Novel Hepatocyte Technologies for the Evaluation of Adverse Drug Properties

Transcription

1 Novel Hepatocyte Technologies for the Evaluation of Adverse Drug Properties Albert P. Li, Ph. D., President and CEO In Vito ADMET Laboratories Inc. Columbia, MD and Malden, MA

2 Novel Hepatocyte Technologies IVAL Mission Hepatocyte cryopreservation Novel hepatocyte tools and assays Ongoing research

3 In Vitro ADMET Laboratories (IVAL) Columbia, MD and Malden, MA Incorporation: November, Mission: Develop and apply in vitro human and animal based experimental systems for the accurate assessment of human drug properties Focus: Primary human and anoimal cells from key organs

4 In Vitro ADMET Laboratories (IVAL) Columbia, MD and Malden, MA Activities: Products: Cryopreserved human and animal hepatocytes and enterocytes 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)

5 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

6 Cryopreservation of Hepatocytes as an Enabling Technology Advantages of cryopreserved hepatocytes 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

7 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. Chem Biol Interact 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 Metab Pharmacokinet. 2003;18(1): First demonstration of effectiveness of CHRM and plateability Li. Human hepatocytes: isolation, cryopreservation and applications in drug development. Chem Biol Interact May 20;168(1):16-29.

8 Universal Cryopreservation Recovery Medium (UCRM ) for Recovery of Hepatocytes Human Lot Age Gender Race Yield Viability M H F C /10/11 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

9 Reproducibility of Hepatocytes Recovery in UCRM HH1031 HH1036 Experiments Yield Viability Yield Viability mean sd mean sd Yield Viability Yield: Millions viability cells/vial Viability: Percent of Trypan blue excluding hepatocytes

10 Plateability of Cryopreserved Hepatocytes

11 Why plateability is important Plateable hepatocytes are higher in quality than nonplateable hepatocytes: less membrane damage Extension of applications: Hepatocytes in suspension would rapidly lose viability (T1/2 about 6 hours) and therefore are limited to short-term (hours) studies Drug uptake Metabolic stability Metabolite profiling Plated hepatocytes are viable for days/weeks Long-term drug metabolism In vitro hepatotoxicity Metabolism-dependent drug toxicity Time-dependent inhibition studies Enzyme induction Efflux transporters

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

13 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 Hepatocytes from transgenic and humanized animals

14 IVAL Plateable Cryopreserved Human Hepatocytes High viability: Routinely >90% High plateability: near 100% 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 (>500, up to >3000 vials per isolation)

15 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

16 Relative expression Relative expression Latest Inducible Lot: HH1121, approx vials Induction of CYP1A2 in HH1121 Induction of CYP3A4 in HH Ome_50µM Ome_25µM Ome_12.5µM Ome_6.25µM Ome_3.13µM Ome_1.56µM Ome_0.78µM Ome_con Phe_1000µM Phe_500µM Phe_250µM Phe_125µM Phe_62.5µM Phe_31.3µM Phe_15.6µM Phe_con Rif_40µM Rif_20µM Rif_10µM Rif_5µM Rif_1µM Rif_0.2µM Rif_0.04µM Rif_con 0 Ome_50µM Ome_25µM Ome_12.5µM Ome_6.25µM Ome_3.13µM Ome_1.56µM Ome_0.78µM Ome_con Phe_1000µM Phe_500µM Phe_250µM Phe_125µM Phe_62.5µM Phe_31.3µM Phe_15.6µM Phe_con Rif_40µM Rif_20µM Rif_10µM Rif_5µM Rif_1µM Rif_0.2µM Rif_0.04µM Rif_con 16

17 IVAL Human Hepatocytes Lot Number Gender Ethnicity Age Viability Yield 1A2 2A6 2B6 2C8 2C9 2C19 2D6 CYP2E1 3A4- MDZ 3A4- TEST ECOD 7HCG 7HCS HH1016 F H HH1028 M H HH1030 M C HH1031 M H HH1032 F C HH1033 F C HH1037 M C HH1044 M C HH1041 M C HH1042 M C HH1043 M H HH1045 M H HH1046 M H HH1047 M H HH1051 M C TBD HH1052 M C TBD HH1055 F C HH1058 M AA TBD HH1059 M C HH1060 M C TBD HH1061 M C HH1063 F C TBD HH1064 F C HH1079 F C HH1068 F C TBD HH1069 F AA HH1071 M H HH1072 F C TBD HH1076 M C TBD HH1078 F C TBD HH1083 F C HH1084 M C HH1089 F C HH1085 F H HH1086 F H HH1093 F C HH1094 F C

