Hepatocyte Metabolic Kinetics Assays

Transcription

1 Chapter 7 Hepatocyte Metabolic Kinetics Assays The idea pkexpress TM Physiological Metabolism Model predicts drug metabolism using in vitro metabolic kinetic data from human cryopreserved hepatocytes (HCH) as a primary input. The model can predict the fraction dose of drug absorbed to the hepatic vein (F H ) based on intrinsic clearance K m and V max /K m calculated or measured independently of pkexpress. However, measured kinetic data will give a more reliable metabolism result. If you provide measured kinetic data to the Metabolism Model, the model s Data Expert automatically calculates K m and V max /K m from the turnover data. You must provide at least two concentrations or 16 data points for this calculation. Hepatocyte Selection When collecting metabolism data using HCH, LION pools six donors to provide a better representation of an "average" or "normal" population. In order to reproduce the results shown here, you must create a similarly generalized hepatocyte pool. To facilitate your selection of HCH, a stand-alone LION software utility called Hepatocyte Selector has been bundled with pkexpress. See The Hepatocyte Donor Pool Selection Utility on page 159 for information about using this tool. Selecting a Metabolic Kinetics Assay Two assay procedures are provided in this chapter. Use the metabolic kinetics assay appropriate to your needs, based on the following considerations. STANDARD HEPATOCYTE METABOLIC KINETICS ASSAY The standard hepatocyte metabolic kinetics assay (page 102) specifies six concentrations with three replicates. Pros: More complete understanding of the kinetics of a compound Enables better prediction of in vivo hepatic extraction by Metabolism Model Cons: Low throughput - 4 compounds/ day Not good for the early drug discovery setting 2003 LION bioscience (and its affiliates) v1.0 Rev B December 2003

2 102 Hepatocyte Metabolic Kinetics Assays ALTERNATE HEPATOCYTE METABOLIC KINETICS ASSAY The alternate hepatocyte metabolic kinetic assay (page 117) requires four concentrations with n=1. Comparison of results has shown that the absence of replicates has minimal affect on the accuracy of pkexpress K m and CL int calculations. Pros: High throughput - 44 compounds/day Good for the early drug discovery setting Cons: Less complete kinetics Standard Hepatocyte Metabolic Kinetics Assay Goal To measure substrate depletion profiles with time up to 4 hr at 6 substrate concentrations. Set-up INSTRUMENTS: Liquid N 2 storage Centrifuge (100xg force capabilities) Water bath Tissue culture CO 2 incubator with humidity control (5% CO 2, 95% humidity) Hemacytometer (Fisher Scientific, mm cell depth, Brightline, Cat. # ) REAGENTS: The following reagents and materials are required. Human cryopreserved hepatocytes (In Vitro Technologies, Baltimore, MD) 96-well (deep well) plate (non-sterile, non-treated, Marsh Biomedical, Cat. # KAB-0661) Trypan blue (Sigma, Cat. # T8154) Metabolic activity markers: 7-Ethoxycoumarin (Sigma, Cat. A# E1379) v1.0 Rev B December LION bioscience (and its affiliates)

3 Hepatocyte Metabolic Kinetics Assays Hydroxycoumarin (or Umbelliferone) (Sigma, Cat. # U-7626) 7-Hydroxycoumarin Glucuronide (Cedra, Cat. # RS0102 or Ultrafine, Cat. # UC0263) 7-Hydroxycoumarin Sulfate (Cedra, Cat. # RS0101 or Ultrafine, Cat. # UC-283) 10X Dulbecco s Phosphate Buffered Saline (10X PBS, Sigma, Cat. # D-1283) 1X WME (Sigma, W-1878, without phenol red) WME including 10% DMSO, 0.05% DMSO or 0.5% MeOH as cosolvent Percoll (Sigma, P-4937) Acetonitrile, methanol, DMSO, trifluoroacetic acid MeOH:WME=1:1 (v/v) Protocol Summary Human Cryopreserved Hepatocyte Cells: 6 IVT donors pool (usually 3 males & 3 females)/ pool needs to be passed through LION s G2 donor selection program Cell process: a quick thaw at 37 o C, 25% iso-percoll centrifugation Initial cell viability: determined by trypan blue exclusion method Yield: ~ 25 M cells yield after Percoll centrifugation Cell suspension in WME: 0.5 million viable cells/ml Kinetics of Parent Compound Incubation: CO 2 incubator with humidity control at 37 C 6 substrate concentrations: 0.4, 2.0, 10, 50, 125 and 250 µm/ n=3 for each concentration Williams Medium E (WME) with cosolvent of 0.025% DMSO or 0.25% MeOH 96 deep well format: 200 µl final volume (50,000 cells/ well) Sampling time: 0, 30, 120, and 240 min Termination of reaction: 200 µl chilled MeOH 2003 LION bioscience (and its affiliates) v1.0 Rev B December 2003

