Enhancement of Physiological Relevance of In Vitro Assays

Transcription

1 Novel Applications of Primary Cultured Cryopreserved Human Hepatocytes in DDI Evaluation Albert P. Li, Ph. D. Laboratories LLC Columbia, Maryland and Malden, Massachusetts Enhancement of Physiological Relevance of In Vitro Assays IVAL Mission Human hepatocytes as a relevant in vitro DDI experimental system for assessment of clinical findings Hepatocyte cryopreservation Novel hepatocyte tools OnDemand human hepatocytes Pooled plateable human hepatocytes Genetically engineered mouse hepatocytes Novel hepatocyte assays Plated hepatocyte relay assay for slowly metabolized compounds High throughput plated hepatocyte time-dependent inhibition assay Long-term cultured of human hepatocytes in human plasma 1

2 Laboratories (IVAL) Columbia, MD and Malden, MA Incorporation: November, 24. Mission: Develop and apply in vitro human and animal based experimental systems for the accurate assessment of human drug properties Focus: Human and animal hepatocytes Laboratories (IVAL) Columbia, MD and Malden, MA Activities: Products: Cryopreserved human and animal hepatocytes Hepatocyte cultured plates and media IdMOC experimental system for co-culturing of hepatocytes and nonhepatic cells (e.g. cardiomyocytes) Service: hepatocyte-based contract research service to aid drug discovery and development In vitro drug metabolism and drug-drug interaction studies In vitro toxicity studies Research: Advancement of scientific knowledge in pharmacology, drug metabolism, and toxicology Communication: Workshops and conferences (at IVAL Boston Hepatocyte Technology Center) 2

3 Grand Opening of IVAL Boston Hepatocyte Technology Center, Jan. 214 Inaugural Hepatocyte Conference, January, 214 3

4 Hepatocyte Technology Conference, IVAL-BHTC, April, 215 Hepatocytes: Gold Standard for In Vitro Hepatic Metabolism Studies, DDI and Hepatotoxicity Studies Intact plasma membrane Complete, uninterrupted enzymes at physiological conditions Uptake/efflux transporters 4

5 The intact plasma membrane, active uptake transport, and complete drug metabolizing enzymes and cofactors allows the assessment of relevant intracellular drug concentrations and effects based on extracellular concentrations Mission 1: Develop technologies to allow routine use of hepatocytes for experimentation 5

6 Hepatocyte Cryopreservation: Enabling Technology for Routine Use of Hepatocytes Liver availability and hepatocyte preparation eliminated as requirements for experimentation Experiments can be scheduled Can repeat experiments with cells from the same donors Can use pre-characterized hepatocytes Can compare multiple donors/multiple species in the same experiment Can pool hepatocytes from multiple donor to create a generalized composite human Overcoming challenges in hepatocyte cryopreservation First demonstration of successful cryopreservation Loretz, Li et al. Optimization of cryopreservation procedures for rat and human hepatocytes. Xenobiotica May;19(5): First demonstration of retention of drug metabolizing enzymes after cryopreservation Li, Lu et al. Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential. ChemBiolInteract Jun 1;121(1): First international consensus on utility of cryopreserved human hepatocytes Li et al. Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel. Chem Biol Interact Jun 1;121(1): First demonstration of retention of uptake transporter activities after cryopreservation Shitara, Li et al. Function of uptake transporters for taurocholate and estradiol 17beta-D-glucuronide in cryopreserved human hepatocytes. Drug MetabPharmacokinet. 23;18(1): First demonstration of effectiveness of CHRM and plateability Li. Human hepatocytes: isolation, cryopreservation and applications in drug development. ChemBiolInteract. 27 May 2;168(1):

