THE FAST SKELETAL MUSCLE TROPONIN ACTIVATOR TIRASEMTIV INCREASES MUSCLE FUNCTION
PERFORMANCE
AND
B6SJL-SOD1G93A ALS MOUSE MODEL
IN THE
Fady I. Malik, Darren T. Hwee, Adam Kennedy, Julie Ryans and Jeffrey R. Jasper
Cytokinetics Inc, South San Francisco, CA
RESULTS
ABSTRACT
Body mass and muscle performance decreases
over time in B6SJLSOD1G93A mice
OBJECTIVE: To evaluate the effects of tirasemtiv on muscle function in a mouse model of ALS.
BACKGROUND: Tirasemtiv is a fast skeletal troponin activator that sensitizes the sarcomere to calcium and amplifies the
response of muscle to neuromuscular input.
B
A
H i n d li m b G r ip
F o r e lim b G r ip
0 .1 0
0 .1 0
22
0 .0 8
G r ip S t r e n g t h ( K g )
G rip S tre n g th
Body M ass
*
G r ip S t re n g t h ( K g )
B
A
0 .0 8
0 .0 6
0 .0 4
0 .0 2
0 .0 8
0 .0 6
NS
0 .0 4
0 .0 2
21
18
17
0 .0 6
16
0 .0 2
F o r e lim b
0 .0 0
9
10
11
12
13
14
15
16
17
G r id H a n g T i m e
R o ta ro d P e rfo rm a n c e
50
200
H in d lim b
CONCLUSIONS: Tirasemtiv increased both isometric sub-maximal muscle force in situ and in vivo muscle performance in
B6SJLSOD1G93A mice. Tirasemtiv also increased respiratory function. These results indicate that tirasemtiv has the potential to
improve muscle function and respiration in patients suffering from ALS.
D
C
0 .0 4
18
9
10
11
12
A g e (W e e k s )
*
13
14
15
16
17
T im e t o f a ll ( s e c )
19
0 .0 0
0 .0 0
H a n g T im e ( s e c )
20
M a s s (g )
RESULTS: Following a single oral dose (10 mg/kg, PO) of tirasemtiv, forelimb grip strength (p<0.05), grid hang time (P<0.01),
and rotarod performance (p<0.01) all significantly increased in the B6SJL-SOD1G93A transgenic mice. Compared to vehicle
treatment, tirasemtiv significantly increased tidal volume in mice breathing room air following a 30 minute hypercapnic
challenge (p < 0.001). In 90-100 day-old B6SJL-SOD1G93A mice, tirasemtiv treatment (10 mg/kg, IV) significantly increased
submaximal (30Hz) isometric tension in situ from 5.81 ± 0.46 N/cm2 to 10.8 ± 0.71 N/cm2 (p=0.0028). In 110-115 day-old mice,
tirasemtiv increased submaximal tension from 3.34 ± 0.73 N/cm2 to 5.05 ± 1.4 N/cm2 (p=0.064).
G r ip S tre n g th (K g )
DESIGN/METHODS: Forelimb grip strength in female B6SJL-SOD1G93A transgenic mice was monitored weekly starting at
10 weeks of age (baseline). In order to minimize disease variability, mice were subjected to a battery of functional assessments
when their forelimb grip strength declined by 25% from baseline. Mice were dosed once with either vehicle or tirasemtiv (10
mg/kg, PO) and forelimb grip strength, grid hang time, or rotarod performance were evaluated. Respiratory parameters of
WT and B6SJL-SOD1G93A mice were assessed by whole body plethysmography following treatment with vehicle or tirasemtivtreated (10 mg/kg, PO). In a separate cohort of B6SJL-SOD1G93A mice, isometric extensor digitorum longus (EDL) muscle force
output was assessed in situ at 90-100 days and 110-115 days of age.
