UNC/UCSF

Frank M. Longo, Stanford University

Stanford Academic/mechanisticstudies

ADDFAlzheimer’s AssociationNIA U01 NINDS R21Taube FamilyKoret FdnJean Perkins FdnHorngren Family

Targeting the p75 Receptor to Inhibit Degenerative Signaling and Tau Phosphorylation/Misfolding/Missorting: Preclinical through Phase 1

ADC Directors Meeting, April 18th 2015

Also: Steve Massa, MD, PhD at UCSF

• receptors • Ca2+ /Ox mito • membrane effects

p-tau / mf-tau / tau-o / ml-tau

amyloid

AKT

Synaptic dysfunction Spine loss Neurite degeneration

cdk5 p38 GSK3β JNK

calpain MLKRho A PKA

p-CREB

p-fyn

p-NR2B

p-cofilin

Other factors:age, inflammation

• receptors • Ca2+ /Ox mito • membrane effects

p-tau / mf-tau / tau-o / ml-tau

amyloid

PI3K

AKT

p75

Synaptic dysfunction Spine loss Neurite degeneration

Survivaladaptors

cdk5 p38 GSK3β JNK

Deathadaptors

calpain MLKRho A PKA

p-CREB

p-fyn

p-NR2B

p-cofilin

Other factors:age, inflammation

Rho/GDI

proNGF/NGF

• receptors • Ca2+ /Ox mito • membrane effects

p-tau / mf-tau / tau-o / ml-tau

amyloid

PI3K

AKT

p75

Synaptic dysfunction Spine loss Neurite degeneration

Survivaladaptors

cdk5 p38 GSK3β JNK

Deathadaptors

calpain MLKRho A PKA

p-CREB

p-fyn

p-NR2B

p-cofilin

Other factors:age, inflammation

Rho/GDI

p75 enables Aβ-induced degeneration

mea

n di

ffer

entia

l ang

le

1012141618202224

CM Aβ1-42

Aβ1-42

p75 -/-

CM

p75 +/+

*** ***

Knowles et al, J Neurosci 2009Coulson Lab 2009Bartlett Lab 2015

APP-L/S X p75 -/-oligomeric-Aβ + 21 div HC neurons dystrophic neurites(APP IHC)

p75+/+ p75-/- p75+/+ P75-/-

Aβ-induced degeneration is p75 dependent

• receptors • Ca2+ /Ox mito • membrane effects

p-tau / mf-tau / tau-o / ml-tau

amyloid

PI3K

AKT

p75

Synaptic dysfunction Spine loss Neurite degeneration

Survivaladaptors

cdk5 p38 GSK3β JNK

Deathadaptors

calpain MLKRho A PKA

p-CREB

p-fyn

p-NR2B

p-cofilin

Other factors:age, inflammation

Rho/GDI

proNGF/NGF

• receptors • Ca2+ /Ox mito • membrane effects

p-tau / mf-tau / tau-o / ml-tau

amyloid

PI3K

AKT

p75

Synaptic dysfunction Spine loss Neurite degeneration

Survivaladaptors

cdk5 p38 GSK3β JNK

Deathadaptors

calpain MLKRho A PKA

p-CREB

p-fyn

p-NR2B

p-cofilin

Other factors:age, inflammation

Rho/GDI

Signaling effects:

• receptors • Ca2+ /Ox mito • membrane effects

p-tau / mf-tau / tau-o / ml-tau

amyloid

PI3K

AKT

p75

Synaptic dysfunction Spine loss Neurite degeneration

Survivaladaptors

cdk5 p38 GSK3β JNK

Deathadaptors

calpain MLKRho A PKA

p-CREB

p-fyn

p-NR2B

p-cofilin

Other factors:age, inflammation

Rho/GDI

Signaling effects:

Massa et al J Neurosci 2006

CM Aβ1-42 Aβ1-42 + ligand (100 nM)

