Post on 12-Jan-2017
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