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

Yvonne.Will@Pfizer.com

48 Drugs Were Withdrawn for Safety Reasons

Between 1990-2007

18 Hepatotoxic 21 Cardiotoxic 9 Others

Cerivastatin Troglitazone

Tolcapone

Nefazodone

Hepatotoxicity Cardiovascular

Mitochondrial Impairment of Drugs Receiving Black Box Warnings

Antivirals

Abacavir

Didanosine

Emtricitabine

Entecavir

Emtricitabine

Lamivudine

Nevirapine

Telbivudine

Tenofovir

Tipranavir

Stavudine

Zalcitabine

Zidovudine

Anti-Cancer

Flutamide

Dacarbazine

Gemtuzumab

Methotrexate

Pentostatin

Tamoxifen

Antibiotics

Isoniazid

Ketoconazole (oral)

Streptozocin

Trovafloxacin

CNS

Dantrolene

Divalproex Sodium

Felbamate

Naltrexone

Nefazodone

Valproic Acid/

Hypertension

Bosentan

Anthracyclines

Daunorubicin

Doxorubicin

Epirubicin

Idarubicin

NSAIDs

Celecoxib

Diclofenac

Diflunisal

Etodolac

Fenoprofen

Ibuprofen

Indomethacin

Ketoprofen

Mefenamic acid

Meloxicam

Naproxen

Nabumetone

Oxaprozin

Piroxicam

Salsalate

Sulindac

Thioridazine

Tolmetin

Anaesthetic

Bupivacaine

Anti-Cancer

Arsenic Trioxide

Cetuximab

Denileukin diftitox

Mitoxantrone

Tamoxifen

Beta-Blocker]

Atenolol

Antiarhythmic

Amiodarone (oral)

Disopyramide

Dofetilide

Ibutilide

CNS

Amphetamines

Atomoxetin

Droperidol

Methamphetamine

Pergolide

Diabetes

Pioglitazone

Rosiglitazone

Early mitochondrial assessment

allows the identification of

compounds with the desired

efficacy profile, but without

ancillary liabilities.

Dykens et al. (2007) Expert Rev. Mol. Diagn. 7,161-175, with permission

Many Different Mechanisms Lead to Mitochondrial

Dysfunction

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• Drug withdrawn from the market exhibit mitochondrial liabilities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

All ADP phosphorylated

Basal Respiration

Maximum Respiration

ADP-Driven O2

Uncoupling

Inhibition

Time (min) 20min

• Phosphorescent

• Water-soluble

• Cell non-invasive

• Non-cytotoxic

• Stable

• Time resolved or prompt

• Compatible with any reader

• Large stoke shift allows for

high signal to noise ratio

• multiplex with “green dyes”

No

rma

lis

ed

In

ten

sit

y

Wavelength (nm)

0

0.2

0.4

0.6

0.8

1

300 400 500 600

0

1

2

3

4

5

600 620 640 660 680 700

Wavelength (nm)

Fo

ld I

nc

rea

se

Deoxygenated

Air-saturated

Oxygen consumption Measurement in

Isolated Mitochondria

Assay Set-Up

PC

Mitochondria with

substrate +/- ADP

96 Well Plate

Probe

Mineral Oil

Plate reader

Basal respiration

Output of Fluorescent Data from the Oxygen-Sensing

Probe with Isolated Mitochondria

Vehicle

Uncoupler

Inhibitor

Dykens et al. (2007) Expert Rev. Mol. Diagn. 7,161-175, with permission

State 2

0 2 4 6 8 10 12 14 16 18 20

0

2000

4000

6000

8000

10000

Muraglitazar

Rosiglitazone

Ciglitazone

Darglitazone

Pioglitazone

Troglitazone

Control

Time (min)

RF

U(m

ean

+ S

E)

State 3

0 2 4 6 8 10 12 14 16 18 20

0

3000

6000

9000

12000

15000

Muraglitazar

Rosiglitazone

CiglitazoneDarglitazone

Pioglitazone

Troglitazone

Control

Time (min)

RF

U(m

ean

+ S

E)

Drugs present at 25nmol/mg mitochondrial protein. N=4, except for controls N=48.

