Isolation of Partially Purified P450 2D18 and Characterization of Activity toward the Tricyclic Antidepressants Imipramine and Desipramine

doi:10.1006/abbi.1998.0892

Archives of Biochemistry and Biophysics

Volume 359, Issue 1, 1 November 1998, Pages 115-121

https://doi.org/10.1006/abbi.1998.0892 Get rights and content

Abstract

Previous reports have shown that rat brain microsomes are capable of metabolizing tricyclic antidepressants such as imipramine. Subsequent studies have shown that the protein products of several clones isolated from rat brain cDNA libraries are capable of metabolizing imipramine to both its active metabolite, desipramine, and its inactive hydroxylated metabolites. We report here the overexpression and partial purification of P450 2D18 using the baculovirus expression system and the incorporation of a C-terminal [His]₄tag. P450 2D18 was partially purified to a specific content of 4.8 nmol/mg protein and shown to be electrophoretically pure. The apparentK_Mvalues for P450 2D18 toward imipramine and desipramine were 374 and 314 μM, respectively. While apparentK_Mvalues were similar, P450 2D18 was shown to have a fivefold increasedV_max(2.2 nmol/min/nmol P450) for imipramine compared to desipramine (0.44 nmol/min/nmol P450), suggesting a primary involvement in the activation of imipramine to desipramine. We also examined the effect of theCYP2D6 inhibitor quinidine, theCYP3A inhibitor ketoconazole, and the dopamine reuptake inhibitor GBR-12935 for their ability to inhibit P450 2D18-mediated metabolism of imipramine. These results, when compared with previous studies using rat brain microsomes, suggest that P450 2D18 may play an important role in the conversion of imipramine to its active metabolite desipramine in the rat brain.

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Brain CYP2D isoforms exhibit enzymatic activity towards endo- and xenobiotics including psychotropics. The metabolism of tricyclic antidepressant drugs (TADs) with contribution of rat brain CYP2D has been described (Hansson et al., 1992; Kawashima et al., 1996; Sequeira and Strobel, 1996; Thompson et al., 1998; Voirol et al., 2000). On the other hand, CYP2D isoforms in the brain seem to play an important role in the local metabolism of neurosteroids (Hiroi et al., 2001; Kishimoto et al., 2004), arachidonic acid (Kawashima et al., 1996; Thompson et al., 2000), as well as the neurotransmitters serotonin (Yu et al., 2003) and dopamine (Hiroi et al., 1998b; Bromek et al., 2010).
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^☆: This work was supported by Grant MH 58297 from the National Institute of Mental Health DHHW.

^☆☆: Johnson, E. F.Waterman, M. R.

²: The data presented here comprise part of the dissertation research of Chad M. Thompson presented to the Faculty of the Graduate School of Biomedical Science, University of Texas Health Science Center at Houston in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

³: To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77225. Fax: 713.500.0652.

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Regular ArticleIsolation of Partially Purified P450 2D18 and Characterization of Activity toward the Tricyclic Antidepressants Imipramine and Desipramine☆,☆☆

Abstract

Brain Res.

Brain Res.

Brain Res.

Trends Pharmacol. Sci.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Chromatogr. B

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Lancet

Biochem. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Regular Article
Isolation of Partially Purified P450 2D18 and Characterization of Activity toward the Tricyclic Antidepressants Imipramine and Desipramine☆,☆☆