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Chemico-Biological Interactions 168 (2007) 1629

Human hepatocytes: Isolation, cryopreservation


and applications in drug development
Albert P. Li a,b,
a

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-

Tel.: +1 410 869 9037; fax: +1 410 869 9560.


E-mail address: lialbert@apsciences.com.

ment as an experimental model for the evaluation of key


human-specific drug properties such as metabolic fate,
drugdrug interactions, and drug toxicity. The applications range from the early screening the most appropriate
new chemical entities for further development, to the
determination of key drug properties for New Drug
Applications (e.g. drugdrug interactions) to U.S. FDA.
Successful application of human hepatocytes in drug
development requires a thorough understanding of the
strengths and weaknesses of this valuable experimental
system. This review is an effort to present a comprehensive review on the state-of-the-art of the isolation,
cryopreservation, and applications of human hepatocytes

0009-2797/$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2007.01.001

A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

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.

unavailability of a matched recipient;


physical damage to the liver;
pre-existing liver diseases;
breach of sterility during the procurement process;
high liver fat content;
inappropriate age (too young or too old);
inappropriate warm ischemic time;
inappropriate cold storage time.

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

Livers that are procured for transplantation are


usually flushed extensively with a cold preservation
solution, with the University of Wisconsin (UW)
or histidinetryptophanketoglutarate (HTK) solutions
being the most common [1]. The rule of thumb is that
viable hepatocytes can be obtained from human livers
with up to 24 h of cold preservation. There are, however,
cases that highly viable hepatocytes can be obtained from
livers stored beyond this 24-h period.
Hepatocyte isolation from human livers is now
universally performed with a two-step collagenase
procedure developed by Berry and Friend [2]. Originally developed for the isolation of rat hepatocytes, this
procedure has been modified by various laboratories for
the isolation of hepatocytes from sevreal animal species,
including human (e.g. [3,4]). The procedure involves the
initial perfusion of the liver with a warm (37 C) divalent ion-free, EGTA-containing, isotonic buffer (Step 1)
to remove blood and to loosen cellcell junctions, followed by perfusion with a warm, isotonic, collagenase
solution (Step 2) to dissociate the liver parenchyma into
single cells. In general, a higher amount of collagenase
is required for the isolation of hepatocytes from human
livers than that required for rat livers. As collagenase
is a mixture of proteases, its composition can affect its
effectiveness in the dissociation of the hepatocytes as
well as its cytotoxicity. It is a common practice to evaluate multiple lots of collagenase to select the one lot
yielding the highest number of viable hepatocytes from
a liver. After digestion, the cells are harvested by lowspeed centrifugation. A density gradient such as Percoll
is commonly used to enrich for viable cells. The isolated

Hepatocytes, especially human hepatocytes, are now


routinely used after they are cryopreserved [7,8]. The
general procedures for hepatocyte cryopreservation have
not deviated extensively from the original procedures
[9]. Via the use of equipments to control freezing rates
(e.g. programmable control-rate freezer) and appropriate
cryopreservation agents (e.g. dimethyl sulfoxide), hepatocytes now can be stored in liquid nitrogen (lower than
150 C) for an extensively time period (years) with
the retention of high viability and drug metabolizing
enzyme activity [7]. The most recent advancement of
human hepatocyte cryopreservation is the ability of the
thawed hepatocytes to be plated as monolayer cultures
(plateable hepatocytes) [10,11]. In our laboratory, we
routinely prepare cryopreserved human hepatocytes with
most of the lots having post-thaw viability of >90%
(Table 1) and with approximately half of the lots yielding over 50% confluent monolayer cultures when plated
onto collagen-coated plates (Table 1; Fig. 1). In our
laboratory, we believe that the key to successful cryopreservation is to ensure that the hepatocytes are isolated
from the human liver with minimal damages to the
plasma membrane.
It is important to fully understand the properties
of cryopreserved human hepatocytes as compared to
freshly isolated cells. The following are general conclusions on the drug metabolizing enzyme activities of
cryopreserved human hepatocytes:
1. P450 isoform activities: Since the first publication
showing similar P450 isoform activities between

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A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

Table 1
Post-thawed viability and attachment efficiency of human hepatocytes isolated and cryopreserved in APSciences Inc.
Date of preparation

Lot

Yield (cells/vial)

Viability, % (trypan blue)

