Objective
We sought to determine the bidirectional transfer and distribution of vancomycin and telavancin across the dually perfused term human placental lobule.
Study Design
The technique of dually perfused placental lobule was used in its recirculating mode to determine the maternal to fetal (M→F) (n = 20) and fetal to maternal (n = 18) transfer of each antibiotic, which were coperfused with their radioactive isotopes. The concentrations of drugs were determined by liquid scintillation spectrometry.
Results
In the M→F direction, the transfer of vancomycin (9.6 ± 4%) and telavancin (6.5 ± 2%) were low; however, telavancin retention by the perfused lobule was greater than that of vancomycin ( P < .01). The normalized transplacental transfer of telavancin across the placental lobule in the fetal to maternal direction was higher than in the M→F direction ( P < .01), suggesting the involvement of placental efflux transporters.
Conclusion
The ex vivo perfusion experiments revealed low transfer of vancomycin and telavancin to the fetal circuit.
Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging problem in women during pregnancy and the postpartum period. From 2000 through 2004, the rate of MRSA infections in pregnant women increased 10-fold. Postpartum infections due to MRSA are often serious and potentially life threatening, and until recently, vancomycin has been the antibiotic of choice for treatment. However, the development of vancomycin-resistant strains of S aureus have compromised its use and led to the development of its derivative, telavancin.
Telavancin is a lipoglycopeptide antibiotic with improved activity against a wide range of gram-positive organisms with reduced susceptibility to vancomycin. Telavancin has at least 2 mechanisms of action. The first, similar to vancomycin, is inhibition of late-stage peptidoglycan synthesis by binding to the D-ala-D-ala terminus of the peptidoglycan intermediate, thus preventing cross-linking of the cell wall. The second mechanism of action of telavancin increases the permeability of bacterial cell membranes. Owing to telavancin’s multifunctional mechanism of action, it has enhanced (over vancomycin) bactericidal activity, activity against bacterial strains with reduced susceptibility to vancomycin, and lower frequency of resistance. However, due to the limited data on the adverse effects of telavancin to the fetus as well as its pharmacokinetics during pregnancy, it is designated as a Class B drug and is currently not approved by the Food and Drug Administration for treatment of pregnant patients. Therefore, additional preclinical and clinical information on its effectiveness and safety during pregnancy is required. The first step in obtaining this information is to determine the role of human placenta in the disposition of telavancin and, consequently, its concentration in the fetal circulation.
Vancomycin (1485 d) and telavancin (1792 d) have higher molecular weights than most medications. To our knowledge, transplacental transfer of telavancin has not been reported. On the other hand, transplacental transfer of vancomycin has been investigated by utilizing the technique of dual perfusion of placental lobule and in vivo. The transplacental passage of vancomycin was observed in women with amnionitis as well as in noninfected pregnant women. The data obtained from in vivo investigations demonstrated that vancomycin readily crosses the infected (second and third trimesters) and noninfected (term) placentas and suggested that intrauterine exposure of the fetus to vancomycin is possible. However, these data did not agree with those obtained from ex vivo experiments indicating that vancomycin was not transferred across placenta. The observed difference between the in vivo and in vitro/ex vivo data was explained by the formation of a heparin-vancomycin complex in the perfusion medium that prevented its transfer from the maternal to fetal (M→F) circuit. To clarify these controversial data, it was imperative to determine the extent of transplacental transfer of vancomycin and telavancin as well as their retention by placental tissue.
Therefore, the aim of this investigation was to determine the bidirectional transfer of vancomycin and telavancin and the distribution of these drugs in placental tissue using the dually perfused placental lobule. This technique retains the anatomic and functional integrity of placental tissue and has been validated for determining the bidirectional transfer, distribution, and metabolism of numerous drugs.
