poster_ACRIMS-ECTRIM..

Comparative Efficacy between a Generic (M356) and Brand Copaxone®
(glatiramer acetate injection) in an Animal Model of Multiple Sclerosis
Christopher Honan, Tanmoy C. Ganguly, Ian Fier, and Ganesh V. Kaundinya—Momenta Pharmaceuticals, Inc., Cambridge, MA
BACKGROUND
RESULTS
Multiple sclerosis (MS) is an autoimmune mediated inflammatory disease of the CNS
in which the myelin sheaths of nerve cells are damaged, resulting in a wide range of
clinical symptoms including varying degrees of paralysis. MS takes several forms with
new symptoms either occurring in isolated attacks (relapsing-remitting; RRMS) or
building up over time (chronic/progressive).
In the model using active induction with PLP139-151, both M356 and RLD
(Copaxone) significantly delayed the mean day of onset relative to vehicle (Figure
1). Statistically significant differences between M356 and RLD were not observed
for disease onset, disease intensity, and peak disease score (Table 1).
Experimental autoimmune encephalomyelitis (EAE) is the most commonly used
animal model to mimic MS in humans and to test efficacy of potential therapies.
EAE models of both relapsing/remitting as well as chronic/progressive forms of MS
have been developed and are generally predictive of clinical efficacy of new therapies
for MS1. In fact, the EAE model is currently used as a release test to confirm the
biological activity of Copaxone®. EAE is induced with various immunogenic myelin
neuroantigens either directly by immunization with these antigens (active induction)
or passively following transfer of lymphocytes specific to these neuroantigens.
Figure 1 and Table 1: Clinical Scores—Active Induction with PLP139-151
Copaxone is approved for the treatment of RRMS and has been reported to affect
multiple aspects of this autoimmune disease2. The use of three EAE models, two
of active induction and one adoptive transfer, and two different antigens (PLP and
MOG), permitted a thorough comparison of M356 and RLD (Copaxone) in this
experimental model.
Figure 3: Histological Analysis—Active Induction with MOG35-55
The active induction PLP139-151 model was chosen as it is a well-established
model of RRMS. It models the various steps of autoimmune antigen recognition
and presentation, T cell activation and polarization, trafficking of auto-reactive
inflammatory cells, initiation of inflammation in the CNS, and eventual resolution
of inflammation3,4. The active induction MOG35-55 model is also a well-established
model for primary progressive MS. Similar to the PLP139-151 model, it mimics the
various steps of autoimmune response but exhibits more neurodegeneration. It was
therefore chosen as a confirmatory model to compare the neuroprotective effects
of M356 and RLD5.
M356
Demyelination Score (Luxol Blue)
Demyelination Score
(Mean ± SEM)
3
2
There were 12 animals per treatment group in each study. All mice were scored for
disease progression using a standard scale ranging from 0 (normal; no overt signs of the
disease) to 5 (complete hind limb paralysis with front limb involvement; moribund state;
euthanasia required). The following parameters were calculated from the score data:
• Disease Incidence: The sum of animals that attained a score of 1 or greater
for 2 consecutive days / number of animals per group.
• Disease Intensity: The average of daily scores from Day 7 through study
completion.
• Mean Peak Score: The average of the highest score attained by each animal
during the study.
• Mean Day of Onset: The average of the first day each animal reached a score
of 1 or greater.
Histological evaluation was conducted for the MOG35-55 (chronic/progressive) model.
On Day 15, at the peak of EAE in the vehicle group, half the mice were sacrificed. On
Day 28 (end of study), the remaining mice were sacrificed. Mice were perfused with
PBS and spinal cords were collected in 10% buffered formalin. For each mouse, three
Luxol fast blue stained sections (for demyelination) and three H&E stained sections (for
apoptotic cell count and inflammatory foci) from lumbar, thoracic, and cervical spinal cord
were prepared. A total of nine Luxol fast blue and 9 H&E sections for each mouse were
analyzed by a trained pathologist blinded to the experimental groups and all readouts.
1.0
0.5
0.0
One-way ANOVA followed by Tukey’s Multiple Comparison Test
P-values in table are relative to vehicle controls; ns = not significant
No significant differences were observed between RLD and M356
In the model using active induction with MOG35-55, both M356 and RLD
significantly delayed the mean day of onset relative to vehicle (Figure 2).
Statistically significant differences between M356 and RLD were not observed for
disease onset, disease intensity, and peak disease score (Table 2).
