PKTX AAGP Presentation_v13.pptm

AAGPsTM –
Anti-Aging Glyco Peptides
Enhancing Cell, Tissue and Organ Integrity
Molecular and biological attributes
of lead AAGP molecule
1
Acknowledgements
This presentation was prepared by Dr. Samer Hussein and based on the results of
testing conducted on the family of AAGPs created by Dr. Geraldine DeliencourtGodefroy.
Dr. Hussein received his Ph.D. from McGill University in Montreal. His expertise is in the area of
Molecular and Cellular biology with an emphasis on the signals required to direct early stem
cells to differentiate into neural cells. His main focus is on reprogramming skin fibroblasts
into induced pluripotent stem cells (iPS cells). He works at Samuel Lunenfeld Research
Institute at Mount Sinai Hospital in Toronto as a Post-doctoral Fellow in the Nagy Lab.
Dr. Deliencourt-Godefroy received her Ph.D. from the University of Rouen, France. She is the
Founder of TF Chem, a drug discovery company which uses the fluorine atom properties to
develop new glycomimetic compounds: the GlycoMim® technology. TF Chem is a leader in
the area of fluorinated glycosides and their applications as new, more stable and potent and
safer therapeutics.
Dr. Deliencourt-Godefroy has received a number of awards: Vermeil Medal of Innovation
and Research, Francinov (Versailles); Award from the “Contest of Innovative and
Technological Companies” of the French Ministry of Research and Technology; Award from
the “Tremplin Enterprise” competition from the French Senate.
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Agenda
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! 
! 
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ProtoKinetix and the development of
AAGPTM
Molecular Structure
Testing and Evaluation of AAGPTM effects on
cellular viability
Functional Studies using AAGPTM
3
A look at ProtoKinetix Inc.
! 
Biotechnology Company www.protokinetix.com
! 
Research Focus
! 
! 
Development and testing of a family of patented Anti-Aging
Glycopeptides (AAGPs) which enhance cell and tissue survival
against environmental stresses
Commercial Targets
! 
! 
! 
! 
! 
! 
Cell and tissue protection
Stem cell preservation
Cell therapy and regenerative medicine
Organ Transplants
Blood and blood derivative preservation
Anti-aging for skin care/cosmetics
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Structure of Lead AAGP™ Molecule
! 
! 
Several molecules have been studied and
tested
Example of Lead Structure
! 
! 
! 
! 
! 
Ala-Ala-Lys-F-C-F-Galactose
Formula – C20H35CF2N4O11
Molecular Mass– 580.96 Daltons
Highly water soluble– 650 grams/liter
Peptides in the structure suggests lipid solubility
5
History behind AAGPTM
! 
AAGPs™ were derived from work on AntiFreeze Glycoproteins (AFGPs)
! 
! 
! 
AFGPs are natural proteins produced in living
organisms such as fish, plants, and insects
AFGPs enable survival under freezing temperatures
They protect the cellular membrane from freezing
damage
6
History behind AAGPTM (cont.)
! 
Research on AFGPs showed great promise.
! 
Some research areas
! 
! 
! 
! 
Large scale applications of AFGPs are impractical
! 
! 
! 
! 
Preservation of biological materials
Food preservative
Organ transplantation
Costly extraction from unsustainable sources
Very large molecule in its native forms (2,600 – 33,000 Daltons)
Cannot be purified beyond 85%
Solution: Development of AFGP synthetic
analogs
7
History behind AAGPTM (cont.)
! 
AFGP analogs
! 
! 
! 
! 
Unstable
Very susceptible to
degradation
Limited bioactivity
Large complex molecules
(2,600 – 33,000 Daltons)
! 
AAGP Technology
! 
Stable
Not susceptible to degradation
! 
Highly bioactive
! 
! 
Small Molecule (580.96
Daltons)
Summary: New proprietary stable analogs found
to have cell survival and anti-aging properties
8
Agenda
! 
! 
! 
! 
ProtoKinetix and the development of AAGPTM
Molecular structure
Testing and Evaluation of AAGPTM effects on
cellular viability
Functional Studies using AAGPTM
9
Stability Evaluation of AAGPTM
! 
AAGPTM remains highly stable under the
following conditions:
! 
! 
pH range: ~2 (highly acidic) to ~14 (highly alkaline)
No enzymatic degradation from:
! 