Materials and Methods
Chemicals
Tritium isotope ([ 3 H])-vancomycin (specific activity, 4.2 Ci/mmol or 0.1554 TBq/mmol) and [ 3 H]-telavancin (specific activity, 16.3 Ci/mmol or 0.6031 TBq/mmol) were purchased from Vitrax Co (Placentia, CA). High-performance liquid chromatography-grade methanol, acetonitrile, sodium acetate, and hydrochloric acid were obtained from Fisher Scientific (Fair Lawn, NJ). Telavancin was obtained as Vibativ (marketed by Astellas Pharma US Inc, Deerfield, IL) from the pharmacy of the University of Texas Medical Branch, Galveston. Paclitaxel [ o -benzamido- 3 H] (38 Ci/mmol or 1.406 TBq/mmol) was purchased from Moravek Biochemicals Inc (Brea, CA). All other chemicals including radioactive carbon-14 isotope ([ 14 C])-antipyrine (AP) (specific activity, 6.5 mCi/mmol or 0.2405 GBq/mmol) and vancomycin hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO).
Clinical material
Placentas from uncomplicated term (37-42 weeks) pregnancies (n = 38) were obtained immediately following vaginal or abdominal deliveries at the Labor and Delivery Ward of the John Sealy Hospital at the University of Texas Medical Branch, Galveston, according to a protocol approved by the institutional review board. Any evidence of maternal infection, systemic disease, and drug or alcohol abuse during pregnancy excluded the placenta from this investigation.
Dual perfusion of human placental lobule
The technique of dual perfusion of human placental lobule was used as described in detail in reports from our laboratory and originally by Miller et al. Briefly, each placenta was examined for tears and 2 chorionic vessels (1 artery and 1 vein) supplying a single intact peripheral cotyledon were cannulated with 3F and 5F umbilical catheters, respectively. The cotyledon was trimmed and placed in the perfusion chamber with the maternal surface upward. The intervillous space on the maternal side was perfused by 2 catheters piercing the basal plate. The flow rate of the medium in the fetal and maternal circuits was 2.8 and 12 mL/min, respectively. The perfusion medium was made of tissue culture medium M199 (Sigma-Aldrich) supplemented with: dextran 40 (7.5 g/L in the maternal and 30 g/L in the fetal reservoir), 25 IU/mL heparin, 40 mg/L gentamicin sulfate, 80 mg/L sulfamethoxazole, and 16 mg/L trimethoprim. The maternal perfusate was equilibrated with a gas mixture made of 95% O 2 , 5% carbon dioxide, and the fetal perfusate with a mixture of 95% nitrogen, 5% carbon dioxide. Sodium bicarbonate was added to the maternal and fetal circuits to maintain the pH at 7.4 and 7.35, respectively. All experiments were carried out at a temperature of 37°C.
Each placenta was perfused for an initial control period of 1 hour to allow the tissue to stabilize to its new environment. Perfusion was terminated if one of the following occurred: fetal arterial pressure >50 mm Hg, a volume loss in fetal circuit >2 mL/h, or an oxygen partial pressure difference between fetal vein and artery <60 mm Hg, indicating inadequate perfusion overlap between the 2 circuits.
Transplacental transfer and distribution of vancomycin and telavancin
Following the control period, the maternal and fetal perfusates were replaced with fresh medium. Human serum albumin was added to both the maternal and fetal circuits in its physiological concentrations of 30 mg/mL. The nonionizable, lipophilic marker compound AP 20 μg/mL and its [ 14 C]-isotope (1.5 μCi) were cotransfused with vancomycin or telavancin to account for interplacental variations and to normalize the transfer of antibiotics.
AP and one of the antibiotics were added to either the maternal or fetal reservoir according to the transfer direction investigated, from the M→F or fetal to maternal (F→M), respectively. The initial concentration of vancomycin and telavancin in the donor circuit was 25 μg/mL, which corresponds to the therapeutic level of the antibiotics in patients’ plasma.
