primary human cell systems analysis of drug mechanisms
DESCRIPTION
Presentation given at the SBS 15th Annual Conference, Lille, France, 28 April 2009TRANSCRIPT
Primary Human Cell Systems Analysis of Drug Mechanisms
Ellen L. Berg, PhDBioSeek, Inc.
SBS 15th Annual ConferenceLille, France28 April 2009
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Presentation Overview
• BioMAP Human Cell Systems Platform
Primary human cell-based disease models
• Analysis of PPAR agonists
Discriminate clinical-stage compounds
• Class and compound-specific activities
Explore alternative clinical indications
• Prioritize compounds for indications and/or safety related activities
2
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• Covers a lot of biology
Targets, pathways, therapeutic areas, diseases
• Covers the right biology
Human disease biology
• Is quantitative, reproducible, robust, high throughput
Standardized, amenable to database generation
• Is useful to broad range of stakeholders
Project leaders, biologists, chemists, preclinical scientists, clinicians
• Is predictive
Biomarkers
Clinical indications, efficacy, toxicity
Goals for Human Cell Systems Biology Platform
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BioMAP® Technology Platform
Assays
Human primary cells Disease-like culture conditions
LPS
BF4T
SM3C
Profile Database Informatics
Biological responses to drugs and stored in the database
Specialized informatics tools are used to mine and analyze biological data
Complementary to biochemical target and phenotypic screening Complementary to biochemical target and phenotypic screening
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• BioMAP Systems are cell-based assays BioMAP Systems are cell-based assays engineered to model complex human engineered to model complex human disease biologydisease biology
• Human primary cells
• Co-cultures, multiple stimulation factors, activated cells
• Quantitative protein readouts - biomarkers
• Pharmacologically relevance - validated with known
drugs
BioMAP® Technology Platform
Assays
LPS
BF4T
SM3C
Human primary cells Disease-like culture conditions
>25 BioMAP Systems
• Assay endpoints include human clinical Assay endpoints include human clinical biomarkers and risk factors (proteins)biomarkers and risk factors (proteins)
Cytokines, chemokines
Adhesion and growth receptors
Biological mediators (prostaglandins, etc.)
Proteases, enzymes (MMPs, plasminogen activators)
Others (hemostatic factors, matrix components)
Clinically relevant
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AssaysAssays
Human primary cells Disease-like culture conditions
LPS
BF4T
SM3C
Profile DatabaseProfile Database
Biological responses to drugs and stored in the database
BioMAP® Technology Platform
• > 2000 agents> 2000 agents
• Approved drugsApproved drugs
• Clinical stage & Clinical stage &
failed drugsfailed drugs
• Experimental Experimental
compoundscompounds
• BiologicsBiologics
• ToxicantsToxicants
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Assays are Robust and Highly Reproducible High Correlation of Experimental Replicates
5 M dose
Pearson Correlation Coefficient
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12
R1 1
R2 0.95 1
R3 0.96 0.94 1
R4 0.98 0.98 0.96 1
R5 0.93 0.94 0.91 0.94 1
R6 0.96 0.96 0.93 0.97 0.98 1
R7 0.94 0.91 0.9 0.93 0.89 0.9 1
R8 0.95 0.98 0.94 0.98 0.94 0.98 0.92 1
R9 0.91 0.92 0.88 0.92 0.89 0.91 0.93 0.93 1
R10 0.88 0.9 0.81 0.89 0.93 0.93 0.85 0.91 0.83 1
R11 0.94 0.97 0.9 0.94 0.91 0.93 0.94 0.96 0.91 0.89 1
R12 0.92 0.9 0.84 0.89 0.96 0.96 0.89 0.91 0.87 0.92 0.91 1
Consistent data experiment-to-experiment Pearson correlation >0.8 (perfect match = 1)
Consistent data experiment-to-experiment Pearson correlation >0.8 (perfect match = 1)
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MicrotubuleStabilizers
Mitochondrial ET chain
Retinoids
Hsp90
CDK
NFB
MEK
DNAsynthesis
JNK
Proteinsynthesis
MicrotubuleDestabilizers
Estrogen R
PI-3K
Ca++
Mobilization
