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  • A cocktail approach can detect the activities


    A cocktail approach can detect the activities of multiple CYP 450 isoforms following administration of multiple CYP-specific substrates in a single experiment. Caffeine, losartan, omeprazole, dextromethorphan and midazolam are often used as substrates in “cocktail probes” of human cytochrome CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, respectively (Ryu et al., 2007). The object of our study was to assess the DDI magnitude of SLT and identify the attribution of its herbal components on the overall effect of SLT with respect to five major CYP450 isoforms in rats.
    Materials and methods
    Result and discussion
    Conflict of interests
    Acknowledgments This study was financially supported by “Major New Medicine Project” in Megaprojects of Science Research of China (No. 2009ZX09502-006), the National Natural Science Foundation of China (30701101) and the National Basic Research Program of China (973 program, 2015CB554400)
    Introduction Finding compounds with good oral bioavailability is a crucial advancement in drug discovery and development. Factors closely associated with it include the potential of drug incomplete release from the formulation, degradation in stomach or gut lumen, permeability through the gut wall, and metabolism by enzymes and efflux or uptake by transporters occurred in gut and liver. Among all these factors, metabolizing enzymes and efflux transporters play the leading roles attributing to poor bioavailability if a punicalagin has extensive metabolism and poor absorption. Within the drug metabolizing enzymes and efflux transporters, CYP enzymes and P-gp transporter should have the highest impact on bioavailability. Hence, deconvolution of factors that influence the oral-systemic exposure is of great value for improving compounds with poor bioavailability (Caldwell et al., 2005). On the other hand, distinguishing the toxicity mediated by the parent drug or its metabolites using a pan-CYP inhibitor is also essential during safety evaluation early on (Mico et al., 1988; Boily et al., 2015). 1-aminobenzotriazole (ABT, Fig. S1) was found to be able to block CYP activities both in vitro and in vivo three decades ago. Upon oxidation, ABT forms a reactive benzyne intermediate that inactivates CYP by alkylation of the prosthetic heme (Ortiz de Montellano and Mthews, 1981). Thus, it has become a popular tool to block CYP enzymes mediated metabolism both in in vitro and in vivo studies. During an in vitro study, ABT requires a preincubation first in human or animal liver microsomes at high concentrations (~1 mM) to generate reactive metabolite, then test compounds are added to distinguish metabolism mediated by P450 from non-P450 (Dalmadi et al., 2003; Williams et al., 2003; Klieber et al., 2014). In an in vivo study, 15–16 h pretreatment of ABT at a single oral dose of 100 mg/kg for rats is recommended (Meschter et al., 1994; Strelevitz et al., 2006). Although it is acknowledged that ABT is a useful tool compound for determining P450-mediated metabolism, multiple findings proved that it was ineffective in inactivating human CYP2C9, that is, ABT is much less selective for certain P450s (Sodhi et al., 2014). In an early study, it was found that 58% of diclofenac 4′-hydroxylase activity (CYP2C9) still remained after a 30-min preincubation with 1 mM ABT in human liver microsomes (Linder et al., 2009). CYP2C9 is the second abundant CYP enzyme after CYP3A (Chow et al., 2010), many therapeutic drugs are metabolized by CYP2C9. Using ABT as a nonselective inactivator against CYP-mediated oxidative metabolism for reaction phenotyping or mechanistic toxicity studies has its limitation, as such if one takes it for granted that a complete inhibition would be achieved by concomitant test compounds with ABT, CYP mediated fraction would be underestimated. Another available non-specific CYP inhibitor is SKF-525A (Chung et al., 2000; Emoto et al., 2003; Lee et al., 2007). However, it does not strongly inhibit all CYPs as well. In particular, SKF-525A has been reported to weakly inhibit human CYP1A2 and CYP2E1. SKF-525A is also a time-dependent inhibitor (TDI) of CYPs with enhanced inhibition in NADPH-fortified human liver microsomal incubations. However, the IC50 values toward CYP1A2 and CYP2E1 are still far >100 μM after preincubation (Emoto et al., 2003). Furthermore, the reported in vitro or in vivo study protocols of SKF-525A as a pan-P450 inhibitor is limited in literature. Thus, a potent and non-specific CYP inhibitor is desired to enable an accurate determination of drug metabolism by CYPs from non CYPs.