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SEACSAFETY & ENVIRONMENTAL ASSURANCE CENTRE
Date: 19/07/2017
Slow Clearance Xenobiotic MetabolismSlow clearance xenobiotic metabolism in primary human liver microtissue for better understanding of exposure science.
The transition to a pathways-based approach for risk assessment will require better understanding of exposure science. One of those approaches is to understand the bioavailability of chemicals in the
human body and define toxicologically relevant approaches that permit the estimation of internal dose in humans. Physiologically-based biokinetic (PBBK) modelling is recognised as a means to achieve
this, simulating whole-body toxicokinetic profiles by integrating chemical-independent (physiological) and chemical-dependent (absorption, deposition, metabolism and excretion, (ADME)) parameters.
Early prediction of hepatic clearance and metabolite formation in humans is often challenging, in particular for low-clearance compounds. Widely used approaches using human liver microsomes or human
primary hepatocytes (PHHs) suspensions allow only short term metabolism studies due to drastically reduced activity of xenobiotic metabolizing enzymes (e.g. in PHH assays the activity declines within 4
hours of hepatocyte isolation). Therefore the depletion of compounds or formed major metabolites cannot be determined with any significant turnover rate.
Novel 3D cell culture models have been shown to accurately mimic in vivo hepatic phenotypes at both the transcriptomic and protein level. A recent publication, Vorrink et al, shows a significant change of
endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid culture. In SEAC we are currently evaluating the metabolic profiles and intrinsic clearance rates
(CLint) of a range of chemicals across different in vitro PHH culture systems including the co-cultured liver spheroid (InSphero AG), suspensions, plated monoculture, and micropatterned co-cultures
Hepatopac. The aim of this work is to assess the potential application of 3D cellular models for prediction of CLint and metabolite profiling for low clearance compounds through longer duration incubations,
in comparison to other in vitro systems.
In conclusion, 3D spheroid models are applicable for the evaluation of the metabolic clearance of slowly metabolized xenobiotics that otherwise cannot be readily evaluated using current hepatocyte
suspension assays.
ChemicalsKnown Metabolism
Routes
Group(unit: ml/min per
kg)
In Vivo Clearance (ml/min per kg) Treated concentration
CL nonrenal Ref.
Diclofenac CYP2C9, CYP3A4, UGTintermediate CL
5< CL <15 7.6 Lin, 2015 1, 8, 10, 50 µM
Tolbutamide CYP2C9 probe,2C19 Low CL<5
0.38 Lin, 2015 10 µM
Warfarin CYP2C9,3A4 0.05 Lin, 2015 1, 10 µM
Read-out
Depletion rates
Metabolite profile
In single concentration (1µM), In vitro clearance (to be extrapolated to in vivo CL)
Metabolites formation (Quantitative and qualitative data)
Cell viability ATP production during 7 days incubation period without media change
Formation of major metabolite of slow CL chemicals, tolbutamide and warfarin in 168 hours
CYP2C9
tolbutamide
4-hydroxytolbutamide
R-warfarin S-warfarin
Diclofenac acyl
glucuronide
Diclofenac
Hydroxydiclofenac
acyl glucuronide
Absent in human hepatocytes
Major in vivo in human
High in human hepatocytes
Major in vivo in human
major metabolite formation
major metabolite formation ATP assay
Diclofenac metabolism across in vitro culture system
suspension PHH 1uM
Time (hour)
Ln
% r
em
ain
ing
0 1 2 3 4 5 60
1
2
3
4
5
1/slope -0.3338
R square 0.9994
2D collagen PHH 1uM
Time (hour)
Ln
% r
em
ain
ing
0 1 2 3 4 5 60
1
2
3
4
5
1/slope -2.675
R square 0.9817
2D monoculture PHH 1uM
Time (hour)
Ln
% r
em
ain
ing
0 1 2 3 4 5 62.5
3.0
3.5
4.0
4.5
5.0
1/slope -4.189
R square 0.9086
Spheroid [diclofenac 8 µM] TS7
Time (hour)
Ln
% r
em
ain
ing
0 24 48 72 96 120 1444.0
4.2
4.4
4.6
4.8
5.0
1/slope -577.3
R square 0.8678
Suspension culture 2D monoculture 2D collagen culture 3D cultureIn vivo intrinsic
clearance
in vitro half-life (hr) 0.23 1.85 2.90 400.15
Intrinsic CL (ml/min/kg) 308.0 25.6 16.4 17.8 583*
Depletion rate of diclofenac
3D liver spheroid
• Human primary hepatocyte with Kupffer cells
• Diameter: 225.7 ± 11.1 µm
• Cell number per one spheroid: 1,000 cells
Mi-Young Lee, Juliette Pickles, Sue Lovell, Richard Cubberley
One well in 96 well plate
Well diameter: 3.65 mm
OH-D
DAG
D
OH-D
DAG
D
OH-D
DAG
DOH-D
DAGD
(DAG)
(OH-D)
(D)
D. co-cultured spheroid
Metabolite formation rate [nmol per million cells, 10 µM]
B. 2D monoculture
Metabolite formation rate
[nmol per million cells]
were measured in different
in vitro hepatocyte culture
system. Hepatocytes were
treated with 10 µM
diclofenac.
