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)

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