SECOND RESEARCH FUNDING AND INTELLECTUAL PROPERTY ASSIGNMENT AGREEMENT BETWEEN UNIVERSITY OF MELBOURNE AND PRANA BIOTECHNOLOGY LTD (ABN 37 080 699 065)
SECOND
RESEARCH FUNDING AND INTELLECTUAL PROPERTY
ASSIGNMENT
AGREEMENT
BETWEEN
UNIVERSITY
OF MELBOURNE
AND
PRANA
BIOTECHNOLOGY LTD (ABN 37 080 699 065)
1
This
Second Research Funding and Intellectual Property Assignment Agreement is made:
BETWEEN
THE
UNIVERSITY OF MELBOURNE [ABN
84
▇▇▇ ▇▇▇ ▇▇▇] of ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇ ▇▇▇▇, a body politic and corporate pursuant
to the provisions of the Melbourne
▇▇▇
▇▇▇▇▇▇▇▇▇▇
▇▇▇
▇▇▇▇
("the
University").
AND:
PRANA
BIOTECHNOLOGY LTD (ABN 37 080 699 065) having
its principal office at ▇▇▇▇▇ ▇, ▇▇▇ ▇▇▇▇▇▇ ▇▇▇▇▇▇, ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇
▇▇▇▇ ("Prana")
RECITALS:
|
A.
|
Prana
and the University are parties to an undated Research Funding and
Intellectual Property Assignment Agreement, entered into on or about
1
December 2000 as amended from time to time, which expired on 1 December
2003 ("The Research Agreement").
|
|
B.
|
Since
the expiration of The Research Agreement, the parties have continued
to
conduct Projects and work together in accordance with the terms and
conditions of the Original Research Agreement as if it continued
to have
full force and effect.
|
|
C.
|
The
parties now wish to enter into this Second Research Funding and
Intellectual Property Assignment Agreement ('Second Research Agreement')
which is deemed to have come into effect on and from the date of
expiration of The Research
Agreement.
|
NOW
IT IS AGREED:
|
1.
|
DEFINITIONS
& INTERPRETATION. Unless
otherwise specified in this Second Research Agreement, all defined
terms
used in this Second Research Agreement shall have the same meaning
as
given to those terms in The Research
Agreement.
|
'Further
Term'
means a
period of three years deemed to have commenced on and from the expiration of
The
Research Agreement and expiring on 1 December 2006.
‘The
Research
Agreement' means
the
undated Research Funding and Intellectual Property Assignment Agreement, entered
into on or about 1 December 2000 as amended from time to time, which expired
on
1 December 2003.
'Research
Projects' has
the
meaning given to that term in The Research Agreement.
'Second
Research Agreement' means
this Agreement.
|
2.
|
INCORPORATION
OF TERMS AND CONDITIONS OF THE RESEARCH
AGREEMENT
|
2
The
terms
and conditions of The Research Agreement are incorporated into this Second
Research Agreement and are deemed to have had full force and effect as and
from
the expiration of The Research Agreement, save and except for any terms and
conditions specifically amended, replaced or supplemented by this Second
Research Agreement.
|
3.
|
AMENDMENT
OF SCHEDULE
|
The
Parties agree that the Schedule to The Research Agreement shall be amended
as
provided by this Second Research Agreement.
| 4. |
EFFECTIVE
DATE OF THIS AGREEMENT AND EARLY
EXPIRATION
|
This
Second Research Agreement shall be deemed to have come into effect on and from
the date of expiration of The Research Agreement and shall remain in effect
for
a Further Term, unless the parties agree in writing to an earlier expiration
date or termination occurs in accordance with clause 19 of The Research
Agreement.
SIGNED
for
and
on behalf of THE
UNIVERSITY OF
MELBOURNE
In
the
presence of:) ______________
_______________ Authorised
Officer
Witness
signature
_______________
Name
(printed)
SIGNED
for and
on behalf of PRANA
BIOTECHNOLOGY
LTD
In
the
presence of:) ______________
_______________ Authorised
Officer
Witness
signature
_______________
Name
(printed)
3
SCHEDULE
A
Replacement
to Part B
| 1. |
PROPOSED
RESEARCH PROGRAM
|
The
Research comprises the following areas of development of possible therapeutic
or
diagnostic
applications
into Alzheimer's Disease and other neurodegenerative disorders and psychoses
characterised by metal mediated oxidative stress and/or toxic gain of function
by a protein.
Reference
to beta amyloid (AB) below is understood to include other functionally related
proteins such as APP, a -synuclein, a -crystalline, polyglutamine expansions,
SOD and Prion proteins.
For
clarity the following areas are defined:-
| 1. |
The
development of chelators or modulators of AB
activity.
|
|
2.
|
The
development of therapeutic or diagnostic agents arising from research
into
the structural and functional studies of AB metal binding sites,
characterization of AB neurotoxic species that modulate A-B redox
activity, and new molecular targets of AB defined by peptidomimetic
and
biosensor studies.
|
| 3. |
The
development of proteins that bind to and mediate the clearance of
AB.
|
|
4.
|
The
development of therapeutic or diagnostic applications arising from
the
elucidation of the behaviour of zinc, copper, iron and other metal
ions
with respect to redox activity at the
synapse.
|
| 5. |
The
development of compounds with pharmaceutical potential which directly
interfere with redox-mediated
damage.
|
| 6. |
The
development of markers of biochemical oxidative stress and metal
homeostasis in the human
brain as a model for the development of neurodegenerative disorders
or
psychoses as above defined.
|
|
7.
