![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, March 2005, p. 1023-1028, Vol. 49, No. 3
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.3.1023-1028.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Facility for Anti-Infective Drug Development and Innovation, Monash University,1 Department of Medicine, Austin Health,2 Pharmacy Department, The Alfred Hospital,3 Department of Perinatal Medicine, Royal Women's Hospital, Melbourne, Australia4
Received 23 June 2004/ Returned for modification 29 September 2004/ Accepted 27 October 2004
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PIs within highly active antiretroviral therapy are used increasingly in pregnancy due to their potency (21, 24, 26); however, there are limited data on placental transfer of these drugs. Previous studies with isolated perfused human placentae demonstrated low maternal-to-fetal transfer of amprenavir, ritonavir, and saquinavir (4, 6, 11). It is not clear whether this low placental transfer was the result of poor diffusional permeability or of some other mechanism. Because these perfusions were performed in the maternal-to-fetal direction only, comparison with fetal-to-maternal transfer was not possible. However, the low maternal-to-fetal transfer observed in the perfused human placenta models are consistent with the results of studies using matched maternal and umbilical cord blood samples collected at the time of delivery from women who had been medicated previously, which revealed the PI concentration in the fetal circulation to be very low (21, 23).
Huisman et al. (15) have implicated placental P-glycoprotein (P-gp) in limiting maternal-to-fetal transfer of PIs. P-gp is a membrane transporter that facilitates active efflux of a wide range of xenobiotics, including PIs (19), from certain cells. It is expressed in several epithelial barriers, including the trophoblast cells of the placenta (20), where it is present in the maternal facing cell membrane. Placental P-gp may extrude xenobiotics from trophoblast cells back into the maternal circulation and thus limit fetal exposure (37). Inhibition of placental P-gp in mice increases the maternal-to-fetal transfer of saquinavir and fetal exposure to this PI (34). It is postulated that P-gp may also influence the transfer of PIs across the placenta in humans, but this has not yet been investigated. An improved understanding of the mechanism of placental transfer of PIs, including the potential role of placental membrane transporters, is potentially important for the development of strategies involving preloading of the fetus with PIs to reduce MTCT of HIV. Therefore, the purpose of this study was to determine the placental transfer of the PI indinavir, vinblastine (a P-gp substrate) (36), and antipyrine (a marker of passive diffusion) (8, 33). We have used the dually perfused isolated human cotyledon, a technically demanding but powerful model that allows determination of transfer of these compounds in both the maternal-to-fetal and fetal-to-maternal directions within the same placenta.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Placentae. The study protocol was approved by the Ethics Committee of the Royal Women's Hospital and Monash University (Melbourne, Australia). Written, informed consent was obtained from participants prior to delivery. Women were excluded if they had HIV/AIDS or any other chronic diseases or were treated with indinavir or other chronic drug therapy. Placentae were obtained from women undergoing elective caesarean sections from uncomplicated, term pregnancies. Prior to perfusion, placentae were carefully inspected and those with visible tears were discarded.
Perfusion technique. The dual in vitro perfusion of the isolated human placenta was based on the method of Penfold et al. (30) and subsequently modified by our group (7, 8, 13). Briefly, placentae were obtained within 5 min of delivery, and independent fetal and maternal circulations were established in a single, nontraumatized peripheral cotyledon within 20 min. A fetal chorionic artery and vein pair were cannulated by using polyvinyl chloride tubing (inside diameter, 1.0 mm; outside diameter, 1.5 mm; Dural Plastics and Engineering, Sydney, Australia) to establish the fetal circulation. The cotyledon was cleared of blood by flushing with heparinized (10 IU/ml) compound sodium lactate injection (at 37°C) at 3 ml/min for 5 min. The whole placenta was mounted, fetal side uppermost, onto a Perspex platform (containing a cut-out circle, 10 cm in diameter). The perfused cotyledon was centered over the opening and supported by plastic mesh (3-mm grid). The maternal circulation was established by insertion of two blunt-ended cannulas (made from 19-gauge needles) approximately 0.5 cm through the maternal decidual basal plate into the intervillous space. The maternal and fetal perfusate flow rates were increased slowly to approximately 14 and 7 ml/min, respectively.