4 104 Hepatocyte Metabolic Kinetics Assays Bioanalysis: LC-UV, LC-MS or LC-MS-MS Output: conc (µm) vs T (min) profiles Amount required: Approximately 10 mg Instruments: CO 2 incubator with humidity control and hemacytometer (Fisher Scientific, mm cell depth, Brightline, Cat. # ) 4 compounds full kinetics/ 1 FTE/ day Positive Control 7-Ethoxycoumarin (7-EC) metabolic profile (phase I & II) at 75 µm Formation of metabolites: 7-hydroxycoumarin (7-HC), 7-hydroxycoumarin glucuronide (7-HCG), and 7-hydroxycoumarin sulfate (7-HCS) Sampling time: 0, 30, 120, and 240 min with cell; N=3 replicates per time point Cell viability without substance: 0, 120, 240 min Preparation WORKING SOLUTIONS Stock solution: 100 mm drug substance in 10% DMSO/WME or 100% Methanol 2x dosing solution: Prepare 0.8, 4.0, 20, 100, 250 and 500 µm 2x dosing solution with WME including 0.05% DMSO or 0.5% MeOH as cosolvent Standard solution: Prepare 0.05, 0.2, 0.4, 0.8, 2, 4, 10 and 20 µm standard solution with WME:MeOH = 1:1 (Table 36) 2x positive control: 1 7-EC with WME including 0.2% MeCN as a metabolic viability marker HEPATOCYTE DONOR POOL SELECTION Human cryopreserved hepatocytes (HCH) will be used (In Vitro Technologies (IVT), Baltimore, MD). Cells will be obtained from multiple individual human donors and will be pooled (N = 6 donors). HCH will be stored in liquid nitrogen container at < -150 o C until use. Hepatocyte donors will be chosen based on the enzyme activities reported on IVT homepage ( See The Hepatocyte Donor Pool Selection Utility on page 159. v1.0 Rev B December LION bioscience (and its affiliates)

5 Hepatocyte Metabolic Kinetics Assays 105 Current donors that LION G2 uses are 91, RKB, GUY, MAN, PFM, OQD, and were selected by LION s G2 donor selection program, Hepatocyte Selector. Our historical donors selected are shown in Table 30, and showed comparable enzyme activities. Table 30. Hepatocyte Pools Used for pkexpress Metabolism Module Original Pool Activity (pmol/min/million cells) a Lot # Viability DEX MEPH TEST TOLB 7-HCG 7-HCS COUM ECOD PHEN CZX 2D6 2C19 3A4 2C9 GT ST 2A6 2E1 1A2 2E1 63 b c BQL 30 MEAN a Original hepatocyte pool (enzyme activities were reported on IVT website, Oct. 2001) b Values were reported in July, 2001 c Values were reported in June, 2000 Table 31. Mirror Hepatocyte Pool (1) Mirror Pool (1) Activity (pmol/min/million cells) a Lot # Viability DEX MEPH TEST TOLB 7-HCG 7-HCS COUM ECOD PHEN CZX 2D6 2C19 3A4 2C9 GT ST 2A6 2E1 1A2 2E BQL 30 MEAN Difference from Original Pool a Original hepatocyte pool (enzyme activities were reported on IVT website, Oct. 2001) 2003 LION bioscience (and its affiliates) v1.0 Rev B December 2003

