assessment of the anti-parkinsonian effects of the potent and … · 2018-12-27 · assessment of...

Assessment of the Anti-parkinsonian Effects of the Potent and Selective LRRK2 Kinase Inhibitor PF-360 in the AAV-A53T Mouse Model of Parkinson’s DiseaseRobert Hodgson1, Teija Parkkari1, Terina Martinez2, Matthew Fell3, Diane Bryce3, Taneli Heikkinen1, Timo Bragge1, Leena Tähtivaara1, Miia Taavitsainen1, James Koprich,5 Thomas Lanz4, Marco Baptista2

1Charles River Discovery, Kuopio, Finland; 2The Michael J. Fox Foundation, New York, NY; 3Merck & Co. Inc., Boston, MA; 4Pfizer Inc, Cambridge, MA; 5Atuka Inc, Toronto, Canada 755.21

1 BACKGROUND• Mutations in the Leucine-rich repeat kinase 2 (LRRK2), including G2019S, enhance kinase activity and lead to Parkinson’s disease (PD),

therefore, there is a strong therapeutic focus on the discovery of potent and safe LRRK2 kinase inhibitors capable of slowing theprogression of the disease.

• AAV alpha synuclein (AAV-A53T) delivery to mice induces multiple effects that mimic Parkinson’s disease (PD), and therefore, holdspotential to serve as a preclinical model to assess the effect of chronic administration of novel LRRK2 inhibitors.

• In this study, dose-response efficacy of LRRK2 inhibitor, PF-360, in AAV-A53T infused C57Bl/6J and LRRK2-G2019S mice wasinvestigated. In addition, the effect of single dose of PF-360 in 5-6 months old AAV-A53T infused LRRK2-G2019S mice was studied. PF-360 was administered in diet.

2 MATERIALS AND METHODSData are from three independent experiments. Two studies were conducted in 10-12 weeks old C57BL/6J and LRRK2-G2019S mice and thethird study was performed using 5-6 months old LRRK2-G2019S mice.Animals. In total of 90 male C57Bl/6J mice (Charles River, Sulzfeld, Germany) and 105 male C57Bl/6J-LRRK2-G2019S mice (G2019S, n=7510-12 weeks old; n=30 5-6 months, donated by Michael J Fox Foundation) were used in the studies. All mice received unilateral injection ofAAV-A53T in the substantia nigra. The treatment with a diet containing PF-360 or vehicle started 7 days prior to injections. Four weeks afterthe injection, the motor functions of the mice (10-15 mice / group) were evaluated using fine motor kinematic analysis. Five weeks after theinfusions, the brains were collected and processed for stereology and HPLC measurements, and the lungs were collected forhistopathological evaluation. Plasma bile acid levels of all study mice were analysed prior to the study to exclude mice with portal liver shunts.Surgery. All mice received unilateral infusion of AAV1/2 expressing human A53T alpha-synuclein (5 x 1012 vg/ml) to the substantia nigra atfollowing coordinates (relative to the bregma): AP = 3.0 mm posterior; ML = 1.3 mm; DV = 4.2 mm to the skull surface. A total of 2 μl of thevector was infused at a speed of 0.4 μl/min.Fine motor kinematics and gait. The mice were evaluated using an apparatus (Motorater, TSE-systems GmbH, Bad Homburg, Germany)designed for the detection of fine motor skills in rodents. The equipment consists of a brightly illuminated plexiglas corridor (153 x 5 x 10 cm)under which is situated a high-speed camera. The performance of the mice was assessed during walking along the corridor and wererecorded with a high speed video-camera (300 fps). The gait and fine motor skills were analyzed from three dimensions (from below and bothsides), first using the Simi Reality Motion Systems (Unterschleissheim, Germany) and the obtained raw data was further analyzed by acustom analysis system, resulting 97 gait parameters. PCA based approach was utilized. Historical data from AAV-Null vector has been usedfor analysis.Quantification of PF-360 in brain and plasma. An aliquot of each plasma sample was precipitated using acetonitrile. The resultingsupernatant was diluted 5x and then injected onto the LC-MS/MS. Brain tissue samples were homogenized by sonication in PCA solution (6µL per each mg tissue) and centrifuged. The resulting supernatant was used as brain extract sample and diluted 26x prior to injection onto theLC-MS/MS. Chromatographic separation was performed on a reversed phase analytical column (100 x 2.1 mm, 3.5 µm) held at a temperatureof 35 °C. Components were separated using a gradient of acetonitrile-methanol (1:1) containing 0.1% formic acid in ultrapurified H2Ocontaining 0.1% formic acid at a flow rate of 0.2 mL/min. The MS analyses were performed using an API 4000 MS/MS system, consisting of aAPI 4000 MS/MS a Turbo Ion Spray interface (both from Sciex, USA). The instrument was operated in multiple-reaction-monitoring (MRM)mode. The acquisitions were performed in positive ionization mode, with optimized settings for PF-360. Data were acquired and processedusing the Analyst™ data system (v 1.6.2, Sciex, USA).Total and pSer935 LRRK2 protein lung assessment. Total and phosphorylated LRRK2 were assessed using Time-Resolved FluorescentEnergy Transfer from lung homogenates. Equal amounts of protein were loaded in assay for normalization. Data was further normalized to thetotal LRRK2 protein levels of vehicle treated groups. Total and phosphorylated LRRK2 pSer935 levels were analyzed by Cytation3 accordingto the manufacturer’s instructions (Cisbio, #6FNRKPEG and #FLRKPEG).TH+ fiber density assessment using unbiased stereology. Fixed, cryoprotected and frozen midbrain samples were sectioned as 20 µmcoronal sections at 100 µm intervals through substantia nigra (SN) and ventral tegmental area (VTA) and mounted on slides. Sectioning wasstarted at 2.8 mm from bregma and continued to -3.4 mm from bregma in AP axis. The sections were first thawed and air dried. Anti-THimmunohistochemistry as performed with a standard IHC protocol at CRL DRS. number of TH-positive neurons were determined by countingimmunopositive cells through the SN and VTA.