! 
! 
Proteases (peptide cleaving enzymes)
Galactosidases (sugar degrading enzymes)
Temperature
! 
Range: normal body temperature (37˚C) to cryotemperatures(-197˚C)
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Toxicity Evaluation of AAGPTM
! 
! 
Non-toxic to cells in vitro (up to 5 mg/
ml tested)
Transplanted AAGP treated islet cells
in vivo (mice) showed no toxicity
11
Bioactivity Testing of AAGPTM
! 
AAGPTM demonstrated an unprecedented spectrum of
applicability under harsh cellular stresses
! 
Cellular Stress Tests:
!  Nutrient Deprivation
! 
! 
! 
! 
Temperature and Cryopreservation
Oxidative Stress from Hydrogen Peroxide (H2O2)
UV Radiation
Inflammation
12
Nutrient Deprivation
AAGP maintains viability of adult skin fibroblasts during nutrient
depletion and nutrient deprivation
Control
AAGP
160%
Cell Viability (%)
• 
140%
120%
100%
80%
60%
40%
20%
0%
d0
d1
d2
d3
Low Serum Conditions
(minimal nutrients)
d4
d0
d1
d2
d3
d4
Starvation Conditions
(no nutrients)
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Bioactivity Testing of AAGPTM
! 
Cellular Stress Tests:
! 
Nutrient Deprivation
! 
Temperature and Cryopreservation
! 
! 
! 
UV radiation
Oxidation stress by Hydrogen Peroxide (H2O2)
Inflammation
14
Protection from Moderate to Cold
Temperatures
• 
AAGP maintains 100% viability of adult skin fibroblasts under
moderate and cold temperatures
Control
Survival Rate (%)
140%
120%
*
***
15°C
AAGP
***
***
***
100%
80%
60%
40%
20%
0%
Control
140%
Survival Rate (%)
3°C
**
120%
100%
AAGP
***
***
***
80%
60%
40%
20%
0%
d1
d2
d3
d4
d5
d1
d2
d3
d4
d5
*, p< 0.05; **, p< 0.01; ***, p< 0.001
Viability Test: Trypan Blue Exclusion
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AAGP maintains stem cell
survival at 3°C
24 hrs
AAGP enhances survival of
mEScells at low temperatures
• 
Cells were placed at 3°C for either 24 or 48
hrs., then at 37°C for an additional 24hrs prior
to cell counting, as a measure of colony
formation capacity post 3°C.
• 
Media: ES qualified media
• 
Viability assay: Trypan Blue Exclusion
Control
AAGP
300%
200%
100%
0%
10,000
48 hrs
Survival Rate (%)
• 
Survival Rate (%)
400%
Mouse ES cells at 3°C
40%
50,000
250,000
Initial Cell Density (Cells/mL)
Control
AAGP
30%
20%
10%
0%
10,000
50,000
250,000
Initial Cell Density (Cells/mL)
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Human ES Cell Cryopreservation
Human ES Cells Cryopreservation
AAGP enhances cryo-preservation
of Human Embryonic Stem Cells
regardless of their density
• 
AAGP’s effect is concentration
dependent
1 mg/ml AAGP
5 mg/ml AAGP
100%
Cell Viability (%)
• 
120%
Control
2.0 mg/ml AAGP
76%
71%
80%
87%
74%
62% 58%
60%
40%
49%
40%
20%
0%
2.2 Million cells/mL
3.2 Million cells/mL
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Effect of AAGP on Cord Blood
Cryopreservation
AAGP (1mg/ml)
• 
AAGP treatment doubles the
CD34+ stem cell count within Cord
Blood samples post
cryopreservation
• 
• 
AAGP’s effect is concentration
dependent
Optimal Concentration: 4.0 mg/
ml
% Change
(AAGP relative to Dextran Control)
250%
AAGP (2mg/ml)
AAGP (4mg/ml)
200%
150%
100%
50%
0%
Total Nucleated Progenitor
Colony
Cells
(CD34+) cells Forming Units
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AAGP during Cryo-preservation
*, p < 0.05; **, p < 0.01; ***, p < 0.001
AAGP enhances cryopreservation of mammalian
cell lines with DMSO
•  Effect is consistent across all
cell lines tested
• 
AAGP concentration: 2mg/ml
• 
10% DMSO freezing media
AAGP
120%
*
100%
Cell Viability (%)
• 
Control
*
80%
60%
***
40%
53%
93%
68% 79%
78%
93%
78%
20%
0%
24%
mES
Skin
Fibroblasts
HEK293
Hela
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Bioactivity Testing of AAGPTM
! 
