Abstract
The release of aromatic amines from drugs and other xenobiotics resulting from the hydrolysis of metabolically labile amide bonds presents a safety risk through several mechanisms, including geno-, hepato- and nephrotoxicity. Whilst multiple in vitro systems used for studying metabolic stability display serine hydrolase activity, responsible for the hydrolysis of amide bonds, they vary in their efficiency and selectivity. Using a range of amide-containing probe compounds (0.5–10 µM), we have investigated the hydrolytic activity of several rat, minipig and human-derived in vitro systems - including Supersomes, microsomes, S9 fractions and hepatocytes - with respect to their previously observed human in vivo metabolism. In our hands, human carboxylesterase Supersomes and rat S9 fractions systems showed relatively poor prediction of human in vivo metabolism. Rat S9 fractions, which are commonly utilised in the Ames test to assess mutagenicity, may be limited in the detection of genotoxic metabolites from aromatic amides due to their poor concordance with human in vivo amide hydrolysis. In this study, human liver microsomes and minipig subcellular fractions provided more representative models of human in vivo hydrolytic metabolism of the aromatic amide compounds tested.
Highlights
Exposure to aromatic amines (AA) is of concern in pharmaceutical development, as well as in occupational and wider environmental contexts, due to their potential toxicities; some AA structures have been shown to be responsible for genotoxicity[1], hepatotoxicity[2] and the induction of methaemoglobin formation[3]
Key members of this family include the membrane bound carboxylesterases CES1 and CES2 which are present in the cytosol, along with arylacetamide deacetylase AADAC which is only found within the endoplasmic reticulum[16,17]
An additional set of aromatic amides with unknown in vivo metabolism were selected using a systematic approach to investigate substituent effects of e.g., electron withdrawing (-NO2, -F) and electron donating groups (-OH, -CH3) which were varied between compounds around the formanilide ring for future quantitative structure-activity relationship (QSAR) modelling
Summary
Exposure to aromatic amines (AA) is of concern in pharmaceutical development, as well as in occupational and wider environmental contexts, due to their potential toxicities; some AA structures have been shown to be responsible for genotoxicity[1], hepatotoxicity[2] and the induction of methaemoglobin formation[3]. A good example of this is given by flutamide, an antiandrogen used in the treatment of prostatic carcinoma, which undergoes substantial metabolism in vivo with hydrolysis of the amide bond liberating 3-trifluoromethyl-4-nitroaniline This aniline has been detected in the plasma of patients after oral administration of the drug[5] and exposure has been associated with hepatotoxicity[2]. One challenge for scientists working in the area of drug discovery is accurately and rapidly predicting the metabolic susceptibility of aniline-containing candidate drugs to the actions of serine hydrolases, the main enzymes responsible for amide hydrolysis. We assessed the ability of these in vitro systems to predict the in vivo hydrolysis of eight aromatic amide-containing compounds (Fig. 1) for which the human in vivo metabolic fate of the amide bond had previously been characterised, alongside 15 exemplar aromatic amides
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