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).

freshly and cryopreserved human hepatocytes [7],


there are various independent confirmation of this
observation (e.g. [10,13]). In general, the major
human P450 isoform activities: CYP1A2, CYP2A6,
CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1,
and CYP3A4 are retained in cryopreserved human
hepatocytes. Direct comparison of activities between
freshly isolated and cryopreserved human hepatocytes from the same preparation showed no
substantial differences [7].
2. UDP-dependendent glucuronosyl transferase (UGT)
and sulfotransfase (ST) activities: UGT and ST are

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.).

two major Phase II metabolism pathways, leading


to conjugation of xenobiotics or metabolites with
the highly water-soluble glucose (UGT) and sulfate
(ST). There is no apparent deterioration of these
Phase II conjugating pathways upon cryopreservation
([13,14]).
3. Glutathione (GSH) level and glutathione transferase
activities (GST): It is now well-established that while
GST activities are similar between freshly isolated
and cryopreserved human hepatocytes, intracellular
GSH levels are drastically reduced upon cryopreservation and thawing [15].
4. Transporter activities: Transporter research is the
latest exciting area in drug metabolism. Transporter activities are now known to be involved in
drug metabolism, drugdrug interactions, and drug
toxicity. Some lots of cryopreserved human hepatocytes are known to retain transporter activities
and therefore can be used for transporter research.
The transporters that have been reported to be
active in human hepatocytes include human Na(+)taurocholate cotransporting polypeptides (NTCP)
and organic anion transporting polypeptides (OATP)
[16,17].
The general consensus is that cryopreserved human
hepatocytes can be used routinely for the evaluation
of drug metabolism ([7,8,14]). The strengths of the
cryopreserved human hepatocytes over freshly isolated hepatocytes as an experimental model are as
follows:
1. Ease of experimentation: Unlike freshly isolated hepatocytes which is dependent on the availability of a
human liver for research, experimentation with cryopreserved human hepatocytes can be planned.

A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

2. Repeat experimentation: Cryopreserved hepatocytes


from a single donor (or combination of multiple
donors) can be used at different times to allow the
performance of repeat studies. This is not possible
with freshly isolated hepatocytes.
3. Choice of donors: Experimentation with freshly isolated cells are performed with the liver from the
donor available at a specific point of time. Limiting
the choices of donors may lead to a prolonged waiting period for experimentation. Experimentation with
cryopreserved human hepatocytes, however, allows
the researcher to select hepatocytes from donors
with properties most appropriate to the experimental
objectives.
The following are the limitations:
1. Incubation time for metabolism studies: Most lots of
cryopreserved human hepatocytes cannot be cultured
as attached, monolayer cultures. For these cells that
cannot be cultured, their applications will be limited
to those compatible with suspension cultures. Hepatocytes in suspension have a finite lifespan (hours),
and probably should not be used for longer than 5 h.
For hepatocytes that can attach, their viability is prolonged (days). It is known, however, that activities for
P450 isoforms such as CYP1A2 and CYP3A4 would
decrease (approximately 50% per day) in culture. It
is probably most appropriate to use the plateable
cryopreserved hepatocytes for not more than 3 days
in culture for metabolism studies.
2. GSH conjugation: Because of the decreased intracellular GSH level in cryopreserved hepatocytes,
GSH conjugation may be limited in this experimental system, especially when used as short-term
incubations in suspension. It may be more appropriate to use freshly isolated or cultured cryopreserved
human hepatocytes (e.g. for 24 h to allow recovery of intracellular GSH) for the evaluation of GSH
conjugation.
3. Transporter studies: Not all lots of human hepatocytes retain intact transporter activities. It would be
prudent to evaluate multiple lots of cryopreserved
human hepatocytes to select the lots with active transporters.

