translational biomarker

47
Novel Translational Safety Biomarkers Peter O’Brien DVM, PhD, DVSc MRCVS, FRCPath Diplomate ACVP / ECVCP Pathology Department, Veterinary Sciences Center University College Dublin, Ireland The New England DRUG METABOLISM DISCUSSION GROUP SUMMER SYMPOSIUM Wednesday, June 4, 2008 Formerly @ Pfizer, Sandwich 2001-2006 Director ADL, NovaUCD Belfield Innovation Park, University College Dublin, Belfield, Dublin 4, Ireland.

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Novel TranslationalSafety Biomarkers

Peter O’BrienDVM, PhD, DVScMRCVS, FRCPathDiplomate ACVP / ECVCP Pathology Department,

Veterinary Sciences CenterUniversity College Dublin,Ireland

The New EnglandDRUG METABOLISM DISCUSSION GROUP

SUMMER SYMPOSIUMWednesday, June 4, 2008

Formerly @ Pfizer, Sandwich 2001-2006

Director ADL, NovaUCDBelfield Innovation Park,University College Dublin,Belfield, Dublin 4, Ireland.

Definition ofTranslational Safety Biomarker

Biomarker: a characteristic that is objectively measuredand evaluated as an indicator of normal biologic processes,pathogenic processes, or pharmacologic responses to atherapeutic intervention.“

Translational Biomarker: a biomarker that can beapplied in both a preclinical and clinical setting.

Safety Biomarker: a biomarker that reports atoxicological effect of a drug on an in vitro or in vivosystem.

Definitions (cont’d)

Validation: "characterization demonstrating to user “fit for purpose."

Distinctive features of safety biomarkers: Usually not drug or therapeutic area specific Typically measures unintended effect and off-target process More indicative of damage than function More representative of chemotype than pharmacologic effects More conservative and slower evolution More biofluid based Safety biomarker for a drug may be an efficacy biomarker for another

(& vv)

Overview of Case Presentationson Safety Biomarkers

Case 1: “Rules Based Medicine” – Luminex-based, multi-parameter, plasma profiling

Case 2: “The Troponins” – reverse translation – universalbiomarker for cardiac and muscle injury

Case 3: Glutamate dehydrogenase – universal biomarker forhepatic injury

Case 4: Tissue / serum biomarkers – oxidative stress andintermediary metabolism

Case 5: High content analysis – screening / monitoringhuman toxicity

Antigens Autoimmune/ID Serology

1. Apolipoprotein A12. â-2 Microglobulin3. C-Reactive Protein4. D-Dimer5. EGF6. Endothelin-17. Eotaxin8. Factor VII9. FGF-basic10. FGF-911. Fibrinogen12. GCP-213. GM-CSF14. Growth Hormone15. GST16. Haptoglobin17. IFN-ã18. IgA19. IL-1á20. IL-1â21. IL-222. IL-323. IL-424. IL-525. IL-626. IL-727. IL-1028. IL-1129. IL-12p7030. IL-1731. IL-1832. Insulin33. IP-10

34. KC/GROá35. Leptin36. LIF37. Lymphotactin38. MCP-139. MCP-340. MCP-541. M-CSF42. MDC43. MIP-1á44. MIP-1â45. MIP-1ã46. MIP-247. MIP-3â48. Myoglobin49. OSM50. RANTES51. SCF52. SGOT53. TIMP-154. Tissue Factor55. TNF-á56. TPO57. VCAM-158. VEGF59. von Willebrand Factor

60.â-2 Glycoprotein61. Insulin62. JO-163. Mitochondrial64. MPO65. PCNA66. PR3 (cANCA)67. Ribosomal P68. RNP69. Scl-7070. Smith71. SSA72. SSB

73. Adenovirus74.Clostridium piliforme(Tyzzer’s)75. Cytomegalovirus76. Ectromelia virus77. EDIM (Epidemic diarrhea of infant mice)78.Encephalitozoon cuniculi79. Hepatitis virus80. Lymphocytic choriomeningitis virus81. Minute virus82.Mycoplasma pulmonis83. Parvovirus84. Pneumonia virus of mice85. Polyoma virus86. Reovirus-387. Sendai virus88. Theiler’s mouse encephalomyelitis virus

