In order to demonstrate the therapeutic potential of the DHP dione chemical class, the team next tried to identify a suitable candidate to probe PK/PD relationship in vivo. As mentioned earlier in the SAR, most analogs showed moderate permeability and low efflux ratio in the MDCK-MDR1 cell line. They showed excellent stability in the human and mouse liver microsome assay. The compounds have also shown good stability in human and mouse hepatocytes, as no significant turnover was observed for most compounds under the assay conditions employed. The in vivo drug metabolism and pharmacokinetic (DMPK) properties were assessed in cassettes of five compounds each, including 17 as the reference. Cassette administration is an extremely useful approach to generate in vivo PK data quickly in a cost effective and animal sparing fashion. A cassette dosing strategy also enabled direct comparison of drug brain penetrability among a set of compounds within the same set of animals. In practice, cassette doses were prepared for both intravenous (IV) and oral (PO) administration utilizing a standard dose formulation for each route throughout the project.