Daniel S Lorrain, Michael Poon, Karin J Stebbins, Geraldine Cabrera, Alex Broadhead, Jon Seiders, and Jeff Roppe Pipeline Therapeutics, San Diego, CA

Home , Clemastine

Discovery of PIPE-505, a small molecule therapeutic for the treatment of sensorineural 304.01 hearing loss (SNHL) associated with cochlear synaptopathy *Daniel S Lorrain, Michael Poon, Karin J Stebbins, Geraldine Cabrera, Alex Broadhead, Jon Seiders, and Jeff Roppe Pipeline Therapeutics, San Diego, CA

Introduction Neurite outgrowth mediated by Netrin/DCC pathway PIPE-505 increases hair cells via Notch inhibition Ex vivo explant In vivo drug treatment Isolated SGN PIPE-505 is a gamma secretase inhibitor in development PIPE-505 DMSO 3 Atoh1 Hes5Hes5 ↓ ↑ Atoh1 ** for the treatment of SNHL associated with cochlear 1.0 synaptopathy. A series of in vitro and in vivo studies in PIPE-505 2

γ-secretase 1 Atoh Hes5 Hes5 0.5

animal models of auditory loss have demonstrated two 1

)

(fold increase/vehicle) (fold

9 Fold Increase/vehicle Fold distinct mechanisms of action (MOA) leading to decrease/vehicle) (fold 1 1 .0 l 0 p 0.0

R PF2 0.2% 2% vehicle 0.2% 2%

o t

restoration of hearing function. Specifically, PIPE-505 1) m

r ↓Hes5 0 .5

Brain Research Bulletin 2016 Notch o

n (

facilitates SGN neurite growth via the Netrin/DCC pathway 5 s e OHC *** H ↑ 4 *** P<0.001, 1-way ANOVA, Tukeys post-hoc analysis 0 .0

leading to regeneration of inner hair cell ribbon synapses v e h ic le .0 3 .3 3 1 0 ** * n

P I P E - 5 0 5  M o

i 3

m µ

and 2) increases Atoh1 expression via reduced Notch e

r / Hes5↓ 0 55

PIPE-505 restores synapses in vivo, mouse model 1

r 2

e OHC

↑ H p

signaling leading to outer hair formation. These cellular

O s

C #

H 1 O

# IHC synapses DCC α-fragment

regenerative effects together, restore auditory function. + 45

P=0.0013 A 7

O 0

P<0.0001 Y 16 M 40 untreated Veh 0.02% 0.2% 2% ↑Atoh1 .001 .01 .1 1

C PIPE-505 M PIPE-505 H

I 14

a t

c PIPE-505 improves auditory measures n

u 12

2 P

B 10 t

C ABR Wave 1 ABR Threshold 8 PIPE-505 restores ABR PIPE-505 improves ABR e B 5 5 vehicle 2% PIPE-505 iv d 0 0 a 8 -5 -5 wave 1 amplitude, a measure threshold, a measure of OHC N 9 E E IP IP P P % % of AN function function .2 2 0 PIPE-505 restores auditory nerve synapses and hair cells CtBP2/GluR2 98dB 1 .5 9 0 V e h ic le

P I P E - 5 0 5 , 2 %

) B I N a iv e a n im a l

PIPE-505 causes dose dependent type I spiral PIPE-505 restores synapses in vivo, guinea pig model

d (

e 1 .0 8 0

d a ganglion neurite outgrowth * l

o * * * *

h R s * * * *

# IHC synapses autoradiography * * * * e B 0 .5

r 7 0 A

G041 h

Notch- T C o n t r o l e a r NXT1505 neurite outgrowth DRC sparing 20 P I P E - 5 0 5 , 2 % 0.008 0.010 0 .0 6 0 *** 1 0 2 0 3 0 4 0 . 5 0 6 0 7 0 . 8 0 . 4 8 1 6 2 4 3 2 0.008 0.006 h 225 Human PK prediction t BACEi 15 *** d B S P L

g 5% dose level F r e q u e n c y ( k H z )

n >30d sustained exposure

e 0.006

l g

0.004 n

e In summary, PIPE-505 restores SGN connections to inner hair

i d

r 0.004 10 150

neurite length neurite i

e cells and regenerates outer hair cells. These effects are b

0.002 N

* Neurite length Neurite

0.002 I S

Synapses/IHC 5 mediated via two distinct γ-secretase substrates, DCC and G

***

0.000 0.000 ] 75

H Notch, respectively. A first-in-human study is planned to 1 le 5 3 9 9 9 9 9 9 3 3 3 3 3 3 0.1 10 c 0 T 3 3 3 3 3 3 6 6 6 6 6 6

