WP2: Mechanisms, diagnosis and treatment of acute stroke

WP2: Mechanisms, diagnosis and treatment of acute stroke

INVICTUS. THERAPEUTIC TARGETS FOR STROKE: FROM
BIOMARKERS TO PATENT
WP2: Mechanisms, diagnosis and treatment of acute stroke
Global objective: to investigate some of the most relevant stroke-involved
mechanisms in order to develop, at preclinical level, new neuroprotective
therapies for the treatment of stroke during its acute phase.
Specific objectives:
WP2a: Reperfusion and oxidative stress (Dávalos).
WP2b: Excitotoxicity (Castillo).
WP2c: Immunity (Lizasoain).
WP2d: Intracerebral Hemorrhage (Martí).
WP2: Mechanisms, diagnosis and treatment of acute stroke
(Lizasoain)
WP2a
Reperfusion and
oxidative stress
(Dávalos)
WP2b
Excitotoxicity
(Castillo)
WP2c
Immunity
(Lizasoain)
WP2d
Hemorrhage
(Martí)
RU2. Dávalos
RU1. Castillo
RU3. Chamorro
RU3. Chamorro
RU3. Chamorro
RU2. Dávalos
RU5. Lizasoain
RU7. Martí
RU4. Montaner
RU5. Lizasoain
RU5. Lizasoain
RU6. Almeida
RU6. Almeida
RU11. Alborch
RU8. Castellanos
RU9. Díez-Tejedor
RU8. Castellanos
WP2: Mechanisms, diagnosis and treatment of acute stroke
WORKING PLAN:
1- In vitro models:
- Primary cell cultures of neurons, astrocytes, endothelial cells or their co-cultures.
- POG will be used as in vitro model of ischemia.
2- In vivo models:
- Animal models of focal ischemia (tMCAO-90 min; pMCAO) in WT and KO animals
- Animal models of hemorrhagic transformation
- Animal models of ICH (by injection of bacterial collagenase)
- Animal model of HE (by the administration of anticoagulant and/or vasopressor drugs).
healthy rats, hypertensive rats and transgenic rats with amyloid angiopathy.
WP2: Mechanisms, diagnosis and treatment of acute stroke
WORKING PLAN:
3- Human subjects:
- Glutamate clinical studies (WP2b)
“in patients with acute cerebral infarction conventionally treated with or without 24 h
dialysis for glutamate”.
- Hematoma expansion and uric acid studies (WP2d)
“in patients with spontaneous ICH within 6 and 24 hours from the onset of stroke,
respectively”.
4- Target validation:
- Phase I and IIa clinical trials of novel scavenging molecules of plasmatic glutamate
WP2: Mechanisms, diagnosis and treatment of acute stroke
WP2a: Reperfusion and oxidative stress (Dávalos).
a)the predictive capacity of MRI and blood biomarkers for hemorrhagic transformation (HT),
b)the molecular pathways of Hollotransferrin (HTf) neurotoxicity,
c)the effect of exposition to H2S on lesion outcome
d)developing clinical studies:
d.1) to find out biological signatures of futile revascularization and reperfusion injury
in stroke patients,
d.2) to describe a novel profile of biological signatures of futile recannalization,
d.3) to characterize the histological and biochemical composition of thrombus
associated with resistance to systemic lysis or mechanical thrombectomy,
d.4) to evaluate the neuroprotective effect of Uric Acid in acute stroke patients
treated with intravenous tPA
d.5) to report evidence of the treatment effect of mechanical thrombectomy in
patients with acute ischemic stroke on biological surrogates, neuroimaging markers
and proteomic changes.
WP2a: Reperfusión y estrés oxidativo
Hospital Universitario La Paz
Proyecto: Isquemia cerebral aguda, hipotermia e hibernación: papel de los
inhibidores de la fosforilación oxidativa
-
Objetivos:
Estudiar el efecto de la exposición a sulfuro de hidrógeno (H2S) tras infarto cerebral (IC) en
modelo animal experimental sobre:
Recuperación funcional
Tamaño de lesión
-
Analizar en el IC no lacunar de pacientes y en modelo animal experimental:
Marcadores (estrés oxidativo y muerte celular )
Posible aplicación como predictor pronóstico de recuperación funcional
Realizado: La exposición a H2S fue efectiva en mejorar la recuperación funcional a través de:
- mecanismos de protección ( volumen de lesión y muerte celular)
- pero no de reparación tras 14 días del IC
Pendiente: Estudiar marcadores de estrés oxidativo y muerte celular en modelo animal
experimental a las 24h tras oclusión permanente de arteria cerebral media (pMCAO) y en
pacientes con IC no lacunar.
Entregable: Publicación 2º semestre 2013
WP2: Mechanisms, diagnosis and treatment of acute stroke
WP2b: Excitotoxicity (Castillo).
a) target validation by in vivo optimization of therapeutic conditions for oxaloacetate,
b) lead finding by in vitro testing and hit to lead process of oxaloacetate-like compounds,
c) lead optimization by synthesis and in vitro testing of novel oxaloacete derivatives up to a
preclinical candidate,
d) preclinical-clinical development and Phase I and IIa clinical trials with selected molecules.
