franziska boess, adrian roth pharmaceutical sciences roche...

Comparative Assessment of 3D (Liver)

Models for Predictive Toxicology

Franziska Boess, Adrian Roth Pharmaceutical Sciences Roche Innovation Center Basel

Early Safety: Exclusion of a drug candidate before

expenditure of resources , time and animals:

“Fail early – fail cheap”

(Bass, A. S.et al., (2009). J. Pharmacol. Toxicol. Methods 60, 69–78.)

Status Today: Specific endpoints addressed early on using

in vitro models:

• Metabolism & Induction

• Genetic Toxicity

• Cardiac Function (hERG)

• Embryotoxicity (EST)

Well established specific endpoints which allow

to make decisions based on in vitro assay

Thorough validation of assay required

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Predictive In Vitro Toxicology:

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Major Challenge for Early Safety Prediction:

Organ Toxicity

• Complex in nature –

develops over longer time

• Involves multitude of factors

& interplay of different cell

types

• Often displays species

dependency

→ Difficult to address in vitro!

«…These events are seldom recapitulated in molecular detail, kinetics, dynamics or cellular metabolic processing in

simplified in vitro models (…) no in vitro model completely mimics all complexities of (…) organtoxicity in vivo…”

(Astashkinaa et al., Pharmacology & Therapeutics Volume 134(1), April 2012)

“…may escape detection via simple screening tools when using e.g. cell line & cytotoxicity endpoint…”

(Zhiwu Lin and Yvonne Will, Toxiclogical Sciences Volume 126(1): 114–127, 2012)

Predictive Toxicology in Roche’s Mechanistic

Safety Group

Safety Target assessment

• Target expression across species

and organs, incl. pathway analysis

in cell models

Predictive in vitro assays for

Organ Toxicities

• Liver Toxicity

• Embryo Toxicity

• Bone-marrow Toxicity

• Mitochondrial Toxicity

• Kidney Toxicity

Mechanistic in vitro assays on-demand

• Issue resolution & de-risking by customized approaches

• Assess human relevance of pre-clinical in vivo findings by

use of human cell models

Prioritization of series

• Compare to competitors

• Differentiate candidates

learnings

Case example: pre-neoplastic liver foci

Putative non-genotoxic carcinogen which induced liver proliferation and later on

neoplastic changes in vivo in rats, but not in non-rodent tox species (dog):

Early signals:

• Proliferation

• Glycogen

accumulation

• Gene expression

changes

Project Team requested elucidation of mechanism and/or in vitro species comparison with

emphasis on proliferation as most important endpoint


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MOUSE HUMANRAT DOG

1) Glycogen-

Accumulation

In Vivo Observation:

- Early Markers In Vivo:

In Vitro Species Comparison of early markers:

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CCNB1 CCNG1 CDKN3 MDM20

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0Ccnb1 Ccng1 Cdkn3 Mdm2

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2) Early gene expression

changes (cell cycle

regulators)

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MOUSE HUMANRAT DOG

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Recapitulation of early features of the finding in «simple» primary hepatocyte cultures


3) Hepatocyte

proliferation

In Vivo Observation: In Vitro Species Comparison of early markers:

vehicle

30 µM

150 µM

EGF

Recapitulation of proliferation not reliably possible in the primary hepatocyte cultures nor

hepatocyte/NPC co-cultures

3D culture system


3) Hepatocyte

proliferation

In Vivo Observation: In Vitro Species Comparison of early markers:

vehicle

30 µM

150 µM

EGF

Recapitulation of proliferation

reliably possible only in a more

complex multi-cell type 3D

culture system

Transwell-based in vitro 3D Liver Model

(Regenemed)

9

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Synthesis of

Albumin,

Transferrin,

Fibrinogen

Synthesis of

Urea

Synthesis of

Glycogen

after

Insulin

stimulation

Validation: Basic Liver Functions

Human Liver:

Albumin: 60μg/106 Hepatocytes/Day (Khalil et al., 2001)