Tirasemtiv increased forelimb grip strength, grid hang time,
and rotarod performance in B6SJL-SOD1G93A mice
150
100
50
18
**
40
30
20
SUMMARY
10
0
0
AND
CONCLUSIONS
A g e (W e e k s )
1 0 w e e k S O D 1 b a s e lin e
D
C
R o ta ro d
G r i d H a n g Ti m e
100
50
0
40
Infusion of tirasemtiv increases in situ EDL muscle force in WT and
B6SJL-SOD1G93A transgenic mice at mid-stage and late stage disease
20
A g e (W e e k s )
Figure 1: Body mass and muscle performance decreases over time in B6SJL-SOD1G93A mice: A) Body mass, B) Forelimb and hind
limb grip strength, C) Grid-hang time, and D) Rotarod performance over 10 to 18 weeks of age. Tests were performed on a weekly
basis. Data from mice under assessment with either vehicle or tirasemtiv were excluded from these figures for that week. Data
are expressed as mean ± SEM. n=24/group.
B
In situ muscle testing in a separate cohort of female mice occurred at two stages of disease: at 90-100 days of age,
when signs of weakness were becoming apparent (weakness and trembling of hindlimbs) and a later stage,
110-115 days where signs of single or dual limb paralysis were evident. Muscle contractile properties were assessed
as described previously (4).
0 .0 5
A
**
* **
* **
0 .0 0
D
V e h ic le
0 .2
1. G. Logroscino et al., Incidence of amyotrophic lateral
sclerosis in Europe. Journal of Neurology,
Neurosurgery, and Psychiatry 81, 385 (Apr, 2010).
0 .1
ry
e
ry
c
R
R
e
e
c
%
o
o
v
v
e
O
C
C
%
s
a
B
2
2
O
n
li
e
s
a
Figure 2: B6SJL-SOD1G93A mice exhibit significant functional deficits prior to tirasemtiv administration. Performance values of (A)
Forelimb grip strength, (B) Hindlimb grip strength, (C) Hang test performance, and (D) Rotarod performance at point of selection
for 25% and 40% deficit milestones in forelimb grip compared to the 10-week old baseline. (E) Numerical values for WT, 10 week
SOD1 baseline, 25% and 40% milestones. Data are expressed as mean ± SEM. n=18-24/group. **= p<0.01, *** = p<0.001 vs. WT
by one way ANOVA with post-hoc Tukey’s test.
o f S tim u la tio n
(H z )
F re q u e n c y
o f S tim u la tio n
0
5
1
1
0
0
0
8
0
6
0
0
0
6
1
F re q u e n c y
5
0
0
0
4. A. J. Russell et al., Activation of fast skeletal muscle
troponin as a potential therapeutic approach for
treating neuromuscular diseases. Nature Medicine 18,
452 (Mar, 2012).
4
2 0
Vehicle
0
2 0
+
3
4 0
tirasemtiv
S O D
0
4 0
tirasemtiv
1µM
2
6 0
3µM
+
0
6 0
+
S O D
1
8 0
S O D
5
8 0
0
1 0 0
0
1 0 0
0
S O D 1 40%
1 2 0
tirasemtiv
Vehicle
5
SO D 1 25%
1µM
0
S O D 1 - B a s e lin e ( 1 0 w e e k s )
+
4
***
0
+
W T
0
W T - (1 0 w e e k s )
* **
W T
3
0
50
* **
1 2 0
0
* **
100
3. J. Hegedus, C. T. Putman, T. Gordon, Time course of
preferential motor unit loss in the SOD1 G93A mouse
model of amyotrophic lateral sclerosis. Neurobiology
of Disease 28, 154 (Nov, 2007).
C
B
150
2
100
B
200
8
300
5
e
R o ta ro d P e rfo rm a n c e
**
2. M. E. Gurney et al., Motor neuron degeneration in mice
that express a human Cu,Zn superoxide dismutase
mutation. Science 264, 1772 (Jun 17, 1994).
0 .0
G r id H a n g T im e
200
1 0 m g /k g t i r a s e m t i v
**
*
0 .3
e
C
REFERENCES
***
0 .4
5
***
100
1
Diaphragm contractile force was measured by electrical field stimulation in an organ bath system. Diaphragms from
WT and B6SJL-SOD1G93A mice were excised, and placed in a temperature controlled water-jacketed chamber
(26-27 °C) containing Krebs-Henseleit Buffer, supplemented with 50mg/L tubocurarine and 50U/L insulin. The forcefrequency profile was obtained by stimulating the muscle at frequencies between 10-150 Hz (Grass Stimulator,
0.6 pulse width, 800 ms train duration). Tirasemtiv was dissolved in DMSO (1 or 3µM) and directly added into the bath.
