Comparative metabolism of DDAO benzoate in liver microsomes from various species
Introduction
Carboxylesterases (CEs) belong to a superfamily of α/β-fold proteins, which could hydrolyze chemicals containing carboxylic acid ester, amide, or thioester (Satoh and Hosokawa, 1998). Numerous natural compounds including fatty acids, flavonoids, tanshinones and triterpenoids can inhibit enzymatic activities of hCEs (Wang et al., 2017). In human, two primary CEs including human carboxylesterase 1 (hCE1) and human carboxylesterase 2 (hCE2) have been identified and extensively studied in the past decade (Satoh and Hosokawa, 2006, Williams et al., 2010). hCE1 and hCE2 share 47% amino acid sequence identity, but exhibit differential tissue distribution and distinct substrate and inhibitor specificities (Imai et al., 2006, Hosokawa et al., 2007). Generally, hCE1 is primarily expressed in the liver, and prefers to hydrolyze the ester substrates with a small alcoholic group and a large, bulky acyl groups, such as enalapril and clopidogrel (Zhu et al., 2013, Thomsen et al., 2014). In contrast, hCE2 is expressed at relatively high levels in the small intestine, colon and liver, and prefers to hydrolyze the esters with a relatively large alcohol group and a small acyl group, such as anticancer drugs capecitabine and irinotecan (Quinney et al., 2005, Hatfield et al., 2011, Landowski et al., 2006).
As the major CEs isoform in the human intestine and tumor tissues, hCE2 exerts profound effects on oral bioavailability of ester drugs, the treatment outcomes as well as the adverse effects of ester anti-cancer agents (Hsieh et al., 2015). Of interest, the relative abundance variation of hCE2 between the tumor and normal tissues has been reported (Sanghani et al., 2003). Therefore, the investigation on the tissue-dependent and tumor-associated variations in both expression and function of CE2 is very helpful for the deeper understanding of the biological roles of this key enzyme in the detoxification and metabolic activation of xenobiotic compounds. As the primary catalyzing enzyme responsible for intestinal hydrolysis of irinotecan, CE2 is deemed as an important toxicity biomarker, correlating with the occurrence of severe or life-threatening toxicities of irinotecan, including diarrhea and neutropenia (Di-Paolo et al., 2011). CE2 may protect the central nervous system from toxic esters and maybe a component of blood-brain barrier (Zhang et al., 2002). CE2 has broad and overlapping xenobiotic substrate specificities, attracting the attention of the researchers in academia, the pharmaceutical industry, and regulatory agencies (Ross and Crow, 2007). However, limited by the shortcomings of the available CE2 probe such as in vivo imaging specificity or tissue penetration, it is still difficult to investigate the biological function of CE2 in living animals, including the functional changes in physiological and pathological states as well as the complex interaction between CE2 and xenobiotic.
DDAB is a newly developed colorimetric NIR fluorescent probe of hCE2 and has been successfully used for the rapid, selective and sensitive detection of hCE2 in living cells and living animals (Lei et al., 2017, Jin et al., 2016). Distinguished from the previous fluorescent sensors of CE2, DDAB is of high specificity, good tissue penetration, as well as minimal interference from background in complex biological systems (Jin et al., 2016). DDAB displays high efficiency in measuring the real activities of hCE2 as well as screening hCE2 modulators in complex biological samples. With low cytotoxicity and satisfactory cell membrane permeability, DDAB can be used not only for fluorescence imaging in living cells, but also to visualize endogenous CE2 in living mouse and various CE2-expressing tissues (Jin et al., 2016). Overall, DDAB offers a possibility for efficient monitoring of the biological functions of CE2 in vivo (Fig. 1).
Animal models are indispensable tools for further studies of pharmacological and physiological roles of CE2, and must be used in the pharmacological and toxicological tests of the drug candidate whose in vivo metabolism is prominently mediated by CE2 (Nishimuta et al., 2014, Mukai et al., 2015, Marques et al., 2014). The interspecies similarity and difference on the hydrolysis of DDAB, in spite of its superiority as the CE2 substrate, has not been systematically investigated, limiting the application of DDAB and interpretation of the relevant results in animals. In order to explore the applicability of DDAB in commonly used animal species, the interspecies difference in DDAB hydrolysis was investigated using liver microsomes of human and experimental animals including mouse, rat, dog, minipig, and cynomolgus monkey. The hydrolysis behaviors of DDAB in liver microsomes from different species were characterized with respect to the similarities and differences of metabolic profiles, involved enzymes, catalytic efficacy and inhibitory potency by known chemical inhibitors.
Section snippets
Chemicals and reagents
DDAO (9H-(1,3-Dichloro-9,9-Dimethylacridin-2-One-7-yl) benzoate (also termed DDAB) was synthesized by the authors, and the purity is above 98% determined by HPLC-UV. DDAO was purchased from Tianjin Biolite Biotech (Tianjin, China). Bis(p–nitrophenyl)phosphate (BNPP), ethylene diamine tetraacetic acid (EDTA), huperzine A (HA), and loperamide (LPA) were purchased from TCI (Tokyo, Japan). Ethylene diamine tetraacetic acid (EDTA) and huperzine A (HA) were obtained from J&K Chemical Ltd. (Beijing,
DDAB hydrolysis in liver microsomes from different animal species
As shown in Fig. 2, the metabolic profiles of DDAB hydrolysis in liver microsomes from mouse, rat, dog, minipig, monkey and human (50 μg protein/ml) were plotted, following 15 min incubation under physiological conditions. It was evident from Fig. 2 that DDAB could be hydrolyzed in all tested liver microsomes (Fig. 2). The single metabolite of DDAB was identified as DDAO, by comparison of the LC retention times, UV spectra and MS spectra with the help of the authentic standard (Supplementary Fig.
Discussion
As one of the most important hydrolases distributed in human intestine and tumor tissues, CE2 has significant impacts on the oral bioavailability of ester drugs, the treatment outcomes and the toxicities of various CE2 substrates as anti-cancer agents (Imai, 2007, Hatfield et al., 2010, Imai and Ohura, 2010, Kobayashi et al., 2012, Laizure et al., 2013, Xiao et al., 2013, Hsieh et al., 2015, Pratt et al., 2013). The key roles of CE2 in the activation of anticancer drugs and the detoxification
Conclusion
In summary, the interspecies difference of DDAB hydrolysis was investigated in liver microsomes from different animal species, with respect to the similarities in metabolic profiles, involved enzymes, the kinetic behaviors and the response towards known esterase inhibitors. The mammalian CEs are the predominant enzymes responsible for DDAB hydrolysis in liver microsomes from human and five common experimental animals. Kinetic analyses showed that DDAB hydrolysis in liver microsomes from all
Conflict of interest
The authors declare that there are no conflicts of interest.
Transparency document
Acknowledgments
This work is supported by the Natural Science Foundation of China (81503152, 81460572 & 81473181), Natural Science Fund of Liaoning Province (2015020663), National Basic Research Program of China (2013CB531805), Liaoning postgraduate education & teaching reform project (2016) and Innovative entrepreneurship program of high-level talents in Dalian (2016RQ025).
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These authors contributed equally to this work.