Alternative Hepatocyte Models for In Vitro ADME/Tox Study and a New Hepatocyte Cell Line

35 Pages·2015·2.85 MB·English

Alternative Hepatocyte Models for In Vitro
ADME/Tox Study and a New Hepatocyte
Cell Line – Corning® HepatoCells
Rongjun Zuo, Ph.D.
Senior Scientist
Corning Life Sciences
May 12, 2015
Presentation Outline
• Hepatocyte models
– Primary human hepatocytes
– Renewable hepatocyte sources
– Novel hepatocyte culture technology
• Corning® HepatoCells and characterization
• Summary
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Current Models for Drug ADME Studies
• Easy to use; amenable to • Easy to use; amenable to • Allow better cell-cell & • In vivo architecture • Most suitable model to
high throughput platform high throughput platform cell-matrix interactions reserved study different organ-
• Express phase I & II • Used for toxicity & DDI • Represent the in vivo-like • In vivo-like expression of interaction
enzymes studies conditions in terms of cell drug metabolizing • In vivo studies are a
• Used for clearance, • Co-culture with other cells function & morphology, enzymes, transporters, and requirement for drug
inhibition, covalent binding • Able to be cryopreserved nutrition, oxygenation, functional bile canaliculi approval
• Pool of large number of • Available for multiple configuration. • Zone specific metabolism • Have inter-species
donors species • Enabling better prediction and toxicity may be studied differences
• Can be recovered from • De-differentiate in culture of drug toxicity • Hepatic function reserved • High cost
frozen tissue • Lack representation of • Maintain long term culture; for <24 hrs. • Present ethical issues
• Low cost intact liver structure good for chronic drug • Complicated to use
• Available for multiple treatment • Not a high throughput
species • Cell line: lower cost, • Low to medium throughput system
• Need co-factors renewable source. • Some 3D formats difficult • Difficult to obtain human
• Lack a set of liver functions • Need to be evaluated for for imaging tissue
• Limited assay time target applications
EODMT 2014 Costa. e al
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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
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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
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Renewable Hepatocyte Sources
Human Hepatoma Cell Line
• HepG2
– Widely used for toxicity screening
– Low metabolic capability
Arch Toxicol
(2012) Lin, et al.
• 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
Cell BiolToxicol(2012) Gerets, et al.
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Renewable Hepatocyte Source
Hepatocyte-like Cells Differentiated from Human ESC/iPSC
Differentiation driven by viral transfection of transcription factors, recombinant
growth factors, or small molecules (or transdifferentiation from fibroblast cells)
Protocol
Characterization of Differentiation Efficiency
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.
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Hepatocyte-like Cells Differentiated from hESC/iPSC
Expression of Hepatocyte-specific Genes/Proteins
Efficiency of Phase III Differentiation
Stem Cell Reports (2015) Siller R, et al.
• High expression level of fetal genes,
e.g., AFP.
• Low expression of adult genes, i.e.,
CYP3A4.
PlosOne (2015) Ishikawa, T., et al.
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Hepatocyte-like Cells Derived from hESC/iPSC
Functional Characterization for Drug Metabolism
Metabolism capability, drug inducibility, and transporter activity
Black: hESC
pluripotent
control
Blue: basal
activity
Orange:
induced activity
with either
omeprazole
(1A2) or
rifampicin (3A4)
Cell Stem Cell (2014)
Huang, et al.
Stem Cell Report (2015) Siller et al
• 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
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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
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