NT3 0.01 i 5 N 8 8 8 8 8 8 1 1 1 1 1 1 3 0.001 h 1 3 3 3 3 3 3 8 8 8 8 8 8 e 0 0 0 0 0 0 [ vehicle 0.0001 M D D D D D D 7 7 7 7 7 7 V µ Z Z Z Z Z Z ------1 A A A A A A S S S S S S 0 [NXT1505](µM) M M M M M M evaluate PIPE-505 in patients with SNHL associated with M M M M M M p p n n n µ B B B B B B PIPE-505 (μM) 0 0 1 0 0 1 M M M M M M 1 0 1 0 p p n n n µ 1 1 0 0 1 0 0 1 1 0 1 0 naive vehicle 0.02% 0.2% 2% 0 1 1 Vehicle 0.02% 0.2% 2% cochlear synaptopathy. Measures of audibility as well as

PIPE% NXT1505-505 IT injection (30l) speech intelligibility will be assessed.

P <0.05 1-way ANOVA, Tukey's post-hoc Small molecule inhibition of the muscarinic M1 acetylcholine receptor by potent, selective antagonists facilitate 206.19 OPC differentiation

*Michael M Poon1, Kym I Lorrain1, Ariana O Lorenzana1, Karin J Stebbins1, Alexander Broadhead1, Thomas O Schrader2, Yifeng Xiong2, Jill Baccei2, Ari J Green4, Jonah R Chan4, Daniel S Lorrain3; 1Biol., 2Chem., 3Pipeline Therapeutics, San Diego, CA; 4Neurol., UCSF, San Francisco, CA

Introduction Calcium mobilization Lysolecithin mouse brain slice Human brain slice

Inhibition of the muscarinic acetylcholinergic receptors by Fold selectivity against M1 Compound M1 IC50 (nM) M2/M1 M3/M1 M4/M1 non-selective muscarinic antagonists (e.g., clemastine, 3.19 Benztropine 16.9 11.2 4.78 Compound EC50 (nM) MBP M1 Figure 4 Pipeline M1 benztropine) accelerates the differentiation of Compound 57 0.716 343 763 430

5 antagonists induced Mbp ) PIPE-359 7.9 t PIPE-359 1.69 102 43 26 C d * in a naïve human cortical oligodendrocyte precursor cells (OPCs) into oligodendrocytes d

- *

2.1 2 Compound 77 98.8 1270 212 ( 4

PIPE-307 10 brain slice assay. Slices e

(OLs). Subsequent work has implicated the M1 isoform as l

PIPE-307 2.35 555 64.2 54.2 c

i were incubated in MT7 or h

being a key driver of this phenomenon. In-house chemistry 6.69 Compound 107 30 e 3 Compound 29 57.4 347 91.9 v

compound for 9 days prior

s v

PIPE-683 7.45 698 175 292

efforts have identified a number of potent, selective M1 PIPE-683 36.2 e 2 to RNA isolation and g

Compound 107 8.91 178 117 313 n

a QPCR.

antagonists. Using these, we have characterized the effects of h

13.5 c

Compound 51 417 1590 24.5 Compound 77 44.3 1 d [PIPE-307](logM) l inhibiting M1 in a diverse set of in vitro assays, including OPC Compound 25 19.6 128 199 241 o F 0 differentiation, cortical myelination, and organotypic brain Compound 14 51.5 124 217 58.4 300nM 11µMµM 11µMµM Vehicle MT7 OPC359 OPC797 slice. Our data show that a selective, small molecule inhibitor Table 2 Pipeline compounds are potent and selective in a cellular PIPE-359 Cmpd 77 of M1 is sufficient to drive OPCs towards differentiation and setting. Compounds were evaluated in CHO-K1 cells overexpressing one of M1-4 receptors for inhibition of ACh-induced calcium release at EC Figure 2 Pipeline compounds induced Mbp in cultured cortical mouse that the resulting oligodendrocytes express myelin basic 80 Dunnett's multiple concentrations. brain slice demyelinated with lysolecithin. Slices were cultured at comparisons test Significant? Summary Adjusted P Value protein. Moreover, these OLs are functional, i.e., capable of postnatal day 17, demyelinated and treated with compound. Mbp was Vehicle vs. MT7 Yes * 0.0136 axonal wrapping and induction of nodes of Ranvier. Of note, Rat OPC differentiation measured by quantitative PCR. The highly M1 selective peptide MT7 was Vehicle vs. PIPE-359 No ns 0.1802 an M3 selective antagonist (Sagara et al., 2006) was not active used as a positive control.

