ADME/Tox Study and a New Hepatocyte Cell Line – Corning ......• Minimal drug inducibility, i.e.,

Alternative Hepatocyte Models for In Vitro ADME/Tox Study and a New Hepatocyte Cell Line – Corning® HepatoCells

Rongjun Zuo, Ph.D.Senior ScientistCorning Life Sciences

May 12, 2015

2Life Sciences © 2015 Corning Incorporated

Presentation Outline

• Hepatocyte models – Primary human hepatocytes– Renewable hepatocyte sources– Novel hepatocyte culture technology

• Corning® HepatoCells and characterization• Summary


Current Models for Drug ADME Studies

EODMT 2014 Costa. e al

• Easy to use; amenable to high throughput platform

• Used for toxicity & DDI studies

• Co-culture with other cells• Able to be cryopreserved• Available for multiple

species• De-differentiate in culture• Lack representation of

intact liver structure

• Cell line: lower cost, renewable source.

• Need to be evaluated for target applications

• Easy to use; amenable to high throughput platform

• Express phase I & II enzymes

• Used for clearance, inhibition, covalent binding

• Pool of large number of donors

• Can be recovered from frozen tissue

• Low cost• Available for multiple

species• Need co-factors• Lack a set of liver functions• Limited assay time

• Allow better cell-cell & cell-matrix interactions

• Represent the in vivo-like conditions in terms of cell function & morphology, nutrition, oxygenation, configuration.

• Enabling better prediction of drug toxicity

• Maintain long term culture; good for chronic drug treatment

• Low to medium throughput• Some 3D formats difficult

for imaging

• In vivo architecture reserved

• In vivo-like expression of drug metabolizing enzymes, transporters, and functional bile canaliculi

• Zone specific metabolism and toxicity may be studied

• Hepatic function reserved for <24 hrs.

• Complicated to use• Not a high throughput

system• Difficult to obtain human

tissue

• Most suitable model to study different organ-interaction

• In vivo studies are a requirement for drug approval

• Have inter-species differences

• High cost• Present ethical issues


Primary Human Hepatocytes

• Primary human hepatocytes are considered the gold standard model for in vitro drug metabolism/toxicity studies

• Contain all the hepatic enzymes, co-factors (NADPH, UDPGA, GSH, PAPS, etc.), transporter proteins needed for drug metabolism studies and hepatotoxicity studies.

• Eliminate species difference for IVIVE compared to animal hepatocytes. • Contain full machinery for enzyme regulation, important for CYP induction

and inhibition study.• Can form in vivo-like hepatobiliary network for drug transport study


Need for Renewable Hepatocyte Sources• Inherent limitations of primary human hepatocytes

– Large lot-to-lot variability – Need to qualify every lot (time consuming)– Limited supply of high quality lots/limited lot sizes– Tendency to de-differentiate in culture– High cost

• Increasing need for renewable hepatocytes sources– Human hepatoma cell lines

• HepG2• HepaRG

– Stem cell or induced pluripotent stem cells derived hepatocyte-like cells– Immortalized hepatocyte-like cell lines

• Corning® HepatoCells


Renewable Hepatocyte SourcesHuman Hepatoma Cell Line

• HepG2– Widely used for toxicity screening– Low metabolic capability

• HepaRG– Pros:

• Possessing most hepatocyte-specific functions, compared to other hepatoma cell lines

• Metabolism competent• Show comparable sensitivity to a group of

hepatotoxic compounds similar to primary human hepatocytes

– Cons:• Mixture of 2 cell types; difficult to control the ratio• Large lot-to-lot variation observed• Need high concentration of DMSO to maintain

differentiated status• Relatively low expression of some uptake

transporters

Arch Toxicol(2012) Lin, et al.

Cell Biol Toxicol (2012) Gerets, et al.


Renewable Hepatocyte SourceHepatocyte-like Cells Differentiated from Human ESC/iPSC

Protocol

Gene & Protein Expression

Cellular Morphology

Functional Characterization: Albumin secretion, urea synthesis, glycogen storage, indocyanine green uptake, low density lipoprotein uptake, gene expression of hepatocyte specific genes, drug metabolism, etc.

