1

Steven W. Andrews, Ph.D. Associate Director of Drug Discovery

Allosteric Small Molecule Inhibitors of the NGF/TrkA Pathway A New Approach to Treating Inflammatory Pain

Challenges Faced with Discovery of New Drugs in the Pain Space

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Translation of pre-clinical results to clinical outcome

Efficacy

Poor translation from animal models to man and vice versa

Safety

Mechanism specific toxicities are not always apparent until reaching the clinic

Bar for safety for new pain drugs is very high

Economic Considerations

While existing SOC have deficiencies and liabilities, these treatments are quite cost effective

and offer many convenient dosage forms

Our approach: small molecule inhibitors for antibody validated pathways

AR-797 P38 inhibitor – targeting NSAID resistant pain (Phase II)

Trk inhibitors – targeting the NGF pathway (pre-clinical)

3

Tanezumab- anti-NGF Antibody has Validated Clinical Efficacy

Similarly impressive efficacy reported in Chronic Low Back Pain2

but concerns of joint findings in OA patients with chronic dosing, particularly with NSAIDs

Lane et al. (2010) NEJM 363:1521

Katz et al. (2011) Pain 152:2248

Key Question- is there an alternative and perhaps safer approach to inhibit this pathway

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Neurotrophin / Trk Signaling Mediates Peripheral Pain Response

NGF TrkA

Peripheral Hypersensitization

Local release

Proinflammatory cell recruitment and degranulation

Central Hypersensitization

Neurotrophin Receptors- TrkA and B are critical

signaling partners in the NGF pain cascade

5

Mechanisms for Inhibiting Neurotrophin / Trk Signaling Cascade

C1

C2 Ig1 Ig2

LRR1-3

Trk kinase domain inhibitors

100% homology in ATP site ATP site

Our ATP site inhibitors selectively inhibit the pan-Trk axis, but not other kinases TrkC TrkB Trk A

P75NTR

CR1 CR2 CR3 CR4

NGF BDNF NT-4 NT-3

Growth Factor Antibodies

Trk Receptor Antibodies

6

Key Scientific Questions- Small Molecule Pan-Trk Kinase vs. anti-NGF

Efficacy for Pain Relief?

• Added efficacy from the TrkB component?

• Differences related to mechanism of inhibition?

• Differences related to duration of inhibition?

– Long term vs intermittent target knockdown?

Safety?

• Differences related to mechanism of inhibition?

• Differences related to duration of inhibition?

– Long term vs intermittent target knockdown?

• Safety concerns for added TrkB and TrkC inhibition?

Our approach: in vivo evaluation

with selective small molecule pan-Trk inhibitors –

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Approach to Finding Highly Selective Trk Chemical Matter

High Throughput Screen ARRAY diversity

and kinase focused

Trk potency

Promising Kinase

Selectivity

and “drug-likeness”

~80TrkA ATP site x-ray structures solved to date

Medicinal Chemistry Optimization

>1500 designed compounds

~many compound hits in > 20 chemical series

<50 compounds in 2 novel chemical series

Trk X-ray Crystallography

DMPK Pharmacology

Toxicology

pan-Trk series (ATP site) • Tuned for high kinase selectivity outside of Trk family • equipotent for TrkA, TrkB and TrkC

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Select Properties of Pan-Trk Leads

Program Pan-Trk Lead ARRY-470 AR-772 AR-523

hTrkA cell IC50 9.7 nM 1.6 nM 10 nM

hTrkA free cell IC50

23 nM 12 nM 42 nM

hTrkB / hTrkC cell 24 nM 1.6 nM 12 nM

230 member Kinase Panel

Clean @ 1 µM

Clean @ 1 µM

Clean @ 1 µM

Predicted hepatic Cl

Human, Rat

10, 18 (med, low)

13, 32 (med, med)

8, 13 (med, low)

Plasma protein binding

Human, Rat 68%, 82%, 79%, 79%, 75%, 89%,

Solubility (ng/mL)

pH 1.2 / 6.5 / 7.4

>1000, >1000, >1000

>1000, 780, 820

>1000, 780, 820

peripheral to CNS exposure 16 to 1 25 to 1 28 to 1

Peripherally Selective: only peripheral

inhibition in efficacy studies

High kinase selectivity

Very potent for inhibiting neurotrophin driven TrkA, TrkB and TrkC

signaling in cell

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Kinase Selectivity of AR-470 compared to literature pan-Trk Kinase Inhibitors

At 1 µM Potent on Trks Plus 83 off targets

AR-470 ATP site Pan TRK inhibitor

Diaryl urea Type II DFG out

Amino pyrimidine ATP site

at1 µM Potent on Trks One weak off target

at 0.5 µM Potent on Trks Plus ~7 off targets

No activity at 10 µM against other pain targets:

Clean pharmacology for inhibiting TrkA, B and C

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Summary of in vivo Efficacy Observed for Pan-Trk Leads

Acute Pain

UV burn model (thermal hyperalgesia)

CFA paw model (thermal hyperalgesia, gait analysis)

CFA joint model (gait analysis)

Fracture pain (flinching and guarding)

Bone cancer pain (flinching, guarding and nerve budding)

Surgical Incision

Chronic Pain

CIA Model of rheumatoid arthritis (pain and histological evaluation)

MIA Model of osteoarthritis (pain)

CFA paw model (mechanical allodynia)

Excellent efficacy is observed in pre-clinical models of inflammatory pain

ARRY-470 Broad Efficacy Observed in Multiple Pre-Clinical Models

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ARRY-872ARRY-872ARRY-872

p p

vehicle ARRY-470 (3 mg/kg) ARRY-470 (10 mg/kg) ARRY-470 (30 mg/kg) ibuprofen (100 mg/kg)

Mantyh et al. Bone 48 (2) , pp. 389-398 Mantyh et al. Molecular Pain 6 , art. no. 87

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ARRY-470 is Superior to NSAIDs in the CFA Joint Model

Rofecoxib

Naive Vehicle 30 100 300 0

100

200

300

400

Dose (µMol/kg p.o. twice daily)

Naproxen

Naive Vehicle 7.5 30 900

100

200

300

400

Results shown 2-3 days after start of treatment

Naive Vehicle 3 10 30 0

100

200

300

400

Dose (mg/kg p.o. twice daily)

(

Valdecoxib

Naive Vehicle 10 30 100 0

100

200

300

400

Gua

rdin

g in

dex

Gua

rdin

g in

dex

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Safety of Pan-Trk Inhibitors – Neuronal Safety

No changes in functional observations in mice, rats, or monkeys at therapeutic doses / exposures

No histological changes in peripheral neuronal density in brain, spinal cord, sciatic nerve or skin neurons to 300 mg/kg with 28 days of dosing

No changes in normal pain response at therapeutic doses

Von Frey (Mechanical: A-delta fibers)Day 48 post fracture

Fracture + vehicle FRX + 30mg/kg AR470

50%

With

draw

l lat

ency

(g)

0

1

2

3

4

5

6

n=6 n=3

Col 3 Col 3

Hot Plate (Thermal: C-fibers)Day 48 post fracture

Fracture + vehicle FRX + 30mg/kg AR470

Hin

dlim

b w

ithdr

awl r

espo

nse

time

at 5

5C (s

ec)

0

5

10

15

20

n=6 n=3

Mantyh et al. Bone 48 (2) , pp. 389-398 No observed effects on neuronal safety

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Safety of Pan-Trk Inhibitors – On Target Effects

Unger et al. JNEUROSCI, 2007, 27(52): 14265 Lin JC, et al. PLoS ONE 3(4): e1900 (2008)

Hyperphagia / weight gain • Increased food consumption –

peripheral effect • Increase weight gain- even when

food consumption is controlled • Likely BDNF / TrkB effect –

rodent specific?

Dose of Pan-Trk

inhibitor

Ataxia Score (max) Incidence

30 mg/kg 1 1 of 3

100 mg/kg 1 3 of 3

300 mg/kg 2 3 of 3

Ataxia Scoring System 1 -"swimming” through litter, flattened or splayed on cage bottom 2 - head bobbing, jittery or hyperactive, head bobbing, nervous 3 - head rearing, disoriented, lethargy, agitated 4 - plus falling over when on hind legs, sleeping on back Correlates with pan-Trk target coverage in the CNS

Potential narrow therapeutic window for broad clinical pain treatment

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Small Molecule Pan-Trk Leads -

Efficacy

Great efficacy across pre-clinical pain models

Equivalent to historical anti-NGF in the same models

Intermittent target knockdown is sufficient for efficacy

Partial pathway knockdown is sufficient for efficacy

• No apparent added effect for TrkB

• (In the clinic- concerns of too much pain relief with anti-NGFs)

Safety

No observed adverse effects on peripheral neuronal health or function

• Hyperphagia and weight gain – rodent specific?

• Reversible Ataxia when CNS target coverage is achieved. TI related to plasma to brain ratio

Would selective TrkA inhibition provide a broader TI?