Antigens Autoimmune/ID Serology

1. Apolipoprotein A12. â-2 Microglobulin3. C-Reactive Protein4. D-Dimer5. EGF6. Endothelin-17. Eotaxin8. Factor VII9. FGF-basic10. FGF-911. Fibrinogen12. GCP-213. GM-CSF14. Growth Hormone15. GST16. Haptoglobin17. IFN-ã18. IgA19. IL-1á20. IL-1â21. IL-222. IL-323. IL-424. IL-525. IL-626. IL-727. IL-1028. IL-1129. IL-12p7030. IL-1731. IL-1832. Insulin33. IP-10

34. KC/GROá35. Leptin36. LIF37. Lymphotactin38. MCP-139. MCP-340. MCP-541. M-CSF42. MDC43. MIP-1á44. MIP-1â45. MIP-1ã46. MIP-247. MIP-3â48. Myoglobin49. OSM50. RANTES51. SCF52. SGOT53. TIMP-154. Tissue Factor55. TNF-á56. TPO57. VCAM-158. VEGF59. von Willebrand Factor

60.â-2 Glycoprotein61. Insulin62. JO-163. Mitochondrial64. MPO65. PCNA66. PR3 (cANCA)67. Ribosomal P68. RNP69. Scl-7070. Smith71. SSA72. SSB

73. Adenovirus74.Clostridium piliforme(Tyzzer’s)75. Cytomegalovirus76. Ectromelia virus77. EDIM (Epidemic diarrhea of infant mice)78.Encephalitozoon cuniculi79. Hepatitis virus80. Lymphocytic choriomeningitis virus81. Minute virus82.Mycoplasma pulmonis83. Parvovirus84. Pneumonia virus of mice85. Polyoma virus86. Reovirus-387. Sendai virus88. Theiler’s mouse encephalomyelitis virus

1. Apolipoprotein A12. â-2 Microglobulin3. C-Reactive Protein4. D-Dimer5. EGF6. Endothelin-17. Eotaxin8. Factor VII9. FGF-basic10. FGF-911. Fibrinogen12. GCP-213. GM-CSF14. Growth Hormone15. GST16. Haptoglobin17. IFN-ã18. IgA19. IL-1á20. IL-1â21. IL-222. IL-323. IL-424. IL-525. IL-626. IL-727. IL-1028. IL-1129. IL-12p7030. IL-1731. IL-1832. Insulin33. IP-10

34. KC/GROá35. Leptin36. LIF37. Lymphotactin38. MCP-139. MCP-340. MCP-541. M-CSF42. MDC43. MIP-1á44. MIP-1â45. MIP-1ã46. MIP-247. MIP-3â48. Myoglobin49. OSM50. RANTES51. SCF52. SGOT53. TIMP-154. Tissue Factor55. TNF-á56. TPO57. VCAM-158. VEGF59. von Willebrand Factor

60.â-2 Glycoprotein61. Insulin62. JO-163. Mitochondrial64. MPO65. PCNA66. PR3 (cANCA)67. Ribosomal P68. RNP69. Scl-7070. Smith71. SSA72. SSB

73. Adenovirus74.Clostridium piliforme(Tyzzer’s)75. Cytomegalovirus76. Ectromelia virus77. EDIM (Epidemic diarrhea of infant mice)78.Encephalitozoon cuniculi79. Hepatitis virus80. Lymphocytic choriomeningitis virus81. Minute virus82.Mycoplasma pulmonis83. Parvovirus84. Pneumonia virus of mice85. Polyoma virus86. Reovirus-387. Sendai virus88. Theiler’s mouse encephalomyelitis virus

• Luminex• multiplexed• immunoassay• rat & human• cytokines,chemokines,hormones,acute phaseproteins,cancer,cardiovascular,infectious• 88 for rat, 166for human• 50 uL plasma

Case 1: Rules Based MedicineO'Brien PJ, Chevalier S, Schenck E, Pawlowski V, Dagues N, Ledieu D. Multianalyte immunoassay profiles of plasmabiomarkers of inflammatory change in a rat model of early mesenteric vascular injury. Vet Clin Path 34:306-7, 2005.

Multi-Analyte Immunoassay Profiles of PlasmaBiomarkers of Inflammatory Change in a Rat Model of

Early Mesenteric Vascular Injury

Background: Phosphodiesterase inhibitors may induce mesentericvascular injury in rats. We studied early plasma biomarkers ofvascular injury using a novel, multi-analyte, immunoassay profile of60 acute phase reactants, cytokines, chemokines, growth factors,and hormones using Luminex technology (Rules Based Medicine)that has recently become commercially available (Charles RiverLaboratories, Manston, UK).