Differentiation driven by viral transfection of transcription factors, recombinant growth factors, or small molecules (or transdifferentiation from fibroblast cells)

Characterization of Differentiation Efficiency


Hepatocyte-like Cells Differentiated from hESC/iPSCExpression of Hepatocyte-specific Genes/Proteins

Efficiency of Phase III Differentiation

• High expression level of fetal genes, e.g., AFP.

• Low expression of adult genes, i.e., CYP3A4.

Stem Cell Reports (2015) Siller R, et al.

Plos One (2015) Ishikawa, T., et al.


Hepatocyte-like Cells Derived from hESC/iPSCFunctional Characterization for Drug Metabolism

Metabolism capability, drug inducibility, and transporter activity

• Significant metabolism activity compared to pluripotent cell control • Minimal drug inducibility, i.e., <3 fold for CYP3A4. Not suitable for induction assay• Transporter activity shown in the lower range of primary human hepatocytes

Black: hESCpluripotent control

Blue: basal activity

Orange: induced activity with either omeprazole (1A2) or rifampicin (3A4)

Stem Cell Report (2015) Siller et al

Cell Stem Cell (2014) Huang, et al.


Considerations in Using hESC/iPSC Derived Hepatocytes• Majority of the improved differentiation protocols can achieve significant expression of

hepatocyte-specific genes/proteins compared to their parental cells; however the differentiated cells are far different from mature hepatocytes in terms of important drug metabolism relevant functions

– Need better understanding of development of an adult phenotype, the mechanisms of cell reprogramming, and the role of the tissue culture microenvironment.

• Although much higher differentiation efficiency obtained with improved protocols (e.g., using small molecules), technical challenges still remain

– Scalability, fully defined conditions, cost, and reproducibility • In order to validate hepatocytes derived from hESC/iPSC for drug metabolism application, it

is critical to use:– The right assays, e.g., LCMS or HPLC (vs. P450-Glo™) for metabolism capacity– The right reference control cells, e.g., primary adult hepatocytes (vs. cell line such as

HepG2) with culture format appropriate for target assays


Novel Hepatocyte Culture Technology- 3D Hepatocyte Culture

• Primary hepatocytes tend to dedifferentiate in 2D in vitro culture, which results in limited life span, greatly reduced hepatic specific functions, such as metabolic activity and transporter activity.

• There is an increasing need for a more in vivo-like hepatocyte culture format for better in vitro to in vivo prediction of drug ADME features.

• 3D hepatocyte culture can maintain hepatocyte polarization and functionality better than conventional 2D model due to a more in vivo-like environment created by optimal cell-ECM and cell-cell contacts

Altered cytoskeleton structure

Reduced cell-cell & cell-ECM contact

Reduced polarization & signal pathways

De-differentiation over culture time

Deterioration of function


Static 3D Cultures - Examples

Adopted from Exp Biol Med (2014) Bale et al

RegeneMed InSphere HepreGen• Co-culture of non-parenchymal cells

and hepatocytes on nylon scaffold Transwell® seeded at near physiological ratio.

• Co-culture induces ECM/GF production to form tissue structure that supports long-term function.

• Maintain up to 3 months hepatic functions of both rat & human hepatocytes, including, albumin, transferrin, fibrinogen secretion, and urea synthesis; maintain stable CYP3A4, 1A1 and 2C9 activity.

• Demonstrated inflammatory response of the liver tissue upon exposure to LPS and release of pro-inflammatory cytokines measured.

• Human 3D liver tissue has been used to test drug toxicity.

• Expensive

• Spheroids are formed using hanging drop technology with hepatocytes and non-parenchymal cells introduced into the drop, transferred into a 96-well microplate and cultured.

• Maintain stable function for up to 5 weeks. • Cellular phenotype of endothelial and

Kupffer cells are maintained within the spheroids.

• Better acetaminophen and diclofenac TC50values in toxicity assays when compared with 2D cultures

• High reproducibility in spheroid size, dimension and density.

• Forming spheroids is time consuming without robot

• Instrument sensitivity may be an issue with analysis of small sample amounts.

• Micro-patterned plate with hepatocytes surrounded by stromal cells (3T3-J2 mouse fibroblast cells).