Transferrin: 6-14μg/106 Hepatocytes/Day (Acharya and Dimichele, 2008)

Fibrinogen: 5-9μg/106 Hepatocytes/Day (Bates and McClain, 1981)

Urea: 182 μg/106 Hepatocytes/Day (Khalil et al., 2001; Rudman et al.,1973)

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• Stable P450 enzyme activity and

induction for >90 days of continuous

culture (similar data for CYP2C9, CYP1A1)

• Despite disappearance of parent

compound no retrieval of metabolites

Validation: Inflammatory reaction & Drug

metabolism

Validation using Reference Drugs

Troglitazone 1997: First PPAR- Agonist for Treatment of Type 2 Diabetes

1999: Removed from Market after Reports of >40 cases of acute Liver failure

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Additional Readouts

Caspase3/7 >no Signal

ALT >no Signal

Therapeutic

DoseTroglitazone

Cmax=7 µM

Additional Readouts:

Caspase3/7 >activated

ALT >increased

No Toxicity

Toxicity

3D based assessement

rat

human


Trovafloxacin

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Therapeutic Dose

Trovafloxacin: Cmax=7 µM

Lovofloxacin: Cmax=23.8 µM

Trovafloxacine

Lovofloxacine

• antibacterial drug from the class of fluoroquinolones

• withdrawn from the market due to hepatotoxicity at least in 140, from which14

patients had acute hepatic failure

Key Features of this 3D Liver System: - Different Species available including Human

- Long term stability

- Drug Metabolism

- Inflammatory Cell Types

- Practical aspects (scaffolds, logistics)

- No retrieval of metabolites, despite disappearance of parent

suited for mechanistic investigations but not for more

routine testing of compounds

non-specific compound binding seems to be a problem

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Almost all of the test compounds showed high non-specific binding which needs

to be overcome before device can be used for DMPK applications

Drug binding to microfluidic device to assess likelihood of non-

specific binding affecting drug clearance measurements

Nicole Kratochwil, Patrick Heim; Screening, Enzymology and Automation Group, Non-Clinical Safety,

Basel.

Method:

• Compounds submitted in duplicate to inlet wells

of microfluidic device.

• Concentrations of remaining substance in inlet

chamber and that which flowed through to

outlet chamber assessed 24h later

• BLQ=Below Limit of Quantitation

Non-specific binding

CellASIC’s Pearl

Model Suitablity still Limited:

* Microgranuloma “collection” of immune cells (macrophages), which may indicate need to remove precipitates/damaged cells.

Case: Microganuloma* in 2

week cynomolgus study

(no finding in rat)

Q: Assess/compare

backup candidates

Use of assay systems containing

immuno-competent cells (KCs)

Regenemed Sytem

No “one fits all” model

Predictive Toxicology in our Roche’s mechanistic

Safety Group

Safety Target assessment

• Target expression across species

and organs, incl. pathway analysis

in cell models

Predictive in vitro assays for

Organ Toxicities

• Liver Toxicity

• Embryo Toxicity

• Bone-marrow Toxicity

• Mitochondrial Toxicity

• Kidney Toxicity

Mechanistic in vitro assays on-demand

• Issue resolution & de-risking by customized approaches

• Assess human relevance of pre-clinical in vivo findings by

use of human cell models

Prioritization of series

• Compare to competitors

• Differentiate candidates

learnings

Predicting Hepatotoxicity – Exposure Time

matters

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Hepregen’s

HepatoPac™ (2D)

hanging drop hepatocytes

spheroids (3D)

In vitro «POS» labelling based on effects on readouts (ALB, UREA, GSH, ATP) below a certain concentration threshold

Repeated/prolonged treatment time may decrease effect concentrations

Xiaoman Ang, Claudia McGinnis;

Mechanistic Safety Group, Roche

Innovation Center, Basel.

