HTL0014242 a novel metabotropic glutamate receptor 5 (mGlu 5 ) negative allosteric modulator (NAM) limits glial activation and slows late stage disease progression in the SOD1 G93A mouse model of motor neuron disease Heledd Brown-Wright Kirstie Bennett, Pamela Shaw, Alastair Brown, Matt Barnes & Richard Mead
Metabotropic glutamate receptor 5 (mGlu 5 ) is a novel therapeutic target for MND • Glutamate-mediated toxicity is recognised as a mechanism of both neuronal and glial injury +/- in SOD1 G93A mice (Bonifacino et al. mGlu 5 Blocking mGlu 5 with the NAM MPEP in SOD1 G93A 2017 Neuropharmacology ) mice (Rossi et al. 2008 Cell Death and Differentiation ) Pharmacological and genetic evidence to support role for mGlu 5 in the SOD1 G93A mouse model of • MND
Sosei Heptares ’ mGlu 5 NAM HTL0014242 – highly potent and selective World leader in GPCR drug design MPEP (mGlu 5 NAM ) 3D structure of mGlu 5 receptor HTL0014242 (mGlu 5 NAM) Acknowledgements to Dr Ben Tehan at Sosei Heptares for this image Christopher et al. 2015 J Med Chem
HTL0014242 has an excellent pharmacokinetic profile in the SOD1 G93A transgenic mice 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 1 0 0 0 0 H T L 0 0 1 4 2 4 2 ( n M ) F r e e in s p in a l c o r d 1 0 0 0 1 0 0 The estimated mGlu 5 receptor 1 0 occupancy (RO) in SC was in the 1 I C 5 0 region of 40% - 90% at Cmax for 0 . 1 doses of 3,10 and 30mg/kg of HTL0014242 0 . 0 1 0 1 0 2 0 T i m e ( h )
HTL0014242 90D dose response study in the SOD1 G93A mouse model of MND • Inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6) (Mead et al. 2011 PLoS ONE) Average onset of Decline in motor clinical signs of Standard Weaning performance disease end-point Phenotype age 21 40 60 75 90 140 - 150 Glial activation in lumbar 59% reduction in α -motor neurons SC HTL0014242 dose-response study design Tissue collected for age 25 35 60 75 90 histology HTL0014242 daily oral dosing at 3,10, 30mg/kg (11 mice per group ) Vehicle = 0.5% methyl cellulose Rotarod testing once per week Neuroscored twice per week
Dose-dependent reduction of astrocyte activation at 90D in VH of lumbar SC Vehicle 10mg/kg 3mg/kg 30mg/kg G F A P S t a i n i n g a r e a 1 0 0 0 0 * * * * * 8 0 0 0 2 ) 6 0 0 0 GFAP ( m 4 0 0 0 Staining 2 0 0 0 0 GFAP GFAP GFAP GFAP 3 m g / k g 1 0 m g / k g 3 0 m g / k g V e h i c l e DAPI DAPI DAPI DAPI 2 5 0 0 I b a 1 S t a i n i n g a r e a * 2 0 0 0 2 ) 1 5 0 0 Iba1 ( m 1 0 0 0 Staining 5 0 0 Iba1 Iba1 Iba1 Iba1 0 DAPI DAPI DAPI DAPI e g g g l k k k c / / / i g g g h e m m m V 3 0 0 1 3 Scale bars = 50µm
mGlu 5 receptor occupancy from HTL0014242 dose- response study inversely correlates with GFAP staining GFAP Staining Vehicle 3mg/kg G F A P S t a i n i n g a r e a 1 0 0 0 0 * * * * * 8 0 0 0 2 ) 6 0 0 0 GFAP GFAP ( m DAPI DAPI 4 0 0 0 2 0 0 0 10mg/kg 30mg/kg 0 e g g g l k k k c / / / i g g g h e m m m V 3 0 0 1 3 Data presented as mean ± SEM Data presented as mean ± SD GFAP GFAP DAPI DAPI Scale bars = 50µm
HTL0014242 survival study in the SOD1 G93A mouse model of MND Average onset of Decline in motor clinical signs of Standard performance Weaning disease end-point Phenotype age 21 40 60 75 90 140-150 First signs of glial 59% reduction in α -motor neurons activation in lumbar SC Tissue HTL0014242 survival study design collection age 25 35 60 75 90 140-150 30mg/kg HTL0014242 dosed daily (25D cohort) 30mg/kg HTL0014242 dosed daily (75D cohort) cohort Rotarod testing twice per week Neuroscored three times per week
Significant increase in lumbar SC ChAT stained motor neurons ChAT Staining in VH Lumbar SC 9 0 D a y s Non-transgenic Vehicle l u m b a r S C / v e n t r a l h o r n c l i n i c a l s i g n s o f d i s e a s e M o t o r n e u r o n c o u n t s i n 6 * * * 9 0 n s A v e r a g e o n s e t o f * 8 5 4 ( D a y s ) 8 0 ChAT 2 ChAT DAPI DAPI 7 5 25D Cohort 75D Cohort 0 7 0 t c V e h i c l e e t 2 5 D C o h o r t r i r l o n c o h e i h h o g o e c s C V n D a D 5 r 5 t 7 - 2 n o N Data presented as mean ± SD Data presented as mean ± SD ChAT ChAT DAPI DAPI Scale bars = 50µm
