The ADMET Group LLC and In Vitro ADMET Laboratories LLC, 15235 Shady Grove Road,
Suite 303, Rockville, MD 20850, United States
Advanced Pharmaceutical Sciences Inc., 9221 Rumsey Road, Suite 8, Columbia, MD 21045, USA
Available online 9 January 2007
Abstract
The recent developments in the isolation, culturing, and cryopreservation of human hepatocytes, and the application of the cells in
drug development are reviewed. Recent advances include the improvement of cryopreservation procedures to allow cell attachment,
thereby extending the use of the cells to assays that requires prolong culturing such as enzyme induction studies. Applications
of human hepatocytes in drug development include the evaluation of metabolic stability, metabolite profiling and identification,
drugdrug interaction potential, and hepatotoxic potential. The use of intact human hepatocytes, because of the complete, undisrupted
metabolic pathways and cofactors, allows the development of data more relevant to humans in vivo than tissue fractions such as
human liver microsomes. Incorporation of key in vivo factors with the intact hepatocytes in vitro may help predictive human in vivo
drug properties. For instance, evaluation of drug metabolism and drugdrug interactions with intact human hepatocytes in 100%
human serum may eliminate the need to determine in vivo intracellular concentrations for the extrapolation of in vitro data to in vivo.
Co-culturing of hepatocytes and nonhepatic primary cells from other organs in the integrated discrete multiple organ co-culture
(IdMOC) may allow the evaluation of multiple organ interactions in drug metabolism and drug toxicity. In conclusion, human
hepatocytes represent a critical experimental model for drug development, allowing early evaluation of human drug properties to
guide the design and selection of drug candidates with a high probability of clinical success.
2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Human hepatocytes; Hepatocyte isolation; Hepatocyte cryopreservation; Drug development; Drug metabolism; Drugdrug interactions;
Drug toxicity; Toxicogenomics; IdMOC
1. Introduction
Primary cultures of hepatocytes represent an experimental tool that has been used extensively in biomedical
research, both in academia as well as for commercial
purposes such as drug development. The most exciting advances are the successful isolation, culturing,
and cryopreservation of hepatocytes from human livers.
Human hepatocytes are used routinely in drug develop-
0009-2797/$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2007.01.001
in drug development, with emphasis on the research performed in our laboratories in the past 20 years.
2. Human hepatocyte isolation
One of the major advances in human hepatocyte technology is the availability of human livers for research. In
the United States, livers procured but not used for transplantation are allowed to be used in research. The major
reasons that procured livers are not used for transplantation are as follows:
1.
2.
3.
4.
5.
6.
7.
8.
17
cells can be used in suspension for experiments requiring a relatively short time duration (hours), plated on
tissue culture surfaces pretreated with attachment substrates (e.g. collagen; Matrigel) for longer term studies,
or cryopreserved for future use.
The method of isolation of human hepatocytes from
a human liver is by no means optimized. Currently, a socalled good yield of human hepatocytes from a human
liver is approximately 1030 billion viable cells when a
whole liver is perfused. Using an approximation of 1.5 kg
as an average weight of a human liver, this leads to a yield
of or approximately 720 million hepatocytes per gram
of liver (e.g. [5,6]), which is considerably less that the
total number of hepatocytes (approximately 300 billion)
in the human liver. It is to be noted that the yield of
human hepatocytes (in terms of number of hepatocytes
per gram liver) is in general higher from smaller (e.g.,
10 to 300 g) liver fragments then whole livers or lobes.
3. Cryopreservation
18
Table 1
Post-thawed viability and attachment efficiency of human hepatocytes isolated and cryopreserved in APSciences Inc.