6 106 Hepatocyte Metabolic Kinetics Assays Table 32. Mirror Hepatocyte Pool (2) Mirror Pool (2) Activity (pmol/min/million cells) a Lot # Viability DEX MEPH TEST TOLB 7-HCG 7-HCS COUM ECOD PHEN CZX 2D6 2C19 3A4 2C9 GT ST 2A6 2E1 1A2 2E MEAN Difference from Original Pool a Original hepatocyte pool (enzyme activities were reported on IVT website, Oct. 2001) Table 33. Mirror Hepatocyte Pool (3) Mirror Pool (3) Activity (pmol/min/million cells) a Lot # Viability DEX MEPH TEST TOLB 7-HCG 7-HCS COUM ECOD PHEN CZX 2D6 2C19 3A4 2C9 GT ST 2A6 2E1 1A2 2E TBD b NA MEAN Difference from Original Pool a Original hepatocyte pool (enzyme activities were reported on IVT website, Oct. 2001) b Values were reported in July, 2001 v1.0 Rev B December LION bioscience (and its affiliates)

7 Hepatocyte Metabolic Kinetics Assays 107 Table 34. Mirror hepatocyte pool (4) Mirror Pool (4) Activity (pmol/min/million cells) a Lot # Viability DEX MEPH TEST TOLB 7-HCG 7-HCS COUM ECOD PHEN CZX 2D6 2C19 3A4 2C9 GT ST 2A6 2E1 1A2 2E TBD RKB MEAN Difference from Normal Pool a Original hepatocyte pool (enzyme activities were reported on IVT website, Oct. 2001) Mirror hepatocyte pool (5): 91, 102, 117, 120, RKB, GUY Mirror hepatocyte pool (6): 91, RKB, GUY, MAN, PFM, OQD Experimental Procedure % DMSO/WME EC Table 35. Dosing plate format and preparation (in 48 deep well: 4 ml): Comp A Comp B Comp C Comp D µm 0.8 µm 0.8 µm 0.8 µm 4 4 µm 4 µm 4 µm 4 µm 5 20 µm 20 µm 20 µm 20 µm µm 100 µm 100 µm 100 µm µm 500 µm 500 µm 500 µm 1. Prepare 500 µm dosing solution: 10 µl stock (100 mm in 10% DMSO) ml WME. 2. Prepare 2 dosing solution: 5 µl stock (100 mm in 10% DMSO) + 5 µl 10% DMSO/WME ml WME LION bioscience (and its affiliates) v1.0 Rev B December 2003

8 108 Hepatocyte Metabolic Kinetics Assays 3. Serial dilution with 0.05% DMSO/WME 100 µm 400 µl of 500 µm ml of 0.05% DMSO/WME 20 µm 400 µl of 100 µm ml of 0.05% DMSO/WME 4 µm 400 µl of 20 µm ml of 0.05% DMSO/WME 0.8 µm 400 µl of 4 µm ml of 0.05% DMSO/WME 4. Prepare 1 7-EC with WME including 0.2% MeCN (2 ml). 5. Add 0.05 % DMSO/WME (2 ml). Table 36. Calibration plate format and preparation (in 96 deep well: 1.2 ml): Comp A Blk 0.05 µm 0.2 µm 0.4 µm 0.8 µm 2 µm 4 µm 10 µm 20 µm 500 µm Comp B Blk 0.05 µm 0.2 µm 0.4 µm 0.8 µm 2 µm 4 µm 10 µm 20 µm 500 µm Comp C Blk 0.05 µm 0.2 µm 0.4 µm 0.8 µm 2 µm 4 µm 10 µm 20 µm 500 µm Comp D Blk 0.05 µm 0.2 µm 0.4 µm 0.8 µm 2 µm 4 µm 10 µm 20 µm 500 µm 1. Prepare 500 µm standard solution: 5 µl stock (100 mm in 10% DMSO) µl MeOH:WME=1:1. 2. Serial dilution with MeOH:WME=1:1 20 µm calibrator 40 µl 500 µm µl MeOH:WME=1:1 10 µm calibrator 20 µl 500 µm µl MeOH:WME=1:1 4 µm calibrator 200 µl 20 µm µl MeOH:WME=1:1 2 µm calibrator 200 µl 10 µm µl MeOH:WME=1:1 0.8 µm calibrator 200 µl 4 µm µl MeOH:WME=1:1 0.4 µm calibrator 200 µl 2 µm µl MeOH:WME=1:1 0.2 µm calibrator 250 µl 0.8 µm µl MeOH:WME=1: µm calibrator 125 µl 0.4 µm µl MeOH:WME=1:1 v1.0 Rev B December LION bioscience (and its affiliates)

