Aromatase Is the Major Enzyme Metabolizing Buprenorphine in Human Placenta SUJAL V. DESHMUKH, TATIANA N. NANOVSKAYA, and MAHMOUD S. AHMED Department of Obstetrics & Gynecology, University of Texas Medical Branch, Galveston, Texas Received April 18, 2003; accepted June 5, 2003 ABSTRACT Buprenorphine (BUP) is a partial opiate agonist used for treat- ment of the adult and the pregnant addicted to this class of narcotics. The kinetic parameters for transplacental transfer and the metabolism of BUP during its perfusion in a placental lobule were the subject of an earlier report from our laboratory. The aim of this investigation is to identify and characterize the enzyme catalyzing the metabolism of BUP in term human pla- centa. Norbuprenorphine (norBUP) is the only metabolite formed as determined by high performance liquid chromatog- raphy and mass spectrometry. The activity of the enzyme re- sponsible for BUP metabolism is highest in the microsomal fraction and lowest in the cytosolic, with the mitochondrial in between. Compounds with selective affinity to the enzyme aromatase (CYP 19), namely 4-hydroxyandrostenedione and aminoglutethimide, caused 70% inhibition of norBUP forma- tion. Monoclonal antibodies raised against CYP 19 were the most potent inhibitors of BUP dealkylation. A comparison be- tween the data obtained from the saturation isotherm for BUP dealkylation by placental microsomes and a commercially available system of cDNA-expressed CYP 19 indicated similar kinetic parameters, with apparent K m values of 12  4.0 and 14  8.0 M, respectively. Therefore, aromatase is the major enzyme catalyzing the biotransformation of BUP to norBUP in term human placentas obtained from healthy pregnancies. The minor involvement of other cytochrome P450 isoforms or en- zyme(s) in the metabolism of BUP in placental tissue cannot be ruled out. Methadone is, and has been for decades, the drug of choice in treatment of the adult and the pregnant opiate addict. In the last decade, data obtained from clinical trials on treat- ment of the pregnant woman with BUP indicated that the drug was well tolerated by the patient and that the incidence of neonatal abstinence was low to nonexistent (Marquet et al., 1997; Fischer et al., 2000; Eder et al., 2001), suggesting that BUP might be an alternative to methadone for mainte- nance of this patient population. BUP levels in the fetal circulation can have a direct effect on its normal growth and development. On the other hand, maternal circulating levels of the opiate could affect placen- tal physiology and, consequently, the fetus. The kinetics for transplacental transfer of BUP, its concentration in the fetal circuit, and effects on placental functions were determined in our laboratory utilizing the technique of dual perfusion of placental lobule (Nanovskaya et al., 2002). Our data indi- cated that the tissue accumulated 13 times the amount of BUP present in the maternal circuit, and less than 5% of it was metabolized to norBUP. However, in the perfusion sys- tem mentioned, BUP was delivered in the intervillous space, thus bypassing the maternal myometrium and endometrium, and its access to the metabolizing enzymes was less. There- fore, it is likely that in vivo metabolism of BUP in the pla- centa is higher. In humans, BUP is biotransformed to norBUP by hepatic enzymes during first pass metabolism. The major enzyme responsible for N-dealkylation of BUP to norBUP in the liver is cytochrome P450 3A4 (CYP 3A4) (Iribarne et al., 1997; Kobayashi et al., 1998). The formation of the glucuronide conjugates of BUP and norBUP was also reported (Cone et al., 1984). During pregnancy, the human placenta plays an important role in the metabolism of endogenous compounds, xenobiotics, and environmental pollutants (Juchau 1980; Contractor, 1983; Nandakumaran et al., 1983, Blanck et al., 1983; Roe et al., 1990; Pienimaki et al., 1997). The expression and activity of various P450 isoforms depends on placental gestation age and tissue maturity but is lower than that in the liver (Hakkola et al., 1996a,b). Aromatase/CYP 19 is one of the cytochrome P450 isozymes with a well established role in the biosynthesis of steroids in human placenta (Thompson Supported by a grant from the National Institute on Drug Abuse to M.S.A. (DA 13431). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.053199. ABBREVIATIONS: BUP, buprenorphine; norBUP, norbuprenorphine; P450, cytochrome P450; EFC, 7-ethoxy-4-trifluoromethylcoumarin; HFC, 7-hydroxy-4-trifluoromethylcoumarin; LC/MS, liquid chromatography/mass spectrometry; HPLC, high-performance liquid chromatography; ANOVA, analysis of variance. 0022-3565/03/3063-1099 –1105$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 306, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 53199/1088983 JPET 306:1099–1105, 2003 Printed in U.S.A. 1099 at ASPET Journals on August 2, 2017 jpet.aspetjournals.org Downloaded from