Increase Functionality over Time:

Micropatterning

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Increased and prolonged functionality

of hepatocytes:

• Viability over weeks

• Albumin and Urea production in

physiological range over weeks

• Retention of some metabolic

function (P450); P450 inducibility

establishing as current best model

for low clearance drug assessement

• Different species available

Medium Throughput (96-well)

Close to current cell culture technique:

cell numbers, handling, automation,

Imaging

Non-liver derived cell type present

(even different species)

Hepregen’s HepatoPac™

Xu et al., Tox. Sci. 2008,105(1):97–105

Khetani et al.,

Tox. Sci. 2014:132(1):107pp

Increase functionality over Time:

Microspheroids

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Increased and prolonged functionality

of hepatocytes:




• Retention of some metabolic

function (P450); P450 inducibility

Including Kupffer Cells !! Response

to LPS

Different species available

Medium Throughput (96-well)

Easy to handle, amenable to

automation

Low number of cells per tissue

Dense structure; speed of «perfusion»;

Immaging

InSphero’s InSight™

Microtisssues


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Rat 3D

>100

Rat 2D

<100

Hu 3D

<100

Hu 2D

>100

1 2

Hu 3D

<100

Hu 2D

>100

Hu 2D

>100

Hu 3D

>100

Preliminary data

1

2

DILI prediction

Thomson et al., Chem Res Toxicol. 2012 , 25(8):1616-32

DILI prediction in vitro panel (AZ)

Threshold concentrations:

EC50 below threshold additional hazard score

Current approach based on hazard identification/accumulation

qualitative predictions works reasonably well (70-80 sensitivity, >90% sensitivity)

Qualitative vs Quatintiative prediction

Gastroenterology 2014 146, 914-928.e1DOI: (10.1053/j.gastro.2013.12.032)

«Idiosyncratic» DILI

“Lessons learned from the fate of AstraZeneca’s drug pipeline: a five-

dimensional framework”

Cook et al., Nature Reviews Drug Discovery ,2014, 13:419-431

Non-idiosyncratic human or pre-

clinical DILI

Lack of quantitative predictive approach*, which would be needed for non-idiosyncratic

human DILI or pre-clinical DILI prediction

* Quantitative predictive approach = predicting whether and at which concentrations/doses pre-

clinical DILI (in animals) or human DILI (in clinical development) will be seen

Need for even more physiogical models and PK

– Addition of flow?

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Increased and prolonged functionality of

hepatocytes:




• Metabolic function (P450) remains

relatively high; P450 inducibility

Complex cell composition,

compartmentalization

Shear stress

PK aspects

Scaffolds material, tubing and high

surfaces often high unspecific

compound binding

Often low troughput and «complicated

handling»

Zyoxel’s Liver Chip

Gerlach’s minituarized

bioreactor

Summary

3D Cell Models do show improved physiological parameters

• Longterm culture, interplay of different cell types considered key for drug safety aspects

Improved physiology allows better in vitro to in vivo correlations

• Detection of drug candidates with unfavorable characteristics (qualitative: broader MoA

spectrum)

Technical Challenges:

• Ease of use

• Reproducibility (replacment of primary cells?)

• Throughput

• Non-specific binding

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From Cell Lines to «Organ-like» Models

Status: Application defines suited in vitro model

By use of M&S

However:

Struggling with prospective, quantitative predictions wrt to liver toxicity

outcomes in vivo

Roche:

Stefan Kustermann

Cristina Bertinetti

Claudia McGinnis

Sabine Sewing

Marcel Gubler

Annie Moisan

Radina Kostadinova

Xiaoman Ang

Nicole Kratochwil

Patrick Heim

Christoph Funk

Franz Schuler

Thomas Singer

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Acknowledgements

Regenemed:

Dawn Applegate

MIT:

Linda Griffith

InSphero:

Jens Kelm

Simon Messmer

Doing now what patients need next

3D Multicellular Systems are dynamic

Comparison of gene signatures at 1day, 2-, 4- & 6-weeks:

Benchmark against reference genes from human tissues

Earlier Time points Later Time points

Dif

fere

nt

tissu

e t

iyp

es

Doing now what patients need next

franziska boess, adrian roth pharmaceutical sciences roche...

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