Involvement of CYP2D6, CYP3A4, and other cytochrome P-450 isozymes in N-dealkylation reactions

ABSTRACT:CYP2D6 and CYP3A4, two members of the cytochrome P450 superfamily of monooxygenases, mediate the biotransformation of a variety of xenobiotics. The two enzymes differ in substrate specificity and size and characteristics of the active site cavity. The aim of this study was to determine whether the catalytic properties of these isoforms, reflected by the differences observed from crystal structures and homology models, could be confirmed with experimental data. Detailed metabolite identification, reversible inhibition, and time-dependent inhibition were examined for levorphanol and levallorphan with CYP2D6 and CYP3A4. The studies were designed to provide a comparison of the orientations of substrates, the catalytic sites of the two enzymes, and the subsequent outcomes on metabolism and inhibition. The metabolite identification revealed that CYP3A4 catalyzed the formation of a variety of metabolites as a result of presenting different parts of the substrates to the heme. CYP2D6 was a poorer catalyst that led to a more limited number of metabolites that were interpreted in terms to two orientations of the substrates. The inhibition studies showed evidence for strong reversible inhibition of CYP2D6 but not for CYP3A4. Levallorphan acted as a time-dependent inhibitor on CYP3A4, indicating a productive binding mode with this enzyme not observed with CYP2D6 that presumably resulted from close interactions of the N-allyl moiety oriented toward the heme. All the results are in agreement with the large and flexible active site of CYP3A4 and the more restricted active site of CYP2D6.

Section: Exploration Of Catalytic Properties Of Cyp2d6 and Cyp3a4supporting

confidence: 77%

Exploration of Catalytic Properties of CYP2D6 and CYP3A4 Through Metabolic Studies of Levorphanol and Levallorphan

Bonn¹,

Masimirembwa²,

Castagnoli³

2009

“…N-Dealkylation is a common metabolic pathway for drugs containing secondary and tertiary amines and, in many cases, CYP3A4, CYP1A2, or CYP2C19 are involved in the catalysis (Coutts et al, 1994). This reaction also occurs in CYP2D6 but it is far less common than in the above-mentioned isoforms.…”

Section: Discussionmentioning

confidence: 99%

“…The main reason is that the electrostatic interaction between the basic nitrogen in the substrate and the carboxylic acid group of Glu216 on top of the active site cavity would favor an orientation of the substrate with the nitrogen directed away from the heme. Nevertheless, N-dealkylation does occur in CYP2D6 but is far less common than in, for example, CYP3A4 (Coutts et al, 1994). In a previous study, the influence and importance of the positively charged nitrogen on the orientation of the substrates in the active site cavity was explored (Upthagrove and Nelson, 2001).…”

mentioning

confidence: 99%

The Molecular Basis of CYP2D6-Mediated N-Dealkylation: Balance between Metabolic Clearance Routes and Enzyme Inhibition

Bonn¹,

Masimirembwa²,

Aristei³

et al. 2008

ABSTRACT:N-Dealkylation is a commonly observed metabolic reaction for drugs containing secondary and tertiary amines. On searching the literature, it is obvious that this reaction is far less common among cytochrome P450 2D6 catalyzed reactions compared with other cytochromes P450. The CYP2D6 pharmacophore and characteristic features in the active site cavity suggest a favored substrate orientation that prevents N-dealkylation from occurring. In this study, the literature was searched for N-dealkylated and non-Ndealkylated CYP2D6 substrates. The hypothesis that was suggested and confirmed demonstrated that N-dealkylation occurs by this enzyme when the preferred site of metabolism is blocked toward other oxidative metabolic pathways. An interesting observation was also that addition of stable groups at preferred sites of metabolism generally improved the metabolic stability but also resulted in retained or increased inhibition of the enzyme. In addition, the effect of pH on N-and O-dealkylation of dextromethorphan was shown to be consistent with the hypothesis that an ionized amino function favored substrate dockings resulting in O-dealkylation.Cytochrome P450 2D6 (CYP2D6) is a polymorphic member of the cytochrome P450 superfamily important for the metabolism of a variety of xenobiotics (Guengerich, 2003). In drug discovery research it is of great interest to identify whether a compound is a substrate or not, and considerable effort has been put into the development of models to identify substrates and/or inhibitors of CYP2D6. Several pharmacophore models have been published identifying common features in CYP2D6 substrates and inhibitors (Koymans et al., 1992;de Groot et al., 1997de Groot et al., , 1999Lewis et al., 1997). The pharmacophore comprises basic nitrogen atom 5-7 or 10 Å from the site of oxidation, a flat hydrophobic region near the site of oxidation and a negative molecular electrostatic potential coplanar with this hydrophobic region (see examples in Fig. 1). The intramolecular distances of 5 and 7 Å were combined by Koymans et al. (1992) in a model assuming a carboxylate group as an anchor point in the protein with one of the oxygen atoms interacting with the "5-Å substrates" and the other with the "7-Å substrates." Since several substrates with approximately 10 Å between the nitrogen atom and the site of oxidation were also known, refined models were constructed including this intramolecular distance (Lewis et al., 1997). Even though the early pharmacophore models are rather crude, they have been successful in predicting the influence of CYP2D6 in the metabolism of xenobiotics as well as predicting possible sites of metabolism [e.g., 75% predictability (de Groot et al., 1999)].Structural information of CYP2D6 from homology models (de Groot et al., 1996;De Rienzo et al., 2000) and a crystal structure of ligand-free CYP2D6 (Rowland et al., 2006) revealed two acidic amino acids, Glu216 and Asp301, and two phenylalanine residues, Phe120 and Phe483, in the active site cavity. This was compatible with t...