Both the maternal and the fetal perfusate were composed of Krebs-Ringer (Henseleit) phosphate-bicarbonate electrolyte solution (pH 7.4), with the addition of 3% dextran and 0.1% glucose, as described by Penfold et al. (30). The placenta, together with the maternal and fetal perfusate reservoirs, was housed in a thermostatically controlled cabinet at 37°C. The maternal and fetal circuits were pumped separately by a peristaltic pump (Minipals 2; Gilson Medical Electronics, Villiers le Bel, France). Perfusate passed from the reservoirs, through Millipore filters (pore size, 1 µm), silastic membrane oxygenators (receiving 95% O2-5% CO2), and bubble traps (to which separate maternal and fetal manometers were attached), and finally to the inflow cannulae.
Each perfusion was commenced by using drug-free perfusate for 20 min to stabilize the placenta and determine viability. The maternal circuit, containing 10 IU of heparin in 350 ml of perfusate, was in an open, single-pass mode, while the fetal circuit was in a closed, recycling mode. The viability of the placenta was assessed by monitoring the pressure in the fetal circuit and the loss of perfusate from the fetal reservoir. The placental preparation was discarded if there was a change in pressure (>10 mm Hg) or a loss of perfusate (>3 ml/h). Temperature and pressure were monitored continuously for the entire perfusion.
Study design. Following the stabilization period, both maternal and fetal circuits were operated in an open, single-pass mode for the remainder of the perfusion (90 min). In each placenta, the maternal-to-fetal and fetal-to-maternal transfer across the placenta of indinavir, [3H]vinblastine (a P-gp substrate) (36) in tracer quantities, and antipyrine (a marker for passive diffusion) (8) was examined.
For determining maternal-to-fetal transfer, the three analytes were added to the maternal reservoir at the following concentrations: indinavir, 7.6 mg/liter; [3H]vinblastine, 0.03 mg/liter (5 µCi/liter); antipyrine, 20 mg/liter. The fetal inflow perfusate was drug free. The perfusate indinavir concentration (7.6 mg/liter) is similar to the maximum unbound concentration in plasma encountered in a population pharmacokinetic study (8.2 mg/liter) (10) but somewhat higher than the mean maximum unbound concentration in plasma derived from the product information (22) by using a protein binding value of 54% (1) (3.6 mg/liter). Samples (3 ml) were collected from the maternal inflow reservoir and the fetal outflow perfusate at time zero and every 5 min for 40 min in order to determine the maternal-to-fetal transfer clearance at steady state. Fetal-to-maternal transfer was assessed after addition of the three analytes to the fetal reservoir at the concentrations described above; while the maternal inflow perfusate was drug free. Fetal inflow from the reservoir and the maternal outflow perfusate were sampled as before. A washout period (10 min) separated the two phases. Five out of 19 placentae were successfully perfused. The order of transport studies (maternal donor first [n = 3] or fetal donor first [n = 2]) was randomly assigned for each perfusion. Maternal and fetal perfusate samples were stored at –20°C until the time of analysis.
Analytical methods. (i) Indinavir. Perfusate indinavir concentrations were determined by using high-performance liquid chromatography (HPLC). Briefly, indinavir and the internal standard, verapamil, were extracted from the perfusate by using solid-phase extraction (Strata X; Phenomenex, Torrance, Calif.). The cartridges were first conditioned with 2 ml of methanol followed by 2 ml of water. Perfusate samples (1 ml) were added to the cartridges and washed with 3 ml of 10% methanol in water. Indinavir and verapamil were eluted from cartridges by using 2 ml of methanol. The eluate was evaporated to dryness by using a centrifuge evaporator. The residue was reconstituted in 100 µl of methanol-water (55:45), and 30 µl was injected onto a Waters (Milford, Conn.) Symmetry C8 analytical HPLC column (3.9 by 150 mm). The mobile phase contained 25 mmol of tetramethylammonium perchlorate/liter, 0.2% trifluoroacetic acid, and methanol (50:50), at a flow rate of 0.8 ml/min. Indinavir and verapamil were detected at 205 nm by using a UV detector. Calibration standards (0.2 to 10.0 mg/liter) and quality control (QC) samples (0.25, 1.0, and 9.0 mg/liter) were used in each analytical run. The measured concentrations of the QC samples were within 15% of the nominal concentrations, and reproducibility was within 15% as assessed by coefficients of variation at 1.0 and 9.0 mg/liter. For the low QC, the corresponding values were within 20%.
