The number of women treated with HAART during pregnancy is increasing [1,2]. The current guidelines of the United States recommend a combination antiretroviral therapy (ART) as a standard procedure even without clinical criteria for an antiretroviral treatment and low plasma viral load . However, even less is known about pharmcokinetics during pregnancy and the placental passage of non-nucleoside reverse transcriptase inhibitors and protease inhibitors (PI).
Several physiological changes during pregnancy can potentially affect drug disposition, including altered protein binding, enlargement of plasma volume and changes of gastrointestinal function [4,5]. If these pharmacological alterations lead to lower plasma levels of certain antiretroviral agents, then their efficacy could deteriorate in pregnancy.
There are contradictory results in a few studies [6–8] regarding the difference of the pharmacokinetics of nevirapine (NVP) between pregnant and non-pregnant women. Regarding the pharmacokinetics of PI, there are limited data from small series for the different drugs [9–13].
Most results concerning placental transfer of antiretroviral agents derives from the ex vivo placenta perfusion model [14–16]. Only few in vivo studies have been published which reflect the clinical situation much better. The non-nucleosidic reverse transcriptase inhibitor NVP was shown to cross the placenta rapidly, reaching equivalent plasma levels in maternal and cord blood [17,18]. The in vivo transplacental passage of PI was found to be very low in a limited number of patients [19,20]. Chappuy et al.  was the only investigator who looked also at the concentration of PI in the amniotic fluid, and found low drug levels in this compartment as well.
This prospective study was conducted at the 1st Department of Obstetrics and Gynecology, Ludwig-Maximilians-University of Munich, Germany. The protocol was approved by the local ethics committee. Written informed consent was obtained from all participants.
Eligible HIV-infected pregnant women who attended prenatal care in our outpatient clinic for HIV-infected women were enrolled in the study. Inclusion criteria were antiretroviral combination therapy including NVP and/or different PI (nelfinavir, NFV; saquinavir, SQV; lopinavir, LPV; ritonavir, RTV). All women had Caesarean sections according to the German/Austrian guidelines for pregnancy care of HIV-infected women . On the day of delivery maternal blood samples were collected pre-dose, 1 h until 6 h post-dose and at the time of birth. The time of tablet intake on the day of delivery and the evening before were recorded as well as the times of maternal and foetal blood specimen collection. During Caesarean section amniotic fluid was taken by careful preparation of the amniotic membrane with the least possible blood contamination. Then the amniotic sac was opened and amniotic fluid was extracted using a syringe with a urine catheter to avoid contamination of the fluid with maternal blood. Cord blood was drawn from the placental part of the umbilical cord (artery) shortly after the placenta was removed from the uterus.
The pharmacokinetics of NVP and different PI under steady-state conditions were performed by Prof. Kurowski, Berlin, Germany. Plasma samples were analysed by validated liquid chromatography/tandem mass spectrometry . This method has been described previously .
HIV-1 RNA concentration in patient plasma specimen was measured by COBAS AMPLICOR HIV-1 MONITOR Test, v1.5 (ROCHE, Basel, Switzerland), which allows an equivalent quantification of HIV-1 group M viruses (subtypes A–G) in the range of 50–750 000 copies/ml. The test was used according to the manufacturer's recommendations. Viral load was analysed in the mother at delivery (± 2 days) and also in the amniotic fluid.
Lymphocyte immunophenotyping was performed on peripheral blood mononuclear cells (PBMC) isolated from whole blood by Lymphoprep density centrifugation (Nycomed Pharma AS, Oslo, Norway). After undergoing two washing steps, the cells were immediately stained with the following fluorochrome-labelled antibodies (Becton Dickinson, Beiersdorf, Germany): anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD56, and anti-CD25. FACS analysis was performed with CellQuest FACScan research software (BD Immunocytometry Systems, San Jose, California, USA). The absolute number of each T-cell subpopulation (cells/μl) was calculated by multiplying the fraction of cells for which staining revealed positive findings by the absolute lymphocyte count, which was derived from the differential white blood cell count.
