Elsevier

Biochemical Pharmacology

Volume 92, Issue 4, 15 December 2014, Pages 690-700
Biochemical Pharmacology

Identification of CYP3A7 for glyburide metabolism in human fetal livers

https://doi.org/10.1016/j.bcp.2014.09.025Get rights and content

Abstract

Glyburide is commonly prescribed for the treatment of gestational diabetes mellitus; however, fetal exposure to glyburide is not well understood and may have short- and long-term consequences for the health of the child. Glyburide can cross the placenta; fetal concentrations at term are nearly comparable to maternal levels. Whether or not glyburide is metabolized in the fetus and by what mechanisms has yet to be determined. In this study, we determined the kinetic parameters for glyburide depletion by CYP3A isoenzymes; characterized glyburide metabolism by human fetal liver tissues collected during the first or early second trimester of pregnancy; and identified the major enzyme responsible for glyburide metabolism in human fetal livers. CYP3A4 had the highest metabolic capacity towards glyburide, followed by CYP3A7 and CYP3A5 (Clint,u = 37.1, 13.0, and 8.7 ml/min/nmol P450, respectively). M5 was the predominant metabolite generated by CYP3A7 and human fetal liver microsomes (HFLMs) with approximately 96% relative abundance. M5 was also the dominant metabolite generated by CYP3A4, CYP3A5, and adult liver microsomes; however, M1–M4 were also present, with up to 15% relative abundance. CYP3A7 protein levels in HFLMs were highly correlated with glyburide Clint, 16α-OH DHEA formation, and 4′-OH midazolam formation. Likewise, glyburide Clint was highly correlated with 16α-OH DHEA formation. Fetal demographics as well as CYP3A5 and CYP3A7 genotype did not alter CYP3A7 protein levels or glyburide Clint. These results indicate that human fetal livers metabolize glyburide predominantly to M5 and that CYP3A7 is the major enzyme responsible for glyburide metabolism in human fetal livers.

Introduction

During pregnancy, 5–14% of women will be diagnosed with gestational diabetes mellitus (GDM) [1], [2]. Glyburide is an oral hypoglycemic drug that is prescribed more commonly for the treatment of GDM than before [3]. Though glyburide exhibits nearly comparable efficacy to insulin, the incidence rates of adverse fetal effects associated with glyburide verses insulin therapy, such as neonatal hypoglycemia and large for gestational age infants, are conflicting [4], [5], [6], [7], [8]. At present, there are no long-term safety data for infants whose mothers were treated with glyburide. In addition, studies are needed to understand the mechanistic determinants of fetal exposure that impact the fetal safety of glyburide.

Glyburide is extensively metabolized in the maternal liver by CYP3A4, CYP2C9, and CYP2C19 to several metabolites, such as 4-trans-hydroxycyclohexyl glyburide (M1), 4-cis-hydroxycyclohexyl glyburide (M2a), 3-cis-hydroxycyclohexyl glyburide (M2b), 3-trans-hydroxycyclohexyl glyburide (M3), 2-trans-hydroxycyclohexyl glyburide (M4), and ethylene-hydroxylated glyburide (M5); though there may be more [9], [10]. As treatment of GDM with glyburide is optimized, there will be several factors that could be important determinants of fetal exposure to glyburide and its metabolites, (and ultimately its safety profile), including maternal exposure, transplacental clearance, placental metabolism, and fetal elimination. Maternal exposure to conventional doses of glyburide during human pregnancy is reduced compared to non-pregnant women, suggesting the need for increased doses [11]. Glyburide concentrations, which are measurable in umbilical cord blood at the time of delivery, suggest that glyburide can cross the placenta [11]. ATP-binding cassette (ABC) efflux transporters, particularly breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) are highly expressed in the placenta and may limit fetal glyburide exposure to some extent [12], [13], [14], [15], [16], [17], [18]. Since the expression of ABC transporters in the placenta changes as gestation progresses [15] and the placenta grows larger with gestational age, penetration of glyburide into the fetal compartment across the placental barrier may also change over gestation. In addition, glyburide can be metabolized in human term placenta [9], [10], [19], [20], specifically by the cytochrome P450 (CYP) enzyme CYP19 (aromatase). Although the relative contribution of placental drug-metabolizing enzymes to the overall maternal disposition of glyburide may in fact be minimal, glyburide metabolism in such close proximity to the fetus could possibly affect fetal exposure to glyburide and its metabolites. Metabolite transplacental clearances have not been determined, however. Finally, fetal exposure to glyburide may also be influenced by fetal liver metabolism upon entrance into the fetal circulation. At present, little is known about glyburide metabolism in human fetal livers.