(ii) Vinblastine. Concentrations of [3H]vinblastine in perfusate were analyzed by using liquid scintillation counting (Packard Tricarb 1900CA scintillation counter) against an external quench curve. With this system, 1 ml of placental perfusate was mixed with 10 ml of Ultima Gold liquid scintillation cocktail and disintegrations per minute were counted for 15 min.
(iii) Antipyrine. Perfusate antipyrine concentrations were determined by using the HPLC assay of Ghabrial et al. (13). The measured concentrations of the QC samples (0.5, 5.0, and 20.0 mg/liter) were within 8% of the nominal concentrations, and reproducibility was also within 8% for all QCs.
Data analysis.
When the maternal perfusate was spiked with indinavir, vinblastine, and antipyrine, samples were collected to determine the transplacental maternal-to-fetal clearance at steady state (CLM
F) as described previously (7). CLMF was calculated as (Fout · Qf)/Min, where Qf is the fetal perfusate flow rate, Fout is the fetal outflow concentration, and Min is the maternal inflow concentration of the respective analyte.
When the fetal perfusate was the donor circuit for all analytes, samples were collected and the transplacental fetal-to-maternal clearance at steady state (CLFM) was calculated as (Mout · Qm)/Fin, where Qm is the maternal perfusate flow rate, Mout is the maternal outflow concentration, and Fin is the fetal inflow concentration of the respective analyte.
The transmembrane clearances of indinavir and vinblastine in the maternal-to-fetal direction and vice versa were normalized to the respective transmembrane clearances of antipyrine, the marker of passive diffusion, which was the same in both directions (see below). The ratio of the clearance of indinavir or vinblastine to that of antipyrine was termed a clearance index. Clearance indexMF was calculated as (CLMF of X)/(CLMF of antipyrine), and clearance indexFM was calculated as (CLFM of X)/(CLFM of antipyrine), where X is indinavir or vinblastine.
Statistical analysis.
Group data are expressed as means ± standard deviations. Differences between the CLMF and CLFM for each analyte were analyzed by using the paired Student t test. The CLMF/CLFM ratio for antipyrine was compared to unity by using the one-sample t test. Student t tests (accepting a P value of <0.05 as statistically significant) and determination of 95% confidence intervals (95% CIs) were performed by using SPSS for Windows, version 11.5 (SPSS Inc., Chicago, Ill.).
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
F and CLFM for each analyte were determined over the period from 25 to 40 min in the respective perfusion phases.
|
F:CLFM) for antipyrine (0.98 ± 0.15; 95% CI, 0.77 to 1.15) was not significantly different (P = 0.76) from unity.
|
F (± standard deviation) was 0.97 ± 0.09 ml/min while the corresponding CLFM was 2.53 ± 0.51 ml/min (P = 0.001). The mean maternal-to-fetal clearance index for indinavir (0.39 ± 0.09) was different (P < 0.001) from the fetal-to-maternal clearance index (0.97 ± 0.12) (95% CI of the difference, –0.70 to –0.45) (Fig. 3A).
|
F was 0.79 ± 0.20 ml/min, and the mean vinblastine CLFM was 1.75 ± 0.54 ml/min (P = 0.01). The mean vinblastine clearance index in the maternal-to-fetal direction (0.31 ± 0.05) was significantly different (P = 0.017) from the vinblastine clearance index in the fetal-to-maternal direction (0.67 ± 0.17) (95% CI of the difference, –0.61 to –0.11) (Fig. 3B). |
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, the maternal-to-fetal clearance index of indinavir was significantly less than the fetal-to-maternal clearance index. This is not a result of differential distribution in the placenta, since indinavir transfer clearance was calculated at steady state, after distribution was complete. Differential protein binding in maternal and fetal perfusate cannot explain the differential bidirectional clearance, since the perfusate did not contain human serum albumin as an oncotic agent and perfusions were performed in an open, single-pass mode. It is also not likely to be due to indinavir metabolism, since the term placenta has a very limited metabolic capacity (28), and there is no reason to expect that any influence of metabolism would be dependent on the direction of placental transfer.
