Automated high throughput ADME assays for metabolic stability and cytochrome P450 inhibition profiling of combinatorial libraries.

Early determinations of pharmaceutical properties can serve as predictors of a compound's likely development success. Our laboratory has implemented high throughput in vitro absorption, distribution, metabolism and excretion (ADME) assays which address absorption, metabolism, and physico-chemical properties in an effort to identify potential development liabilities early, thereby minimizing discovery to market attrition. In response to the throughput demands of parallel synthesis, we have incorporated a SAGIAN core robotics system for the determination of both metabolic stability in human liver microsomes (HLMs) and cytochrome P450 (CYP450) inhibition. This automated solution has led to an increase in capacity, throughput and reliability for both in vitro assays. The SAGIAN core robotics system integrates devices such as liquid handlers, plate hotels and incubators through the use of an ORCA robotic arm. The HLM stability assay utilizes a Multimek 96-channel pipettor for liquid handling. The incubation plates are transferred off-line for final semi-quantitative analysis using high throughput parallel LC/MS. The CYP inhibition method combines both liquid handlers and an integrated fluorescence plate reader to perform single concentration percent inhibition assays for 88 compounds. Cytochrome P450 inhibition is measured for both CYP3A4 and CYP2D6 isozymes. This system represents a fully integrated approach to high throughput ADME evaluation in support of drug discovery. The core system concept creates a plug-and-play approach, which combines a series of modular stations to build a robotic platform, which is flexible, upgradable, and easily reconfigured when assays change or are newly developed. The application of these strategies as a means of assessing metabolic stability and CYP inhibition of synthetic libraries is discussed.

Application of parallel liquid chromatography/mass spectrometry for high throughput microsomal stability screening of compound libraries.

Solution-phase and solid-phase parallel synthesis and high throughput screening have enabled biologically active and selective compounds to be identified at an unprecedented rate. The challenge has been to convert these hits into viable development candidates. To accelerate the conversion of these hits into lead development candidates, early assessment of the physicochemical and pharmacological properties of these compounds is being made. In particular, in vitro absorption, distribution, metabolism, and elimination (ADME) assays are being conducted at earlier and earlier stages of discovery with the goal of reducing the attrition rate of these potential drug candidates as they progress through development. In this report, we present an eight-channel parallel liquid chromatography/mass spectrometry (LC/MS) system in combination with custom Visual Basic and Applescript automated data processing applications for high throughput early ADME. The parallel LC/MS system was configured with one set of gradient LC pumps and an eight-channel multiple probe autosampler. The flow was split equivalently into eight streams before the multiple probe autosampler and recombined after the eight columns and just prior to the mass spectrometer ion source. The system was tested for column-to-column variation and for reproducibility over a 17 h period (approximately 500 injections per column). The variations in retention time and peak area were determined to be less than 2 and 10%, respectively, in both tests. The parallel LC/MS system described permits time-course microsomal incubations (t(o), t5, t15, t30) to be measured in triplicate and enables estimations of t 1/2 microsomal stability. The parallel LC/MS system is capable of analyzing up to 240 samples per hour and permits the complete profiling up to two microtiter plates of compounds per day (i.e., 176 test substrate compounds + sixteen controls).