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Hepatocytes and Enterocytes Technologies for Drug Development

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1 Hepatocytes and Enterocytes Technologies for Drug Development
IVAL HERO Workshop, Dec. 4, 2017 Hepatocytes and Enterocytes Technologies for Drug Development Albert P. Li, Ph. D., President and CEO In Vito ADMET Laboratories Inc. Columbia, MD and Malden, MA

2 Species Differences in Hepatic P450 isoforms
Family Human Mouse Rat Dog Monkey CYP1A 1A1/2 CYP2A 2A6 2A5 2A13/25 2A23/24 CYP2B 2B6 2B9/10 2B1 2B11 2B17 CYP2C 2C8/9/19 2C29/37/38/40/44 2C6/7/11, 2C21/41 2C20/43 CYP2D 2D6 2D22 2D1 2D15 2D17 CYP2E 2E1 CYP3A 3A4/5 3A11/13 3A1/2 3A12/26 3A8

3 Drug metabolism as a key determinant of species-species differences in drug properties
Metabolic stability Drug-drug interactions Drug-toxicity An animal species which does not metabolize a parent drug to form metabolites found in human may under or over estimate drug toxicity in human Underestimation of toxicity if the major toxic metabolites are human-specific Overestimation of toxicity if the major toxic metabolites are animal-specific

4 In vitro in vivo strategy (IVIVS)
Predict human in vivo drug toxicity with Human-specific information developed from in vitro human-based experimental systems Key in vivo parameters obtained from early in vivo studies such as human clinical studies and/or relevant preclinical studies in experimental animals Human-based in vitro experimental systems for the evaluation of human drug safety. Li AP, Curr Drug Saf (3):193-9.

5 In Vitro In Vivo Strategy (IVIVS)
Human in vitro Animals in vivo Li AP (2007). Current Drug Safety

6 Parallelogram Approach for the Preclinical Assessment of Clinical Adverse Drug Effects
Human in vivo Extrapolation Animal in vivo PBPK IVIVC Genetic polymorphism Animal Species Selection Risk Factors Human in vitro Animal in vitro Species Comparison

7 Human-specific Metabolism (M) Human Targets (T) Relevant Endpoints (E)
Key Components of an In Vitro Experimental System for the Evaluation of Human Toxicants: The human MTE Requirement Human-specific Metabolism (M) Human Targets (T) Relevant Endpoints (E)

8 Key IVAL Experimental Systems: Hepatocytes and Enterocytes
Enterocytes: First-pass metabolism of orally-administered drugs Hepatocytes: First-pass metabolism of absorbed orally-administered drugs

9 Hepatocytes

10 Cryopreservation of Human Hepatocytes as an Enabling Technology
Advantages of cryopreserved cells Experiments can be scheduled Can repeat experiments with cells from the same donors Can use pre-characterized hepatocytes Can compare multiple donors in the same experiment Can pool hepatocytes from multiple donors SafeSciMet Course 5.3 Introduction to Human Liver Metabolism, Albert Li, PhD

11 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 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):17-35. 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):33-41. 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.

12 Recovery of Cryopreserved Hepatocytes
Universal Cryopreservation Recovery Medium (UCRM): Same as CHRM® from GIBCO)

13 Universal Cryopreservation Recovery Medium (UCRM™) for Recovery of Hepatocytes
Human Lot Age Gender Race Yield Viability 1 1006 35 M H 7.90 94.60 2 1007 26 F C 6.00 97.60 3 1009/10/11 47 5.30 95.41 4 1012/13 25 5.80 78.62 5 1016/17 64 7.00 93.33 6 1014/18 15 75.47 7 1015/19 17 4.40 96.70 8 1020/21/22/23 21 6.40 88.89 9 1025/26/27 59 8.25 97.13 10 1028 56 7.50 96.79 11 1029 51 5.75 89.06 12 1031 42 6.90 96.50 13 1033 40 92.00 14 1035 4.35 92.55 1036 55 8.00 97.58 16 1037 45 5.10 98.08 mean sd Yield 6.28 1.22 Viability 92.52 6.72 Yield: Millions viability cells/vial Viability: Percent of Trypan blue excluding hepatocytes

