Comparative metabolism of sildenafil in liver microsomes of different species by using LC/MS-based multivariate analysis

Abstract

Sildenafil metabolism in liver microsomes obtained from different species was studied in vitro and compared using liquid chromatography–mass spectrometry (LC/MS) and multivariate statistical analysis. Sildenafil (1, 5, and 25 μM) was incubated with rat, mouse, dog, monkey, and human liver microsomes along with NADPH, and the reaction mixtures were analyzed by LC/MS to obtain species-specific metabolic profiles of sildenafil. A total of 12 metabolites were detected and their peak area ratio values were used as variables for multivariate analyses to evaluate the interspecies differences in sildenafil metabolism. Principal components analysis of the metabolic profiles showed that the mouse samples were generally clustered closer to the human samples on the principal component score plot. Similarity index (SI) indicated that sildenafil metabolism in mice, compared to the other animals, was highly analogous (SI = 0.764 at 25 μM) to that in humans. These results suggest that LC/MS-based multivariate analytical approaches are useful for the evaluation of interspecies differences in the metabolism of xenobiotics.

Introduction

Drug metabolism and pharmacokinetic properties of new chemical entities are extensively investigated at various stages of drug discovery and development [1]. In particular, drug metabolism is important in that it reveals the pharmacological activity and toxicological implications of a drug to ensure that it can be used safely in humans. In the lead discovery and optimization phases, drug metabolism properties are screened using in vitro systems such as microsomes, S9 fractions, and/or hepatocytes, and compounds with favorable metabolic stability are selected [2], [3], [4], [5]. The in vitro systems are used to characterize and compare metabolic pathways across species to determine whether major metabolites in humans are appropriately reflected in nonclinical species selected for safety or toxicological studies [6]. Based on in vitro metabolism profiles, in vivo metabolism studies are further conducted through the late preclinical and phase I/II clinical trials [1].

Liquid chromatography–mass spectrometry (LC/MS) provides a powerful solution for investigating the metabolism of xenobiotics and is commonly used in various phases of drug discovery and development. Recently, LC/MS analysis has been combined with multivariate chemometric techniques such as principal component analysis (PCA) and used for metabolomic and metabonomic studies [7], [8], [9], [10], [11]. These approaches can be used to visualize the enormous datasets collected from biological samples by describing an n-dimensional space of the variable metabolic profile as a smaller number of dimensions. Additionally, by using a mathematical technique such as a similarity analysis, the similarity between multi-dimensional datasets can be represented by a score, and the datasets be clearly compared with each other [12], [13]. In this context, LC/MS analysis coupled with multivariate techniques can be successfully employed for investigation of xenobiotic metabolism, particularly, comparative metabolism of xenobiotics with numerous metabolites in different species, in which case multiple datasets should be analyzed and compared simultaneously.

Sildenafil is a cGMP-specific phosphodiesterase type 5 (PDE5) inhibitor and well known as a drug used to treat erectile dysfunction [14]. The in vitro and in vivo metabolism of sildenafil has already been extensively investigated in various species [15], [16]. According to a previous study [15], 10 metabolites were detected in experimental animal and human samples, and the major metabolic pathways of sildenafil were piperazine N-demethylation, pyrazole N-demethylation, loss of a 2-carbon fragment from the piperazine ring (N,N′-deethylation), oxidation of the piperazine ring, and aliphatic hydroxylation. The metabolic profile of sildenafil metabolism differs among species. Therefore, a multivariate analysis approach could help to comprehensively understand and clearly compare sildenafil metabolism in different species.

In the present study, a novel application of PCA and similarity index (SI) was studied to evaluate the interspecies differences in metabolism of a xenobiotic by using sildenafil as a model compound. For a comparative metabolism study, sildenafil was incubated with liver microsomes collected from 5 different species, the reaction mixtures were analyzed by LC/MS, and the metabolic profiling data based on the LC/MS chromatogram were subjected to PCA and similarity analysis.