18 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

19 QuickFreeze Pooled Donor Cryopreserved Human Hepatocytes Patent allowed in United States (2016), China (2017) 19

20 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

21 Pooled Cryopreserved Human Hepatocytes Pooled Suspension Human Hepatocytes: 5, 10, 20 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

22 Morphology of Re-cryopreserved Human Hepatocytes (Individual Donors) HH1023 HH1031 HH1033 HH1036 HH1049 HH1052

23 Pooled Plateable Human Hepatocytes PHP8002 Morphology Pool of HH1031, HH1033, HH1036, HH1049, HH1052

24 Relative expression Pooled Plateable Human Hepatocytes PHP8002 CYP1A2 Induction Pool of HH1031, HH1033, HH1036, HH1049, HH Induction of CYP1A2 in pool hepatocytes Omeprazole Phenobarbital Rifampin Rif 40μM Rif 20μM Rif 10μM Rif 2μM Rif 0.4μM Rif 0.08μM Rif 0.016μM Control Phe 1000μM Phe 500μM Phe 250μM Phe 100μM Phe 50μM Phe 20μM Phe 4μM Control Ome 100μM Ome 50μM Ome 25μM Ome 10μM Ome 2μM Ome 0.4μM Ome 0.08μM Con *96-well plate format, 3 day induction

25 Relative expression Pooled Plateable Human Hepatocytes PHP8002 CYP2B6 Induction Pool of HH1031, HH1033, HH1036, HH1049, HH Induction of CYP2B6 in pool hepatocytes 10 8 Omeprazole Phenobarbital Rifampin Rif 40μM Rif 20μM Rif 10μM Rif 2μM Rif 0.4μM Rif 0.08μM Rif 0.016μM Control Phe 1000μM Phe 500μM Phe 250μM Phe 100μM Phe 50μM Phe 20μM Phe 4μM Control Ome 100μM Ome 50μM Ome 25μM Ome 10μM Ome 2μM Ome 0.4μM Ome 0.08μM Con *96-well plate format, 3 day induction

26 Relative expression Pooled Plateable Human Hepatocytes PHP8002 CYP3A4 Induction Pool of HH1031, HH1033, HH1036, HH1049, HH Induction of CYP3A4 in pool hepatocytes Omeprazole Phenobarbital Rifampin Rif 40μM Rif 20μM Rif 10μM Rif 2μM Rif 0.4μM Rif 0.08μM Rif 0.016μM Control Phe 1000μM Phe 500μM Phe 250μM Phe 100μM Phe 50μM Phe 20μM Phe 4μM Control Ome 100μM Ome 50μM Ome 25μM Ome 10μM Ome 2μM Ome 0.4μM Ome 0.08μM Con *96-well plate format, 3 day induction

27 Relative expression Pooled Plateable Human Hepatocytes PHP8002 CYP3A4 Induction Pool of HH1031, HH1033, HH1036, HH1049, HH Induction of CYP3A4 in pool hepatocytes Omeprazole Phenobarbital Rifampin Rif 40μM Rif 20μM Rif 10μM Rif 2μM Rif 0.4μM Rif 0.08μM Rif 0.016μM Control Phe 1000μM Phe 500μM Phe 250μM Phe 100μM Phe 50μM Phe 20μM Phe 4μM Control Ome 100μM Ome 50μM Ome 25μM Ome 10μM Ome 2μM Ome 0.4μM Ome 0.08μM Con *96-well plate format, 3 day induction

28 P450 Induction in Individual vs Pooled Cryopreserved Human Hepatocytes Inducers CYP HH1031 HH1033 HH1036 HH1049 HH1051 Mathematical Average Pooled PHP8002 Ome (50 μm) CYP1A PB (1000 μm) CYP2B Rif (20 μm) CYP3A