0 .0 5
*
**
0 .1 0
5
Respiratory function was assessed in WT and B6SJL-SOD1G93A mice once all motor function tests at 40% decrements
were complete. Mice were orally dosed with vehicle or 10 mg/kg tirasemtiv and placed in unrestrained whole body
plethysmography chambers for 30 minutes of acclimation. Mice were then monitored for 10 minutes at room air,
followed by exposure to a 5% CO2 gas mixture for 30 minutes before being returned to room air for 10 minutes.
***
T im e to F a ll (s e c )
Rotarod performance was evaluated by placing mice on the rotarod (San Diego Instruments, San Diego, CA)
at 12 RPM for a maximum of ten minutes, time to fall was recorded.
* **
0 .0 0
H a n g T im e (s e c )
A modified cage grid apparatus was designed to allow mice to grab the cage wire mesh grid and then the grid was
inverted 180 degrees. The time before the mice dropped off of the grid on to a soft pad below was recorded for
each assessment with a maximum time of 300 seconds.
G r ip S tre n g th (K g )
G r ip S tr e n g th (k g )
Forelimb and hindlimb grip measurements were acquired in triplicate with a 250 gram Dual Sensor Grip Meter
(DFS-R-250G, Transcat, Inc., Rochester, New York, USA).
0 .1 5
0 .1 0
****
150
These results support the potential
of fast skeletal muscle troponin
activators to improve muscle
function in neuromuscular diseases.
**
Tirasemtiv increased tidal volume and submaximal diaphragm
tension in late stage female B6SJL-SOD1G93A mice
H i n d li m b G r ip
0 .1 5
****
200
Figure 4: Infusion of tirasemtiv increases in situ EDL muscle force in wild-type and B6SJL-SOD1G93A transgenic mice at mid-stage
(90-100 days) and late stage (110-115 days) disease. The EDL muscle was stimulated every 2 minutes with a 5 Hz stimulus
(1 ms pulse width, 350 ms train duration) via the peroneal nerve. Tirasemtiv was then administered as a 2 minute i.v. bolus in four
cumulative doses up to a total of 10 mg/kg. (WT mice, n=8; mid-stage B6SJL-SOD1G93A mice, n=5; late-stage B6SJL-SOD1G93A mice, n=6
Data are expressed as mean tension ± SEM. *p<0.05, **p<0.01, ****p<0.0001 vs. respective baseline force).
Wild-type background strain B6SJLF1/J mice and B6SJL-SOD1G93A mice were received from Jackson Labs, Inc.
(strains 100012 and 002726, respectively; Bar Harbor, ME) at approximately seven weeks of age.
F o r e lim b G r ip
***
Dose tirasemtiv (mg/kg)
Functional assessments of muscle performance
A
Late-Stage
Disease
****
n
Animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory
Animals of the Institute (Seventh Edition, National Research Council) and under the supervision of the Cytokinetics
Institutional Animal Care and Use Committee.
250
0
B6SJL-SOD1G93A mice exhibit significant functional deficits
prior to tirasemtiv administration
Mid-Stage
Disease
li
Animals
Baseline measurements of mouse body weight, forelimb grip strength, grid hang-time and rotarod performance
were recorded initially at ten weeks of age and weekly thereafter for the following eight weeks. Once forelimb grip
strength declined by 25% (as prespecified by protocol), each mouse performed a battery of functional tests in the
presence of tirasemtiv or vehicle treatment. Tirasemtiv was administered by oral gavage (10 mg/kg dose in 0.5%
HPMC/0.2% Tween 80) 30 minutes prior to each test. This dose was expected to provide a peak plasma concentration
of approximately 3-4 µg/ml between 20-30 minutes after dosing. The investigator was blinded to treatment and the
mice were given the same treatment on successive days, with each assessment occurring on a separate day. Mice
were re-randomized to treatment between 25% and 40% test periods. Statistical significance was calculated by
t-test between vehicle and treated group at specific animal age and p < 0.05 was considered significant.
