Supplemental Figures s1

Supplemental Figures

Has inhibition of Aβ production adequately been tested as therapeutic approach in mild

AD? A model-based meta-analysis of γ-secretase inhibitor data

Camilla Niva1, Joanna Parkinson2, Fredrik Olsson1, Erno van Schaick3, Johan Lundkvist1, Sandra A.G. Visser4

1) Neuroscience, CNSP Innovative Medicines, AstraZeneca Södertälje

2) Modeling & Simulation, DMPK, CNSP Innovative Medicines, AstraZeneca Södertälje

3) Exprimo N.V., Mechelen, Belgium

4) Global DMPK Centre of Excellence, Innovative Medicines AstraZeneca Södertälje

Corresponding Author:

Sandra A.G. Visser. [email protected] Figure S1: A). Left: Mean plasma Aβ40 (upper panels) and plasma Aβ42 (lower panels) over time, after a single dose of 600 mg Begacestat in healthy volunteers and 450 mg in AD patients (left upper panel). Right: Concentration-response relationships for Aβ40 and Aβ42 levels for Begacestat in plasma. The Aβ rise was more pronounced for Aβ42 compared to Aβ40 upon dosing of 600 mg Begacestat which was similar for healthy volunteers and AD patients. Begacestat plasma exposure up to 600 ng/mL resulted in an increase in plasma Aβ42, whereas for plasma Aβ40 a robust decrease was observed for exposure levels above 100 ng/mL. B). Mean CSF Aβ40 (upper panels) and CSF Aβ42 (lower panels) for single doses of Semagacestat: Left: CSF Aβ levels (% of placebo and pre-dose value) over time; Middle: Plasma concentration vs CSF Aβ levels; Right: CSF concentration vs CSF Aβ levels. Concentration-effect relationships for Aβ42 demonstrate that the increase in Aβ42 for the 280 mg is not observed for the lower doses at similar plasma or CSF concentrations.

Begacestat PD plasma PK-PD 250 250 600 mg H 600 mg H Placebo H Placebo H ) ) 200 200 % % 450 mg AD ( 450 mg AD (

4 Placebo AD 4 150 Placebo AD 150   A A

a A40 a 100 100 m m s s a a l l

0 0 0 6 12 18 24 30 36 42 48 0.1 1 10 100 1000 Time (h) Plasma Concentration (ng/ml)

250 250 600 mg H 600 mg H Placebo H

) Placebo H ) 200 200

450 mg AD % %

( 450 mg AD (

2 2 Placebo AD 4 4 150 150 Placebo AD   A A

a A42 a 100 100 m m s s a a l l

0 0 0 6 12 18 24 30 36 42 48 0.1 1 10 100 1000 Time (h) Plasma Concentration (ng/ml)

Semagacestat PD plasma PK-PD CSF PK-PD

100 mg 100 mg 100 mg 200 200 200 140 mg 140 mg 140 mg ) )

) 280 mg 280 mg 280 mg % % ( (

% 150 150

0 0 0 4 4 4   

A 100 A 100

A40 F F F S S S C C C 50 50 50

0 0 0 0 6 12 18 24 30 36 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 Time (h) Plasma concentration (ng/mL) CSF concentration (ng/mL)

100 mg 100 mg 100 mg 200 200 200 140 mg 140 mg

140 mg ) ) )

% 280 mg % 280 mg ( (

% 150 150

280 mg ( 150

2 2 2 4 4 4   

A 100 A 100

A42 F F F S S S C C C 50 50 50

0 0 0 0 6 12 18 24 30 36 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 Time (h) Plasma concentration (ng/mL) CSF concentration (ng/mL) Figure S2: Mean human plasma and CSF exposure (left panels), CSF Aβ40 over time (middle panels) and plasma concentration versus CSF Aβ40 (right panels) for 5 GSI after single dose in human volunteers. Limited effects, incomplete time-courses and concentration-effect relationships were observed in CSF for GSIs.