Red laser excites specific dyes toidentify the analyte; green laserexcites a different dye to quantifythe result.

Microspheres pass singlefile past two lasers.Each microsphere set is covered

with capture antibodies that reactwith the target protein

Specific fluorescent dyespermeate the polystyrenebiospheres.

Rats (25 SpraqueDawley 7 wk males)treated with singledose - 0, 160, or320 mg/kg of potentPDE4 inhibitor givenby oral gavage. At16 h, plasmaanalysed.

= no overlap betweentreated and controls

different for high doseonly

CRP

500

700

900

1100

ug/m

L

**

Eotaxin

0

50

100

150

pg/m

L

*

Fibrinogen

200

400

600

800

ug/m

L **

Haptoglobin

300

400

500

600

ug/m

L **IL-10

050

100150200250

pg/m

L **MDC

0

100

200

300

400

pg/m

L

**

Rantes

0

25

50

75

pg/m

L

*

VCAM-1

150

200

250

300

350

ng/m

L

* *

VEGF

100

150

200

250

300

pg/m

L

* *

SCF

Contro

ls

Low Dose

HighDose

0

25

50

75

100

pg/m

L

*

M-CSF

Contro

ls

Low Dose

HighDose

0.50

0.75

1.00

1.25

ng/m

L

GCP-2

Contro

ls

Low Dose

HighDose

0.00

0.02

0.04

0.06

0.08

ng/m

L

*

vWF

Contro

ls

Low Dos

e

HighDos

e500

700

900

1100

ug/m

L

**Luminex, Multi-analyte,

Plasma Biomarkers inVasculitis

Methods andResults

5 Most Significant ANOVA and AnalytesSignificant between High and Low Dose t-Test

• Haptoglobin• Fibrinogen• CRP• VEGF• VCAM-1• SCF• GCP-2• M-CSF• Eotaxin• vWF

Blue – Control, Green – Low Dose, Red – High Dose

ANOVA

t-Test

-50 0 50 100

IaHp

IL-10CRPvWG

VEGFM-CSFVCAM

EotaxinSCF

MDC

% Difference from Controls

Profile of Inflammatory Changes

Anal

yte

Cross-species reactivity occurred in 35% of immunoassays. However,21 assays were effective in identifying a major effect of treatment: mild, acuteinflammatory response, with increased release of acute phase proteins (Ia, Hp,CRP, vWF) and altered concentrations of cytokines and chemokines (eg IL-10,eotaxin, GCP-2, and MDC) modulatory of inflammation. VEGF, an angiogenesisfactor induced by inflammation was also slightly affected.

Conclusions

vWF

• 1963 Ebashi discovers Tn• globular protein in muscle &heart• 3 subunits: C binds Ca,I inhibits actin & myosininteraction at low Ca,T binds tropomyosin• separate genes for muscle& heart TnT and TnI, but notfor TnC where cardiac genealso found in slow muscle• highly conserved acrossspecies

Muscle Troponin

Myosin

Actin

Case 2: Cardiac Troponin

• Gold standard biomarker ofmyocardial injury in man.

• Myofibrillar protein regulatingcontraction that leaches out ofinjured cardiac cells.

• Applicability in toxicologyrapidly becoming recognised.

ActinMyosin

IT

C

IC

Ca “Thin Filament”Contraction

Relaxation

Ca

Use of cTnT in Lab Animals

0

5

10

Controls Males Females

Ser

umcT

nT

(ng/

ml)

Doxorubicin Toxicity in Mice

Cardiac Ischemia in Dogs

0

20

40

1 min 45 min

Ser

umcT

nT(n

g/m

l)

Cardiac Puncture in Ferrets

Ischemia

Reperfusion

10 1300 45

Time (min)

Ser

um

cTn

T(n

g/m

l)0.1

1

10

100

0 5 10 15 20-10

0

10

20

30

Ser

um

cTn

T(n

g/m

l)

r = 0.96p < 0.0001

Infarct Size (g)

Cardiac Ischemia in Rats

O'Brien PJ, Dameron GW, Beck ML, Erickson BK, Di Battista TH, Miller KE, Jackson KN, Mittelstadt S. Cardiac troponin T is asensitive and specific biomarker of cardiac injury in laboratory animals. Lab Anim Sci 47:486-495, 1997.

