Heledd Brown-Wright Kirstie Bennett, Pamela Shaw, Alastair Brown, - - PowerPoint PPT Presentation

HTL0014242 a novel metabotropic glutamate receptor 5 (mGlu5) negative allosteric modulator (NAM) limits glial activation and slows late stage disease progression in the SOD1G93A mouse model of motor neuron disease

Heledd Brown-Wright

Kirstie Bennett, Pamela Shaw, Alastair Brown, Matt Barnes & Richard Mead

Metabotropic glutamate receptor 5 (mGlu5) is a novel therapeutic target for MND

• Glutamate-mediated toxicity is recognised as a mechanism of both neuronal and glial injury

Blocking mGlu5 with the NAM MPEP in SOD1G93A mice (Rossi et al. 2008 Cell Death and Differentiation) mGlu5

+/- in SOD1G93A mice (Bonifacino et al.

2017 Neuropharmacology)

• Pharmacological and genetic evidence to support role for mGlu5 in the SOD1G93A mouse model of

MND

Sosei Heptares’ mGlu5 NAM HTL0014242 – highly potent and selective

MPEP (mGlu5 NAM ) HTL0014242 (mGlu5 NAM)

Acknowledgements to Dr Ben Tehan at Sosei Heptares for this image Christopher et al. 2015 J Med Chem

World leader in GPCR drug design 3D structure of mGlu5 receptor

HTL0014242 has an excellent pharmacokinetic profile in the SOD1G93A transgenic mice

1 0 2 0 0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

T i m e ( h ) H T L 0 0 1 4 2 4 2 ( n M )

T o t a l in s p in a l c o r d T o t a l in b lo o d F r e e in s p in a l c o r d I C 5 0

The estimated mGlu5 receptor

• ccupancy (RO) in SC was in the

region of 40% - 90% at Cmax for doses of 3,10 and 30mg/kg of HTL0014242

HTL0014242 90D dose response study in the SOD1G93A mouse model of MND

• Inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6)

(Mead et al. 2011 PLoS ONE)

59% reduction in α-motor neurons Weaning Average onset of clinical signs of disease Standard end-point Decline in motor performance Glial activation in lumbar SC

age 21 40 75 90 140 - 150 60

Phenotype HTL0014242 dose-response study design age 25 35 75 90 60

HTL0014242 daily oral dosing at 3,10, 30mg/kg (11 mice per group ) Rotarod testing once per week Neuroscored twice per week

Tissue collected for histology

Vehicle = 0.5% methyl cellulose

Dose-dependent reduction of astrocyte activation at 90D in VH of lumbar SC

Scale bars = 50µm

V e h i c l e 3 m g / k g 1 0 m g / k g 3 0 m g / k g

2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0

G F A P S t a i n i n g a r e a (  m

2 )

* * * * * V e h i c l e 3 m g / k g 1 m g / k g 3 m g / k g

5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0

I b a 1 S t a i n i n g a r e a (  m

2 ) *

GFAP Staining Iba1 Staining

GFAP DAPI

10mg/kg 30mg/kg Vehicle 3mg/kg

Iba1 DAPI GFAP DAPI GFAP DAPI GFAP DAPI Iba1 DAPI Iba1 DAPI Iba1 DAPI

mGlu5 receptor occupancy from HTL0014242 dose- response study inversely correlates with GFAP staining

Data presented as mean ± SEM Data presented as mean ± SD

Scale bars = 50µm 10mg/kg 30mg/kg Vehicle 3mg/kg

GFAP Staining

V e h i c l e 3 m g / k g 1 m g / k g 3 m g / k g

2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0

G F A P S t a i n i n g a r e a (  m

2 )

* * * * *

GFAP DAPI GFAP DAPI GFAP DAPI GFAP DAPI

HTL0014242 survival study in the SOD1G93A mouse model of MND

59% reduction in α-motor neurons Weaning Average onset of clinical signs of disease Standard end-point Decline in motor performance First signs of glial activation in lumbar SC

age 21 40 75 90 140-150 60

Phenotype HTL0014242 survival study design age 25 35 75 90 60

30mg/kg HTL0014242 dosed daily (25D cohort) Rotarod testing twice per week Neuroscored three times per week Tissue collection 30mg/kg HTL0014242 dosed daily (75D cohort) cohort

140-150

Significant increase in lumbar SC ChAT stained motor neurons

V e h i c l e 2 5 D C o h o r t

7 0 7 5 8 0 8 5 9 0

A v e r a g e o n s e t o f c l i n i c a l s i g n s o f d i s e a s e ( D a y s )

n s

N

• n
• t

r a n s g e n i c V e h i c l e 2 5 D C

• h
• r

t 7 5 D c

• h
• r

t

2 4 6

9 0 D a y s M o t o r n e u r o n c o u n t s i n l u m b a r S C / v e n t r a l h o r n

* * * *

ChAT DAPI ChAT DAPI ChAT DAPI

25D Cohort Vehicle 75D Cohort Non-transgenic Scale bars = 50µm

ChAT DAPI

ChAT Staining in VH Lumbar SC

Data presented as mean ± SD Data presented as mean ± SD

HTL0014242 slows late stage disease progression for 75D Cohort

75D Cohort vs. Both Vehicle Group and 25D Cohort 75D Cohort vs. Vehicle Group

Overall effect on rotarod (10.5 weeks – 20.5 weeks) 75D cohort retain motor function for longer

