8 years of life (23) were observed in a few patients. In addition, neurological abnormalities have been found in a very limited proportion of children (<0.5%) (4, 9). Concerns regarding long-term mitochondrial toxicity in these infants have been raised, and mitochondrial DNA depletion was assessed in 2-year-old infants exposed perinatally to NRTI (4, 10, 43). However, other reports of large populations of patients did not confirm these observations of long-term toxicity (18, 22, 42). This discrepancy could be partially explained by the differences in methods of identification of mitochondrial deficiency (11). Prospective follow-up and long-term surveillance of children exposed to antiretroviral agents, as well as drug evaluation in newborns, are therefore needed to ensure efficient and fully safe prophylaxis.

Since drug metabolism and elimination in newborns differ from those in infants and adults, the safety and pharmacokinetics of antiretrovirals administered to newborns must be investigated as independent issues.

The pharmacological behaviors of AZT and 3TC were previously partially addressed using data for plasma from newborns (12, 39). Plasma levels in neonates are high but reach those of treated adults after 1 week. However, NRTI like AZT and 3TC are prodrugs, which do not exert any therapeutic effect by themselves. They are metabolized through an intracellular multistep phosphorylation process, through monophosphate (MP) and diphosphate intermediates, to their corresponding active triphosphates (TP), which are found only within the cells. Moreover, in vitro experiments showed that NRTI-related mitochondrial toxicity involves intracellular phosphorylated metabolites such as AZT-MP (47) and AZT-TP (34). It is therefore essential to consider drug exposure in terms of intracellular metabolite concentrations.

To date, intracellular NRTI pharmacological data for children have been restricted to small-scale studies of cord blood (45) or older infants (35), excluding newborns. Despite the lack of precisely defined therapeutic ranges for NRTI metabolites, their determination would provide new insight into NRTI-related toxicity in newborns.

The objective of the present study was to determine, for the first time, intracellular concentrations of AZT and 3TC metabolites in newborns, together with AZT and 3TC plasma levels. In particular, intracellular levels of AZT-MP, AZT-TP, and 3TC-TP were investigated. Comparisons with previously reported pharmacological plasma data in newborns and plasma and intracellular data in HIV-infected adults as well as correlations with the available biological data were used to evaluate potential NRTI toxicity in these newborns.


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MATERIALS AND METHODS
 
Patients and study design. Data were obtained from newborns of HIV-infected mothers from outpatient clinics consulting at the Armand Trousseau Hospital (AP-HP, Paris, France) between 1998 and 2002.

Prospective follow-up was done according to French Perinatal Study guidelines (38) (Enquête Périnatale Française), with parental consent. The follow-up included regular proviral HIV DNA quantification and monitoring of potential drug toxicity (including hematological parameters and lactate) for 2 years.

During the 6-week duration of their antiretroviral prophylaxis, blood from the newborns was sampled twice at the occasion of morning follow-up consultations (in the first week of life and beyond 3 weeks) for proviral HIV DNA detection and safety evaluation. Any remaining aliquot was stored at the Virology Department. Among the 216 newborns in care over the 4-year period, remaining peripheral blood mononuclear cell (PBMC) samples (>106 cells) were available for 52 of them. Samples from three preterm newborns (<37 weeks) were excluded because they had received a reduced AZT or AZT-3TC dosage according to national guidelines. Thus, finally, the study involved samples from 49 newborns receiving MTCT oral prophylaxis including AZT (8 mg/kg of body weight/24 h in 4 daily doses) either alone (n = 29) or combined with 3TC (4 mg/kg/24 h in 2 daily doses) (n = 20) for 6 weeks after delivery. Doses were based on birth weight. Seven newborns also received a single dose of nevirapine at birth in the situation of increased risk of transmission.

For nine newborns, PBMC samples from two different time points were available. For the 40 others, only one sample was available, yielding a total of 58 PBMC samples and their corresponding 58 plasma samples.

