1. 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 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. Results 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 assay 1,2.  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  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 Methods: Microsome Assay Specifics Overview Introduction 0 MIN 5 MIN 10 MIN 20 MIN 30 MIN 60 MIN NO CoF TIP BOX ACN & IS A B C D E 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.  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. References 1.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. 2.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. Compound Assay (8) 2.8 µl Microsomes & Cofactor 25 µl ACN (Protein Precip.) 75 µl 1. Incubate @ 37°C 0, 5, 10, 20, 30, 60 min. 2. Conclusions: Key Learning’s  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! 34 µl/sec100 µl/sec 3. 4. 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. D C A B Figure 1. 27.8 µl assay. Figure 4. Intrinsic clearance values for 12 compounds (n=96) run on both automated workstations were similar to reported values. y = 0.9416x + 1.3742 R 2 = 0.9226 0.00 50.00 100.00 150.00 200.00 250.00 0.0050.00100.00150.00200.00250.00 Sciclone ALH 3000 Cl int Biomek FX Cl int y = 1.2534x - 2.1081 R 2 = 0.8603 y = 1.2401x - 2.5148 R 2 = 0.8171 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 0.0050.00100.00150.00200.00250.00 Experimental Cl int Literature Cl int ALHFX Figure 5. Different pipetting techniques and on- deck incubation provided similar Cl int values on either workstation. Reaction Temp. (°C) MecourPeltier Average35.0131.4 Range34.5 – 35.731.4 – 36.8 STDEV0.2951.13 Table 1. Measured temperature variation for on-deck incubation hardware. Table 2. Excel scheduling macro used with Sciclone for time-point incubations. Written by Jim Batchelor, Caliper Life Sciences. Figure 6. Non-contact mixing. Diazepam Quinidine Amitriptyline Desipramine Propranolol Dilitiazem Diclofenac Prednisone VerapamilMidazolam PropafenoneNicardipine B A A+B

2. Success & Key Learning's from Microsome Assay Automation 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 34 ul/sec100 ul/sec Success: 1.Automated dilution during Mic prep 2.1 hour unattended completion of assay