100 Compound EC50 (nM) Vehicle vs. Compound 77 Yes * 0.0444

n )

in a rat OL differentiation assay. In concert with our in vivo o

t T

a PIPE-683 18.9

data (also presented at this meeting), a strong case can be t

t n

e PIPE-359 20.3 Rat cortical myelination d

made that the development of an M1 selective small r

e f

z Cortical coculture (myelination) Conclusion

i i

molecule antagonist is a promising approach for treating l PIPE-307 25.4

a D

) 40

0 L

O r

demyelinating diseases such as multiple sclerosis. Compound 107 78.5 /

h P

o MT7

t MT7 Selective inhibition of M1 results in the differentiation of g IC 25.4 x

O EC 25.4 nM

n 50 50

(

e d

Compound 77 175 l 30

% OPCs into mature oligodendrocytes. Here, we described the

i t

-50

i t

-9 -8 -7 -6 -5 M3 selective* > 3.00E+03 m identification of potent, selective small molecule M1

n e 3 [PIPE-307](logM) i l 3

log[OPC-0001307], M s

[ H]NMS membrane binding e 20 antagonists as evaluated by [ H]NMS binding and calcium

y B

M vehiclevehicle M Vehicle T3 1µM PIPE-307 mobilization assays and further showed that these molecules

g EC50 16.2nM

v a

Fold selectivity against M1 ( 10 induce myelination-competent oligodendrocytes. These Compound M1 Avg Ki (nM) M2/M1 M3/M1 M4/M1 M5/M1 PIPE-307 -11 -10 -9 -8 -7 -6 -5 molecules also induced Mbp in mouse and human Benztropine 1.14 16 2.67 8.21 2.7 [PIPE[OPC1-307](307](lologMgM) ) PIPE-359 0.144 130 14.4 45.1 17.4 organotypic slice models. Together, this provides compelling PIPE-307 0.349 73 18.5 38 259 evidence that inhibition of M1 with small molecule Compound 57 1.13 22 7.11 29.9 5.37 MBP antagonists developed at Pipeline have a positive impact in Hoechst Figure 3 Differentiated Vehicle Compound 25 1.41 160 8.81 189 736 OLs are myelination treating demyelinating disorders such as multiple sclerosis. At Compound 77 1.48 8.8 41.1 13.5 54.5 OPPIPEC 6-86833 p pulseulse dosedose [ACh] in OPC conditioned media (rOPC diff; well average) competent. Myelination this point, a clinical development candidate has been Compound 51 2.34 390 113 148 538 (rOPC diff; well average) 40000 ) 2.0 was evaluated in a rat e n.s. n.s. P=0.002 P=0.048 identified and IND-enabling studies have been initiated.

Compound 29 2.55 90 17.4 1.95 6.07 l c i cortical myelination assay

h ** * 30000 Compound 14 3.6 >7692 59.5 174 583 e

v 1.5

as described previously

PIPE-683 4.04 87 13.3 121 167 U

L 20000 (Lariosa-Willingham et al

m r R 1.0

Compound 107 7.55 120 38.4 93.1 n.d. cells/well nM ACh o

n 2016). Myelin segments ( 10000 60,000 12nM References 40,000 5nM P were identified by MBP PIPE-307 B 0.5

Table 1 Pipeline compounds are potent and selective for human M1 in an 30,000 2nM M