All mothers, except for one newborn's mother (HIV type 2 [HIV-2]), were infected with HIV-1. Forty-seven of 49 women received antepartum antiretroviral therapy in various combinations. At the onset of the labor, 44/49 women received intravenous AZT (2-mg/kg bolus followed by 1 mg/kg/h until delivery). All mothers were discouraged from breastfeeding.

Toxicity data. We chose to focus on hematological parameters and lactates that were systematically collected and analyzed as part of the infants' routine care during the first 6 months of life, as they have been more consistently linked to perinatal drug exposure (40). Severity was graded based on the WHO pediatric classification. For lactates, values above 3 mmol/liter were considered to be significant for toxicity (3), keeping in mind that hyperlactatemia could be asymptomatic and an artifact of the collection technique (53) and that values of 2.5 mmol/liter (21) and 5 mmol/liter (1) have been used by others as cutoff values.

Preparation of plasma and PBMC samples. Blood samples of approximately 4 ml were collected from newborns into EDTA and diluted three times in RPMI 1640 medium before separation on a Ficoll-Hypaque gradient. Plasma and PBMC were collected. PBMC were washed (including ammonium chloride-mediated red blood cell lysis) (20). All samples were stored at –80°C in the Virology Department of Trousseau Hospital from the time of the collection until retrospective pharmacological analysis in 2005 at the Pharmacology and Immunoanalysis Unit of the CEA.

Plasma and PBMC samples required for calibration standards and quality control samples from blood from healthy volunteers were prepared (EFS, Rungis, France) identically to the preparation of patient samples.

PBMC counting. The number of PBMC was determined following cell lysis by using a validated biochemical test (8). This assay relies on a DNA-based fluorescence signal directly proportional to the number of cells. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), it allows the precise evaluation of intracellular concentrations, expressed in pmol/106 cells or fmol/106 cells.

Stock and diluted solutions. 3TC, 3TC-TP, AZT-MP, and AZT-TP were obtained from Moravek Biochemicals (Brea, CA). 2-Chloroadenosine, 2-chloroadenosine 5'-TP (ClA-TP), zalcitabine, AZT, and deoxythymine (dT)-TP were obtained from Sigma-Aldrich.

Stock solutions of 3TC and AZT (about 1 mg/ml) and 3TC-TP and AZT-TP (1 to 100 µg/ml) in ultrapure water were stored at –20°C and –80°C, respectively.

Diluted solutions for calibration standards and quality control were prepared from these stock solutions by serial dilution in human plasma-RPMI (1/2) (for NRTI) or in ultrapure water (for NRTI-MP/TP) and stored frozen until analysis.

PBMC lysis and NRTI-MP/TP and dT-TP extraction. Sample preparation and extraction were fully described in previous publications (7, 14). In particular, PBMC were lysed at the time of analysis. All samples were spiked with ClA-TP as an internal standard. Lysis and nucleotide TP extraction from frozen PBMC were carried out with 0.5 ml of Tris-HCl (0.05 M Tris-HCl [pH 7.4])-methanol (3/7). After lysate centrifugation (30 min at 18,000 x g at 4°C), supernatants were evaporated to dryness (37°C) (TurboVap; Zymark, France) and reconstituted with 120 µl Tris (0.05 M, pH 7.4) before analysis using standard LC-MS/MS conditions (7, 14).

LC-MS/MS analysis of NRTI-MP/TP and dT-TP. An 1100 liquid chromatography system (Agilent Technology, France) connected to a TSQ Quantum Ultra tandem mass spectrometer with an electrospray source (Xcalibur V.1.4; Thermo-Electron, France) was used for the intracellular nucleotide analysis.

The LC-MS/MS method for PBMC samples was adapted from previously reported assays (7, 14), with minor modifications concerning AZT-MP monitoring. ClA-TP was used as an internal standard for AZT-MP quantification. With this setting, lower limits of quantification (LLOQ) (and upper limits of quantification [ULOQ]) were established at 150 fmol/sample (ULOQ of 5,000 fmol/sample). To avoid a matrix effect on AZT-MP detection, low-cell-number samples (1 x 106 to 8 x 106 cells) and high-cell-number samples (8 x 106 to 30 x 106 cells) were analyzed separately using standard and quality control samples containing either 4 x 106 or 107 PBMC.

