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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
2
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
4
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 –
7
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
8
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
9
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
10
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
11
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
12
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
13
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
14
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
15
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?
16
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
18
>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
19
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
20
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
21
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
22
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
23
Equivalent and Compelling efficacy for TrkA Selective and Pan-Trk in a model of inflammatory pain
24
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
25
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
26
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
28
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
29
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?
30
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
31
TrkA free IC90
Intermittent target knockdown is sufficient for efficacy, required levels seem to vary by model
32
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
33
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