Objective
Substrates of placental efflux transporters could compete for a single transporter, which could result in an increase in the transfer of each substrate to the fetal circulation. Our aim was to determine the role of placental transporters in the biodisposition of oral hypoglycemic drugs that could be used as monotherapy or in combination therapy for gestational diabetes.
Study Design
Inside-out brush border membrane vesicles from term placentas were used to determine the efflux of glyburide, rosiglitazone, and metformin by P-glycoprotein, breast cancer resistance protein, and multidrug resistance protein.
Results
Glyburide was transported by multidrug resistance protein (43 ± 4%); breast cancer resistance protein (25 ± 5%); and P-glycoprotein (9 ± 5%). Rosiglitazone was transported predominantly by P-glycoprotein (71 ± 26%). Metformin was transported by P-glycoprotein (58 ± 20%) and breast cancer resistance protein (25 ± 14%).
Conclusion
Multiple placental transporters contribute to efflux of glyburide, rosiglitazone, and metformin. Administration of drug combinations could lead to their competition for efflux transporters.
Normal pregnancy is associated with metabolic changes leading to decreased insulin sensitivity and reduced glucose tolerance, however, 3-5% of pregnant women proceed to develop gestational diabetes mellitus (GDM). Treatment of GDM includes dietary regulation, exercise, home blood glucose monitoring, and in some cases, pharmacotherapy with insulin or oral hypoglycemic agents. However, metabolic changes occurring during pregnancy to accommodate the development of the fetoplacental unit usually alter the pharmacokinetics of administered medications, thus requiring dose adjustment. One of the goals of the Obstetric-Fetal Pharmacology Research Unit (OPRU) is to investigate the pharmacokinetics of oral hypoglycemic agents during pregnancy. As a center of the OPRU, our laboratory is investigating the role of the human placenta in the disposition of the oral hypoglycemic drugs: glyburide, metformin, and rosiglitazone.
Placental disposition of a drug depends on many factors, including its physicochemical properties (charge, molecular weight, and protein binding), physiological properties of the placenta (blood flow and gestational age), the expression and activity of metabolizing enzymes, and efflux transporters. In recent years, a large number of transporters were identified in human placental syncytiotrophoblast. In general, efflux transporters localized in the apical membranes of the syncytiotrophoblast extrude their substrates (endogenous compounds or medications) from the fetoplacental unit to the maternal circulation thus decreasing fetal exposure. P-glycoprotein (P-gp; ABCB1 gene product), multidrug resistance-associated protein 1 (MRP1; ABCC1 gene product), and the breast cancer resistance protein (BCRP; ABCG2 gene product) are highly expressed in placental tissue. The activities of P-gp, BCRP, and MRP1 in the fetal-to-maternal efflux of substrates across the apical membrane appear to have a major role in protecting the fetus from exposure to xenobiotics and endogenous metabolites present in the maternal circulation.
P-gp, BCRP, and MRP1 have wide and overlapping substrate specificity, and drugs—if coadministered—could compete with each other for 1 or more of the transporters. Therefore, human placental disposition of a drug and its efflux in the fetal-to-maternal direction may differ depending on whether it is used as monotherapy or in combination therapy. The use of combinations of oral hypoglycemic drugs for the management of type 2 diabetes mellitus (DM) proved to be effective. For example, glucovance, a combination of metformin and glyburide, is considered safe for use during pregnancy. Furthermore, in patients with inadequate glycemic control despite established glyburide/metformin therapy, the addition of rosiglitazone improves glucose tolerance. Because of their effectiveness in the nonpregnant patient, it is plausible that the coadministration of glyburide, rosiglitazone, and metformin may be a therapeutic option for pregnant patients with uncontrolled GDM.
The transplacental transfer of glyburide, rosiglitazone, and metformin have been individually documented using dual perfusion of human placental lobule (DPPL). Asymmetric placental transfer of these drugs favoring fetal-to-maternal transport and/or low placental tissue accumulation revealed during DPPL suggest the involvement of efflux transporters in their distribution. Indeed, recent investigations indicated a role for placental ABC transporters in the transplacental distribution of glyburide. However, it remains unclear whether glyburide, rosiglitazone, and metformin are transported by 1 or more of the same placental transporters. Two medications transferred by the same transporter could, in the case of combination therapy, introduce competition for their efflux and consequently increase their concentration in the fetal circulation. Thus, the aim of this investigation was to identify the involvement of human placental ABC transporters responsible for the efflux of glyburide, rosiglitazone, and metformin.
Materials and Methods
Chemicals
[ 3 H]-rosiglitazone (specific activity, 50 Ci/mmol) and [ 14 C]-metformin (specific activity, 50 mCi/mmol) were purchased from American Radiolabeled Chemicals, Inc (St. Louis, MO), and [ 3 H]-glyburide (specific activity, 44.6 Ci/ mmol) from Perkin-Elmer (Boston, MA). All other chemicals were purchased from Sigma-Aldrich (Dallas, TX) unless otherwise noted.