Statistical analysis was performed using SPSS version 12.0 software (descriptive analysis).
All children were administered postnatally a standard regimen of zidovudine (ZDV) syrup 2 mg/kg every 6 h over 6 weeks; two children received ultrashort prophylaxis with NVP in addition, and two other children received lamivudine (3TC) as an escalation of the postnatal prophylaxis because of a high-risk situation (e.g., premature rupture of the membranes).
The newborns were considered to be HIV-1 uninfected if no HIV-RNA was detectable in three repeated blood samples until the age of 3 months. All newborns are in follow-up until they are 18 months old.
Forty pregnant women were enrolled in this study between 2000 and 2004, including two twin pregnancies. The mean age of the women at delivery was 31.4 years (range, 19–42 years) with a median body mass index at the beginning of pregnancy of 23 kg/m2 (range, 17–34 kg/m2) and at delivery of 27.5 kg/m2 (range, 19–42 kg/m2).
Thirty-five women were delivered by elective Caesarean section at a mean gestational age of 38 weeks (range, 33–40 weeks). Five women were delivered immediately by emergency Caesarean section because of premature rupture of the membranes, premature birth or starting of contractions at a mean gestational age of 38 weeks (range, 37–39 weeks). No maternal postnatal complications were observed. The 40 pregnancies resulted in 42 live births with a mean birth weight of 2952 g (range, 1045–3480 g). None of the babies were HIV infected. However, one child had neuroblastoma, one had a ventricular septum defect, and one had a vertically acquired toxoplasmosis.
The participants of our study were taking 21 different antiretroviral drug combinations. Twenty-one (53%) were on an antiretroviral regimen that included NVP. The combination of ZDV, 3TC and NVP was used most frequently (n = 13). Seven patients were taking NFV, four SQV, 11 LPV and 15 RTV as part of their antiretroviral drug regimen. In several cases up to three drugs were measured in the plasma samples. All regimens were given twice daily. Daily doses of NVP were 400 mg/day, NFV 2500 mg/day. When given alone the daily dose of SQV soft-gel capsule (SGC) was 3200 mg/day, and when used in combination with RTV it was 2000 mg/day. LPV was always given combined with RTV 800 mg/day. In one patient RTV was given as a mono-PI at 1200 mg/day. RTV in combination with SQV or LPV was always given in a boosting dose of 200 mg/day. All patients were taking the medication for at least 4 weeks, 11 of them for the whole pregnancy.
The maternal viral load measured at a time close to the scheduled day of delivery (± 2 days) was below the minimum level of detection (< 50 copies/ml) in 32 women. In seven samples the viral load was below 1000 copies/ml, and in one sample it was 110 000 copies/ml (regimen, AZT/3TC/NVP). The median CD4 cell count of the mothers at the same time point was 437/μl (range 84–1084/μl).
Pharmacokinetic parameters were evaluated in the 35 women who delivered by elective Caesarean section. For the five women who underwent emergency Caesarean section, only one blood sample from the time of birth was available. The cumulated pharmacokinetic parameters are given in Table 1.
Figure 1 shows the individual pharmacokinetic profiles of the different PI (NFV, n = 5; SQV-SGC, n = 3; lopinavir, n = 10; RTV babydose, n = 13). SQV-SGC and LPV were each boosted with RTV. PI pharmacokinetic parameters are given as well in Table 1.
A foetal cord blood sample was available from all 42 newborns and a satisfactory amniotic fluid sample was obtained for 35. In seven Caesarean sections no suitable amniotic fluid sample was available for different reasons such as oligohydramnion, transplacental uterotomy due to anterior wall placentation or technical failure. The cord-to-mother (C: M) and the amniotic fluid-to-mother (A: M) ratios of drug levels was calculated as a parameter for the placental passage of the different antiretroviral drugs into the foetal compartment. The results are shown in Table 2. NVP has a very high C: M ratio (1.02) as well as a high A: M ratio (0.75). As shown in Table 2 corresponding ratios for PI were much lower. The highest values were detected for NFV/LPV while SQV showed the weakest placental passage.
