Results

1. 0 MIN 30 MIN 5 MIN 60 MIN 10 MIN 60 MIN NO CoF 20 MIN Incubate @ 37°C 0, 5, 10, 20, 30, 60 min. 350.00 300.00 y = 1.2534x - 2.1081 2 R = 0.8603 250.00 200.00 Literature Clint 150.00 y = 1.2401x - 2.5148 2 R = 0.8171 100.00 50.00 ALH FX 0.00 0.00 50.00 100.00 150.00 200.00 250.00 Experimental Clint A B A+B ON DECK INCUBATION AND 384-WELL FORMAT SIMPLIFY AND INCREASE THROUGHPUT FOR HUMAN LIVER MICROSOMAL SCREENING IN AN HT-ADME SCREENING LABORATORY M. Snyder, C. Taylor, J. Janiszewski & K. Whalen - PDM, Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340 Overview Introduction Results E • Introduction • To develop an automated 384-well microsomal metabolic stability assay to support rapid screening of 2,000 discovery compounds per week. • Methods • Biology: • Twelve compounds with a broad range of clearance rates and P450 pathways were chosen to validate assay1,2. • Eight plates were generated per 384-well master plate: a matrix blank; 0, 5, 10, 20, 30, 60 minute timepoints; and a 60 minute no co-factor control. • Assay volume: 27.8 µl. Automation/Bioanalytical: • Caliper Life Sciences Sciclone ALH3000 and Beckman Coulter Biomek FX workstations equipped with 384-channel disposable tip array were used to perform all liquid handling steps. • An AB/Sciex API3000 triple quadrupole mass spectrometer, operating in selected reaction monitoring (SRM) mode, was used for analyte and internal standard detection. • Results • Validation results were assessed relative to published clearance values. • Key learning’s: • Rapid Dispense (100 µl /sec) • On-deck Incubation • Scheduling with Excel Macro • The experimental results correlated well to the literature values (Figure 4). The larger discrepancies may be attributed to the differing incubation conditions (e.g. protein concentration). • Little difference in workstations (Figure 5). • The combination of 384-well format and automated liquid handling allows for 720 test compounds to be assayed in a single morning. This format equates to a potential 3-day throughput of over 4300 compounds. • Further throughput gains are restricted by LC/MS/MS analysis. (i.e. 4300 compounds = 43,000 LC injections) • The large capacity allows us to maintain discovery chemistry’s capacity of over 2000 compounds per week. In drug discovery, the number of chemical compounds requiring in-vitro ADME screening is ever increasing. To keep pace with compound submissions, new methods must be developed to increase the throughput of current in-vitro ADME assays. Herein, we present our group’s efforts towards increasing throughput of the Human Liver Microsome metabolic stability assay using a compressed 384-well format and an automated liquid handler. B D C ACN & IS TIP BOX A Methods: Microsome Assay Specifics 250.00 Amitriptyline Figure 2.Sciclone Deck Layout. (A) Two 6-position Mecour heating blocks, (B) Recirculating reservoir containing cold acetonitrile and IS, (C) Microsome reservoir, (D) 384-well tip-wash station, (E) Extra deck space where crashed plates are moved off of heat. Desipramine Propafenone Propranolol Nicardipine Midazolam Prednisone Verapamil Diclofenac Dilitiazem Quinidine Diazepam 200.00 • P450 concentration: 0.25 µM (0.76 mg protein/mL) • Compound substrate concentration: 1 µM • Co-Factor utilizes NADPH regeneration system w/ 1 mM MgCl2 • Buffer concentration: 100 mM KPO4, pH 7.4 • Time-points: 0, 5, 10, 20, 30 and 60 minutes • Internal standard used to ensure LC/MS/MS performance and reproducibility • Liquid handling performed on Caliper Life Sciences Sciclone ALH3000 or Beckman Coulter Biomek FX workstation 150.00 Biomek FX Clint 100.00 y = 0.9416x + 1.3742 2 R = 0.9226 50.00 0.00 0.00 50.00 100.00 150.00 200.00 250.00 Sciclone ALH 3000 Clint D Figure 5.Different pipetting techniques and on-deck incubation provided similar Cl int values on either workstation. Figure 4. Intrinsic clearance values for 12 compounds (n=96) run on both automated workstations were similar to reported values. B C 2. 4. ACN (Protein Precip.) Microsomes & Cofactor A Table 1.Measured temperature variation for on-deck incubation hardware. 25 µl 75 µl 1. 34 µl/sec 100 µl/sec Figure 3.FX Deck Layout. (A) 3 X 4 Peltier heating ALP, (B) Recirculating reservoir containing cold acetonitrile and IS, (C) Microsome reservoir, (D) 384-well tip-wash station, (E) Extra deck space where crashed plates are stacked off of heat not shown. Figure 6. Non-contact mixing. Table 2.Excel scheduling macro used with Sciclone for time-point incubations. Written by Jim Batchelor, Caliper Life Sciences. 2.8 µl Assay (8) Compound Conclusions: Key Learning’s References • Rapid Dispense/ Non-contact Mixing 100 µl/sec - Savings in Tips, Time, & Deck Space (Figure 6). • On-Deck Incubation @ 37°C • Savings in Hardware/ Human Intervention (Table 1). • Easy Scheduling with Excel Macro (Table 2). • 27.8 µl Assay in 384 well plate • Savings in Microsomes - 720 Compounds in 3 hours! 3. • Riley Robert J; McGinnity D F; Austin R P. A unified model for predicting human hepatic, metabolic clearance from in vitro intrinsic clearance data in hepatocytes and microsomes. Drug Metab and Dispos (2005), 33(9), 1304-11. • Obach, R. Scott. Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: an examination of in vitro half-life approach and nonspecific binding to microsomes. Drug Metab and Dispos (1999), 27(11), 1350-1359. Figure 1. 27.8 µl assay.

2. Rapid Dispense Speed 100 ul/sec Deck space, tips ($$$$) On-deck Incubation Mecour Thermal Blocks Temp range 5.4 vs. 1.2°C Easy Scheduling Excel Macro Success & Key Learning's from Microsome Assay Automation 34 ul/sec 100 ul/sec • Success: • Automated dilution during Mic prep • 1 hour unattended completion of assay