The perfusion system was used in its closed-closed configuration (recirculation of the medium). The concentrations of the compounds were determined in 0.5-mL aliquots taken from the maternal and fetal arteries and veins at 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 minutes. The amounts of [ 3 H] and [ 14 C] radioactivity in the maternal and fetal perfusates were determined simultaneously by liquid scintillation spectrometry (1900TR; Packard Instruments Inc, Shelton, CT). The concentration of vancomycin or telavancin in all samples was calculated after correcting for the specific activity as previously reported.
At the end of each experiment, the perfused area of the tissue was dissected, weighed, and homogenized in a volume of saline equal to 4 times its weight. A total of 1 mL of 1 mol/L sodium hydroxide was added to 1 mL of the homogenate and the samples were incubated for 12 hours at 60°C in the dark to allow for luminescence decay. Scintillation cocktail was added to each sample and the concentration of each drug was determined.
Effect of telavancin on the uptake of [ 3 H]-paclitaxel by the membrane vesicles
The method for preparing inside-out vesicles (IOV) from placental brush border membranes was modified in our laboratory as previously reported from that originally described by Ushigome et al. The effect of a range of telavancin concentrations (0-300 μmol/L) on the uptake of [ 3 H]-paclitaxel by IOV was determined in 140 μL of uptake buffer (40 mmol/L 3-(N-morpholino)propanesulfonic acid, 70 mmol/L potassium chloride, 10 mmol/L magnesium chloride, pH 7.4) containing 40 μg of membrane protein, and 40 nmol/L of [ 3 H]-paclitaxel. The reaction was initiated by the addition of adenosine triphosphate (ATP), and adenosine monophosphate (AMP) was used for control. The reaction was terminated after 1 minute by the addition of 3 mL of ice-cold buffer followed by rapid filtration (Brandel Cell Harvester; Brandel, Gaithersburg, MD) to isolate the vesicles. The amount of [ 3 H]-paclitaxel retained on the filter was determined by liquid scintillation analysis. Active uptake was calculated as the difference in the amount of tritium isotope [ 3 H]-paclitaxel in the presence of ATP and AMP and expressed as pmol.mgprotein —1 min —1 . The half maximal inhibitory concentration (IC 50 ) was determined from the plots of the percent of the [ 3 H]-paclitaxel uptake vs the log of telavancin concentration.
Data and statistical analysis
Statistical analyses were performed using GraphPad Prism, version 5.01 (GraphPad Software Inc, La Jolla, CA). All reported values are expressed as mean ± SD. Statistical significance of the differences observed between groups were calculated by the Mann-Whitney U test and considered significant when P < .05.
Results
Bidirectional transfer of AP
The bidirectional transfer of vancomycin and telavancin across term human placentas was normalized to the transfer of the highly diffusible marker compound AP. The transfer of AP coperfused with vancomycin in the M→F and F→M directions did not differ from its transfer when coperfused with telavancin. These data indicate that irrespective of direction, transplacental transfer of the 2 antibiotics was studied under the same experimental conditions, ie, the efficiency of the exchange between maternal and fetal circuits and vice versa was identical for vancomycin and telavancin.
M→F transfer of vancomycin and telavancin
Both antibiotics crossed the placenta and appeared in the fetal circuit within the first 5 minutes of perfusion. The concentration of vancomycin in the fetal circuit at the end of the experiment reached 2.4 ± 0.9 μg/mL and accounted for 9.6 ± 4% of its initial concentration in the maternal circuit. However, under the same experimental conditions the transfer of telavancin to the fetal circuit was lower (1.6 ± 0.3 μg/mL, 6.5 ± 2% of its initial concentration). The lower transfer of telavancin to the fetal circuit was also confirmed after transfer of each antibiotic was normalized to that of the marker compound AP ( Figure 1 , A). The normalized transfer of telavancin to the fetal circuit (0.14 ± 0.04) was significantly lower than normalized transfer of vancomycin (0.23 ± 0.08; P < .01). Furthermore, at the end of the experimental period the retention of telavancin by the perfused lobule (11 ± 1 μg/g) was higher than the retention of vancomycin (7.5 ± 1 μg/g; P < .001).