Variable Tn Assay Sensitivity and Species-Specificity

Cont

rols

Asym

ptom

atic

Sym

ptom

atic

Pace

d0

1

2

3

4

Dogs with Symptomatic Heart Disease(dysrhythmia, dyspnea, LVH, valvular disease)

cTnI

(ug/

L) DPC Immulite cTnI suboptimalin rats, Bayer cTnI works best,cTnT works well, ELISA insensitive

cTnI increases with dysrhythmia,dyspnea, valvular disease, cardiachypertrophy, atrial pacing

0 2 4 6 240

10

20

30

40

cTnI (DPC Immulite)cTnI (Bayer Centaur) cTnT (Roche)

Analyser Comparison: IsoproterenolInduced Cardiotoxicity in Rats

Time (hours)

Trop

onin

(ng/

mL)

cTnI (Trichem ELISA)

O’Brien PJ, Smith DEC, Knechtel TJ, Marchak MA, Pruimboom-Brees I, Brees DJ, Spratt DP, Archer FJ, Butler P, Potter AN, Provost JP,Richard J, Snyder PA, Reagan WJ. Cardiac troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals. Lab An 40:153-171, 2006.

0 10 20 30 40 500

150,000

300,000

450,000

600,000 Rat

Human

Rela

tive

Fluo

resc

ence

Units

(RFU

)

cTnI as cTnI-T-C

Human Dog Rat

Mouse

0.00

0.02

0.04

0.06

cTnI

(ug/

L)

Female Male0.000.030.06

0.1

0.3

0.5

cTnI

(ug/

L)

High Sensitivity cTnI AssayCorrelation with HistopathologyRat vs Human Reactivity

8-mo SD Male RatsHave Hi Serum cTnI

ReferenceRanges

O’Brien PJ. Blood cardiac troponin in toxic myocardial injury: archetype of a translational, safety biomarker. Expert Rev Molec Diagnostics6 (5): 685-702, 2006.

0 1 2 3 4 50

10

20

30

40

r = 1.0

Cent

aurc

TnI(

ug/L

)

Histo Score

PreDos

e2 Hrs

4 Hrs8 Hrs

24Hrs

0

3

6

9

12 skTnI

skTn

I(ug

/L)

0

2000

4000

6000

CK(IU

/L)

CK

Serum skTnI, cTnI and CK withPropylene Glycol Toxicity

0.0

0.2

0.4

0.6

0.8 cTnIsk

TnI(u

g/L

)

0

3

6

9

12CK

CK(fo

ldin

crea

se)

PreDose 2 Hrs

4 Hrs8 Hrs

24Hrs

0

3

6

9

12 skTnI

skTn

I(fo

ldin

crea

se)

Muscle TnI Validation Study• 30 rats dosed with 0.2 mL / kg propylene glycolwere analysed for skTnI, cTnI, and CK• skTn l correlated with CK & histological findings(but not cTnI), with max damage 2 to 8h post dose

• skTnI increase up to7x more (at 8 h) since itpersists 2-3x longer• cardiac injury in 17%rats, indicating need torun both skTnI and cTnI

Case 3: ALT Issue in Hepatotoxicity Preclinical use of ALT the “universal biomarker of hepatotoxicity”

is frequently ineffective at predicting hepatic effects in man e.g.

1) Glitazones: ALT in dogs but not rats nor man, exceptingtroglitazone

2) XXXX: ALT in man but not rat nor monkey at comparabledoses

3) No increase in acetaminophen toxicity at end of 4-day repeatdose study

4) ALT inducible by corticosteroids and certain chemicals(cyproterone) and inhibited by various drugs (isoniazid)

Pathological significance of a mild to moderate, or a transient, orsporadic ALT is unknown (no histopathological correlate)

Glutamate Dehydrogenase (GLDH)(EC 1.4.1.3)

• Conserved structure, distribution & function• Liver: oxidizes amino acid releasing urea• Kidney - excretes NH3, Nerve - glu an excitatory

neurotransmitter, Pancreas - sensor for protein-mediated insulin release. Note: release into urine, CSF orgut

• mitochondrial location & large size (330 kDa) inhibitrelease making it more necrosis specific)

• mainly pericentral (centrolobular) matching drugdistribution & xenobiotic-metabolism

0 3 6 91

10

1001

2

34

5

1. GLD

4. ALT3. AST2. SDH

5. ALP

Act

ivity

(fold

incr

ease

)

Relative Increase in Plasma Activityof Liver Enzymes after Hepatectomy

Time (days)O'Brien PJ, Slaughter MR, Swain A, Elcock F, Bugelski PJ. Adaptive response of hepatic antioxidantsystem to repeated dosing with acetaminophen in rats. HUMAN EXP TOXICOL 19: 277-283, 2000.

