Identification of a Novel in Vitro Metabonate From Liver Microsomal Incubations

Abstract

ABSTRACT:In vitro drug metabolism studies during the early drug discovery stage are becoming increasingly important. With the increasing demand for high throughput and quick turnaround time for in vitro metabolism studies, however, careful examination of the results and proper design of the experiments are still crucial. In this communication, we report the identification and mechanism of formation of a novel metabonate from incubations of a diamine-containing compound with liver microsomes. The metabonate appeared to be the major product, and its formation was NADPH-and microsomal protein-dependent. Liquid chromatography/mass spectrometry and NMR analysis of the metabonate indicated an extra carbon and unusual formation of an imidazolidine ring. Further studies revealed that this metabonate was not a true biotransformation product from the diamine compound itself in the microsomal incubation, but rather a product resulting from a condensation reaction between the compound and a metabolite of the solvent (alcohol) used in the incubation. When the microsomal incubations contained a small amount of methanol or ethanol as solvent, the alcohols were metabolized to formaldehyde or acetaldehyde, which then condensed with the diamine compound through an imine intermediate to form the metabonate. The compound itself was metabolically stable in vitro when acetonitrile or dimethyl sulfoxide was used as solvent. During the study of in vitro microsomal stability and metabolite identification of amine-containing compounds, the use of alcohol as solvent should be avoided.

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

“…Yin et al (2001) reported that the concentration of formaldehyde formed from methanol metabolism reached up to 600 M over 60 min. In a second report describing formation of similar methylene artifacts from 1,2-ethylene diamine-containing compounds, the source of the aldehyde component was again proposed as alcohol solvent, established through experiments with ethanol, methanol, and deuterium-labeled methanol (Li et al, 2006). However, alcohol solvents were not used in the present study, so methanol was not the source of the formaldehyde supporting the methylenation reaction.…”

“…Consequently, some understanding of the metabolism of a new drug is desirable before Phase 1 clinical studies in humans. To aid selection of appropriate toxicology species as well as the design of appropriate safety studies, metabolite formation is assessed in in vitro incubations of drug candidates with liver microsomes, S9 fraction, or hepatocytes from humans and nonclinical species (Li et al, 2006;Tiller et al, 2008). These same in vitro test systems are commonly used to assess metabolic clearance of the parent drug (Yin et al, 2001) and to investigate the formation of reactive (electrophilic) metabolites (Park et al, 2011), although the formation of reactive metabolites itself does not necessarily imply toxicity (Prakash et al, 2008).…”

Glycerolysis of Acyl Glucuronides as an Artifact of in Vitro Drug Metabolism Incubations

“…Several examples of this phenomenon exist in the scientific literature and include oximes (Beckett et al, 1971), imidazolines (Li et al, 2006), phenothiazones (Beckett and Navas, 1978), esters (Dickinson and King, 1989), and many entities arising from internal cyclization reactions (reviewed in Erve, 2008). In the course of the study of the glucuronidation of [ 14 C]benoxaprofen in human liver S-9 fraction, in addition to observation of the expected acyl glucuronide metabolite, an unusual drugrelated entity was also observed (Fig.…”

Contribution of Artifacts to N-Methylated Piperazine Cyanide Adduct Formation In Vitro from N-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.…”