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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 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 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/sec 100 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 0.00 50.00 100.00 150.00 200.00 250.00 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 0.00 50.00 100.00 150.00 200.00 250.00 Experimental Cl int Literature Cl int ALH FX Figure 5. Different pipetting techniques and on-deck incubation provided similar Cl int values on either workstation. Reaction Temp. (°C) Mecour Peltier Average 35.01 31.4 Range 34.5 – 35.7 31.4 – 36.8 STDEV 0.295 1.13 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. Diazep am Quinidi ne Amitriptyli ne Desiprami ne Propranol ol Dilitiaz em Diclofen ac Predniso ne Verapam il Midazol am Propafeno ne Nicardipi ne B A A+B M icrosom e Assay_12_11_2006_11_16_33.xls 12_11_2006 StartTim e End Tim e Actual Incubation R equested Incubation tim erflags cell counters 60 M inutes no drug,C m pnd A 10:09:21 ################# 0:59:45 1 17 17 60 M inutes no drug,C m pnd B 10:09:40 ################# 0:59:45 1 60 M inutes no C oF,C m pnd A 10:10:37 ################# 0:59:45 1 60 M inutes no C oF,C m pnd B 10:10:56 ################# 0:59:45 1 60 M inutes CoF,C m pnd A 10:11:28 ################# 0:59:45 1 60 M inutes CoF,C m pnd B 10:11:46 ################# 0:59:45 1 30 M inutes CoF,C m pnd A 10:12:16 10:42:12 0:29:56 0:29:45 30 M inutes CoF,C m pnd B 10:12:34 10:42:31 0:29:57 0:29:45 20 M inutes CoF,C m pnd A 10:13:04 10:33:00 0:19:56 0:19:45 20 M inutes CoF,C m pnd B 10:13:24 10:33:19 0:19:55 0:19:45 10 M inutes CoF,C m pnd A 10:13:53 10:23:48 0:09:55 0:09:45 10 M inutes CoF,C m pnd B 10:14:12 10:24:07 0:09:55 0:09:45 5 M inutes C oF,C m pnd A 10:14:25 10:19:20 0:04:55 0:04:45 5 M inutes C oF,C m pnd B 10:14:42 10:19:38 0:04:56 0:04:45 0 M inutes C oF,C m pnd A 10:17:13 10:17:13 0:00:00 0 M inutes C oF,C m pnd B 10:18:01 10:18:01 0:00:00
