Direct and quantitative evaluation of the major human CYP contribution (fmCYP) to drug clearance using the in vitro Silensomes™ model.

1. We have applied the concept of using MBIs to produce CYP-Silensomes to quantify the contribution of the major CYPs to drug metabolism (fmCYP). 2. The target CYPs were extensively and selectivity inhibited by the selected MBIs, while non-target CYPs were inhibited by less than 20% of the homologous control activities. Only CYP2D6-Silensomes exhibited a CYP2B6 inhibition that could be easily and efficiently encountered by subtracting the fmCYP2B6 measured using CYP2B6-Silensomes to adjust the fmCYP2D6. 3. To validate the use of a panel of 6 CYP-Silensomes, we showed that the fmCYP values of mono- and multi-CYP metabolised drugs were well predicted, with 70% within ± 15% accuracy. Moreover, the correlation with observed fmCYP values was higher than that for rhCYPs, which were run in parallel using the same drugs (<45% within ±15% accuracy). Moreover, the choice of the RAF substrate in rhCYP predictions was shown to affect the accuracy of the fmCYP measurement. 4. These results support the use of CYP1A2-, CYP2B6-, CYP2C8-, CYP2C9-, CYP2D6 and CYP3A4-Silensomes to accurately predict fmCYP values during the in vitro enzyme phenotyping assays in early, as well as in development, phases of drug development.

Direct and quantitative evaluation of the human CYP3A4 contribution (fm) to drug clearance using the in vitro SILENSOMES model.

1. Among the different in vitro studies recommended by the regulatory agencies, no gold-standard model can easily and directly measure the quantitative CYP450 contributions to drug biotransformation. In this article, we propose an original strategy, called SilensomesTM, to produce human liver microsomes silenced for one specific CYP450, thanks to specific mechanism-based inhibitors (MBI). 2. Using azamulin as a specific CYP3A4 MBI, we demonstrated the proof of concept that CYP3A4 can be totally, specifically (even against 3A5) and permanently (at least for six years) inhibited by our process. Thus, comparing clearance in control and CYP3A4-SilensomesTM, CYP3A4 contributions were determined for 11 CYP3A4 substrates which correlated with known in vivo contributions and revealed accuracy with less than 10% error. In comparison, contributions determined using recombinant human CYP450 (rhCYP450s) were less accurate (more than 10% error for 30% of the tested CYP3A4 substrates). 3. This easy and ready-to-use in vitro method combines the advantages of existing models (specificity of rhCYP450s and representativeness of HLM) without their drawbacks. The same strategy could be used to silence other major CYP450s one-by-one to provide a complete direct CYP450 quantitative phenotyping kit.