Characterization of cyanide‐trapped methylated metabonates formed during reactive drug metabolite screening in vitro

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.

High-Resolution Mass Spectrometry Elucidates Metabonate (False Metabolite) Formation from Alkylamine Drugs during In Vitro Metabolite Profiling

“…A report on cyanide-trapped metabonate formation under reactive metabolite screening incubation conditions was recently published (Rousu and Tolonen, 2011). Consistent with our findings, the authors described structural changes to amine moieties of three drugs (propranolol, amlodipine, and ciprofloxacin), resulting in the addition of a CH 2 group and cyano conjugation, and ascribed these structural changes to metabonate formation.…”

The Aggravation of Clozapine-Induced Hepatotoxicity by Glycyrrhetinic Acid in Rats

“…CLZinduced hepatotoxicity was considered to be related to the chemically reactive metabolites during CLZ metabolism (3,4,30,31). Our results demonstrated that GA enhanced CLZinduced hepatotoxicity, accompanied by inhibition of CLZ metabolism indexed as formation of CLZ's two metabolites norCLZ and CLZ Noxide.…”

Contribution of Artifacts toN-Methylated Piperazine Cyanide Adduct Formation In Vitro fromN-Alkyl Piperazine Analogs

“…Furthermore, our results demonstrated that this one-carbon addition to the alicyclic secondary amines (I and IX) was NADPH dependent, suggesting the involvement of cytochrome P450 enzyme catalyzed bioactivation. We hypothesized that when using potassium carbonate as the one-carbon source in the liver microsomal incubation, the carbamic acid formation would be the first step of this methylation, Iminium ions from piperazine a-carbon oxidation (pathway 1) have been considered a bioactivation process and those from pathway 2 as experimental artifact resulting from the potential interaction with formaldehyde in the incubation buffer (Gorrod and Sai, 1997;Li et al, 2006;Rousu and Tolonen, 2011;Barbara et al, 2012). However, the biologic and toxicological implications of iminium ions via pathway 2 require further investigation for the following reasons.…”

“…In our CN-trapping screening on Ndealkylated alicyclic compounds, significant CN adducts with an additional 14 Da were also observed and confirmed as the addition of a methylene group (unpublished data). It has been suggested that this type of N-methylated CN adduct from the secondary alicyclic amines was an experimental artifact, or "metabonate," from an in vitro microsomal incubation system (Gorrod and Sai, 1997;Li et al, 2006;Rousu and Tolonen, 2011;Barbara et al, 2012). In the case of N-methyl piperazines, there are two possible pathways for the formation of Nmethyl piperazine CN adducts (Fig.…”

“…In pathway 2, Ndealkylation results in the formation of a secondary alicyclic amine, which can react with formaldehyde and eventually lead to the formation of an iminium ion. The CN adduct from pathway 2 has been considered the artifact from the in vitro system and irrelevant to in vivo (Gorrod and Sai, 1997;Rousu and Tolonen, 2011;Barbara et al, 2012). In the study reported here, we have evaluated the mechanism of CN adduct formation in several piperazine-containing compounds ( Fig.…”