A

BV eh ic

le

1 mg /k

g PF -3

6 0

3 mg /k

g PF -3

6 0

1 0 mg /k

g PF -3

6 0

3 0 mg /k

g PF -3

6 0

6 0 mg /k

g PF -3

6 00

5 0

1 0 0

1 5 0

2 0 0

% o

f C

on

tro

lp

LR

RK

2/T

ot

LR

RK

2 *

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

3 LRRK2 PHOSPHORYLATION IN BRAIN AND LUNGS

C57Bl/6J 8-10 weeks old

G2019S 11-12 weeks old

G2019S 5-6 months old

V eh icle

3 mg /k

g PF -3

6 0

1 0 mg /k

g PF -3

6 0

3 0 mg /k

g PF -3

6 0

6 0 mg /k

g PF -3

6 00

5 0

1 0 0

1 5 0

2 0 0

% o

f C

on

tro

lp

LR

RK

2/T

ot

LR

RK

2

****

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

V e h icle

3 0 mg /k

g PF -3

6 00

5 0

1 0 0

1 5 0

2 0 0

% o

f C

on

tro

lp

LR

RK

2/T

ot

LR

RK

2

****

V e h icle

3 0 mg /k

g PF -3

6 00

5 0

1 0 0

1 5 0

2 0 0

% o

f C

on

tro

lp

LR

RK

2/T

ot

LR

RK

2

****

V e h icle

1 mg /k

g PF -3

6 0

3 mg /k

g PF -3

6 0

1 0 mg /k

g PF -3

6 0

3 0 mg /k

g PF -3

6 0

6 0 mg /k

g PF -3

6 00

5 0

1 0 0

1 5 0

2 0 0

% o

f C

on

tro

lp

LR

RK

2/T

ot

LR

RK

2

****

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

Figure 1. Phosphorylated Ser935 LRRK2 levels in cortex (A) and lungs (B) of C57Bl/6J (8-10 wk old) and G2019S mice (11-12 wk and 5-6 mo)measured by HTRF assay. The treatment with PF-360 diet was started 7 days before the AAV injection. Mice were terminated 5 weeks afterAAV injection. Target engagement is observed both in cortex and lungs. Data is represented as Mean ± SEM, cortex n=14-15 per group andlungs n=9-10 per group. Statistical analysis with one-way ANOVA and Dunnet’s post-hoc compared to Vehicle group or Welch’s t-test. * p <0.05 **** p < 0.0001.