|
The
development of compounds and processes which modulate A-B membrane
interactions, AB mitochondrial biology and other cellular biochemical
neurotoxic processes mediated by
AB.
|
|
8.
|
The
development of any possible therapeutic or diagnostic applications
arising
from the characterization of mechanisms involved in transmembrane
APP
dimerization.
|
|
9.
|
The
development of a rational based drug design programme arising from
the
elucidation of the biophysical characteristics and structure of
AB.
|
|
10.
|
The
development of possible therapeutic or diagnostic applications arising
from studies of prion proteins in Creutzfeldt-Jakob
Disease.
|
|
11.
|
The
development of possible therapeutic or diagnostic applications arising
from studies of SOD in Motor Neuron
Disease
|
|
12.
|
The
development of assays arising from studies of polyglutamine expansions
in
Huntington's
Disease and other related
disorders.
|
|
13.
|
The
development of assays arising from studies of a-crystallin in
neurodegenerative disorders and
cataracts.
|
|
14.
|
The
development of assays arising from studies of a-synuclein in ▇▇▇▇▇▇▇▇▇'▇
Disease and related disorders.
|
4
|
15.
|
The
development of the technology, assay, cell line, animal model or
patient
database involved with the detection and screening of compounds developed
under this Agreement for use in the above field of
Research.
|
| 2. |
STAFF/PROJECTS/BUDGET
ESTIMATES
|
Research
Projects being conducted during the period 2 December 2003 to 31 December 2004.
The
details of each Research Project are provided in the Appendix to this Second
Research Agreement.
| 4. |
FUNDING
FOR PERIOD 2 DECEMBER 2003 — 1 DECEMBER 2006:
|
Funding
for 2 December 2003 to 31 December 2004, all figures are ex GST:
| Research Project Title. |
Budget
Period.
|
|
2
Dec 2003 — 31 March 2004.
|
|
|
'AB
interactions with
mitochondrial
respiratory
chain complexes'*.
(Trounce)
|
$ 156,645 |
|
'AB
clearance mechanisms’**.
(Cappai)
|
$ 179,021 |
|
'AB
binding ligands for imaging
of
Alzheimer's
Disease’***.
(Masters)
|
$ 134,266 |
*Research
Project formerly denoted as Project 7 in the Letter Amendment of 7 March 2003
to
the original Research Funding and Intellectual Property Assignment
Agreement.
**Research
Project formerly denoted as Project 8 in the Letter Amendment of 7 March 2003
to
the original Research Funding and Intellectual Property Assignment
Agreement.
***Research
Project formerly denoted as Project 9 in the Letter Amendment of 7 March 2003
to
the original Research Funding and Intellectual Property Assignment
Agreement.
|
2
Dec 2003 — 2 Dec 2004.
|
|
|
Project
1.
|
|
|
'Structure
Based Drug Design'
|
$150,000
|
|
Project
2.
|
|
|
'Drug
Screening & Development'
|
$300,000
|
|
Project
3.
|
|
|
'Cell
Based Drug Discovery’
|
$150,000
|
|
Project
4.
|
|
|
'APP
Metal Binding'
|
$12,000
|
|
Project
5.
|
|
|
'Blood
Brain Barrier Studies'
|
$0
to $24,000 invoice only on
needs-be-basis.
|
5
|
Commencing
1 April 2004- 31 Dec 2004.
|
|
|
Project
6.
|
|
|
'Amyloid
AB Imaging ligands:
|
$101,250
|
|
high
throughput screen approach
|
|
|
—
development of screening assays'
|
|
|
Project
7.
|
|
|
'Expression
profiling of
|
$50,625
|
|
AD
imaging targets'
|
|
|
Project
8.
|
|
|
'Amyloid
AB monoclonal antibodies
|
$50,625
|
|
as
imaging ligands'.
|
|
|
Project
9.
|
|
|
'AB
binding proteins'
|
$151,875
|
|
Project
10.
|
|
|
'Amyloid
imaging with PBT-1'
|
$174,300****
|
|
Project
11.
|
|
|
'AB
binding metallocomplexes'
|
$101,250
|
****A
12
month
budget for Project 10 of $275,000 (net GST) to be administered under the Austin
Health Sub contract to The University of Melbourne commenced 1 January 2004
under the Project 'AB binding ligands for imaging of Alzheimer’s Disease',
accordingly the budget for the period 1 April 2004 to the final payment to
31
Dec 2004 is calculated: $275,000 -0.75($134,266) = $174,300.
Funding
for each subsequent calendar year 2005 and 2006 is to be set by the Management
Committee one month before each calendar year in accordance with the funding
for
each annual Research Plan.
| 5. |
UNIVERSITY
REPRESENTATIVES
|
▇▇▇▇▇▇▇▇▇
▇▇▇▇▇ ▇▇▇▇▇ or a representative nominated by the University from time to
time.
| 6. |
PRANA
REPRESENTATIVE
|
▇▇
▇▇▇▇▇▇
▇▇▇▇▇, Prana or a representative nominated by Prana from time to
time.
| 7. |
MINIMUM
PERFORMANCE LEVELS
|
$2,000
per annum for the period beginning on the date an amount first becomes payable
under clause 12 of the Original Research Agreement until the end of the Further
Term of this Agreement.
6
APPENDIX
-TO SECOND RESEARCH AGREEMENT
Project
1. Research Prefect: ‘Structure Based Druq Design'
| General Aim : |
Characterizing
the biophysical interactions between metals and other co-factors
with: a -
synuclein with dopamine, APP and AB
to:-
|
| (i) | elucidate new therapeutic targets, |
|
(ii)
|
study
the effect of such biophysical interactions on the structure and
potential
oligomerisation of AB, APP and a -
synuclein
|
|
(iii)
|
develop
screens for PD and AD therapeutic
agents.
|
Proposed
Objectives:
(1) Develop
a
dopamine / a-synuclein / metal model to screen compounds for ability to moderate
ROS production and ability of agent to compete with a-synuclein -
dopamine.