colocalization with Tuj1 d

mAChR recombinant membrane binding assay. 0 l Sagara, Y. et al. Identification of a novel 4-aminomethylpiperidine class of M3 muscarinic o -10 -9 -8 -7 -6 -5 f 0.0 (axonal marker) and Veh 683 Veh 683 Veh 683 Veh 683 receptor antagonists and structural insight into their M3 selectivity. J Med log[ACh], M Chem, 2006;49(19), 5653–5663. 2h 6h 24h 72h averaged per OL. Time in compound Students t-test MBP Lariosa-Willingham, K.D., et al. Development of a central nervous system axonal myelination Figure 1 Pipeline compounds induce OL differentiation in rat OPCs at nM Tuj1 assay for high throughput screening. BMC Neuro, 2016;17(6). potencies. Compounds were evaluated by immunocytochemistry in rat OPCs Hoechst (Mei et al 2016). ACh levels in OPC conditioned media measured by calcium Mei, F. et al. Accelerated remyelination during inflammatory demyelination prevents axonal loss and improves functional recovery. eLife, 2016; 5: e18246. *Small molecule M3 selective antagonist from Banyu (Sagara et al 2006). flux in hM1-CHO. Pulse dosing using PIPE-683, a structural analog of PIPE-307, Ki (nM) for M1: 4670, M2: 6730, M3: 25.5, M4: 3600 in 3HNMS membrane binding shows 6h exposure is sufficient to initiate OPC differentiation. 206.18 PIPE-359, a novel, potent and selective M1 muscarinic receptor antagonist as a therapeutic approach for remyelination in multiple sclerosis

* Karin J Stebbins1, Michael M Poon1, Alexander Broadhead1, Geraldine Edu1, Ariana Lorenzana1, Kym I Lorrain1, Thomas Schrader1, Yifeng Xiong1, Jill Baccei1, Christopher Baccei1, Ari J Green2, Jonah R Chan2 and Daniel S Lorrain1 ., 1 Pipeline Therapeutics, San Diego, CA; 2UCSF, Department of Neurology, San Francisco, CA

Introduction Orally administered PIPE-359 occupies M1 receptors and inhibits PIPE-359 improves behavioral score, VEP N1 latency and Novel small molecule approaches aimed at stimulating remyelination M1 function in mouse forebrain g-ratios in a murine MOG-EAE model would greatly complement immunotherapies and provide significant PIPE-359 Exposure in rat 30 mg/kg PIPE-359 provides full M1 coverage for 8h in mouse brain Visual Evoked Potential (baseline, d7, d14, d21) AUCP.O. 0.14 10 (hr*mg/mL) PPB (%) 20 Induce Vehicle or PIPE-359 QD TEM on spinal Image ) 2 mg/kg IV 10

neural protection in demyelinating conditions such as multiple sclerosis M

m 1 10 mg/kg PO t1/2, P.O. (hr) 8.6 BTB (%) 13 MOG daily behavioral score cord and optic analysis (

) ) 3 C (mM) 0.09 @ 1h

- max

M M E 0.1 M 2 M 2 (d0) (d0-d21) nerves (g-ratios)

(MS). Recently, we described the muscarinic M1 receptor (M1R) as an 0.1 m

(

P F (%) 4.5

M 4 M 4

9 a 9 0.01 Slice EC50

5 M 5 M 5

s 3

0.01 AUC (hr*mg/mL) 0.5 3

- a BLQ for IV is 0.005 mM (PO 0.009 mM) I.V. -

l 0.001

E P

important regulator of oligodendrocyte precursor cell (OPC) E M 3 M 3 30 mg/kg improves VEP N1 latency P

P 30 mg/kg improves behavioral score I

Cl (mL/min/kg), ER 61, 0.87 I

0.0001 M1 Ki P 0.001 P M 1 M 1 0 4 8 12 16 20 24 V (L/kg) 2.2 Brain Total differentiation and a promising target for drug discovery. We developed d 0.00001 Plasma Total 4.0 75 **** Time (h) Plasma Unbound Brain Unbound Veh

Plasma Unbound Brain Unbound e Vehicle ) t1/2, I.V. (hr) 0.9 0.000001 r 3.5 PIPE-359 s

o 70 PIPE-359 c m 0 4 8 12 16 20 24 0 4 8 12 16 20 24 m some Cl 23 3.0 (

int S

PIPE-359, a novel, potent and selective M1R antagonist and highlight its * y [Brain] (mM) 0.026 (mL/min/mg) Time (h) Time (h) y

2h t

2.5 **** c 65

i e

[Plasma]2h (mM) 0.080 PPB (%) 20 b 2.0 t

a 60

potential for remyelination. a

s i [CSF] (mM) <0.004 BTB (%) 13 1.5 L

l 1.0 55 N

K , K 0.32, 0.21 a baseline N1 latency

p p,uu

c P

i 0.5

n E

i 50 l

0.0 V M1 occupancy ED ~ 2 mg/kg 30 mg/kg PIPE-359 provides ≥ 50% M1 occupancy EC ~ 3 nM 30 mg/kg PIPE-359 fully inhibits C 50 50 45 PIPE-359 binds to M1 with high affinity and demonstrates M1 occupancy for ~20h M1-mediated IP1 accumulation 0 2 4 6 8 10 12 14 16 18 20 22 pre-MOG 7d 14d 21d Days post MOG induction Days post MOG induction selectivity over other muscarinic receptors 300 **** p<0.0001, mixed-effects model with Sidak's multiple comparisons test * P <0.05, **** P < 0.0001 vs. vehicle, 2way ANOVA, Tukey's

multiple comparison test

)