75D cohort demonstrate a 3-week extension in the median time (weeks) to reach a score of 0 ( P < 0.001)

Data presented as mean ± SD ( 2-way ANOVA)

5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6

A g e i n W e e k s L a t e n c y t o F a l l i n s e c o n d s ( a v e r a g e  S D ) 2 5 D C o h o r t V e h i c l e 7 5 D C o h o r t

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

HTL0014242 has no effect on survival

Data presented as mean ± SD

Reduction of astrocyte and microglial activation at 90D in VH of lumbar SC

Scale bars = 50µm

GFAP Staining Iba1 Staining 75D Cohort Non-transgenic Vehicle 25D Cohort

GFAP DAPI Iba1 DAPI GFAP DAPI GFAP DAPI GFAP DAPI Iba1 DAPI Iba1 DAPI Iba1 DAPI

V e h i c l e 2 5 D C o h o r t 7 5 D C o h o r t N o n - t r a n s g e n i c

1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0

T o t a l G F A P S t a i n i n g A r e a (  m

2 )

* * * * * * * * * * * * V e h i c l e 2 5 D C o h o r t 7 5 D C o h o r t N o n - t r a n s g e n i c

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

T o t a l I b a 1 S t a i n i n g A r e a (  m

2 )

* * * * * * * * * *

Riluzole does not reduce glial activation at 90D in VH of lumbar SC

• Riluzole dosed at 240µg/ml in drinking water (~70mg/kg) from 25 days of age
• HTL0014242 at 30 mg/kg, orally, from 25 days of age

V e h i c l e R i l u z o l e ' 2 4 2 + R i l u z o l e

2 0 0 4 0 0 6 0 0 8 0 0

T o t a l I b a 1 S t a i n i n g A r e a (  m

2 ) * * * *

V e h i c l e R i l u z o l e ' 2 4 2 + R i l u z o l e

2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0

T o t a l G F A P S t a i n i n g A r e a (  m

2 ) * * * * * * * *

Scale bars = 50µm

GFAP Staining Iba1 Staining Riluzole + HTL0014242 Vehicle Riluzole

GFAP DAPI Iba1 DAPI GFAP DAPI GFAP DAPI Iba1 DAPI Iba1 DAPI

Summary

Readouts Vehicle HTL0014242 25D Cohort HTL0014242 75D cohort Riluzole Effect on onset of clinical signs of disease ✕ ✕

• Increased number of motor neurons at 90D

✕ ✕ ✓

• Reduction in GFAP staining at 90 days in SC

✕ ✓ ✓ ✕ Reduction in Iba1 staining at 90 days in SC ✕ ✓ ✓ ✕ Improvement in motor function as seen on rotarod ✕ ✕ ✓

• Effect on survival

✕ ✕ ✕

Acknowledgements

SITraN Dr Richard Mead Prof Dame Pamela Shaw Yuri Ciervo Amy Keerie Dr Jodie Stephenson Dr Matthew Stopford Sosei Heptares Dr Kirstie Bennett Dr Matt Barnes Dr Alastair Brown Dr Fiona Marshall Dr Eimear Howley

Heptares is a trademark of Heptares Therapeutics Limited Sosei is a trademark of Sosei Group Corporation

mGlu5 is expressed on a sub- population of motor neurons in lumbar SC

SOD1G93A lumbar SC

ChAT mGlu5 mGlu5 ChAT ChAT mGlu5

ChAT mGlu5 Merge Scale bars = 500µm Scale bars = 100µm

Rotarod performance is remarkably consistent between vehicle dosed mice in

• ur SOD1G93A mouse model of MND

Control data from 6 survival studies over multiple years

8 1 0 0 2 0 0 3 0 0 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

A g e i n W e e k s L a t e n c y t o F a l l i n s e c o n d s ( a v e r a g e  S D ) H is t o r ic a l m e t h y l c e llu lo s e d o s e d o r a lly f r o m 7 5 D 2 5 D C o h o r t V e h ic le 7 5 D C o h o r t

2 1 0 0 2 0 0 3 0 0 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

R o t a r o d d a t a f r o m h i s t o r i c a l v e h i c l e d o s e d m i c e

A g e i n W e e k s L a t e n c y t o F a l l i n s e c o n d s ( a v e r a g e  S D ) O r a l g a v a g e f r o m 4 5 D D r in k in g w a t e r I P f r o m 4 5 D V ir u s in m u s c le a t 2 1 D I n c h o w f r o m 2 5 D O r a l g a v a g e f r o m 7 5 D 7 5 D C o h o r t

HTL0014242 Survival Study data

A g e (d a y s ) A v e ra g e s p e e d (k m /h r)

2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 2 3

R ilu z o le V e h ic le

Riluzole at clinically relevant plasma exposures improves running wheel performance but does not improve survival in SOD1G93A mice

Two way ANOVA overall p value for effect of treatment <0.0001

A g e (d a ys) P e rc e n t s u rv iv a l

2 0 4 0 6 0 8 0 1 0 0

1 0 0 1 2 0 1 4 0 1 6 0

R ilu zo le V e h ic le

Riluzole dosed at 240µg/ml (~72mg/kg/day) in drinking water

Riluzole concentrations (uHPLC/TOF mass spec)

Blood (nM) Spinal Cord (nM) SC/Blood ratio Peak 116.2 (±54.4) 307.4 (±173.6) 2.5 (±0.6) Trough 78.7 (± 8.3) 220.8 (±43.8) 2.8 (±0.4)