Establishing in vivo-in vitro correlations for metabolic clearance in Discovery is important in selecting compounds for further assessment. Since non-specific microsomal binding can lead to underestimation of microsomal clearance, determining the unbound intrinsic clearance becomes important for developing meaningful correlations. An automated method using a Tecan liquid handling system has been developed to rapidly assess microsomal binding of all newly synthesized compounds using a 96-well equilibrium dialysis apparatus. A high throughput has been achieved by the employment of a cocktail approach where three compounds are allowed to bind to microsomal proteins in the same well. To further increase the throughput, matrix blending was used, where the protein side of one well is mixed with the buffer side of a second well and vice versa. The unbound fractions for the six compounds are analyzed in a cassette fashion by a fast LC-MS-MS methodology with 45s injection-to-injection time. This method enables the determination of microsomal binding for close to 800 compounds in 2 days. The availability of the unbound microsomal fraction has enabled better predictions of in vivo clearance, and better ADME support for Discovery projects.

Automated high-throughput assay for microsomal binding with ultra fast liquid chromatography-tandem mass spectrometry

243rd National Meeting (2012)

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