(2) Develop
a
PD-specic cell-based tox. assay (dopaminergic neurons) to screen
for:
-
compounds positive in test (1),
-
to
detect inhibition of toxicity otherwise induced by 6-OHDa +Dopamine +test
agent
-
neuroprotection effects.
(3) Develop
a
PD animal model to test compounds positive in test (2) via 6-OHDa model and
transgenic PD models.
(4) Investigate
and characterise biophysical relationship of a-synuclein - dopamine interaction.
[ie. determination of a-synuclein binding site by mutagenesis, effect on
a-synuclein folding and aggregation of a-synuclein, effect on redox activity
of
a-synuclein] via:
-
protein
production
-mitochondrial
assays
-
CD,
ERR, NMR
(5) Investigate
and characterise biophysical relationship of APP and/or AB - metal interaction.
[ie, determination of binding sites by mutagenesis, effect on protein
folding
and aggregation and redox activity via;
-
protein
production
-mitochondrial
assays
-
CD,
ERR, NMR
Budget:
$150,000
Personnel:
K
Bamham:
Project Leader
▇.
▇▇▇▇▇
7
Project
2. Research Project: 'Drug Screening &
Development'
General
Aims : To
screen
and characterize Prana compounds in models of (i) AD and (ii) PD related
pathology and to develop new
screens based on the emerging knowledge of the underlying pathogenic
mechanisms of these diseases. To characterize
AB oligomers.
Proposed
Objectives:
|
·
|
The
hydrogen peroxide assay. To test the ability of candidate compounds
to
inhibit the copper catalysed generation of H2O2 by AB and a-synuclein
via
a 96 well format fluorometric
assay.
|
|
·
|
The
brain amyloid solubilisation assay (BAS). To test the ability of
candidate
compounds to solubilise protein deposits in a sample of human brain
tissue
or development of an assay using synthetic AB oligomers to detect
dissaggregation.
|
|
·
|
Haemolysis
assay: AB is incubated with red blood cells (RBC) in the presence
of
copper. Resultant lysis is assayed by UV absorption spectrometry.
Candidate compounds are assayed for their ability to inhibit
haemolysis.
|
|
·
|
Cholesten
4 assay. Assay development to identify the oxidation of cholesterol
as a
consequence of AB toxicity.
|
|
·
|
Membrane
perturbation assay. The insertion of protein into artificial lipid
micelles is associated with pore formation. Candidate compounds will
be
assessed for their ability to inhibit pore
generation.
|
|
·
|
Acute
toxicity assay for AD and PD. Incremental doses of a drug candidiate
which
has passed through the in
vitro efficacy
and toxicity screens are administered to mice to establish a tolerable
dose range for in-vivo
animal
trials.
|
|
·
|
Whole
mouse plasma pharmocology assessment for PD and AD test
agents.
|
|
·
|
Animal
trials. AD and PD Drug candidates to be administered to a cohort
of the
appropriate animal model for each disease. At completion of the trial,
brain and other tissues may be collected and assayed for Target Protein
content, changes to Target Protein and other markers, including
metals.
|
|
·
|
Characterisation
of endogenous AB oligomeric species, incl. ADDLs and their role in
AD
pathology.
|
Budget:
$300,000
Personnel: ▇.
▇▇▇▇▇▇: Project Leader
D.Carringtom,
I. Volitakis
8
Project
3. Research Project: ‘Cell Based Drug Discovery'
General
Aims:
To
screen
and characterize Prana compounds in
cell
based systems that measure toxicity and cellular dysfunction
associated with (i) AD & (ii) PD pathology and to develop new screens based
on the emerging knowledge of the
underlying
pathogenic mechanisms of these diseases.
Proposed
Objectives:
|
·
|
The
screening/testing of therapeutic or diagnostic agents in cell based
assays
for (i) AD and (ii) PD that measure toxicity and cellular dysfunction.
These assays will measure the agent's cellular
toxicity
|
|
·
|
Develop
a PD-specific cell-based toxicity assay to screen
for:
|
-
compounds which moderate ROS production,
-
to
detect inhibition of toxicity otherwise induced by 6-OHDa +Dopamine
+test
agent
-
neuroprotection effects.
|
·
|
The
screening/testing of therapeutic or diagnostic agents in cell based
AD
assays that measure their ability to inhibit metal mediated oxidative
stress interactions of AB, the ability to modulate cellular survival,
the
ability to modulate cellular survival following a toxic
insult/stress.
|
|
·
|
The
screening/testing of therapeutic or diagnostic agents in cell based
assays
that measure the cell penetration and cell transport properties of
the
agents. These assays will provide a measure of the pharmacokinetic
properties of the agents. The CaCo2 model and Menkes
model.
|
|
·
|
Screening/testing
for toxicity induced by 6OHDa and Dopamine with PD test agents.
|
|
·
|
Screening/testing
of agents to determine effect on APP
processing.
|
Budget:
$150,000
Personnel:
▇.
▇▇▇▇▇▇: Project Leader
▇.▇▇▇▇▇
9
Project
4. Research Project: 'APP Metal Binding'
General
Aims:
Adoption
of an in
silico screening
methodology based on the NMR structure of the copper binding domain on
APP to investigate this domain as an AD drug target.