Classification of Drugs By Mechanism Pairwise Correlation of BioMAP Reveals Functional Similarities
mTOR
PKC Activation
p38 MAPK
HMG-CoAreductase
Calcineurin
Transcription
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BioMAP Systems are Validated Corticosteroids (Prednisolone) Are Active in Inflammation Systems
BioMAP Systems
Readout Parameters (Biomarkers)Cytotoxicity Readouts
Lo
g e
xpre
ssio
n r
atio
(Dru
g/D
MS
O c
ontr
ol)
Control (no drug)
99% significance envelope
DoseResponse
Profiles retain shape over multiple concentrationsProfiles retain shape over multiple concentrations
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E-selectin
TNF-
IL-8
BioMAP Systems are Validated Activities of Corticosteroids Match Clinical Effects
PGE2
IL-8MCP-1
MCP-1, IL-8, E-sel. decreaseLeukocyte recruitment
Many, e.g. Jilma et al., 2000
PGE2 decreasePain, swellingSebaldt et al., 1990
Readouts in BioMAP show the same pattern as has been reported for patients receiving steroid therapy
Readouts in BioMAP show the same pattern as has been reported for patients receiving steroid therapy
Collagen I & III
Collagen I, III decreaseSkin atrophyAutio et al., 1994
MMP-1
Lo
g e
xpre
ssio
n r
atio
(Dru
g/D
MS
O c
ontr
ol)
PAI-1SAA
PAI-1, SAA increaseCV complications
Sartori et al., 1999Fyfe et al., 1997
PAI-1
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Project Goal
• Characterize PPAR agonists by BioMAP profiling
Compare and contrast PPAR agonists (anti-inflammatory
activities)
Identify shared and unique pathway effects
Identify potential new indications
PPAR PPARRosiglitazone (Avandia)
Troglitazone (Resulin)
Pioglitazone (Actos)
Fenofibrate (Tricor)
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BioMAP Systems
Eot3IP-10I-TAC
VCAM
E-sel
Monocyte activation
Macrophageactivation
T cell activation
MCP-1
TNFMCSF
CD40IL-8
BioMAP Profile of Rosiglitazone
IP-10I-TAC
• Rosiglitazone has strong anti-inflammatory activities anti-inflammatory activities Inhibition of monocyte and T cell activation (T cell proliferation ) & recruitment Inhibition of inflammatory chemokines (Eotaxin3, IP-10, ITAC, IL-8) Consistent with inhibition of NFB pathway by rosiglitazone
• Consistent with efficacy in vivo Mouse models of colitis (Shah, Y.M., et al., Am. J. Physiol. Gastrointest. Liver Physiol. 2007,
292:G657; Saubermann, L.J., Inflamm. Bowel Dis., 2002, 8:330). Animal model of exposure-induced asthma (Lee, J. Immunol, 2006 117:5248). MCP-1 and TNF are clinical biomarkers
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BioMAP Profile of Rosiglitazone
BioMAP Systems
Eot3IP-10I-TAC
MMP9
VCAM
E-sel
Monocyte activation
Macrophageactivation
T cell activation
MCP-1
TNFMCSF
CD40IL-8
uPARCol III
Col IV
PAI-1
• Rosiglitazone has strong effects on tissue remodeling parameters Inhibition of MMP9, PAI-1, uPAR, Collagen III; upregulation of Collagen IV; Strong inhibition
of myofibroblast activation Consistent with modulation of TGF pathway by rosiglitazone
• Consistent with results from in vivo studies Rosigitazone is effective in models of neointimal hyperplasia (MMP9 is a biomarker in vivo) Rosiglitazone protects in scleroderma model (myofibroblast accumulation and Collagen III)
Col III
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BioMAP Systems
• Rosiglitazone upregulates prostaglandins In both bronchial epithelial and leukocyte-containing systems Potent activity
PGJ2PGF1a
PGD2PGF2a PGD2
PGF2aPGJ2
PGF1aPGJ2PG1a
PGE2PGD2PGF2a
PGF2a
BioMAP Profile of Rosiglitazone
Leukocyte-containing systemsBronchial epithelial cell-containing systems
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Upregulation of Prostaglandins by Rosiglitazone
• Are prostaglandin effects PPAR-dependent? Not reversed by PPAR antagonists
Reversed by COX1/2 inhibitors
Non-TZD PPAR agonists do not upregulate prostaglandins
• Consistent with secondary activity / activities Rosiglitazone has been reported to inhibit 15-hydroxy-
prostaglandin dehydrogenase and CYP450 2C8
Q: What about other TZDs, PPAR ligands?