• Maintain stable function for up to 6 weeks, including albumin secretion, urea synthesis, Phase I and II drug metabolism, and formation of bile canaliculi.


3D Perfusion Cultures - Examples

CellAsic Zyoxel Hurel• A pseudo-3D culture in microfluidic plate• Micro-structure pattern mimics sinusoidal

barrier function of liver, shielding the hepatocytes from shear stress of medium.

• Nutrient exchange achieved with media flow.

• Showed high cell viability up to 7 days and response to drug.

• In vivo structure not maintained.• Can not control flow rate; need extensive

training on cell loading.• Need to establish protein synthesis and

drug metabolism.• Expensive system

• Consists of two reservoirs with one for cells and the other for media for recirculation

• Perfusion using pump to provide nutrient exchange; oxygen concentration similar to a sinusoid.

• Maintained high viability and phenotype for up to 13 days in co-culture of hepatocytes and NPC.

• Not amenable to imaging due to presence of scaffold .

• Further characterization are needed on NPCs since they are liver sinusoidal endothelial cell-enriched fraction.

• Cell Culture Analog (CCA) with cell seeding area and multiple devices in parallel

• Able to achieve high-density seeding of hepatocytes and maintain viability and metabolic functions under flow condition for 24 hours in initial short duration studies.

• Co-culture of hepatocyte with non-parenchymal cells shows high-viability and in vivo-like clearance for various drugs up to 8 days.

Additional studies to evaluate important liver functions (albumin secretion, urea excretion, drug toxicity) are needed to realize their full potential.


Corning Solutions to Provide Renewable HepatocytesCorning Solution…..• Develop a “renewable” hepatocyte-like product with hepatocyte morphology and functions

similar to primary human hepatocytes Consistent lot-to-lot performance (eliminates need to pre-qualify lots) Large lot sizes for long-term comparative studies (500 to 1,000 vials) Easy to use format: “Thaw and Go” reagent

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• Fresh plated human hepatocytes, with or without Corning® Matrigel®matrix overlay

• Induction-qualified cryopreserved human hepatocytes

• Metabolism-qualified cryopreserved human hepatocytes

• Transporter-qualified cryopreserved human hepatocytes

• Metabolism-qualified cryopreserved human kepatocyte suspension

• Transporter-qualified Cryopreserved human hepatocyte suspension

• Cryopreserved animal hepatocyte suspension (rat, mouse, dog, monkey)

Corning Hepatocyte Portfolio to Support ADME Studies

Suspension Hepatocytes Plated Hepatocytes

Corning HepatoCellsA Renewable Hepatocyte Product

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• Mature hepatocyte-like morphology; homogeneous population • Wild type genotype for important Cytochrome P450s

− CYP2D6, CYP2C9, CYP2C19

• >8 million cells per vial

• >80% post thaw viability

• No percoll density gradient required

Product Feature

• Robust CYP3A4 induction (>10 fold), as well as CYP1A2 and 2B6 induction

• Better than current alternative models and some lots of PHH

• Consistency− Large lot size (500 to 1,000 vials) helps to ensure low lot-to-lot

variation, and no back orders

• Convenience/easy to use− No special additives required for differentiation− One medium for all phases of culturing (plating, maintenance,

induction)


Mature Hepatocyte-Like Morphology

100% confluence Pure population Typical cuboidal

morphology Double/Multi

Nucleation Distinct nucleoli Distinct cell‐cell

contact Bile canaliculi


Application: CYP Induction

• Regulatory agencies recommend to use primary human hepatocytes in in vitro test for CYP3A4, 1A2, and 2B6 induction.

• Any alternative hepatic cell models are expected – To possess all 3 induction regulation pathways – To respond to prototypical inducers in a similar pattern to primary human

hepatocytes– To perform consistently to save time and cost on screening

Corning® HepatoCells are evaluated against primary human hepatocytes for their performance in induction applications.

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EYFP-hCAR Expression and LocalizationData courtesy from Prof. Hongbing Wang, University of Maryland

Phenobarbital-responsive CAR Nuclear Translocation

Corning HepatoCellsNon-treated

PB treated

Human Hepatocytes

PB treated

Non treatedNo nucleus expression

Translocated to nucleus after PB treatment

No nucleus expression

Translocated to nucleus after PB treatment

CAR translocation upon PB treatment in Corning HepatoCells was similar to primary hepatocytes; not seen in many cell lines.


