Emiliano Trias1, Sofía Ibarburu1, Romina Barreto-Núñez1, Joël Babdor2, Thiago Maciel2, Pablo Díaz-Amarilla3, Patricia Cassina4, Laura Martínez-Palma4, Colin Mansfield5, Alain Moussy5, Ivan Moura2, Joseph Beckman6, Olivier Hermine2,7, Luis Barbeito1
1 Institut Pasteur de Montevideo, Uruguay; 2 Imagine Institute, Hôpital Necker, Paris, France;3 Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay; 4 Departamento de Histología, Facultad de Medicina, UdelaR, Montevideo, Uruguay; 5ABScience 6Linus Pauling Institute, Department of Biochemistry and Biophysics, Oregon State University 7Department of Hematology, Necker Hospital; INSERM UMR 1163, Laboratory of cellular and molecular mechanisms of hematological disorders and therapeutic implications; Paris Descartes - Sorbone Paris Cité University, Imagine Institute; CNRS ERL 8254; Laboratory of Excellence GR-Ex; Centre national de référence des mastocytoses (CEREMAST), Paris, France
IntroductionPhenotypic
Proliferation
Masitinib
Linfocyte
Nerve
2 Masitinib reduces aberrant glial cells in thedegenerating spinal cord
5 Masitinib reduces macrophages and mast cell cell inflitration in sciatic nerve
transformation
pre-AbA
AbA
CSF-1R
CSF1
Microglia
Ventral Horn
Monocyte
SOD1G93AAsympt
SOD1G93A post-paralysis
Symptomatic hSOD1G93A
GFAP/Ki67
Masitinib
Astro
Olig
Spinal Cord
NMJs
Macrophages
CSF1
Masitinib
Mast cells
Vehicle
Masitinib 30mg/Kg
Motor neuron
CSF1
Proliferating "aberrant glial cells" in the degenerating spinal cord
Masitinib
CSF-1R PDGF-R c-Kit Lyn
Methods. Daily masitinib 30 mg/kg vs Vehicle, orally.
Treatment starting day 1 post-paralysis and lasting 20 days.
Hindlimb onset only
GFAP/S100
50 M
that induce neurotoxicity
Downregulate macrophages/microglia
Downregulate mast cells
Hypothesis. Tyrosine kinase inhibition with masitinib might prevent the appearance of aberrant glial cells, regulate inflammation in both central and peripheral nervous systems, and slow paralysis progression.
Why masitinib?
AB1010 is highly selective kinase inhibitor shown to prevent neuroinflammation in multiple sclerosis, stroke and Alzheimer's disease. Is presently being evaluated in clinical trials for oncologic and neurologic diseases.
Why post-paralysis?
T8/T9 L1/L2
Nº of AbA cells/mm2
10 M
50
40
AbA
30 *
Motor 20
neuron
10
6 Masitinib increases post-paralysis survival
3
Masitinib targets aberrant glial cells that emerge only after paralysis onset in the SOD1G93A rat model, which is characterized rapidly progressing disease progression.
GFAP/S100
0
Vehicle Masitinib
Tg Symptomatic
Probability of survival post-paralysis
1.0
Methods. Daily oral masitinib ( 3 0 m g / k g ) o r v e h i c l e administration, starting after paralysis onset.
Survival was increased even when treatment was started 7 days after paralysis onset (7d onset).
0.9
0.8
Vehicle
Masitinib (gait onset)
Masitinib (7d onset)
ResultsMasitinib prevents microglia activation and motor
neuron pathology in ALS rats
0.7
0.6
35
Mean survival
post-paralysis
30 * **
1 Masitinib inhibits M-CSF induced aberrant glial cell
SOD1G93A post-paralysis
SOD1G93A post-paralysis
25
20
0.5 15
proliferation and inflammatory phenotype
Masitinib + M-CSF
SOD1G93A Asympt
Vehicle
Masitinib
Control
Symp. Vehicle Symp. Masitinib
SOD1G93A Asympt
Vehicle
Masitinib
0.4
0.3
10
5
0
Vehicle Masitinib
Masitinib
Vehicle
M-CSF
0.1 M 1 M
140
120
Cell/mm2
100
80
60
40
20
0
SOD1
ChAT
*
120
Number of MTNs (% respect to NonTg)
100
80
G93A
60
90
SMTN soma diameter (m)
Control 80
Symp. Vehicle 70
Symp. Masitinib 60
**
50
40
Iba1
* Conclusions
0.2
0.1
0.0
0 5 10 15 20 25 30 35 40 45
Days
(gait onset)
(7d onset)
DAPI/BrdU
post-paralysis
40 *
20
30 Masitinib can slow paralysis progression by targeting CNS
10
20 aberrant glial cells and inflammatory cell infiltration in the PNS.
50
100
%BrdU+ cells (respect to Vehicle)
Kinaes activity (%)
40
CSF-1R inhibition
IC50 (nM) = 90 ± 35 4
0 0
Protective effect would be at least in part mediated by masitinib
30
* 50
20
*
10
*
M-CSF expression in the degenerating sciatic nerve
inhibition of CSF-1R (microglia/macrophages) and c-Kit (mast cells)
0 Vehicle M-CSF 0.1 0.5 1.0
Masitinib(M)
0
10-4
10-2
100
102
Remarkably the protection can be obtained when masitinib is
mRNA levels (% of Vehicle)
100
40 *
20
+
M-CSF (nM)
Masitinib 1M
*
* *
Masitinib (M)
Vehicle
*
*
*
*
mRNA analysis of aberrant glial cells after Masitinib treatment during 72 hs. Data are expressed as mean ± m.s.e. *p
administered post-paralysis.
Masitinib represents a new drug candidate for the treatment of ALS.
References
Díaz-Amarilla P, Olivera-Bravo S, Trias E, Cragnolini A, Martínez-Palma L, Cassina P, Beckman J, Barbeito L. Phenotypically aberrant astrocytes that promote motoneuron damage in a model of inherited amyotrophic lateral sclerosis. Proc Natl Acad Sci USA. 2011 Nov 1;108(44):18126-31.
0 MCP1 IL1 Iba1 Cox2 IL6 TNF iNOS PU.1
Trias E, Díaz-Amarilla P, Olivera-Bravo S, Isasi E, Drechsel DA, Lopez N, Bradford CS, Ireton KE, Beckman JS, Barbeito L. Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS. Front Cell Neurosci. 2013 Dec 24;7:274.
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