In Vitro Screening Approaches to Estimate Human Hepatic Responses to Xenobiotic Exposures Stephen S. Ferguson, Ph.D. National Toxicology Program (NTP) Division National Institute of Environmental Health Sciences (NIEHS) • I have no financial relationships to disclose. • The statements, opinions or conclusions contained therein do not necessarily represent the statements, opinions or conclusions of NIEHS, NIH or the US government.
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In Vitro Screening Approaches to Estimate Human Hepatic Responses to Xenobiotic Exposures
Stephen S. Ferguson, Ph.D.
National Toxicology Program (NTP) Division
National Institute of Environmental Health Sciences (NIEHS)
• I have no financial relationships to disclose.
• The statements, opinions or conclusions contained therein do not necessarily represent
the statements, opinions or conclusions of NIEHS, NIH or the US government.
Tissues and Organs: a text of scanning electron microscopy, Kessel, RG and Kardon,RH, 1979.
Immortal liver cells
Physiological architecture of liver
=?
Cell reporter assays
Human-derived In Vitro Liver Models for Estimation of Human DMPK/DDI
• Hepatic clearance
− Suspensions of PHHs (typically pooled donor)
• Retain liver-like xenobiotic metabolism & uptake transport
• Ineffective models of efflux transport
• Very short longevity (few hours)
• Liver enzyme induction - DDI
− Sandwich-cultured primary human hepatocytes
• Effective to model liver enzyme induction via AhR,
CAR, PXR, and PPAR pathways
• Useful models of biliary excretion
• ~90% reduced baseline metabolic competence
• High variabilities
− donor prep variations
− technically variations
− Short longevities (days, sporadic cell death) Time (hrs)
Blo
od C
oncentr
ati
on
Ineffective level
Therapeutic Window
(drug efficacy)
Toxic / side-effect level
Seeding Density
1.5
0.75 0.
50.
25
0.17
5
CY
P1A
2 A
cti
vit
y
(pm
ol/m
in/1
06 c
ells)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Impact of cell density on cytoskeleton and xenobiotic metabolism with
primary cultures of human hepatocytes
Hamilton et al., Cell Tissue Res, 2001; 306: 85-99.
CY
P3
A4
/5 I
nd
uce
d
En
zym
atic A
ctivity
LeCluyse et al., 2010. Chapter 8: Cytochrome P450 Induction. Enzyme Inhibition in Drug
Discovery and Development: The Good and the Bad. John Wiley & Sons.
Mechanism-based CYP3A Inhibitors/Inducers Over Time in SC-PHHs
CYP3A4 mRNA
0
2
4
6
8
10
12
2 4 8 24 48 72 96
Time (Hrs)
(CY
P3A
4 m
RN
A C
on
ten
t}
Rela
tiviv
e F
old
-Over-
Co
ntr
ol
TAO 0.1 µM
TAO 10 µM
CYP3A4 Activity
0
50
100
150
200
250
2 4 8 24 48 72 96
Time (Hrs)
(Testo
ste
ron
e 6
ß-H
yd
roxyla
tio
n)
Perc
en
t o
f V
eh
icle
Co
ntr
ol
TAO 0.1 µM
TAO 10 µM
0
20
40
60
80
100
120
2 4 8 24 48 72 96Time (Hrs)
(Testo
ste
ron
e 6
ß-H
yd
roxyla
tio
n)
Perc
en
t o
f V
eh
icle
Co
ntr
ol
Ritonavir 0.1 µM
Ritonavir 10 µM
CYP3A Activity CYP3A4 mRNA
0
5
10
15
20
25
30
35
2 4 8 24 48 72 96
Time (Hrs)
(CY
P3A
4 m
RN
A C
on
ten
t}
Rela
tiviv
e F
old
-Over-
Co
ntr
ol
Ritonavir 0.1 µM
Ritonavir 10 µM
0
0.5
1
1.5
2
2 4 8 24 48 72 96
In Vitro Disposition of Omeprazole in SC-PHHs
50 µM Omeprazole in Cultures of Primary Human Hepatocytes
0
20
40
60
80
100
120
0.25 0.5 2 6 24 48 72
Time (hr)
[OM
P]
µM
Extracellular
Intracellular
Total
Time[Inducer]
Added
Percent in
Monolayer
Supernatant
Concentratio
n (µM)
Est. Monolayer
Concentration
(µM)†
15 min OMP (50 µM) 24.4 80.9 26
30 min OMP (50 µM) 23.4 75.2 23
2 hr OMP (50 µM) 21.40 75.2 20
6 hr OMP (50 µM) 22.1 48.6 14
24 hr OMP (50 µM) 21.47 15.66 4.28
48 hr OMP (50 µM) 17.28 7.51 1.57
72 hr OMP (50 µM) 14.36 2.12 0.36
†Assumed 4 µL volume per million cells
YALE JOURNAL OF BIOLOGY AND MEDICINE 69 (1996), pp. 203-209.
Smith et al. J. Pharm. Sci. 2012. v.101(10):3898.
Isolated Primary Liver Cells
Rapidly De-differentiate Once
Removed from Liver Tissue
Tox21 Evolution: Predictive Toxicology Screening
• Physiologically-relevant in vitro screening models