Protokinetix, Inc. · OTC:PKTX
Protokinetix — Technical Presentation (March 2010)
30 slides from Protokinetix's technical presentation, March 2010
Slide 1: AAGPs – Anti-Aging Glyco Peptides
AAGPsTM – Anti-Aging Glyco Peptides Enhancing Cell, Tissue and Organ Integrity Molecular and biological attributes of lead AAGP molecule 1
Slide 2: Acknowledgements
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 Deliencourt- Godefroy. Dr. Hussein received his Ph.D. from McGill University in Montreal. His expertise is in the area of Molecular and C
Slide 3: Agenda
Agenda ! ProtoKinetix and the development of AAGPTM ! Molecular Structure ! Testing and Evaluation of AAGPTM effects on cellular viability ! Functional Studies using AAGPTM 3
Slide 4: A look at ProtoKinetix Inc.
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
Slide 5: Structure of Lead AAGP Molecule
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
Slide 6: History behind AAGP
History behind AAGPTM ! AAGPs™ were derived from work on Anti- Freeze 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
Slide 7: History behind AAGP (cont.)
History behind AAGPTM (cont.) ! Research on AFGPs showed great promise. ! Some research areas ! Preservation of biological materials ! Food preservative ! Organ transplantation ! Large scale applications of AFGPs are impractical ! Costly extraction from unsustainable sources ! Very large molecule in
Slide 8: History behind AAGP (cont.)
History behind AAGPTM (cont.) ! AFGP analogs ! AAGP Technology ! Unstable ! Stable ! Very susceptible to ! Not susceptible to degradation degradation ! Highly bioactive ! Limited bioactivity ! Large complex molecules ! Small Molecule (580.96 (2,600 – 33,000 Daltons) Daltons) Summary: New proprietary
Slide 9: Agenda
Agenda ! ProtoKinetix and the development of AAGPTM ! Molecular structure ! Testing and Evaluation of AAGPTM effects on cellular viability ! Functional Studies using AAGPTM 9
Slide 10: Stability Evaluation of AAGP
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
Slide 11: Toxicity Evaluation of AAGP
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
Slide 12: Bioactivity Testing of AAGP
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
Slide 13: Nutrient Deprivation
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 d4 d0 d1 d2 d3 d4 Low Serum Conditions Starvation Conditions (minimal nutrients) (no nutrien
Slide 14: Bioactivity Testing of AAGP
Bioactivity Testing of AAGPTM ! Cellular Stress Tests: ! Nutrient Deprivation ! Temperature and Cryopreservation ! UV radiation ! Oxidation stress by Hydrogen Peroxide (H2O2) ! Inflammation 14
Slide 15: Protection from Moderate to Cold Temperatures
Protection from Moderate to Cold Temperatures • AAGP maintains 100% viability of adult skin fibroblasts under moderate and cold temperatures 3°C Control AAGP 15°C Control AAGP 140% 140% * *** *** *** *** 120% ** *** 120% *** *** Survival Rate (%) Survival Rate (%) 100% 100% 80% 80% 60% 60% 40% 40% 2
Slide 16: AAGP maintains stem cell survival at 3°C
AAGP maintains stem cell survival at 3°C Mouse ES cells at 3°C 24 hrs Control AAGP 400% Survival Rate (%) • AAGP enhances survival of 300% mEScells at low temperatures 200% 100% • Cells were placed at 3°C for either 24 or 48 0% hrs., then at 37°C for an additional 24hrs prior 10,000 50,000 250,000 t
Slide 17: Human ES Cell Cryopreservation
Human ES Cell Cryopreservation Human ES Cells Cryopreservation Control 1 mg/ml AAGP 2.0 mg/ml AAGP 5 mg/ml AAGP 120% • AAGP enhances cryo-preservation of Human Embryonic Stem Cells 100% 87% regardless of their density 76% 71% 74% Cell Viability (%) 80% 62% 58% • AAGP’s effect is concentration 60% 49
Slide 18: Effect of AAGP on Cord Blood Cryopreservation
Effect of AAGP on Cord Blood Cryopreservation AAGP (1mg/ml) 250% AAGP (2mg/ml) (AAGP relative to Dextran Control) • AAGP treatment doubles the AAGP (4mg/ml) CD34+ stem cell count within Cord 200% Blood samples post cryopreservation % Change 150% • AAGP’s effect is concentration 100% dependent 50% •
Slide 19: AAGP during Cryo-preservation
AAGP during Cryo-preservation *, p < 0.05; **, p < 0.01; ***, p < 0.001 Control AAGP • AAGP enhances cryo- 120% preservation of mammalian 100% * cell lines with DMSO * Cell Viability (%) 80% • Effect is consistent across all cell lines tested 60% *** 93% • AAGP concentration: 2mg/ml 40% 78% 93% 78%
Slide 20: Bioactivity Testing of AAGP
Bioactivity Testing of AAGPTM ! Cellular Stress Tests ! Nutrient Deprivation ! Temperature and Cryopreservation ! UV radiation ! Oxidative Stress from Hydrogen Peroxide (H2O2) ! Inflammation 20
Slide 21: AAGP protects against UVA
AAGP protects against UVA Skin Fibroblasts 105% **, p< 0.01 100% 95% Control Cell Viability (%) 88% 87% 85% AAGP-1 83% 83% 10 J/cm2 75% 75% 10 J/cm2 + **70% AAGP-1 65% 59% 55% 0h "-24h" 1h 24h 21
Slide 22: Protection against UVC
Protection against UVC AAGP added immediately BEFORE irradiation AAGP added immediately AFTER irradiation Control AAGP Control AAGP 180% 140% 160% ** p < 0.01 123% * p< 0.05 150% 120% 143% Viable Cells (%) 140% 104% Viable Cells (%) 100% 100% 120% 117% 117% 100% 100% 80% 80% 64% 60% 60% 64% 51% 40%
Slide 23: Protection against Oxidative Stress
Protection against Oxidative Stress 1mM H2O2 at 37°C Effects of AAGP on H2O2 treated 10hrs Neonatal Skin Fibroblasts at 37°C Control AAGP 120% Control 100% Cell Viability (%) 80% 60% 40% AAGP 20% 0% 0h 2h 10h RED: Dead cells/Eth-Br derivative GREEN: Live cells/Calcein 23
Slide 24: Bioactivity Testing of AAGP
Bioactivity Testing of AAGPTM ! Cellular Stress Tests ! Nutrient Deprivation ! Temperature and Cryopreservation ! UV radiation ! Oxidation stress by Hydrogen Peroxide (H2O2) ! Inflammation 24
Slide 25: Anti-Inflammatory Effect
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
Slide 26: Agenda
Agenda ! ProtoKinetix and the development of AAGPTM ! Testing and Evaluation of AAGPTM effects on cellular viability ! Functional Studies using AAGPTM 26
Slide 27: Functional assays on Platelets and Islets Cells
Functional assays on Platelets and Islets Cells • Platelets • Preservation and Aggregation • Islet cells • Diabetes and Islet cell transplantation 27
Slide 28: Preservation of Platelets
Preservation of Platelets Untreated 1 mg/ml AAGP 4 mg/ml AAGP 22°C • 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 15°C 28
Slide 29: AAGP Treated Islet Cell Transplantation
AAGP Treated Islet Cell Transplantation Results by Dr. Ray V. Rajotte, University of Alberta • AAGP increases viable mouse islet cells within the transplant * • Model: Streptozocin-induced untreated * * diabetic mouse * AAGP * * • Method: Islet cell transplantation from AAGP treated and untreated is
Slide 30: AAGP Presentation Summary
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 tissu