19

4.1. Drug metabolism


As liver is the major organ for drug metabolism, hepatocytes represent a valuable experimental system for
drug metabolism studies. Unlike subcellular fractions,
intact hepatocytes contain all the drug metabolizing
enzymes and cofactors at physiological levels. The key
experiments in drug metabolism are as follows:
4.1.1. Metabolic stability
Estimation of intrinsic hepatic clearance is an important discipline in drug development. It allows the
selection of drug candidates with the most appropriate
metabolic stability in vivo. In the past, metabolic stability studies are performed predominantly with liver
microsomes supplemented with NADPH+ cofactors,
with the assumption that Phase I oxidation represent
the most important pathway for metabolic clearance.
This is obviously not universally true as a large number of structures are metabolized by non-microsomal
enzymes (e.g. cytosolic dehydrogenases, mitochondrial
monoamine oxidases), and not always by Phase I oxidation (e.g. UGT and ST conjugating activities).
Intrinsic hepatic clearance studies with hepatocytes
are performed via the incubation of the chemical in question with intact hepatocytes and the quantification of the
chemical at various times after incubation. Intrinsic hepatic clearance (CL) can be estimated using one of the
following three equations:
1. CL = {Vmax /(Km + [S])}/number of hepatocytes (or
=(Vmax /Km )/number of hepatocytes, if [S]Km);
2. CL = 0.693/(t1/2 hepatocyte concentration);
3. CL = (initial concentration)/(AUC hepatocyte concentration).

4. Application of human hepatocytes in drug


development

CL is in general expressed in the units of ml/min/


million hepatocytes. CL estimation using equation 1
requires the performance of the study with multiple concentrations of the drug substrate, while CL estimation
using equation 2 or 3 requires a simpler procedure of the
incubation of hepatocytes with a single concentration
of the drug substrate. In most laboratories, the simpler
procedure of incubation of hepatocytes with a single concentration of the drug substrate is used with CL derived
using equations 2 or 3. The results can further scaled up
to hepatic intrinsic clearance per liver and per human
using the following assumptions:

The key areas in drug development that can benefit


from the use of human hepatocytes are drug metabolism,
drugdrug interaction, and drug toxicity evaluation.

1. hepatocytes (1.2 million) per gram of human liver


[18];
2. liver (1.8 kg) per 70 kg human body weight [19].

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A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

Hepatocytes are usually used as suspension cultures,


with a cell concentration of 0.52.0 million cells/ml, and
incubation times up to 5 h. Cryopreserved human hepatocytes used as suspension cultures in a 96-well plate,
using 150,000 cells/well, have been shown to be useful as
a screening assay for human hepatic metabolic stability
during early drug discovery [48].
Due to the known decreases in drug metabolizing
enzyme activities with primary cultures of hepatocytes, it
is generally recommended that metabolic stability studies to be performed with freshly isolated hepatocytes or
thawed cryopreserved hepatocytes from fresh isolates. If
prolonged incubation (e.g. days) is required, it is recommended that freshly isolated or plateable cryopreserved
hepatocytes are allowed a short attachment period (e.g.
4 h) and then used for incubation with the drug substrate.
It is not advisable to use hepatocytes that have been cultured for over 48 h for the estimation of in vivo hepatic
clearance (e.g. [20,21]).
An interesting advancement in the application of
intact hepatocytes in the evaluation of metabolic stability is the incubation of the test article and hepatocytes in
100% human serum, therefore providing an experimental condition similar to humans in vivo. For instance,
it has been reported that in vivo hepatic clearance can
be predicted more accurately from data obtained with
hepatocytes incubated in serum than from data obtained
in the absence of serum using rat hepatocytes [20] and
human hepatocytes [21,22]. The combination of human
hepatocytes and 100% human serum may represent a
more physiologically relevant experimental model. It
eliminates the need to consider plasma protein binding
and intracellular concentration of the chemical studied for the estimation of in vivo metabolic rates. This
approach therefore is conceptually attractive and should
be carefully evaluated for universal applications. One
puzzling observation made in our laboratory was that
testosterone, a naturally occurring steroid that is readily metabolized in human in vivo, was metabolized
by cryopreserved human hepatocytes in suspension in
the absence of serum, but was virtually not metabolized in the presence of 100% serum (unpublished
observation).
Metabolic stability studies are usually conducted during earlier stages of drug development to allow the
selection of structures with the most appropriate stability for further development. Because of the presence of
all hepatic drug metabolizing enzymes and cofactors at
physiological levels, intact hepatocytes, especially cryopreserved human hepatocytes represent a more relevant
experimental system than human liver microsomes for
general screening for metabolic stability. Human liver