GLD

ALT

ALT

GLD is Superior to ALT forHepatotoxicity in Rats

Glucose-AlanineCycle and

Urea Formation

1

10

100

1000

Log

Act

ivity

(IU/L

) SDH

*

AST *

Contro

ls

Cypro

teron

e

Wye

th-146

43

Isonia

zid

Dexam

ethas

one

Methap

yrilen

e1

10

100

1000GLD

*

*

*

Contro

ls

Cyprot

erone

Wye

th-14

643

Isonia

zid

Dexam

ethaso

ne

Methapy

rilene

ALT

*

***

1

10

100

1000ALP

***

ALP: mild with PPAR &methapyriline; with dex

SDH & AST: moderate with methapyriline

ALT: mild to moderate with dex, cyproterone,methapyriline; withisoniazid

GLD: moderate to marked with dex, cyproterone,methapyriline

Hepatotoxicant-inducedIncrease in Plasma Activity of

Hepatic Enzymes in Rats

Automated Chemistry Analyser (ADVIA 1650)for automated enzyme, protein, & metabolite analyses

in biofluids, tissue, cellsCase 4:

Tissue Biochemistry Biomarkers forIntermediary Metabolism

-in addition to widearray of serumchemistryparameters there are~30 tissuebiomarkers availablefor differentmetabolic substratesand metabolicpathway activities

Use of Automated Clinical Chemistry Analysers toAssay Frozen Tissue and Cell Culture Biomarkers

Tissue Preparation

• flash-freeze fresh tissue in liquid N2& store at –80 oC

• thaw when ready to assay, transportin liquid N

• weigh 200 mg

• homogenise in physiological, bufferedsaline and centrifuge

• dilute and run

1.7 2.0 2.3 2.610

20

30

40

Cow

- 50

bpm

Shee

p- 7

5D

og- 1

10Pi

g- 1

20

Mou

se- 4

70

Gui

nea

Pig

- 280

Rat

- 352

r = 0.97p < 0.0001

HADH

Rab

bit -

260

Log Heart Rate (bpm)

Activ

ity(U

/g)

50

100

150

200

r = 0.98p < 0.0001

ATPaseAc

tivity

(U/g

)

Fatty Acid Oxidation

Tissue BiochemicalBiomarkers of Metabolism:

Relationship to FunctionalDemand Across Species

Conclusion: Cardiac fatty acidoxidation, ATP cycling, and oxidativestress increase in proportion to aerobicmetabolic activity in the heart.

2.0 2.3 2.60

50

100

150

r = 0.80p < 0.003

MDA

Log Heart RateCo

nten

t(nm

ol/g

)

Mitochondrial Activity

Oxidative Stress

Application of TissueBiochemistry in Drug Discovery compound X causes

accumulation of hepatic fat fat accumulation associatedwith liver enzymes in serumInvestigative studies showedX G6PD and Krebs cycle Reversal by Y which had oppositebiochemical effects

X-induced Steatosis

Mitochondrial Krebs Cycle(CS = Citrate Synthase )

0.0

0.5

1.0

1.5

2.0YX

X + Y

Rel

ativ

eA

ctiv

ity

Pentose Phosphate Pathway(G6PD = Glucose-6-phosphate dehydrogenase)

0.0

0.5

1.0

1.5

2.0YX

X + Y

Rel

ativ

eA

ctiv

ity

Opposing Effects of X and Y on Oxidative Stress andMitochondrial Parameters Prevent Hepatotoxicity

0 25 50 75 100 1251.0

1.5

2.0

2.5r = 0.88p < 0.0001

Log (Serum ALT)

TG

0 100 200 300 4000

3000

6000

9000

12000 r = 0.64

Serum ALT

Live

rALT

Correlation BetweenLiver Fat Contentand ALT Release

into Plasma

Oxidative Stress and the Antioxidant System

Reactive oxygen species (ROS) generated throughnormal cellular metabolism (e.g., electron transportchain, oxidases) and drug metabolism

Local ROS production can fluctuate (e.g., UV light,xenobiotic metabolism, inflammatory responses),increasing risk of oxidative stress

ROS controlled by a complex antioxidant network,which includes enzymes that are modulated byredox sensitive transcription factors

Oxidative Stress in Drug-Induced Toxicity

- transitions metals (eg Fe, Cu)