HTL0014242 slows late stage disease progression for 75D Cohort 75D cohort retain motor Overall effect on rotarod function for longer (10.5 weeks – 20.5 weeks) 3 5 0 L a t e n c y t o F a l l i n s e c o n d s 3 0 0 * * * * * * 2 5 0 S D ) V e h i c l e 2 0 0 2 5 D C o h o r t ( a v e r a g e 7 5 D C o h o r t 1 5 0 * * * * * * * * 1 0 0 * * 5 0 0 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 75D cohort demonstrate a 3-week Data presented as mean ± SD ( 2-way ANOVA) extension in the median time (weeks) to 75D Cohort vs. Both Vehicle Group and 25D Cohort reach a score of 0 ( P < 0.001) 75D Cohort vs. Vehicle Group
HTL0014242 has no effect on survival Data presented as mean ± SD
Reduction of astrocyte and microglial activation at 90D in VH of lumbar SC Vehicle 25D Cohort 75D Cohort Non-transgenic * * * * T o t a l G F A P S t a i n i n g A r e a * * * * 4 0 0 0 * * * * 3 0 0 0 2 ) GFAP ( m 2 0 0 0 Staining 1 0 0 0 0 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 GFAP GFAP GFAP GFAP DAPI DAPI DAPI DAPI T o t a l I b a 1 S t a i n i n g A r e a * * * * * * 5 0 0 * * * 4 0 0 Iba1 * 2 ) 3 0 0 Staining ( m 2 0 0 Iba1 Iba1 Iba1 Iba1 1 0 0 DAPI DAPI DAPI DAPI 0 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 Scale bars = 50µm
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 Riluzole + Vehicle Riluzole HTL0014242 T o t a l G F A P S t a i n i n g A r e a 8 0 0 0 * * * * * * * * 6 0 0 0 2 ) ( m 4 0 0 0 GFAP Staining 2 0 0 0 0 GFAP GFAP GFAP 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 DAPI DAPI DAPI T o t a l I b a 1 S t a i n i n g A r e a 8 0 0 * * * * Iba1 6 0 0 2 ) Staining ( m 4 0 0 2 0 0 Iba1 Iba1 Iba1 DAPI DAPI DAPI 0 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 Scale bars = 50µm
Summary Vehicle HTL0014242 HTL0014242 Readouts Riluzole 25D Cohort 75D cohort ✕ ✕ - 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 Sosei Heptares Dr Richard Mead Dr Kirstie Bennett Prof Dame Pamela Shaw Dr Matt Barnes Yuri Ciervo Dr Alastair Brown Amy Keerie Dr Fiona Marshall Dr Jodie Stephenson Dr Eimear Howley Dr Matthew Stopford Heptares is a trademark of Heptares Therapeutics Limited Sosei is a trademark of Sosei Group Corporation
mGlu 5 is expressed on a sub- population of motor neurons in lumbar SC SOD1 G93A lumbar SC mGlu 5 Merge ChAT ChAT mGlu 5 ChAT mGlu 5 ChAT mGlu 5 Scale bars = 100µm Scale bars = 500µm
Rotarod performance is remarkably consistent between vehicle dosed mice in our SOD1 G93A mouse model of MND Control data from 6 survival studies over multiple years HTL0014242 Survival Study data 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 3 0 0 L a t e n c y t o F a l l i n s e c o n d s 3 0 0 L a t e n c y t o F a l l i n s e c o n d s O r a l g a v a g e f r o m 4 5 D V e h ic le S D ) 2 5 D C o h o r t D r in k in g w a t e r S D ) 2 0 0 7 5 D C o h o r t 2 0 0 I P f r o m 4 5 D ( a v e r a g e H is t o r ic a l m e t h y l c e llu lo s e ( a v e r a g e V ir u s in m u s c le a t 2 1 D d o s e d o r a lly f r o m 7 5 D 1 0 0 I n c h o w f r o m 2 5 D 1 0 0 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 0 0 0 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 0 2 4 6 8 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 A g e i n W e e k s
Riluzole at clinically relevant plasma exposures improves running wheel performance but does not improve survival in SOD1 G93A mice 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) 3 A v e ra g e s p e e d (k m /h r) 1 0 0 P e rc e n t s u rv iv a l 8 0 2 6 0 4 0 1 R ilu zo le R ilu z o le V e h ic le 2 0 V e h ic le 0 0 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 0 0 1 2 0 1 4 0 1 6 0 0 A g e (d a y s ) A g e (d a ys) Two way ANOVA overall p value for effect of treatment <0.0001
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