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Approach to Finding Highly Selective TrkA Chemical Matter

High Throughput Screen ARRAY diversity

and kinase focused

Trk potency

Promising Kinase

Selectivity

and “drug likeness”

>120 TrkA allosteric + small molecule x-ray structures solved to date

Medicinal Chemistry Optimization

>2000 designed compounds

~many compound hits in > 20 chemical series

<50 compounds in 2 novel chemical series

Trk X-ray Crystallography

DMPK Pharmacology

Toxicology

TrkA Selective Series (allosteric site) • high kinase selectivity outside of Trk family • high selectivity for TrkA over TrkB and C

2 TrkA vs TrkB selective hits

Induced Fit

17 confidential

Challenges of Drug Design in an Induced Fit Site

existing pocket ATP site

Allosteric site optimization is enabled by X-ray crystallography

induced fit allosteric site

ATP site Potency is easy

Selectivity is hard

Allosteric site Potency is hard

Selectivity is easy

TrkA Selective Inhibitors- Identifying the Allosteric Site

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>100 TrkA Constructs Cloned

Triaged by Expression, Purification, and Binding

Thousands of Crystal Screens

>120 TrkA/Inhibitor Structures

>120 Selective TrkA Inhibitor Structures Median Resolution: 2.8 Å

Range: 2.3 Å – 3.3 Å

Several Crystal Forms

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Mechanism for Inhibiting Neurotrophin / Trk Signaling Cascade

C1

C2 Ig1 Ig2

LRR1-3

Trk kinase domain inhibitors

high homology in ATP site

low homology in the allosteric site

ATP site

allosteric site

Allosteric site inhibitors selectively inhibit TrkA, but not TrkB, TrkC, or other kinases

TrkC TrkB Trk A P75NTR

CR1 CR2 CR3 CR4

NGF BDNF NT-4 NT-3

Growth Factor Antibodies

Trk Receptor Antibodies

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Select Properties of Pan-Trk and TrkA Selective Leads

Program TrkA Selective

Lead AR-786 AR-256 AR-618

hTrkA cell IC50 0.6 nM 0.9 nM 5.6 nM

hTrkA free cell IC50

12 nM 18 nM 30 nM

hTrkB / hTrkC cell >1000 nM >1000 nM >1000 nM

230 member Kinase Panel

Clean @ 10 µM

Clean @ 10 µM

Clean @ 10 µM

Predicted hepatic Cl

Human, Rat

10, 38 (med, med)

12, 31 (med, med)

7, 17 (med, low)

Solubility (ng/mL)

pH 1.2 / 6.5 / 7.4

750 60 1

>1000 5 1

>1000 590 130

peripheral to CNS exposure 10 to 1 48 to 1 48 to 1

Peripherally Selective: Only peripheral

inhibition in efficacy studies

High selectivity over TrkB / C High kinase selectivity

Very potent for inhibiting NGF driven TrkA signaling

in cell

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Kinase Selectivity of Array pan-Trk and TrkA Selective Inhibitors

AR-786 Allosteric Selective TRKA inhibitor

at 10 µM Potent on TrkA weak on TrkB/C

At 1 µM Potent on Trks Plus 83 off targets

AR-470 ATP site Pan

Diaryl urea Type II DFG out

Amino pyrimidine ATP site

at1 µM Potent on Trks One weak off target

at 0.5 µM Potent on Trks Plus ~7 off targets

No activity at 10 µM: against other pain targets

Clean TrkA pharmacology

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Key Scientific Questions- Small Molecule pan-Trk vs TrkA Selective

Do TrkA selective (allosteric) inhibitors show similar pain efficacy to Pan-Trk (ATP site) inhibitors?

• Is blocking TrkA upstream of BDNF / TrkB sufficient to alleviate various modalities of pain / hypersensitization?

• Is ATP site and allosteric site inhibition functionally equivalent in vivo?

0

10

20

30

40

50

60

70

80

Vehicle 30 mg/kg ARRY-470(pan-Trk)

30 mg/kg AR786(TrkA selective)

Mea

n D

iffer

ence

in P

rint A

rea

(%) ±

SEM

**

*p<0.05 by One-Way ANOVA with Bonferroni's correction compared to vehicle

***

TrkA Selective and pan-Trk Inhibitors are Equivalent in the Rat CFA Paw Model

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Equivalent and Compelling efficacy for TrkA Selective and Pan-Trk in a model of inflammatory pain

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Duration of Action of a TrkA Selective Inhibitor in the Rat CFA Paw Model

-10

0

10

20

30

40

50

60

70

80

1HR 2HR 4HR 8HR

Mea

n D

iffer

ence

in P

rint A

rea

(%)

±SEM

30 mg/kg AR786

Vehicle 30 mg/kg AR-786

* **

***

Promising onset and duration of action

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TrkA Selective and pan-Trk in a Surgical Incision Model