- hyperoxia, ethanol, ozone, nitrogen dioxide, asbestos

- pyridyls (eg diquat), carbon tetrachloride

- anthracyclines (eg doxorubicin)

- quinones (menadione, acetaminophen, primaquine, eugenol)

- bleomycin, halothane, nitrofurantoin

- peroxisome proliferators

- NRTI’s

Key Antioxidant(AOS) SystemComponents

G6PD / 6PGD

NADP+ NADPH

GSH GSSGGR

GPx

O2- H2O2

SOD CAT

O2

Fe

•OH

H2O

GCS

Glu + Cys

CAT - catalaseGSH - glutathione

GR - GSH reductaseGPx - GSH peroxidase

SOD - superoxide dismutaseGCS - glutamyl-cysteine synthase

G6PD - glucose-6-phosphatedehydrogenase

• Single dose of 1400 mg/kgin rats depleted liver GSH by75% from 6h to 1d• Induction of G6PD to 3xand GR to 1.5x control wasassociated with GSHrecovery, despite GPxdecrease by 25% at 2d

GLD = glutamate dehydrogenaseGSH = glutathioneGR = GSH reductaseGPx = GSH peroxidaseG6PD = glucose-6-phosphatedehydrogenaseSOD = superoxide dismutase

0

2500

5000

7500

10000Liver GSH

**

IU/L

0

1000020000

3000040000 Liver G6PD *

*IU/L

0 25 50 750

4000

8000

12000

16000Liver GR

*

Time (h > 1400 mg/kg)

IU/L

Acetaminophen HepatotoxicityGSH Depletion Causes Hepatic Necrosis

with Induction of G6PD & GRx

0

2500

5000

7500

10000Liver GPx

*

IU/L

-5 20 45 700

50100150200250 Serum GLD

Time (h > 1400 mg / kg)

IU/L

0

1500

3000

4500

Liver SOD

IU/L

O’Brien PJ, Cleall P, Towell P, Brees D, Pruimboom-Brees I. Protective, compensatory, hepatic adaptations of the antioxidant system, mitochondria and intemediary metabolism to non-toxic andtoxic doses of acetaminophen. Vet Clin Path 34:305-6, 2005.

-50 0 50 100

150

GSHG6PD

GRGPxSODCAT

HADHPFK

LactateCS

% Difference Peroxisomal proliferation Mito fatty acid oxidation Mito Krebs cycling Oxidative stress Glycolysis

PPARalpha Effects onMouse Liver

-50 50 150

250

GSHG6PD

GRGCSGPxSODCAT

HADHPFK

GSH GSH system Mito oxidative activity Glycolysis anti ROS enzymes

Acetaminophen Effectson Rat Liver

% Difference-10

010

030

050

070

0

GSHG6PD

GRGPxSODCATMTTGLDLDH

Peroxisomal proliferation Mito oxidative phosphorylation Mito mass Antioxidant enzymes and GSH Glycolysis

Diquat Effects

% Difference

Characteristic Profiles of Different Drug’s Effects onMitochondrial Metabolism and Oxidative Stress

Slaughter MR. Thakkar H. O'Brien PJ. Effect of diquat on the antioxidant system and cell growth in human neuroblastoma cells. Toxicology & AppliedPharmacology. 178:63-70, 2002.

Case 5: High Content Screening in Toxicology

♦ Single cell, cell populations or well♦ Multiparametric structural and functional

Automated fluorescencemicroscopy of culturedcells

96-well plate in incubator

Live-cell imaging

Real-time, kinetic

Intracellular location

O’Brien PJ, Haskins JR. In vitro cytotoxicity assessment. In: High Content Screening: A Powerful Approach to Systems Cell Biology andDrug Discovery. Human Press: Totowa. Chapter 30. pp 415-425, 2006.

17

Healthy Hepatocytes

18

Unhealthy Hepatocytes

Standard HCA Cytotoxicity Assay

5 mechanistic, kinetic biomarkers simultaneously1) Cell number – proliferation (Hoechst 33342)2) Nuclear area – apoptosis, cell cycle inhibition (Hoechst 3334)3) Mitochondrial membrane potential – mito tox (TMRM)4) Intracellular ionised Ca – Ca homeostasis (Fluo 4)5) Membrane permeability – membrane leakiness (Toto3)

Blue nuclei & normal red mitochondria replaced by green calciumand red of high membrane permeability stain.