14 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 64 88 5.7 154.0 81.4 10.1 1.7 44.2 20.9 4.4 41.3 67.1 454.7 137.3 203.3 25.5 HH1028 M 43 94 5.3 54.3 39.3 6.2 4.8 27.7 55.9 6.3 22.7 33.5 300.7 42.2 126.0 17.6 HH1030 C 31 85 5.2 68.9 8.6 4.1 3.7 29.6 14.0 4.6 51.7 19.1 106.7 71.5 78.7 19.5 HH1031 42 92 7.2 12.7 8.4 81 2.3 59.8 0.9 18 38 14.8 179.2 145.2 528.2 26.5 HH1032 68 95 7.8 7.0 20.0 8.1 0.7 21.4 4.9 7.3 29.8 16.3 102.7 87.4 435.3 41.2 HH1033 40 31.1 1.5 18.3 1.3 66 19.4 35.9 73.9 108.5 493 HH1037 45 3.3 83.7 45.2 6.8 8.2 20.6 9.7 9.4 23.4 256.7 67.8 74.0 24.5 HH1044 67 86 5.0 115 5.6 52.9 55.8 0.5 46.2 41.1 21.3 477.7 153.8 699 48.7 HH1041 53 87 21.7 41.4 15.7 6.4 34.7 0.3 27.8 3.4 33.1 71.9 174.0 31.8 HH1042 57 18.9 11.1 5.4 1.6 10.2 234.7 14.9 36.9 9.0 HH1043 37 89 6.7 50.8 39.8 8.5 23.3 4.7 17.7 10.5 180.0 60.2 301.7 48.4 HH1045 9 51.9 1.4 90.3 4.3 35.5 38.9 17.2 311.4 114.8 392 18.1 HH1046 93 38.2 17.3 6.6 34.1 11.0 14.3 15.0 5.8 128.4 48.6 267.7 52.1 HH1047 44 69.6 3.0 1.0 47.4 14.6 320.3 136.9 440.2 27.9 HH1051 23 96 85.09 TBD 51.15 3.21 5.46 1.08 3.6 10.49 1.48 38.46 24.37 19.23 2.57 HH1052 399 68.7 249.9 8.28 27.3 35.8 5.1 46.3 155.8 572.4 40.2 HH1055 33 84 7.9 78.3 7.1 5.5 36.0 4.2 10.0 81.7 530.7 32.4 391.0 26.1 HH1058 AA 17 91 3.8 88.98 7.78 3.59 6.73 9.57 9.69 3.14 48.39 6.12 4.50 0.81 HH1059 65 80 109.0 37.3 37.7 16.9 190.0 163.0 24.0 HH1060 52 155.04 216.01 29.93 27.38 20.61 22.96 10.75 149.56 3.96 2.18 0.89 HH1061 20 4.0 50.6 22.5 35.3 12.8 14.5 11.2 239.3 61.4 122.6 23.0 HH1063 109.13 19.66 8.08 2.76 2.26 7.03 16.27 12 145.11 36.22 31.07 2.58 HH1064 59.5 16.8 15.6 2.7 19.2 130.7 45.3 171.3 28.0 HH1079 83 38.5 2.2 10.7 10.8 11.4 9.2 249.3 74.3 8.7 HH1068 55 149.23 214.47 26.67 26.94 20.53 40.33 39.93 86.66 570.49 19.68 8.31 5.69 HH1069 61 23.9 124.9 1.9 2.0 6.0 21.9 20.4 142.5 11.8 HH1071 31.3 14.2 13.2 1.1 80.5 100.7 HH1072 90 59.59 14.35 14.89 4.46 39.97 6.50 29.54 180.97 16.58 12.13 2.22 HH1076 34 82 2.1 63.78 234.87 9.19 4.31 2.72 19.43 15.73 17.8 209.11 5.42 4.41 0.51 HH1078 174.13 4.98 12.75 1.19 1.07 21.80 10.05 162.76 67.20 51.80 7.70 HH1083 7.4 11.6 58.8 10.6 15.1 14.7 10.4 230.7 15.2 191.3 9.8 HH1084 52.8 69.5 22.0 16.4 47.5 13.7 25.9 626.7 32.2 214.3 HH1089 15.5 2.5 11.3 13.5 280.0 HH1085 77 4.5 63.2 45.5 12.9 107.1 11.9 31.6 39.5 53.6 540.7 110.0 585.7 HH1086 28.5 77.2 8.0 19.8 29.9 400.7 95.1 204.3 HH1093 62 53.5 8.9 20.7 350.7 56.4 137.7 HH1094 38.8 49.2 18.8 69.4 24.7 15.4 390.0 63.5 160.0 24.3