Semagacestat PD Semagacestat PKPD Semagacestat PK 100 mg 100 mg 10000 100 mg CSF 200 140 mg 200 ) 140 mg L 140 mg CSF ) 280 mg ) m

% % 280 mg /

1000 ( 150 ( 150 g

280 mg CSF n 0 0 (

100 mg Plasma 4 4 n   o i 100 140 mg Plasma A 100 A 100

t a F F r t S S n C C e 50 50

c 10 n o C 1 0 0 0 6 12 18 24 30 36 0 6 12 18 24 30 36 0.01 0.1 1 10 100 1000 10000 Time (h) Time (h) Plasma concentration (ng/mL)

Avagacestat PD Avagacestat PKPD Avagacestat PK Placebo Placebo 10000 50 mg plasma 200 200 50 mg ) 50 mg L 200 mg plasma ) ) 200 mg m % %

/ 200 mg

1000 ( 150 ( 150 g

n 0 0 (

4 4 n   o i 100 A 100 A 100

t a F F r t S S n C C e 50 50

c 10 n o C 1 0 0 0 6 12 18 24 30 36 0 6 12 18 24 30 36 42 0.1 1 10 100 1000 10000 Time (h) Time (h) Plasma concentration (ng/mL)

Begacestat PD Begacestat PKPD Begacestat PK 1000 Placebo 600 mg plasma 200 200 ) 600 mg L 600 mg CSF ) ) m /

100 % % g

( 150 ( 150

n ( 0 0

4 4 n   o i 10 t A 100 A 100

a r F F t S S n C C e

c 1 50 50 n o C 0.1 0 0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 0.1 1 10 100 1000 10000 Time (h) Time (h) Plasma concentration (ng/mL)

PF-3084014 PD PF-3084014 PKPD PF-3084014 PK 1000 120 mg 120 mg Plasma 200 200 ) L

120 mg CSF ) ) m 100 / % % g ( 150 ( 150

n 0 0 (

10 4 4 n   o i

A 100 A 100 t

a F F r 1 t S S

n Placebo C C e 50 50 c

n 0.1 120 mg o C 0.01 0 0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 0.01 0.1 1 10 100 1000 Time (h) Time (h) Plasma concentration (ng/mL)

MK-752 PD MK-752 PKPD MK-752 PK Placebo 100000 200 110 mg 200 Placebo )

L 110 mg

) 300 mg ) m / % %

g 10000 300 mg ( 150 500 mg ( 150

n ( 0 0 500 mg 4 4

n 750 mg   o i 1000 750 mg A A t 100 100

1000 mg a F F r

t 1000 mg S S n C C e 50 50 c 100 1000 mg Plasma n o 1000 mg CSF C 10 0 0 0 2 4 6 8 10 12 0 4 8 12 16 20 24 1 10 100 1000 10000 100000 Time (h) Time (h) Plasma concentration (ng/mL) Figure S3. Mean observations for Semagacestat (combination of published and in-house data), Avagacestat (in- house data only) and Begacestat (published data only) after single dose in mice. Left panels show show plasma Aβ40 time profiles (% of vehicle). Right panels show concentration-effect relationships.

Semagacestat PD Semagacestat PKPD 250 250 11 mg/kg 11 mg/kg e e

l 200 22 mg/kg l 200 22 mg/kg c c i i

h 30 mg/kg h 30 mg/kg e e

v 150 36 mg/kg v 150 36 mg/kg % %

0 54 mg/kg 0 54 mg/kg

4 100 4 100 a 150 mg/kg a 150 mg/kg t t e e

B 50 B 50 Prediction A A

0 0 0 4 8 12 16 20 24 0.1 1 10 100 1000 10000 time (h) concentration (ng/mL)

