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 Automation/Bioanalytical: Ø Caliper Life Sciences Sciclone ALH 3000 and Beckman Coulter Biomek FX workstations equipped with 384 -channel disposable tip array were used to perform all liquid handling steps. Ø An AB/Sciex API 3000 triple quadrupole mass spectrometer, operating in selected reaction monitoring (SRM) mode, was used for analyte and internal standard detection. • Results Ø Validation results were assessed relative to published clearance values. Ø Key learning’s: ØRapid Dispense (100 µl /sec) ØOn-deck Incubation ØScheduling with Excel Macro Ø P 450 concentration: 0. 25 µM (0. 76 mg protein/m. L) Ø Compound substrate concentration: 1 µM Ø Co-Factor utilizes NADPH regeneration system w/ 1 m. M Mg. Cl 2 Ø Buffer concentration: 100 m. M KPO 4, p. H 7. 4 Ø Time-points: 0, 5, 10, 20, 30 and 60 minutes Ø Internal standard used to ensure LC/MS/MS performance and reproducibility Ø Liquid handling performed on Caliper Life Sciences Sciclone ALH 3000 or Beckman Coulter Biomek FX workstation 2. Microsomes & Cofactor 4. 10 MIN 20 MIN X TIP BO 60 MIN NO Co. F A 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. 350. 00 250. 00 300. 00 y = 1. 2534 x - 2. 1081 2 R = 0. 8603 250. 00 200. 00 150. 00 y = 1. 2401 x - 2. 5148 2 R = 0. 8171 100. 00 1. Compound 2. 8 µl Assay (8) 100. 00 y = 0. 9416 x + 1. 3742 2 R = 0. 9226 50. 00 ALH FX 0. 00 50. 00 100. 00 150. 00 200. 00 250. 00 Figure 4. Intrinsic clearance values for 12 compounds (n=96) run on both automated workstations were similar to reported values. B 0. 00 50. 00 100. 00 150. 00 Figure 5. Different pipetting techniques and ondeck incubation provided similar Cl int values on either workstation. C Reaction Temp. (°C) 35. 01 31. 4 Range B 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. Peltier Average A Mecour 34 µl/sec STDEV A+B 100 µl/sec Figure 6. Non-contact mixing. 34. 5 – 35. 7 31. 4 – 36. 8 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. Conclusions: Key Learning’s References 3. Incubate @ 37°C 0, 5, 10, 20, 30, 60 min. Figure 1. 27. 8 µl assay. 200. 00 Sciclone ALH 3000 Clint Experimental Clint D ACN (Protein Precip. ) 75 µl 150. 00 A 25 µl 200. 00 Nicardipine 5 MIN Midazolam Propafenone 30 MIN Prednisone IS Verapamil 0 MIN Diclofenac ACN & D C Desipramine Propranolol Dilitiazem Methods: Microsome Assay Specifics B Ø 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). Ø Little difference in workstations (Figure 5). Ø 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. Ø Further throughput gains are restricted by LC/MS/MS analysis. (i. e. 4300 compounds = 43, 000 LC injections) Ø The large capacity allows us to maintain discovery chemistry’s capacity of over 2000 compounds per week. Quinidine Amitriptyline 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 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. Diazepam • Methods Biology: Ø Twelve compounds with a broad range of clearance rates and P 450 pathways were chosen to validate assay 1, 2. Ø 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. Ø Assay volume: 27. 8 µl. E Results Biomek FX Clint • Introduction Ø To develop an automated 384 -well microsomal metabolic stability assay to support rapid screening of 2, 000 discovery compounds per week. Introduction Literature Clint Overview 1. Riley Robert J; Mc. Ginnity 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), 130411. 2. Obach, R. Scott. Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: an examination of in vitro halflife approach and nonspecific binding to microsomes. Drug Metab and Dispos (1999), 27(11), 1350 -1359. ØRapid Dispense/ Non-contact Mixing 100 µl/sec - Savings in Tips, Time, & Deck Space (Figure 6). ØOn-Deck Incubation @ 37°C Savings in Hardware/ Human Intervention (Table 1). ØEasy Scheduling with Excel Macro (Table 2). Ø 27. 8 µl Assay in 384 well plate Savings in Microsomes - 720 Compounds in 3 hours! 250. 00
Success & Key Learning's from • Microsome Assay Automation Rapid Dispense Speed – 100 ul/sec 34 ul/sec 100 ul/sec • Deck space, tips ($$$$) • On-deck Incubation – Mecour Thermal Blocks Success: 1. Automated dilution during Mic prep 2. 1 hour unattended completion of assay
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