7 Optimized Recovery of Cryopreserved Hepatocytes CHRM : APSciencesproduct sold through Gibco Life Technologies UCRM : IVAL product Simplifies thawing and recovery of highly viable hepatocytes from cryopreservation Universal Cryopreservation Recovery Medium (UCRM ) for Recovery of Hepatocytes Human Lot Age Gender Race Yield Viability M H F C /1/11 47 F C /13 25 M C /17 64 F H /18 15 M C /19 17 M C /21/22 /23 21 F C /26/27 59 F C M H M C M H F C F C M C M C Yield: Millions viability cells/vial Viability: Percent of Trypan blue excluding hepatocytes mean sd Yield Viability

8 Reproducibility of Hepatocytes Recovery in UCRM HH131 HH136 Experiments Yield Viability Yield Viability mean sd mean sd Yield Viability Yield: Millions viability cells/vial Viability: Percent of Trypan blue excluding hepatocytes Key publications on cryopreserved human hepatocytes by non-ival researchers Prediction of hepatic clearance Brown et al. (27). Evaluation of cryopreserved human hepatocytes as an alternative in vitro system to microsomes for the prediction of metabolic clearance. Drug MetabDispos. 35(2): Lu et al (27). A novel model for the prediction of drug-drug interactions in humans based on in vitro cytochrome p45 phenotypic data. Drug MetabDispos. 35(1): Jouinet al (26) Cryopreserved human hepatocytes in suspension are a convenient high throughput tool for the prediction of metabolic clearance. Di et al (212). A novel relay method for determining low-clearance values. Drug Metab Dispos 4(9):186-5 Discovery of novel genetic polymorphism Li et al. (27). New cytochromep45 2D6*56 allele identified by genotype/phenotype analysis of cryopreserved human hepatocytes. Drug MetabDispos. 34(8): Efflux transporter Chang et al. (26). The role of P-glycoprotein in the bioactivation of raloxifene. Drug MetabDispos. 34(12): Bi et al. (26). Use of cryopreservedhuman hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab Dispos. 34(9):

9 Plateability of Cryopreserved Hepatocytes Why plateability is important Plateable hepatocytes are higher in quality than non-plateable hepatocytes: less membrane damage Hepatocytes in suspension would rapidly lose viability (T1/2 about 6 hours) and therefore are limited to short-term (hours) studies Plated hepatocytes are viable for days/weeks Long-term drug metabolism In vitro hepatotoxicity Enzyme induction Efflux transport 9

10 IVAL Plateable Cryopreserved Human Hepatocytes High viability: Routinely >9% High plateability: near 1% confluent Responsive to CYP1A2, CYP2B6 and CYP3A4 induction (mrna and activity) Intact uptake and efflux transporter activities Long duration in culture: Over 7 days in culture (prolonged cytotoxicity and metabolism studies) Large number of vials per lot (>5, up to >3 vials per isolation) Large Vial Numbers per Human Hepatocyte Isolation: Plateable/inducible Grade HH17(26 years old female Caucasian): 5 vials HH12/121/122/123(21 years old female Caucasian): 35 vials (Sold out) HH124(48 years old male Caucasian): 5 vials (Sold out) HH125/126/127(59 years old female Caucasian): 2 vials (HH125 sold out; approx. 2 vials of HH126/HH127 available) HH131(52 year old male Hispanic): 75 vials HH133(43 year old female Caucasian): 95 vials HH145/149/15 (9 year old male Hispanic): 3 vials HH152 (44 year old male Caucasian): 1 vials HH153/155/157 (33 year old female Caucasian): 2 vials 1

11 Value for Large Vial Numbers Allows the use of hepatocytes from the same donor for multiple experiments Minimize validation efforts: Validate data from a single lot can be applied towards a large number of studies Sharing of hepatocytes by different investigators for integration of multiple studies High throughput screening (HTS) assays Enabling Reagent for Hepatocyte and IdMOC Assays: IVAL Plateable Cryopreserved Hepatocytes Human CD-1 mouse SD rat Wistar rat Beagle dog Cynomolgus monkey Rhesus monkey New: Taconic GEM hepatocytes 11

12 IVAL Plateable Cryopreserved Hepatocytes Human Cynomolgus Monkey Beagle Dog CD-1 Mouse SD Rat Wistar Rat New Hepatocyte Tools 12

