8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
Annie Borgne-Sanchez, PhD
CEO-CSO
Combinaison de tests sur mitochondries isolées et
cellules HepaRG pour la prédiction des risques
d’hépatotoxicité chez l’homme
Hôpital Robert Debré- 48, Bd Sérurier - 75019 Paris
Parc Biocitech - 102, Av Gaston Roussel - 93230 Romainville
Innovative screening platforms
MiToxView®
Predictive toxicity assays
MitoPathway®
Efficacy assays on tumor mitochondria
(Bcl-2 antagonists, RC inhibitors…)
Propidium iodide
Propidium iodide
MitoXpert®
Mitochondrial targeting and
protection assays
AnnexinV-fitc
(mPTP, MitoK-ATP, OS)
AnnexinV-fitc
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
2
Drug-induced Mitochondrial Injuries
FAO Inhibition
Swelling & Dym Loss
MMP
Cyto c Release
ATP Production
RC Complex Activity
(COX, F0-F1 ATPase)
mtDNA Quantification
Respiratory Chain Activity
ROS Production
From Begriche et al. J. Hepatol. (2011)
Mitochondrial dysfunctions = key mechanism of hepatotoxicity (DILI)
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
Assays require high quality mitochondria
Isolating Pure, Functional and Stable Mitochondria
Whatever the Source
Rodent organs
Isolated mouse liver mitochondria
Liver
Heart
Kidney
Brain
Purity: 95%
Integrity: >90%
Functional PTP:
swelling with Ca2+
Functional PTP:
Ca2+ + CsA
Respiratory control index (4,2)
Human cell lines
HepaRG
Wi-38
HT-29
PC3
Jurkat
HepG2
…
Isolated tumor mitochondria
Purity: 95%
Integrity: >90%
Functional PTP:
swelling with Ca2+
Functional PTP:
Ca2+ + CsA
Respiratory control index (2,5)
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
Screening assays on isolated mitochondria
Process to detect acute and direct compound mitochondrial toxicity
Compound
Predicting
Drug-induced
Organo-toxicity
Isolated mitochondria
Rodent organs
or
Cell lines
Isolated & Functional
Mitochondria
in Human
Multiparametric assays
(Spectrofluorimetry)
200 compounds on 5 parameters / week
Small molecules, peptides, recombinant proteins, natural extracts
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
Membrane Integrity & Functionality
First stage screening to assess compound toxicity
+
Time (min.)
DY m
Dy
loss
m loss
Swelling
Swelling
Ctrl 100% :
RFU
RFU
O.D.
Time (min.)
Functionality
Cyto c (%)
Membrane Integrity
Membranes
permeabilization
Proton flow alteration
Calcium
ClCCP
Time (min.)
Cyt
Cyt. c release
release
Outer membrane
permeabilization
Apoptosis marker
Alamethicin
Oxygen consumption
Oxygen
consumption
(CI,
CII)I & II
Complex
Respiratory
chain function
Rotenone
Oligomycin A
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
Additional Functional Parameters
Second stage screening to assess compound toxicity
Fatty acid b-oxidation
Lipid metabolism
(Subtrate: palmitoyl L–carnitine)
100% Ctrl :
Rotenone
ROS production
ATP production
Oxidative stress
Metabolic activity
(superoxide anion O2∙–)
Antimycin A
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
Antimycin A
7
Respiratory chain complex activity
Complex I (NADH quinone oxidoreductase)
Complex II (succinate deshydrogenase)
Complex III (coenzyme Q - cytochrome c oxidoreductase)
Complex IV (cytochrome c oxidase)
Complex V (F0-F1 ATP synthase)
Cox activity
O.D 550nm
(KCN)
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
8
High Predictivity Demonstrated on 124 Compounds
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
DILI Prediction Study Design
124 Drugs
87 Hepatotoxic drugs
37 Non-Hepatotoxic Drugs
Screening of mitochondrial dysfunction with multiparametric assays
on isolated mouse liver mitochondria:
swelling, membrane potential, cytochrome c release, respiration (CI and CII)
