BioSeek Presentation SBS 26Apr20099final

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

BioSeek

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


Assays

LPS

BF4T

SM3C


>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


LPS

BF4T

SM3C

Profile DatabaseProfile Database

Biological responses to drugs and stored in the database


• > 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 Systems

Eot3IP-10I-TAC

MMP9

VCAM

E-sel

Monocyte activation


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


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


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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