13 New Hepatocyte Tools OnDemand pre-plated cryopreserved human hepatocytes Pooled plateable human hepatocytes Cryopreserved Taconic genetically engineered mouse hepatocytes OnDemand PreplatedHuman Hepatocytes Plated from pre-characterized human hepatocyte lots Delivered to laboratories based on investigator s schedule 7 days a week Ready to use hepatocytes P45 induction Plated hepatocyte Time-Dependent Inhibition In vitro hepatotoxicity studies Plated metabolism studies 13

14 OnDemand Advantages Eliminates technical challenges in hepatocyte plating in use laboratory Board selection of cryopreserved human hepatocytes Large number of vials per lot Option to reserve lots for multiple studies on different dates HH131: 42 Hour Shipment 14

15 OnDemand PrePlated Human Hepatocytes CYP1A2 mrna Induction Relative expression Induction of CYP1A2 in OnDemand human hepatocytes Omeprazole (5 um) Con Phe Ome Rif OnDemand PrePlated Human Hepatocytes CYP2B6 mrna Induction 7 Induction of CYP2B6 in OnDemand human hepatocytes 6 Phenobarbital (1 um) Relative expression Con Phe Ome Rif

16 OnDemand PrePlated Human Hepatocytes CYP3A4 mrna Induction 1 Induction of CYP3A4 in OnDemand human hepatocytes Rifampin (1 um) Relative expression (log) 1 1 Con Phe Ome Rif New Hepatocyte Tools OnDemand pre-plated cryopreserved human hepatocytes Pooled plateable human hepatocytes Cryopreserved Taconic genetically engineered mouse hepatocytes 16

17 Pooled Cryopreserved Human Hepatocytes Re-cryopreserved human hepatocytes pooled from cryopreserved hepatocytes from multiple donors Rationale: Minimize individual-differences in drug properties Technology: Proprietary QuickRefreeze technology (patent pending) for recryopreservation with minimal cellular damage Pooled Cryopreserved Human Hepatocytes Pooled Suspension Human Hepatocytes: 5, 1, 2 donor pools for metabolism and uptake transporter studies Pooled Plateable Human Hepatocytes: 3 and 5 donor pools for plated metabolism, uptake, induction and cytotoxicity studies 17

18 Transporter-mediated Uptake of Rosuvastatinin Pooled Cryopreserved Human Hepatocytes (PHP81) (Data from Caroline Lee) Rosuvastatin Uptake (pmol/ml) Rosuvastatin Uptake (pmol/ml) Pooled Cryopreserved Human Hepatocytes Pooled Suspension Human Hepatocytes: 5, 1, 2 donor pools for metabolism and uptake transporter studies Pooled Plateable Human Hepatocytes: 3 and 5 donor pools for plated metabolism, uptake, induction and cytotoxicity studies 18

19 Morphology of Re-cryopreserved Human Hepatocytes (Individual Donors) HH123 HH131 HH133 HH136 HH149 HH152 Pooled Plateable Human Hepatocytes PHP82 Morphology Pool of HH131, HH133, HH136, HH149, HH152 19

20 Pooled Plateable Human Hepatocytes PHP82 CYP1A2 Induction Pool of HH131, HH133, HH136, HH149, HH Induction of CYP1A2 in pool hepatocytes Relative expression Omeprazole Phenobarbital Rifampin Rif 4µM Rif 2µM Rif 1µM Rif 2µM Rif.4µM Rif.8µM Rif.16µM Control Phe 1µM Phe 5µM Phe 25µM Phe 1µM Phe 5µM Phe 2µM Phe 4µM Control Ome 1µM Ome 5µM Ome 25µM Ome 1µM Ome 2µM Ome.4µM Ome.8µM Con *96-well plate format, 3 day induction Pooled Plateable Human Hepatocytes PHP82 CYP2B6 Induction Pool of HH131, HH133, HH136, HH149, HH Induction of CYP2B6 in pool hepatocytes Relative expression Omeprazole Phenobarbital Rifampin Rif 4µM Rif 2µM Rif 1µM Rif 2µM Rif.4µM Rif.8µM Rif.16µM Control Phe 1µM Phe 5µM Phe 25µM Phe 1µM Phe 5µM Phe 2µM Phe 4µM Control Ome 1µM Ome 5µM Ome 25µM Ome 1µM Ome 2µM Ome.4µM Ome.8µM Con *96-well plate format, 3 day induction 2