The differential transfer of indinavir across the placenta is consistent with a transport system(s) retarding diffusion from the mother to the fetus. PIs are substrates for the efflux transporters P-gp and multidrug resistance-associated protein (MRP1) (35), which are expressed in placental trophoblast cells (2, 37). P-gp is present on the apical membrane domain (maternal), while MRP1 is located predominantly on the fetal facing basal membrane (2). The difference in localization suggests that P-gp may be more important than MRP1 in protecting the fetus from maternal xenobiotics (2). The results of the present study provide circumstantial evidence that the placental transfer of indinavir is modulated by P-gp, since the fetal-to-maternal transfer clearance of indinavir exceeded the maternal-to-fetal transfer clearance. Extended exposure to PIs has been shown to induce expression of P-gp, with indinavir being the weakest inducer (31). Such induction is not likely in the present study, since the mothers were not treated with PIs prior to delivery and the placentae were exposed to indinavir for only 90 min during the perfusion.
Vinblastine was selected as the reference P-gp substrate (36) in this study. As with indinavir, a significant difference in the bidirectional placental transfer clearance of vinblastine was observed: the maternal-to-fetal clearance index of vinblastine was appreciably less than the fetal-to-maternal clearance index. These findings for the intact placenta are in agreement with those of studies with the BeWo cell line (human placental choriocarcinoma epithelial cells), where vinblastine transport in the apical-to-basolateral direction was significantly less than that in the opposite direction due to the action of P-gp (36). Recently, vinblastine was discovered to be a substrate for MRP1 also (2). As for indinavir, the results of the present study for vinblastine are consistent with the action of P-gp predominating over that of MRP1 in limiting maternal-to-fetal transfer.
Antipyrine diffuses rapidly across the placenta, and its transfer is flow sensitive (33). For these reasons, it was chosen as the marker of passive diffusion in the present study (8). In contrast to both indinavir and vinblastine, there was no significant difference between the maternal-to-fetal and fetal-to-maternal transfer clearances of antipyrine, verifying that the placental transfer of antipyrine occurs exclusively by passive diffusion in both directions. In addition, the antipyrine clearance observed in this study was similar to values reported previously (8).
The role of P-gp in the placental transfer of PIs has been investigated by using the in vivo mouse model. Maternal-to-fetal transfer of saquinavir was sevenfold higher in P-gp knockout mice than in wild-type mice. In addition, pharmacological inhibition of P-gp in wild-type mice was shown to increase the placental transfer of saquinavir to values similar to those observed in P-gp knockout mice (34). This indicates that placental P-gp limits fetal exposure to saquinavir, and possibly other PIs, in mice. Evidence from the present study, conducted with intact human placentae, and from cultured placenta cells (36) suggests that placental P-gp in humans has a similar role in retarding PI transfer from mother to fetus. Thus, the action of placental P-gp may create the fetus as a pharmacological sanctuary to which xenobiotics, including PIs, have limited access (15).
Amazingly, the transfer clearance index for indinavir in the maternal-to-fetal direction in the present study was identical to that reported for amprenavir (0.39 ± 0.09) (4). Both of these studies were conducted using a perfusate without added proteins. In contrast, in studies involving ritonavir (6) and saquinavir (11) in the presence of human serum albumin in the perfusate, the clearance indices in the maternal-to-fetal direction were substantially lower (0.05 ± 0.05 for saquinavir and 0.09 ± 0.05 for ritonavir). Saquinavir and ritonavir are both highly protein bound (
98%) (25), and the lower maternal-to-fetal clearance indices for these drugs may result from protein binding in the maternal perfusate. Unfortunately, in the studies with ritonavir (6), saquinavir (11), and amprenavir (4), the transfer in the fetal-to-maternal direction was not determined. Therefore, it is not possible to conclude whether the lower maternal-to-fetal clearance indices for ritonavir and saquinavir, relative to those for amprenavir and indinavir, result from greater affinity for placental transport systems pumping the compounds in the fetal-to-maternal direction (e.g., via P-gp) or from binding of these PIs to protein added to the perfusate.
