Comparative metabolism of sildenafil in liver microsomes of different species by using LC/MS-based multivariate analysis
Highlights
► Comparative metabolism of sildenafil in liver microsomes from 5 species was studied. ► Liquid chromatography–mass spectrometry and multivariate statistical analysis were used. ► Peak area ratio for 12 metabolites was used as variables for multivariate analyses. ► Mice showed the most similar pattern with humans regarding sildenafil metabolism.
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.
Section snippets
Chemicals
Sildenafil citrate (purity, 99.9%) and mirodenafil HCl (purity, >99%) were donated by SK Ltd. (Suwon, Korea). Pooled human, rat, and mouse liver microsomes were obtained from BD Gentest (Woburn, MA, USA), and pooled monkey and dog liver microsomes, from In Vitro Technologies, Inc. (Baltimore, MD, USA) (Supplemental Table S1). Glucose-6-phosphate, β-NADP+ and glucose-6-phosphate dehydrogenase were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were of analytical
In vitro metabolic profiling
The incubation of sildenafil with human, mouse, rat, monkey, and dog liver microsomes (HLM, MLM, RLM, MkLM, and DLM) in the presence of NADPH generated 12 metabolite peaks (S/N > 5), shown in Fig. 1. When sildenafil was incubated without microsomes, no metabolites were observed, indicating that all metabolites were generated in the presence of microsomal enzymes (data not shown). In HLM, 11 metabolites, except M4, were found at a concentration of 1–25 μM. In MLM and MkLM, all the metabolites were
Discussion
In this study, we attempted to use chemometric approaches i.e., PCA, a type of pattern-recognition analysis, and SI measurement, to investigate interspecies differences in sildenafil metabolism. The metabolic profile at 3 concentration levels of sildenafil (1, 5, and 25 μM) was characterized and applied to PCA and SI measurement. The concentrations investigated in the present study were chosen on the basis of the Km value of sildenafil for the major metabolic reaction and its toxic
Conclusion
In conclusion, a study of interspecies differences in the metabolism of sildenafil was conducted using an LC/MS analysis combined with multivariate analysis approaches such as PCA and similarity analysis. This approach allowed us to elucidate the similarity of metabolic patterns of different species, which may be a useful tool to evaluate the comparative metabolism of xenobiotics.
Acknowledgement
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0012319).
References (22)
- et al.
J. Pharmacol. Toxicol. Methods
(2000) - et al.
J. Biol. Chem.
(2005) - et al.
J. Pharm. Biomed. Anal.
(2007) - et al.
J. Chromatogr. B
(1997) - et al.
- et al.
Toxicol. Pathol.
(1995) - et al.
Nat. Rev. Drug Discov.
(2007) - et al.
Curr. Drug Discov. Technol.
(2010) Expert Opin. Drug Metab. Toxicol.
(2008)- et al.
Rapid Commun. Mass Spectrom.
(2002)
Chem. Res. Toxicol.
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