In Vitro Approaches to Assess Mitochondria-Mediated Drug Toxicity: Advantages and Limitations and a Decade of Learning's
Yvonne Will, Ph.D Pfizer Research & Development Groton, CT
48 Drugs Were Withdrawn for Safety Reasons Between 1990-2007
Cerivastatin Troglitazone Tolcapone Nefazodone
18 Hepatotoxic 21 Cardiotoxic 9 Others Mitochondrial Impairment of Drugs Receiving Black Box Warnings Hepatotoxicity Cardiovascular
Antivirals Antibiotics Anthracyclines Anti-Cancer Abacavir Isoniazid Daunorubicin Arsenic Trioxide Didanosine Ketoconazole (oral) Doxorubicin Cetuximab Emtricitabine Streptozocin Epirubicin Denileukin diftitox Entecavir Trovafloxacin Idarubicin Mitoxantrone Emtricitabine Tamoxifen Lamivudine CNS NSAIDs Nevirapine Dantrolene Celecoxib Beta-Blocker] Telbivudine Divalproex Sodium Diclofenac Atenolol Tenofovir Felbamate Diflunisal Tipranavir Naltrexone Etodolac Antiarhythmic Stavudine Nefazodone Fenoprofen Amiodarone (oral) Zalcitabine Valproic Acid/ Ibuprofen Disopyramide Zidovudine Indomethacin Dofetilide Hypertension Ketoprofen Ibutilide Anti-Cancer Bosentan Mefenamic acid Flutamide Meloxicam CNS Dacarbazine Naproxen Amphetamines Gemtuzumab Nabumetone Atomoxetin Methotrexate Oxaprozin Droperidol Pentostatin Piroxicam Methamphetamine Tamoxifen Salsalate Pergolide Sulindac Thioridazine Diabetes Tolmetin Pioglitazone Rosiglitazone Anaesthetic Bupivacaine Early mitochondrial assessment allows the identification of compounds with the desired efficacy profile, but without ancillary liabilities. Many Different Mechanisms Lead to Mitochondrial Dysfunction
Dykens et al. (2007) Expert Rev. Mol. Diagn. 7,161-175, with permission Objectives/Outline:
• Drug withdrawn from the market exhibit mitochondrial liabilities
• Assays to detect mitochondrial toxicity
• Assay for measuring Oxygen consumption of isolated mitochondria.
• Cell viability assay in (a) Glucose medium, (b) Galactose medium.
• Assay for measuring Oxygen consumption and extracellular acidification of cells.
• Assays for measuring changes in mtDNA and mtDNA-encoded protein levels in cells.
• Summary
Polarographic Mitochondrial Respiration
Drug Mitos Substrate ADP
Basal Respiration Inhibition
Maximum Respiration O2 ADP-Driven All ADP phosphorylated
Uncoupling Time (min) 20min Oxygen consumption Measurement in Isolated Mitochondria
• Phosphorescent • Water-soluble 1 5 Deoxygenated • Cell non-invasive
0.8
4 • Non-cytotoxic
0.6 3 • Stable
Intensity
0.4 2 • Time resolved or prompt
Fold Fold Increase Air-saturated
0.2 1 • Compatible with any reader Normalised • Large stoke shift allows for 0 0 300 400 500 600 600 620 640 660 680 700 high signal to noise ratio Wavelength (nm) Wavelength (nm) • multiplex with “green dyes”
Assay Set-Up
96 Well Plate
Mitochondria with Probe substrate +/- ADP
Mineral Oil
PC
Plate reader Output of Fluorescent Data from the Oxygen-Sensing Probe with Isolated Mitochondria Basal respiration
Uncoupler
Inhibitor
Vehicle Dykens et al. (2007) Expert Rev. Mol. Diagn. 7,161-175, with permission Mitochondrial Effects of Thiozolidinediones Vary
State 2 BasalState respiration 2 Ciglitazone Ciglitazone Troglitazone 10000 Troglitazone 10000 Darglitazone Withdrawn Darglitazone
) 8000 Rosiglitazone
) 8000
Rosiglitazone
SE + SE 6000 Pioglitazone + 6000 MuraglitazarPioglitazone RFU Muraglitazar Blackbox warning RFU 4000
(mean 4000
(mean Control 2000 Control 2000 0 0 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 Time (min) Time (min)
State 3 ADPState-Driven 3 15000 15000 Control Control 12000
12000
) )
SE 9000 + SE 9000 Pioglitazone + Pioglitazone Blackbox warning Rosiglitazone RFU 6000 Rosiglitazone RFU 6000 Muraglitazar
