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  • Based on our studies presented above


    Based on our studies presented above, atipamezole can be used as a new in vitro and in vivo tool 7-Nitroindazole as a pan-CYP inhibitor for CYP mediated metabolism study. It carries many unique characteristics compared to ABT which is currently being widely used for this purpose. These new characteristics include: 1) Atipamezole is a fast acting and reversible pan-CYP inhibitor without discrimination of the 7 major CYPs. 2) It works well at 10–100 μM and 0.141 mmol/kg for in vitro and in vivo rat study that is much lower than that of ABT with 1000 μM and 0.746 mmol/kg for in vitro and in vivo study. 3) It requires a very simple laboratory procedure, as it doesn't need preincubation followed by another step of activity measurement which often needs to add substrate and additional cofactors and dilute the protein concentration (e.g. microsomes) in the first reaction. 4) It worked well in preclinical species, such as rat and dog liver microsomes tested in this study. Rat and dog are two preclinical species popularly used as the toxicological species. Thus, high dose study of the test drug with good bioavailability is desired in these species. When a low bioavailability is observed, atipamezole would be a good tool compound to differentiate whether metabolism by CYP was the cause of low bioavailability. 5) Compared to ABT, atipamezole provides a complete inhibition of CYP2C9 mediated diclofenac 4′-hydroxylation, not only in human liver microsomes, but also in preclinical species. Since CYP2C9 is a major CYP in drug metabolism, it is important to cover its contribution in the overall CYP-mediated metabolism. 6) Atipamezole is rapidly eliminated in vivo, which has its advantages and disadvantages. The advantage is that is can be used as a potent pan-CYP inhibitor with fast on and fast off effects to enhance the bioavailability without long lasting inhibition. In other words, the inhibitory effect can be quickly withdrawn when desired. The disadvantage is again from the high in vivo clearance, if one needs to inhibit CYP activity for a long term, e.g. 24 h, twice daily dose would be required. Our in vivo study of atipamezole and diclofenac is a good example of applying atipamezole in an in-life study setting.
    Conclusion In this study, we provided data for a fast on and fast off reversible pan-CYP inhibitor which has potent inhibition toward 7 major human CYPs. It is a good tool in drug discovery to differentiate the contribution of CYPs toward the total metabolism (e.g. conjugation or protein mediated hydrolysis); as well as differentiate CYP mediated metabolism from P-gp mediated efflux in first-pass effect. It works well in preclinical species and can be applied to in vivo studies. It provides a better and easier alternative for ABT in mechanistic drug metabolism and toxicity studies.
    Acknowledgment This work was supported by the National Science & Technology Major Special Project on Major New Drug Innovation, China [2018ZX09711003-006].
    Introduction Polycyclic aromatic hydrocarbons (PAHs) are widely present in various aquatic ecosystems, particularly in sediments (Li et al., 2006, Guo et al., 2007, Oliva et al., 2010). They are derived from various sources, such as fossil fuels, burning organic matter, and oil spill accidents (Banni et al., 2010). Due to persistence and bioaccumulation, they pose a threat against aquatic organisms. Benzo[a]pyrene (B[a]P) is a high molecular weight (5-ring) PAHs, and is a known carcinogen and/or mutagen (Shaw and Connell, 1994). In aquatic environments, B[a]P concentration is reported to be in the range of pg/L–ng/L in clean surface waters, and up to μg/L in polluted rivers (Maciel and Zaldivar, 2005). Several reports demonstrated that B[a]P has a negative impact on survival (Collier and Varanasi, 1991, Hawkins et al., 1991, Palalnikumar et al., 2012), growth (Jifa et al., 2006, Kim et al., 2008), behavior (Lawrence and Poulter, 2001, Oliveira et al., 2012), and reproduction (Monteverdi and DiGiulio, 2000, Choy et al., 2007) in aquatic organisms. B[a]P can easily diffuse within the cell through the cellular membrane, bind to aryl hydrocarbon receptor (AhR), and subsequently induce the transcription of many genes, including cytochrome P450 (CYP450) (Safe, 2001). B[a]P is metabolized by CYP450, a phase I enzyme, into a reactive metabolite, B[a]P-diolepoxide which forms a DNA adduct and leads to mutation (Wahidulla and Rajamanickam, 2009). B[a]P-diolepoxide is transformed into less harmful substances by phase II enzymes, such as glutathione S-transferase (GST) (Trushin et al., 2012).