) y

y 150

]

( c

100

1 n

e ** n

y 100

I c

o 200

[

n ***

u ****

)

p n

a 100

i 30 mg/kg PIPE-359 remyelinates axons in the spinal cord

a c

Potency (nM) c extrapolated

(

50 l

e 1

50 1 r

o 100 O

M 50

Membrane binding, Ki 0.144 1

(

M % 0 Calcium flux, IC50 1.69 0 0 0 veh MT7 0.1 1 10 30 0 4 8 12 16 20 24 0.00001 0.0001 0.001 0.01 0.1 Vehicle BQCA PIPE-359 MT7

PIPE-359 (mg/kg) Time (h) Free brain PIPE-359 (mM) 100 mg/kg LiCl ** p<0.01, *** p<0.001, **** p<0.0001 vs. BQCA, Fold-selectivity ANOVA with Dunnett's multiple comparison test M2/M1 M3/M1 M4/M1 M5/M1 PIPE-359 enhances remyelination in a murine cuprizone model Membrane binding, Ki 130 14 45 17 Naive Vehicle PIPE-359 unmyelinated axons 0.2% cuprizone diet 1.0 1.0 unmyelinated axons 1.0 unmyelinated axons remyelinated axons remyelinated axons remyelinated axons

o 0.8 o

0.8 o 0.8

i i

Calcium flux, IC 102 43 26 315 i

t t

50 x 5 weeks t

g g demyelination remyelination Free floating brain sections Confocal microscopy 0.6 0.6 0.6

0.4 0.4 0.4 PIPE-359 promotes OPC differentiation in vitro and 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 Normal chow Quantify ROI in cortex axon diameter (mm) axon diameter (mm) axon diameter (mm) increases remyelination ex vivo Vehicle or M1 antagonist x 2 weeks PIPE-359 increases the percentage of remyelinated axons and decreases PIPE-359 dose-dependently differentiates rat OPCs to oligodendrocytes in vitro the percentage of unmyelinated axons ****

100 30 ** 80 s 1.0 unmyelinated axons s

n **** ****

o o 3 ****

IC : 20.3 nM x 50 MBP x

T remyelinated axons 60

20 d

e o

0.8 e

t a

Normal chow a

a n

r i

50 i -

Vehicle l

l b

PIPE-359 dose-dependently enhances remyelination in a g

u y

0.6 PIPE-359 y 10

m m

non-immune-mediated cuprizone model 20 n

25 e

r l

Naive u

% %

h ** 0.4 0 0 e 0 x 0 2 4 6 8

V Naive Vehicle PIPE-359 Naive Vehicle PIPE-359 e

vehicle T3 300 nM PIPE-359 t axon diameter (mm) -25 r mean  SEM, 2 1500x TEM images of the distal lumbar spinal cord region were quantified per animal, n=5-6 animals per group 100 ** p < 0.01, **** p < 0.0001 vs. vehicle, ANOVA with Dunnett's multiple comparisons test MBP Hoechst o **** P<0.0001 vs. Vehicle and vs. Naive, ANOVA,

-9 -8 -7 -6 -5 c ** Tukey's multiple comparisons test 5 weeks CPZ diet

log[PIPE-359], M n * i

PIPE-359 increases MBP and Caspr protein in mouse cortical slices PIPE-359 also promotes remyelination in optic nerves from this study (see Edu et al. poster)