29 Applications of Pooled Plateable Human Hepatocytes P450 induction screening Metabolic stability/metabolite profiling, especially for long term (days) incubation In vitro hepatotoxicity screen Plated hepatocyte uptake Plated hepatocyte time-dependent inhibition

30 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

31 New Hepatocyte Tools

32 New Hepatocyte Tools OnDemand pre-plated cryopreserved human hepatocytes P450 Knocked-Down human hepatocytes MetMax pooled cryopreserved human hepatocytes 32

33 HH1031: 42 Hour Shipment 33

34 Relative expression OnDemand PrePlated Human Hepatocytes CYP1A2 mrna Induction Induction of CYP1A2 in OnDemand human hepatocytes Omeprazole (50 um) Con Phe Ome Rif

35 Relative expression OnDemand PrePlated Human Hepatocytes CYP2B6 mrna Induction 70 Induction of CYP2B6 in OnDemand human hepatocytes 60 Phenobarbital (1000 um) Con Phe Ome Rif

36 Relative expression (log) OnDemand PrePlated Human Hepatocytes CYP3A4 mrna Induction 1000 Induction of CYP3A4 in OnDemand human hepatocytes Rifampin (10 um) Con Phe Ome Rif

37 OnDemand Preplated Human Hepatocytes Ready to use hepatocytes P450 induction studies Cytotoxicity studies Plated metabolism studies Plated from pre-characterized human hepatocyte lots Delivered to laboratories based on investigator s schedule 7 days a week 37

38 New Hepatocyte Tools OnDemand pre-plated cryopreserved human hepatocytes P450 Knocked-Down human hepatocytes MetMax pooled cryopreserved human hepatocytes 38

39 P450 KO Human Hepatocytes Isolation of hepatocyte from human livers Treatment with mechanism-dependent P450 inhibitors (causing irreversible enzyme inaction) before cryopreservation Pan inhibitor CYP3A4-specific inhibitor Cryopreservation to retain plateability 39

40 Drug Metabolizing Enzyme Activities: Control vs Knockdown Human Hepatocytes Metabolic Pathway Activity (pmol/min/million hepatocytes) Pan P450 KD Control (HH1081) KD (HH1091) CYP3A4 KD Control (HH1097) KD (HH1098) CYP1A CYP2A CYP2B CYP2C CYP2C CYP2C CYP2D CYP2E CYP3A CYP3A ECOD UGT SULT

41 Applications of P450-KD Human Hepatocytes Aid delineation of the roles of P450 in drug properties Uptake transport vs P450 metabolism in hepatic clearance P450 specific metabolites Metabolic activation/detoxification of toxicants Prodrug activation 41

42 Application of P450 KD Human Hepatocytes in the Evaluation of Drug Toxicity 42

43 Human Hepatocyte Reactive Metabolite Assay

44 Introduction Reactive metabolite formation is a major mechanism of drug toxicity, especially in drug induced liver injuries (DILI) Individual differences in drug metabolizing enzyme activities may lead to individual differences in toxic responses Reactive metabolite formation is one of the hypothetical mechanisms for idiosyncratic DILI A physiologically-relevant in vitro screening assay for reactive metabolite formation would aid the elimination of drug candidates with hepatotoxic liability from further development

45 Hypothesis Drug-induced decrease in reduced L-glutathione in human hepatocytes can be used to identify drugs that form reactive metabolites The accuracy of the human hepatocyte reactive metabolite assay can be enhanced via the following: Use of normal and P450-knock down human hepatocytes to confirm role of metabolism in reactive metabolite formation Assay in the presence and absence of GSH depletion to confirm reactive metabolite formation

46 Expected Results Drugs that form reactive metabolites would show the following in GSH depletion: Dose-dependent GSH depletion Depletion more pronounced in BSO treated (lower basal GSH level) than in untreated hepatocytes Depletion more pronounced in P450 competent than in P450-knockdown hepatocytes