WT
18
10
17
6
16
4
15
2
14
0
13
10
12
6
11
4
10
2
9
0
18
e
17
0
16
5
15
1
14
10
13
A g e (W e e k s )
6
12
4
11
T id a l V o lu m e (m l/b re a th )
10
2
9
A single dose of tirasemtiv
significantly increases submaximal
isometric force, forelimb grip
strength, grid hang time, and rotarod
performance in a transgenic mouse
model (B6SJL-SOD1G93A) of ALS with
functional deficits.
Diaphragm force and tidal volume
are significantly higher in tirasemtivtreated B6SJL-SOD1G93A mice.
0
0
METHODS
150
Percent of Baseline Force
at 30 Hz
Tirasemtiv is a novel small molecule activator of the fast skeletal muscle troponin complex. Tirasemtiv selectively sensitizes fast
skeletal muscle troponin to calcium (Ca2+), and slows the rate of Ca2+ release from the regulatory troponin complex of fast
skeletal muscle (4). In intact skeletal muscle in situ, the compound amplifies the response of muscle to nerve input and
increases force generation at sub-maximal levels of nerve stimulation. In the present studies, B6SJL-SOD1G93A mice were treated
with single doses of tirasemtiv to investigate its potential effects on skeletal muscle function in vitro and in vivo, including
assessments of muscle strength and endurance as well as respiratory function.
200
Tirasemtiv
Figure 3: Tirasemtiv increased forelimb grip strength, grid hang time, and rotarod performance in B6SJL-SOD1G93A mice. Effect
of tirasemtiv and vehicle at the 25% (approx. 90-110 days) forelimb grip strength loss milestone. (A) Forelimb grip strength, (B)
Hindlimb grip strength, (C) Grid hang time, and (D) Rotarod performance. Dotted line shows mean B6SJL-SOD1G93A transgenic
mouse performance assay values at baseline (10 weeks of age). Data are expressed as mean ± SEM. n=10-12/group. *= p<0.05,
**= p<0.01 vs. WT by one way t-test.
60
T im e o n ro ta r o d (s e c )
Amyotrophic Lateral Sclerosis (ALS) is a debilitating and fatal disease characterized by the selective and progressive loss of
motor neurons in the brain and spinal cord leading to atrophy, weakness, and eventually complete paralysis of skeletal muscle.
ALS is the most common motor neuron disease in adults, approximately affecting 22,000 individuals in the United States
alone (1). Transgenic mice carrying ALS-associated mutant human superoxide dismutase (SOD1) genes, including the
B6SJL-SOD1G93A mouse, parallel many features of the human disease (2). B6SJL-SOD1G93A mice develop progressive limb and body
weakness at approximately 80 days of age, culminating in full limb paralysis, morbidity and death at around 135-140 days (2).
Many of the histological features of disease in the B6SJL-SOD1G93A mice are similar to those observed in ALS patients, although
there appears to be less robust enlargement and sprouting of neighboring motor units towards denervated muscles as
compensation for the loss of the primary motor neuron (3).
H a n g T im e (s e c )
250
% o f W T V e h ic le M a x im a l T e n s io n
INTRODUCTION
Vehicle
(H z )
Figure 5: Tirasemtiv increased tidal volume and submaximal diaphragm tension in late stage female B6SJL-SOD1G93A mice. Respiratory
function and diaphragm tension output were assessed in B6SJL-SOD1G93A mice at 18 weeks of age. (A) B6SJL-SOD1G93A mice dosed with
tirasemtiv had significantly higher tidal volume than vehicle-dosed mice at baseline and during recovery following a 5% CO2 exposure.
Compared to its baseline tidal volume, vehicle-treated B6SJL-SOD1G93A mice had significantly lower tidal volume during recovery. (B,C)
Harvested WT and B6SJL-SOD1G93A diaphragms treated with tirasemtiv produced greater tension in response to submaximal frequencies
of electrical stimulation. Data are expressed as mean ± SEM. N ≥ 5/group.