Toxicity

Toxicity of Cerivastatin (25 uM)

CerivastatinControls

Composite: Blue for DNA, Orange for mito membrane potential, Green forCa, Pink for membrane permeation 1. mito potential; 2. Ca; 3. Permeabilizedwith Ca & mito potential; 4. ruptured

3

3

3

2

2

2

2

2

23

1

1

4

1

Diaz D, O’Brien PJ. Defining the sequence of events in cerivastatin toxicity using a high-content multi-parametric cytotoxicity assay. EurPharm Rev 11:38—45, 2006.

Membrane permeability increases,leading to LDH release, ATP depletion,and cell rupture

Data graphed as mean SEM of all cells in a field of view (mean = 43cells / field, range = 30-56 cells / field)

Kinetic Changes in Cell Function inResponse to Dantrolene (100 uM, 24 h)

Ca

MitochondrialPotential

300

250

200

150

100

50

0

Fluo

resc

ence

Inte

nsity

Time (min)

0 25 50 75 100 125 150 175

Single Cell Changes

TOTO3

0

100

200

300

400

500

0

100

200

300

400

500

010002000300040005000

0

1.95

3.91

7.81

15.6

3

31.2

5

62.5

0

125

250

500

1000

2000

C o n c e n t r a t i o n

10

15

20

25

30

0

50

100

150

200

10

15

20

25

30

1000

1500

2000

2500

3000

3500

400

800

1200

0

100

200

300

0

2.0

3.9

7.8

15.6

31.3

62.5

125.

0

250

500

1000

2000

C o n c e n tra tio n

0.00

0.06

0.12

0.18

1st Quad Probe Assay 2nd Quad Probe AssayNuclear Area Nuclear Area

Ca2+ (Fluor4) Mitochondrial DNA (picogreen)

Mitochondrial membrane potential (TMRM_ Oxidative Stress (DHE Oxidation Rate)

Plasma Membrane Integrity (Toto-3) Mitochondrial Mass (MitoTracker Deep Red

Cell Count (10x objective 10 fields) Cell Proliferation (cell count, 20x objective 6 fields)

HCA of Fenofibrate Toxicity: dose-response

uM

IC50 Curves for Cerivastatin-inducedCytotoxicity

Sequence of cellular events: increased proliferation (0.1 uM) antiproliferation and mitochondrial hyperpolarisation (~0.4 uM) Cadyshomeostasis (0.9 uM) apoptosis (1.2 uM) permeabilization (2.3 uM) mitochondrial dysfunction (3 uM)

0

150

300

450

600IC50=0.90 uMSE= 0.19 uMr2 = 0.88

Cyto

solic

Calci

um

-9 -8 -7 -6 -5 -45

10

15

20

25

IC50=1.20 uMSE= 0.16 uMr2 = 0.97

Cerivastatin (M)

Nucle

arAr

ea

-9 -8 -7 -6 -5 -40

500

1000

1500

2000 IC50=2.3 uMSE= 0.11 uMr2 = 0.99

Mem

bran

ePe

rmea

bility

Cerivastatin (M)-9 -8 -7 -6 -5 -4

0

150

300

450

600IC50= 3.0 uMSE= 0.3 uMr2 = 0.87

Cerivastatin (M)

TMRM

Sign

al

20

40

60

80

100

IC50= 0.42 uMSE= 0.16 uMr2 = 0.92

Cells

perF

ield

Assessment of Mitochondrial Toxicity ofNucleoside Analogues for HIV Treatment

Nucleoside reverse transcriptase inhibitors used for HIVtreatment, eg zidovudine (AZT), zalcitabine. Chronic myopathy firstreported in 1990, 5 years after approval of AZT (also neuropathy,anemia, pancreatitis). Attributed in part to inhibition of mitochondrialDNA polymerase.

-9 -7 -5 -30

25

50

75

100

Sensitisation to Cytotoxicity byProlonged Exposure (days 4, 7, 10 right to left)

Cel

lcou

nt(%

cont

rol)

4d

10d

Zalcitabine Concentration (M)

7d

Oligomycin Sensitisation to Cytotoxicity

HCA Better Correlated with HumanHepatotoxicity than Conventional Assays or

Animal TestingSensitivity Specificity

DNA synthesis 10 % 92Protein synthesis 4 97Glutathione depletion 19 85Superoxide induction 1 97Caspase - 3 induction 5 95Membrane integrity 2 99Cell viability 10 92

Cell viability or GSH or DNA Syn 25 ~90Regulatory animal toxicity tests 52 -

%

Cellomics Assay 93 97O’Brien PJ, Irwin W, Diaz D, Howard-Cofield E, Krejsa CM, Slaughter MR, Gao B, Kaludercic N, Angeline A, Bernardi P,Brain P, Hougham C. High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in anovel cell-based model using high content screening. Archives Toxicology Sep;80 (9):580-604, 2006.