(mean Muraglitazar (mean 3000 Ciglitazone 3000 DarglitazoneCiglitazone Darglitazone Withdrawn 0 Troglitazone 0 Troglitazone 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 Time (min) Time (min) Nadanaciva et al. (2007) Toxicol. Appl. Drugs present at 25nmol/mg mitochondrial protein. N=4, except for controls N=48. Drugs present at 25nmol/mg mitochondrial protein. N=4, except for controls N=48. Pharmacol. 223, 277-287, with permission Dykens JA, Jamieson J, Marroquin L, Nadanaciva S, Billis PA, Will Y. Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro. Toxicol Appl Pharmacol. 2008 Dec 1;233(2):203-10. Bioaccumulation Will Y, Dykens JA, Nadanaciva S, Hirakawa B, Jamieson J, Marroquin LD, Hynes J, Patyna S, Jessen BA. Effect of the multi-targeted tyrosine kinase inhibitors imatinib, dasatinib, sunitinib, and sorafenib on mitochondrial function in isolated rat heart mitochondria and H9c2 cells. Toxicol Sci. 2008 Nov;106(1):153-61. Liver toxicity of sorafenib Dykens JA, Jamieson JD, Marroquin LD, Nadanaciva S, Xu JJ, Dunn MC, Smith AR, Will Y. In vitro assessment of mitochondrial dysfunction and cytotoxicity of nefazodone, trazodone, and buspirone. Toxicol Sci. 2008 Jun;103(2):335-45. rank order correct, need additional risk factors Nadanaciva S, Dykens JA, Bernal A, Capaldi RA, Will Y. Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration. Toxicol Appl Pharmacol. 2007 Sep 15;223(3):277-87. rank order correct, Cmax , accumulation
Summary: Oxygen Consumption of Isolated Mitochondria
• Values: • Identifies inhibitors and uncouplers of the electron transport chain • High-throughput; highly reproducible; easy to use • May be used to identify structure-activity-relationships • Learnings: Can rank order compounds within a series for their mitochondrial toxicity effects. Rankorder in most cases correlates with toxicity profile in the clinic. Cmax information strengthen readout ( exceptions like statins and biguanides)
• Limitations: • Can potentially overestimate toxicity since the isolated organelle is being used • Identifies only immediate (acute) effects; may need to pre-incubate mitochondria with drug • Does not take into account conversion of parent drug reactive/inactive metabolites
Aerobically Poised Cell Models oxygen & pH sensors Objectives/Outline:
• Drug withdrawn from the market exhibit mitochondrial liabilities
• Assays to detect mitochondrial toxicity
• Assay for measuring Oxygen consumption of isolated mitochondria.
• Cell viability assay in (a) Glucose medium, (b) Galactose medium.
• Assay for measuring Oxygen consumption and extracellular acidification of cells.
• Assays for measuring changes in mtDNA and mtDNA-encoded protein levels in cells.
• Summary Circumventing the Crabtree Effect: The “Glucose- Galactose” Model
Crabtree Effect (1929): inhibition of respiration by elevated glucose.
Warburg Effect (1929): aerobic glycolysis yields lactate despite competent mitochondria.
Contemporary cell culture often uses 25mM glucose media (5X physiological!)
Transformed cells are often characterized by low rates of O2 consumption & resistance to mitotoxicants.
Marroquin et al. (2007) Toxicol. Sci., 97, 539-547 Cells Grown in Galactose Become Susceptible to Mitochondrial Toxicants 120 120
100 100
80 * 80
* *
60 60 ATP
ATP 40 40
(% Control) (% (% Control) (% % ATP Control % ATP 20 20 0 0 0.001 0.01 0.1 1 0.0001 0.001 0.01 0.1 1 10 [Rotenone] M [Antimycin] M
120 120
100 100
* *
80 80
60 60 ATP
40 ATP 40 * (% Control) (% 20 Control) (% 20 0 0 0.0001 0.001 0.01 0.1 1 10 0.01 0.1 1 10 100 1000
[Oligomycin] M [FCCP] M Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y. Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol Sci. 2007 Jun;97(2):539-47. Cells Grown in Galactose are More Susceptible to Mitochondrial Toxicants such as Nefazodone 120
100
80
50 45 38.4 40 35 60 30 25 20
IC50(µM) 15
% ATP Control ATP % 8.98 40 10 5 0 Galactose Glucose 20 Cell Culture Media
0 0.01 0.1 1 10 100 Concentration (µM)
Dykens et al. (2008) Toxicol. Sci., 103, 335-345 Correlating The RST and HepG2 Glu-Gal Assays
A compound could belong to any of the following categories:
RST assay HepG2 Glucose-Galactose assay Mechanism of Toxicity
More toxic in Gal than Glu Toxicity primarily through mitochondrial effects (<5% of compounds) Not toxic in either medium Compound may be converted to inactive metabolite or does not get into cells Equally toxic in both media Multiple mechanisms of toxicity (most of RST positives) - More toxic in Gal than Glu Compound may affect apoptosis, impair fatty acid transport, activate HIF-1a - More toxic in Glu than Gal Compound may impair glycolysis
- Equally toxic in both media Toxicity primarily through non-mitochondrial “off-targets” Objectives/Outline:
• Drug withdrawn from the market exhibit mitochondrial liabilities
• Assays to detect mitochondrial toxicity
• Assay for measuring Oxygen consumption of isolated mitochondria.