21 Pooled Plateable Human Hepatocytes PHP82 CYP3A4 Induction Pool of HH131, HH133, HH136, HH149, HH152 Relative expression Con Ome.8µM Induction of CYP3A4 in pool hepatocytes Omeprazole Phenobarbital Rifampin Ome.4µM Ome 2µM Ome 1µM Ome 25µM Ome 5µM Ome 1µM Control Phe 4µM Phe 2µM Phe 5µM Phe 1µM Phe 25µM Phe 5µM Phe 1µM Control Rif.16µM Rif.8µM Rif.4µM Rif 2µM Rif 1µM Rif 2µM Rif 4µM *96-well plate format, 3 day induction P45 Induction in Individual vs Pooled Cryopreserved Human Hepatocytes Inducers CYP HH131 HH133 HH136 HH149 HH151 Mathematical Average Pooled PHP82 Ome (5 μm) CYP1A PB (1 μm) CYP2B Rif (2 μm) CYP3A

22 Applications of Pooled Plateable Human Hepatocytes P45 induction screening Metabolic stability/metabolite profiling, especially for long term (days) incubation In vitro hepatotoxicity evaluation Plated hepatocyte uptake Plated hepatocyte time-dependent inhibition New Hepatocyte Tools OnDemand pre-plated cryopreserved human hepatocytes (not available in Japan) Pooled plateable human hepatocytes Cryopreserved Taconic genetically engineered mouse hepatocytes 22

23 GEM Hepatocytes Manufactured Uptake transporter knockout: Oatp 1a/1b KO mice (model 177) Humanized human CYP3A4: hpxr/hcar/hcyp3a4 (model 11585) mice Mouse Cyp3a knockout FVB/N-Cyp3a13tm1Ahs Del(5Cyp3a57-Cyp3a59)1Ahs (model 911) Humanized CYP3A4 (mouse Cyp3a knockout) FVB.129P2-Cyp3a13,tm1Ahs.Del(5Cyp3a57- Cyp3a59)1Ahs Tg(APOE-CYP3A4)A1Ahs (model 948) Cryopreserved GEM Mouse Hepatocytes 23

24 Applications of GEM Hepatocytes In vitro-in vivo bridge Bridge between in vitro studies performed using human hepatocytes and in vivo studies performed using GEM mouse models. Selection of GEM in vivo models Prescreening of GEM hepatocytes to aid evaluation of the utility of the corresponding in vivo mouse models. Enhance efficiency of GEM in vivo studies Develop in vitrodata to aid experimental design and data interpretation of in vivogem studies. Tier approach of first performing in vitro studies with GEM hepatocytes, followed by confirmation of critical observations using in vivo GEM. Mechanistic studies Investigate the role of specific proteins such as transporters and P45 isoforms in drug metabolism and toxicity Investigate species difference of specific proteins (e.g. P45; transporters) Mission 2: Develop Practical and Physiologically Relevant Hepatocyte Assays for Preclinical Assessment of Human Drug Properties 24

25 Plated Hepatocyte Relay Assay (PHRA) for Slowly Metabolized Chemicals (patent pending) Current Challenge in Drug Metabolism: Slowly Metabolized Compounds Routine screening for metabolic stability led to the accumulation of compounds that are resistant to metabolism Current in vitro HLM and hepatocyte metabolism assays are not effective for the evaluation of SMC for key drug properties Hepatic clearance estimation Metabolite profiling Metabolic phenotyping 25

