Transport of Glyburide by Placental ABC Transporters: Implications in Fetal Drug Exposure

doi:10.1016/j.placenta.2005.11.012

Placenta

Volume 27, Issues 11–12, November–December 2006, Pages 1096-1102

https://doi.org/10.1016/j.placenta.2005.11.012 Get rights and content

Abstract

Much evidence has demonstrated that a number of ATP-binding cassette (ABC) efflux transporters including P-glycoprotein (PGP), the multidrug resistance-associated proteins (MRPs) and the breast cancer resistance protein (BCRP) are highly expressed in placental tissues and are believed to profoundly limit the passage of therapeutic or toxic xenobiotics to the fetus. Recent studies indicate that the oral hypoglycemic glyburide does not cross the human placenta to an appreciable extent. Our objective was to identify placental transporters potentially involved in limiting the transplacental transfer of glyburide to the fetus. Thus, [³H]-glyburide transport was examined in BCRP, PGP, MRP1, MRP2 and MRP3 over-expressing cell lines in the presence or absence of specific inhibitors. Our results demonstrated significant increases in the intracellular accumulation of [³H]-glyburide in BCRP and MRP3 over-expressing cells in the presence of the inhibitors novobiocin and indomethacin, respectively. PGP inhibition with verapamil or MRP inhibition with indomethacin did not affect [³H]-glyburide accumulation in the PGP or MRP2 over-expressing cell lines and only limited changes were seen in the MRP1 over-expressing cell line. On the other hand, glyburide was found to significantly inhibit MRP1-, MRP2- and MRP3-mediated efflux of 5-carboxyfluorescein diacetate and PGP-mediated transport of rhodamine 123. Our evidence is the first to clearly indicate that glyburide is preferentially transported by BCRP and MRP3.

Introduction

The placenta plays an important role as a site for nutrient and waste exchange between the mother and the fetus and may serve as a protective barrier for the fetus against maternal blood-borne toxins. Much evidence has demonstrated that the placenta expresses a number of transporters, some of which function in the removal of endogenous compounds and xenobiotics. Specifically, several of the ATP-binding cassette (ABC) transporters, P-glycoprotein (PGP), the multidrug resistance-associated proteins 1–3 (MRP1, MRP2, MRP3) and the breast cancer resistance protein (BCRP) are highly expressed in placental tissues [20]. It is believed that these transporters may profoundly limit the passage of therapeutic or toxic xenobiotics into the fetus. Fundamental studies in PGP and BCRP deficient mice, demonstrating increased fetal drug accumulation and increased teratogenicity, have clearly established the importance of placental transporters in fetal drug exposure [9], [10]. However, to date the impact of transporters on fetal exposure has been studied for only a few xenobiotics.

Gestational diabetes affects up to 20% of pregnant women. Untreated, it is associated with serious increases in perinatal risks [1]. The mainstay of management is diet and insulin injections. However, many women do not comply with insulin injections and the cost of insulin is prohibitive in many countries. Due to fears of neonatal hypoglycemia, there has been only limited use of oral hypoglycemics in pregnancy. Interestingly a drug used to treat gestational diabetes, glyburide (also known as glibenclamide), does not appear to demonstrate significant maternal to fetal transfer. In vitro studies by Elliot et al. [28] demonstrated negligible levels of glyburide in the fetal compartment even when maternal concentrations were eight times greater than therapeutic concentrations. Moreover, offspring of mothers treated with glyburide reportedly do not exhibit hypoglycemia or other adverse events [1]. Glyburide's poor maternal to fetal transfer may be due to its high protein binding (99.8%) but it may also be due to interaction of glyburide with ABC efflux transporters expressed within the placenta [11], [23]. Recently, we have demonstrated in placental perfusion studies that glyburide is effluxed against its concentration gradient from the fetal to the maternal circulation, and that inhibition of PGP activity with verapamil does not impact its transport across the placenta [23].

Presently, there is little knowledge on the mechanisms by which glyburide is transported across epithelial cell membranes and there is a lack of clinically-relevant studies regarding its transport. Although it has been recently shown by Payen et al. [18] that glyburide inhibits MRP1 and MRP2 activity in human cancer cells, the transport of glyburide itself has not been characterized. Hence, the objective of the present study was to identify placental transporters which are involved in the transport of glyburide in the context of understanding its efflux from the fetus to the mother.

Section snippets

Materials

5-Carboxyfluorescein diacetate (5-CFDA), verapamil, novobiocin, rhodamine 123 (Rh123), indomethacin, mitoxanthrone and tissue-culturing plates were purchased from Sigma–Aldrich (Toronto, ON). Culture media, trypsin, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Canadian Life Technologies (Hamilton, ON). [³H]-Glyburide (specific activity = 44.6 Ci/mmol) was purchased from Perkin Elmer (Toronto, ON).

Tissue culture

PGP over-expressing cells A2780/Adr (ECACC, UK) and BCRP over-expressing

[³H]-Glyburide transport

Inhibition of BCRP by novobiocin resulted in pronounced increases in the intracellular accumulation of [³H]-glyburide in BCRP over-expressing MCF7-MX cells (p = 0.008, Fig. 1). Significant increases were seen in the uptake of [³H]-glyburide for all time points in novobiocin-treated cells, as compared to controls, resulting in an overall 3-fold increase in the total intracellular accumulation over the 60 min time period (AUC_0–60). Likewise, inhibition of MRP activity with indomethacin resulted in

Discussion

Glyburide is a second generation sulfonylurea that has been shown to effectively treat gestational diabetes while exhibiting marginal placental transfer [1]. Indeed, in a recent placental perfusion study we have documented that glyburide is transferred from the fetal to the maternal circulation against its concentration gradient [23]. To elucidate mechanisms involved in this phenomenon, we examined the potential involvement of several highly expressed placental ABC transporters in glyburide

Acknowledgements

G.K. and M.P.M. are supported by grants from CIHR. G.K. hold the Ivey chair in Molecular Toxicology at the University of Western Ontario. C.G. is a recipient of the Ontario Graduate Scholarship.

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Transport of Glyburide by Placental ABC Transporters: Implications in Fetal Drug Exposure

Abstract

Introduction

Section snippets

Materials

Tissue culture

[3H]-Glyburide transport

Discussion

Acknowledgements

Int J Biochem Cell Biol

Reprod Toxicol

J Biol Chem

Placenta

Adv Enzyme Regul

Am J Obstet Gynecol

A comparison of glyburide and insulin in women with gestational diabetes mellitus

N Engl J Med

Suppression of drug metabolizing enzymes and efflux transporters in the intestine of endotoxin-treated rats

Drug Metab Dispos

Camptothecin resistance: role of the ATP-binding cassette (ABC) mitoxantrone-resistance half-transporter and potential for glucuronidation in MXR-expressing cells

Cancer Res

Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone and topotecan-resistant cell lines

Cancer Res

Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2)

Oncogene

Subcellular localization and distribution of breast cancer resistance protein transporter in normal human tissues

Cancer Res

Multidrug resistance transporters and modulation

Curr Opin Oncol

Absence or pharmacological blocking of placental P-glycoprotein profoundly increases fetal drug exposure

J Clin Invest

[³H]-Glyburide transport