Non-targeted metabolomics-guided sildenafil metabolism study in human liver microsomes
Introduction
The study of the biotransformation of drugs is essential for understanding and predicting the generation of active metabolites, toxic mechanisms, and excretion pathways. Therefore, it is very important to identify all generated metabolites of a drug in biofluids, such as urine and plasma, and in liver microsomes, which contain the xenobiotic metabolizing enzymes [1]. For the study of drug metabolism, liquid chromatography-mass spectrometry (LC–MS) is used as a common platform in nonclinical safety testing in novel drug development processes.
During drug metabolism studies, generated metabolites can be filtered using an extracted ion monitoring (EIC) mode for values of mass increase or decrease by structurally based, known metabolic reactions, and chemical structures of identified metabolites can be postulated from fingerprinting of detected product ions after MSn fragmentation [1]. However, this approach has a limitation in that it cannot detect all generated products following metabolism, usually involving biotransformation or structural rearrangement [2]. Because methodology using filtering of shifted mass values frequently gives false-positive results or misses real metabolites, it is the bottleneck of current metabolic identification methods.
Metabolomics is the systemic study of low molecular weight chemicals profiled through an unbiased analysis of various biofluids [3]. It allows chemical fingerprinting of small molecule metabolites that are generated by specific cellular processes. Moreover, by combining high resolution mass spectrometry (HR-MS) with multivariate data analysis, metabolomics has broad applications in the study of xenobiotic metabolism [4], [5]. This new technology has been used to identify the metabolic profiles of melatonin, acetaminophen, isoniazid, and griseofulvin, and has allowed the discovery of several novel metabolites in human, mouse, and rat [6], [7], [8].
Sildenafil (5-(2′-alkoxyphenyl)-pyrazolo-[4,3-d]-pyrimidin-7-ones) is a potent and selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) in the corpus cavernosum [9], [10]. Sildenafil citrate (Viagra™) is currently used for the oral therapy of erectile dysfunction in the clinic. Sildenafil is known to be metabolized to various metabolites by biotransformation and was thus supposed to show diversity of metabolism between species [11]. In humans, 9 phase 1 metabolites were generated by a combination of four principle pathways of metabolism, namely piperazine N-demethylation, loss of a two-carbon fragment from the piperazine ring, oxidation of the piperazine ring, and aliphatic hydroxylation [11], [12].
Here, we have combined non-targeted metabolomics approaches in a metabolism study of sildenafil to comprehensively understand its metabolic fates in human liver microsomes (HLMs). We incubated the sildenafil with or without a reduced β-nicotinamide adenine dinucleotide phosphate (β-NADPH) generation system in pooled HLMs, and the LC–MS results were analyzed with multivariate analytical methods for pattern recognition analysis, such as principal component analysis (PCA) [3], [4], [13]. Seven novel sildenafil metabolites were identified, and the related isoforms of cytochrome P450 (CYP) were examined in this study. Overall, we successfully updated the phase 1 metabolic pathway of sildenafil in HLMs to achieve a global picture of whole sildenafil metabolism using a metabolomics approach.
Section snippets
Chemicals
Sildenafil HCl, glucose 6-phosphate, β-NADPH, and glucose 6-phosphate dehydrogenase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pooled HLMs (BD Ultra Pool™ HLM 150®) and human recombinant CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were obtained from Corning Gentest (Woburn, MA).
Biotransformation of sildenafil in human liver microsomes
Sildenafil at final concentrations of 5 and 20 μM was incubated with 1 mg/mL of microsomal protein in 0.1 M potassium phosphate buffer, pH 7.4, at 37 °C for 60 min in a
Research strategy
To identify novel phase 1 metabolites of sildenafil, sildenafil (5 and 20 μM) was incubated with HLMs with or without an NGS system (Fig. 1). LC–HR–MS analyses of microsomal incubations were performed with global MS profiling, and MS spectral data were suitably processed with chemometric analysis, such as chromatogram deconvolution, isotope grouping, and peak alignment. Initially 1502 peaks were identified and filtered to 423 peaks having less than 20% coefficient of variation within triplicate
Discussion
In this study, phase 1 metabolism of sildenafil was revisited using LC–HR–MS-based metabolomics approaches. We identified twelve phase 1 metabolites in HLMs, including seven novel metabolites (M6-M12) and five previously reported metabolites (M1-M5) [11], [12]. LC–MS-based metabolomics in the study of xenobiotic metabolism was first proposed by Plumb et al. [18] and has been reviewed recently [3], [4]. Combining this with other techniques including the introduction of stable isotopes and the
Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Acknowledgement
This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Export Promotion Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number 316017-3) and was supported by a grant from the National Research Foundation of Korea, Ministry of Science, ICT and Future Planning (NRF-2014M3A9D9069714), Republic of Korea.
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These authors contributed equally.