Masitinib significantly slows disease progression in post-paralysis SOD1G93A rats and reduces inflammation in both CNS and PNS

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

Introduction

Phenotypic

Proliferation

Masitinib

Linfocyte

Nerve

2 Masitinib reduces aberrant glial cells in the

degenerating 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)

Results

Masitinib 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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