LM11A-31 inhibits Aβ-induced dystrophy

0-5 6-1011

-1516

-2021

-2526

-3031

-3536

-40

% o

f neu

rites

0

25

50

75CM

AβAβ+ C2

Modified from Yang et al PLoS One, 2008

E17 div 21maturedhippocampalneurons

• receptors • Ca2+ /Ox mito • membrane effects

p-tau / mf-tau / tau-o / ml-tau

amyloid

PI3K

AKT

p75

Synaptic dysfunction Spine loss Neurite degeneration

Survivaladaptors

cdk5 p38 GSK3β JNK

Deathadaptors

calpain MLKRho A PKA

p-CREB

p-fyn

p-NR2B

p-cofilin

Other factors:age, inflammation

Rho/GDI

Signaling effects:

• receptors • Ca2+ /Ox mito • membrane effects

p-tau / mf-tau / tau-o / ml-tau

amyloid

PI3K

AKT

p75

Synaptic dysfunction Spine loss Neurite degeneration

Survivaladaptors

cdk5 p38 GSK3β JNK

Deathadaptors

calpain MLKRho A PKA

p-CREB

p-fyn

p-NR2B

p-cofilin

Other factors:age, inflammation

Rho/GDI

Signaling effects:

Yang et al PLoS One 2008Nguyen et al, JAD 2014Yang et al, SNS 2014

AT8 p-tauSer202

tau0

20

40

60

80

100

****** *** ***

p-ta

u202

/ tau

Aβ1-42

Aβ1-42 (5μM)

p75NTR ligands block Aβ-induced p-tau / AT8 epitope

E17 div 21hippocampalneurons

Modified from Yang et al PLoS One, 2008

Inhibition of Aβ-induced tau mislocalization

**

**

MAP2 (dendrites)

Tau (axons)

CM Aβ Aβ + C31 (100 nM)

yellow µm / 100 µm

CM Aβ Aβ + C31(100 nM) Yang, Longo SNS 2014

E17 DIV 21hippocampalneurons

FYN-NR2Bactivation?

Inhibition of Aβ-induced FYN and NR2B activation21 DIV HC neurons

p-FY

N in

tens

ity /

neur

on (%

con

trol)

CM Aβ + LM11A-31Aβ

Yang, Longo SNS 2014

p-FYN:

**

**

CM Aβ + LM11A-31

Inhibition of Aβ-induced FYN and NR2B activation21 DIV HC neurons

p-N

R2B

inte

nsity

/ne

uron

(% c

ontro

l)p-

FYN

inte

nsity

/ne

uron

(% c

ontro

l)

Aβ

CM Aβ + LM11A-31Aβ

Yang, Longo SNS 2014

p-FYN:

p-NR2B:

**

**

CM Aβ + LM11A-31

Inhibition of Aβ-induced FYN and NR2B activation21 DIV HC neurons

p-N

R2B

inte

nsity

/ne

uron

(% c

ontro

l)p-

FYN

inte

nsity

/ne

uron

(% c

ontro

l)

Aβ

CM Aβ + LM11A-31Aβ

Yang, Longo SNS 2014LTP/behavior ?

p-FYN:

p-NR2B:

In vivo Studies

Orally bioavailable CNSWater soluble, stableBrain/plasma > 1Brain T1/2 ~ 1 hrFDA limit dose 2000 mg/kg non-toxicAMES / hERG / CYP each clear

ADMET favorable

CNS target engagement: p75 cleavage

Intraneuronal p-tau

WT veh WT L

Tg veh Tg L

Reduced tau phosphorylation (AT8)

Nguyen et al, JAD 2014

WT-V WT-L Tg-V Tg-L

p-tau Ser202 (AT8) ctx

Mo: 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

APPL/S mice; 50 mg/kg po qd X 3 mo

Reduced tau aberrant folding (MC-1)

Mo: 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

APPL/S mice; 50 mg/kg po qd X 3 mo

-L

Nguyen et al JAD 2014

ctx:

WT veh WT-L

Tg veh Tg-L

Reduction of cortex ChAT neurite loss

Mo: 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

APPL/S mice; 50 mg/kg po qd X 3 mo

WT-V WT-L Tg-V Tg-L

Nguyen et al JAD 2014

Reduced loss of LC noradrenergic projection fibers to prefrontal ctx

Mo: 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

APPL/S mice; 50 mg/kg po qd X 3 mo

Nguyen, Longo

TH IHC

WT-V WT-L Tg-V Tg-L

p75NTR ligands: LTP and behavior

NOR Y-maze

CFC DMP-MWM

Time (min)0 15 30 45 60 75 90 105 120 135fE

PSP

slop

e (%

of b

asel

ine)