26 Limited Incubation Durations of Current Metabolic Stability Assays Human liver microsomes: 2 hrs Human hepatocyte suspension: 4 hrs Continuous incubation with primary human hepatocyte cultures for SMC metabolism is not appropriate: spontaneous down-regulation of P45 gene expression and activity 3 Culture Duration Dependent Down Regulation of P45 Gene Expression 2 1 CYP2B6 CYP2C8 CYP2C9 CYP2D6 CYP3A4 CYP3A5 Relative expression 2hrs 6hrs 12hrs 24hrs 48hrs 26

27 Relay Assay with Hepatocytes in Suspension (Di et al., 212)* Incubation for 4 hrs using pooled human hepatocytes in suspension Centrifuge to remove hepatocytes Re-incubate for 4 hrs with new hepatocyte suspension (Relay) 5 relays = 5 x 4 or 2 hrs *Di, L., P. Trapa, et al. (212). "A novel relay method for determining lowclearance values." Drug Metab Dispos 4(9): Plated Human Hepatocyte Relay Assay Similar to the Relay Assay of Di et al (212), except that plated human hepatocytes are used, with each relay extended to 24 hrs. Each relay: 24 hrs 5 relays: 5 x 24 = 12 hrs 27

28 Plate hepatocytes in Cryopreserved Hepatocytes Plating Medium (CHPM) Removal of medium by aspiration 28

29 Addition of medium containing test article and incubate for 24 hrs Removal of medium, pool for analysis and use for relay incubation Acetonitrile addition (estimate nonspecific binding) Pool Incubated medium Relay: Addition of incubated medium to a new hepatocyte plate Old plate LC/MS-MS New hepatocyte plate prepared 4 h before use 29

30 Validation Study: Collaboration with Chi-Chi Peng and Chandra Prakash (Biogen) Procedures Pool of HH17, HH123, HH131, and HH well plates: 5K cells per well in1 µl HIM (.5 million cells/ml) 1 µm substrate concentration,, 8, 24, 48, 72, 96 and 12 h time points Evaluation of 15 drugs with known in vivo hepatic clearance 3

31 Compounds Evaluated in PHRA Diazepam Tolbutamide Prednisolone Voriconazole Quinidine Tenoxicam Ibuprofen Disopyramide Dexamethasone Tenidap Prednisone Warfarin Meloxicam Riluzole Clozapine Glimepiride SMC requiring hr metabolism LN % remaining LN % remaining 4 2 dexamethasone LN % remaining 5 time (hr) prednisolone time (hr) tolbutamide time (hr) LN % remaining LN % remaining 4 2 disopyramide LN % remaining 5-5 time (hr) prednisone time (hr) voriconazole time (hr) LN % remaining meloxicam LN % remaining LN % remaining 5 time (hr) tenoxicam time (hr) warfarin time (hr) 31

32 SMC requiring shorter incubation times LN % remaining LN % remaining 5 clozapine time (hr) ibuprofen time (hr) LN % remaining LN % remaining glimepiride (I) time (hr) quinidine time (hr) LN % remaining LN % remaining glimepiride (II) time (hr) riluzole time (hr) LN % remaining risperidone time (hr) Plated Human Hepatocyte Relay Assay for Slowly Metabolized Compounds Estimation of in vivo hepatic clearance Estimation of key metabolic pathways* Metabolite profiling Use of inhibitors to identify of drug metabolizing enzyme pathways for slowly metabolized compounds *critical for the estimation of DDI victim potential 32

33 Time-Dependent CYP3A4 Inhibition Time-Dependent Inhibition of Drug Metabolizing Enzymes Important mechanism of DDI Persistent DDI after perpetrator drug removal Possible mechanism of idiosyncratic hepatotoxicity Traditionally performed with HLM IC5 shift (pre-incubation +/-NADPH) for identification of TDI Pre-incubation/2X dilution for KI and Kinact determination 33