Cellular Stress Tests
! 
! 
Nutrient Deprivation
Temperature and Cryopreservation
! 
UV radiation
Oxidative Stress from Hydrogen Peroxide
(H2O2)
! 
Inflammation
! 
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AAGP protects against UVA
Skin Fibroblasts
105%
**, p< 0.01
100%
95%
Cell Viability (%)
Control
87%
85%
83%
88%
83%
AAGP-1
10 J/cm2
75%
75%
**70%
10 J/cm2 +
AAGP-1
65%
59%
55%
0h
"-24h"
1h
24h
21
Protection against UVC
AAGP added immediately BEFORE irradiation
Control
AAGP
180%
140%
120%
123%
100%
80%
51%
UVC: 400J/m2
29%
0%
100%
100%
104%
117%
80%
64%
60%
40%
20%
40%
29%
UVC: 400J/m2
0%
0h
• 
p< 0.05
64%
60%
20%
*
120%
117%
100%
AAGP
143%
Viable Cells (%)
Viable Cells (%)
150%
Control
140%
** p < 0.01
160%
40%
AAGP added immediately AFTER irradiation
24h
48h
96h
AAGP protects adult skin fibroblasts • 
against UVC when added before
and after irradiation
0h
24h
48h
96h
AAGP treatment post irradiation
suggests a repair effect
22
Protection against Oxidative Stress
1mM H2O2 at 37°C
Effects of AAGP on H2O2 treated
Neonatal Skin Fibroblasts at 37°C
10hrs
Control
AAGP
AAGP
Cell Viability (%)
Control
120%
100%
80%
60%
40%
20%
0%
0h
2h
10h
RED: Dead cells/Eth-Br derivative
GREEN: Live cells/Calcein
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Bioactivity Testing of AAGPTM
! 
Cellular Stress Tests
! 
! 
! 
! 
! 
Nutrient Deprivation
Temperature and Cryopreservation
UV radiation
Oxidation stress by Hydrogen Peroxide (H2O2)
Inflammation
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Anti-Inflammatory Effect
• 
AAGP protects against in vitro
IL 1-! induced inflammatory
response (Cox2 expression)
p < 0.05
*
• 
Cell line: Hela cells
• 
Quantification from RT-PCR
analysis
*
25
Agenda
! 
! 
! 
ProtoKinetix and the development of
AAGPTM
Testing and Evaluation of AAGPTM effects
on cellular viability
Functional Studies using AAGPTM
26
Functional assays on Platelets
and Islets Cells
• 
• 
Platelets
• 
Preservation and Aggregation
Islet cells
• 
Diabetes and Islet cell transplantation
27
Preservation of Platelets
Untreated
22°C
15°C
1 mg/ml AAGP
4 mg/ml AAGP
• 
Untreated platelets, no AAGP added, show
strong and consistent aggregation
• 
In the presence of AAGP, platelet activation
is prevented (i.e. low aggregation count)
4°C
28
AAGP Treated Islet Cell
Transplantation
• 
AAGP increases viable mouse islet
cells within the transplant
• 
• 
Model: Streptozocin-induced
diabetic mouse
• 
Method: Islet cell transplantation
from AAGP treated and untreated
islets
• 
Measure of response: Blood
Glucose Level
Results by Dr. Ray V. Rajotte,
University of Alberta
*
*
untreated
AAGP
*
*
*
*
Islets treated with AAGP stabilized
blood glucose level in diabetic mice
much more efficiently than controls
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AAGP™ Presentation Summary
• 
Lead AAGP molecule is stable and bioactive
• 
It has protective properties for cells and platelets under multiple
stress conditions
• 
It enhances their viability and preserves their function, both in vitro
and in vivo
• 
These protective properties may be extended to tissues and organs
in general
• 
AAGP™technology has potential applications in a variety of
therapeutic areas involving cell, tissue and organ preservation and
possibly preserving body tissue integrity
30