CYP3A7 is the predominant CYP enzyme in the human fetal liver [21], [22], [23]. CYP3A5 has also been found in the human fetal liver [24], but at much lower amounts relative to CYP3A7. In a panel of human fetal livers (n  10), the mRNA levels of CYP3A5 were approximately 700-fold lower than those of CYP3A7, and CYP3A5 protein was detected in just one liver (CYP3A5 genotype was not determined) [23]. Previous studies using adult human liver microsomes (HLMs) and recombinant enzymes have shown that glyburide is a substrate of CYP3A4 and CYP3A5 [9], [25], suggesting the plausibility of glyburide metabolism by other CYP3A isoforms such as CYP3A7. Indeed, CYP3A7 has been shown to metabolize endogenous substrates of CYP3A4, such as testosterone and dehydroepiandrosterone (DHEA), but with different oxidation site preferences [22], [26]. Several in vitro studies have also shown that both embryonic (less than 60 days) and fetal livers can metabolize endogenous compounds (i.e. testosterone, retinoic acid, and DHEA) and xenobiotics (i.e. warfarin, benzyloxyresorufin, and coumarin), as well as activate promutagens and procarcinogens [27], [28]. CYP3A7 preferentially metabolizes testosterone to its 2α–OH metabolite, rather than its 6β–OH metabolite, which is primarily produced by CYP3A4 [22], suggesting the metabolic profile of glyburide in human fetal livers could differ from that in adult livers. Given that glyburide can cross the placental barrier [11], [29] and that CYP3A7 expression/activity is variable in human fetal livers [22], it is important to investigate CYP3A7-mediated metabolism of glyburide and characterize the variability of glyburide metabolism in human fetal livers. Understanding fetal metabolism of glyburide is clinically relevant because this may govern fetal exposure to not only glyburide, but its major metabolites such as M1 and M2b, which are believed to be pharmacologically active [30].

In this study, we determined the kinetics of glyburide depletion by CYP3A4, CYP3A5, and CYP3A7 supersomes, measured the Clint of glyburide in human fetal liver microsomes (HFLMs), and compared the metabolite profiles generated by CYP3A supersomes, HFLMs, and HLMs. We examined the correlation between glyburide Clint and metabolism of DHEA (a known CYP3A7 probe substrate) as well as midazolam (a CYP3A substrate) in human fetal livers. To better understand sources of variability in fetal metabolism, we also investigated the relationship between glyburide Clint and CYP3A7 protein levels in human fetal livers, fetal liver gestational age, fetal sex, CYP3A7 genotype, and other fetal demographics. Results from this study will have important clinical implications for informing fetal exposure and the fetal safety profile of glyburide in early and mid-gestation.

Section snippets

Materials

Glyburide and glipizide (internal standard) were purchased from Sigma–Aldrich (St. Louis, MO). The following glyburide metabolites were purchased from TLC PharmaChem (Vaughan, Ontario, Canada): 4-trans-hydroxycyclohexyl glyburide (M1), 3-cis-hydroxycyclohexyl glyburide (M2b), and 3-trans-hydroxycyclohexyl glyburide (M3). 4-cis-hydroxycyclohexyl glyburide (M2a) was a generous gift from the University of Texas Medical Branch in Galveston. [Cyclohexyl-2,3-3H(N)]-glyburide ([3H]-Gly) (50 Ci/mmol)

Glyburide depletion kinetics of CYP3A supersomes

We first compared the kinetics of glyburide metabolism with recombinant CYP3A4, CYP3A5 and CYP3A7 supersomes. Substrate depletion kinetic parameters were estimated and the data are shown in Table 2. The kinetic profiles from a representative experiment are depicted in Fig. 1. Km and Vmax values were estimated from three independent experiments. The fraction unbound (fu) remained constant for each CYP3A isoform over 0.01–20 μM glyburide. The fu was 5–7% for CYP3A4 and CYP3A7 and ∼15% for CYP3A5 (

Discussion

Glyburide is frequently used to treat GDM. However, a major concern of pregnant women using glyburide is the safety of the drug for the developing fetus. It is critical that we understand the mechanisms that control fetal exposure to glyburide, such as placental penetration and placental and fetal metabolism of glyburide. Previous studies including those from our laboratory have identified that ABC transporters such as BCRP play an important role in limiting transfer of glyburide across the

Acknowledgements

We greatly acknowledge Drs. Abhinav Nath and Laura Shireman for their discussions and expertise regarding substrate depletion experimental design and data analysis. This study was supported in part by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) [Grant U10HD047892] and the National Center for Advancing Translational Sciences (NCATS) [Grant TL1 RR025016]. The content is solely the responsibility of the authors and does not necessarily represent the

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