Avagacestat PD Avagacestat PKPD 250 1.6 mg/kg 250 1.6 mg/kg e

l 200 10 mg/kg e

l 10 mg/kg c 200 i c

15.6 mg/kg i h 15.6 mg/kg h e e v 150 30 mg/kg v 150 30 mg/kg %

0 150 mg/kg 150 mg/kg 0 4 100 4

156 mg/kg a t 156 mg/kg t e e B 50 B Prediction

A 50 A

0 0 0 4 8 12 16 20 24 0.1 1 10 100 1000 10000 time (h) concentration (ng/mL)

Begacestat PD Begacestat PKPD 250 250 30 mg/kg 30 mg/kg e e

l 150 mg/kg l 200 150 mg/kg 200 c c i i

h Prediction h e e v v 150 150 % %

0 0 4 4 100 100 a a t t e e B B 50 50 A A

0 0 0 4 8 12 16 20 24 1 10 100 1000 10000 100000 time (h) concentration (ng/mL) Figure S4. Mean Semagacestat, Avagacestat and Begacestat effects on CSF Aβ40 and brain Aβ40 levels after single dose in mice. Upper rows: CSF Aβ40 (% of vehicle) time-profile for (left, middle and right, respectively and plasma concentration vs. effect relationship for CSF Aβ40). Lower rows: brain Aβ40 (% of vehicle) time profile and plasma concentration vs. effect relationship for brain Aβ40.

Semagacestat CSF PD Avagacestat CSF PD Begacestat CSF PD 250 250 250 30 mg/kg 30 umol/kg 30 mg/kg 200 150 mg/kg 200 150 umol/kg 200 150 mg/kg ) ) ) % % % ( ( (

0 150 0 150 0 150 4 4 4    A A A

100 100 100 F F F S S S C C 50 50 C 50

0 0 0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 time (h) time (h) time (h)

Semagacestat CSF PKPD Avagacestat CSF PKPD Begacestat CSF PKPD 250 250 250 30 mg/kg 30 mg/kg 30 mg/kg 200 150 mg/kg 200 150 mg/kg 200 150 mg/kg ) ) ) % % Prediction % Prediction Prediction ( ( (

0 0 150 0 150 150 4 4 4    A A A

100 100 100 F F F S S S C 50 C 50 C 50

0 0 0 10 100 1000 10000 100000 1 10 100 1000 10000 10 100 1000 10000 100000 Plasma concentration (ng/mL) Plasma concentration (ng/mL) Plasma concentration (ng/mL)

Semagacestat Brain PD Avagacestat Brain PD Begacestat Brain PD 250 250 36 mg/kg 250 30 mg/kg 30 mg/kg 30 mg/kg 150 mg/kg ) 200 ) 200 150 mg/kg

) 200 % % (

150 mg/kg ( %

150 150 0 4 4 150 4    A A

n 100 100 n i

n 100 i i a a a r r r B B 50 50 B 50

0 0 0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 time (h) time (h) time (h)

Semagacestat Brain PKPD Avagacestat Brain PKPD Begacestat Brain PKPD 250 250 250 11 mg/kg 16 mg/kg 30 mg/kg 1.6 mg/kg 150 mg/kg )

22 mg/kg ) 200 ) 200 200 % % % (

( Prediction ( 36 mg/kg 156 mg/kg

0 0 0 150 150 150 4 4 4 54 mg/kg 30 mg/kg    A A A 30 mg/kg 150 mg/kg

n 100 n 100

n 100 i i i 150 mg/kg Prediction a a a r r r B B B 50 Prediction 50 50

0 0 0 10 100 1000 10000 100000 1 10 100 1000 10000 10 100 1000 10000 100000 Plasma concentration (ng/mL) Plasma concentration (ng/mL) Plasma concentration (ng/mL) Figure S5. Mean observations for Semagacestat (combination of published and in-house data), Avagacestat (in- house data only) and PF3084014 (published data only) after single dose in guinea pig. Left panels show plasma Aβ40 vs. time profiles (% of vehicle). Right panels show predicted plasma concentration vs effect relationships.