34 Advantages of Human Hepatocyte TDI Assay with Plated Cells Efficient removal of inhibitors before substrate metabolism, thereby minimizing competitive inhibition No decrease in hepatocyte CYP3A activity during pre-incubation period Active gene expression and protein synthesis allowing evaluation of recovery of enzyme activity via replacement of inactivated enzyme by newly synthesized enzymes, thereby allowing the evaluation of recovery Evaluation of TDI that are also inducers (e.g. ritonavir) High Throughput, 384-well Plate Assay for Time Dependent CYP3A4 Inhibition Evaluation in Human Hepatocytes 384 well assay 34

35 Materials Human Hepatocytes (Lot # HH157) Universal Cryopreserved cells Recovery Medium (UCRM) Collagen Coated 384-well plate (Cellaffix) Hepatocyte Induction Medium (HIM). CYP3A4 inhibitors: Erythromycin: 25µM, 125µM, 62.5µM, 31.25µM, 15.6µM, 7.8µM and 3.9µM Aminobenzotriazole: 1µM, 5µM, 25µM, 125µM, 62.5µM, 31.25µM and 15.6µM Time points: 2 min, 15 min, 1 min, 5 min, 4 min, 2 min, 1min and min CYP3A4 Substrate: 3µM Luciferin-IPA (Promega) Method Thaw cryopreserved human hepatocytes using UCRM, Resuspendcellsatdensityof1x1 6 cells/mlinhim. Add1,cells/well(1µL)andallowtoattachfor4hrs. Add1µLof2Xconcentrationsofinhibitors,startingwiththelongesttimepointi.e2minall thewaytotime. Add 8µL of the induction medium to all the wells to dilute the inhibitor immediately following time using automatic multi-well dispenser(microtek) followed by aspiration using 384-well aspirator(aquamax 4). Add 3µL of Luciferin-IPA(3µM) to each well manually using 16-channel pipettor. Incubate for 4 min and aliquot 15 ul and transfer to a fresh opaque white 384-well plate. Add equal volume of detection reagent and measure luminescence using Victor Multilabel Plate Reader. Measure ATP in the wells containing cells using ATPlite (Perkin Elmer) as per the manufacturer s instructions. Correct CYP3A4 activity luminescence with ATP luminescence for each well and calculate % activity remaining correcting for control and calculate the inhibition parameters (K I and kinact) using Graph Pad Prism. 35

36 Results Inactivation Parameters Erythromycin 1-Aminobenzotriazole K I (µm) k inact (min -1 )

37 Long-term Culturing of Human Hepatocytes in Human Plasma Rationale for Culturing Human Hepatocytes in Human Plasma Inthehumanbody,allcells are nourishedby plasma. Human plasma should therefore be the most appropriate medium for the culturing of human cells. This idea, however, has notyet beentested. 37

38 Experimental Approach Human hepatocytes: HH153/57/62 (1% confluent plateable cryopreserved human hepatocytes) Culture format: 96 well plate Culture media: 1% human plasma and William s E medium Human plasma: minimally modified human plasma pooled from 5 donors Culture regiment: medium change every Monday, Wednesday, and Friday Culture duration: 29 days days withmedium changeevery 2 3 days. Cell Morphology(HH162) Innormalculturemedium (William se),humanhepatocytes started todisintegrateatday 14and continuedtodeteriorate. In humanplasma,thecells remainedintactwith beautiful,epithelial cell morphology all through Day7 Day14 William s E Medium Day24 Human Plasma 38

39 Day 29: The human hepatocytes were nearly all dead in William s E but remained beautiful in human plasma William s E Medium Human Plasma Summary Humanhepatocytescan be culturedfor an extendedperiod(over4 weeks)inhuman plasma Innormal medium(william se medium, the most commonlyusedmediumfor hepatocyte culturing),thecellsstartedto deteriorateat week2 39