• Cell viability assay in (a) Glucose medium, (b) Galactose medium.
• Assay for measuring Oxygen consumption and extracellular acidification of cells.
• Assays for measuring changes in mtDNA and mtDNA-encoded protein levels in cells.
• Summary The O2 Consumption Rate of Cells is a Measure of Mitochondrial Respiration
Glucose O2
G6P O2
+ NAD H2O Acetyl-CoA ATP Glycolysis NADH ATP ADP
ADP Pyruvate
Lactic Acid
Extracellular Acidification Rate of Cells is a Measure of Glycolysis Phenformin and Buformin Decrease Oxygen Consumption in HepG2 cells
Oxygen Consumption Rate
125M 125M 125M 125M
DMSO Metformin Buformin
Phenformin % change in % oxygen consumption
HepG2 cells
Dykens et al. (2008) Toxicol. Appl. Pharmacol. 233, 203-210, with permission Phenformin and Buformin Cause Media Acidification
Extracellular Acidification Rate 125M 125M 125M 125M
Phenformin
Buformin % change in % pH
Metformin DMSO
HepG2 cells
Dykens et al. (2008) Toxicol. Appl. Pharmacol. 233, 203-210, with permission Correlating Cellular Respiration and Cell Viability
Extracellular
O2 consumption acidification Cell viability Comment (Glycolysis) (ATP levels)
Decrease Increase Decrease Compound inhibits mitochondrial respiration
Increase Increase Decrease Compound uncouples mitochondrial respiration
Decrease Increase No impairment Cells compensate for mitochondrial impairment
Increase Increase No impairment Cells compensate for mitochondrial impairment
Decrease Decrease Decrease Compound impairs non-mitochondrial
Decrease Decrease No impairment Cells compensate for mitochondrial impairment Objectives/Outline:
• Drug withdrawn from the market exhibit mitochondrial liabilities
• Assays to detect mitochondrial toxicity
• Assay for measuring Oxygen consumption of isolated mitochondria.
• Cell viability assay in (a) Glucose medium, (b) Galactose medium.
• Assay for measuring Oxygen consumption and extracellular acidification of cells.
• Assays for measuring changes in mtDNA and mtDNA-encoded protein levels in cells.
• Summary High content screening approach for identifying antibacterials and anti-retrovirals that cause mitochondrial toxicity
mtDNA-encoded protein Nuclear DNA-encoded protein in cells grown in 40 M Linezolid in cells grown in 40 M Linezolid
mtDNA-encoded protein Nuclear DNA-encoded protein in vehicle-treated cells in vehicle-treated cells Summary:
• Many, but not all, drugs causing organ toxicity have a mitochondrial liability. • Elevated serum liver enzymes = hepatocyte death • Lactic acidosis is classic hallmark.
