Application of higher throughput screening (HTS) inhibition assays to evaluate the interaction of antiparasitic drugs with cytochrome P450s.

In this study we have evaluated the application and reliability of using fluorescence (FLUO)-based high throughput screening assays with recombinant CYPs (rCYP). This was accomplished by screening 29 clinically important antiparasitic drugs for inhibition of the five major drug-metabolizing CYPs (-1A2, -2C9, -2C19, -2D6, and -3A4). Data from FLUO/rCYP assays were compared with that obtained by conventional HPLC assays using human liver microsomes (HLM) and rCYPs. The K(i) values showed good correlations: FLUO/rCYP and HPLC/rCYP (r(2) = 0.81), HPLC/rCYP and HPLC/HLM (r(2) = 0.82), and FLUO/rCYP and HPLC/HLM (r(2) = 0.72). Niclosamide had substrate-dependent contrasting effects on CYP2C9 activity with an apparent activation (400%) of 7-methoxy-4-trifluoromethylcoumarin demethylase activity and potent inhibition (K(i) = 6.00 microM) of diclofenac 4-hydroxylase activity. Potent inhibitors of CYP1A2 were artemisinin, dihydroartemisinin, thiabendazole, primaquine, and niclosamide (K(i) = 0.43, 3.67, 1.54, 0.22, and 2.70 microM, respectively). Proguanil, cycloguanil, amodiaquine, and desethylamodiaquine inhibited CYP2D6 (K(i) = 6.76, 5.97, 2.1, and 4.13 microM, respectively). Considering the C(max) of these drugs, artemisinin, thiabendazole, primaquine, amodiaquine, and desethylamodiaquine may cause clinically important interactions because they are predicted to inhibit 67 to 99% of the activities of the CYPs they interact with. In addition, our results suggest CYP1A2 inhibition as the mechanism behind the observed thiabendazole/theophylline and primaquine/antipyrine interactions in vivo.