Cynomolgus Monkey CYP2D44 Newly Identified in Liver, Metabolizes Bufuralol, and Dextromethorphan

Abstract

ABSTRACT:The cynomolgus monkey is used in drug metabolism studies, because of its evolutionary closeness to human, including cytochrome P450. Cynomolgus monkey CYP2D17, highly homologous to human CYP2D6, has been identified and characterized. Here, we report characterization of another CYP2D, CYP2D44, identified in cynomolgus monkey liver. The CYP2D44 cDNA contained an open reading frame of 497 amino acids sharing high sequence identity (87-93%) with other primate CYP2Ds. CYP2D44 mRNA was predominantly expressed in liver, similar to CYP2D17 mRNA. CYP2D17 and CYP2D44 form a gene cluster in the genome, similar to human CYP2Ds. Metabolic assays of the CYP2D17 and CYP2D44 proteins heterologously expressed in Escherichia coli indicated that CYP2D44 metabolized human CYP2D6 substrates, bufuralol and dextromethorphan (bufuralol 1-hydroxylation and dextromethorphan O-demethylation) but to a lesser extent than CYP2D17. Kinetic analysis of dextromethorphan metabolism indicated that the apparent K m and V max of CYP2D17 and CYP2D44 catalyzed O-demethylation were similar, and, the V max values of CYP2D17 and CYP2D44 catalyzed Ndemethylation (which human CYP2D6 catalyzes much less effectively) were similar, but the apparent K m of the CYP2D44 reaction was higher. Western blot analysis showed that CYP2D proteins were expressed in cynomolgus and rhesus monkey liver as well as in human and marmoset liver. Similar to CYP2D6, CYP2D44 copy number varied among the eight cynomolgus monkeys and four rhesus monkeys used in this study. These results indicated that CYP2D44, together with CYP2D17, had functional characteristics similar to those of human CYP2D6 but measurably differed in dextromethorphan N-demethylation, suggesting its importance for CYP2D-dependent drug metabolism in macaque.

Species Difference between Cynomolgus Monkeys and Humans on Cytochromes P450 2D and 3A-Dependent Drug Oxidation Activities in Liver Microsomes

“…9) Both CYP2D17 and CYP2D44 mRNA were expressed predominantly in the liver, and CYP2D44 mRNA expression level was 4.4-fold lower than that of CYP2D17. 9) The functional characteristics of both CYP2D17 and CYP2D44 enzymes were similar to those of human CYP2D6 but measurably differed in dextromethorphan Ndemethylation. 9) In this study, recombinant monkey CYP2D17 and CYP2D44 effectively catalyzed dextromethorphan O-and N-demethylation, as monkey mfCYP3A4 and mfCYP3A5 did, but recombinant human CYP2D6 did not mediate dextromethorphan…”

“…10) Pooled liver microsomes from humans (H150) and mon- in Escherichia coli membranes was prepared as described previously. 9,11) Microsomal protein concentrations were estimated using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, U.S.A.). Concentration of total P450 in membranes was estimated as described previously by Omura and Sato.…”

“…6) The homologies of cDNA and amino acid sequences between monkey P450 2D17 and human P450 2D6 were 94% and 93%, respectively. Recently, Uno et al 9) successfully identified a novel P450 2D44 cDNA in cynomolgus monkey. The homologies of cDNA and amino acid sequences between P450 2D44 and P450 2D6 were 93% and 91%, respectively.…”

Immunochemical Detection of Cytochrome P450 Enzymes in Liver Microsomes of 27 Cynomolgus Monkeys

“…CYP2D17/44 content was 3.2 pmol/mg protein, similar to that of human CYP2D6 (5 pmol/mg protein) (Shimada et al, 1994). Thus, the higher rate of CYP2D-dependent reaction in cynomolgus monkey liver might be partly caused by the faster rate of cynomolgus CYP2D enzyme, as shown previously (Mankowski et al, 1999;Uno et al, 2010d). In human, CYP2D6, involved in the metabolism of approximately 25% of known drugs in the market, is highly polymorphic, leading to interindividual variations in response to drugs that are metabolized by CYP2D6 (Ingelman-Sundberg, 2005).…”

“…The cynomolgus CYP3A subfamily includes CYP3A4 and CYP3A5, which are predominantly expressed in liver (Uno et al, 2007a) enzymes involved in the metabolism of human CYP3A substrates, such as midazolam and nifedipine (Iwasaki et al, 2010;Uno et al, 2010c). Likewise, other cynomolgus P450 subfamilies including CYP1A (CYP1A1 and CYP1A2), CYP2A (CYP2A23, CYP2A24, and CYP2A26), CYP2B (CYP2B6), CYP2D (CYP2D17 and CYP2D44), and CYP2E (CYP2E1), are also predominantly expressed in liver and encode the proteins that metabolize the substrates of orthologous human P450s (Uno et al, 2007a(Uno et al, , 2009b(Uno et al, , 2010d(Uno et al, , 2011dUehara et al, 2010). Cynomolgus CYP1D1 is orthologous to human CYP1D1P and is expressed in liver at a comparable level to CYP1A1, but is much more abundant than CYP1A2 (Uno et al, 2011d).…”