Semagacestat PD Semagacestat PKPD 250 250 2 mg/kg 2 mg/kg ) 200 6 mg/kg ) 200 6 mg/kg % % ( (

20 mg/kg 20 mg/kg 0 0 4 150 0.2 µmol/kg 4 150 0.2 µmol/kg   A A

0.6 µmol/kg 0.6 µmol/kg a 100 a 100 m 2 µmol/kg m 2 µmol/kg s s a a l 6 µmol/kg l 6 µmol/kg

P 50 50 20 µmol/kg P 20 µmol/kg 0 60 µmol/kg 0 60 µmol/kg 0 4 8 12 16 20 24 0.1 1 10 100 1000 10000 Prediction Time (h) Plasma concentration (ng/mL)

Avagacestat PKPD Avagacestat PD 250 250 30 mg/kg 30 mg/kg ) 200 100 mg/kg ) 200 100 mg/kg % (

% 300 mg/kg ( 0 300 mg/kg 4 0 150 Prediction  4 150  A

A a 100 a 100 m s m a s l a l

P 50 0 0 0.1 1 10 100 1000 10000 0 4 8 12 16 20 24 Plasma concentration (ng/mL) Time (h)

PF3084014 PD PF3084014 PKPD 250 250 0.1 mg/kg 0.1 mg/kg ) 200 0.3 mg/kg ) 200 0.3 mg/kg % % ( (

1 mg/kg 1 mg/kg 0 0 4 150 3 mg/kg 4 150  3 mg/kg  A A

10 mg/kg 10 mg/kg a 100 a 100 m m Prediction s s a a l l

0 0 0 4 8 12 16 20 24 0.1 1 10 100 1000 10000 Time (h) Plasma concentration (ng/mL) Figure S6. Mean Semagacestat, Avagacestat and PF3084014 effects on CSF Aβ40 and brain Aβ40 levels after single dose in guinea pig. Upper rows: CSF Aβ40 (% of vehicle) time-profile for (left, middle and right, respectively and plasma concentration vs. effect relationship for CSF Aβ40). Lower rows: brain Aβ40 (% of vehicle) time profile and plasma concentration vs. effect relationship for brain Aβ40. CSF

Semagacestat PD CSF Avagacestat PD CSF 250 250 2 mg/kg 30 mg/kg 200 6 mg/kg 200 100 mg/kg ) ) % 20 mg/kg % 300 mg/kg ( (

0 150 0.2 mg/kg 0 150 4 4  0.6 mg/kg  A A

100 100 F 2 mg/kg F S S C 6 mg/kg C 50 50 20 mg/kg 0 60 mg/kg 0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 Time (h) Time (h)

Semagacestat PKPD CSF Avagacestat PKPD CSF 250 250 0.2 mg/kg 30 mg/kg 200 0.6 mg/kg 200 100 mg/kg ) ) % 2 mg/kg % 300 mg/kg ( (

0 150 6 mg/kg 0 150 Prediction 4 4  20 mg/kg  A A 100 100 F 60 mg/kg F S S

C Prediction C 50 50

0 0 0.01 0.1 1 10 100 1000 10000 1 10 100 1000 10000 Plasma concentration (ng/mL) Plasma concentration (ng/mL) Brain

Semagacestat PD brain Avagacestat PD Brain PF-3084014 PD Brain 250 250 250 2 mg/kg 30 mg/kg 0.1 mg/kg 6 mg/kg 100 mg/kg ) ) 200 200 0.3 mg/kg ) 200 % % ( ( 20 mg/kg 300 mg/kg %

( 1 mg/kg 0 0 150 150 4 4 0.2 mg/kg 0 150   4 3 mg/kg  A 0.6 mg/kg A

A n 100 n 100 10 mg/kg

i 2 mg/kg i

a a 100 n i r r a

B 6 mg/kg B

50 50 r

20 mg/kg B 50 0 60 mg/kg 0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 Time (h) Time (h) 0 4 8 12 16 20 24 time (h)