“…The structure of M4 was somewhat unexpected, as C-demethylation is not common during metabolism, whereas O-and N-demethylations are frequently observed in the biotransformation of xenobiotics (Burke et al, 1994;Coutts et al, 1994;Ertl et al, 1999). In the case of terfenadine possessing a tert-butyl moiety on the backbone of the compound, similarly to LC15-0133, both alcohol and acid metabolites have been identified, but the C-demethylated metabolite has never been reported ( Terhechte and Blaschke, 1995).…”

Section: Resultsmentioning

confidence: 99%

EnzymaticC-Demethylation of 1-[2-(5-tert-Butyl-[1,3,4] oxadiazole-2-carbonyl)-4-fluoro-pyrrolidin-1-yl]-2-(2-hydroxy-1,1-dimethyl-ethylamino)-ethanone (LC15-0133) in Rat Liver Microsomes

Yoo¹,

Chung²,

Lee³

et al. 2007

ABSTRACT:The in vitro metabolism of 1-[2-(5-tert-butyl-[1,3,4] oxadiazole-2-carbonyl)-4-fluoro-pyrrolidin-1-yl]-2-(2-hydroxy-1,1-dimethyl-ethylamino)-ethanone (LC15-0133), a novel dipeptidyl peptidase-4 inhibitor, was investigated using a hepatic microsomal system. The structures of the metabolites were characterized using mass spectral analysis and by comparison with synthetic references. The in vitro incubation of LC15-0133 with rat liver microsomes resulted in the formation of six metabolites, with the major metabolic reactions being hydroxylation and carbonyl reduction. Of the metabolites, a C-demethylated metabolite (M4) was identified, but was only detected in rat liver microsomes; experimental evidence revealed that the C-demethylated metabolite was generated by nonenzymatic decarboxylation of the carboxyl metabolite (M1). Nonenzymatic decarboxylation is postulated to occur due to the resonance stabilization by the oxadiazole ring attached to the tert-butyl moiety., a novel dipeptidyl peptidase-4 inhibitor, is under development as a therapeutic agent for diabetes. Dipeptidyl peptidase-4 inhibitors have been shown to lower blood glucose in diabetic rodents via prolongation of glucagon-like peptide-1 and the action of gastric inhibitory polypeptide (Ahren and Schmitz, 2004;Demuth et al., 2005;Barnett, 2006;Campbell, 2007;Pratley and Salsali, 2007).As a part of the preclinical study of LC15-0133, the in vitro metabolism of LC15-0133 was investigated using rat, dog, and human liver microsomes. LC15-0133 was metabolized to 4 to 6 metabolites, via hydroxylation and carbonyl reduction, with considerable species differences noted in rat liver microsomes. C-Demethylated and carboxyl metabolites were observed only in the rat liver microsomal incubation. Because the C-demethylation reaction is not a common enzymatic reaction, the metabolic pathway for the generation of the C-demethylated metabolite requires further investigation. The purpose of the present study was to characterize the in vitro metabolism of LC15-0133 and identify the mechanism of the rat liver microsomal enzyme catalyzed C-demethylation reaction of LC15-0133. Materials and MethodsChemicals. LC15-0133 and LC15-0133-d 6 (internal standard), with a chemical purity Ͼ99%, were provided by LG Life Sciences R&D (Daejeon, Korea). Authentic standards of LC15-0133 metabolites (M3, M4, and M5), with chemical purity Ͼ95%, were also provided by LG Life Sciences R&D. All other chemicals were the highest grade commercially available.Microsomes. Human liver microsomes were purchased from BD Biosciences (Bedford, MA). Rat liver samples were taken from Sprague-Dawley rats (Daehan Animal, Daejeon, Korea) and dog liver samples from Beagle dogs (Daehan Animal). The microsomal fraction was prepared according to the method previously described elsewhere (Guengerich et al., 1986).In Vitro Microsomal Incubation. LC15-0133 (100 M final concentration) was incubated with 2 mg/ml liver microsomes at 37°C for 2 h, in the presence of an NADPH-generating system (10 mM glucose 6-p...