Date of preparation
Lot
Yield (cells/vial)
Plateability
Confluency (%)
8 September 2006
13 September 2006
14 September 2006
28 September 2006
3 October 2006
5 October 2006
8 October 2006
18 October 2006
30 October 2006
30 October 2006
HU4019/20/21
HU4017/18/22
HU4026
HU4027
HU4028
HU4023
HU4024/25/29
HU4030
HU4032
HU4034
5.4 106
5.5 106
5.9 106
5.9 106
3.2 106
2.1 106
8.5 106
3.5 106
4.8 106
5.8 106
89
91
91
92
83
89
88
88
99
97
Yes
Yes
No
No
No
No
Yes
Yes
Yes
Yes
70
80
<10
30-40
30-40
20
80
50
70
85
In September and October of 2006. Using proprietary procedures for cryopreservation and post-thaw recovery, the human hepatocytes were found to
have post-thaw viability values (based on trypan blue exclusion) of >80%, with over 50% of the lots found to be plateable monolayer hepatocyte
cultures (with >50% confluency at 24-h after plating).
Fig. 1. Photomicrograph of human hepatocytes cultured after cryopreservation. Historically, human hepatocytes in general would lose
their ability to attach for monolayer culturing. One rule of thumb is that
approximately 1 out of 1020 lots (each lot representing hepatocytes
from a single isolation) of cryopreserved human hepatocytes lots would
be plateable. In our laboratory, we have developed proprietory technologies with which approximately 50% of the cryopreserved human
hepatocytes would be plateable as defined by their ability to form
monolayer cultures with over 50% confluency (photograph obtained
from CellzDirect Inc.).
19
20
21
test article into the hepatocytes. Using cell free systems such as liver microsomes, one usually estimate
in vivo effect (e.g. by the ratio of plasma Cmax ([I])
to the inhibitory constant, Ki , with [I]/Ki of <0.1 considered as unlikely to yield DDI [25]) using plasma
concentration assuming that it is equal to the concentration of the inhibitor at the site of the inhibited enzyme.
This may not be correct if the inhibitor has a low permeability across the plasma membrane (leading to an
intracellular concentration lower than plasma concentration), or if the inhibitor is actively uptaken (leading
to an intracellular concentration higher than the plasma
concentration).
It was discussed earlier that intact hepatocytes
cultured in 100% human serum may provide physiologically relevant data for metabolic clearance studies.
The same approach has also been evaluated for
inhibitory DDI studies [28]. Lu et al. applied a novel
approach using cryopreserved human hepatocytes suspended in human plasma for the evaluation of P450
inhibitory effects. They proposed that this approach
would allow prediction of P450 inhibitory potential in
vivo without the need of estimating the inhibitor concentrations at the enzyme active site or the Ki . They
reported success in the estimation of the magnitude
of the clinical DDI of ketoconazole, an investigational compound MLX, and several marketed drugs
(theophylline, tolbutamide, omeprazole, desipramine,
midazolam, alprazolam, cyclosporine, and loratadine),
with a correlation coefficient (r2 ) of 0.992.
4.2.1.3. Enzyme induction
As described earlier, induction of drug metabolizing
enzyme is a second major mechanism of pharmacokinetic drugdrug interactions. P450 isoforms represent
the most important drug metabolizing enzymes for
enzyme induction studies.
Primary human hepatocytes have always been the
gold standard for enzyme induction studies. As of this
writing, the following statement apparently is true:
All known human P450 inducers in vivo are inducers
in human hepatocytes in vitro
The reverse of this statement, i.e., if all in vitro inducers are in vivo inducers, is not yet established.
It has been long established that primary human
hepatocytes are known to be responsive to prototypical P450 inducers such as omeprazole (for CYP1A2)
and rifampin (for CYP3A4) [29,30,49]. It is interesting that while other P450 isoforms such as CYP2A6,
CYP2B6, CYP2C8, CYP2C9, CYP2C19 and CYP2E1
are also inducible (e.g. [30]), there is not a known
22
23
Fig. 2. Heat map summarizing the toxicogenomics results of the treatment of primary human hepatocytes with hepatotoxic drug, troglitazone and
its less toxic analogs, rosiglitazone and pioglitazone. The results of genes that are differentially suppressed and induced by troglitazone are shown
in A and B, respectively. Genes are grouped by physiological mechanisms of action, and each line represents the results of a single gene. Significant
response is defined as >50% induction or suppression over solvent control. Microarray used was the TOX-chip from Phase I Molecular Toxicology
Inc. (data reconstructed from [43]).