Proposed
Objectives:
|
·
|
Section
of top ranked in
silico screened
putative binders to CuBD of APP.
|
|
·
|
Development
of an in
vitro fluorescence
binding assay.
|
|
·
|
Development
of a secondary cell based assay to identify test agent(s) agonist
activity.
|
|
·
|
Coordination
of possible decision to undertake crystal structure analysis and
medicinal
chemistry.
|
Budget:
$12,000*.
Personnel:
▇.
▇▇▇▇▇▇
and ▇.▇▇▇▇▇▇▇ (Projects leaders),
Prana personnel: G Kok
[SVIMR:
▇. ▇▇▇▇▇▇, G.
Kong]
*(Budget
not inclusive of Prana payments to SVIMR in
silico screening
and crystal structure and Prana Medicinal Chemistry program).
10
Project
5. Research Project: 'Blood Brain Barrier Studies'
General
Aims: To
assess
the ability of new Prana test compounds to pass the Blood Brain Barrier in
a rat
model.
Proposed
Objectives:
|
·
|
Work
undertaken by ▇. ▇▇▇▇▇▇▇ on a "needs be” basis in the Department of
Pharmacology as directed by Prana.
|
|
·
|
Assay
with a short duration direct perfusion of the rat brain with compounds
at
nominal 50uM concentrations in PBS buffered saline. Brain tissue
sample
collection and analysis of brain tissue
uptake.
|
Budget
Upto
$24,000
Personnel:
▇.
▇▇▇▇▇▇▇ Dept. Pharmacology. Liasing with ▇. ▇▇▇▇▇▇▇ of Prana
11
Project
6. Research Project: 'Amyloid AB imaging Iigands: a high throughput
screen
approach
- development of screening assays'
General
Aim:
-Development
of two assays used for HTS and medium throughput screen (MTS) to identify
selective AB binding molecules for the imaging of Alzheimer's Disease.
-In
vivo
imaging of AB in the brain for diagnosis and monitoring of therapy.
Proposed
Objectives: Research
Plan for the coming 6 months:
A)
High throughput screen (Melb.
Uni., SAG)
Two
different assays will be evaluated for high throughput screening. Both assays
are competition assays with fluorescent dye labeled AB1-42
as
ligand. One assay will use AB1-42
synthetic plaque as target (developed by Melb. Uni, assay 1) and the other
assay
will use soluble oligomers of AB1-42
as
target (SAG, assay 2). Fluorescence Anisotropy (FP) will be use to detect the
signal in both assays. Decision criteria for assay selection will be feasibility
(costs per well), reproducibility, and stability under high throughput
conditions (96 or 386 well format). If both assays fulfil these minimal
HTS-decision criteria then HTS will be carried out with assay 2, because
oligomers reflect the human situation in early stage AD better than synthetic
plaques. Both assays will be transferred to Assay Development (SAG) for the
assessment of the HTS suitability 3 month after project start. Decision will
be
taken 6 month after project start.
Assay
1:Competition
assay with fluorescent labeled AB1-42
on
synthetic AB1-42
plaques
(conducted by Melb. Uni)
|
>
|
Define
condition for plaque formation to get as close as possible to the
human
situation (Zn-concentration, pH)
|
| > | Determine the concentration of AB-binding sites in the plaque |
| > | Assay: |
| 1. | Formation of the AB1-42-plaques |
| 2. | Fixation of the AB1-42-plaque to the well |
| 3. | Incubation of compound with the AB1-42-plaque |
|
4.
|
Incubation
of Dye- AB1-42
at
the same time as the compound or after the incubation with the
compound
|
| 5. | Detection via FP or fluorescence |
>
Positive
control: unlabeled AB1-42
>
Negative
control: random aB1-42
>
Transfer
assay to SAG
Assay
2:Competition
assay with fluorescent labeled AB1-42
on
soluble AB1-42
oligomers (SAG)
> Define
condition for formation of oligemer to get as close as possible to the
human
situation
> Determine
the concentration of AB-binding sites at the oligomers
>
Assay:
1.
Formation
of the AB1-42-oligomers
2. Incubation
of compound with the AB1-42-oligomers
3.
Incubation
of Dye- AB1-42
at the
same time as the compound or after the incubation with the compound
4.
Detection
via FP
>
Positive
control: unlabeled AB1-42
>
Negative
control: random AB1-42
B)
Medium throughput screen (conducted by Melb. University)
Medium
throughput screen (MTS) will be carried out using a competition assay with
S-35
labeled AB1-42 on
human
late stage AD homogenates.
>
Assay
will be developed by Melb. Uni. and transferred to SAG (6
month).
12
>
MTS
will
be carried out by SAG.
>
Human
late stage AD brain tissue samples will be provided by Melb. Uni.
>
S-35
AB1-42
will be
produced by Melb. Uni. and delivered to SAG.
>
Assay:
1.
Incubate
compound with the homogenate.
2.
Incubation
of S-35- AB1-42 at
the
same time as the compound or after the incubation
with the compound.
3.
Separate
unbound from bound S-35 AB1-42
by
filtration.
4.
Detection
via radioactivity
>
Positive
control: unlabeled AB1-42
>
Negative
control: random AB1-42
Milestones
(month after project start):
>
HTS
assay
development 3 month
>
HTS
assay
transfer to assay development SAG 3 month
>
MTS
assay
development 6 month
>
MTS
assay
transfer to SAG 6 month
>
Delivery
of human late stage AD tissue sample (400 mg) and S-35 AB1-42
12
month
Critical
Issues:
>
Difficulties
in AB1-42
handling
-> shift to AB1-40
>
Low
signal to noise ratio in the MTS assay
>
Set
up
stable and reproducible high throughput screen within 6 month. Identified
hits
may be not suitable for radio labeling/imaging.
