Harmonised High Throughput Microsomal Stability Assay

Introduction: Prediction of human pharmacokinetics from in vitro assays and pre-clinical data is an integral part of drug discovery. In vitro stability metabolic studies can provide an estimate of in vivo hepatic intrinsic clearance through inclusion of biological scaling factors. Many labs have personalised stability protocols including marker compounds and have adopted QC criteria and assay limits to ensure data integrity. Contract research organisations (CRO's) provide integrated drug discovery support to academic and pharmaceutical/biotechnology institutions to progress their in-house projects. The majority of these clients have established in-house protocols with associated criteria to ensure data consistency between in-house and external labs.

Methods: In this study, numerous assay variables were condensed into one harmonised assay format and a range of compounds with diverse physicochemical properties were evaluated. The protocols were diverse with respect to the following attributes: buffer, microsomal concentration and species strain.

Results: Comparison of human lots in vitro CL between the traditional and consolidated assay formats showed a good correlation with no significant difference. A clear relationship was demonstrated between strains. Interpretation of in vitro intrinsic clearance between the strains for each species was consistent. Using strict classes of in vitro hepatic intrinsic clearance values (<50, 50-100, >150μL/min/mg protein) comparisons across different conditions such as, assay variables, human lots, mouse and rat strains showed >80% agreement.

Discussion: A high throughput assay was developed that enables the simultaneous measurement of CL using mouse, rat and human hepatic microsomes (consolidated assay). By condensing all possible variables into one assay format, consistent data were obtained during head to head tests.