This paper provides data on TFV pharmacokinetics in plasma and PBMCs of the neonate. As no pharmacokinetic data were available for studying TFV in neonates, the TEmAA (Tenofovir/Emtricitabine in Africa and Asia) ANRS 12109 Trial study was built in two steps. In the first one, TDF was administered only to the mother at the onset of labor (16
). A cord sample allowed the estimation of the dose that reached to the fetus and a few neonatal samplings allowed estimation of the TFV neonatal half-life. The first-order kinetic was used to describe maternal/fetal transfer; however, at higher concentrations of the drug, this assumption of linearity may not be checked. Indeed, tenofovir is a substrate of influx transporters human organic anion transporter type 1 (OAT1) and OAT3 and of efflux transporter MRP4 in the kidney. OAT1 and OAT3 are also expressed in the placenta and could transport tenofovir according to a saturable process. Furthermore, biological processes affecting placental blood flow and placental transfer are also not captured in the first-order kinetics presented. Using the data from step 1 and making a hypothesis about neonatal absorption and distribution, we evaluated the neonatal dose of TDF that would produce the same plasma drug exposure and minimal concentrations in neonates as those observed in adults with the recommended dose (6
). To apply this strategy, a population approach was developed to determine a neonatal dose in the first part of the study, which allowed us to progress to the second step. This approach was validated by the results obtained in the second part reported here.
Based on the results obtained in the first step, a readministration of two tablets of TDF-FTC to the mother was proposed after 12 h if the mother had not delivered yet, and a first dose of 13 mg/kg at birth was proposed for the neonate. These propositions could be evaluated here. First, the two mothers and fetuses, for which the mother was readministered TDF 12 h after the first intake, had comparable concentrations compared to the mothers and fetuses for which the mother had a single administration at the onset of labor. Second, the dose of 13 mg/kg administered to the neonate within 12 h after birth produced plasma TFV concentrations close to the adult values: 3.73 mg/liter · h versus 2.88 and 2.65 mg/liter · h for AUC, 0.076 mg/liter versus 0.060 and 0.053 mg/liter for Cmin
, and 0.29 mg/liter versus 0.31 and 0.29 mg/liter for Cmax
). In conclusion, as expected by our simulations based on the first step of the study, with the neonatal TDF dose of 13 mg/kg, plasma drug concentrations were comparable between neonates and adults.
The method used to obtain similar adult plasma drug exposure is currently used to determine the dosage in children. However, concentrations of the drug at the site of action could be different in children and adults for the same plasma concentrations (14
). Indeed, as HIV acts inside the lymphocyte and as TFV is not active by itself but undergoes intracellular phosphorylation by various cellular kinases to give the active diphosphate TFV-DP, we aimed to compare not only plasma drug concentrations but also intracellular drug concentrations between adults and neonates in the second step. A previous study showed that newborns are likely to be overexposed to AZT/AZT-MP/AZT-TP and 3TC-TP during the first 2 weeks of life (10
). These observations are consistent with short-term hematological disturbances seen in clinics (7
). However, no correlation was found between intracellular nucleoside reverse transcriptase inhibitor monophosphate (NRTI-MP)/TP concentrations and short-term toxicity data at birth and at 1, 3, and 6 months of age. The authors suggested that this overexposure could be related to differences in the PBMC activation state between adults and neonates, some NRTIs being more efficiently phosphorylated in activated cells (12
). Thus, by collecting and dosing TFV and TFV-DP in PBMCs extracted from one fetal and one neonatal sample and by using a population approach, TFV cell transfer and TFV phosphorylation were described.
In our study, TFV-DP in PBMCs was measured 22 to 38 h after the neonatal drug intake for 20 children and 10 h and 45 h after neonatal drug intake for two other children, and neonates were not likely to be overexposed to the intracellular phosphorylated TFV-DP. Indeed, median TFV-DP concentrations were 146 fmol/106
cells (IQR, 53 to 430 fmol/106
cells), these concentrations were comparable to adult therapeutic values obtained at steady state and were lower than the values obtained after administration of a single TDF dose to an adult (15
). As TFV-DP elimination could not be estimated in the neonates due to the lack of points after 50 h, the exposition between neonates and adults could not be compared. Although it was not evidenced by the model, we think that the difference between NRTI phosphorylation (TFV-DP versus AZT-TP or 3TC-TP) could be due to the enzymes implicated. The last phosphorylation is performed by the same enzyme (5′ diphosphatase kinase), but the enzymes implicated differ for the first or second phosphorylation of the compound: adenylate kinase for TFV, thymidine and thymidylate kinase for ZDV, and deoxycytidine kinase and monophosphate kinase for 3TC (1
). Furthermore, it seems that PBMC activation in neonates is more effective by some enzymes than by others (12
Two children had very high TFV-DP concentrations (1,530 and 2,963 fmol/106
cells—i.e., 6 to 12 times the median TFV-DP concentration) but normal plasma drug concentrations, so these children could not be identified by a simple plasma sample. None of them had serious adverse events, while four serious adverse events were reported in this study (24
Both fetal and neonatal intracellular drug concentrations were measured and allowed estimation of TFV-DP generation. In the fetus, samples were taken from 0.4 to 12 h after maternal administration, and in all the neonates except one, samples were taken from 22 to 45.5 h after neonatal administration. The lack of information on the neonate before 24 h has been compensated for by information on the fetus for which concentrations were collected between 0.4 and 12 h. We have a hole between 12 and 22 h, but we could estimate TFV-DP generation. With the exception of two samples, no TFV-DP was quantifiable in fetal PBMCs. This could be due to too-low fetal plasma drug concentrations, a low rate of cell transfer, or a delay before the phosphorylation. Although drug concentrations were quite low in the plasma of the fetus, TFV was present and quantifiable in fetal PBMCs, and thus the low TFV-DP concentrations at birth seem to be attributable to a delay before phosphorylation. This delay could not be estimated precisely due to the lack of TFV-DP concentrations between 12 and 22 h. Although in the first step of the TEmAA Study, we recommended administration of TDF to the neonate at birth, for practical reasons, it was given at the same time as emtricitabine (at a median delay of 9.2 h after birth). Taking into account the relatively low plasma TFV concentrations in the fetus and the delay before the drug starts to be active, we again recommend giving the 13-mg/kg dose of TDF to the neonate at birth.
In conclusion, the 13-mg/kg TDF dose given to the neonate produced similar plasma TFV and intracellular active TFV-DP concentrations in neonates compared to those in adults. This dose should be given immediately after birth to reduce the delay before the active compound TFV-DP appears.