• Depending on potency, if a drug has a mitochondrial liability, it will have consequences. Importance of Cmax – Bioaccumulation alters PK. – >10,000-fold concentration of some cations in matrix over plasma. – Additional factors increase risk (BSEP) not discussed today – Phys-chem property space (promiscuity) drives mitotox • Severity of such adverse effects is idiosyncratic. – Function of organ history and genetics (incl. mtDNA). • Preclinical assessments are done in young, perfectly healthy animals. – Threshold effects and physiological scope. Risk Factors Converge to Yield Idiosyncratic Toxicity
Mitochondrial make up MEFs from different mouse strains respond . differently to nefazodone when grown in glucose and galactose
Toxicology and Applied Pharmacology, Volume 264, Issue 2, 2012, 167 - 181 Acknowledgements • Lisa Marroquin, MS • Dr. James Dykens
• Dr. James Hynes • Dr Richard Fernandes
• Dr. Roderik Capaldi • Autumn Bernal, BS
• Dr David Ferrick
• Dr. Sashi Nadanaciva • Rachel Swiss, Payal Rana
References
1. Nadanaciva S, Aleo MD, Strock CJ, Stedman DB, Wang H, Will Y. Toxicity assessments of nonsteroidal anti-inflammatory drugs in isolated mitochondria, rat hepatocytes, and zebrafish show good concordance across chemical classes. Toxicol Appl Pharmacol. 2013 Oct 15;272(2):272-80. 2. Swiss R, Niles A, Cali JJ, Nadanaciva S, Will Y. Validation of a HTS-amenable assay to detect drug-induced mitochondrial toxicity in the absence and presence of cell death. Toxicol In Vitro. 3. James Hynes, Sashi Nadanaciva, Rachel Swiss, Conn Carey, Sinead Kirwan, Yvonne Will. A high- throughput dual parameter assay for assessing drug-induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays. Toxicol.Vitro, Toxicol In Vitro. 2013 Mar;27(2):560-9 4. Dykens, J and Will Y, 2012. Mitochondrial Toxicity Encyclopedia of Toxicology, 3rd Edition, Elsevier 5. Nadanaciva S, Rana P, Beeson GC, Chen D, Ferrick DA, Beeson CC, Will Y. Assessment of drug-induced mitochondrial dysfunction via altered cellular respiration and acidification measured in a 96-well platform. J Bioenerg Biomembr. 2012 Aug;44(4):421-37. 6. Naven RT, Swiss R, McLeod JK, Will Y, Greene N. The Development of a Structure-Activity Relationships for Mitochondrial Dysfunction: Uncoupling of Oxidative Phosphorylation. Toxicol Sci. 2012 Sep 13. 7. Pereira CV, Oliveira PJ, Will Y, Nadanaciva S. Mitochondrial bioenergetics and drug-induced toxicity in a panel of mouse embryonic fibroblasts with mitochondrial DNA single nucleotide polymorphisms. Toxicol Appl Pharmacol. 2012 Aug 4. 8. Rana P, Anson B, Engle S, Will Y. Characterization of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes: Bioenergetics and Utilization in Safety Screening. Toxicol Sci. 2012 Jul 27. 9. Nadanaciva S, Will Y. The role of mitochondrial dysfunction and drug safety. IDrugs. 2009 Nov;12(11):706-10. 10. Dykens JA, Jamieson J, Marroquin L, Nadanaciva S, Billis PA, Will Y. Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro. Toxicol Appl Pharmacol. 2008 Dec 1;233(2):203-10.
References Continued: 11. Will Y, Dykens JA, Nadanaciva S, Hirakawa B, Jamieson J, Marroquin LD, Hynes J, Patyna S, Jessen BA. Effect of the multi-targeted tyrosine kinase inhibitors imatinib, dasatinib, sunitinib, and sorafenib on mitochondrial function in isolated rat heart mitochondria and H9c2 cells. Toxicol Sci. 2008 Nov;106(1):153-61. 12. Dykens JA, Jamieson JD, Marroquin LD, Nadanaciva S, Xu JJ, Dunn MC, Smith AR, Will Y. In vitro assessment of mitochondrial dysfunction and cytotoxicity of nefazodone, trazodone, and buspirone. Toxicol Sci. 2008 Jun;103(2):335-45. Dykens JA, Will Y. The significance of mitochondrial toxicity testing in drug development. Drug Discov Today. 2007 Sep;12(17- 18):777-85. Review. 13. Nadanaciva S, Dykens JA, Bernal A, Capaldi RA, Will Y. Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration. Toxicol Appl Pharmacol. 2007 Sep 15;223(3):277-87. 14. Will Y, Hynes J, Ogurtsov VI, Papkovsky DB. Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes. Nat Protoc. 2006;1(6):2563-72. 15. Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y. Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol Sci. 2007 Jun;97(2):539-47. 16. Nadanaciva S, Bernal A, Aggeler R, Capaldi R, Will Y. Target identification of drug induced mitochondrial toxicity using immunocapture based OXPHOS activity assays. Toxicol In Vitro. 2007 Aug;21(5):902-11 17. Dykens JA, Marroquin LD, Will Y. Strategies to reduce late-stage drug attrition due to mitochondrial toxicity. Expert Rev Mol Diagn. 2007 Mar;7(2):161-75. 18. Hynes J, Marroquin LD, Ogurtsov VI, Christiansen KN, Stevens GJ, Papkovsky DB, Will Y. Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen- sensitive probes. Toxicol Sci. 2006 Jul;92(1):186-200.