Characterization of cyanide-trapped methylated metabonates formed during reactive drug metabolite screening in vitro.
Rapid Commun Mass Spectrom
; 25(10): 1382-90, 2011 May 30.
Article
in En
| MEDLINE
| ID: mdl-21504003
ABSTRACT
Reactive metabolites are estimated to be one of the main reasons behind unexpected drug-induced toxicity, by binding covalently to cell proteins or DNA. Due to their high reactivity and short lifespan, reactive metabolites are analyzed after chemical trapping with nucleophilic agents such as glutathione or cyanide. Recently, unexplained and uncharacterized methylated reaction products were reported in a human liver microsome based reactive metabolite trapping assay utilizing potassium cyanide as a trapping agent. Here, a similar assay was utilized to produce mono- or dimethylated and further cyanide-trapped reaction products from propranolol, amlodipine and ciprofloxacin, followed by ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS) and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) experiments for their more detailed structural elucidation. Formation of all observed cyanide-trapped products was clearly NADPH-dependent and thus metabolism-mediated. The suggested reaction pathways included N-methylation leading to iminium formation in primary and/or secondary amines preceded by cytochrome P450 (CYP)-mediated reactions. As the methylation reaction was suggested to be involved in formation of the actual reactive iminium ion, the observed cyanide-trapped products were experimental artifacts rather than trapped reactive metabolites. The results stress that to avoid overestimating the formation of reactive metabolites in vitro, this methylation phenomenon should be taken into account when interpreting the results of cyanide-utilizing reactive metabolite trapping assays. This in turn emphasizes the importance of identification of the observed cyano conjugates during such studies. Yet, metabolite identification has a high importance to avoid overestimation of in vitro metabolic clearance in the cases where this kind of metabonate formation has a high impact in the disappearance rate of the compound.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Potassium Cyanide
/
Pharmaceutical Preparations
/
Drug Evaluation, Preclinical
/
Metabolomics
/
Isotope Labeling
Type of study:
Diagnostic_studies
/
Screening_studies
Limits:
Female
/
Humans
/
Male
Language:
En
Journal:
Rapid Commun Mass Spectrom
Year:
2011
Document type:
Article
Affiliation country:
Finland