Introduction cHTS Screening Platform Steroid Dissociation ...Introduction cHTS Screening Platform...

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1. We have developed a technology to systematically andefficiently find synergistic combinations that target multiplebiological pathways and that can be developed into newimmuno-modulatory therapeutics.

2. A number of novel combination drug candidates have emerged from this approach and we have advanced them into clinical trials in immuno-inflammatory disease settings.

3. The platform has also revealed novel, synergistic pathwayinteractions that form the basis for future development of multi-targeted therapeutics.

cHTS Systematic discovery of novel anti-inflammatory combination therapeutics E. Roydon Price, Palaniyandi Manivasakam, Grant Zimmermann, Joseph Léhar, Alexis A. Borisy, Curtis T. Keith. CombinatoRx Inc., Boston, MA 02118, USA

Introduction cHTS Screening Platform Steroid Dissociation through Combinations Translates in Animal Models

Summary

ACR, San Diego, CA, USA, 2005

Master X PlateConc: 1000 units

Master Y PlateConc: 1000 units

+ Combined and Diluted

CombinationDilution Plate

Conc: 10 units

Approved drugs and target probes in pair-

wise combination

QC

Analyze

Screen

Explore

Combine low-dose steroid with an enhancer

• Low-dose steroid lower effect and lower side effect

• Enhancer selectively synergize on efficacy targets no synergy on side effect targets

SSA Example: CRx-119Synergies between established anti-inflammatories such as cyclosporine and glucocorticoids are observed when each target separate parallel pathways.

Compound Library• 2000 FDA and late-stage compounds• ADME/Tox and targets are often known

Cell Based Assay• Primary human lymphocytes• Many induction protocols

• LPS, PMA/Ionomycin, CD3/CD28, etc.• Many downstream endpoints

• TNFα, IL1, IL2, IFNγ, etc.• Can focus on a particular cell type or pathway

Synergy Analysis• Proprietary tools to classify and measure

combination effect• An ideal combination has unknown

ratio/concentration• Both potency shifts and effect boosts possible• Therefore one needs to cover all possibilities

with a Dose Response Matrix

CombinatoRx has developed a platform to systematically screen for synergisticinteractions between any two drugs, not limited to existing anti-inflammatorytherapies.

In addition, we have discovered compounds which when combined with effective but dose limited anti-inflammatory therapies (e.g. glucocorticoids or calcineurin inhibi-tors) allow dose reduction but maintain anti-inflammatory effect.

Approach

Selective Steroid Amplifiers (SSA): Distinct Cytokine Profiles

CRx-119: a non-obvious in-vitro synergyPrednisolone with AmoxapineAmoxapine– Tricyclic Antidepressant– Not used as an anti-inflammatory

Dissociated target and side effect profiles– Synergistic anti-TNFα effect Positive

isobologram– Amoxapine does not potentiate

glucocorticoid’s ability to transactivateGRE containing promoters

– Amoxapine does not potentiateprednisolone’s ability to translocate theglucocorticoid receptor

Prednisolone Amoxapine

Amoxapine does not potentiateGRE dependent transactivation

Amoxapine does not potentiateGlucocorticoid Receptor translocation

Good Efficacy: TNFα inhibitionGR translocation

Rat Adjuvant Model of ArthritisFreund’s adjuvant injected in tail of Lewis rats (male/female) on Day 0, 48.Double placebo animals received mineral oil injections at the base of thetail. Between Days 10 and 20, the animals were treated daily via gastricgavage with compound(s). After Day 10, joint diameters were assessedevery other day.

Also active in endotoxemia

Rat Safety StudyEight Lewis rats per arm were dosed p.o. daily for seven days. Fastingblood glucose levels were measured along with body weight, and serumcorticosterone

TNFα

GR

GC

CsAIKK

NFκB

IkB

CN

P

Ca++

JNK p38ERK

MKK3/6 MKK4/7MEK1/2

MLK MEKK4Raf

NFAT

RasPKC

P

ATF2AP1

P

Prednisolone

CP

Rheumatological diseases are mediated by the intersection of multiple biological pathways and networks. Consequently, a multi-targeted therapeutic approach may provide superior clinical benefit. Historically, combination treatments have been discovered through trial and error using drugs with proven disease modifying effect. We have developed a technology to systematically and efficiently find synergistic combinations of compounds that target multiple pathways underlying disease biology and that can be developed into new anti-inflammatory therapeutics.

Pred High

AmoxapinePred Low

60

70

80

90

100

110

120

*

#

#

#

NormalizedBlood

Glucose(mg/dL/

200 grams)

Active in adjuvant models GC toxicity not enhanced

No synergy observed with prednisolone forblood glucose, body weight or serumcorticosterone

Amoxapine

T cell Monocyte

TNFα

PMA/Iono PMA/Iono

LPS

Combos of singlytherapeutic drugsfor target disease~2 million possible

pair wise combos

~2,0

00ap

prov

eddr

ugs

~2,000 approved drugs~10

~10

Combos of singlytherapeutic drugsfor target disease~2 million possible

pair wise combos

~2,0

00ap

prov

eddr

ugs

~2,000 approved drugs~10

~10

Dose Matrix

100

0

Effe

ct

[Drug A]

[Dru

gB]

100

0

Effe

ct

[Drug A]

[Dru

gB]

100

0

Effe

ct

100

0

Effe

ct

100

0

Effe

ct

CRX-102

CRX-119

CRX-139

CRX-160

CRX-163

CRX-164

CRx-102

CRx-119

CRx-139

CRx-160

CRx-163

CRx-164

CRx-119

CRx-102

TNF-PI IL2-PI IFN-PI TNF-LPS

Synergy

Synergy SynergyPrednisolone (uM) Prednisolone (uM) Prednisolone (uM) Prednisolone (uM)

Prednisolone (uM) Prednisolone (uM) Prednisolone (uM) Prednisolone (uM)Synergy

Synergy

Synergy

Pro-inflammatory genes GC side effect genes

GCHigh Dose GCLow Dose

Reduced GC side effect

Combination Therapy allowsreduction of GC dose and sideeffects but maintains control overpro-inflammatory gene expression

Enhancer

GR

GR

GRE

GR

Pro-Inflammatory Genes Toxicity-Associated Genes

TNFα

Robust Pipeline of Anti-Inflammatory Therapies

TM

Effe

ct

Concentration

Drug B

Effe

ct

Drug A

Response Surface

Drug A

Dru

gB 1:1?

10:1?

1:10?

Drug A

Dru

gB 1:1?

10:1?

1:10?

OHHO

O

OH

O

H

H

H

N

N

O

Cl

NH

Control Prednisolone 3.0 mg/Kg CRx-119

Adjuvant Alone Prednisolone 0.1 mg/Kg Amoxapine 3.0 mg/Kg

Dip

yrid

amol

e (u

M)

Dip

yrid

amol

e (u

M)

Dip

yrid

amol

e (u

M)

Dip

yrid

amol

e (u

M)

Am

oxap

ine

(uM

)

Am

oxap

ine

(uM

)

Am

oxap

ine

(uM

)

Am

oxap

ine

(uM

)

PrednisoloneAmoxapineEnhancer 2Cyclosporine

Prednisolone

Pred/Amox

Pred/Enh2

Cyclosporine

Control Pred 10nM Pred 100nM

Amoxapine Pred 10nM + Amoxapine Pred 100nM + Amoxapine

[Drug A]

[Dru

gB]

Immunofluorescence for GR-alpha in Hela, 10um Amoxapine

Prednisolone (uM)

Am

oxap

ine

(uM

)

Prednisolone (uM)

Am

oxap

ine

(uM

)