47 Experimental Approach Human hepatocytes: Normal (HH1082) and P450-knockdown hepatocytes (HH1091). Both lots were prepared from the same liver. Culture condition: The human hepatocytes were thawed, recovered in Universal Cryopreservation Recovery Medium (UCRM; IVAL) and plated in a collagen-coated 384 well plate (10 ul/well; 5000 cells) in Hepatocyte Incubation Medium (HIM; IVAL) with or without 300 mm of buthionine sulfoximine (BSO; Sigma-Aldrich), an inhibitor of de novo GSH synthesis. The hepatocytes were cultured in a 37 deg. C. cell culture incubator maintained at a highly humidified atmosphere of 5% CO2 and 95% air. The cells were cultured for 4 hrs before the initiation of drug treatment. Treatment: At the end of the 4 hr. BSO pretreatment, medium was changed to that containing the desired concentrations of test substances, with and without BSO for a treatment duration of 6 hrs. Endpoint: At the end of treatment, cellular GSH content was quantified based on luminescence using a GSH kit (Promega GSH-Glo ).

48 Data Analysis 1. Calculation of Relative Cellular GSH Contents: Relative Cellular GSH Contents (%) = [GSH (treated)/gsh (control)] x 100% 2. Estimation of IC 50 for GSH reduction (GraphPad Prism 4.0) 3. Calculation of Reactive Metabolite Index (RMI) RMI = IC 50 (-BSO)/ IC 50 (+BSO)

49 Control and P450 Knock-Down Human Hepatocytes Collagenase perfusion of a human liver Isolated hepatocytes divided into two separate lots: P450 Knock-Down (HH1091): Treatment of the hepatocytes with a non-specific time-dependent P450 inhibitor Normal (HH1082): No treatment Cryopreserved and stored in liquid nitrogen

50 Both lots are plateable (>90% confluent; 24 hr. Cultures) HH1082 HH1091

51 Drug Metabolizing Enzyme Activities Drug Metabolizing Enzyme Substrate (µm) Incubation Time (minutes) Metabolite Quantified HH1082 Activity (pmol/minute/ million cells) HH1091 Activity (pmol/minute/ million cells) CYP1A2 Phenacetin (100) 15 Acetaminophen CYP2A6 Coumarin (50) 30 7-Hydroxycoumarin CYP2B6 Bupropion (500) 15 Hydroxybupropion CYP2C8 Paclitaxel (20) 15 6α-Hydroxypaclitaxel CYP2C9 Diclofenac (25) 15 4-Hydroxydiclofenac CYP2C19 CYP2D6 CYP2E1 CYP3A4 S-Mephenytoin (250) Dextromethorphan (15) Chlorzoxazone (250) 30 4-Hydroxymephenytoin Dextrorphan Hydroxychlorzoxazone Midazolam (20) 10 1-Hydroxymidazolam Testosterone (200) 15 6β-Hydroxytestosterone ECOD 7-Ethoxycoumarin (100) 30 7-Hydroxycoumarin UGT 7-Hydroxycoumarin (100) 30 7-Hydroxycoumarin glucuronide Sulfotransferase 7-Hydroxycoumarin (100) 30 7-Hydroxycoumarin sulfate

52 Acetaminophen HH1082 HH1091

53 Cyclophosphamide HH1082 HH1091

54 Leflunomide

55 Leflunomide

56 Flutamide

57 Nefazodone

58 Ketoconazole (Cytotoxic Negative Control)

59 Aspirin (Noncytotoxic Negative Control)

60 Reactive Metabolite Index Control Hepatocytes HH1082 P450 KD HH1091 Drug IC 50 (Control) IC 50 (BSO) RMI IC 50 (Control) IC 50 (BSO) RMI Acetaminophen Cyclophosphamide Flutamide Leflunomide Nefazodone Ketoconazole Aspirin NA NA NA NA NA NA

61 Expected Results Observed Drugs that form reactive metabolites would show the following in GSH depletion: Dose-dependent GSH depletion Depletion more pronounced in BSO treated (lower basal GSH level) than in untreated hepatocytes Depletion more pronounced in P450 competent than in P450-knockdown hepatocytes