Comparison to Flow Cytometry

Flow cytometry well established for assessing blood cells withrespect to immunophenotype, viability, various mechanisticbiomarkers of toxicity

Potential complementarity of HCA Subcellular localisation eg

– mitochondrial vs nuclear DNA– micronuclei and centromeres– transcription factor translocation– endocytosis

Morphometric parameters eg nuclear – cytoplasmic ratios,cell types, shape

Kinetic parameters – oxidative stress, enzyme, ion transport

12.5 uM ImipramineControls

Cells triple-stained with Hoechst, Mitotracker Far Red, Lysotracker Green

Translational Safety Biomarker forPhospholipidosis

• Micronuclei (± centromere) formation inlymphocytes isolated from peripheral blood canbe used for assessment of genotoxicity potential(cells are cultured for 3 days with cytochalasin Bblock of cytokinesis)

Translational Safety Biomarker forGenotoxicity

Chromosomal aberrations in arsenic-exposed human populations: a review with specialreference to a comprehensive study in West Bengal, India Mahataa, M. Chakia, P. Ghosha,L.K. Dasb, K. Baidyac, K. Raya, A.T. Natarajand, e, A.K. Giria.Cytogenetic and Genome Research 104:359-364

2004.

1.6 uM Zalcitabine (ddC)

Translational Safety Biomarker forMitochondrial DNA Depletion by NRTI’s

Control Cells

Cells stained with Picogreen

Davila JC, Xu JJ, Hoffmaster KA, O'Brien PJ, Strom SC. Current In Vitro Models to Study Drug-Induced Liver Injury In:Hepatotoxicity: From Genomics to In Vitro and In Vivo In press

Dihydro-ethidium

(DHE)

TranslationalSafety

Biomarkerfor Oxidative

StressDiquat Erythromycin

Controls

TranslationalSafety

Biomarker forMitochondrialProliferation

(Mitotracker DeepRed 633)

(human hepatocytestreated 3 days; AZTcauses ragged red

fibers in AIDSpatients)

Diquat AZT

HCAof Non-Adherent,

HumanLymphocytes

(Hut 78)

Red = TMRMGreen = Fluo 4Blue = Nuclear DNA

Ca increase TMRM decrease

Controls

Calcium (Fluo 4)

0

100

200

300

400

Fluo

resc

ence

Mitochondria (TMRM)

0

100

200

300

400

Fluo

resc

ence

Nuclear Area

Contro

ls

Propran

olol

0.05%

Triton

10uM

FCCP

100u

MFCCP

0255075

100125

Fluo

resc

ence

Arsenic Trioxide - TMRM

0

250

500

Fluo

resc

ence

Arsenic Trioxide - Fluo4

0

250

500

Fluo

resc

ence

Arsenic Trioxide - Nuclear Area

0.02 0.2 2 20 20

00

50

100

uM Concentration

Fluo

resc

ence

Mitozanthrone - Fluo4

0

250

500

Fluo

resc

ence

Mitozanthrone - TMRM

0

250

500

Fluo

resc

ence

Mitozanthrone - Nuclear Area

0.02 0.2 2 20 20

00

50

100

150

200

uM Concentration

Fluo

resc

ence

HCA of Non-adherent Lymphocytes

0.01 0.1 1 10 10

010

00150

350

550 Arabinoside CArsenic TrioxideDoxorubicinMitozanthone

Dose-Dependent Effect on MitochondrialMembrane Potential of Human Lymphocytes

uM Concentration

Fluo

resc

ence

Mitochondria (TMRM)

Contro

l 1

Contro

l 2

Contro

l 3

Treated

1

Treated

20

1000

2000

Fluo

resc

ence

In vivo Effect of Anti-cancer DrugTreatment on Canine Blood

Leukocytes

Note: preliminary studies; no other parameters affected

Conclusions

Peripheral blood cells can be translational safetybiomarkers of drug toxicity (archetypical ex:phospholipidosis, mitochondrial DNA depletion)

HCA can be conducted on non-adherent cells Dose-response relationships can be determined on

lymphocytes HCA can be conducted on peripheral blood cells with

potential for monitoring a wide range of mechanisticbiomarkers and non-specific cytotoxicity and altered drugsusceptibility