9 Hepatocyte Metabolic Kinetics Assays 109 PREPARATION OF CELL SUSPENSION Warm up tube A1, A2, B1 & B2 in 37 o C water bath. Take the cell out of the liquid nitrogen storage tank and do a quick thaw in 37 o C water bath (1-1.5 min). Pour the cells (~ 3 ml total) into Tube A1 & B1. Rinse the cell vials (~1 ml medium/vial) using medium from tube A2 & B2 and combine the rinses with media in Tube A1 & B1, respectively. Transfer 3 ml medium from A2 & B2 into A1 & B1, respectively. Total volume of A1 and B1 is 49 ml, and the final Percoll concentration is 25%. Invert the tube A1 & B1 gently to mix the cells and the media. Then centrifuge the tubes at 100 xg for 5 min. A low brake setting should be used for all centrifugation steps. Discard the supernatant and resuspend the cells using fresh media in Tube A2 & B2. Centrifuge the tubes at 50 xg for 3 min. Resuspend the cell pelletes in fresh WME, combine all the cells together and count the cells. Percoll Volume (ml) WME Volume (ml) Total Volume (ml) A A B B VIABILITY: PRE-INCUBATION 1. Cell Suspension Dilution w/ Trypan Blue: 100 µl cells µl dye 2. % Viability: (viable cells/total cells)* Cell Yield (cell#/ml):(viable cells/# quadrants)*(10 4 ) *dilution factor Final re-suspension of cells in WME should allow for 50,000 cells/ 100 µl (0.5 M/ml). CELL DILUTION Approximate expected yield is 25,000,000 viable cells for 6 donor vials; therefore initially re-suspend cell pellet with 50 ml WME to allow for a final dilution of cells that will allow 100 µl to be equivalent to 50,000 cells. However, for safety purposes, re-suspend cell pellet with 30 ml WME. 1. If cell #: 100, then cell yield (viable cells/# quadrants)*(10 4 ) *dilution factor): (100/4)*(10 4 )*2 = 0.5M/ml, total cell #: 0.5M* LION bioscience (and its affiliates) v1.0 Rev B December 2003

10 110 Hepatocyte Metabolic Kinetics Assays 2. If cell #: NN, then total volume for 0.5M/ml: (NN/100)*30. Example, cell #: 200, then total volume: 60 ml; cell # 120, then total volume: 36 ml Table 37. Incubation Plate Format and Preparation (96 Deep Well: 1.2 ml): 0, 30, 120, and 240 min Comp A Comp A Comp A Comp B Comp B Comp B Comp C Comp C Comp C Comp D Comp D Comp D % DMSO/ WME 2 75 µm 7-EC 0.025% DMSO/ WME 75 µm 7-EC 0.025% DMSO/ WME 75 µm 7-EC µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 4 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 5 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm µm 125 µm 125 µm 125 µm 125 µm 125 µm 125 µm 125 µm 125 µm 125 µm 125 µm 125 µm Dispense 100 µl dosing solutions. 2. Dispense 100 µl cell suspension. 3. Start incubation at 37 C/95% humidity. All incubation plates will be pre-incubated for 5 min in a cell culture incubator at 37 o C with 5% CO 2 and 95% humidity. After 5 min pre-incubation, T 0 sample plate will be taken out of the incubator. The rest of the plates will remain until the appropriate incubation time comes up (30, 120 & 240 min). 4. At the end of incubation, dispense 200 µl MeOH containing IS/ (0.1% ZnSO 4 ) except 0.025% DMSO/WME. (Inclusion of an internal standard is optional.) 5. Add 100 µl trypan blue to 0.025% DMSO/WME wells for viability determination. 6. Centrifuge the plates 30 min at 2000g/4 C. v1.0 Rev B December LION bioscience (and its affiliates)