62 Reactive Metabolite Assay A high through 384-well assay has been developed for the estimation of reactive metabolite formation Endpoint: Cellular GSH contents upon drug treatment with and without BSO depletion of basal GSH levels Calculation of Reactive Metabolite Indices as ratio of IC50 of control to BSO treated hepatocytes Assay can be used as routine screening in drug development to rank order drug candidates based on RMI 62

63 New Hepatocyte Tools OnDemand pre-plated cryopreserved human hepatocytes P450 Knocked-Down human hepatocytes MetMax pooled cryopreserved human hepatocytes 63

64 MetMax Hepatocytes (patent pending) Permeabilized, cofactorsupplemented, cryopreserved hepatocytes pooled from multiple donors Maximized drug metabolizing enzyme activities

65 Easy storage: MetMax Advantage Long-term storage in a -80 degree freezer (no need for LN2 storage) Easy usage: Thaw and use No centrifugation, No microscopic examination No cell counting and viability determination

66 Activity (pmol/min/million hepatocytes) Pooled Normal Human Hepatocytes (PHH) Vs MetMax (PHHX) in Drug Metabolizing Enzyme Activities PHH PHHX Drug Metabolizing Enzyme Pathway 66

67 MetMax Hepatocytes and Enterocytes (patent pending) Maximized drug metabolizing enzyme activities for the evaluation of human hepatic (hepatocytes) and enteric (enterocytes) metabolism Easy to use: Thaw and use directly. No centrifugation, no microscopic examination, no cell counting. Maximized phase 1 and phase 2 drug metabolizing enzyme activities Applications include Metabolic stability Metabolite profiling and identification Enzyme inhibition Metabolic activation of pro-toxicants and pro-mutagens

68 In Vitro Hepatic Clearance Metabolic Stability Screening: MetMax Pooled Donor Human Hepatocytes accurately predict human hepatic clearance in vivo (PHS9001: Pooled Human Hepatocytes; PHX 8001: MetMax Pooled Human Hepatocytes; Collaboration with Gary Hingorani and Karin Brown, Array Biopharma) PHS9001 y = x R² = 0.13 MetMax PHHX 8001 y = x R² = Test article concentration: 1 um Hepatocyte: 1 million cells/ml Time points: 0, 5, 15, 30, 45 Endpoint: T1/2; Prediction: In vivo intrinsic hepatic clearance In Vivo Systemic Clearance

69 Metabolite Profiling MetMax Generates Metabolites at Cytotoxic Acetaminophen (APAP) Concentrations Metabolic Pathway Substrate Conc. (µm) Marker Metabolite PHHX8001 (MetMax) PHS9001 (Normal) GST UGT SULT 100 mm APAP- 857 ± ± mm Glutathione 677 ± ± mm APAP ± ± mm Glucuronide 1362 ± ± mm 150 ± ± 3.36 APAP-Sulfate 200 mm 156 ± ±

70 MetMax as Exogenous Metabolic Activation System for the Evaluation of Pro-toxicants 70

71 MetMax Activation of Protoxicants Cytotoxicity of drugs were evaluated in HEK 293 cells in the absence of activation in the presence of exogenous activation (with MetMax hepatocytes), or in the presence of an inactivated exogenous metabolic system (boiled MetMax hepatocytes) 71

72 MetMax Activation of Acetaminophen Experiment 1: 0.5 million hepatocytes per ml Without Hepatocytes With Boiled MetMax Hepatocytes With MetMax Hepatocytes Experiment 2: 1.0 million hepatocytes per ml 72

73 MetMax Activation of Ifosfamide and Cyclophosphamide Without Hepatocytes With Boiled MetMax Hepatocytes Without Hepatocytes With Boiled MetMax Hepatocytes With MetMax Hepatocytes With MetMax Hepatocytes 73

74 MetMax Human Hepatocytes A convenient human hepatocyte reagent for the evaluation of drug metabolism One step reagent: thaw and use Effective as an exogenous metabolic system for the evaluation of metabolic activation of drugs forming toxic metabolites 74

75 The MetMax Advantage Easy storage: Long-term storage in a -80 degree freezer (no need for LN2 storage) Easy usage: Thaw and use No centrifugation, No microscopic examination No cell counting and viability determination

76 Ongoing Research Enhance in vivo characteristics

77 Culturing of Human Hepatocytes in Human Plasma Hepatocyte 77

78 Long-term Culturing of Human Hepatocytes in Human Plasma

79 Rationale for Culturing Human Hepatocytes in Human Plasma In the human body, all cells are nourished by plasma. Human plasma should therefore be the most appropriate medium for the culturing of human cells. This idea, however, has not yet been tested.

