Metoclopramide is a widely used clinical drug in a variety of medical settings with rare acute dystonic events reported. The aim of this study was to assess a previous report of inactivation of CYP2D6 by metoclopramide, to determine the contribution of various CYPs to metoclopramide metabolism, and to identify the mono-oxygenated products of metoclopramide metabolism. Metoclopramide interacted with CYP2D6 with Type I binding and a Ks value of 9.56 ± 1.09 μM. CYP2D6 was the major metabolizer of metoclopramide and the two major products were N-deethylation of the diethyl amine and N-hydroxylation on the phenyl ring amine. CYPs 1A2, 2C9, 2C19, and 3A4 also metabolized metoclopramide. While reversible inhibition of CYP2D6 was noted, CYP2D6 inactivation by metoclopramide was not observed under conditions of varying concentration or varying time using Supersomes™ or pool human liver microsomes. The major metabolites of metoclopramide were N-hydroxylation and N-deethylation formed most efficiently by CYP2D6 but also formed by all CYPs examined. Also, while metoclopramide is metabolized primarily by CYP2D6, it is not a mechanism-based inactivator of CYP2D6 in vitro.

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of more than half of pharmaceutic drugs, and inactivation of CYP3A4 can lead to adverse drug-drug interactions. The substi-(EMTPP) have been previously identified as mechanism-based inactivators (MBI) of CYP2D6. The present study shows that both SCH 66712 and EMTPP are also MBIs of CYP3A4. Inhibition of CYP3A4 by SCH 66712 and EMTPP was determined to be concentration, time, and NADPH dependent. In addition, inactivation of CYP3A4 by SCH 66712 was shown to be unaffected by the presence of electrophile scavengers. SCH 66712 displays type I binding to CYP3A4 with a spectral binding constant (K s ) of 42.9 6 2.9 mM. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. Whole protein mass spectrum analysis revealed 1:1 binding stoichiometry of SCH 66712 and EMTPP to CYP3A4 and a mass increase consistent with adduction by the inactivators without addition of oxygen. Heme adduction was not apparent. Multiple monooxygenation products with each inactivator were observed; no other products were apparent. These are the first MBIs to be shown to be potent inactivators of both CYP2D6 and CYP3A4.

CYP2D6 is a major drug metabolizing enzyme responsible for the metabolism of ~20% of pharmaceutical drugs. Inactivation of CYP2D6 is rare, but ‐ due to its importance in drug metabolism, can be clinically significant. Thus, understanding of the mechanisms of inactivation of CYP2D6 is important for devising strategies to avoid undesirable drug‐drug interactions. Metoclopramide is a drug that has previously been reported to be a mechanism‐based inactivator of CYP2D6. In order to better understand possible inactivation of CYP2D6 by metoclopramide, we expanded these initial findings by determining the spectral binding constant, partition ratio, metabolite profile, enzyme activity after an inactivation phase, and metabolite profile. While we did observe reversible competitive inhibition, our studies did not show metoclopramide to be an inactivator of CYP2D6 (Support: NIH 1R15‐GM086767–01 & ‐01S1).

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of over fifty percent of pharmaceutical drugs on the market today. Inhibition of CYP3A4 can lead to adverse drug‐drug interactions. 5‐Fluoro‐2‐[4‐[(2‐phenyl‐1H‐imidazol‐5‐yl)methyl]‐1‐piperazinyl]pyrimidine (SCH66712) has been previously identified as a mechanism based inactivator (MBI) of CYP2D6 and preliminarily data also suggested it could be an inactivator of CYP3A4 [Palamanda, Casciano, Norton, Clement, Favreau, Lin, Nomeir (2001) Drug Metab Dispos 29, 863‐867]. In the current study SCH66712 was shown to be an MBI of CYP3A4. Inhibition of CYP3A4 by SCH66712 was determined to be concentration‐, time‐ and NADPH‐dependent. In addition, inactivation of CYP3A4 by SCH66712 was shown to be unaffected by the presence of electrophile scavengers. SCH66712 displays type II binding to CYP3A4 with a spectral binding constant (Ks) of 43 ± 2 μM. The partition ratio was determined to be 11, suggesting SCH66712 is a potent MBI of CYP3A4. Analysis of possible mechanism of inactivation are also described. Grant Funding Source: Supported by NIH 1R15‐GM086767‐02