11 Hepatocyte Metabolic Kinetics Assays 111 Table 38. Bioanalytical Plate Format and Preparation (96 Shallow Well): Each Compound Blk 0.05 µm 0.2 µm 0.4 µm 0.8 µm 2 µm 4 µm 10 µm 20 µm wash 2 wash wash wash wash wash wash wash wash µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 0.4 µm 4 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 2 µm 5 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm µm µm µm µm µm µm µm µm µm µm µm 2 T240 min T120 min T30 min T µm 2 1. Transfer 200 µl calibrators. 2. Transfer 200 µl wash (water). 3. Dispense 180 µl of MeOH:WME=1:1 to rows 6, 7, 8 for 10 fold dilution. 4. Transfer 200 µl supernatant of 0.4, 2, and 10 µm incubation samples to rows 3, 4, Transfer 20 µl supernatant of 50, 125, and 2 incubation samples to rows 6, 7, HLPC injection sequesce of each compound: Calibrators: blk-20µm, wash/ 0.4 µm:c1-c12, wash/ and high conc. 7. Dilution correction in the calculation of concentrations: 0.4, 2, and 10 µm: x2 50, 125, and 2: x LION bioscience (and its affiliates) v1.0 Rev B December 2003

12 112 Hepatocyte Metabolic Kinetics Assays Table 39. Bioanalytical Plate Format and Preparation (96 Shallow Well): Control 7-EC Blk wash wash wash wash wash wash wash wash wash wash wash wash Batch 1 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm Batch 2 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm Batch 3 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm Batch 4 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm Batch 5 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm Batch 6 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm 75 µm T240 min T120 min T30 min T 0 1. Keep 7-EC samples at 80ºC up to 6 batch studies for LC/UV analysis. 2. When 6 batch samples are ready, transfer 200 µl supernatant of samples. 3. Prepare an analytical standard curve containing 7-EC and 3 metabolites (7-HC, 7-HCG, 7-HCS) using 1:1 (v/v) Methanol/ WME mixture. 4. Nominal concentrations for the standard curve are as follows: Blk, , , , , 3.125, 6.25, 12.5, 25, 50 µm. 5. Transfer 200 µl calibrators to row 1. OUTPUT DATA 1. Kinetic concentration profiles for each compound 2. % remaining of positive control, 7-EC 3. Formation profiles for the metabolites of 7-EC 4. Cell viability/ yield for batch v1.0 Rev B December LION bioscience (and its affiliates)

13 Hepatocyte Metabolic Kinetics Assays 113 Sample Analysis of Metabolic Viability markers 7-EC and its metabolites (7-HC, 7-HCG, and 7-HCS, Figure 35) will be monitored by using HPLC/UV. Percentage of remaining 7-EC will be determined as compared to the concentration at T 0. Formation rates of metabolites will be expressed as pmol/minute/ 10 6 cells. Figure 35. Chemical Structures of 7-EC, 7-HC, 7-HCG, 7-HCS TO PERFORM ANALYSIS 1. Prepare an analytical standard curve using 1:1 (v/v) Methanol/WME mixture. Nominal concentrations for the standard curve are as follows: , , , , 3.125, 6.25, 12.5, 25,. 2. Inject 40 µl aliquots of the individual samples and reference standards using a suitable HPLC instrument. 3. Perform quantitative analysis for each individual sample. 4. Evaluate the performance of the analytical assays, and determine the actual concentration of the samples from the standard curve. GENERAL RECOMMENDATIONS For sample analysis, the general recommendations are as follows: EQUIPMENT Hewlett Packard 1100 Series High Pressure Liquid Chromotographic (HPLC) system or equivalent consisting of: 2003 LION bioscience (and its affiliates) v1.0 Rev B December 2003

14 114 Hepatocyte Metabolic Kinetics Assays Solvent delivery system with at least two solvent channels or equivalent Autosampler with a 100 µl syringe or equivalent Diode-array detector or equivalent ChemStation software or equivalent Phenomenex C18 reverse phase HPLC column, 4.6 x 250 mm, 5mm (Part# 00G-4041-E0), or Equivalent Phenomanex Guard cartridge, 4.6 x 12.5 mm, 5mm (Part# AJ0-4321), or equivalent SYSTEM SET-UPS Pump: Autosampler: Column: Column Temp: HP1100 Series Quaternary Pump CTC Analytical PAL System from Leap Technology Phenomenex C18 50 o C Solvent: Solvent A: Solvent B: 0.5% TFA in H 2 O 100% Acetonitrile Injection Volume: 40 µl Flow Rate: 1.0 ml/min Gradient: Time (min) A (%) B(%) Run Time: Post Time: Detection: 19 min 2 min UV absorbance 320 ± 30 nm v1.0 Rev B December LION bioscience (and its affiliates)