80 Experimental Approach Human hepatocytes: HH1053/57/62 (100% confluent plateable cryopreserved human hepatocytes) Culture format: 96 well plate Culture media: 100% 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 with medium change every 2 3 days.

81 Experimental Approach Human hepatocytes: HH1053/57/62 (100% confluent plateable cryopreserved human hepatocytes) Culture format: 96 well plate Culture media: 100% 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 with medium change every 2 3 days.

82 Cell Morphology (HH1062) In normal culture medium (William s E), human hepatocytes started to disintegrate at day 14 and continued to deteriorate. In human plasma, the cells remained intact with beautiful, epithelial cell morphology all through Day 7 Day 14 Day 24 William s E Medium Human Plasma

83 Cell Morphology (HH1062) In normal culture medium (William s E), human hepatocytes started to disintegrate at day 14 and continued to deteriorate. In human plasma, the cells remained intact with beautiful, epithelial cell morphology all through Day 7 Day 14 Day 24 William s E Medium Human Plasma

84 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

85 Summary Human hepatocytes can be cultured for an extended period (over 4 weeks) in human plasma In normal medium (William s E medium, the most commonly used medium for hepatocyte culturing), the cells started to deteriorate at week 2

86 CYP3A4 Induction: HIM versus Human Plasma

87 Induction Protocol 96-well format HIM: 2-day culture, 3 day treatment with rifampin (highest concentration: 20 um) Human Plasma: 5 day culture, 3 day treatment with rifampin (highest concentration: 20 um (HIM), 200 um (plasma)

88 Rifampin Induction of CYP3A4 (mrna) HIM vs Human Plasma (HH1051)

89 Rifampin Induction of CYP3A4 (mrna) HIM vs Human Plasma (HH1051)

90 Rifampin Induction of CYP3A4 (mrna) HIM vs Human Plasma (HH1051)

91 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 200 um

92 Human Plasma Induction: Ongoing Research Optimize induction protocol Culture duration Treatment duration: 14 days Evaluate multiple inducers in multiple human donors for IVIVC

93 Carb_con Carb_17µM Carb_34µM Carb_51µM Carb_68µM Nif_con Nif_0.14µM Nif_0.29µM Nif_0.43µM Nif_0.58µM Phe_con Phe_26µM Phe_52µM Phe_78µM Phe_104µM Phen_con Phen_35µM Phen_70µM Phen_105µM Phen_140µM Piog_con Piog_1.4µM Piog_2.8µM Piog_4.2µM Piog_5.6µM Ple_con Ple_1.7µM Ple_3.3µM Ple_5.0µM Ple_6.7µM Rif_con Rif_5µM Rif_10µM Rif_15µM Rif_20µM Rosi_con Rosi_0.7µM Rosi_1.4µM Rosi_2.1µM Rosi_2.8µM Trog_con Trog_3.1µM Trog_6.3µM Trog_9.4µM Trog_12.6µM Relative expression Induction of CYP3A4 in plasma: Multiple Inducers (2 days induction) Collaboration with Chuang Lu, Ph. D. 93

94 Ongoing Research: 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

95 Integrated Discrete Multiple Organ Co-culture: IdMOC Patent allowed: USA, China, Japan, EU 95

96 An Ideal In Vitro System for Human Toxicity Evaluation (MTE Requirement) Human hepatic metabolism (M) Interacting, multiple human target organs: liver and nonhepatic organs (T) Physiologically relevant endpoints (E)

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101 RM* Drug RM*

102 IdMOC-96

103 IdMOC: Demonstration of Metabolism-dependent Toxicity Cyclophosphamide cytotoxicity towards 3T3 cells in 3T3/Human Hepatocyte Co-culture