Mitochondrial Effects of Thiozolidinediones Vary

Nadanaciva et al. (2007) Toxicol. Appl.

Pharmacol. 223, 277-287, with permission

State 2

0 2 4 6 8 10 12 14 16 18 20

0

2000

4000

6000

8000

10000

Muraglitazar

Rosiglitazone

Ciglitazone

Darglitazone

Pioglitazone

Troglitazone

Control

Time (min)

RF

U(m

ean

+ S

E)

State 3

0 2 4 6 8 10 12 14 16 18 20

0

3000

6000

9000

12000

15000

Muraglitazar

Rosiglitazone

CiglitazoneDarglitazone

Pioglitazone

Troglitazone

Control

Time (min)

RF

U(m

ean

+ S

E)

Drugs present at 25nmol/mg mitochondrial protein. N=4, except for controls N=48.

Withdrawn

Blackbox warning

Basal respiration

ADP-Driven

Withdrawn

Blackbox warning

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

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.

Circumventing the Crabtree Effect: The “Glucose-

Galactose” Model

Marroquin et al. (2007) Toxicol. Sci., 97, 539-547

0

20

40

60

80

100

120

0.001 0.01 0.1 1

[Rotenone] M

% A

TP

Co

ntr

ol

*

AT

P

(% C

on

tro

l)

[Antimycin] M

*

AT

P

(% C

on

tro

l)

0

20

40

60

80

100

120

0.01 0.1 1 10 100 1000

[FCCP] M

*

*

AT

P

(% C

on

tro

l)

0

20

40

60

80

100

120

0.0001 0.001 0.01 0.1 1 10

[Oligomycin] M

*

AT

P

(% C

on

tro

l)

Cells Grown in Galactose Become Susceptible to

Mitochondrial Toxicants

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.

0

20

40

60

80

100

120

0.0001 0.001 0.01 0.1 1 10

*

Cells Grown in Galactose are More Susceptible to

Mitochondrial Toxicants such as Nefazodone

0

20

40

60

80

100

120

0.01 0.1 1 10 100Concentration (µM)

% A

TP

Co

ntr

ol

8.98

38.4

0

5

10

15

20

25

30

35

40

45

50

Galactose Glucose

Cell Culture Media

IC5

0 (

µM

)

Dykens et al. (2008) Toxicol. Sci., 103, 335-345

Correlating The RST and HepG2 Glu-Gal Assays

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”

A compound could belong to any of the following categories:

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

Glycolysis

NAD+

NADH ATP

O2

Glucose

Pyruvate

G6P

Acetyl-CoA

Lactic

Acid

H2O

ADP

O2

ATP

ADP

Extracellular Acidification Rate of Cells is a Measure of Glycolysis

Phenformin and Buformin Decrease Oxygen

Consumption in HepG2 cells

DMSO

Metformin

Buformin

Phenformin

125M 125M 125M 125M

Dykens et al. (2008) Toxicol. Appl. Pharmacol. 233, 203-210, with permission

Oxygen Consumption Rate

HepG2 cells

% c

han

ge i

n o

xyg

en

co

nsu

mp

tio

n

Phenformin and Buformin Cause Media Acidification

Phenformin

Buformin

Metformin DMSO

125M 125M 125M 125M

HepG2 cells

Extracellular Acidification Rate %

ch

an

ge i

n p

H

Dykens et al. (2008) Toxicol. Appl. Pharmacol. 233, 203-210, with permission

Correlating Cellular Respiration and Cell Viability

O2 consumption

Extracellular

acidification

(Glycolysis)

Cell viability

(ATP levels)

Comment

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

in vehicle-treated cells

mtDNA-encoded protein

in cells grown in 40 M Linezolid

Nuclear DNA-encoded protein

in cells grown in 40 M Linezolid

Nuclear DNA-encoded protein

in vehicle-treated cells

• 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.

Summary:

Risk Factors Converge to Yield

Idiosyncratic Toxicity

Mitochondrial make up

Toxicology and Applied Pharmacology, Volume 264, Issue 2, 2012, 167 - 181

.

MEFs from different mouse strains respond

differently to nefazodone when grown in glucose and galactose

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.