The calibration curve best fits were linear regressions (1/x weighted for 3TC-TP, AZT-MP, and dT-TP and 1/x2 weighted for AZT-TP) and allowed quantification within the analytical range (0.9 to 503 pmol/sample and 150 to 5,000, 250 to 40,000, and 150 to 5,000 fmol/sample, respectively). For all analytes, intra- and interday precisions and accuracies tallied with previously reported values (7, 14).

All values were then divided by the number of PBMC to obtain the concentration in fmol/106 cells.

Extraction and LC-MS/MS analysis of NRTI from plasma samples. The procedure for extraction and LC-MS/MS analysis of NRTI from plasma samples was an improved version of a previously reported assay (15), with minor modifications concerning the working volume of samples. Only 250 µl of plasma was taken, spiked with 50 µl of internal standard (2-chloroadenosine and zalcitabine), but the preparation process remained unchanged. The resulting solutions were filtered and analyzed using standard LC-MS/MS conditions (calibration range, 0.5 to 200 ng/ml). Intra- and interday precisions and accuracies tallied with previously reported recommendations (48) and previously reported values (15). No interlaboratory tests have been performed, since this kind of quality control exists only for protease inhibitors and nonnucleoside reverse transcriptase inhibitors (19, 33) but not for NRTI.

Data analysis. Plasma samples with values above the quantification range were reassayed after dilution. This was not possible for PBMC samples, as no duplicate tube was available. In order to avoid rejecting values, we applied the following rule (often used in similar cases and in pharmacokinetics) for calculation of the means, medians, and group comparison: when the result for a PBMC sample was above the ULOQ, the result was set to the ULOQ. It is worth noting that the ULOQ, when expressed in mol/106 cells, is different for each sample, since it corresponds to the highest quantifiable amount in the standard curve divided by the cell count. For example, the highest quantifiable amount for AZT-MP is 5,000 fmol/sample, and for a sample containing 5 x 106 cells, the ULOQ is 1,000 (i.e., 5,000/5) fmol/106 cells. When the result for a sample was below the LLOQ but the newborns received the corresponding drug, the value was set to LLOQ/2, which is also different for each sample. For newborns not treated with 3TC, levels of 3TC and 3TC-TP below the LLOQ were set to zero.

To ensure the statistical independence of the data, when two samples were available for one patient, we decided to include only the one corresponding to the earlier time point. GraphPad Prism 3.02 (GraphPad Software) was used to highlight any statistical difference between the data groups (Mann-Whitney U test) or any correlation (Spearman test). A P value of <0.05 was considered to be significant. Chi-square and Fisher exact tests were performed using SigmaStat 3.0 (Sysstat Software Inc.).


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RESULTS
 
Intracellular phosphate metabolite concentration in PBMC. AZT-MP, AZT-TP, 3TC-TP, and dT-TP concentrations could be analyzed in 57 of the 58 PBMC samples (Table 1).


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  		TABLE 1. Intracellular AZT-TP, 3TC-TP, AZT-MP, and dT-TP concentrations in samples from newborns from HIV-infected mothers on AZT monotherapy or AZT-3TC combination therapya

Overall, median AZT-MP and AZT-TP concentrations were 436 fmol/106 cells (range, from the LLOQ to 4,562 fmol/106 cells) and 98 fmol/106 cells (range, from the LLOQ to 385 fmol/106 cells), respectively. Thirty-five percent of the tested samples had AZT-MP concentrations above the ULOQ, particularly during the first 2 weeks.

No difference in AZT metabolite concentrations was found when the AZT-treated group was compared to the AZT-3TC-treated group, or within each group, according to gender (P > 0.05 by Mann-Whitney U test).