C. 2D collagen culture
A. Suspension culture
4-OH-diclofenacHigh in human hepatocytes
Major in vivo in human
CYP2C9
7OH-warfarin X-hydroxywarfarin
(6OH-warfarin(?)**)
Liver spheroids were treated with 1 µM tolbutamide for 168 hours without
changing media. During incubation, cell viability assay was performed and the
results were above the defined threshold of 8 pmol ATP/microtissue.
Warfarin 10 µM was treated in liver spheroid for 168 hours without
changing media. During incubation, cell viability assay was performed
and the results were above the defined threshold of 8 pmol
ATP/microtissue.
ATP assay
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CENTRE OF EXCELLENCE – SAFETY &ENVIRONMENTAL SUSTAINABILITY SCIENCES
Based on in vivo clearance rates, compounds cleared above 15 ml/min per kg are high clearance, compounds cleared between 5 and 15
ml/min per kg are intermediate clearance, and compound cleared below 5 ml/min per kg are slow clearance. In this study, intermediate and
slow clearance compound groups were examined to determine in vitro clearance by using liver microtissue (spheroid),
OUR EXPOSURE-DRIVEN, NON-ANIMAL SAFETY RISK ASSESSMENT APPROACH
Is the product exposure
safe for the consumers?
What is the
internal
exposure to
the
ingredient?
Could this
ingredient
cause a toxic
effect? If so, at
what level of
exposure?
EXPOSURE
SCIENCE
What ingredient
exposure occurs
through product
use?
Which biological
process could the
ingredient stimulate
or block?
MIEs into
PATHWAYS
7 days, without change media
In vitro half-life = "LN(2)/slope"
Intrinsic CL= "Ln(2)/T1/2 *inc vol (ml)/cells*10^6*120*10^6 hepatocyte/gLiver * 25.7g Liver/kg BWT”
*, Hieronymus H et al, ‘A comparison of suspension, plated monoculture, and HµREL co-culture hepatocyte models for estimating intrinsic clearance of low-turnover drugs
2D monoculture(100 donors)
2D collagen culture (100 donors)
3D spheroid(10+1donors)
Suspension culture(50 donors)
EXPOSURE Research Priorities in SEAC
Current exposure assessment
In vivo kinetics
In vitro kinetics
Applications for risk assessment
Refinement to exposure based waiving (under construction)
Approaches to Exposure
Next Generation Methods
References.
Lin, 2015: AAPS J.17(2): 352–357.
Vorrink et al., 2017, FASEB J 31 (6), 2696-2708.
Safety and Environmental Assurance Centre, Unilever, Colworth Science Park, Sharnbrook, Bedford MK44 1LQ, United Kingdom
Comparison of scaled intrinsic clearance (CLint) values (mL/min/kg)
** Based on in vitro kinetic data in HepG2 recombinant of human P450s by Kaminsky LS. 1997