Cytochrome P450s (CYPs) are heme‐containing enzymes that metabolize small organic molecules including drugs. CYP3A4 and CYP2D6 are responsible for more than 70% of pharmaceutical drug metabolism and inactivation of these enzymes can lead to drug‐drug interactions. The substituted imidazole compounds, 5‐fluoro‐2‐[4‐[(2‐phenyl‐1H‐imidazol‐5‐yl)methyl]‐1‐piperazinyl]pyrimidine (SCH 66712) and 1‐[(2‐ethyl‐4‐methyl‐1H‐imidazol‐5‐yl)methyl]‐4‐[4‐(trifluoromethyl)‐2‐pyridinyl]piperazine (EMTPP), have been previously identified as mechanism‐based inactivators (MBIs) of CYP2D6. The current study shows SCH 66712 and EMTPP are also MBIs of CYP3A4. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. The rates of inactivation, kinact, and inhibition constant, Ki,were 0.21 min‐1 and 1.6 µM for SCH 66712, and 0.046 min‐1 and 11.7 µM for EMTPP, respectively. Whole protein MS analysis was consistent with a binding stoichiometry of 1:1 for both MBIs on CYP3A4 apoprotein. To determine the site of modification of the enzymes, MS analysis of digested peptides of inactivated enzymes was performed. Additionally, inactivation mechanisms of enzymes by possible electrophiles of SCH 66712 and EMTPP are proposed based in part on metabolite studies (Support: NIH 1R15‐GM086767‐02).

CYP2D6 metabolizes approximately 20% of pharmaceutical drugs. The important role that CYP2D6 plays in drug metabolism makes inactivation events clinically relevant. Thus, understanding the inactivation of CYP2D6 may prevent adverse drug‐drug interactions. Metoclopramide, a drug commonly prescribed to counteract nausea in chemotherapy patients, has previously been reported in the literature to be a mechanism‐based inhibitor of CYP2D6. We sought to expand this initial finding by constructing a metabolite profile and employing molecular modeling programs in an attempt to better understand the interactions between metoclopramide and CYP2D6. While we did not observe mechanism‐based inhibition with metoclopramide, we did observe predicted and new metabolites of metoclopramide. Furthermore, we were able to establish the relative contribution of specific P450 enzymes to the metabolic profile of metoclopramide and the role of CYP2D6 as the major metabolizer of metoclopramide. (Support: NIH 1R15‐GM086767–01 & ‐01S1).

Human cytochrome P450 enzymes (CYPs) are monooxygenases that are responsible for metabolism of a variety of small compounds including many pharmaceutical drugs. The main drug metabolizing CYP is CYP3A4 that metabolizes around half of drugs. Some drugs are known to inhibit CYP3A4, lowering the ability of CYP3A4 to metabolize other compounds leading to adverse events. 1‐[(2‐Ethyl‐4‐methyl‐1H‐imidazol‐5‐yl)‐methyl]‐4‐[4‐trifluoromethyl‐2pyrinyl]piperazine (EMTPP), while not a pharmaceutical drug, contains a piperazine group and substituted imidazole ring frequently found in pharmaceutical compounds. EMTPP is also similar in structure to SCH66712 and both EMTPP and SCH 66712 have previously been described as inactivators of CYP2D6 [Hutzler, Steenwyk, Smith, Walker, Wienkers (2004) Chem Res Toxicol 17, 174‐184; Nagy, Mocny, Diffenderfer, Hsi, Butler, Arthur, Fletke, Palamanda, Nomeir, Furge (2011) Drug Metab Dispos 39, 974‐983]. Our group has also recently shown SCH66712 to be a potent inactivator CYP3A4 and hypothesized that EMTPP would be as well. In this study, time‐ and concentration‐dependent inactivation of CYP3A4 by EMTPP was observed. Furthermore, the partition ratio for potency of EMTPP inactivation of CYP3A4 was measured along with possible routes of inactivation. This study lends insight into similarities between CYP2D6 and CYP3A4 for interaction with inactivators. Grant Funding Source: Supported by NIH 1R15‐GM086767‐02