24
of the first to define transporter-mediated DDI interaction with human hepatocytes. Several subsequent studies
were performed based on the methods developed in this
pivotal study, providing critical information concerning
human drugtransporter interactions [17,37,38].
While the study of transporter-mediate uptake can
be evaluated with hepatocytes in suspension, the study
of efflux transporter is more difficult. It is now known
that the efflux transporters in hepatocytes are internalized in hepatocytes in suspension, and therefore cannot
be studied effectively (e.g. by evaluating the excretion
of a compound into the extracellular medium). Hepatocytes are found to form functional bile canaliculi when
cultured between two layers of collagen gels (collagen
sandwich), an observation firstly observed in rat hepatocytes [39,40] and later extended to cryopreserved human
hepatocytes [36]. It is to be noted that while most efflux
transporters are located in the liver canaliculi, there is
also evidence that the transporters that are responsible for the uptake of some drugs may be involved in
the sinusoidal efflux of xenobiotics. For instance, the
rat Oatp-1, Oatp-2 have been identified as efflux transporters for reduced glutathione, and therefore may also
have activities towards the efflux of certain drugs [41].
Human hepatocytes, used as either freshly isolated
or cryopreserved cells, therefore can be use effectively
for the evaluation of hepatic transporter functions. An
important application of human hepatocytes will be
the evaluation of the complicated roles of drug uptake,
metabolism and efflux on drug metabolism, drugdrug
interactions, and drug toxicity.
4.4. Drug toxicity
An important application of human hepatocytes is
to evaluate human-specific hepatotoxicity that may not
be possible with nonhuman preclinical animal species.
Unanticipated drug toxicity, especially hepatotoxicity,
has always been a major challenge of the drug industry [42]. As drug metabolism is known to be a critical
parameter for hepatotoxicity, and that speciesspecies
differences in drug metabolism is a well-established phenomenon, human hepatocytes may represent a relevant
in vitro experimental system for the evaluation of human
hepatotoxic potential of drugs and drug candidates.
In vitro toxicity assays with human hepatocytes can
be applied in various during phases of drug development
[44,50]. The two major applications are as follows:
4.4.1. Early screening of intrinsic toxicity
Human hepatocytes, especially cryopreserved hepatocytes, can be used for rapid screening of new chemical
entities to allow the selection of structures with low hepatotoxic liability for further development. The screening
assay can allow logical evaluation of structures responsible for toxicity (toxicophore) which, hopefully, can be
separated from structures for pharmacological activity
(pharmacophore). Toxicity screening with human hepatocytes (e.g. using ATP content as an endpoint [7])
can be performed in 96-well or 384-well plate formats,
requiring as little as 1500 cells/well, and would need a
relatively small amount of test articles (e.g. 0.1 mg), and
is rapid and quantitative.
4.4.2. Mechanistic evaluations
Besides screening, in vitro experimental systems such
as human hepatocytes can be used to define toxic mechanisms. The defined experimental conditions and the
availability of reagents and approaches for multiple endpoints allow one to define the key pathways involved
in a toxicological phenomenon. High-content endpoints
such as toxicogenomics [43,45] have been applied in
human hepatocytes for the evaluation of toxic mechanisms. Mechanistic understanding is critical to drug
development. It allows a better understanding of human
health risks, defines potential risk factors, and the relationship between efficacy and adverse effects.
4.4.3. Toxicogenomics
High-content assays such as toxicogenomics for gene
expression, proteomics for protein expression, metabonomics for natural metabolite synthesis, and cellomics
for cell morphology changes, are relative novel technologies with potential application in the definition of
drug toxicity. It is anticipated that these high-content
assays, which allow one to evaluate the effect of a test
substance on myriad events (e.g. expression of 20,000
human genes), one has the potential to achieve the following:
1. discovery of response patterns (response profiles) that
can be used to define toxicity;
2. discovery of new biomarkers for specific types of
toxicity;
3. discovery of early indicators of chronic toxicity;
4. discovery of indicators of idiosyncratic toxicity.