Vehicle Control
4
RLD
M356
3
2
0
15
10
20
25
)
(D
M
35
6
ay
(D
L
RD
28
)
28
)
(D
28
)
Avg Number od Foci
(Mean ± SEM)
(D
M
35
6
ay
(D
L
28
28
)
)
)
28
(D
RD
cl
hi
Ve
M
e
(D
35
6
ay
(D
L
RD
15
15
)
)
)
15
(D
e
cl
hi
Ve
Figure 4 and Table 3: Clinical Scores—Adoptive Transfer from PLP139-151
Vehicle Control
FTY720
RLD
M356
2
REFERENCES
5
5
15
10
Vehicle Control
RLD (2mg,sc,qd)
M356 (2mg,sc,qd)
30
20
25
30
DAY
FTY720 (3mg,po,qd)
Percent Disease
Incidence
Disease
Intensity
Mean Peak
Score
Mean Day
Onset
Treatment
Group
Percent Disease
Incidence
Disease
Intensity
Mean Peak
Score
Mean Day
Onset
Vehicle
100
2.2 ± 1.3
3.4 ± 0.3
11.8 ± 1.9
Vehicle
92
0.8 ± 1.0
2.5 ± 1.2
12.4 ± 2.3
25
0.1 ± 0.5
p<0.001
0.5 ± 1.0
p<0.001
27.6 ± 3.0
p<0.001
FTY720
0
0.0 ± 0.0
p<0.001
0.0 ± 0.0
p<0.001
>29.0
ND
17
0.0 ± 0.2
p<0.001
0.3 ± 0.8
p<0.001
28.7 ± 0.8
p<0.001
55
0.5 ± 1.0
ns
1.5 ± 1.5
ns
17.3 ± 3.3
p<0.05
83
0.4 ± 0.8
ns
1.6 ± 1.2
ns
18.4 ± 3.5
p<0.001
One-way ANOVA followed by Tukey’s Multiple Comparison Test
P-values in table are relative to vehicle controls
No significant differences were observed between RLD and M356
CONCLUSIONS
1
Treatment
Group
M356
These EAE models were part of a larger set of equivalence assays; similarity
between M356 and RLD was evaluated by demonstrating sameness of starting
materials, control of process, and equivalence of physicochemical, biological, and
immunological properties (e.g., multiple methods for amino acid composition,
molar mass distribution, N- and C-terminal analysis, and potency, T cell, B cell,
APC biology, gene expression profile, etc.).
These results were supportive of and consistent with results from a larger
program to demonstrate equivalence of M356 and RLD across biological and
physiochemical aspects of glatiramer acetate.
DAY
RLD
All three EAE models demonstrated equivalent efficacy between M356 and RLD.
Significant delays in onset of disease were observed in all models. In addition,
histological examination of the MOG35-55 study confirmed a strong inhibition of
inflammation as measured by immune cell infiltration and damage to myelin
sheaths. Importantly, there was no significant difference between M356 and RLD
in any parameter.
The biological equivalence of M356 and RLD (Copaxone) across several EAE
models was demonstrated using different antigens and dosing regimens.
0
0
Ve
(D
6
35
M
In the PLP139-151/adoptive transfer model, both M356 and RLD significantly
delayed the mean day of symptom onset relative to vehicle (Figure 4). Statistically
significant differences between M356 and RLD were not observed for disease onset,
disease intensity, and peak disease score (Table 3). FTY720 (fingolimod) was used
as a positive control in this study and inhibited disease when given daily at 3 mg/kg.
0
1
hi
cl
e
(D
M
35
6
ay
(D
L
RD
hi
cl
e
28
28
)
)
)
ay
ay
(D
RD
L
cl
hi
Ve
One-way ANOVA followed by Tukey’s Multiple Comparison Test
***p<0.001; **p<0.01; *p<0.05; ns = not significant
3
Figure 2 and Table 2: Clinical Scores—Active Induction with MOG35-55
15
)
15
15
)
M356
(D
14.2 ± 1.3
p < 0.01
L
3.3 ± 1.0
ns
28
92
1.6 ± 1.4
p < 0.05
RLD
(D
14.3 ± 1.6
p < 0.01
RD
3.7 ± 0.7
ns
e
100
1.9 ± 1.3
ns
cl
12.3 ± 0.9
)
3.8 ± 0.5
hi
2.3 ± 1.1
15
100
5
0
(D
Vehicle
ns
10
–2
Ve
Mean Day
Onset
6
Mean Peak
Score
0
)
Disease
Intensity
ns
15
2
(D
Percent Disease
Incidence
The adoptive transfer PLP139-151 model is another well-established model of RRMS
that bypasses the T cell activation process and focuses on more downstream aspects
of the disease, such as lymphocyte trafficking, homing to the CNS, and resolution of
inflammation. It was chosen to compare effects of M356 and RLD on these aspects
of the autoimmune response, using a different (daily) therapeutic dosing regimen4.