V e h icle

3 mg /k

g PF -3

6 0

1 0 mg /k

g PF -3

6 0

3 0 mg /k

g PF -3

6 0

6 0 mg /k

g PF -3

6 00

5 0

1 0 0

1 5 0

2 0 0

% o

f C

on

tro

lp

LR

RK

2/T

ot

LR

RK

2

**

**** ****

4 MOTOR PHENOTYPE IN KINEMATIC GAIT ANALYSIS

Figure 2. Overall gait scores for C57B6l/J (A) and G2019S (B) mice, and discriminant vector bar graphs, (E) and (F), respectively. The overallscores are based on PCA, emphasizing those gait features which differentiate the AAV-A53T Vehicle from AAV-Null (historical data). TheDiscriminant vector bar graph illustrates how much weight each gait parameter has in the score – the bar graph can also be seen as “a kinematicfingerprint” characterizing the kinematic changes due to AAV-A53T infusion in both strains. Individual gait parameters for C57 (D) and G2019S(C) are presented as boxplots. The corresponding parameters are highlighted orange in the bar graphs (E and F). Data are presented as mean ±SEM, and considered significant if p < 0.05 (unpaired t-test, (#) AAV-A53T vehicle vs. AAV-Null (3 months old), (*) AAV-A53T PF-360 vs. AAV-A53T vehicle). n=10-14 (C57 and G2019S AAV-A53T), n=8 (C57 and G2019S AAV-null).

Gait differences associated to AAV-A53T delivery in:1. C57B6l/J : Overall speed slower, tail tip position lower. Decrease in hip and tail base height.2. G2019S young: Change in hip orientation, manifested in tail base (↓), hip height (↑), and most prominently in protraction (↓). Tail tip position isalso decreased.3. G2019S old: Overall speed is decreased due to both stride duration (↑) and length (↓), as well as slower swing speeds.Beneficial PF-360 treatment effects in G2019SIn gait overall scores beneficial treatment effect of PF-360 at 10 mg/kg was seen, namely in younger cohort recovered hindlimb protraction andretraction. In older cohort PF-360 at 30 mg/kg improved the overall speed (stride duration and swing speeds).

c o n traip

s i

c o n traip

s i

c o n traip

s i

c o n traip

s i

c o n traip

s i

c o n traip

s i0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

Es

tim

ate

d p

op

ula

tio

n o

fT

H p

os

itiv

e c

ell

s

#

c o n tra

ips i

c o n tra

ips i

c o n tra

ips i

c o n tra

ips i

c o n tra

ips i

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

Es

tim

ate

d p

op

ula

tio

n o

fT

H p

os

itiv

e c

ell

s

c o n tra

ips i

c o n tra

ips i

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

Es

tim

ate

d p

op

ula

tio

n o

fT

H p

os

itiv

e c

ell

s

*# V e h ic le

1 m g /k g P F -3 6 0

3 m g /k g P F -3 6 0

1 0 m g /k g P F -3 6 0

3 0 m g /k g P F -3 6 0

6 0 m g /k g P F -3 6 0

5 TH+ CELL COUNTS IN SN

Figure 3. Estimated TH positive cell population in substantia nigra of C57Bl/6 (A) and G2019S mice at the age of 11-12 wk (B) and 5-6 months(C) 5 weeks after unilateral infusion of AAV1/2-A53T-aSyn. PF-360 dosing increased ipsilateral TH cell population compared to vehicle group inC57Bl/6J mice with dose 3 mg/kg. Higher TH cell count was also observed in ipsilateral G2019S, 5-6 mo old with 30 mg/kg dose. Student’s t-test, Vehicle ipsi to contra side comparison (# p < 0.05). Two-way ANOVA, Bonferroni’s post-hoc ipsi- or contralateral compared to vehicledosed group. Data presented as mean + SEM, Ipsilateral side n=13-15, contralateral side n=13-15 except C57Bl/6J 8-10 wk old n=5-6. * p <0.05.