15 Hepatocyte Metabolic Kinetics Assays 115 Table 40. Typical Chromatographic Retention Time Data Compound ID F.W. Retention Time (min) 7-Hydroxycoumarin Glucuronide Hydroxycoumarin Sulfate Hydroxycoumarin Ethoxycoumarin Table 41. Standard Curves of 7-HCG, 7-HCS, 7-HC and 7-EC µm µm µm µm µm 6.25 µm 12.5 µm 25 µm 50 µm 7-HCG (AUC) Mean SD HCS (AUC) Mean SD HC (AUC) Mean SD EC (AUC) Mean SD LION bioscience (and its affiliates) v1.0 Rev B December 2003

16 116 Hepatocyte Metabolic Kinetics Assays Figure 36. Standard Curve of 7-EC Figure 37. Standard Curve of 7-HC Figure 38. Standard Curve of 7-HCG v1.0 Rev B December LION bioscience (and its affiliates)

17 Hepatocyte Metabolic Kinetics Assays 117 Figure 39. Standard Curve of 7-HCS Positive control data Table 42. Cell Viability Cell Viability (%) Cell Yield T0 T120 T240 Mean million SD million Range million Exp (N) Table EC at 4 hr Incubation 7-EC % Remaining Formation Rate (pmole/min/million viable cells) 7-HC 7-HCG 7-HCS Mean SD Range Exp (N) Alternate Hepatocyte Metabolic Kinetics Assay Goal Compared to conventional format (4 compounds throughput), the alternate assay format has reduced sample inputs with higher throughput (44 compounds) LION bioscience (and its affiliates) v1.0 Rev B December 2003

18 118 Hepatocyte Metabolic Kinetics Assays Set-up MATERIALS AND STANDARD SOLUTIONS 96 deep well for dosing solution plate (2 for 44 compounds) 96 shallow well for incubation plate (8 for 44 compounds) 96 shallow well for bioanalytical plate (14 for 44 compounds) Percoll WME 20 mm stock in DMSO Protocol summary Human Cryopreserved Hepatocyte (same as conventional) Kinetics of Parent Compound Incubation: CO 2 incubator with humidity control at 37 C 4 substrate concentrations: 2.0, 10, 30, and / n=1 for each concentration Williams Medium E (WME) with cosolvent of 0.25% DMSO 96 shallow well format: 100 µl final volume (25,000 cells/ well) Sampling time: 0, 1, 5, and 6 hr Termination of reaction: 100 µl chilled MeOH Bioanalysis: LC-UV, LC-MS or LC-MS-MS Output: % remaining Amount required: Approximately 1 mg or 50 µl of 20 mm test compound in DMSO Instruments: CO 2 incubator with humidity control and hemacytometer (Fisher Scientific, mm cell depth, Brightline, Cat. # ) 44 compounds kinetics/ 1 FTE/ day Positive Control Parent metabolic profiles at 2 µm Diclofenac: high turn over (% remaining < 10% at 5-6 hr) Propranolol: medium turn over (% remaining: 20-70% at 5-6 hr) v1.0 Rev B December LION bioscience (and its affiliates)

19 Hepatocyte Metabolic Kinetics Assays 119 Atenolol: low turn over (% remaining: >80% at 5-6 hr Cell viability: >40-50% at 5-6 hr Experimental procedures DOSING PLATE FORMAT AND PREPARATION Table Well Dosing Plate µm 20 µm 60 µm 100 µm X µm 20 µm 60 µm 100 µm X X X X X7 5 Diclofen ac 4 µm Propran olol 4 µm Atenolol 4 µm 0.5% DMSO/ WME 8 X8 6 Diclofen ac 4 µm Propran olol 4 µm Atenolol 4 µm 0.5% DMSO/ WME 1. Prepare 100 µm dosing solution (X): 5 µl stock µl WME. 2. Serial dilution with 0.5% DMSO/WME: 60 µm: 300 µl of 100 µm µl of 0.5% DMSO/WME 20 µm: 100 µl of 100 µm µl of 0.5% DMSO/WME 4 µm: 20 µl of 100 µm µl of 0.5% DMSO/WME 3. Instead of 3,24, prepare 4 µm diclofenac (positive control: high turn over), propranolol (positive control: mid turn over), atenolol (negative control: no turn over), and 0.5% DMSO/ WME (cell viability). 40 µl of 100 µm µl of 0.5% DMSO/WME. PREPARATION OF CELL SUSPENSION 1. Same as the conventional method LION bioscience (and its affiliates) v1.0 Rev B December 2003