Induction Assay in 96-well Microplate

Comparable CYP induction response to primary human hepatocytes with much smaller lot-to-lot variability.

All data are mean of several lots for each cell type.

18.5(n=15)

28.7(n=15)

7.8(n=15)

19.6(n=6)

32.4(n=3)

5.0(n=3)

0

10

20

30

40

50

CYP3A4 CYP1A2 CYP2B6

Fold In

duction

Primary humanhepatocytes

Corning HepatoCells


FDA and EMA recommend using PHH to evaluate induction potential• To predict in vivo induction potential:

− Primary hepatocytes need to be previously characterized with a sufficient number of known clinical inducers and non-inducers to determine a threshold.

Corning® HepatoCells were evaluated − As an in vitro screening tool for predicting clinical CYP3A4

• 3 batches of HepatoCells• 13 known clinical CYP3A4 inducers• Enzymatic activity and mRNA expression

− Using Relative Induction Score (RIS) approach

− The prediction was compared with that of primary human hepatocytes

Application: Induction Prediction with the RIS Approach


R2 = 0.99

Phenytoin, M0.1 1 10 100 1000

Fold

Indu

ctio

n

0

2

4

6

8

10

12

R2 = 0.95

Rifampicin, M

0.1 1 10 100

Fold

Indu

ctio

n

5

10

15

20

R2 = 0.96

Carbamazepine, M0.1 1 10 100

Fold

Indu

ctio

n

0

1

2

3

4

5

6

Examples of Concentration-dependent Induction Response Curve

Concentration‐dependent induction response curves were generated for 13 compounds All curves showed good correlation with R2 >0.9 3 pilot lots showed similar concentration‐dependent response curve

Probenecid, M

0.01 0.1 1 10 100

CYP

3A4

Fold

0

5

10

15

20

25

30

35

R2 = 0.98

EC50 = 42 MEmax = 31

Probenecid, M0.01 0.1 1 10 100

CYP

3A4

Fold

0

10

20

30

40

R2 = 0.9

EC50 = 51 M Emax = 39

Probenecid, M

0.01 0.1 1 10 100

CYP

3A4

Fold

0

10

20

30

40

R2 = 0.98

EC50 = 44 MEmax = 36

PR Lot 2 PR Lot 3A PR Lot 3B


RIS Comparison between Corning® HepatoCells and Primary Hepatocytes

Similar RIS data obtained from 3 prototype lots of Corning HepatoCells. HepatoCells showed similar RIS ranking as primary human hepatocytes. HepatoCells showed much smaller variation than primary human hepatocytes

(average % CV is 18.2% for HepatoCells, and 53.3% for primary human hepatocytes).

Compounds Observed AUC change

RIS from Corning HepatoCells RIS from Primary Human Hepatocyte Average RIS %CV

Lot PR2 Lot PR3A Lot PR3B Lot 295 Lot 312 Lot 318 HepatoCells PHH HepatoCells PHH

Dexamethasone 19 0.0042 0.0053 0.0053 0.001 0.001 0.0004 0.0050 0.0008 12.7% 43.3%

Terbinafine* 25 0.047 0.059 0.040 0.027 0.024 0.007 0.0485 0.0193 20.1% 55.8%

Nifedipine* 4 0.048 0.052 0.045 0.019 0.007 0.011 0.0484 0.0123 7.4% 49.5%

Pleconaril* 35 0.087 0.095 0.101 0.025 0.029 0.011 0.0944 0.0217 7.5% 43.6%

Omeprazole ‐25 0.189 0.139 0.154 0.008 0.019 0.019 0.161 0.0153 16.0% 41.4%

Pioglitazone* 26 0.190 0.199 0.178 0.012 0.011 0.025 0.189 0.0160 5.5% 48.8%

Troglitazone* 65 0.513 0.355 0.650 0.12 0.2 0.03 0.506 0.1167 29.2% 72.9%

Phenobarbitol 61 0.902 0.729 1.158 0.88 2.4 1.6 0.930 1.63 23.2% 46.7%

Carbamazepine* 94 1.25 1.074 0.723 1.1 1.1 9.3 1.02 3.83 26.5% 123.5%

Phenytoin* 94 1.50 1.381 1.908 1 1.3 1.8 1.60 1.37 17.3% 29.6%

Rifampicin* 97 10.93 8.175 16.201 7 11 6.4 11.77 8.13 34.7% 30.7%


Prediction of In Vivo DDI: RIS Model

RIS values (generated using EC50 and Emax data from a group of clinical inducers and non‐inducers) correlated well with observed AUC decrease.

