In Vitro Metabolism and Identification of Human Enzymes Involved in the Metabolism of Methylnaltrexone

Abstract

ABSTRACT:Methylnaltrexone (MNTX) is a peripherally acting -opioid receptor antagonist and is currently indicated for the treatment of opioidinduced constipation in patients with advanced illness who are receiving palliative care, when response to laxative therapy has not been sufficient. Sulfation to MNTX-3-sulfate (M2) and carbonyl reduction to methyl-6␣-naltrexol (M4) and methyl-6␤-naltrexol (M5) are the primary metabolic pathways for MNTX in humans. The objectives of this study were to investigate MNTX in vitro metabolism in human and nonclinical species and to identify the human enzymes involved in MNTX metabolism. Of the five commercially available sulfotransferases investigated, only SULT2A1 and SULT1E1 catalyzed M2 formation. Formation of M4 and M5 was catalyzed by NADPH-dependent hepatic cytosolic enzymes, which were identified using selective chemical inhibitors (10 and 100 M) for aldo-keto reductase (AKR) isoforms, short-chain dehydrogenase/reductase including carbonyl reductase, alcohol dehydrogenase, and quinone oxidoreductase. The results were then compared with the effects of the same inhibitors on 6␤-naltrexol formation from naltrexone, a structural analog of MNTX, which is catalyzed mainly by AKR1C4. The AKR1C inhibitor phenolphthalein inhibited MNTX and naltrexone reduction up to 98%. 5␤-Cholanic acid 3␣,7␣-diol, the AKR1C2 inhibitor, and medroxyprogesterone acetate, an inhibitor of AKR1C1, AKR1C2, and AKR1C4, inhibited MNTX reduction up to 67%. Other inhibitors were less potent. In conclusion, the carbonyl reduction of MNTX to M4 and M5 in hepatic cytosol was consistent with previous in vivo observations. AKR1C4 appeared to play a major role in the carbonyl reduction of MNTX, although multiple enzymes in the AKR1C subfamily may be involved. Human SULT2A1 and SULT1E1 were involved in MNTX sulfation.

Preclinical Disposition (In Vitro) of Novel μ-Opioid Receptor Selective Antagonists

“…Reduction of naltrexone to 6␤-naltrexol was observed only in cytosolic fractions but not in microsomal fractions. Enzymes responsible for the metabolism of naltrexone to 6␤-naltrexol are aldo-keto reductase isoforms 1C4, 1C1, and 1C2 (Porter et al, 2000;Breyer-Pfaff and Nill, 2004;Tong et al, 2010). Both NAP and NAQ lack the ketone moiety at position 6 of the morphinan ring structure and are hence not expected to be reduced to the corresponding alcohol.…”

“…The intrinsic clearance (V max /K m ) for the formation of 6␤-naltrexol from naltrexone in human liver cytosolic fractions has been reported to be 12.6 and 5 to 36.7 l ⅐ min Ϫ1 ⅐ mg protein Ϫ1 (Porter et al, 2000;Tong et al, 2010). To obtain an estimate of the hepatic clearance (CL hep ), these values of intrinsic clearance were scaled up using the same method used for NAP and NAQ (metabolism calculations in Data Analysis).…”

Involvement of microsomal NADPH‐cytochrome P450 reductase in metabolic reduction of drug ketones

“…For the reduction of ketone functions of xenobiotics in the organism, mainly two classes of enzymes are thought to be responsible, namely the AKRs and the SDRs [3][4][5]. Therefore, the effect of published inhibitors of these enzymes [10,11] on the reduction of compound 5 was determined in this former study [9]. Since the AKR inhibitors flufenamic acid and phenolphthalein did not affect the formation of the alcohol 6 from ketone 5 (Table 1), a contribution of AKRs in this metabolic reaction was ruled out.…”

“…In rat liver S9 fractions, compound 5 was even completely metabolized to the alcohol 6. Here, cytosolic SDRs (cSDRs) are held mainly to be responsible for this reaction, because the keto-reduction could be inhibited by certain compounds, which were reported in the literature to be inhibitors of cSDR enzymes [10,11].…”

Effect of Experimental Kidney Disease on the Functional Expression of Hepatic Reductases

“…Figure 1 depicts the classes of reductase enzymes and the corresponding chemical inhibitors used in this study. The inhibitors and concentrations (10 and 100 mM) used and their selectivity for reductase enzymes were based on the previous reports as follows: quercetin for cytosolic CBRs (Hermans and Thijssen, 1992;Tong et al, 2010); flufenamic acid for cytosolic AKRs (Atalla et al, 2000;Molnari and Myers, 2012); indomethacin for both cytosolic CBRs and AKRs (Usami et al, 2001;Tong et al, 2010); dicoumarol for cytosolic NQO1 (Tong et al, 2010); and 18b-glycyrrhetinic acid for microsomal 11b-HSD1 (Breyer-Pfaff et al, 2004;Molnari and Myers, 2012). All inhibitors were dissolved in a mixture of dimethylsulfoxidemethanol (50:50, v/v) except for dicoumarol, which was prepared in 1% sodium Fig.…”