Overall, a significant nonlinear decrease in the levels of AZT-MP and AZT-TP with the age of newborns was observed (all P < 0.0001 by Spearman test) (Fig. 1). AZT-MP and AZT-TP concentrations were significantly higher during the first 15 days of life than after, with medians of 732 versus 282 fmol/106 cells and 170 versus 65.1 fmol/106 cells, respectively (P < 0.0001 by Mann-Whitney U test). No correlation was found between intracellular AZT metabolite concentrations and the newborns' weight in either younger (<8 days) or older (>20 days) newborns except for a weak correlation between weight and AZT-MP concentrations (P = 0.01 by Spearman test).


Figure 1
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  		FIG. 1. Relationship between intracellular AZT-TP (A) and AZT-MP (B) concentrations and age of newborns receiving AZT-based prophylaxis (n = 49) (P < 0.0001 by Spearman correlation). The dotted line represents the 95% confidence interval of the nonlinear best-fit regression curve. Note that below-LLOQ (n = 1 and 3 in panels A and B, respectively) and above-ULOQ (n = 18 in panel B) values have been set to the LLOQ/2 and ULOQ values, respectively (see Materials and Methods).

The median 3TC-TP concentration was 18.9 pmol/106 cells (range, from the LLOQ to 40.4 pmol/106 cells) in newborns receiving the AZT-3TC combination. 3TC-TP concentrations were associated with the age of the newborns (P = 0.0163 by Spearman test) but not with their weight. Concentrations in females were higher than those in males by 1.8-fold; however, the difference was not statistically significant (P = 0.0630 by Mann-Whitney U test). This trend cannot be explained by differences in age or weight between the groups.

dT-TP concentrations were simultaneously measured by LC-MS/MS and ranged from 280 to 7,948 fmol/106 cells, with a median of 1,271 fmol/106 cells. The presence of dT-TP in every sample confirmed the quality of storage and analysis. The AZT-TP/dT-TP ratio was significantly higher during the first 15 days of life than after, with a median of 0.11 versus 0.05 (P = 0.0007 by Mann Whitney U test).

Plasma NRTI concentrations. The study involved an analysis of AZT and 3TC in 58 plasma samples from 49 newborns (Table 2). AZT and 3TC measurements were successful for all samples.


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  		TABLE 2. Plasma AZT and 3TC concentrations in samples from newborns from HIV-infected mothers treated with AZT monotherapy or AZT-3TC combination therapya

Overall, AZT was detected in 98% of tested samples (range, from the LLOQ to 1,290 ng/ml; median, 89.1 ng/ml). No difference in AZT levels was seen when the AZT-treated group was compared to the AZT-3TC-treated group or when gender of the newborns was considered (P > 0.05 by Mann-Whitney U test). However, a significant nonlinear decrease in the AZT level was seen with the age of newborns (P < 0.0001) (Fig. 2). AZT concentrations were significantly higher during the first 15 days of life than after (199 versus 52.7 ng/ml; P < 0.0001 by Mann-Whitney U test). No correlation was found between AZT levels and weights of younger (<8 days) or older (>20 days) newborns. Significant correlations were found between AZT plasma concentrations and intracellular AZT-TP concentrations (P < 0.0001) and between AZT-MP and AZT-TP concentrations (P < 0.0001) (Fig. 3).


Figure 2
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  		FIG. 2. Relationship between plasma concentrations of AZT and age of newborns when receiving AZT-based prophylaxis (n = 49) (P < 0.0001 by Spearman correlation). The dotted line represents the 95% confidence interval of the nonlinear best-fit regression curve.


Figure 3
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  		FIG. 3. Relationships between plasma AZT concentrations and intracellular metabolite AZT-MP (A) and between AZT-MP and AZT-TP (B) (both P < 0.0001 by Spearman correlation).