50

100

150

200

250

300

350

400wt-vehwt-C31APP/PS1-vehAPP/PS1-C31

Ottavio Arancio Lab, ColumbiaHC slice preparations

Knowles et al Neurobiol Aging 2013Nguyen et al JAD 2014

APP-L/S mice 3 mo rx:

Late stage reversal of BF neurite degeneration

W TgV TgL

Mo: 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

APPL/S mice; 50 mg/kg po qd X 1 mo

Simmons et al PLoS One 2014

baselinedegen rx

Simmons, Longo

Spines: late stage rx with p75NTR ligands

Mo: 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

APPL/S mice; 50 mg/kg po qd X 1 mo

WTV

T41V

T41L

WTV

T41V

T41L

L

Golgi stainingHC pyramidal

• receptors • Ca2+ /Ox mito • membrane effects

p-tau / mf-tau / tau-o / ml-tau

amyloid

PI3K

AKT

p75

Synaptic dysfunction Spine loss Neurite degeneration

Survivaladaptors

cdk5 p38 GSK3β JNK

Deathadaptors

calpain MLKRho A PKA

p-CREB

p-fyn

p-NR2B

p-cofilin

Other factors:age, inflammation

Rho/GDI

Microglial activation?

WT veh WT 31

Tg veh Tg 31

HippocampusCortex

Reduction of microglial activation

Mo: 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

APPL/S mice; 50 mg/kg po qd X 3 mo

CD68 IHC

Aberrantly folded tau – MG activationZilka et al J Neuroinflammation, 2012

L

L

L L L L

PET imaging ?

Nguyen et al JAD 2014

ChA

T C

ell A

rea

0

50

100

150

200

MS+VDBp=0.001 p=0.012

4 25V 25L 18 mo

Mo: 0 5 10 15 20 25

50 mg/kg qd X 4 mo

Aged WT mice: reversal of BFCN atrophy

ChAT IHC

4 mo 25 mo V 25 mo L

Xie, Meeker, Longo UNC-CH

184

Challenge of mouse to human prediction

• Many ‘AD-relevant’ mechanisms• Many in vivo endpoints: morphology, behavior• Multiple ‘AD’ models:

APP-L/S (morphology, behavior, mechanisms) Ts65Dn (morphology, behavior)Tg2576 (morphology)PS1-APP(LTP)

• Effects in aged wild-type mice• Reversal of late-stage effects• Translatable biomarkers: PET

Our strategies:

5 models

Phase 1: LM11A-31-M Safety and Pharmacokinetics

Single ascending dose (SAD), 6 cohorts: • Young subjects: D1-4 dose ascending• Elderly: D4 (fasted and non-fasted)

Multiple ascending dose (MAD – 10 days), 2 cohorts:• Elderly D5 2x/day • Elderly D6 1x/day

LM11A-31-M is safe and well tolerated in the target elderly population

No serious Adverse Events (AEs)

48 subjects (36 drug / 12 placebo)

20 subjects(16 drug / 4 placebo)

Total: 68 subjects (52 drug / 16 placebo)

PharmatrophiX

Michelle James & Sam Gambhir, Stanford

PET/MRI Imaging TSPO ligand: GE-180

HC:

ctx:

wt-vehicle APPL/S-vehicle10

%ID

/g

wt-C31 APPL/S-C31

*** *

norm

aliz

ed P

ET s

igna

l Cortex

norm

aliz

ed P

ET s

igna

l Hippocampus

** *

0

James et al Longo-GambhirSNS 2014

GE-180 PET: APP-L/S mice; 3 mo rx (start 5.5-7 mo)

Next: Phase 2a study in mild AD - 2016

• Exploratory endpoint• 6 mo rx• Biomarkers: FDG-PET, MRI

CSF p-tau/tau, Aβ, choline acetyltransferase activity • Clinical measures: ADAS-Cog-14, NPI, Cogstate