Twenty-three of the 35 amniotic fluid specimens were available for HIV-1 RNA testing. In 12 samples the volume of amniotic fluid was too low after measuring the drug level (regarded as the priority aim) to determine HIV RNA. In 22 samples no HIV RNA was detectable. In one sample we found a viral load of 1400 copies/ml whereas HIV RNA was undetectable in the parallel maternal plasma sample.
The objective of our prospective trial was to study the pharmacokinetics of different PI and NVP in pregnant women. Another aim was to determine the in vivo transfer of these drugs into umbilical cord and the amniotic fluid as part of the foetal compartment.
In contrast with the few published studies about placental transfer of these compounds which were predominantly based on in vitro experiments [14–16] we obtained ‘real life’ data with defined measuring intervals after drug intake, which better reflect the clinical situation. Pharmacokinetic data in pregnant women are rare and derive mostly from even smaller case series (see [7–10]).
One major finding was a significantly altered pharmacokinetic course of NVP in pregnant compared to non-pregnant women. Recommended minimum concentrations (Cmin) of NVP under steady-state conditions in adults with 400 mg NVP/day are 4.0–4.5 mg/l [25–27]. Mirochnick et al.  described no difference between the pharmcokinetics of NVP in pregnant and non-pregnant women. However, they noted reduced NVP levels in patients after initial doses given during labour. A very small trial (PACTG 1022) confirmed that steady-state plasma NVP concentrations during second and third trimester of expectant mothers are equivalent to those outside pregnancy . By contrast, another study found reduced plasma levels of NVP during pregnancy below the recommendations for adults . As shown in Table 1, in our study none of the patients reached NVP trough levels of 4.0 mg/l on the day of Caesarean section. However, we do not have pharmacokinetic data of the same women while not pregnant to directly compare the drug levels, but we are trying to record those data as an ongoing part of the study.
In an observational cohort analysis low NVP plasma concentrations were predictive of virological failure . In this examination only one of our 21 patients on a combination regimen, which included NVP, showed significant virological failure with a viral load of 110 000 copies/ml on the day of Caesarean section. This virological failure though was due to patient non-compliance. Furthermore there was no vertical transmission in this study in which all women delivered by Caesarean section.
We conclude that, at least from the perspective of prevention of vertical transmission, there is no need for an NVP dose adaptation. However, it cannot be ruled out that the reduced NVP level in pregnant women might be important if only NVP prophylaxis is available, and/or the transmission-risk strategy is not extended with an elective Caesarean section. Moreover, an impact on future therapy options and NVP resistance development for the patient cannot be excluded. Therapeutic drug monitoring of NVP and dose adaptation should be considered for pregnant patients. However, the question remains whether or not it might be necessary to increase the NVP dose in pregnant patients with low plasma levels.
In agreement with the HIVNET 006 study  (single dose NVP during labour with a C: M ratio of 0.75), we found quantitative and quick placental passage of NVP under steady-state conditions with a C: M ratio of 1.02 and an A: M-ratio of 0.75. Therefore NVP is clearly suitable for a rapid therapy escalation, particularly in an emergency situation with a higher risk for vertical transmission of a patient with ZDV monoprophylaxis. Another option is the late perinatal short-term prophylaxis in resource-poor settings. However, combination therapy should be preferred whenever available because of the rapid selection of resistance mutations by NVP.
Plasma concentrations of the PI showed a considerable, patient-dependent variability.
In a twice-daily regimen of NFV (2500 mg/day) the Cmin values are reported to be between 0.7 and 2 mg/l  which only one of our patients (patient 4) did not reach. Cmax values between 3 and 4 mg/l were achieved by only one patient.
A study by Kosel and others  showed a reduction of indinavir levels but more stable levels of NFV in pregnancy whereas Nellen et al.  found low levels of NFV. SQV-SGC levels were below average in pregnant women according to two studies [11,12].