Simultaneously, 3TC was detected in 95% of tested samples from AZT-3TC-treated newborns. 3TC levels (range, from the LLOQ to 1,157 ng/ml; median, 412 ng/ml) were not associated with the newborns' age, gender, or weight (P > 0.05 by Spearman and Mann-Whitney U tests). A significant correlation between plasma 3TC and intracellular 3TC-TP concentrations was noted (P < 0.0001 by Spearman test).

Plasma 3TC and intracellular 3TC-TP concentrations in newborns treated only with AZT. Unexpectedly, 3TC and 3TC-TP signals were observed in plasma and PBMC, respectively, in a few samples from newborns treated with AZT alone. This observation was limited to younger newborns (<8 days) not treated with 3TC but whose mother was receiving 3TC antepartum (n = 7 [including patient 8, whose mother's treatment was unknown]). In patient 8, only the 3TC-TP signal was found. In patient 10, 3TC was detected but 3TC-TP was not. Overall, 3TC and 3TC-TP concentrations also decreased significantly with the age of the newborns (P < 0.0001) (Fig. 4). The curve that gave the best fit to a one-phase exponential decay indicated half-lives of 20 h and 37 h for 3TC and 3TC-TP, respectively.


Figure 4
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  		FIG. 4. Relationship between plasma 3TC or intracellular 3TC-TP concentrations and age of newborns receiving only AZT but whose mother received 3TC antepartum (n = 7, one-phase exponential decay regression) (P < 0.0001 by Spearman correlation).

Clinical and toxicity data. In all the newborns, the drug was given orally and was well accepted; only two cases of mild regurgitations were reported. One infant was diagnosed with HIV infection. No clinical evidence of mitochondrial disorders (i.e., cardiac or neurological) was observed during the infants' follow-up, although two cases of convulsions were recorded (at day 6 and week 6) and two other infants presented withdrawal syndrome. Hematological toxicity was mild and rapidly reversible. Grade 3 anemia was identified in one sample at birth and another sample at 1 month. Grade 3 neutropenia was found in four samples at birth: two samples at 1 month and two samples at 6 months. Increased lactate levels, above 3 mmol/liter, were found in 8 of 14 samples at birth, in 12 of 27 samples at 1 month, and in only 2 of 24 samples at 6 months.

We compared toxicity levels among newborns displaying intracellular drug metabolite concentrations above and below the median. Overall, there were no significant differences in the proportions of toxicity grade, at each time point, between the two groups (P > 0.01 by chi-square or Fisher exact test). The same tests were also performed when the population was limited to younger patients (<15 days old) (n = 19), who exhibit higher drug levels, but no significant association was found.


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DISCUSSION
 
Both plasma NRTI and intracellular NRTI-MP/TP levels were determined in newborns undergoing MTCT prophylaxis using the same 2- to 5-ml blood sample for both pharmacological and virological analyses. LC-MS/MS assays of plasma and intracellular metabolites were able to ensure appropriate accuracy and sensitivity despite the small blood volume.

Considering the period of storage between sampling and pharmacological analysis (between 3 and 7 years), the stabilities of both NRTI and NRTI-MP/TP were questioned. Measured plasma levels of NRTI were in agreement with previous values (12, 39). Stability of the intracellular triphosphate metabolites is a crucial issue and is likely to be related to the TP group. The PBMC samples were stored at –80°C, which is a relevant temperature for storage of nucleotides (6, 13). The storage of unlysed PBMC pellets has been used in most of the intracellular pharmacological studies performed by our group, without any evidence of a stability issue, but in this study, the storage duration of the PBMC samples largely exceeded the 6-month testing period. To circumvent this deficiency, we have simultaneously monitored the endogenous nucleotide dT-TP as a surrogate for nucleotides. The median dT-TP concentration was 1.2 pmol/106 cells and was in the range of dT-TP values obtained using different methodologies in previous studies (31, 32, 46), with all studies exhibiting marked interindividual variability. As additional evidence of stability, we investigated the effect of the sample storage duration on the concentration of both AZT-TP and 3TC-TP (Fig. 5). No significant correlation was found for either AZT-TP or 3TC-TP (P = 0.806 and P = 0.360, respectively, by Spearman test). These combined results are in agreement with the stability of intracellular nucleotides in our samples, but confirmation of these data on fresh samples would be of interest. It is worth noting that even if some degradation has occurred, the high levels of intracellular AZT-TP and plasma AZT observed in this study during the first day of life would not be called into question.