) n

t 140 0.012

P o

n * **

B o

120 r

t 50

M j

s 100 n

0.008 (

u h

PIPE-359 dose-dependently induces Mbp i

/ e

80 t

u H

in cultured mouse cortical slices / 60

y Conclusion

c a

0.004

) r

r 40

l 2.0

e P

c 20 Vehicle i

MT7 C

h M 0 0.000 0

e Vehicle LPC MT7 PIPE-359 Vehicle LPC MT7 PIPE-359 % v • 300 nM 1 M m These data highlight the therapeutic potential of a selective M1R 1.5 300 nM 1 mM r EC50 7.9 nM e LPC PIPE-359 Normal 5 wks Veh 1 3 10 30

v Vehicle o

( naive antagonist to benefit conditions such as MS in which demyelination 1.0

n PIPE-359 (mg/kg)

o i

t plays a role. c

u 2 weeks remyelination

d 0.5 LPC 30 mg/kg PIPE-359 n i • * p<0.05, ** p<0.01, **** p<0.00 vs. vehicle, ANOVA with Dunnett's multiple comparisons test A clinical development candidate has been identified and IND-enabling p ** p<0.05 Normal chow vs. 5 weeks CPZ diet, Student's t-test b 0.0 M -10 -9 -8 -7 -6 -5 studies have been initiated. [PIPE-359](logM) MBP MBP Caspr Hoechst The muscarinic M1 antagonist PIPE-359 demonstrates remyelination in vivo through visual evoked 206.19 potential (VEP) and electron microscopy (EM) of mice with experimental autoimmune encephalitis (EAE)

Geraldine C Edu*1, Karin J Stebbins1, Alexander R Broadhead1, Michael M Poon1, Ariana O Lorenzana1, Thomas Schrader1, Yifeng Xiong1, Jill Baccei1, Ari J Green2, Jonah R Chan2 and Daniel S Lorrain1. 1Pipeline Therapeutics, San Diego, CA; 2Neurol., UCSF, San Francisco, CA

Introduction PIPE-359 reduces N1 latency shifts and N1P2 amplitude degradation Profiling M1 antagonists through VEP

) VEP @ 21d

)

V 

25 B 50 Vehicle # C V s Multiple sclerosis is characterized by immune mediated myelin injury A (

**** Vehicle 

) 100 vehicle 100 (

**** p<0.0001 A y #### C e VEP at 21 days s PIPE-359

* 2 a s

e r =0.4640 PIPE-359

m a

PIPE-359 d

20 s ✱✱ (

40 vehicle (C1)

and progressive axonal loss. Visual evoked potential (VEP) is a clinically a 80

)

i c

*** r

2 ✱✱✱✱ 40 PIPE-307 3mg/kg

h e

15 c e

S 60 

D 30

@ 80 ✱✱✱ ( PIPE-307 30mg/kg

translatable model used in patients with multiple sclerosis due to its

)

e e s vehicle (C2)

n 40 u

10 d

d t

e 20 m

i 30

l (

ability to measure myelin damage of the visual pathway through the t PIPE-683 30mg/kg

i * p=0.04936

l t

2 y L

20 i p

r =0.2085

l vehicle (C3) m

5 c

1 1 10 60

latency of VEP - which reflects the velocity of signal conduction along N PIPE-359 30mg/kg

A e

2 0

t 20

2 a

0 - Control P

0 A

the visual pathway; while the amplitude of VEP is believed to be 0 7 14 21 -20

1 N

0 7 14 21 1 2

N -20 0 20 40 60 80

N 40

days post MOG induction P

7 3 9

e 10 l

days post MOG induction N1 Latency Shift (ms) o

8 5

closely correlated with axonal damage of the retinal ganglion cells 0

3 6 3

- - - m

*** p=0.0008, **** p<0.0001 vs vehicle via ANOVA with Tukey N n

* p=0.0304, # p=0.0260 vs vehicle via ANOVA with Tukey's h

E E

3 E

P P P

v 7

(RGC) . PIPE-359 is a novel, potent and selective M1 antagonist with C

I I I

Fig 2A Differences in N1 latency shift can be detected at 7 days post MOG and becomes more significant overtime suggesting functional remyelination. 2B. N1-P2 amplitude decreases are 0 0

P P P 3 due to axonal damage at the retinal ganglion cells (You, Y. et.al 2011) and PIPE-359 prevents N1P2 amplitude degradation over time. 2C. At 21days in a scatter plot of each eye N1 latency -

E 40 60 80 100 120 good oral exposure and brain penetration which is efficacious in 30mg/kg

shifts and N1-P2 amplitude decreases are significantly correlated measures. PIPE-359 (Y = 1.365*X + 19.88) had a significantly more positive slope than vehicle (Y = 0.4056*X + 29.71). P I