Clinical material
Placentas from uncomplicated term pregnancies were obtained immediately after vaginal or abdominal deliveries from the labor and delivery ward of the University of Texas Medical Branch, Galveston, TX, according to a protocol approved by the institutional review board (IRB 02-106). Any evidence or history of maternal infection, systemic diseases, and drug or alcohol abuse during pregnancy excluded the placenta from this investigation.
Preparation of placental brush border inside-out vesicles
Placental brush border membrane inside-out vesicles (IOVs) were prepared according to a previously reported method. Tissue was dissected from the maternal side and rinsed twice in 0.9% NaCl, transferred to sucrose-HEPES-Tris (SHT) buffer (250 mM sucrose, 10 mM HEPES-Tris, pH 7.4), and stirred for 1 hour to disrupt brush border membranes (all steps in preparation were carried out at 4°C). The tissue lysate was filtered through 2 layers of woven cotton gauze, and the tissue was discarded. The brush border membrane was isolated using differential centrifugation and resuspended in SHT buffer with a 26-gauge needle. To maximize the proportion of IOVs, affinity chromatography was used to separate right-side out vesicles (ROVs) according to a method previously reported from our laboratory.
Vesicles were aliquoted and immediately stored at −80°C until use. ATP-dependent transport activity was verified an aliquot from each placental preparation, and those with low or no detectible ATP-dependent transport after thawing were excluded from the pool. The pool was prepared using membrane preparations of 60 placentas obtained from uncomplicated term pregnancies. The large pool size reduces the confounding variable of interindividual variation in the activity of transporters, and provides multiple and long-term use of the same lot of membranes.
Uptake by membrane vesicles
The activity of placental efflux transporters was determined by uptake of the radiolabeled isotopes of [ 3 H]-glyburide, [ 3 H]-rosiglitazone, and [ 14 C]-metformin, by placental IOVs according to a previously reported protocol. Each reaction was carried out in buffer (250 mM sucrose, 10 mM HEPES-Tris) containing 4 mM MgCl 2 , 10 mM creatine phosphate, 100 μg/mL creatine phosphokinase, either 2 mM ATP or 3 mM NaCl, and placental IOVs at a concentration of 0.05 μg/μL (7 μg total protein). The reaction was initiated by the addition of drug ([ 3 H]-glyburide, [ 3 H]-rosiglitazone, or [ 14 C]-metformin), at a final concentration of 100 nM. The concentration of 100 nM was chosen based on placental tissue concentration of glyburide and rosiglitazone determined from perfusion experiments. The concentration of metformin was selected in the nanomolar range because organic cation transporters of the placenta are known to transport metformin in the micromolar range and we were aiming to minimize interference from these transporters. The reaction was terminated after 1 minute by the addition of 1 mL ice-cold buffer; the 1 minute time point was selected from the initial linear portion of the time-dependent transport curve of P-gp substrate, paclitaxel, in placental brush border membrane vesicle preparations. Vesicles were isolated by rapid filtration using a Brandel Cell Harvester (Brandel, Gaithersburg, MD), and the amount of radiolabeled substrate retained on the filter was determined by liquid scintillation analysis. Active transport was calculated as the difference in the amount of drug uptake in the presence and absence of ATP and expressed as pmol/mg protein × minute. P-gp-mediated transport (T P-gp ) was determined by the addition of P-gp inhibitor, verapamil (600 μM). BCRP-mediated transport (T BCRP ) was determined by the addition of BCRP-selective inhibitor, 25 nM KO143. MRP1-mediated transport (T MRP1 ) was determined by the addition of MRP1 inhibitor, indomethacin (100 μM). Total ABC protein-mediated transport (T ABC ) was determined for P-gp, BCRP, and MRP1 using 1 μM KO143.
The effect of metformin on rosiglitazone transport by P-gp was determined using varying concentrations of cold metformin measuring its inhibition of [ 3 H]-rosiglitazone uptake by placental IOVs.
Results
Glyburide transport
The total ATP-dependent uptake of [ 3 H]-glyburide, at its concentration of 100 nM, by placental IOVs was 3.2 ± 0.3 pmol/mg protein × minute. Inhibition of P-gp by verapamil decreased [ 3 H]-glyburide uptake by 9 ± 5%. Inhibition of BCRP by 25 nM KO143 decreased [ 3 H]-glyburide uptake by 25 ± 5%. Inhibition of MRP1 by indomethacin decreased [ 3 H]-glyburide uptake by 43 ± 4%. Total inhibition of P-gp, BCRP, and MRP1 using 1 μM KO143 decreased [ 3 H]-glyburide uptake by 78 ± 4%. The contributions of each transporter to the total efflux were MRP1>BCRP>P-gp ( Figure 1 , A).
