Elsevier

Biochemical Pharmacology

Volume 68, Issue 3, 1 August 2004, Pages 583-591
Biochemical Pharmacology

Methadone metabolism by human placenta

https://doi.org/10.1016/j.bcp.2004.04.011Get rights and content

Abstract

Methadone pharmacotherapy is considered the standard for treatment of the pregnant heroin/opioid addict. One of the factors affecting the transfer kinetics of opioids across human placenta and their levels in the fetal circulation is their metabolism by the tissue. The aim of this investigation is to identify the enzyme(s) responsible for the metabolism of methadone, determine the kinetics of the reaction and the metabolites formed utilizing placental tissue obtained from term healthy pregnancies. Microsomal fractions of trophoblast tissue homogenates had the highest activity in catalyzing the metabolism of methadone. The product formed was identified by HPLC-UV as 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Inhibitors selective for cytochrome P450 (CYP) isozymes were used to identify the enzyme catalyzing the biotransformation of methadone. Aminoglutethimide and 4-hydroxyandrostenedione inhibited EDDP formation by 88 and 70%, respectively, suggesting that CYP19/aromatase is the enzyme catalyzing the reaction. This was confirmed by the effect of monoclonal antibodies raised against CYP19 that caused an 80% inhibition of the reaction. The apparent Km and Vmax values for the CYP19 catalyzed metabolism of methadone to EDDP were 424 ± 92 μM and 420 ± 89 pmol (mg protein)−1 min−1, respectively. Kinetic analysis of a cDNA-expressed CYP19 for the metabolism of methadone to EDDP was identical to that by placental microsomes. Taken together, these data indicate that CYP19/aromatase is the major enzyme responsible for the metabolism of methadone to EDDP in term human placentas obtained from healthy pregnancies.

Introduction

Methadone is the only approved pharmacotherapy for treatment of the pregnant heroin/opioid addict. Its use, since the late 1960s, has been the subject of numerous clinical trials, reports, and review articles that, for the most part, agree on the benefits of methadone maintenance programs in improving maternal and neonatal outcome. A review of this literature would be out of the scope of this manuscript.

Methadone is a synthetic, long-acting μ-agonist that undergoes first pass metabolism by its sequential N-demethylation to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP) as shown (Fig. 1). Both EDDP and EMDP do not have analgesic activity [1]. Cytochrome P450 (CYP) 3A4 was identified as the major enzyme catalyzing the metabolism of methadone in human liver and intestine though the involvement of other CYP isoforms (namely CYP2D6, CYP1A2, CYP2C9, and CYP2C19) was not ruled out [2], [3], [4], [5].

Several reports indicated that the metabolism of methadone in pregnant women is different from the non-pregnant. The differences were attributed to the changes in maternal physiology that accompany the onset of pregnancy to accommodate the growth and development of the feto-placental unit. During pregnancy, the placenta functions as an additional extra-hepatic site for biotransformation of drugs though the amount and activity of its metabolic enzymes is lower than that in the liver [6], [7], [8], [9]. Therefore, the metabolic activity of the placenta could contribute to the changes in the pharmacokinetic-pharmacodynamic profile (bio-disposition) of drugs administered to the pregnant women. Indeed, methadone plasma concentrations during the second and third trimesters were lower than after delivery and elimination of methadone in pregnant patients was significantly more rapid than in the non-pregnant [10], [11]. These reports suggested that biotransformation of methadone during pregnancy may be accelerated as a result of enhanced maternal and fetal hepatic metabolism and (or) biotransformation of the drug by the placenta. As a result, the dose of methadone during pregnancy may have to be adjusted to enhance compliance and safety of the mother and newborn [10], [11].

Pregnant women in methadone maintenance programs may require therapeutic interventions due to infections or other pregnancy-associated complications that dictate consideration of drug interactions. Changes in methadone clearance were observed in patients treated with cimetidine, erythromycin, ketoconazole, rifampin, and barbiturates [12], [13]. Other sites for drug interactions can include key enzymes in placental biosynthesis of steroid hormones namely, CYP11A1 and CYP19, that are also involved in the metabolism of xenobiotics [14], [15], buprenorphine, and l-α-acetylmethadol (LAAM) [16], [17].

Taken together, these reports emphasize the need for information on the enzyme metabolizing methadone in human placenta and the products formed. This information will also allow a comparison of placental bio-disposition of methadone and buprenorphine as the latter is emerging as an alternative to treatment of the pregnant opioid-dependent woman. Therefore, the goal of this investigation is to identify and characterize the enzyme responsible for the metabolism of methadone in term human placenta.

Section snippets

Chemicals and other supplies

All chemicals were purchased from Sigma Chemical Co. unless otherwise mentioned. Acetonitrile was purchased from Fisher Scientific Co. Methadone, its metabolites, and l-α-acetylmethadol (LAAM) were a gift from the National Institute on Drug Abuse drug supply unit. Monoclonal antibodies to CYP isoforms were a generous gift from Dr. Andrew Parkinson (Xenotech LLC). Rabbit antiserum to human placental aromatase was purchased from Hauptman–Woodward Institute. Properties of the antibodies and their

Methadone metabolite(s)

Separation of methadone and its metabolite EDDP was achieved by HPLC according to the method described. A chromatogram showing the retention times of the opioid and its metabolite is presented in Fig. 2A. Aliquots of the reaction supernatant containing methadone and the microsomal fraction in absence (Fig. 2B) and presence (Fig. 2C) of the NADPH-regenerating system are shown. The chromatogram revealed the formation of the metabolite EDDP (14 min) in the presence of NADPH suggesting that a CYP

Discussion

Methadone maintenance programs are considered the standard for pharmacotherapy of the heroin/opioid addict, including the pregnant. However, information on its bio-disposition by human placenta is scarce to nonexistent. This report provides information on the identification of the major enzyme responsible for the metabolism of methadone in term placentas obtained from healthy pregnancies without obstetrical complications.

Microsomal preparations from human liver catalyzed the N-demethylation of

Acknowledgements

The authors appreciate the support of the National Institute on Drug Abuse drug supply program for providing methadone, EDDP and, LAAM. We also thank Dr. Andrew Parkinson of XenoTech LLC for his generous gift of monoclonal antibodies. We greatly appreciate the assistance of the medical staff, the Chairman’s Research Group, and Publication, Grant, & Media Support Office of the Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas. Supported by a grant from

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