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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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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