microsomes represent a relevant system if microsomal


enzymes are known to be responsible for the metabolism
of the chemicals in question.
4.1.2. Metabolite proling, metabolite identication
and species comparison
Metabolism is a determinant of metabolic stability as
discussed above, and also a key aspect of drug toxicity.
A chemical can be activated from a nontoxic parent
molecule to toxic metabolites. Conversely, a toxic parent
chemical can be detoxified via biotransformation (e.g.
by Phase II conjugation) to nontoxic metabolites.
Metabolite identification therefore is an important
part of drug development. Knowing the structure of the
metabolite, one can design structures with improved
metabolic stability and toxicity, for instance, by eliminating or blocking the critical sites of metabolism.
Early speciesspecies comparison of metabolite profiling is also critical to drug development to allow the
selection of the most appropriate animal species for preclinical drug development. The appropriate species will
be the ones with metabolite profiles similar to human.
Human liver microsomes are generally used in
metabolite profiling. As discussed earlier, this approach
assumes that Phase I oxidation is the most important
pathway of the metabolism of the chemical entity in
question. This is an appropriate approach if one is certain that the predominant route of metabolism is Phase
I oxidation by microsomal pathways. If this is not
well-established, intact hepatocytes represent a more scientifically relevant experimental system.
The use of human liver microsomes versus hepatocytes in metabolite profiling can be illustrated with
ethynyl estradiol (EE2) [12]. EE2 is predominantly
metabolized by human in vivo via Phase II metabolism
into glucuronide and sulfate conjugates. However,
liver microsomes produce primarily the 2-hydroxylation
metabolite. Human hepatocytes produce the conjugate
metabolites, similar to human in vivo. The results with
EE2 illustrate the use the human hepatocytes to provide
an initial evaluation of the major metabolites, while the
use of liver microsomes allows the evaluation of specific
Phase I oxidative metabolites.
4.2. Drugdrug interactions
A major advancement of the application of human
in vitro drug metabolism systems is its application in
drugdrug interaction (DDI) studies. U.S. FDA now
requires in vitro DDI studies to be performed for New
Drug Applications (NDA) [2325]. It is proposed by
U.S. FDA that, if the in vitro DDI studies show no poten-

A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

tial interactions, clinical DDI studies are not required to


be performed [25].
DDI studies involve the following major approaches:
1. Determination of key drug metabolism pathways:
Elucidation of the key pathway of metabolism of a
drug would allow the estimation of its interaction with
existing drugs that are known to inhibit or induce the
pathways involved.
2. Evaluation of drug metabolizing enzyme inhibitory
potential: Evaluation of the ability of a drug to inhibit
the major human drug metabolizing enzymes (e.g.
P450 isoforms) would allow the estimation of its
DDI potential with co-administered drugs that are
substrates of the affected pathway. The result of
inhibitory DDI is the unwarranted increase in systemic burden of the affected drug which may lead to
severe toxicity (e.g. ketoconazole/terfenadine interactions [26]).
3. Evaluation of P450 induction potential: Evaluation of
the ability of a drug to induce the major human drug
metabolizing enzymes (e.g. P450 isoforms) would
allow the estimation of its DDI potential with coadministered drugs that are substrates of the induced
pathway. The major consequence of inductive DDI is
the lowering of the systemic burden to the affected
drug due to accelerated metabolic clearance, leading
to loss of efficacy (e.g. cyclosporine/rifampin interactions [27]).
4.2.1.1. Metabolic pathway elucidation
In general, human liver microsomes and cDNA
expressed microsomes are used for pathway elucidation studies. Human hepatocytes, however, can provide
valuable information which will complement liver
microsomal data. One key experiment that is not always
performed, but should be performed, for pathway elucidation of the metabolism of a test article is the
identification of metabolites from intact hepatocytes.
For instance, if the major metabolites are results of
Phase II conjugation, then evaluation with human liver
microsomes or genetically expressed microsomes are
not necessary. This initial experiment is in fact recommended by FDA [25].
4.2.1.2. P450 inhibition
As for pathway elucidation, liver or cDNA microsomes are routinely used for P450 inhibition studies.
It is now realized that the use of intact hepatocytes
may allow a more relevant estimation of in vivo effects.
Intact hepatocytes provide an intact cell membrane,
which allows the modeling of differential uptake of the