Since several blood samples were not taken exactly at the intended point in time, the area-under-the-curve (AUC) values from 0 to 6 h were interpolated or extrapolated. For the purpose of better comparability, all AUC values are given as the AUC from 0 to 6 h (AUC6). AUC values from 0 to 12 h are available for only a few of our patients. No corresponding AUC6 values for NFV, RTV or SQV are published. A rough extrapolation of our data to AUC8 or AUC12 values yields comparable results with published AUC8 or AUC12 values for these PI.
Compared with NVP, the PI (NFV, SQV-SGC, LPV, RTV) crossed the placenta poorly. However, all PI were detectable in cord blood and in amniotic fluid. C: M ratios ranged from 0.01 to 0.31 (Table 2). Transfer into the amniotic fluid was even smaller with A: M ratios between 0.01 and 0.2. This is consistent with other in vivo data which were, however, recorded in less detail.
To our knowledge, no data for boosted LPV in pregnant women are available. Our series showed that cord blood concentrations of LPV were between 5 and 25% of the maternal plasma concentrations, averaging 16.7%. AUC6 values ranged from 6.8 to 36.3 μg/h/l with a median AUC6 of 23.4. Although LPV and NFV concentrations in amniotic fluid are low, they show a clear positive, linear correlation to maternal plasma levels (data not shown).
Reasons for the limited placental transfer are probably the high protein binding capacity of PI and the large molecule size, being a substrate of p-gylcoprotein .
All of the studies, including ours, are limited due to small patient numbers. Other trials however, are additionally hampered by the fact that either maternal plasma levels are missing , or that there were extremely long time lapses between drug intake and Caesarean section . The latter leads to insufficient maternal plasma levels of the PI at birth. In another study, a similar problem with a high proportion (57%) of maternal plasma level at birth below the target trough concentration was reported . In the same trial very low drug concentrations of PI in the amniotic fluid were found. In our trial the evaluation of the placental transfer would have been more accurate if we had collected infant blood samples as well but there was no parents agreement for this.
PI are potent antiretroviral drugs, can produce a sustained suppression of HIV replication and are considered first-line therapies in HIV-infected individuals. Used as part of combinations for the therapy of pregnant women, they may be additionally advantageous, as unlike for nucleoside reverse transcriptase inhibitors there are no data showing mitochondrial toxicity or possible long-term mutagenic effects. Limited placental transfer may protect the foetus against the potential toxic or mutagenic effects of these agents, since the compounds and/or their metabolites do not reach the foetus to a great extent. On the other hand, we do not know yet if relevant plasma levels of antiretroviral drugs in the foetus could further decrease the risk of vertical transmission, or if a therapeutic plasma concentration at the time of birth would be desirable to provide post-exposure prophylaxis to the newborn.
We therefore conclude that PI are not appropriate drugs for the prophylaxis of vertical transmission in women presenting very late in pregnancy or for short-term escalation of antiretroviral therapy because of a high risk situation. However, they play an important role in the treatment of HIV-infected pregnant women and/or in prophylaxis of mother-to-child transmission of HIV-1.
In this study there was no vertical transmission but all women had combination therapies, including other effective compounds with better placental transfer characteristics. However, there will be some pregnant women with fewer therapy options because of multiple drug resistance. We do not know if the reduction of the viral load due to these compounds (PI) alone would be sufficient for the prevention of vertical transmission in these cases.
We found detectable HIV-1 RNA concentrations in only one of 23 amniotic fluid samples. A similar result was recently published for amniocentesis samples . In the single HIV-positive sample a viral load of 1400 copies/ml was detected (this sample was just visibly contaminated with maternal blood at delivery). However, in maternal plasma drawn during the Caesarean no HIV RNA was detectable. This finding suggests the existence of a uterine compartment with much higher viral load than in the maternal plasma. It is comparable to other known discordances between different compartments, for example HIV virus load in cervical/vaginal smears or lymph nodes versus peripheral blood . The newborn of this particular mother obtained post-exposure prophylaxis and remained uninfected. This phenomenon will be investigated further in the ongoing arm of our study.
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