>
Legal
issues regarding the use of homogenates from AD patients for drug
characterization to be clarified by ▇▇▇▇.▇▇▇▇▇▇▇▇▇▇.
No
-Go
Criteria :
No
suitable HTS assay after 9 month.
No
suitable MTS assay after 12 month.
Budget:
Personnel:
R ▇▇▇▇▇▇ (Project Leader), [▇▇▇▇▇▇ ▇▇▇▇▇▇ (from Schering AG)]
13
Project
7. Research Project: 'Expression profiling of AD imaging
targets:
General
Aims:
|
·
|
To
identify molecules which are differentially expressed in AD brains
|
to
use
as novel AD specific imaging targets
|
·
|
In
vivo imaging of AB in the brain for diagnosis and monitoring of
therapy.
|
Proposed
Objectives: Research
Plan for the coming 24 months:
The
experimental plan is to use gene chip technology to identify gene(s) which
are
differentially expressed in AD. To come up with highly selective diagnostic
markers different experimental settings reflecting different pathology relevant
factors will be used and will allow a pathology driven selection process via
calculation of the affymetrix data set.
Resources
(for the first research year):
Melb.
Uni: Tissue supply, Pathological characterisation, collection of patient data,
animal model,
Validation
by immunohistochemistry (0.5)
[SAG:
RNA
extraction, real time PCR, Affymetrix analysis, Data calculation, Validation
by
immunohistochemistry
(FTE to be allocated after project proposal approval)
Experimental
settings overview: Human patient samples
·
Disease
vs non disease
·
Dementia
AD type vs dementia non AD type (Lewy Body Dementia, fronto temporal
dementia)
·
Brain
vessels isolation will allow analysis of gene expression in vessel wall
endothelial cells
·
MMSE
(mini-mental state examination) will be taken and used for data
calculation/correlation if available
·
Plaque
load, soluble and insoluble AB fraction and NFT (neurofibrillary tangles)
will
be determined and used for data
calculation/correlation
AD
tissue
will not only be compared versus control brain tissue but also versus tissue
from other neurological diseases especially other dementia's to allow
differential diagnosis. For each experimental settings 6 individual probes
will
be analysed, each on its own gene chip, to allow statistical evaluation of
the
results which is the absolute must in Affymetrix studies. In addition to RNA
extraction from total brain tissue also brain vessels will be isolated to allow
the analysis of differential expression in vessel endothelial cells. This is
of
especially importance because:
>
Amyloid
deposits in the vessel wall is one major pathological finding in AD
>
Significant differential expression in brain vessel endothelial cells are
probably not visible without prior purification of the vessel wall
because brain vessels represent less then 1 % of the total brain tissue and
thus
the signal will be dramatically diluted.
>
Cell
surface proteins on the brain vessel wall are very attractive targets for brain
imaging (no BBB)
Experimental
setting in detail:
Tissue
probes
Disease
versus non disease
Human
brain tissue will be taken from
>
non
demented aged control; n=6
>
AD
dementia n=18 (6 early, 6 medium and 6 late disease stages)
>
Non
AD
dementia n=9 (2 Lewy Body Demintia and 7 Fronto temporal
demintia)
14
Affected
area
Different
affected or non affected brain areas will be chosen:
>
frontal
cortex (FL)
>
temporal
cortex (TL)
Number
of
chips: (6+18+9) x 2 (FL/TL)= 66
Brain
parachym versus brain vessels
From
the
non demented aged controls and the AD cases also brain vessels will be purified
for RNA extraction
>
non
demented aged control; n=6
>
AD
dementia n=18 (6 early, 6 medium and 6 late disease
stages)
Number
of
chips: (6+18) x 2 (FL/TL)= 48
Total
number of chips: 48 +
66
= 114
For
the
human tissue the following assessments will be taken MMSE, Amyloid AB load
(Plaque, soluble and insoluble AB) and NFT pathology.
Data
calculation and selection criteria
Using
all
expression and patient information data, relevant AD imaging targets will be
selected based on the following selection criteria:
·
Relevance
to disease -> expressed only in AD
·
Relevance
to phenotypic pathology -> correlation with amyloid AB load and/or NFT
load
·
Relevance
to functional outcome -> correlation with MMSE (early targets may not fit
this criteria)
·
The
differential expressed molecule has to have the potential as diagnostic
marker
(e.g. receptor, cell surface protein)
Before
starting the Affymetrix Experiment the quality of RNA extracted from AD post
mortem tissue will be checked by PCR and a pilot Affymetrix experiment using
4
tissue probes.
Data
validation
Selected
targets will then be validated using real time PCR to get the real quantitative
picture. Thereafter the differential expression of relevant AD imaging targets
will be further validated on the protein and structural level by
immunohistochemistry (depending on availability of antibodies) and in situ
hybridisation using AD brain slices
Milestones
(times after month after start of project)
>
Human
Tissue sampling, collection of patient data and analysis of pathological
parameter 4 month
>
RNA
extraction and quality control 6 month
>
Affymetrix
12 month
>
Data
calculation 13 month
>
Validation
of differential expressed targets 24 month
Critical
Issues :
Get
access to human tissue with appropriate post mortem time and thus RNA quality.
Get
capacity to perform Affymetrix analysis at Schering.
Legal
issues regarding the use of homogenates from AD patients for drug
characterisation has to be clarified by Melb. University.
15
No
-Go Criteria :
Quality
of human tissue does not allow extraction of intact RNA after 9 month
Do
not
get necessary Affymetrix capacity to perform statistics.