40 Conclusion Humanhepatocytescan be maintainedfor over4 weeksinhumanplasmawhile the cells startedtodeteriorateinculture mediumafter 2 weeks. The abilitytoculture humanhepatocytesfor extendedperiodof timeinhumanplasma allows manyusefulapplications,including long-termdrugmetabolism, drug-drug interactions, and drug toxicity which cannot be otherwise studied using cultured hepatocytes. CYP3A4 Induction: HIM versus Human Plasma 4

41 96-well format Induction Protocol HIM: 2-day culture, 3 day treatment with rifampin (highest concentration: 2 um) Human Plasma: 5 day culture, 3 day treatment with rifampin (highest concentration: 2 um(him), 2 um(plasma) Rifampin Induction of CYP3A4 (mrna) HIM vs Human Plasma (HH151) 41

42 Rifampin Induction of CYP3A4 (mrna) HIM vs Human Plasma (HH151) CYP3A4 Induction in Human Plasma Dose-dependent induction of CYP3A4 gene expression by rifampin At the concentrations evaluated, the maximal fold induction in plasma was found to be lower than that observed in HIM However, saturation not observed at highest concentration evaluated of 2 um 42

43 Human Plasma Induction: Ongoing Research Optimize induction protocol Culture duration Treatment duration: 14 days? Evaluate multiple inducers in multiple human donors for IVIVC Long-term Human Plasma Cultured Human Hepatocytes Characterization of hepatic functions upon prolonged incubation Evaluation of lot to lot variations of human plasma DDI Prolonged CYP3A4 induction Time-dependent inhibition: Recovery of inactivated enzyme upon removal of perpetrator Long-term metabolism Long-term hepatotoxicity 43

44 Advancing physiological relevance of in vitro DDI assays Successful cryopreservation of hepatocytes from multiple animal species and human High viability Plateable Large number of vials per isolation (human hepatocytes) Novel hepatocyte tools OnDemand human hepatocytes Pooled plateable human hepatocytes GEM humanized mouse hepatocytes` Novel hepatocyte assays Plated hepatocyte relay assay (PHRA) to evaluate metabolism of slowly metabolized compounds 384-well plated hepatocyte assay for time-dependent inhibition Long-term culturing of human hepatocytes in human plasma P45 induction in human plasma Next Experimental System: Cryopreserved enterocytes 44

45 Acknowledgment IVAL: Utkarsh Doshi, Ph. D., QianYang, Ph. D., Aarti Uzgare, Ph. D., Kirsten Amaral; Nicole Li, Yumiko LaForge Collaborators: FDA-NCTR: Weida Tong; Jie Zhang; Hong Fang; Minjun Chen FDA-CDER Jerry Collins, John Strong, Neil Hartman EPA ToxCast Program: David Dix; Keith Houck U. S. Army: GundaReddy; CaiChang; Mick Major; Stephanie Cole, Jana Madren- Whalley, Jonathan Oyler, Russell Dorsey, Harry Salem CDC: Patricia Richter Penn State University: Raghu Sinha NIH: Menghang Xia Sanofi (Montpellier): Gerard Fabre and colleagues University of Tokyo: Yuichi Sugiyama and colleagues Biogen: Chi-Chi Peng, Chandra Prakash Ex-colleagues Monsanto/Searle team: Yves Verderberghe, Dale Beck, Barry Bode, Tim Whitehead St. Louis U. team: Lilly Xu (now Sanofi), Dale Beck, AsenathRasmussen, John Brems, M. D., Robert Kane, M. D., Donald Kaminsky, M. D. Chuang Lu (previously IVT; now Takeda) Intact human hepatocytes cultured in human plasma as an improved in vitro experimental system for drug metabolism, DDI, and toxicity A physiologically relevant experimental system to model hepatic events based on plasma drug concentrations Appropriate plasma protein binding of parent and metabolites Inclusion of plasma drug metabolizing enzymes: e.g. plasma esterases Intact plasma membrane to model permeability Complete phase (uptake), I (oxidation), II (conjugation), and III (efflux?) metabolic pathways and cofactors Relevant intracellular metabolism Complete, uninterrupted, drug metabolizing enzyme pathways and cofactors 45