Activities for Optimizing CiPA Recommended Protocols in Patch-Clamp Assay

Revising the ICH regulatory guideline, S7B, for the Non-clinical Evaluation of the Potential for Delayed Ventricular repolarization (QT Interval Prolongation by Human Pharmaceuticals) has been suggested and Comprehensive In Vitro Proarrhythmia Assay (CiPA) activities are being implemented. We previously studied the applicability of the CiPA-recommended voltage protocols for hERG, Cav1.2, and late Nav1.5 current patch clamping which was announced last year (this research was presented at the 2019 Japanese Safety Pharmacology Society annual meeting). As a result, inter-facility differences were found for some compounds in the hERG assay on an automated patch-clamp system, QPatch. Such differences were also noted in enhancement ability of ATX-II for late Nav1.5 current. This time, we investigated the factors for the interfacility differences and studied more appropriate protocols. In addition, we present the results of the assays by the CiPA-recommended protocols for each ion channel. Manual patch clamp and QPatch system were selected as the platforms. For the voltage protocols, the CiPA-recommended ones were followed whereas the composition of intra- and extracellular buffers were modified. Preparation procedures and application methods of test solutions (such as application duration or number of replicates) for the QPatch assay were examined. Test compounds were selected from each of the low-, intermediate-, and high-risk categories of the “training drugs” in the CiPA in silico model. We present the comparison results of our data by the CiPA-recommended protocols and the data given by the CiPA. We also discuss buffer compositions, preparation procedures or application methods for test solutions to reduce the inter-facility differences. We would like to show and discuss the more appropriate protocols including the detailed experimental methods, which are not given in the CiPA-recommended protocols, and points to be modified.