Budget
Personnel:
▇▇▇▇▇ ▇▇▇▇▇▇▇ (Project Leader), [▇▇▇▇▇▇ Dyrks (Schering AG)]
16
Project
8. Research Project: 'Amyloid AB monoclonal antibodies as imaglnq
ligands'.
|
General
Aim:
|
-
To develop and test radiolabelled Amyloid specific antibodies as
AD
imaging agents.
|
|
-
In vivo imaging of AB in the brain for diagnosis and monitoring of
therapy.
|
Proposed
objectives: Research
Plan for the coming 15 months:
>
Test
already available monoclonal antibodies with regard to Amyloid AB
pathology.
> Set
up
strategies regarding PK questions: Antibody fragments, BBB-carrier
systems
>
Label
antibody for proof of concept study in tg mice(biotin or fluorescent label
may
be chosen if
Antibody
has to be send over to Melb. Uni.)
>
Make
proof of concept study with one selected already existing high affinity Antibody
(anti AB42) using TG mice.
Resources
(for the first research year):
Melb.
University: supply of human AD tissue, tissue of non-AD amyloidosis, reference
tissue from healthy persons, for immunohistochemistry, production of paraffin
sections of human AD tissue, supply of datab characterizing the pathologic
material. SAG:
Generation and labeling of antibodies, pathological characterization of
monoclonal antibodies, antibody modification with regard to penetration of
BBB.
Organizations
aspects: Input from Melb.
University
Delivery
of human patient tissue for Immunohistochemistry.
First
delivery: June 2004
>
Tissue
selection:
·
early,
medium and late AD
·
non-AD
amyloidosis (Lewy Body dementia, fronto temporal dementia)
·
healthy
Milestones
(month after project start)
>
PK
strategy: 3 month
>
Antibody
label: 6 month
>
Proof
of
concept with whole Antibody 9 month
>
Characterisation
of already existing monoclonal Ab: 9 month
>
Development
of new strategies: other Ab, Ab-fragment/ modified Ab 15 month
>
Label
and
proof of concept study with next generation antibodies (fragments/modified)
24
month
Critical
Issues :
Legal
issues regarding the use of brain tissue from AD patients for drug
characterisation has to be clarified by Melbourne University (July
04)
No
-Go
Criteria :
No
proof
of concept with existing Ab after 12 months.
Budget:
Personnel:
▇▇▇▇▇▇ ▇▇▇▇▇▇▇, [▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇ (Schering AG)]
17
Project
9. Research Project: 'AB binding proteins'
General
Aim:
To
identify proteins which bind to AB as novel AB imaging targets.
Proposed
Objectives: [Research
Plan for the coming 6 months):
The
experimental plan is to identify specific brain proteins that bind to AB and
not
to control peptides and fulfil the criteria as drugable targets.
Resources
(for the first research year):
Melb.
Uni: Affinity isolation and identification of AB binding proteins.
SAG:
Assistance in
proteomics
for identification of AB binding proteins.
1.
Isolation
of AB-complexes from human brain.
1.1
AB-affinity
capture of AB-complexes from human brain: AB will be coupled to Tosyl-activated
magnetic beads and used to affinity purify AB binding proteins from brain
homogenates. Bound proteins are separated by 2D-electrophoresis Control peptides
will be coupled to the Tosyl-activated magnetic beads to determine specific
AB
binding proteins. Control peptides will include AB scrambled, AB-reverse,
a-synuclein and/or Prion peptide 106-126. If specific spots (unique to the
AB)
are obtained they will be identified by mass spectrometry.
1.2
AB-TAP
protein affinity purification: An AB tandem affinity purification (TAP) tag
will
be used to affinity purify AB binding proteins from brain homogenates. A control
TAP fusion protein will be used to determine specific AB binding proteins.
The
TAP controls will be TAP alone and a TAP-a-synuclein fusion protein. If specific
spots (unique to the AB-TAP) are obtained they will be identified by mass
spectrometry.
2.
Verification
of AB binding proteins.
2.1 In
vitro
binding assay: In vitro binding of different AB peptides to recombinantly
expressed candidate binding protein as measured by gel filtration or
co-immunoprecipitation or pull-down assay or BiaCore.
3.
Validation
of AB binding proteins
Immunohistochemistry
to be performed against selected targets at the protein level by
on AD
and non-AD control brain slices to determine expression levels in AD vs
non-AD
controls.
Data
calculation and selection criteria
Relevant
AD imaging targets will be selected based on the following selection
criteria:
·
Relevance
to disease -> increased expression in AD
·
Relevance
to affected area -> expressed only in affected brain area
·
Relevance
to phenotypic pathology -> correlation with amyloid and/or NFT
load
·
The
AB
binding molecule has to have the potential as diagnostic marker (e.g.
receptor,
cell surface protein)
Data
validation
Selected
targets will then be validated as a relevant AD imaging target at the protein
level by immunohistochemistry using AD brain slices (depending on availability
of antibodies).
Organisational
aspects
·
Study
to
be performed by Melb. Uni.
·
Schering
AG to assist if required with protein identification
Milestones
(times after month after start of project)
1°
milestone: Identification of specific AB-binding proteins which are unique
in
either the AB-affinity assay or the AB-TAP assay as compared to the control
peptides on 2D-gels. Unique spots to be identified
by mass spectrometry. (11/2004)).
2°milestone:
Verification (in vitro) of binding partner (12 month)
3°
milestone: Verification that binding protein binds to AB in tissue (24
month)
Critical
Issues :
Confirming
that proposed AB binding proteins are physiologically/pathologically
relevant.
