Difference in desipramine metabolic profile between wild-type and CYP2D6-humanized mice

Abstract

Desipramine (DMI), a CYP2D6 probe, was used as a model drug to test whether CYP2D6-humanized (Tg-CYP2D6) and wild-type control mice could be used as preclinical animal models to identify the effects of CYP2D6 genotype/phenotype on drug metabolic profiles. After the analyses by liquid chromatography coupled with tandem mass spectrometry, DMI biotransformations were compared in Tg-CYP2D6 and wild-type mouse liver microsomes (MLM), and in human CYP2D6 extensive and poor metabolizer liver microsomes. Furthermore, urinary DMI metabolic profiles in Tg-CYP2D6 and wild-type mice were evaluated. Three metabolites, 2-hydroxyl-, 10-hydroxyl, and N-desmethyl-desipramine were identified in the incubations of DMI with both wild-type and Tg-CYP2D6 MLM, as well as in human CYP2D6 extensive metabolizer liver microsomes. Three additional metabolites were found in mouse urine samples, and their chemical structures were elucidated. Although the ratio of individual metabolites produced in Tg-CYP2D6 MLM was closer to that in human CYP2D6 extensive metabolizer liver microsomes, the urinary DMI metabolic profiles did not show much difference between wild-type and Tg-CYP2D6 mice. The results suggest that other mouse enzymes have significant contribution to DMI metabolism.

Fig 1

Extracted ion chromatograms of desipramine and its oxidative metabolites obtained after full-scan data acquisition. (A) Incubation with CYP2D6 poor metabolizer HLM. (B) Incubation with CYP2D6 extensive metabolizer HLM. All incubations were conducted at 37°C for 120 min.

Fig 2

Individual MS/MS spectra of M1 (A), M2 (B), and M3 (C) found in microsomal incubations with desipramine.

Fig 3

Extracted ion chromatograms of desipramine and its oxidative metabolites produced by wild-type (A) and Tg-CYP2D6 (B) mouse liver microsomes after 90-min incubation.

Fig 4

Extracted ion chromatograms of desipramine and its urinary metabolites in wild-type (A) and Tg-CYP2D6 (B) mice treated i.p. with 30 mg/kg of desipramine.

Fig 5

Individual MS/MS spectra of M4 (A), M5 (B), and M6 (C) found in mouse urine samples.

Fig 6

Proposed metabolic pathways of desipramine in wild-type and Tg-CYP2D6 mice. DMI is primarily oxidized to 2-hydroxyl-desipramine (2-OH-DMI, M2), which is subjected to O-glucuronidation to produce desipramine-2-O-glucuronide (DMI-2-O-Gluc, M4). A small fraction of DMI is converted to 10-hydroxyl-desipramine (10-OH-DMI, M1) and desmethyl-desipramine (DDMI, M3). In addition, M1 and M2 might be further N-demethylated to M5 (10-OH-DDMI) and M6 (2-OH-DDMI), respectively.