15 Beautiful Plateable IVAL Cryopreserved Human Hepatocytes

16 Novel IVAL Product: Plateable Pooled Donor Cryopreserved Human Hepatocytes PHH8015A (10 donor pool; 5 male/5 female) Day 1 Day 7 IVAL Pool Cryopreserved Hepatocyte Patent Allowed: U. S., China Patent Pending: European Union

17 MetMax™ Hepatocytes

18 Suspension Cryopreserved Human Hepatocytes

19 Cryopreserved Human Hepatocytes in Drug Develpment
Suspension: Easier to use than plateable cryopreserved hepatocytes Major application: drug metabolism (metabolic stability screening) Drug-drug interactions (P450 inhibition but mainly done with HLM) Uptake transport (now mainly done with plateable hepatocytes)

20 Application of Human Hepatocytes in Drug Metabolism
Advantages (over HLM, S9) Complete drug metabolizing enzyme activities Microsomes lack cytosolic enzymes S9 lacks mitochondrial, lysosomal, plasma membranal enzymes

21 Application of Human Hepatocytes in Drug Metabolism
Disadvantages (over HLM, S9) Laborious Storage in LN2 Centrifugation and microscopic evaluation of cell viability and cell concentration Sensitive to experimental manipulation (not robot friendly) Metabolism may be hindered by drug toxicity (high drug concentration cannot be used for metabolite profiling)

22 MetMax™ Human Hepatocytes
A Novel IVAL Product to Retain the Advantages and to Eliminate the Disadvantages of Cryopreserved Suspension-Cultured Human Hepatocyte

23 MetMax™ Hepatocytes Permeabilized, cofactor supplemented hepatocytes
An experimental system with the advantages of hepatocytes and the ease of operation and robustness of cell free systems

24 MetMax™ Hepatocytes: Permeabilized Hepatocytes
Intact Hepatocyte Hepatocyte In Vivo MetMax™ Hepatocyte

25 Organelle Composition of Key In Vitro Drug Metabolism Experimental Systems
Endoplasmic reticulum: P450, UGT etc. (S9; HLM; Hepatocytes) Cytosol: SULT, NAT etc. (S9; Hepatocytes) Mitochondria: MAO (Hepatocytes)

26 Organelle Composition of Key In Vitro Drug Metabolism Experimental Systems
Plasma membrane: Hepatocytes ATPases, anion transport protein,glyceraldehyde 3-phosphate dehydrogenase,protein kinases, adenylate cyclase,acetylcholinesterase.  Lysosome: Hepatocytes glycosidases, proteases, sulfatases Nucleus: Hepatocytes Nuclear shield enzymes - glutathione transferase, catalase and glutathione peroxidase; up to seven times higher than in the cytosol.

27 MetMax™ Hepatocytes: Retention of All Organelles
Intact Hepatocytes Microsomes S9 Endoplasmic Reticulum Cytosol Mitochondria Lysosomes Golgi Plasma Membranes Nucleus

28 Ease of use of MetMax™ Hepatocytes
Organelles MetMax™ Intact Hepatocytes Microsomes S9 Storage -80 deg. C LN2 Centrifugation No Yes Microscopic Examination Cell Counting Cofactor Addition Thaw and Use

29 MetMax™ Hepatocytes A Thaw and Use Reagent
Cryopreserved Hepatocytes Retrieve from LN2 freezer Thaw in a 37 deg. water bath Add to recovery medium Centrifuge Microscopic quantification of viability and cell number Adjust to 2X final cell density Add at equal volume to 2X test article Incubate Freezer to Incubation: >30 minutes MetMax™ Hepatocytes Retrieve from -80 deg. freezer Thaw in a 37 deg. water bath Add at equal volume to 2X test article Incubate Freezer to Incubation: <5 minutes