No
-Go
Criteria :
No
identified and verified (in vitro) binding partner by 11/2004
Verification
that the AB binding protein does not bind to AB in tissue (24
month)
Budget:
Personnel:
▇▇▇▇▇▇▇ ▇▇▇▇▇▇, [▇▇▇▇▇▇ Dyrks (Schering AG)]
Project
10. Research
Project: ‘Amyloid imaging with PBT-1'
General
Aim: - To test radiolabelled PBT1 as AD imaging agents.
- In vivo imaging of AB in the brain for diagnosis and monitoring of
therapy.
Proposed
Objectives:
Research
Plan for the coming 18 months:
>
The
experimental plan is to fully characterize radiolabeled PBT-1 (Clioquinol)
binding to A[]. 4 month
>
Radiotracers
will be screened as A[] binding tracers through in vitro assays to determinate
binding parameters (Kd, Bmax)
of high specific activity 125l-PBT1
to
synthetic A[] amyloid aggregates
>
Biodistribution
and subsequent ex vivo autoradiographic studies in tg and non tg mice,
with and without competitor
compounds
(DTPA, Congo red, ThT).
>
The
competitor compound ThT will be used to evaluate the tg animal models.
>
In
vivo
human SPECT studies with 125l-PBT1
>
Based
on
full characterization of PBT1.
Resources
(for the first research year):
Melb.
Uni: Proof of concept study with A[] fibers, tg mice, and human SPECT
studies.
1. In
vitro
binding assays
1.1
Synthetic A[] amyloid aggregates will be added to a mixture containing
0.01-5
pM
of
125l-PBT1
(2000 Ci/mmol) in buffer Tris-HCI (pH 7.35; final volume of 1
ml).
The
final concentration of EtOH will be 10%. Nonspecific binding will be defined
in
the presence of 2 uM PBT1.
1.2
Selectivity studies will be determined in the presence of different competitors
(ThT, C red, DTPA, Zn(ll) and glycine). Ki value will be estimated for each
competitor. The competitor concentration range will be ± 3 order of magnitude
the concentration of 125I-PBT1.
2. Ex
vivo
biodistribution studies
2.1.
Tg2576 (n=3) and non-Tg (n=3) mice will be injected i.v. with 125l-PBT1
(1
mCl). The animals will be sacrificed at 5, 15, 30, 60 and 120 min, and the
organs will be dissected, weighted and each organ counted. Blood and urine
samples will be also collected. The percentage of radioactivity in each organ
(%ID/g) will be calculated.
2.2
To estimate the specificity and saturability of the brain-uptake of 125l-PBT1,
mice will be administered i.p. with cold PBT1, C red, DTPA, glycine,
Thioflavin-t 30-60 min previous to 125l-PBT1
i,v. injection.
3. Autoradiographic
studies
3.1
In vitro autoradiography.
3.1.1.
Unfixed brain coronal slices of AD and AC brain tissue, as well as Tg2576 (15-20
mm) and non-Tg mice (15-20 mm), will be adsorbed to gelatin-coated coverslip.
The samples will be incubated with 125l-PBT1
(100,000 cpm) in buffer Tris-HCI (pH 7.35) containing 10% EtOH for 1 h at 20°C.
Samples will be submitted to 3 washing steps with the same buffer. The samples
will be dried to 20° C for 2h. The regional distribution of 125l-PBT1
will be analysed qualitatively by autoradiography using
autoradiographic
films. Quantification of 125l-PBT1
positive binding will be performed using radioactive strips
standards.
3.1.2.
Competition studies will be performed in the presence of cold PBT1, DTPA, ThT
glycine and Congo Red (1,000 times excess).
3.1.3.
To
analyse co-localization of 125l-PBT1
binding amyloid deposits, adjacent slices will be stained for amyloid with
either Congo red, Thioflavin T or A[] Immunohistochemistry.
3.2
Ex vivo autoradiography
At
the
best time from TAC kinetics (5-15 mpi), brain coronal slides from Tg2576 and
non-Tg mice i.v. injected with 125I-PBT1
will be exposed to autoradiography film. Experiments will be performed as
above.
4. Radiometal
binding.
4.1.
Tg2576 (n=3) and non-Tg (n=3) mice will be injected i.v. with 64Cu.
The animals
will be sacrificed at 5 min and the organs will be
dissected, weighted and
each
organ counted. Blood and urine samples will be also collected. The
percentage of radioactivity in
each organ (%lD/g) will be calculate
4.2.
Tg2576 (n=3) and non-Tg (n=3) mice will be injected i.v. with 64Cu-PBT1.
The
animals will be sacrificed at 5 min and the organs
will be dissected, weighted
and each organ counted. Blood and urine samples will be also collected.
The percentage of
radioactivity in each organ (%ID/g) will be calculated.
4.3. Comparison of results Prom. Radiometal binding with and without
chelator transporter.
5.
SPECT
studies
Ten
AD
patients will be recruited to undergo 123l-PBT1
SPECT brain imaging. The scans obtained will be compared with those of ten
healthy, age-matched volunteers.
The
scans
will undergo blinded analysis by two experienced nuclear medicine specialists
who will assign each subject's scan to either a "diseased" or "normal" condition
on the basis of visual analysis, normalized brain radioactivity (SUV) and
kinetic analysis of cortical and subcortical regions of interest (ROI).
Compartmental and graphical analysis using the cerebellum as reference region
will be used.
Formal
whole-body radiation dosimetry calculations for 123l-PBT1
will be performed on three of the control subjects.
Organizations
aspects: Input from Melb. Uni.