P N1 Latency (ms) rodent models of demyelination such as cuprizone and experimental ***p=0.0003, **** p<0.0001, **p=0.0016, #### p<0.0001 autoimmune encephalitis (EAE). Flash VEPs were recorded from EAE EM of spinal cord and optic nerve show remyelination by PIPE-359 vs vehicle via ANOVA with Dunnett B D VEP at 21 days mice to determine if a selective M1 antagonist can demonstrate ) 40 A V 1.0 unmyelinated axons B 2.0 vehicle (merge)  ✱

**** (

30 80 e PIPE-307 3mg/kg

functional remyelination. Spinal cords and optic nerves were collected **** remyelinated axons **

s s d ✱✱ s 30

n **** ****

n u

o PIPE-307 30mg/kg

i x

t 1.5

l o

0.8 60 d

i a

PIPE-683 30mg/kg

for electron microscopy (EM) imaging and g-ratios were calculated to p

t 1

20 d

2 e

e 20

t PIPE-359 30mg/kg

a e

Vehicle A

n 40

i d

l Control

confirm remyelination. l

2 1.0

u y

0.6 P y

PIPE-359 10 t -

10 i

l m

20 1

Naive

m % % 0.5

PIPE-359 is efficacious in VEP of EAE mice A 0 0 0

0.4 l

7 3 9

naive vehicle PIPE-359 naive vehicle PIPE-359 e

0 8 5

p r

0 2 4 6 8 c

3 6 3

- - -

mean  SEM, 2 1500x TEM images of the distal lumbar spinal cord region were quantified per animal, n=5-6 animals per group m

E E

B E o ** p < 0.01, **** p < 0.0001 vs. vehicle, ANOVA with Dunnett's multiple comparisons test e

A axon diameter (m) 0.0

P P P

I I I

P P **** P<0.0001 PIPE-359 and naive vs. Vehicle, ANOVA, Tukey's P 40 60 80 100 120

✱ 3

D - C ✱✱

50 E 30mg/kg N1 Latency (ms) P

unmyelinated axons s

I e

1.0 40 n

o P

a 40

remyelinated axons i

a d

x **p=0.0037, * p=0.0111 vs vehicle ANOVA with Dunnett e

30 M

m a

t 30

x l **** t

E Fig 5A. M1 antagonists all showed significant N1 latency difference from vehicle treated EAE mice by 21 days post MOG induction. B. Not all M1 antagonists but PIPE-307 at

0.8 a 20

20 y

l i

0 both 3 and 30mg/kg showed a significant difference in N1P2 amplitude from vehicle at 21days. C. N1 latency vs N1P2 amplitude linear regression (xy scatter data points not

y 5

a 10

1 shown) at 21 days of each M1 antagonist where the desired profile is low N1 latency and high N1-P2 amplitude. D. N1 latency vs amplitude ratio linear regression (xy scatter

- 10

e 0 data points not shown) at 21 days the desired profile is a positive slope where demyelination is seen with a negative slope (Y = -0.01194*X + 1.446). M1 antagonist PIPE-307 p 0.6 Vehicle % 0 Vehicle PIPE-359 at both 3mg/kg (Y = 0.03111*X - 0.5940) and 30mg/kg (Y = 0.05091*X - 1.282) achieves a positive slope very close to control mice (Y = 0.02540*X - 0.08203) . PIPE-359 Vehicle PIPE-359 1 image quantified per ON from 4 independent animals C D * P < 0.05 vs. Vehicle, unpaired t-test 0.4 1 image quantified per ON from 4 independent animals 4.0 ** P < 0.01 vs. Vehicle, unpaired t-test Conclusions Vehicle 75 Vehicle 0 2 4 6 8 3.5 **** Fig 3A. EM of the spinal cord and g-ratio calculation shows that PIPE-359 shows significant remyelination. PIPE-359 axon diameter (um) 3B . PIPE-359 shows %remyelination and % unmyelinated axons show values close to naïve mice. 3C. EM

3.0 PIPE-359 e

) 70 1 image quantified per ON (n=4 independent animals) of the optic nerve and g-ratio calculation shows that PIPE-359 shows significant remyelination. 3D. PIPE- r

s ➢ **** P < 0.0001 vs. vehicle, unpaired t-test VEP is a sensitive measure of remyelination due to its ability to detect

o 2.5 * 359 treated mice have a greater percentage of remyelinated axons than vehicle treated mice.

m (

S 65

2.0 l

y impairment in the visual pathway before the onset of clinical disability in

a c

c 1.5 n

i 60

n t i VEP amplitude symmetry is preserved by M1 antagonists EAE mice.