20 120 Hepatocyte Metabolic Kinetics Assays Table 45. Incubation Plate Format and Preparation: 0, 1, 5, and 6 hr µm 10 µm 30 µm X µm 10 µm 30 µm X X X X X7 5 Diclofen ac 2 µm Propran olol 2 µm Atenolol 2 µm 0.25% DMSO/ WME 8 X8 6 Diclofen ac 2 µm Propran olol 2 µm Atenolol 2 µm 0.25% DMSO/ WME 1. Dispense 50 µl dosing solutions. 2. Dispense 50 µl cell suspensions. 3. Start incubation at 37 C/ 95% humidity. All incubation plates will be pre-incubated for 5 min. Prepare blk using left over cells: Y ml cell suspension + Y ml 0.5% DMSO/WME + 2Y ml MeOH. 4. At the end of incubation, dispense 100 µl MeOH containing IS/ (0.1% ZnSO 4 ) into all wells except wells containing 0.25% DMSO/WME. 5. Add 100 µl trypan blue to 0.25% DMSO/WME wells for viability determination. 6. Centrifuge the plates 30 min at 2000g/ 4 C. v1.0 Rev B December LION bioscience (and its affiliates)

21 Hepatocyte Metabolic Kinetics Assays 121 Table 46. Bioanalytical Plate Format and Preparation:,, X3, X4 (4 compds/plate): X3 X3 X3 X3 2 X3 X3 X3 X3 3 X3 X3 X3 X3 4 Blk X3 X3 X3 X3 Blk 5 X4 X4 X4 X4 6 X4 X4 X4 X4 7 X4 X4 X4 X4 8 Blk X4 X4 X4 X4 Blk 6 hr 5 hr 1 hr 0 hr 6 hr 5 hr 1 hr 0 hr 1. Dispense 100 µl wash: water. 2. Dispense 100 µl Blk. 3. Dispense 100 µl of water to row 1, 2, 5, 6 for 2 fold dilution. 4. Dispense 180 µl of water to row 3, 4, 7, 8 for 10 fold dilution. 5. Transfer supernatant of comp sample: 100 µl to row 1, 2; 20 µl to row 3, Repeat for -X4. 7. HLPC injection sequence of : R1 (C1-C5) R4 and Blk LION bioscience (and its affiliates) v1.0 Rev B December 2003

22 122 Hepatocyte Metabolic Kinetics Assays Table 47. Bioanalytical Plate Format for Controls (diclofenac, propranolol, atenolol): Diclofen ac Diclofen ac Diclofen ac Diclofen ac Atenolol Atenolol Atenolol Atenolol 2 Diclofen ac Diclofen ac Diclofen ac Diclofen ac Blk Atenolol Atenolol Atenolol Atenolol Blk Propran olol Propran olol Propran olol Propran olol 6 Propran olol Propran olol Propran olol Propran olol Blk hr 5 hr 1 hr 0 hr 6 hr 5 hr 1 hr 0 hr 1. Dispense 100 µl wash. 2. Dispense 100 µl Blk. 3. Dispense 100 µl of water to row 1, 2, 5, 6 for 2 fold dilution. 4. Transfer supernatant of control sample: 100 µl to row 1, 2, 5, HLPC injection sequence: Diclofenac: R1 (C1-C5) R2 and Blk. OUTPUT DATA 1. Bioanalysis: AUC 2. Percent remaining kinetic profiles for each compound 3. Percent remaining kinetic profiles for each controls (diclofenac, propranolol, and atenolol)/ n=2 4. Cell viability/ yield for batch v1.0 Rev B December LION bioscience (and its affiliates)

23 Hepatocyte Metabolic Kinetics Assays 123 Table 48. Positive control data (% remaining) 0 hr 1 hr 5 hr 6 hr Diclofenac Mean SD Range Exp (N) Propranolol Mean SD Range Exp (N) Atenolol Mean SD Range Exp (N) LION bioscience (and its affiliates) v1.0 Rev B December 2003

24 124 Hepatocyte Metabolic Kinetics Assays Example Alternate HCH Assay Template See Figure 40 for an example of the Alternate HCH Assay template and results graph. Figure 40. Alternate HCH Assay Template v1.0 Rev B December LION bioscience (and its affiliates)