Figure 5
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  		FIG. 5. Intracellular AZT-TP and 3TC-TP concentrations versus duration of biological sample storage.

We observed significantly higher concentrations of AZT and its intracellular phosphate metabolites (AZT-MP and AZT-TP) and also higher ratios of AZT-TP to dT-TP during the first 2 weeks of life than afterwards. These findings were in agreement with previous studies showing a decrease in plasma AZT concentrations with the age of newborns (12, 39) and proved that the same observation was true at the intracellular level.

Plasma AZT levels, collected 4 to 6 h after dosing and measured here during the first 2 weeks (range, 89.1 to 1,290 ng/ml), are in agreement with those previously reported for neonates (trough concentration of around 400 ng/ml at birth) (39) and are higher than those measured in adults (trough concentration, 100 to 300 ng/ml) (2, 26, 27, 52). Intracellular AZT-MP/TP measurements in newborns are reported here for the first time. Concentrations were initially high (medians of 732 and 170 fmol/106 cells for AZT-MP and AZT-TP, respectively) and declined to reach those observed in adults within a month (300 and 30 to 120 fmol/106 cells for AZT-MP and AZT-TP, respectively) (2, 27, 41).

High levels of AZT and intracellular metabolites during the first 2 weeks could hardly be related to a heavy drug dosing of the mother by infusion during labor, as all but three samples were sampled beyond 48 h after birth. AZT has elimination half-lives of 4 h in neonates (12, 39) and 1 to 1.5 h in adults (51), with the drug being rapidly eliminated at all ages. Similarly, the AZT-TP elimination half-life is around 7 h in adults (2), and concentrations observed in newborns after 48 h should be not related to the dose received by the mother during labor.

A hypothesis for the high levels of AZT and intracellular metabolites could be the contribution of membrane transporters, since AZT uptake is well-known to be mediated and regulated by membrane transporters (44). However, previous data on rats have demonstrated that the full maturation of transporter expression required several weeks (28), which could lead only to a reduction of intracellular levels of AZT during the first weeks of life and subsequent metabolites. Therefore, this hypothesis seems unlikely, and complementary works would be needed to better address this issue.

The most likely reason for these high levels in newborns is the low glucuronidation activity in newborns, consistent with a lower clearance of AZT, as previously highlighted in premature and term newborns (39). It seems that high levels of plasma AZT are thus responsible for high levels of intracellular metabolites.

A significant correlation between plasma AZT and intracellular AZT-MP/TP concentrations was observed (Fig. 3). However, due to the huge variability of both plasma and intracellular data and the poor fit of the data to any of the tested models, individual plasma AZT concentrations do not offer a precise evaluation of intracellular phosphate levels. Among previous studies addressing this issue, some of them have shown in adults a relationship between total phosphate metabolites (thus, mainly AZT-MP) and plasma AZT area under the curve despite high variability in the data, either plasma or intracellular (5). On the other hand, no significant correlation between plasma AZT and AZT-TP concentrations was found (41). In adults, AZT-TP concentrations are limited by the saturation of thymidylate kinase (TK), which catalyzes the phosphorylation of AZT-MP into AZT-DP, and higher concentrations of AZT-TP cannot be reached by increasing the dose (5). Figure 3 suggests a similar saturation in the phosphorylation process in newborns. However, the higher phosphorylated AZT species levels in neonates than in adults may be an indication of a higher TK saturation threshold in this population. This could be related to differences in the PBMC activation state between adults/newborns and neonates, with AZT being more efficiently phosphorylated in activated cells (29). When AZT plasma concentrations decreased after several weeks of life due to glucuronidation enzyme maturation, a similar expected decay of intracellular AZT-MP was observed. This could enable AZT-MP to reach levels below the TK saturation threshold, leading to a symmetrical decrease in AZT-TP levels. In addition, by studying a subset of data (AZT-MP levels ranging from 300 to 800 fmol/106 cells) in neonates (n = 12) and older newborns (n = 14) (i.e., before and after 15 days), which displayed similar AZT-MP levels (P = 0.231 by Mann-Whitney U test), we found significantly lower AZT-TP concentrations in older newborns (P = 0.003 by Mann-Whitney U test). This difference indicates that the TK saturation threshold decreased with the age of the newborns. In all cases, it is worth noting that when the concentration at birth was compared to that at day 30 (Fig. 1 and 2), the mean AZT level decreased by 10-fold, but the AZT-TP level decreased by only fourfold, which is consistent with TK activity partially limiting the phosphorylation cascade.