l 1.0

a C L 55 0.5 Naïve Controls Vehicle PIPE-307 1 A B C PIPE-307 ➢ M1 antagonists demonstrate robust remyelination and axonal protection as 0.0 N D E 50 3mg/kg 30mg/kg Right seen by reduced N1 latency shifts and preserved VEP amplitude waveform 0 2 4 6 8 10 12 14 16 18 20 22 45 Day 0 7 14 21 symmetry . days post MOG induction ➢ Multiple compounds screened through this in vivo discovery paradigm have * p=0.0196, **** p<0.0001 vs vehicle via ANOVA with Tukey's VEP @ 21d F VEP at 21days demonstrated remyelination thus confirming a small molecule selective M1 Fig 1A. Methods study timeline of PIPE-359 in the EAE model with 40 flash VEP recordings performed in parallel. 1B. EAE mice are dark *** p=0.0003392 vehicle ✱ antagonist is a promising approach to treat multiple sclerosis. ) r2=0.3728 adapted in a room with red lights for 1hr prior to recording. Mice are EV anesthetized with ketamine/xylazine (75/10mg/kg) and eyes are 2.0 ##

 PIPE-359

( ➢ A clinical development candidate has been identified and IND-enabling

30 dilated with 1% Tropicamide for 2 minutes. Genteal eyedrops were o ✱✱

i e

control t d

applied for lubrication and mice are placed on the Diagnosys Celeris a 1.5 u

R studies have been initiated

t Model D430 (Lowell, MA) 37°F heated platform. Electrodes are

i Left

e l

carefully placed and flash VEP execution is performed 10 minutes d

p 20 u

post anesthesia wherein each exam consists of at least 3 runs, with t 1.0

l A

** p= 0.009181 pulse intensity 3 cd.s/m2, frequency 1 Hz, on time 4 ms, pulse color: p

2 m

2 r = 0.3211 white-6500K, 100 sweeps per result. Flash VEPs are recorded from A P 10 0.5 References

- each eye independently and simultaneously. N1 latency is

1 determined by the average N1 from 3 VEP traces. 1C. Clinical signs of N

EAE are first detected at day 11 and by the end of the study PIPE-359 0.0 1. Green AJ, Gelfand JM, Cree BA, Bevan C, Boscardin WJ, Mei F, Inman J, Arnow S, Devereux M, Abounasr A, Nobuta H, Zhu A, Friessen M, Gerona R, von

3 9

0 7 l

significantly alleviates EAE clinical disability. 1D. In same animals in o

0 8 5 p

r Büdingen HC, Henry RG, Hauser SL, Chan JR. Clemastine fumarate as a remyelinating therapy for multiple sclerosis (ReBUILD): a randomised, controlled,

3 6 3

- - -

40 60 80 100 120 parallel, flash VEP detects a difference in N1 latency with PIPE-359 as m

n h

3 double-blind, crossover trial. Lancet. 2017 Dec 2;390(10111):2481-2489.

E E E

e o

early as 7days post MOG induction, over time the effect becomes

P P P

I I

N1 Latency (ms) I

P P

more significant out to 21days. 1E. Each individual eye scatter plot P 3

- 2. You Y, Klistorner A, Thie J, Graham SL. Latency delay of visual evoked potential is a real measurement of demyelination in a rat model of optic neuritis.

show that N1-P2 amplitude and N1 latency measures are E

P Invest Ophthalmol Vis Sci. 2011;52(9):6911–6918. significantly correlated measures at 21 days post MOG induction and I 30mg/kg PIPE-359 (Y = -0.03580*X + 2.971) has a significantly more positive Fig 4A-D. Sample VEP waveform examples of each treatment group showing 3 VEP traces and the average trace. Top panel is the right eye, P slope than vehicle (Y = -0.01338*X + 1.656). bottom panel is the left eye. 4E. Standard VEP waveform components. Amplitude ratio description and calculation. 4F. M1 antagonists do not **p=0.0026, ## p=0.0032, *p=0.0356 vs 3. You Y, Klistorner A, Thie J, Gupta VK, Graham SL. Axonal loss in a rat model of optic neuritis is closely correlated with visual evoked potential amplitudes show a degradation of N1P2 amplitude values and thus preserve of the symmetry of P1N1 and N1P2 amplitude waveforms vehicle via ANOVA with Dunnett using electroencephalogram-based scaling. Invest Ophthalmol Vis Sci. 2012;53:3662.