Explore other neuroprotective strategies like:
e) the use of plasmatic glutamate dialysis (based of experimentally demonstrated evidences),
f) the blockage of NR2B-NMDAR glutamate receptors by TAT or iRNA technologies
(GIPC/DAPK1), and by anti-GIPC antibodies
g) to identify new molecular targets involved in apoptotic and autophagic cell dead after
ischemia-reperfusion induced excitotoxic processes.
WP2: Mechanisms, diagnosis and treatment of acute stroke
WP2d: Hemorraghe (Martí).
a)demonstrating that amyloid angiopathy is a risk factor for hematoma expansion
b)demonstrating the role of uric acid as a neuroprotectant in ICH.
WP2: Mechanisms, diagnosis and treatment of acute stroke
WP2c: Immunity (RU3: Chamorro y RU5: Lizasoain).
a) the contribution of TLR4 expression in circulating cells
b) the implication of leukocytic TLR4 and hematopoietic TLR4 on infarct outcome,
c) the implication of microglial expression of TLR4 and its activation in leukocyte
recruitment,
d) the implication of TLR4 in the BBB impairment and the development of HT.
e) the analysis of recruitment/activation of immune cells, lymphocyte and antigen
presenting cells (APCs) in the infarcted rodent brain tissue.
Adaptado de (Garay and McAllister, 2010)
Fase aguda del ictus isquémico.
6-24h
Acute
phase
6 months
Chronic
phase
Macrez et al., Lancet Neurol, 2011
INNATE IMMUNITY
damaged tissue
healthy tissue
fibronectin
Heparan sulphate
necrotic cell
HSP
RNA
DNA
Hyaluronic acid
fibrin
fibrinogen
PAMPs “pathogen associated molecular patterns”
DAMPs “damage associated molecular patterns”
damaged blood
vessel
microglia
macrophage
inflammatory response
Iadecola and Anrather. Nature Med. 2011
Kariko et al., J Cereb Blood Flow Metabolism. 2004
ADAPTIVE IMMUNITY
6-24h
Acute phase
6 months
Chronic phase
Iadecola & Anrather, Nat Med, 2011
Resolution of inflammation and tissue repair
3rd phase
Promoting resolution
 anti-inflammatory response
4th phase
Promoting repair
Iadecola and Anrather. Nature Med. 2011
WP2: Mechanisms, diagnosis and treatment of acute stroke
WP2c: Immunity (Lizasoain)
Milestones
1. Contribution of TLR4 expression in circulating
cells on brain ischemic damage.
2. Implication of TLR4 in HT
3. Confirmation of the presence of
neuroantigens in peripheral lymph nodes
4. Contribution of the neuroantigens to the
adaptative immune response and specificity of
this response.
Outcomes
(New therapeutic approaches)
1. Drugs against circulating TLR4
expression (such as aptamers).
Reducing inflammation and leukocyte
recruitment
2. Immunomodulation of adaptative
response.
WP2: Mechanisms, diagnosis and treatment of acute stroke
WP2c: Immunity (Lizasoain).
INDICATORS (TIMEFRAME):
1) Report of involvement of TLR4 expression in circulating cells on:
a) Brain ischemic damage (2nd year)
b) Hemorrhagic transformation (4th year)
2) Report of aptamers against TLR4 (patent) (4th year).
3) Identification of neuroantigens in peripheral lymph nodes (2nd year).
4) Report on the involvement of neuroantigens:
a) in adaptative immune responses
b) on whether they are antigen-specific (4th year).
WP3: Brain repair after stroke
WP3b: Immunological and inflammatory response in brain repair (Lizasoain)
Milestones
1. Contribution of TLR4 on neurogenesis and
brain plasticity after stroke.
Outcomes
(New therapeutic approaches)
2. Implication of TLR4 and neuroantigens in
autoimmunity processes after stroke
1. Drugs against circulating TLR4
expression (such as aptamers).
Reducing inflammation and leukocyte
recruitment
3. Contribution of the presence of peripheral
neuroantigens in repair mechanisms.
2. Immunomodulation of adaptative
response.
WP3b: Respuesta inmunológica e inflamatoria en la reparación cerebral
RU5: Universidad Complutense Madrid. Lizasoain
Proyecto: Doble papel de los receptores toll-like en el ictus:
reguladores de daño y reparación.
Objetivos:
1. Estudiar la contribución de TLR4 en los procesos de neurogénesis.
2. Estudiar la contribución de TLR4 en plasticidad cerebral.
2. Estudiar la implicación de TLR4 en los procesos de autoinmunidad
Realizado: Estudios de proliferación en SVZ y corteza de BrdU+ y de microglia
Pendiente: Analizar plasticidad y procesos de autoinmunidad
Entregable: Publicación 4º trimestre 2013.
WP3b: Respuesta inmunológica e inflamatoria en la reparación cerebral
Hospital Universitario La Paz
Proyecto: Identificación de marcadores de daño y nuevas dianas terapéuticas para potenciar
la reparación y recuperación en el infarto cerebral. Estudio traslacional
Objetivos:
- Analizar marcadores de respuesta inflamatoria en el infarto cerebral (IC) en pacientes y
modelo animal experimental
- Correlación con la recuperación funcional
Realizado: Demostración que la terapia celular modula la respuesta inflamatoria (IL-6, TNF-α) en
modelo animal experimental tras IC.
Pendiente: Analizar marcadores de respuesta inflamatoria en modelo animal experimental y en
pacientes con IC y su correlación con la recuperación funcional.
Entregable: Publicación 1er semestre 2014