Inflamatory Foci
ns
4
35
30
15
25
M
20
)
15
15
0 9 10
Clinical Score (Mean±SEM)
3. Adoptive transfer from PLP139-151 immunized donors: In the adoptive transfer
model of EAE, SJL/J female donor mice were immunized as described above
for the active/PLP model. On Day 10, donor spleens were removed and
splenocytes isolated for culture. Cells were cultured for 3 days at 5x106 cells/
mL in the presence of 20 μg/mL PLP131-159. Cells were then transferred (2030x106 cells per mouse) i.v. to naïve recipient female SJL/J mice. Mice were
treated daily with M356 or RLD at 2 mg/mouse given s.c. on Days 0-9. Symptoms
were typically observed between Days 6-8.
1.5
Ve
ns
6
Treatment
Group
Score (Mean±SEM)
2. Active induction with MOG35-55: The active induction of the MOG model
(chronic/progressive) was initiated by immunization of female C57Bl/6 mice
subcutaneously at three sites on the dorsal surface with 50-75 μg of MOG3555 peptide emulsified in CFA. In addition, mice were injected i.p. with 200
ng of pertussis toxin on Days 0 and 1. For prophylactic treatment, 500 μg of
M356 or RLD was included in the encephalitogenic emulsions. Symptoms were
typically observed between Days 9-11.
2.0
(D
Apoptotic Cell Count
DAY
1. Active induction with PLP131-159: The active induction version of the PLP model
(RRMS) was initiated by immunization of female SJL/J mice subcutaneously at
three sites on the dorsal surface with 75-100 μg of PLP139-151 peptide emulsified
in CFA. For prophylactic treatment, 500 μg of M356 or RLD was included in
the encephalitogenic emulsions. Symptoms were typically observed beginning
between Days 11-13.
ns
–0.5
1
0
ns
2.5
e
Three different mouse EAE models were used to compare the efficacy of M356 and RLD:
RLD
Histological analysis in the MOG35-55 model revealed a significant reduction of
demyelination, apoptotic cell counts, and inflammatory foci at both time points
(Day 15 and 28) in groups treated with RLD or M356 relative to vehicle. No
statistically significant differences between RLD and M356 were detected for any
of these parameters (Figure 3).
Apoptotic Cell Count
(Mean ± SEM)
METHODS
4
Clinical Score (Mean±SEM)
M356 is being developed as a generic version of Copaxone (aka Reference
Listed Drug; RLD) for the treatment of RRMS and is under FDA review.
Equivalence between M356 and RLD was evaluated using a comprehensive set of
physicochemical (structural) and biological assays. The objective of these analyses
was to demonstrate “sameness” between M356 and RLD, and included evaluation
in several mouse EAE models.
Vehicle Control
DISCUSSION
RLD
M356
One-way ANOVA followed by Tukey’s Multiple Comparison Test
P-values in table are relative to vehicle controls; ns = not significant; ND = not done
No significant differences were observed between RLD and M356
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disability in relapsing-remitting multiple sclerosis: results of a phase III multicenter, doubleblind placebo-controlled trial. The Copolymer 1 Multiple Sclerosis Study Group. Neurol
1995;45(7):1268-76.
3. McRae BL, Kennedy M, Miller SD. Induction of active and adoptive relapsing experimental
autoimmune encephalomyelitis (EAE) using an encephalitogenic epitope of proteolipid
protein. J Neuroimmunol 1992;38(3):229
4. Teitelbaum D, Fridkis-Hareli M, Arnon R, et al. Copolymer 1 inhibits chronic relapsing
experimental allergic encephalomyelitis induced by proteolipid protein (PLP) peptides in mice
and interferes with PLP-specific T cell responses. J Neuroimmunol 1996;64(2):209-217.
5. Jee Y, Liu R, Vollmer TL. Do Th2 cells mediate the effects of glatiramer acetate in
experimental autoimmune encephalomyelitis? Int Immunol 2006;18(4):537-44.
Presented at the 2014 Joint ACTRIMS-ECTRIMS Meeting
September 12, 2014, Boston, MA

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