Global Gene Expression Changes Induced In Primary Human Hepatocytes By Thiazolidinediones Upon Repeat Dosing of HepatoPac™ Cultures

Global
gene
expression
changes
as
assessed
using
whole
genome
microarrays
can
provide
insights
into
the
mechanism
of
ac4on
of
drug
candidates.
Furthermore,
signatures
of
gene
expression
may
dis4nguish
compounds
with
poten4al
to
cause
4ssue
injury
with
diverse
mechanisms
from
the
non-­‐toxic
compounds1,2.
In
the
case
of
the
liver,
primary
human
hepatocytes
are
widely
considered
to
be
the
most
suitable
to
assess
drug-­‐induced
gene
expression
changes
in
vitro
since
they
contain
the
full
repertoire
of
regulatory
pathways;
however,
hepatocytes
display
a
precipitous
decline
in
phenotypic
func4ons
when
lel
in
suspension
or
cultured
in
a
sandwich
of
extracellular
matrix
proteins.
Therefore,
hepatocytes
in
these
conven4onal
systems
are
generally
unsuitable
for
assessment
of
gene
expression
changes
upon
chronic
exposure
in
order
to
mimic
clinically
relevant
dosing
regimens.
We
have
previously
developed
a
microscale
model
of
the
human
liver
in
which
primary
human
hepatocytes
are
organized
in
colonies
of
empirically
op4mized
dimensions
and
subsequently
surrounded
by
suppor4ve
stromal
cells
(HepatoPac™).
Here,
we
sought
to
discern
the
effects
of
acute
(24
hours)
and
chronic
(7
to
14
days)
drug
exposure
on
the
transcriptome
of
primary
human
hepatocytes
using
the
hepatotoxic
and
non-­‐toxic
drug
pair,
Troglitazone
and
Rosiglitazone,
respec4vely.
HepatoPac
cultures
were
dosed
from
24
hours
to
14
days
at
a
sub-­‐lethal
and
clinically
relevant
C[max]
dose.
Messenger
RNA
was
collected
at
24
hours,
7
days,
and
14
days
aler
dosing,
and
hybridized
to
whole
genome
human
Affymetrix
GeneChips™.
Subsequent
analysis
of
gene
expression
data
with
GeneSpring
solware
revealed
a
4me-­‐dependent
increase
in
sta4s4cally
significant
gene
expression
changes
induced
by
both
Troglitazone
and
Rosiglitazone
as
compared
to
vehicle
controls.
However,
Rosiglitazone
significantly
changed
the
expression
of
2,
16,
and
221
genes,
while
Troglitazone
changed
the
expression
of
17,
409
and
928
genes
over
24
hours,
7
days,
and
14
days,
respec4vely.
In
par4cular
Troglitazone
caused
up-­‐regula4on
of
several
genes
involved
in
cellular
pathways
such
as
oxida4on-­‐reduc4on,
lipid
metabolism
and
biosynthesis,
and
stress/wounding
response.
Studies
are
currently
underway
to
evaluate
drug-­‐induced
gene
expression
changes
of
mul4ple
toxic/non-­‐toxic
pairs
in
order
to
evaluate
common
pathways.
In
the
future,
HepatoPac
cultures
may
be
u4lized
with
both
gene
expression
and
biochemical
assays
to
select
op4mal
drug
candidates
in
both
drug
discovery
and
development.