30 MetMax™ Human Hepatocytes
Advantages over human liver S9 and microsomes: Complete drug metabolism enzyme pathways Advantages over intact human hepatocytes for application in drug metabolism studies: Ease of use; robustness; maximized enzyme activities

31 Comparison of MetMax™ and Intact Human Hepatocytes in Drug Metabolizing Enzyme Activities

32 Evaluation of 16 Drug Metabolizing Enzyme Substrates
Metabolic Pathway Substrate Substrate Conc. (µM) Marker Metabolite CYP1A2 Phenacetin 100 Acetaminophen CYP2A6 Coumarin 50 7-HC, 7-HC-Sulfate, 7-HC-Glucuronide CYP2B6 Buproprion 500 Hydroxybuproprion CYP2C8 Paclitaxel (Taxol) 20 6α-hydroxypaclitaxel CYP2C9 Diclofenac 25 4-OH Diclofenac CYP2C19 S-Mephenytoin 250 4-OH S-Mephenytoin CYP2D6 Dextromethorphan 15 Dextrophan CYP2E1 Chlorzoxazone 6-OH Chlorzoxazone CYP3A4-1 Midazolam 1-Hydroxymidazolam CYP3A4-2 Testosterone 200 6β-hydroxytestosterone ECOD 7-Ethoxycoumarin SULT 7-Hydroxycoumarin 7-Hydroxycoumarin Sulfate UGT 7-Hydroxycoumarin Glucuronide FMO Benzydamine HCl Benzydamine-N-Oxide MAO Kynuramine HBr 160 4-hydroxyquinoline AO Carbazeran 10 4-Hydroxycarbazeran

33 Comparison of 16 Drug Metabolizing Enzyme-Selective Substrates Intact (PHH; blue) Vs MetMax™ (PHHX; red) Hepatocytes:

34 Comparison of Intact and MetMax™ Human Hepatocytes in DME Activities
MetMax™ human hepatocytes were similar or higher than intact human hepatocytes in the metabolism of 16 pathway-selective DME pathways Results suggest that MetMax™ human hepatocytes can be used for drug metabolism studies performed routinely with intact human hepatocytes

35 Enterocytes

36 Why Enterocytes Key cell type for oral bioavailability
First pass metabolism before the liver Intestinal DDI with orally co-administered substances (foods; nutrient supplements; drugs) Intestinal DDI may not occur in the liver due to lower hepatic exposure (e.g. grapefruit juice)

37

38 As of now, primary enterocytes are not commercially available for drug metabolism evaluation
Current commercially available enterocytes are cultured for multiple passages with little information on drug metabolizing enzyme activities

39 Isolation and cryopreservation of enterocytes from human small intestines

40 Hepatocytes Vs. Enterocytes: Cellular Protein Contents
Human Hepatocytes (1.45 mg/million cells)

41 Cryopreservation of Human Enterocytes at IVAL
Successful isolation and cryopreservation of enterocytes with high viability (>75%) and reproducible yield (1-3 million cells per vial)

42 Drug Metab Dispos 45:686–691, June 2017
Human Enterocytes as an In Vitro Model for the Evaluation of Intestinal Drug Metabolism: Characterization of Drug-Metabolizing Enzyme Activities of Cryopreserved Human Enterocytes from Twenty-Four Donors

43 DME Activities of Human Enterocytes
Lot Number Gender Ethnicity Age CYP2C9 CYP2C19 CYP3A4 UGT SULT 2J2 CES2 HE3005 Male C 23 1.68 0.56 2.7 8.38 8.72 1.20 0.23 HE3006 Female 44 0.59 0.29 0.13 2.30 2.04 0.73 0.33 HE3007 H 43 0.91 0.39 0.99 3.08 4.04 0.57 0.46 HE3008 18 0.68 0.87 1.80 1.79 0.55 HE3009 1.18 0.35 0.72 4.32 7.78 0.25 0.60 HE3010 47 1.21 0.62 2.56 3.32 0.41 HE3011 50 0.03 0.01 0.09 1.01 1.70 HE3013 AA 57 NA 0.2 HE3014 49 0.44 0.11 0.4 3.55 2.66 0.17 HE3015 24 2.50 0.49 2.55 7.33 5.23 0.95 0.51 HE3016 32 2.05 1.08 1.0 5.71 4.13 0.93 0.34 HE3019 61 0.24 0.30 1.47 1.89 0.26 HE3020 25 0.31 0.14 0.5 5.83 1.84 0.58 HE3021 60 0.20 0.06 1.49 1.64 0.19 0.08 HE3027 53 2.02 0.7 3.68 2.69 0.76 HE3028 34 0.21 0.82 3.84 9.02 0.71 HE3029 41 0.86 0.12 0.6 6.55 3.65 HE3031 0.16 1.60 0.79 0.18