104 Cyclophosphamide Metabolism

105 IdMOC with metabolically incompetent (3T3) and competent cells (human hepatocytes) Medium (no cells) 3T3 cells (metabolically incompetent) Human hepatocytes (metabolically competent)

106 IdMOC 3T3/Hepatocytes: MTT Metabolism after Cyclophosphamide Treatment Cyclophosphamide 0 mm 2.5 mm 5 mm 10 mm No Hep 1 Hep 2 Hep 3 Hep

107 Relative Viability (%) Relative Viability (%) Relative Viability (%) Cyclophosphamide Cytotoxicity to 3T3 Cells in IdMOC: Effects of Varying Number of Wells with Human Hepatocytes Plate No Hepatocytes with hepatocytes (1 well) With hepatocytes (2 wells) With hepatocytes (3 wells) Cyclophosphamide (mm) Plate Plate No hepatocytes 80 No hepatocytes Cyclophosphamide (mm) with hepatocytes (1 well) With hepatocytes (2 wells) With hepatocytes (3 wells) Cyclophosphamide (mm) with hepatocytes (1 well) With hepatocytes (2 wells) With hepatocytes (3 wells)

108 Percent Control Cyclophosphamide immunotoxicity in IdMOC as determined by splenocyte function (PHA induction of proliferation): Hepatocytes were required for toxicity Relative Splenocyte Function Relative ATP content Cyclophosphamide (mm) Single Culture Co-Culture

109 Significance of Metabolism-based Classification Type I: Direct-acting (Tamoxifen): Organ-specific toxicity dependent of route of exposure to parent toxicant Type II: Metabolically activated, localized toxicity (Aflatoxin-B1): Organ-specific toxicity dependent on site of metabolic activation Type III: Metabolically activated, diffusible toxic metabolites (Cyclophosphamide): Organspecific toxicity dependent on metabolite distribution

110 IdMOC: A practical co-culture system Wells-in-a well concept allowing co-culturing of multiple cell types as physically separated (discrete) entities but interconnected (integrated) by a common overlying medium Specially manufactured plates with shallow inner wells to minimize dilution Identical foot-prints as regular 24- and 96-well plates, thereby compatible with routinely used lab equipment such as multichannel pipettes and plate readers

111 IdMOC Most complete in vitro model of a whole organism (human; nonhuman) Hepatic metabolism-dependent xenobiotic effects Paracrine effects Endocrine effects

112 Key IdMOC Publications Li AP, Bode C, Sakai Y. A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcicnoma breast cancer cells. Chem Biol Interact. 2004; 150: Li AP. In vitro evaluation of human xenobiotic toxicity: scientific concepts and the novel integrated discrete multiple cell co-culture (IdMOC) technology. ALTEX. 2008;25(1):43-9. Li AP. Integrated Discrete Multiple Organ Co-culture (IdMOC) Experimental System for the Evaluation of Organ-Specific Toxicity. Altern Lab Anim Sep;37(4): Richter, Li, Roy. Cytotoxicity of eight cigarette smoke condensates in three test systems: comparisons between assays and condensates. Regul Toxicol Pharmacol Dec;58(3): Li, AP, LaForge, Y, Uzgare A. Definition of metabolism-dependent drug toxicity with the novel integrated discrete multiple organ co-culture: Results with model toxicants tamoxifen, aflatoxin B1 and cyclophosphamice. Chem Biol Interact Jul 30;199(1):1-8.

113 IdMOC-Man: Key cell types of human organs cultured in human plasma HEART KIDNEY VASCULAR LIVER LUNG SKELETAL/SMOOTH/ MUSCLE NEURONAL SYSTEM

114 Conclusions

115 Accurate Prediction of Human Drug Properties Relevant experimental models Organ-specific properties Species-specific properties Appropriate experimental protocols Accuracy Reproducibility Physiological relevance Appropriate data analysis IVIVC 115

116 Key Systems: Hepatocytes and Enterocytes 116

117 117

118 sometime in the future Albert P. Li, age 105, pioneered human hepatocytes and enterocytes cropreservation, inventor of IdMOC 118

119 Happy Hepatocytes Happy Enterocytes Happy Scientists 119