All 3 pilot lots generated similar RIS cut‐off value at 20% AUC decrease, suggesting no calibration necessary for each individual lot of Corning® HepatoCells.

CorningHepatoCells

Primary Hepatocytes

PR2 0.116 Lot 295 0.21PR3 0.135 Lot 312 0.054PR4 0.121 Lot 318 0.23Mean 0.12 Mean 0.16%CV 7.5% %CV 58.5%

RIS cut-off at 20% AUC decrease


Prediction Accuracy: Predicted AUC Change vs. Observed AUC Change

Comparable prediction accuracy between Corning® HepatoCells and primary human hepatocytes (all within 20% of observed value)

R2 = 0.96R2 = 0.91


Application: Transporter Characterization

• Drug transporters play an important role in drug-induced hepatotoxicity and adverse drug-drug interactions.

• Many hepatic cell lines are found to express low level of uptake transporters.

Corning® HepatoCells were evaluated for transport of prototypical substrates of important hepatic uptake transporters.


Uptake Transporter Study: Time-dependent and Concentration-dependent Uptake of OCT1 Substrates

Time, min 5 10 20 30

Substrate Lot Uptake Ratio (37C/4°C)

MPP+PR2 12.5 12.5 12.0 11.3PR3A 11.6 11.0 14.1 10.7

TEAPR2 2.3 3.3 2.0 5.2PR3A 1.8 2.3 3.7 2.7

MetforminPR2 2.4 6.0 3.8 3.4PR3A 3.2 2.5 3.1 2.7

0

5

10

15

20

25

30

35

0 10 20 30

MPP

+ uptake (pmol/m

g)

Incubation Time, min

37C_PR2 4C_PR237C_PR3A 4C_PR3A

Time-dependent MPP+ Uptake

[MPP+], M

0 200 400 600 800 1000 1200

Upt

ake

activ

ity, p

mol

/min

/mg

0

50

100

150

200

250

300

Vmax = 266 pmol/min/mgKm = 75 M

Concentration‐dependent MPP+ UptakeTransporter Substrate

Km, µM

CorningHepatoCells

1CorningTransportoCells™

2Human Hepatocytes

3Human Hepatocytes

OCT1MPP+ 75 n.a 101 N/A

TEA 1940 713 N/A 407

Reference data: 1,3 In house data (3suspension hepatocytes); 2Chem Biol Interact. 190:165-70, 2011.

Corning® HepatoCells demonstrated time- and concentration-dependent uptake of 3 prototypic substrates of hepatic transporter OCT1 with good signal to noise ratio

Km values align with literature data.


Uptake Transporter Study: Inhibition of Uptake of OCT1 Substrates

1Zhang L. et al. JPET 51:913-921, 1997 2Zhang L. et al. JPET 286:354-61, 1998

Test system Substrate IC50

Corning® HepatoCells MPP+ 0.27+/-0.046Corning TransportoCells™ MPP+ 2.2

X. laevis oocyte1 MPP+ 4.7HeLa cell line2 TEA 2.7

Inhibition of MPP+ Uptake by Decynium‐22 Lot # IC50, µM

Lot 1 0.26Lot 2 0.34Lot 3 0.22Lot 4 0.24Lot 5 0.27Mean 0.27%CV 17%

Five different lots of Corning HepatoCells showed similar Decynium-22 inhibition of MPP+ uptake with small lot-to-lot variation.

The IC50 values from Corning HepatoCells are comparable with literature values from other cell systems.

IC50 from 5 lots


Uptake Transporter Study: Concentration-dependent Uptake of OATP1B1/1B3 and NTCP Substrates

Corning® HepatoCells demonstrated concentration-dependent uptake of Rosuvastatin and TCA.

