Annie Borgne-Sanchez, PhD CEO-CSO
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1 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 Paris Parc Biocitech - 102, Av Gaston Roussel Romainville
2 Propidium iodide Propidium iodide Innovative screening platforms MiToxView Predictive toxicity assays MitoPathway Efficacy assays on tumor mitochondria (Bcl-2 antagonists, RC inhibitors ) AnnexinV-fitc MitoXpert Mitochondrial targeting and protection assays (mptp, MitoK-ATP, OS) AnnexinV-fitc 2
3 Drug-induced Mitochondrial Injuries Swelling & Dy m Loss FAO Inhibition Cyto c Release MMP ATP Production mtdna Quantification RC Complex Activity (COX, F0-F1 ATPase) Respiratory Chain Activity ROS Production From Begriche et al. J. Hepatol. (2011) Mitochondrial dysfunctions = key mechanism of hepatotoxicity (DILI)
4 Assays require high quality mitochondria Isolating Pure, Functional and Stable Mitochondria Whatever the Source Rodent organs Liver Heart Kidney Brain Isolated mouse liver mitochondria Purity: 95% Integrity: >90% Functional PTP: swelling with Ca 2+ Functional PTP: Ca 2+ + CsA Respiratory control index (4,2) Human cell lines HepaRG Wi-38 HT-29 PC3 Jurkat HepG2 Purity: 95% Integrity: >90% Isolated tumor mitochondria Functional PTP: swelling with Ca 2+ Functional PTP: Ca 2+ + CsA Respiratory control index (2,5)
5 Screening assays on isolated mitochondria Process to detect acute and direct compound mitochondrial toxicity Compound Predicting Drug-induced Organo-toxicity Rodent organs or Cell lines Isolated mitochondria Isolated & Functional Mitochondria Multiparametric assays (Spectrofluorimetry) in Human 200 compounds on 5 parameters / week Small molecules, peptides, recombinant proteins, natural extracts
6 O.D. RFU Cyto c (%) RFU Membrane Integrity & Functionality First stage screening to assess compound toxicity Membrane Integrity Functionality + Time (min.) Time (min.) Time (min.) Swelling Dy DY m loss Cyt. c release Oxygen consumption Complex (CI, CII) I & II Membranes permeabilization Proton flow alteration Outer membrane permeabilization Apoptosis marker Respiratory chain function Ctrl 100% : Calcium ClCCP Alamethicin Rotenone Oligomycin A
7 Additional Functional Parameters Second stage screening to assess compound toxicity Fatty acid b-oxidation ROS production ATP production Lipid metabolism (Subtrate: palmitoyl L carnitine) Oxidative stress (superoxide anion O 2 ) Metabolic activity 100% Ctrl : Rotenone Antimycin A Antimycin A 7
8 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) O.D 550nm Cox activity (KCN) 8
9 High Predictivity Demonstrated on 124 Compounds
10 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) 2 Cut-off: EC x Cmax variability, liver, multiple doses (Xu et al., 2008) EC µm MitochondrioToxic drug when: EC20 cut-off for at least 1 parameter Non MitochondrioToxic drug when: EC20 > cut-off for the 5 parameters
11 Examples of Dose-response Alpidem Diclofenac Perhexiline Troglitazone
12 Correlation with DILI & Mechanisms of Toxicity in vitro mitochondrial toxicity Compound Therapeutic Swelling DY m loss Cyto c O 2 cons CII O 2 cons CI Human class EC 20 µm EC 20 µm EC 20 µm EC 20 µm EC 20 µm hepatotoxicity Amantadine Anti-viral >800 >200 >400 >400 >400 N Amiodarone Anti-arythmic ND 2.6 < ND Y Clotrimazole Anti-fungal ND 23.9 > ND Y Diclofenac NSAID > > Y Gentamicin Antibiotic > >200 >200 ND Y Ketoconazole Anti-fungal > >400 > Y Lumiracoxib NSAID > > Y Manganese chloride Nutritive agent >800 >800 >800 >400 >400 N Methyldopa Antihypertensive >400 >400 ND > Y Molsidomine Anti-anginal >400 >400 >400 >200 >200 N Perhexiline Anti-anginal < Y Saquinavir Antiretroviral ND 10.3 > * 30.4* Y Sucrose Anti-asthenic >800 >800 >800 >800 >800 N Sulindac NSAID >200 >200 >200 > Y Tamoxifen Anti-cancer ND ND Y Tramadol Analgesic > >400 > * 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 Positive predictive value >82% (p<0.001) Sensitivity: 92%
13 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% Functional PTP: swelling with Ca 2+ Functional PTP: Ca 2+ + CsA High respiratory control index Suitable for screening Transmembrane potential O2 consumption (CII)
14 Detection of Hepatotoxicity using HepaRG Cells Assays on HepaRG treated cells for detection of: - long term mitochondrio-toxicity - metabolites mitochondrio-toxicity Dose-response and time-course (2, 7, 12 days) Cell viability Transmembrane potential ADP/ATP ratio mtros and global ROS production Global cellular respiration Quantification of mtdna (qpcr) Long term effects of AZT and ddc (12 days treatment)
15 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 : Moderate effect : Strong effect
16 Mitologics SAS Robert Debre Hospital 48, Bd Serurier Paris France Annie BORGNE-SANCHEZ, PhD For further information, visit our web site
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