Summary of BRAINco Drug Discovery Project in Major Depression. Resumen del Proyecto de Drug Discovery de BRAINco en Depresión Mayor

1.
New treatment for Major Depression
Modulators of Synaptogenesis and Neurogenesis for the
treatment of Major Depression
Advantages:
A novel biological system has been found altered in brain samples
from Depressive patients.
The alteration of a particular cytokine, has been associated with
depressive/anxious behavior in a transgenic mouse model over-
expressing this target specifically in the brain.
Lead compounds acting on some of this specific cytokine receptors
are available.
Background:
Studying post-mortem brain samples (right prefrontal cortex) by genomic and proteomic methods, BRAINco has
found a significant over-expression of a particular cytokine in depressive suicides compared to controls.
Hypothesis:
BRAINco has set up a Drug DEPRESSION
Discovery Program focused on this
new therapeutic target, which is
potentially implicated in the
biological bases of Depression
through its effects on
neuroplasticity and cognition. ALTERATIONS IN:
BRAIN PLASTICITY
BRC Target HPA AXIS
NEUROTRANSMISSION
BEHAVIOR & COGNITION
(HPA: Hypothalamic‐Pituitary Adrenal)
Targeting specific cell signaling pathways modulated by BRAINco target, could render new
antidepressant drugs.

2.
Development Stage: Target Validation
ANIMAL MODEL
BRAINco has generated a transgenic (TG) animal model
that over-expresses the specific cytokine in the brain
(Fig.1). Its comprehensive characterization has pointed it
out as a good model for the development of new
antidepressant drugs with novel mechanisms of action.
Figure 1. Target expression in WT and TG mice, measured
Behavioral features: by in situ hybridization.
At basal conditions… 50
OF 80
EPM 125 LDB
% open arm distance/total
Time spent in central area
40 100
Time spent in lit (s)
TG mice showed a consistent anxious phenotype (Fig.2), 60
**
(mean±sem)
mean±sem
30 75
not attributable to locomotion impairment. No 40 *
50
differences were found in tests classically used to 20
20
**
determine antidepressant efficacy (such as tail 10 25
n=18 n=21 n=16 n=10 n=16 n=10
suspension and forced swimming test). 0
WT TG
0
WT TG
0
WT TG
100 T‐Maze
% entries into new arm
In addition, the over-expression of the candidate induced 80
a decrease in long term potentiation as well as deficits in 60
the T-Maze test (Fig.3), being both results associated
*
40
with a cognitive impairment. 20
n=7 n=6
0
WT TG
After an aversive stimulus… Figure 2. Anxiety-like responses (top) and cognition analysis (botton) of TG
animals and WT siblings. OF: Open Field; EPM: Elevated Plus Maze; LDB:
Light-Dark Box.
TG mice presented important indications of a depressive-like
↓ 26 % ↓↓ 44 % behavior as assessed by Anhedonia test, after being exposed
80 *** ***
to different aversive stimulus (chronic unpredictable mild
% Sucrose preference
stress and olfactory bulbectomy) (Fig.3). These results
60
suggest that TG mice show a higher sensitivity to develop
depression after a stressful situation, than wild-types.
40
20 A B
NSF Anhedonia
500 WT
n=11 n=15 n=16 n=14 TG
***
80
**
Latency to feed (secs)
**
% Sucrose preference
0 400
WT WTO TG TGO 60
300
Figure 3. Sucrose preference in WT and TG mice with
and without olfactory bulbectomy (O). 40
200
100 20
After pharmacological treatment…
n=7 n=7 n=7 n=7 n=11 n=15 n=7 n=16 n=14 n =7
0 0
-Flx +Flx
TG mice exhibited a higher sensitivity to chronic
TO
X
T
TG
O
X
W
FL
FL
TG
W
TO
O
TG
Fluoxetine treatment, measured in normal conditions, as
W
Figure 4. Chronic Fluoxetine treatment in WT and TG animals (A) under basal
well as after Olfactory Bulbectomy (Fig.4). conditions (measure by NSF (Novelty Supressed Feeding) test) and (B) in
bulbectomized animals (measuring sucrose preference or anhedonia).

3.
Development Stage: Target Validation
Biochemical characterization:
TG mice overexpressing the target showed important
alterations in phosphoproteins implicated in dendrite
formation, synaptogenesis and regulation of actin
cytoskeleton.
The over-expression of BRAINco target in hippocampus,
induced significant changes in proteins previously found
altered in Depression such as, Wnt, BDNF, CREB and β-
catenin (Fig.5).
All these data showed the implication of BRAINco target in
crucial biological processes found altered in Depression.
Figure 5. Cell signaling pathways affected in BRAINco TG model.
Characterization of Neurogenesis:
The overexpression of BRAINco target, induced a decrease in
both, cell proliferation (measured by BrdU incorporation) and
neurogenesis (Dcx labeled cells). Interestingly, KO mice for
this target, showed a significant increase in hippocampal cell
Figure 6. BrdU positive cells in TG and KO mouse models, compared to
the respective WT animals. proliferation (Fig.6).
TG mice exhibit several features similar to those observed in human Depressive patients.
Therefore, BRAINco TG mice could be considered as a novel animal model to study Depression and to test the
effectiveness of new drugs as well as existing compounds modulating the system.
FINDING OF NEW CHEMICAL ENTITIES
The main objective of this project is the development of a novel therapy for Depression by finding new drugs directed
to a new target found altered in human specimens. For this purpose, we tested the effect of BRAINco target in
different cellular systems in order to set up assays suitable for High-Throughput Screening.
Different assays including cell proliferation, migration, differentiation and survival of new neurons are being set up,
using human neural progenitor cells.
BIOMARKERS
BRAINco therapeutic target is a secreted cytokine measurable in human serum. A biomarker discovery program is
being set up in order to investigate the alteration of target expression levels in non-invasive samples of the animal
model, as well as in Depressive patients. The final purpose is to use this biomarker as patient selection criteria.

4.
Summary
Depression is a devastating disorder with high prevalence and mortality, resulting in massive
socioeconomic burden (200 Millions in 2010). Current antidepressant treatments are limited by their
efficacy and delay onset of action. Therefore, new therapies based on novel mechanisms of action are
urgently needed.
In 2008, BRAINco discovered that a specific cytokine was significantly over-expressed in the
prefrontal cortex of depressive patients, which became a novel promising therapeutic target for drug
discovery.
With the purpose of functionally validate the target, a transgenic mouse which over-expressed the
cytokine in the brain was generated. A comprehensive characterization of this mouse showed a
consistent anxious-like behavior and cognitive impairment in basal conditions, combined with a
depressive-like phenotype after a stressful event, which was reversed by chronic antidepressant
treatment. Therefore, this transgenic mouse could be considered as a non-classical animal model of
Depression, which may be a very valuable tool for the development of new antidepressants with novel
mechanisms of action.
BRAINco team is also developing a cell based High Throughput Screening program with human
neuronal progenitor cells, to look for new chemical entities targeting the system.
In addition, a biomarker program is ongoing. The objective is to identify non-invasive biomarkers
specific to the mechanism of action of BRAINco compounds and that could be used for patient
selection.
BRAINco has already filed a Patent application on these results.
BRAINco Biopharma S.L
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