EC20  100 x Cmax
2 Cut-off:
variability, liver, multiple doses (Xu et al., 2008)
EC20  200 µM
MitochondrioToxic drug when: EC20  cut-off for at least 1 parameter
Non MitochondrioToxic drug when: EC20 > cut-off for the 5 parameters
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
Examples of Dose-response
Alpidem
Diclofenac
Perhexiline
Troglitazone
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
Correlation with DILI & Mechanisms of Toxicity
in vitro mitochondrial toxicity
Compound
Amantadine
Amiodarone
Clotrimazole
Diclofenac
Gentamicin
Ketoconazole
Lumiracoxib
Manganese chloride
Methyldopa
Molsidomine
Perhexiline
Saquinavir
Sucrose
Sulindac
Tamoxifen
Tramadol
Therapeutic
class
Anti-viral
Anti-arythmic
Anti-fungal
NSAID
Antibiotic
Anti-fungal
NSAID
Nutritive agent
Antihypertensive
Anti-anginal
Anti-anginal
Antiretroviral
Anti-asthenic
NSAID
Anti-cancer
Analgesic
Swelling
EC20 µM
DY m loss
EC20 µM
Cyto c
EC20 µM
O2 cons CII
EC20 µM
>800
ND
ND
>800
>800
>400
>200
>800
>400
>400
14.8
ND
>800
>200
9.0
>800
>200
2.6
23.9
137.9
49.0
243.0
148.8
>800
>400
>400
3.2
10.3
>800
>200
2.9
263.8
>400
<50
>800
>200
>200
>400
>800
>800
ND
>400
<25
>400
>800
>200
7.7
>400
>400
45.92
2.9
9.1
>200
>400
26.3
>400
>800
>200
88.4
21.1*
>800
>400
ND
>400
O2 cons CI
Human
EC20 µM
hepatotoxicity
>400
N
ND
Y
ND
Y
29.8
Y
ND
Y
2.9
Y
12.0
Y
>400
N
63.1
Y
>200
N
87.7
Y
30.4*
Y
>800
N
35.6
Y
ND
Y
238.7*
N
Porceddu M, Buron N, Roussel C, Labbe G, Fromenty B and Borgne-Sanchez A. “Prediction of Liver Injury induced by
chemicals in Human with a Multiparametric Assay on Isolated Mouse Liver Mitochondria”. Toxicol. Sci. 2012
Positive predictive value >82% (p<0.001)
Sensitivity: 92%
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
Detection of hepatotoxicity risks using HepaRG® mitochondria
Isolated HepaRG mitochondria
HepaRG = hepatocyte-like cells

Metabolic performances of primary hepatocytes

Indefinite growth capacity of hepatic cell lines
Purity: 95%
Integrity: >90%
High respiratory control index
Functional PTP:
swelling with Ca2+
Functional PTP:
Ca2+ + CsA
Suitable for screening
Transmembrane potential
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
O2 consumption (CII)
Detection of Hepatotoxicity using HepaRG® Cells
Cell viability
Assays on HepaRG® treated cells for detection of:
Transmembrane potential
- long term mitochondrio-toxicity
ADP/ATP ratio
- metabolites mitochondrio-toxicity
 mtROS and global ROS production
Dose-response and time-course (2, 7, 12 days)
Global cellular respiration
Quantification of mtDNA (qPCR)
Long term effects of AZT and ddC (12 days treatment)
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
Summary
Multiparametric study on isolated mitochondria combined with time-courses on
HepaRG differentiated cells allows identification of compounds with:
•
Direct and acute mitochondrial toxicity of the parent drug
Isolated Mito
HepaRG 2d
HepaRG 7d
HepaRG 12d
Cpd 1
•
Direct mitochondrial toxicity of a metabolite
Isolated Mito
HepaRG 2d
HepaRG 7d
HepaRG 12d
Cpd 2
•
Direct but long term mitochondrial toxicity (mtDNA)
Isolated Mito
HepaRG 2d
HepaRG 7d
HepaRG 12d
Cpd 3
•
Cytotoxic effect with downstream mitochondrial damages
Isolated Mito
HepaRG 2d
HepaRG 7d
HepaRG 12d
Cpd 4
•
no apparent mitochondrial toxicity
8e colloque du réseau MeetOchondrie – 10-13 mai 2015 – Guidel, France
: Moderate effect
: Strong effect
Mitologics SAS
Robert Debre Hospital
48, Bd Serurier
75019 Paris
France
Annie BORGNE-SANCHEZ, PhD
+33 689 728 213
[email protected]
For further information, visit our web site
www.mitologics.com