DMD #048264 1 Discovery and Characterization of Novel, Potent ...

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DMD #048264 CYP2J2 Enzymatic Activity Was Determined by Astemizole O- demethylation Reaction. The astemizole O-demethylation reaction was used to characterize the metabolic/enzymatic activity of CYP2J2 as this biotransformation is well known to be catalyzed primarily by CYP2J2 in human (Matsumoto et al., 2002; Lee et al., 2012). The metabolizing activity of CYP2J2 for astemizole O-demethylation was measured to ensure that substrate concentration used in the follow-up inhibition studies was suitably around the Km value. Under our experimental conditions, the apparent kinetic parameters of astemizole O-demethylation using recombinant human CYP2J2 were determined as the followings: Km = 0.09 ± 0.01 μM and Vmax = 339 ± 13.0 pmol/min/nmol protein (n = 2). Telmisartan and Flunarizine Show Significant and Selective Inhibition against CYP2J2. A total of 69 marketed drugs were screened by using this in vitro astemizole O-demethylation system to characterize their inhibitory effect on CYP2J2 activity. The results are shown in Table 3. Out of the 69 compounds, 20 of them inhibit the CYP2J2 metabolizing activity with an IC50 value less than 50 μM, while 12 compounds even show IC50 values < 10 µM. Three most potent compounds, namely telmisartan, flunarizine, and amodiaquine, exhibit sub-μM potency against CYP2J2 with IC50 values of 0.42, 0.94, and 0.99 μM, respectively. The concentration-dependent inhibition curves for telmisartan and flunarizine are shown in Figure 1. In order to evaluate the selectivity of CYP2J2 inhibition and given its predominant expression in the extrahepatic tissues such as intestine, we examined the inhibitory effect of these 20 compounds against five major human CYP isoforms including CYP3A4, CYP2C9, CYP2C19, and CYP2D6 which are also the most abundantly expressed CYP isoforms in the human intestine (Ding and Kaminsky, 2003; 16
DMD #048264 Paine et al., 2006) and CYP1A2. As shown in Table 3, besides inhibition of CYP2J2 telmisartan also inhibits CYP2C9 (IC50 = 4.8 μM), nearly 10-fold less potent comparing to that of CYP2J2. On the other hand, telmisartan does not exhibit any inhibition against other four major CYPs including CYP3A4 and CYP2D6. Similarly, flunarizine only inhibits CYP2D6 with an IC50 of 7.8 μM, which is also about 10-fold less potent than that of CYP2J2, and shows minimum inhibition for other four key CYPs (IC50 > 50 µM). Moreover, amodiaquine is a potent inhibitor for both CYP2J2 (IC50 = 0.99 μM) and CYP2D6 (IC50 = 0.64 μM). In addition, it is noteworthy that both norfloxacin and metoprolol display excellent selectivity for CYP2J2 with IC50 values of 2.6 and 4.9 μM, respectively and are not active against all five major CYPs (IC50s > 50 μM; Table 3). Telmisartan and Flunarizine Are Non-substrate CYP2J2 Inhibitors. Many CYP inhibitors are also substrates of the isoform they inhibit, especially for those competitive inhibitors that exert their inhibitory power by competing for the same catalytic binding site of the substrate. In this study the metabolic activity of CYP2J2 towards telmisartan and flunarizine was evaluated. The metabolic clearance of astemizole by CYP2J2 was also measured to define the enzyme activity. After incubating for 30 minutes, astemizole was metabolized by CYP2J2 with an intrinsic clearance (CLint) of 3.05 ± 0.07 μL/min/pmol protein (n = 2; Figure 2, panel A). This correlates well with the intrinsic clearance calculated from Vmax and Km (CLint = Vmax/Km = 3.77 ± 0.05 μL/min/pmol protein), indicating excellent consistency of the CYP2J2 activity between these two studies based on percentage remaining of the substrate astemizole and enzyme kinetics. Importantly, the amount of telmisartan and flunarizine remains almost unchanged after incubation for 30 minutes (CLint = 0.0 μL/min/pmol protein, n = 2; Figure 2, panels B and C). This result clearly shows that both telmisartan 17
Page 1 and 2: DMD Fast Forward. Published on Octo
Page 3 and 4: DMD #048264 Abstract Cytochrome P45
Page 5 and 6: DMD #048264 its rather broad substr
Page 7 and 8: DMD #048264 role of CYP2J2 in drug
Page 9 and 10: DMD #048264 CYP2C9, CYP2C19, and CY
Page 11 and 12: DMD #048264 AST, DES-AST, and IS, r
Page 13 and 14: DMD #048264 where v is the reaction
Page 15: DMD #048264 = ΔEelec + ΔEvdW + Δ
Page 19 and 20: DMD #048264 plot does not go throug
Page 21 and 22: DMD #048264 surface for telmisartan
Page 23 and 24: DMD #048264 given its predominant e
Page 25 and 26: DMD #048264 subsequently confirmed
Page 27 and 28: DMD #048264 activity by restraining
Page 29 and 30: DMD #048264 References Baron A, Fri
Page 31 and 32: DMD #048264 Lafite P, Dijols S, Zel
Page 33 and 34: DMD #048264 Young CL, Dias VC, and
Page 35 and 36: DMD #048264 Figure Legends FIGURE 1
Page 37 and 38: DMD #048264 segment (Ile376, Pro377
Page 39 and 40: DMD #048264 Table 2: Probe substrat
Page 41: DMD #048264 Table 4: Binding free e

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