title: Bioactivation of the cannabinoid receptor antagonist rimonabant to a cytotoxic iminium ion metabolite.
abstract: The cannabinoid type 1 receptor (CB1r) antagonist rimonabant was approved in 2006 for the treatment of obesity but was withdrawn in 2008 due to serious drug-related psychiatric disorders. In vitro metabolism studies with rimonabant have revealed high levels of reactive metabolite formation, which resulted in irreversible time-dependent P450 3A4 inhibition and in covalent binding to microsomal proteins. In the present study, an in vitro approach has been used to explore whether metabolic bioactivation of rimonabant might result in cell toxicity. A panel of SV40-T-antigen-immortalized human liver derived (THLE) cells that had been transfected with vectors encoding various human cytochrome P450 enzymes (THLE-1A2, 2C9, 2C19, 2D6, and 3A4) or with an empty vector (THLE-Null) were exposed to rimonabant. Cell toxicity and covalent binding to cellular proteins were evaluated, as was metabolite formation. Rimonabant exhibited markedly potentiated dose and time dependent cytotoxicity to THLE-3A4 cells, compared to that of all other THLE cell lines. This was accompanied by high levels of covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins (1433 pmol drug equivalents/mg protein) and the formation of several metabolites that were not generated by THLE-Null cells. These included N-aminopiperidine (NAP) and an iminium ion species. However, no toxicity was observed when THLE cells were incubated with NAP. Glutathione depletion did not alter the observed potent cell cytotoxicity of rimonabant to THLE-3A4 cells. Preincubation of THLE-3A4 cells with the cytochrome P450 3A4 inhibitor ritonavir blocked the selective toxicity of rimonabant to these cells. In addition, ritonavir pretreatment blocked the metabolism of the compound in the cells and thereby significantly decreased the covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins. We conclude that the potent toxicity of rimonabant in THLE-3A4 cells occurs by a mechanistic sequence, which is initiated by cytochrome P450 3A4 mediated formation of a highly cytotoxic reactive iminium ion metabolite that binds covalently to cellular proteins.
authors.affiliation: Global Safety Assessment, ‡DMPK Innovative Medicine, AstraZeneca , Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom.
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Foster
authors.forename: Lucy H
authors.initials: LH
authors.lastname: Prime
authors.forename: Frida
authors.initials: F
authors.lastname: Gustafsson
authors.forename: David G
authors.initials: DG
authors.lastname: Temesi
authors.forename: Emre M
authors.initials: EM
authors.lastname: Isin
authors.forename: Johanna
authors.initials: J
authors.lastname: Midlöv
authors.forename: Neal
authors.initials: N
authors.lastname: Castagnoli
authors.forename: J Gerry
authors.initials: JG
authors.lastname: Kenna
mesh_terms: D063387:Cannabinoid Receptor Antagonists / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D002250:Carbon Radioisotopes / Q000737:chemistry; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007097:Imines / Q000737:chemistry*; D007477:Ions / Q000737:chemistry; D055442:Metabolome / Q000187:drug effects; D010880:Piperidines / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity; D011190:Potassium Cyanide / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011720:Pyrazoles / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D000077285:Rimonabant; D019438:Ritonavir / Q000737:chemistry / Q000494:pharmacology
chemical_list: D063387:Cannabinoid Receptor Antagonists; D002250:Carbon Radioisotopes; D007097:Imines; D007477:Ions; D010880:Piperidines; D011506:Proteins; D011720:Pyrazoles; C034628:N-aminopiperidine; D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione; D011190:Potassium Cyanide; D019438:Ritonavir; D000077285:Rimonabant