Studies
to be performed by Melb. Uni.
Characterization
of 123/125l-PBT1.
Proof of concept study using tg mice. Biodistribution, In vivo Autoradiography
/
immunohistochemistry
Proof
of
concept study in humans SPECT studies with 123I-PBT1
Schering
AG to assist if required with radiolabelling of BTA-1.
Milestones
(month after project start)
>
>
In
vitro
characterization PBT1 (completed):
>
Ex
vivo
characterization PBT1 (ongoing): 1 month
>
Ex
vivo
autoradiographic studies PBT1 (ongoing): 2 month
>
Ex
vivo
characterization 64Cu
and
64Cu-PBT1:
3 month
>
Ex
vivo
autoradiographic studies BTA-1: 6 month
>
Proof
of
concept with human SPECT studies (n=20) 12 month
No
-Go
Criteria :
If
IP
situation regarding PBT1 and analogues thereof can not be solved after 4 month
preclinical work will be closed. 08/2004
Failure
of proof of concept study with human SPECT after 12 month.
(12/2004)
Current
Project Status:
>
In
vitro
characterization PBT1 (ongoing)
>
Ex
vivo
characterization PBT1 (ongoing)
>
Autoradiographic
studies PBT1 (ongoing)
>
Proof
of
concept with human SPECT studies (n=20)
Lead
criteria:
1. Proven
pharmacological efficacy in secondary in vitro (micro autoradiography
study
using AD brain slices; potency 1-100nM)
2. Proven
accumulation in AD plaques as determined by ex-vivo autoradiography after
i.v. injection of I-125-PBT-1 into tg mice
3. Proven
suitable pharmacokinetic properties (stability, plasma/brain level)
4. Compatible
with PET/SPECT radiochemistry
5. Clear
patent strategy
6. Primary
structure binding relationship data
Budget:
Personnel:
▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇, ▇. ▇▇▇▇▇▇, (Project leaders) [▇▇▇▇▇▇▇▇ ▇▇▇▇▇▇ (Schering
AG))
Project
11. Research Project: 'AP binding metallocomolexes'
General
Aim:
To
test
radiolabelled metallocomplexes as AD imaging agents. In vivo imaging of AB
in
the brain for diagnosis and monitoring of therapy.
Proposed
Objectives:
[Research
Plan for the coming 24 months]:
The
experimental plan is to identify, synthesize and characterize metallocomplexes
that bind to A[] in vivo.
Initial
proof of concept (4 months):
>
Synthesize a Pt(II) complex of a 1,10-phenanthroline derivative (Rphen) ligand
suitable for radioiodination (Melb. Uni)
>
Screen the cold metallocomplex in-vitro for binding affinity to ABfibers (Melb.
Uni)
>
Derive procedures to radiolabel the Pt(Rphen)Cl2
complex
with l-125 (SAG)
>
Perform in-vitro autoradiography with 1-125-Pt(Rphen)Cl2
on AD
slices (Melb. Uni)
Resources
(for the first research year):
Melb.
Uni.: Chemistry and intial screening with AB fibers,
SAG:
Radiolabelling of selected metallocomplexes, toxicity screening
General
research plan (24 months):
>
Candidate metallocomplexes will be synthesized and characterized (MS, NMR,
HPLC)
>
Candidate metallocomplexes will be screened as AB binding tracers through in
vitro assays with AB fibers
>
Selected metallocomplexes will be assessed for bioavailability: a) in vitro
uptake in CaCo 2 cells; b) in vivo brain uptake in
non-transgenic
mice as determined by ICP-MS
> Selected
metallodrugs will be assessed for radiolabeling potential (SAG)
>
Radiolabelled metallocomplexes will be evaluated to ensure that they display
similar
binding profile (affinity, selectivity) as
the cold
complexes using the in vitro assays
with AB fibers
>
Ex
vivo
tissue binding, binding assays, dialysis studies, and autoradiography studies
in
tg and non tg mice, with and
without competitors (DTPA, Congo red, ThT), and human AD/AC brain
slices.
>
Toxicity
screen (SAG)
Organizations
aspects: Input from Melb. Uni.
Synthetic
chemistry and characterization of AB binding metallocomplexes. Bioavailability
studies. Proof of concept study using AB fibers
Milestones
(month after project start)
>
Proof
of
concept in vitro with ▇-▇▇▇ ▇▇(▇▇▇▇▇)▇▇▇:
4
months
>
Identification,
synthesis, and characterization of 30 compounds: 12 months
>
Bioavailability
evaluation (SAR): 5-24 months
>
In
vitro
characterization (10-20/year): 7-24 months
>
Ex
vivo
characterization (6-10/year): 9-24 months
>
Radiolabeling:
7-24 months
>
Autoradiographic
studies: 15-24 months
>
Toxicity
screening: 9-24 months
>
Proof
of
concept with human PET studies: > 24 months
No
-Go
Criteria :
Failure
of ▇-▇▇▇ ▇▇(▇▇▇▇▇)▇▇▇
proof of
concept in-vitro after 4 months
Failure
to show binding of new metallocompiex to amyloid pathology in tg mice after
20
months
Lead
criteria:
1
Proven pharmacological efficacy in secondary in vitro (micro autoradiography
study using AD brain slices; potency 1-100nM)
2
Proven suitable pharmacokinitic properties (stability, plasma/brain
level)
3
Compatible with PET/SPECT radiochemistry
4
Clear patent strategy
5
Primary structure binding relationship data
Budget:
Personnel:
▇▇▇▇▇ Bamham, ▇▇▇▇ ▇▇▇ (Schering AG)