3TC plasma concentrations are consistent with those recorded in a previous pediatric study (39) and are in agreement with available data on 3TC-treated adults (2, 27, 54).

3TC-TP levels (median, 18.9 pmol/106 cells) have been measured in PBMC from newborns and are higher in female newborns than in male newborns. These concentrations are higher than those reported previously for 3TC-treated adults, 3.98 and 3.85 pmol/106 cells (54), 8.7 pmol/106 cells (27), and 8.5 pmol/106 cells (2). This could be related to differences in PBMC activation states between adults/newborns and neonates, with 3TC being more efficiently phosphorylated in activated cells (29). The gender difference, reported previously for adults (2), has no obvious biological explanation and has to be confirmed in larger-scale studies. Unlike other authors, we found no correlation between 3TC and 3TC-TP levels and the age of the newborn. This could be explained by the small number of samples from very young neonates (<10 days) (n = 4).

Unexpectedly, 3TC and 3TC-TP were found in six samples from the seven young newborns (<8 days of age) treated only with AZT but whose mothers received 3TC antepartum (including patient 8, whose mother's treatment is unknown). Their levels were significantly lower than those observed in the group treated with AZT-3TC, and levels of both decreased with the age of the newborns. These findings suggest that 3TC and 3TC-TP in untreated newborns come from maternal treatment crossing the placental barrier and are slowly eliminated in newborns, within the first week.

Overall, newborns are likely to be overexposed to AZT/AZT-MP/AZT-TP and 3TC-TP during the first 2 weeks of life. These observations are consistent with short-term hematological disturbances seen in clinics (12, 25). However, in this pilot study, no correlation was found between intracellular NRTI-MP/TP concentrations and short-term toxicity data at birth and at 1 month, 3 months, and 6 months of age. This could be due to the small and sparse number of patients and/or the rarity of severe events. The long-lasting effects of the exposure of newborns to NRTI are controversial, but in this context, the high levels of 3TC-TP and AZT-related species could be of concern.

In conclusion, with a small blood sample volume, consistent with pediatric requirements, these LC-MS/MS assays gave reliable measurements of plasma NRTI and intracellular NRTI phosphate concentrations and provided relevant results for NRTI phosphate intracellular pharmacology in newborns throughout the course of MTCT prophylaxis. While plasma 3TC levels are close to those seen in treated adults, concentrations of AZT and intracellular metabolites (AZT-MP, AZT-TP, and 3TC-TP) suggest drug overexposure in newborns during the first weeks of life. This should be kept in mind to enlighten, if they occur, possible long-term adverse events.


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ACKNOWLEDGMENTS
 
This work was supported by the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS, France).


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FOOTNOTES
 
* Corresponding author. Mailing address: Service de Pharmacologie et d'Immunoanalyse, CEA, iBiTecS, 91191 Gif-Sur-Yvette Cedex, France. Phone: 33 (0)1 69 08 72 98. Fax: 33 (0)1 69 08 59 07. E-mail: henri.benech{at}cea.fr Back

{triangledown} Published ahead of print on 21 April 2008. Back


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