B CA C57Bl/6J 8-10 wk old

G2019S 11-12 wk old

G2019S 5-6 months old

C

Figure 4. Total dopamine (DA) and metabolites (DOPAC and HVA) of C57Bl/6J (A) and G2019S mice at the age of 11-12 wk (B) and 5-6months (C) measured measured from ipsi- and contralateral striatum 5 weeks after unilateral infusion of AAV-A53T. PF-360 dosing did notimprove DA or DOPAC levels in ipsilateral striatum compared to vehicle group. HVA was elevated in G2019S, 5-6 mo old with 30 mg/kg dose.Welch’s t-test, Vehicle ipsi to contra side comparison (# p < 0.05). Two-way ANOVA, Bonferroni’s post-hoc ipsi- or contralateral compared tovehicle dosed group. Data presented as mean + SEM, n=14-15. * p < 0.05 ** p < 0.01

G2019S 11-12 wk old

co n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

0

1 0 0 0

2 0 0 0

3 0 0 01 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

ng

/g

DA

D OP A C

H VA DA

D OP A C

H VA

**

#

#

#

co n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

0

1 0 0 0

2 0 0 0

3 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

ng

/g

DA

D OP A C

H VADA

D OP A C

H VADA

D OP A C

H VADA

D OP A C

H VADA

D OP A C

H VADA

D OP A C

H VA

#

co n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

c o n traips i

0

1 0 0 0

2 0 0 0

3 0 0 01 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

ng

/g

D OP A CDA

H VA

D OP A CDA

H VA

D OP A C

DAH VA

D OP A CDA

H VA

D OP A C

DAH VA

#

#

#

NEUROCHEMISTRY6

B

AV e h ic le

1 m g /k g P F -3 6 0

3 m g /k g P F -3 6 0

1 0 m g /k g P F -3 6 0

3 0 m g /k g P F -3 6 0

6 0 m g /k g P F -3 6 0

C57Bl/6J 8-10 wk old

G2019S 5-6 months old

CONCLUSIONS 7• In this study, the first attempt was made to study efficacy of LRRK2 inhibitor PF-360 in the AAV-A53T

mouse model of PD. The treatment with a diet containing PF-360 or vehicle was started 7 days prior tounilateral injection of AAV-A53T and continued for 6 weeks. The study was conducted in C57BL6J andG2019S mice. In addition, data between two different age cohorts of G2019S mice were compared.

• Chronic, 42-day in-diet dosing with LRRK2 inhibitor PF-360 resulted in dose dependent inhibition ofSer935 in the brain and periphery both in C57Bl6 and G2019S mice.

• Neurochemical analysis showed that LRRK2 inhibitor is not able to improve dopamine, DOPAC or HVAlevels in striatum in younger cohorts. In older G2019S mice, HVA levels were increased with the treatment.

• Stereological analysis revealed a treatment related trend for increased TH positive cells counts insubstantia nigra of C57Bl6 mice. In G2019S mice, significantly higher TH positive cell counts wereobserved only in the older cohort.

• Fine motor kinematic gait analysis showed the impairment caused by unilateral infusion of AAV-A53T in allthree cohorts. However, a significant treatment effect was observed only in G2019S mice (at dose of 10mg/kg in the younger cohort and at dose of 30 mg/kg in the older cohort).

• Taken together, this data suggests that despite dose-related inhibition of Ser935, PF-360 failed to showrobust improvements in dopaminergic function.

A

B

E F

C

D

Discriminant direction for ”Distance from C57 AAV-Null”

Discriminant vectors for G2019S AAV11-12 wk old 5-6 months old