Identification of glyburide metabolites formed by hepatic and placental microsomes of humans and baboons

doi:10.1016/j.bcp.2006.08.024

Biochemical Pharmacology

Volume 72, Issue 12, 15 December 2006, Pages 1730-1737

https://doi.org/10.1016/j.bcp.2006.08.024 Get rights and content

Abstract

Glyburide (glibenclamide) is a second-generation sulfonylurea used for treatment of type-2 and gestational diabetes mellitus. To date, two glyburide metabolites have been identified in maternal urine: namely, 4-trans-hydroxycyclohexyl glyburide and 3-cis-hydroxycyclohexyl glyburide. The use of glyburide to treat gestational diabetes prompted us to investigate its metabolism by the placenta. The metabolism of glyburide by microsomal preparations from human and baboon placenta was compared with metabolism by their livers. The metabolites formed by the microsomes of the four tissues were identified by high-performance liquid chromatography–mass spectrometry using retention times, ion current (extracted at m/z 510), and selected-ion monitoring. The data obtained revealed the formation of six distinct hydroxylated derivatives of glyburide by each of the four microsomal preparations. However, the amounts of the six metabolites formed by the placentas were a fraction of that formed by the livers. Moreover, the relative quantities of each metabolite formed differed between species as well as between the two tissues. Also, the structure of the unidentified metabolites was determined by comparison with synthesized standards. These metabolites were identified as the 4-cis-hydroxycyclohexyl glyburide, 3-trans-hydroxycyclohexyl glyburide, and 2-trans-hydroxycyclohexyl glyburide. Therefore, one glyburide metabolite remains to be identified, but the data we obtained allowed us to suggest its structure.

Introduction

Glyburide (glibenclamide; N-4-[β-(5-chloro-2-methoxybenzamidoethyl) benzenesulfonyl]-N′-[cyclohexyl] urea) is a second-generation sulfonylurea hypoglycemic drug that has been used successfully for controlling glucose levels in women with pregestational and gestational diabetes mellitus [1], [2], [3], [4], [5]. The pharmacokinetics (PK) of glyburide has been determined in healthy individuals [6], [7], [8], [9], patients with impaired renal function [10], and those with type-2 diabetes [11] but not for women with gestational diabetes mellitus. The physiological changes associated with the onset of pregnancy in addition to various diseases have a significant effect on the bio-disposition of administered therapeutics. One of the factors affecting the PK of a drug during pregnancy is its disposition in the placenta. The latter includes transfer of the drug to the fetal circulation, metabolism by placental enzymes, and efflux by transporters from the tissue back to the maternal circulation. Data obtained from in vivo [5] and in vitro [1], [3] investigations demonstrated a very low transplacental transfer of glyburide. Other related investigations included the effect of human serum albumin on glyburide transfer and distribution [12] as well as the role of efflux transporters [13], [14]. However, to the best of our knowledge, data on the metabolism of glyburide by placental enzymes are scarce to nonexistent.

Glyburide is extensively metabolized by the human liver and two major metabolites identified in the maternal urine: 4-trans-hydroxycyclohexyl glyburide and 3-cis-hydroxycyclohexyl glyburide. These two metabolites are pharmacologically active and are considered potent hypoglycemic agents [15], [16]. Recent reports indicated that microsomes of human, rat, dog, and monkey metabolized glyburide and, depending on the species, up to four new metabolites were formed [17], [18]. Currently, the baboon (Papio cynocephalus), a nonhuman primate, is being characterized as an animal model for investigating placental transfer and metabolism of drugs during pregnancy, which is not possible in humans due to safety considerations. During pregnancy, the human placenta plays an important role in the metabolism of endogenous compounds, xenobiotics, and environmental toxins [19]. However, the expression and activity of various P450 isoforms depends on placental gestational age and is lower than that in the liver [20], [21].

Therefore, the aim of this investigation is to identify the metabolites of glyburide formed by placental microsomes of a human and a nonhuman primate (the baboon) and compare them with those formed by liver microsomes of both species. To achieve this goal, we developed a method to determine the metabolites formed utilizing HPLC–mass spectrometry and described recently in a preliminary report [22] from our laboratory. The newly synthesized standards of the anticipated metabolites were used as an analytical tool to identify those formed in vitro by the four microsomal preparations, namely, human and baboon placentas and livers.

Section snippets

Chemicals and supplies

All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise mentioned. Acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ). Glyburide metabolites were synthesized according to the procedure reported by Hill et al. [23] and made available to our laboratory.

Human and baboon tissues

A pool of 15 donor human liver microsomes was purchased from Cellz Direct (Austin, TX). The pool was made up of livers from males and females between the ages of 24 and 74 years. Human placentas were

Extracted ion current for the six metabolites of glyburide formed by human and baboon liver microsomes

Each of the six glyburide metabolites is referred to in this text by an “M” followed by a number that corresponds to its elution order, i.e., M1 for the metabolite with lowest retention time, M2 for the metabolite with the next lowest retention time, and so on. The six metabolites of glyburide were formed upon incubation with human liver (Fig. 1a) and baboon liver (Fig. 1b) microsomes eluted with identical retention times, suggesting that they are most likely the same compounds. However, the

Discussion

Glyburide is a hypoglycemic drug that could be used for treatment of gestational diabetes; this drug is currently in clinical trial to determine its efficacy and pharmacokinetics. Human placental disposition of a drug is one of the factors affecting the changes in its PK observed during pregnancy. Therefore, the aim of this investigation was to identify the metabolites of glyburide formed by human placental microsomal preparations and compare them with those formed by hepatic microsomes.

Acknowledgements

This investigation was supported by the Obstetric-Fetal Pharmacology Research Units Network (OPRU/NICHD). The assistance of the following personnel is greatly appreciated: the medical staff of the labor and delivery ward of the John Sealy hospital; the Perinatal Research Division; the Publication, Grant, & Media Support Office in the Department of Obstetrics & Gynecology, University of Texas Medical Branch, Galveston, TX.

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Identification of glyburide metabolites formed by hepatic and placental microsomes of humans and baboons

Abstract

Introduction

Section snippets

Chemicals and supplies

Human and baboon tissues

Extracted ion current for the six metabolites of glyburide formed by human and baboon liver microsomes

Discussion

Acknowledgements

Semin Perinatol

Biochem Pharmacol

Am J Obstet Gynecol

J Chromatogr A

Pharmacol Ther

Biochem Pharmacol

Biochem Pharmacol

Bioorg Med Chem

Biochem Pharmacol

J Chromatogr

Insignificant transfer of glyburide occurs across the human placenta

Am J Obstet Gynecol

Comparative placental transport of oral hypoglycemic agents in humans: a model of human placental drug transfer

Am J Obstet Gynecol

Oral hypoglycemic drugs for gestational diabetes

N Engl J Med

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

N Engl J Med

Metabolism and kinetics of hypoglycemic agent glipizide in man—comparison with glibenclamide

J Clin Pharmacol