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

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A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

inducer which would selectively induce these isoforms


without also inducing CYP3A4. For instance, a potent
inducer of CYP2B6, phenobarbital, is also a potent
inducer of CYP3A4 in human hepatocytes [30]. In contrast, CYP1A2 inducers in general (e.g. omeprazole,
3-methylcholanthrene) would not induce CYP3A4 and
vice versa. For the investigation of P450 induction potential of drug candidates, it may be adequate to evaluate
CYP1A2 and CYP3A4 induction. If CYP3A4 induction
is observed, then investigations on the induction potential of the drug candidate towards other isoforms are
warranted. If no CYP1A2 or CYP3A4 is observed, the
drug candidate is not likely to have induction effects
on the other P450 isoforms, and therefore can be considered not to be a potential P450 inducer. As of
this writing, isoniazid is the only inducer that apparently would induce CYP2E1 but with little effects on
the other isoforms, including CYP1A2 and CYP3A4
[30]. It is to be noted while induction of CYP2E1
gene expression can be readily detected, induction
of CYP2E1 activity, (e.g. measured as chloroxazone
6-hydroxylation activity) by inducers, including isoniazid and ethanol, is not a reproducible phenomenon
for human hepatocytes in culture (Li, unpublished
observation).
A major advancement in the P450 induction studies
with human hepatocytes is the use of plateable cryopreserved human hepatocytes. The first observation
of the responsiveness of cryopreserved human hepatocytes to P450 inducers made by Ruegg et al. [31]
is now substantiated by results of several laboratories
[10,32,33], including ours (Fig. 3). It is now generally
believed that there is little qualitative difference between
cryopreserved and freshly isolated human hepatocytes
towards known P450 inducers. U.S. FDA has recently
recommended the use of plateable cryopreserved human
hepatocytes for the evaluation of P450 induction for DDI
studies [25].
P450 activity measurement remains the gold standard for P450 induction studies as activity is responsible
for DDI. Surrogate endpoints such as mRNA and protein
quantification are also used, especially for the evaluation
of induction potential for a compound which is also an
inhibitor of the activity of the induced P450 (e.g. ritonavir [34]). As of now, FDA considers activity as the most
acceptable endpoint for the definition of DDI potential

23

due to P450 induction [25]. A positive inducer has been


defined in practical terms by FDA as the following:
>2 basal activity (activity of solvent control) or >40%
of positive control activity.
4.3. Transporter studies
The field on hepatic transporters has received great
attention lately, mainly due to the activities of several key leading scientists in this area who show that
transporter activities can be critical to drug absorption,
metabolism, and drugdrug interactions. Hepatocytes in
vivo have both uptake and efflux transporters, with the
uptake transporter responsible for the active uptake of
biomolecules and xenobiotic, and the efflux transporters
for the bile excretion of bile salts and conjugated xenobiotics.
Hepatocytes have been used in suspension to evaluate the activities of uptake transporters. For this type of
studies, hepatocytes are incubated in suspension with the
transporter substrate, followed by centrifugation of the
hepatocytes through silicon oil to remove extracellular
substrates, thereby allowing accurate quantification of
intracellular or uptaken substrates.
As with drug metabolizing enzymes, it is now known
that there are substantial species differences in hepatic
transporters. Intact human hepatocytes therefore represent a convenient experimental system for the evaluation
of human transporter activities. Cryopreserved human
hepatocytes are now known to active uptake transporters
[35], and can be cultured to expressed efflux transporters
[36].
An elegant application of intact hepatocytes to evaluate transporter-mediated DDI was the study of Shitara
and coworkers [16]. The study was performed with
cryopreserved human hepatocytes to evaluate if the
known DDI between cerivastatin (CER) and cyclosporin
A (CsA) was due to drug metabolism or transportermediated hepatic uptake. They found that CER uptake
was inhibited by CsA with Ki values approximately 10X
lower than the estimated Ki values the inhibition of
CER metabolism. From these results, it was concluded
that the DDI between CER and CsA is mainly due to
the inhibition of transporter (at least partly OATP2)mediated uptake in the liver. This study represents one

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]).