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Rosiglitazone Upregulation of PGE2 is not a Class EffectSearch of BioMAP Database for Compounds that Increase PGE2
Retinoids
MicrotubuleDestabilizers
TXA2 inhibitorPPAR
PPAR
RNA SynthesisInhibitor
mTORInhibitor
AMPKactivator
JNK Inhibitor
CYP450Inhibitor
CompoundSpecific Effect
MechanismClass Effect
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MMP9
IL-8
Monocyte activation
MCSFCD40
ITAC
MCP-1
CD38
T cell activation
PGJ2PGD2PGF2a PGJ2
PGD2PGE2PGF2aPGJ2
PGD2PGF2a
PGF1a
PGJ2
PGD2PGF2a
PGF1aVCAMCD40
BioMAP Profile of Pioglitazone
• Pioglitazone shows few anti-inflammatory activities shows few anti-inflammatory activities Modest inhibition of VCAM, ITAC Pioglitazone may be a weaker inhibitor of NFB than rosiglitazone or have reduced
cell uptake
• Pioglitazone has modest effects on tissue remodeling parameters has modest effects on tissue remodeling parameters Inhibition of MMP9 Pioglitazone has no effect on myofibroblast activation (in contrast to rosiglitazone)
• Pioglitazone has differential effects on prostaglandins Prostaglandins are inhibited in leukocyte/endothelial cell systems; unaffected in
bronchial epithelial cells
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Eot3IP-10I-TAC
MMP9E-sel
Monocyte activation
Macrophageactivation
T cell activation
TNF uPAR MCP-1TF
Col III
Col IVMMP1
TM
MCP-1
• Troglitazone shows modest anti-inflammatory activities shows modest anti-inflammatory activities Activities are similar to those of rosiglitazone Inhibition of inflammatory chemokines (Eotaxin3, IP-10, ITAC, IL-8) Troglitazone is cytotoxic at higher concentrations
• Troglitazone also affects tissue remodeling parameters also affects tissue remodeling parameters Inhibition of MMP9, PAI-1, Collagen III, some inhibition of myofibroblast activation Upregulation of thrombomodulin in CASM3C system
• Troglitazone affects prostaglandin pathways Upregulation of PGF1a, PGF2a, and PGD2 in bronchial epithelial cells No effect in leukocyte-containing systems (/LPS and /SAg)
PGF1a
PGD2PGF2a
PGF1a
PGD2PGF2a
BioMAP Profile of Troglitazone
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QuickTime™ and a decompressor
are needed to see this picture.
• Fenofibrate shows modest anti-inflammatory activitiesFenofibrate shows modest anti-inflammatory activities Some inhibition of monocyte and T cell activation Inhibition of inflammatory chemokines (Eot3, IL-8, ITAC)
• Modest effects on tissue remodeling parametersModest effects on tissue remodeling parameters Inhibition of MMP9, Collagen III; upregulation of MMP1
• Differential modulation of prostaglandins Inhibition of prostaglandins in leukocyte-containing systems (/LPS and /SAg) No effect on prostaglandins in epithelial cell-containing systems
Eot3 IL-8 MMP9
Monocyte activation
T cell activation
MCSFCD69
uPAR
HLA-DR
TM
Col III
MMP1IL-8
MCP-1
PGJ2PGD2PGF2a PGJ2
PGD2PGF2a
PGD2PGE2PGF2a
PGF1a
PGJ2
PGD2PGF2aPGF1a
Mig
VCAMITAC
VCAM
IL1
IL-8
TM
BioMAP Profile of Fenofibrate - PPAR
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Summary of PPAR Agonists
• BioMAP profiling can discriminate PPAR agonists
Compound-and class-specific effects
• PPAR agonists exhibit anti-inflammatory activities consistent with
inhibition of NFkappaB pathway
Rosiglitazone, Fenofibrate > Troglitazone > Pioglitazone
• Some PPAR agonists inhibit myofibroblast activation (TGF signaling)
Rosiglitazone, Troglitazone, but not Pioglitazone
• PPAR agonists have diverse effects on prostaglandins
Rosiglitazone upregulates prostaglandins in both leukocyte-containing systems and
bronchial epithelial cells
Troglitazone upregulates prostaglandins in bronchial epithelial cells
Pioglitazone and Fenofibrate inhibit prostaglandins in leukocyte-containing systems
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Summary
• Differential activities can suggest prioritization for therapeutic utility
Anti-inflammatory activities ( inhibition of T cell, monocyte activation)
• Autoimmune disease, vascular inflammation, atherosclerosis
Inhibition of myofibroblast activation / TGF signaling
• Fibrotic diseases (IPF, scleroderma)
Upregulation of prostaglandins
• Bronchodilation, potential utility in respiratory disease
• Differential effects may also be associated with potential for side effects
Differential clinical effects of pioglitazone and rosiglitazone with respect to
cardiovascular outcomes (Winkelmeyer, W., 2008, Comparison of cardiovascular
outcomes in elderly patients with diabetes who initiated rosiglitazone vs
pioglitazone therapy. Arch Intern Med 168:2368)
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Acknowledgements
• BioSeek Eric Kunkel Jennifer Melrose Dat Nguyen Elen Rosler Stephanie Tong Jian Yang Antal Berenyi David Patterson Jonathan Bingham
• Stanford Eugene Butcher Rob Tibshirani Trevor Hastie
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