The high-content assays may be extremely valuable
to the definition of toxicity difficult to evaluate with conventional approaches. For instance, prediction of chronic
toxicity in human that eludes detection in preclinical
nonhuman animals may be possible using human cells in
vitro (e.g. human hepatocytes for hepatotoxicity), provided that the key events for chronic toxicity can be
25
Fig. 3. Schematic presentation of the integrated discrete multiple organ co-culture (IdMOC) system. The IdMOC system is based on the concept
that in the human body, there are multiple organs that are physically separated but interconnected by the systemic circulation (top figure). A toxicant
entering the systemic circulation will have interactions with all the interconnected organs. Results of the interactions, which can be the formation
of metabolites or the induction of reactive biomolecules (e.g. cytokines), will have the potential to interact with the multiple organs. The concept is
reduced to practice using a wells-in-a-chamber concept, with multiple wells in a containing chamber, allowing the culturing of cells from different
organs as physically discrete wells interconnected by an overlying medium (bottom figure). Figures reconstructed from [46]).
26
isoform 3A4) which may be responsible for the generation of reactive metabolites [47]. Troglitazone, however,
uniquely induced gene expression of the toxification
pathways such as apoptosis, and inflammation, and suppressed gene expressions of the gene responsible for
detoxification pathways such as acute phase proteins,
phase II conjugation, and stress proteins [43,45]. It is
projected that the genes that are affected differentially
by troglitazone and its less toxic structural analogs may
be used to predict the human hepatotoxic potential of
new troglitazone structural analogs.
The study with troglitazone and its less toxic analogs
illustrates an application of toxicogenomics in the development of hypothesis to allow subsequent mechanistic
investigations. The combination of toxicogenomics and
human cells such as human hepatocytes in vitro can be a
powerful combination to aid the definition or prediction
of human drug toxicity. The key is to develop an in vitro
system to incorporate in vivo factors that are critical
to the manifestation of toxicity. One such system is the
integrated discrete multiple organ co-culture (IdMOC)
system.
4.4.4. IdMOC
One major drawback of in vitro systems is that each
cell type is studied in isolation, while in the human body,
Fig. 5. Differential cytotoxicity of tamoxifen, an anticancer drug with known multiple organ toxicity, in IdMOC with primary cells from five major
organs and a tumor cell line. The primary cells are: human hepatocytes (hepatocytes), representing the human liver; human aortic endothelial
cells (HAEC), representing the vascular endothelium; human astrocytes (astrocytes), representing the central nervous system; human renal proximal
tubule epithelial cells (RPTC), representing the kidney; human small airway epithelial cells (SAEC), presenting the lung; the human breast carcinoma
(MCF-7). The results shown here illustrate an application of the IdMOC as a tumor-bearing man in the discovery of potential anticancer agents
with minimal toxicity towards normal human tissues. Results are adopted from [46].
27
5. Conclusions
In the past decade, human hepatocytes have been
used increasingly in drug development. The experience
as of now is that relevant information can be obtained
with this experimental system, including humanspecific drug properties such as metabolic stability
and fate, drugdrug metabolizing enzyme interactions,
drugtransporter interactions, and drug toxicity. The
availability of human hepatocytes for research has been
dramatically increased due to the increased availability
of commercial sources for the cells.
While it is generally agreed that isolated hepatocytes retain most of the key liver properties (e.g. drug
metabolism), the value of the data obtained with hepatocytes remains limited by the inherent inadequacies
of the in vitro systems (at least as of the current practice), namely, the absence of key in vivo factors. It
is therefore of no surprise that the recent improvements on the use of intact hepatocytes is to incorporate
key in vivo factors (e.g. incubations of intact hepatocytes in 100% serum; IdMOC). Human hepatocytes
represent a valuable experimental system in drug development. It is through the application of the experimental
system within its limitations, while continually developing approaches to overcome these limitations, that
this valuable resource can be utilized to its fullest
potential.
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