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A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

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

A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

defined in the target cells after treatment with the toxicant


in vitro.
Another potential valuable application of highcontent assays is to the definition of idiosyncratic drug
toxicity. Efforts to use a single theory (e.g. reactive
metabolite formation) to universally defined idiosyncratic drug toxicity have proven not yet to be useful.
One possible explanation for the illusiveness of the definition of idiosyncratic toxicants is that there may be
multiple factors involved [44]. These factors are proposed to be the chemical properties of the toxicant, and
the genetic make up of the human victim, as well as the
complex environmental factors surrounding the victim.
It is projected that the factor that can be readily controlled
is the chemical properties. Therefore, a more practical approach to eliminate idiosyncratic drug may be the
definition of the common properties of the toxicants [44].
Towards this goal of defining the properties of drugs
that would cause idiosyncratic hepatotoxiciy, we have
applied toxicogenomics to evaluate troglitazone, a drug
that was withdrawn from the market due to its association

25

with idiosyncratic hepatotoxicity [43]. Trogltiazone was


compared to the relatively nontoxic structural analogs,
rosiglitazone and pioglitazone [43]. Freshly isolated
human hepatocytes from three donors were treated in
vitro with the three chemicals, followed by extraction
of mRNA and hybridization with cDNA microarrays.
The gene expression heat map (using colors to classify the magnitude of response) is presented in Fig. 2.
Dramatic differences were observed in gene expression
profiles between the toxic troglitazone and the nontoxic structural analogues. Troglitazone was found to
selectively down regulate genes with protective functions (Fig. 2A) and up regulate genes with toxification
functions (Fig. 2B). Similar toxicogenomics results with
troglitazone were also obtained by the laboratory of Waring and coworkers [45].
Toxicogenomics studies with troglitazone and its less
toxic analogs, rosiglitazone and pioglitazone provided
clues for the differential hepatotoxicity of these structurally related chemicals. All three chemicals appear
to induce genes for oxidative metabolism (e.g. P450

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

A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

Fig. 4. Photograph of an IdMOC plate based on the format of a standard


96-well plate (IdMOC-96). The plate consists of six individual wells
inside a containing chamber. The wells are filled with colored liquids
(left) to show that they are physically separated. The chambers are filled
(right) to show the connection of the wells. Each chamber therefore
represents a single experimental unit, allowing each plate to be used for
multiple treatment conditions. For instance, one IdMOC-96 plate can
be used to evaluate the effect on six cell types for 15 test articles (using
one chamber as control) at a single concentration, two test articles
at four concentrations in duplicates, or a single test article at four
concentrations in quadruplicates.

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].

A.P. Li / Chemico-Biological Interactions 168 (2007) 1629

there may be multiple organ interactions that are critical


to drug toxicity. An example of multiple organ interactions is a drug which is firstly metabolized by the liver to
form metabolites which may cause toxicity in a distant
organ such as the heart.
To overcome this deficiency, the independent discrete multiple organ co-culture (IdMOC) system has
been developed in our laboratory [46]. The IdMOC
allows the co-culturing of cells from different organs
as physically separated cultures that are interconnected
by an overlying medium, akin to the blood circulation connecting the multiple organs in the human body.
The IdMOC models the multiple organ interaction in
the whole organism in vivo, allowing the evaluation of
organ-specific effects a drug and its metabolites (Fig. 3).
The IdMOC represents an improved in vitro experimental system for routine screening of ADMET drug
properties.
The IdMOC involves the wells-in-a-well concept.
The typical IdMOC plate consists of a chamber within
which are several wells (Fig. 4). Cells of different origins (e.g. from different organs) are initially cultured,
each in its specific medium, in the wells. When the cells
are established, the wells are flooded with an overlying
medium, thereby connecting all the wells. The multiple
cell types now can interact via the overlying medium,
akin to the multiple organs in a human body interacting
via the systemic circulation.
The IdMOC system can be used for the following:

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.
References

1. Differential cytotoxicity: Evaluation of the toxicity


of a substance on different cell types (e.g. cells
from different organs) under virtually identical experimental conditions with multiple cell type interactions.
2. Differential distribution: Evaluation of the differential accumulation/distribution of a substance among
multiple cell types.
3. Multiple organ metabolism: Evaluate the ultimate
metabolic fate of a substance upon metabolism by
cells representing multiple organs with metabolic
functions (e.g. liver, kidney, lung).
We have recently evaluated the differential cytotoxicity of tamoxifen using the IdMOC system [46] which
illustrates the application of IdMOC in the screening of
anticancer agents for their cytotoxicity towards cancer
cells (the desired property) and cells from normal tissues
(the undesired properties) (Fig. 5). Further validation
of the system is now in progress in our laboratory to
evaluate the strengths and limitations of this novel
technology.

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