44 New Enterocyte Development
Preparation and characterization of pooled multiple-donor cryopreserved enterocytes Preparation of MetMax™ Cryopreserved Pooled Human Enterocytes (patent pending) Permeabilized enterocytes supplemented with cofactors Easy to use: Thaw and use – no centrifugation, no cell counting Easy to store: -80 deg. Freezer (Liquid nitrogen not needed) High activity

45 Donors Used for Pooling 10 donors (5 female; 5 male) 20 million cells per donor
Lot No. Gender Race Age (Years) HE3031 F C 49 HE3032 48 HE3006 44 HE3027 53 HE3011 50 HE3021 M AA 60 HE3019 61 HE3028 34 HE3033 H 32 HE3010 47

46 Drug Metabolizing Enzyme Activities of Pooled Cryopreserved Human Enterocytes

47 Enhanced DME Activities of MetMax™ Pooled Human Enterocytes
Metabolite Activity (pmole/106/min) Metabolic Pathway Substrate Marker Metabolite Pooled Enterocytes MetMax Pooled Enterocytes Ratio CYP2C9 Diclofenac 4-OH Diclofenac 4.05 ± 0.16 5.78 ± 1.13 142% CYP2C19 S-Mephenytoin 4-OH S-Mephenytoin 0.55 ± 0.03 3.36 ± 0.32 610% CYP3A4-1 Midazolam 1-OH-midazolam 1.21 ± 0.03 4.23 ± 1.22 349% CYP3A4-2 Testosterone 6βOH-testosterone 10.6 ± 3.3 147 ± 14.5 1386% UGT 7-OH-Coumarin 7-Hydroxycoumarin Glucuronide 16.05 ± 0.32 275 ± 79.5 1713% SULT 7-Hydroxycoumarin Sulfate 7.24 ± 0.34 13 ± 0.69 179% 2J2 Astemizole O-Demethyl Astemizole 0.92 ± 0.43 5.14 ± 1.53 558% CES2 Irinotecan SN38 0.37 ± 0.14 0.38 ± 0.27 102%

48 MetMax™ Pooled Donor Human Enterocytes
MetMax™ human enterocytes were prepared from Pooled Donor Human Enterocytes and evaluated for drug metabolizing activities for multiple pathways MetMax™ enterocytes were equal or more active than Pooled Donor Human Enterocytes in all pathways evaluated

49 MetMax™ Hepatocytes and Enterocytes
MetMax™ hepatocytes and enterocytes represent robust, convenience experimental system for the evaluation of hepatic and enteric drug metabolism, respectively Metabolic clearance Metabolite profiling Prototoxicant activation Drug-drug interactions

50 MetMax™ Hepatocytes and Enterocytes (patent pending)
Superior in vitro system for the evaluation of human hepatic (hepatocytes) and enteric (enterocytes) metabolism with the complete drug metabolizing enzymes of intact cells and the efficiency of cell-free systems 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 Robust: Compatible with automated HTS assays* Applications include Metabolic stability Metabolite profiling and identification: can use cytotoxic concentrations* Enzyme inhibition Metabolic activation of pro-toxicants and pro-mutagens

51 Contact Information Albert P. Li, Ph. D., President and CEO: George Amaral, U. S. Sales and Marketing: Bez Emadi, European Sales and Marketing: Deepak Barot, Indian Sales and Marketing: Nozomi Mizuno, Japanese Sales and Marketing:

52 Thank you for your attention


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