There is only a limited amount of information on the placental transfer of PIs in vivo. The concentrations of PIs in umbilical cord plasma or serum at the time of delivery for women receiving these drugs have been reported to be very low (21, 23), and the ratio of concentrations in umbilical cord plasma to those in matched maternal plasma is also low (21). The authors concluded that the extensive protein binding and relatively high molecular weight of the PIs limited their placental transfer (21, 23). Marzolini et al. also implicated placental P-gp as a possible contributor to the low maternal-to-fetal placental transfer (21); however, they did not provide evidence to support this suggestion. Taken together, the low maternal-to-fetal transfer clearance indices in perfused placentae for indinavir in this study, and reported for other PIs (4, 6, 11), and the low concentrations of PIs found in umbilical cord plasma from in vivo studies are consistent with low transfer from mother to fetus. In our study, we measured the transplacental clearance of indinavir in both directions within each placenta. As a result, we were able to demonstrate that an important contributor to the low maternal-to-fetal clearance index for indinavir was a placental transport system, possibly P-gp, that pumps the PI in the fetal-to-maternal direction.
The limited maternal-to-fetal transfer of indinavir demonstrated in this study may have implications for the management of HIV-infected pregnant women. Preloading the fetus peripartum with antiretroviral agents has the potential to reduce MTCT if transplacental passage of these drugs results in therapeutic concentrations in the fetus (15). Since PIs have low placental transfer, fetal PI concentrations may be ineffective against potential HIV infection and may encourage the rapid development of resistant strains (3). If placental P-gp is proven to restrict the maternal-to-fetal transfer of indinavir and other PIs, coadministration of P-gp inhibitors should be investigated. One approach could be to utilize dual PI therapy with ritonavir (a P-gp inhibitor) (14) in the peripartum period to increase maternal-to-fetal transfer and fetal PI concentrations. While this approach may increase protection against MTCT of HIV, concentration-related toxicities in the fetus must be considered. Indinavir has been associated with dose-related hyperbilirubinemia, which could lead to kernicterus in the neonate (32). Further consideration must also be given to the possibility that P-gp inhibitors such as ritonavir may induce P-gp expression upon chronic dosing (31). The application of possible preloading strategies in the peripartum period would therefore require careful consideration of these factors in addition to the implications for the overall management of the maternal HIV infection.
This study revealed relatively low maternal-to-fetal transfer of indinavir, a result that is consistent with findings for other PIs in the isolated perfused human placenta (4, 6, 11). More importantly, a differential bidirectional transfer was observed whereby the maternal-to-fetal transfer clearance of indinavir was substantially and significantly lower than the corresponding fetal-to-maternal transfer clearance. These results are consistent with the role of a membrane transporter, possibly P-gp, serving to restrict the movement of indinavir in the maternal-to-fetal direction. Further investigation is necessary to determine the clinical impact of these findings. Information from this study may assist in designing strategies for more-effective delivery of PIs to the fetus in order to further reduce MTCT of HIV.
|
ACKNOWLEDGMENTS |
|---|
We are grateful to Merck Research Laboratories for the generous supply of indinavir. Finally, we thank Sue Nisbert of the Department of Perinatal Medicine, and the obstetric and midwifery staff at the Royal Women's Hospital, for assistance in the recruitment of patients.
|
FOOTNOTES |
|---|
This research is dedicated to Lori Esch, HIV pharmacotherapy expert, who recently passed away. She was a true inspiration to all who knew her.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Clin. Vaccine Immunol. | Clin. Microbiol. Rev. |
|---|---|
| J. Clin. Microbiol. | ALL ASM JOURNALS |
), vinblastine (
), and antipyrine (
). Plot of outflow concentration in the receiver circuit as a percentage of inflow concentration in the donor reservoir versus time for a representative placenta.