Km values are comparable to literature data.

High signal-to-noise ratio suggests active expression of functional uptake transporter proteins.

Transporter Substrate Km, M Test system ReferenceNTCP Rosuvastatin 6.5 HeLa

1OATP1B1 Rosuvastatin 7.3 HeLaOATP1B3 Rosuvastatin 9.8 HeLaOATP1B1 Rosuvastatin 0.8 HEK 2OATP1B3 Rosuvastatin 14.2 HEKNTCP TCA 2.1 HEK 3NTCP TCA 7.5 HeLa 4

OATP1B3 TCA 5.8 Oocyte 5NTCP TCA 14 Corning TransportoCells™

6TCA 8.2 Human hepatocytes

Uptake Ratio

Substrate, µM Rosuvastatin TCA

2 8.8 9.910 7.1 8.620 3.6 6.0

[Rosuvastatin], M

0 5 10 15 20 25 30

Upta

ke a

ctiv

ity, p

mol

/min

/mg

0

10

20

30

40

Km = 6.7 MVmax = 36.5 pmol/min/mg

Rosuvastatin Uptake

[TCA], M0 5 10 15 20 25

Upta

ke a

ctiv

ity, p

mol

/min

/mg

0

2

4

6

8

10

12

14

16

18

20

Km = 8.5 MVmax = 23.6 pmol/min/mg

TCA Uptake

1Ho RH et al. Gastro. 130:1793-1806, 2006; 2Kitamura S. et al DMD 36:2014-23, 2008; 3Leslie EM et al. JPET 321:1170-8, 2007; 4Ho RH et al. JBC 279:7213-22, 2004; 5Abe T et al. Gastroenterology 120:1689-99, 2001; 6 Corning in-house data


Applications: Toxicity, Spheroid, and Others

• In vitro assessment of drug induced hepatotoxicity requires using metabolism-competent cell models.

• Primary hepatocytes are not amenable to chronic hepatotoxicity study due to short life span and tendency to de-differentiate in culture.

Corning® HepatoCells are evaluated for assessing metabolism-dependent toxicity, 3D spheroid formation, and other potential applications.

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Aflatoxin B1 Toxicity Test

Corning HepatoCells showed dose‐dependent response to Aflatoxin B1. Ketoconazole inhibited toxicity caused by Aflatoxin B1.

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5

Seed Corning® Matrigel® matrix overlay

Media change

24‐hr Aflatoxin B1 exposure, with or without Ketoconazole

Viability measurement

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Uniform-sized Spheroids from Corning® HepatoCells in 96-well Ultra-Low Attachment Surface Microplates

Potential applications include:• 3D modeling and co-culture with non-parenchymal cells, e.g. Kupffer cells• Extended culture while maintaining functionality• Adaptation of high content imaging assay

6.25K3.13K

1.56K0.78K


Other Potential Applications

• Current beta testing showed better or similar performance of Corning®

HepatoCells compared with primary human hepatocytes in the following applications

– Viral infection– Lipid metabolism


Summary Corning® HepatoCells demonstrated typical mature hepatocyte morphology. Fold induction for 3 CYPs are comparable to PHH. Much smaller lot-to-lot variation than PHH. HepatoCells predicted in vivo CYP3A4 inducers similarly as PHH. HepatoCells showed consistent RIS cut-off values among 3 pilot lots, suggesting a

1-time calibration may be enough, saving time and cost for lot qualification. HepatoCells actively express functional uptake transporters as shown by uptake

of prototypical substrates of OCT1, OATP1B1/1B3, and NTCP. HepatoCells showed dose-dependent response to metabolism-based toxic

compound which can be reversed by CYP450 inhibitor. HepatoCells can form 3D spheroids.

In conclusion, Corning HepatoCells are a very promising renewable hepatocyte model system for ADME/Tox research.


Future/Ongoing Studies

• Gene expression profiling

• CYP, FMO, and UGT (Phase 2) metabolism

• ABC transporters (efflux)

• Tox studies (DILI)

Documents

ADME/Tox Study and a New Hepatocyte Cell Line – Corning ......• Minimal drug inducibility, i.e.,