PF-3084014 PKPD Brain Semagacestat PKPD Brain Avagacestat PKPD Brain 250 250 250 0.1 mg/kg 0.2 mg/kg 30 mg/kg 0.3 mg/kg

) 200 0.6 mg/kg 100 mg/kg %

) 200 ) 200

( 1 mg/kg

( 2 mg/kg ( 300 mg/kg

4 3 mg/kg 0 0

150 150 

4 6 mg/kg 4 Prediction   A 10 mg/kg

A 20 mg/kg A

n 100 n 100 Prediction a i 60 mg/kg i r a a r r B

B Prediction B 50 50 50

0 0 0 0.01 0.1 1 10 100 1000 10000 1 10 100 1000 10000 0.1 1 10 100 1000 Plasma concentration (ng/mL) Plasma concentration (ng/mL) Plasma concentration (ng/mL) Figure S7. Semagacestat, Avagacestat, Begacestat and MK0752 effects on Aβ40 and Aβ42 in primary neurons from mouse (left panels) and guinea-pig (right panels). Symbols represent mean data (n=2-4, including standard deviation) while solid red lines correspond to the population PKPD fits.

Mouse Guinea Pig A40 A42 A40 A42 Semagacestat Semagacestat Semagacestat Semagacestat 150 150 150 150 125 125 125 125 100 )

) ) 100 100 100 ) % ( % % %

( ( 75 (

0 75 2 0 4 75 75 2 4 4 4   

50  A 50 A A 50 50 A 25 25 25 25 0 0 0 0 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 Concentration (µmol/L) Concentration (µmol/L) Concentration (µmol/L) Concentration (µmol/L)

Avagacestat Avagacestat Avagacestat Avagacestat 150 150 150 150

125 125 125 125

100 ) ) 100 ) 100 100 ) % % % % ( ( ( (

75 75 2 0 0 75 75 2 4 4 4 4    50  50 A A A 50 50 A 25 25 25 25 0 0 0 0 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 Concentration (µmol/L) Concentration (µmol/L) Concentration (µmol/L) Concentration (µmol/L)

Begacestat Begacestat Begacestat Begacestat 150 150 150 150

125 125 125 125

100 ) ) 100 ) 100 100 ) % % % % ( ( ( (

75 75 2 0 0 75 75 2 4 4 4 4    50  50 A A A 50 50 A 25 25 25 25 0 0 0 0 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 Concentration (µmol/L) Concentration (µmol/L) Concentration (µmol/L) Concentration (µmol/L)

MK0752 MK0752 MK0752 MK0752 150 150 150 150

125 125 125 125

100 ) ) 100 ) 100 100 ) % % % % ( ( ( (

75 75 2 0 0 75 75 2 4 4 4 4    50  50 A A A 50 50 A 25 25 25 25 0 0 0 0 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 Concentration (µmol/L) Concentration (µmol/L) Concentration (µmol/L) Concentration (µmol/L) Figure S8. Standard goodness of fit for modeling of human plasma Aβ40 levels for 4 γ-secretase inhibitors: Semagacestat, Avagacestat, Begacestat and PF3084014. Figure S9. Standard goodness of fit for modeling of mouse plasma and brain Aβ40 levels for 3 γ-secretase inhibitors: Semagacestat, Avagacestat and Begacestat. Figure S10. Standard goodness of fit for modeling of guinea pig plasma, CSF and brain Aβ40 levels for 3 γ- secretase inhibitors: Semagacestat, Avagacestat and PF3084014. Figure S11. Standard goodness of fit for modeling of monkey plasma and CSF Aβ40 levels for 1 γ-secretase inhibitors: MK0752. Figure S12. Standard goodness of fit for modeling of in vitro Aβ40 and Aβ42 levels using 4 γ-secretase inhibitors: Semagacestat, Avagacestat, Begacestat and MK0752.