Optimizing Higher Throughput Methods to Assess Drug–Drug Interactions for CYP1A2, CYP2C9, CYP2C19, CYP2D6, rCYP2D6, and CYP3A4 In Vitro Using a Single Point IC50

Abstract

Drug-drug interactions involving cytochrome P(450) (CYP) are an important factor in whether a new chemical entity will survive through to the development stage. Therefore, the identification of this potential as early as possible in vitro could save considerable future unnecessary investment. In vitro CYP interaction screening data generated for CYP2C9, CYP2D6, and CYP3A4 were initially analyzed to determine the correlation of IC(50) from 10- and 3-point determinations. A high correlation (r = 0.99) prompted the further assessment of predicting the IC(50) by a single value of percent inhibition at either 10, 3, or 1 microM. Statistical analysis of the initial proprietary compounds showed that there was a strong linear relationship between log IC(50) and percent inhibition at 3 microM, and that it was possible to predict a compound's IC(50) by the percent inhibition value obtained at 3 microM. Additional data for CYP1A2, CYP2C19, and the recombinant CYP2D6 were later obtained and used together with the initial data to demonstrate that a single statistical model could be applicable across different CYPs and different in vitro microsomal systems. Ultimately, the data for all five CYPs and the recombinant CYP2D6 were used to build a statistical model for predicting the IC(50) with a single point. The 95% prediction boundary for the region of interest was about +/- 0.37 on log(10) scale, comparable to the variability of in vitro determinations for positive control IC(50) data. The use of a single inhibitor concentration would enable determination of more IC(50) values on a 96-well plate and result in more economical use of compounds, human liver or expressed enzyme microsomes, substrates, and reagents. This approach would offer the opportunity to increase screening for CYP-mediated drug-drug interactions, which may be important given the challenges provided by the generation of orders of magnitude more new chemical entities in the field of combinatorial chemistry. In addition, the algorithmic approach we propose would obviously be applicable for other in vitro bioactivity and therapeutic target enzyme and receptor screens.

Investigation of miscellaneous hERG inhibition in large diverse compound collection using automated patch-clamp assay

“…For the IC 50 prediction, the compound activity from single concentration would cause more negative predictions [26] , whereas the weighed 2-point method could fix the disadvantage of 1-point prediction. The IC 50 values for the current screening were first calculated by 1-point [32] , and then by weighed 2-point methods [26] . Compared to the 1-point prediction method, weighed 2-point method helps to neutralize the unbalance from the one-concentration's disadvantage.…”

“…The bump shown in Figure 5D is caused by the compounds that inhibited hERG at 10 µmol/L but inactive at 1 µmol/L. Therefore IC 50 values of the hERG inhibitor hits from this set of data were binned into three classes, a high (IC 50 <1 µmol/L), an intermediate (1 µmol/L<IC 50 <10 µmol/L), and a low potential (IC 50 >10 µmol/L) for hERG channel inhibition [26,32,33] . …”

Computational Approaches That Predict Metabolic Intermediate Complex Formation with CYP3A4 (+b5)

“…These studies may be supplemented with additional in vitro assays with more traditional drug substrate probes using liquid chromatography/mass spectrometry analysis (Ekins et al, 2000b). Data from all of these in vitro screens are increasingly applied to predictive algorithm development (Riley et al, 2001;Gao et al, 2002;Ekins et al, 2003a,b). The interest in computational models based on in vitro data for predicting potential drug interactions via this protein and others (Ekins and Swaan, 2004) represents a possible means to improve productivity of the drug discovery process and remove potential bottlenecks caused by in vitro testing.…”

In Vitro Evaluation of Reversible and Irreversible Cytochrome P450 Inhibition: Current Status on Methodologies and their Utility for Predicting Drug–Drug Interactions

“…3 μM) (24,25). However, the requirement to re-test compounds of interest means that when developing a screening strategy including compound supply logistics and short turnaround times it is often more efficient simply to perform an IC 50 determination for each test substance.…”