*

Tim Brennan

Equivalent and compelling efficacy for TrkA Selective and Pan-Trk in a model of surgical pain

TrkA Selective and pan-Trk in the Rat CIA Model of Polyarthritis

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selective

0

0.2

0.4

0.6

0.8

1

Vehicle AR-786TrkA selective

AR-772pan-Trk

Mea

n H

P Pr

int A

rea

(cm

2 )

S

EM

**

Equivalent and compelling efficacy for TrkA Selective and Pan-Trk in a model of rheumatoid arthritis

27 confidential

TrkA Selective Inhibitors are Equivalent to Celecoxib in the Rat MIA Model

Algos

Compelling efficacy for TrkA selective in a model of Osteoarthritis

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Neurotrophin / Trk Signaling Mediates Peripheral Pain Response

NGF TrkA

Peripheral Hypersensitization

Local release

Proinflammatory cell recruitment and degranulation

Central Hypersensitization

Blocking TrkA at the allosteric site

is sufficient to

block the NGF pain cascade

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Key Scientific Questions- Small Molecule pan-Trk vs TrkA Selective

Do TrkA selective (allosteric) inhibitors have a similar safety profile to Pan-Trk (ATP site) inhibitors?

• Hyperphagia and Weight gain?

• Ataxia with CNS target coverage?

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TrkA Selective Inhibitors Do Not Cause Hyperphagia or Ataxia in Rat

Hyperphagia / weight gain • No increased food consumption • No weight gain / hyperphagia • Supports pan-Trk effect was

BDNF / TrkB driven

150

200

250

300

350

400

450

500

0 10 20 30

Mea

n B

ody

Wei

ght (

gm)

SE

M

Time (days)

Vehicle10 mg/kg50 mg/kg300 mg/kg

Pan-Trk Treated Females

Ataxia Score (max)

Incidence TrkA

Selective Treated Females

Ataxia Score (max)

Incidence

30 mg/kg AR523 1 1 of 3 10 mg/kg

AR256 0 0 of 3

100 mg/kg AR523

1 3 of 3 50 mg/kg AR256 0 0 of 3

300 mg/kg AR523

2 3 of 3 300 mg/kg AR256 0 0 of 3

Ataxia Scoring System 1 -"swimming” through litter, flattened or

splayed on cage bottom

2 - head bobbing, jittery or hyperactive, head

bobbing, nervous

3 - head rearing, disoriented, lethargy, agitated

4 - all of above plus falling over when on hind

legs, sleeping on back

Mechanistic Difference- Small Molecule, Oral TrkA kinase vs anti-NGF m-ABs

Potential Clinically Advantages

1. Ability to adjust inhibition by varying dose and schedule

2. Ability withdraw drug

3. Allows flexible titration to address different pain indications

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TrkA free IC90

Intermittent target knockdown is sufficient for efficacy, required levels seem to vary by model

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Small Molecule TrkA Selective Inhibitors: What we know so far

Efficacy

Great efficacy across pre-clinical pain models: similar to pan-Trk

o Looking for Collaborators – new models for mechanistic comparison

Intermittent target inhibition is sufficient for efficacy

Similar efficacy to that reported for anti-NGF antibodies

Safety

No observed adverse effects on peripheral neuronal health

No hyperphagia or weight gain to 28 days

No ataxia even when CNS target coverage is achieved for 28 days

TrkA Selective Kinase Inhibition at the Allosteric Site Represents

a Promising New Mechanism for Modulating the NGF / Trk Pain Pathway

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Acknowledgments Medicinal Chemistry

• Shelley Allen • Julia Haas • Yutong Jiang • Tim Kercher • Gabrielle Kolakowski • Brad Newhouse • Allen Thomas • Steve Andrews • Larry Burgess

Computational Chemistry • Jim Blake

CMC / Formulation • David Fry • Chris Lindemann • Don Corson

Translational • Steve Miller

Enzymology • Lisa Pieti Opie • Francis Sullivan

Cell Biology • Robyn Hamor • Chris Eberhardt • David Chantry

Structural Biology • Tony Morales • Walt Voegtli • Josh Ballard • Guy Vigers • Barb Brandhuber

Executive Leadership • Kevin Koch • David Snitman • Jim Winkler

Pharmacology / Toxicology • Karyn Bouhana • Anna Gomez • Patrice Lee • Jed Pheneger • Vince Murphy • Dale Wright

DMPK • Ross Wallace • Brian Baer • Karin Brown • Dylan Hartley • Gary Hingorani • Jennifer Otten • Jamie Rogan • Susan Rhodes • Ron Franklin

Outsourcing • Cheryl Napier