![Overview ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)
Empfohlen
Empfohlen
Weitere ähnliche Inhalte
Ähnlich wie Pharmacokinetic characterization of angiotensin IV analogs with therapeutic potential for cancer and dementia.
Ähnlich wie Pharmacokinetic characterization of angiotensin IV analogs with therapeutic potential for cancer and dementia. (20)
Pharmacokinetic characterization of angiotensin IV analogs with therapeutic potential for cancer and dementia.
- 1. THESIS PROPOSAL DEFENSE ALENE MCCOY THESIS MENTOR: JOSEPH HARDING APRIL 30, 2009 “ Pharmacokinetic characterization of angiotensin IV analogs with therapeutic potential for cancer and dementia.”
- 4. Is there a molecular target for both, age related dementia and cancer?
- 5. Angiotensins Binds to AT 1 & AT 2 : Blood pressure Vasoconstriction Binds to AT 4 : Cognition Memory
- 7. Angiotensin IV–AT 4 Activity in Cancer
- 9. Effect of AT 4 Antagonists on Human Endothelial Cell Growth Control 10 -6 10 -8 10 -10 10 -12 0 50 100 150 PNB-0718 (M) Absorbance as Percent of Control HUVEC Cells Cell number estimated by MTT Mean +/- SEM, n=8
- 10. Effect of AT 4 Antagonists on Human Endothelial Cell Migration HUVEC Cells Cell Number Estimated by Visual Count Mean +/- SEM, n=8 control -8 -10 -12 -14 0 25 50 75 100 control -8 -10 -12 -14 Concentration (-log M) Average of Five Counts at High Power
- 11. Inhibition of Angiogenesis in the Mouse Aortic Ring Assay Control Treated Control PNB-0718 0.1 nM Angiogenesis (area) n=6 n=8 +/- SEM * p=0.012 *
- 12. Angiogenesis in Cancer Image obtained from roswellpark.org http://www.roswellpark.org/files/1_2_1/cancer_101/Lesson4/Lesson_4.pdf
- 13. Reduction of tumor growth. PNB-0718 Inhibits the Growth of Established Melanoma Tumors Time (Days) Tumor Volume (mm 3 ) Mean +/- SEM N=8 12 13 14 15 16 17 0 1000 2000 3000 Control PNB-0718 (2mg/kg/day) PNB-0718 (0.2 g/kg/day) = Injection
- 17. Development of AT 4 Ligand Molecules Name Structure Activity Angiotensin IV Val-Tyr-Ile-His-Pro-Phe + PNB-0719 Nle -Tyr-Ile-His-Pro-Phe ++ PNB-0718 “ Norleual” Nle -Tyr- Leu- -His-Pro-Phe - - PNB-0405 d-Nle -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH - PNB-0408 Hexanoic acid -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH ++
- 18. Development of AT 4 Ligand Molecules Name Structure Activity Angiotensin IV Val-Tyr-Ile-His-Pro-Phe + PNB-0719 Nle -Tyr-Ile-His-Pro-Phe ++ PNB-0718 “ Norleual” Nle -Tyr- Leu- -His-Pro-Phe - - PNB-0405 d-Nle -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH - PNB-0408 Hexanoic acid -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH ++
- 19. PNB-0718 inhibits HGF-induced proliferation.
- 20. PNB-0718 (“Norleual”) inhibits c-Met signaling.
- 21. PNB-0718 reduces tumor growth.
- 24. Modeling of Physicochemical Properties of PNB-0718 Physicochemical Property Predicted Value Interpretation logP 0.68 slightly hydrophobic Fraction Unbound (to plasma proteins) 13.32 mostly bound to plasma proteins Effective Human Jejunal Permeability 0.28 not well absorbed in the gut (oral administration) Average Intestinal Permeability 0.08
- 26. Pharmacokinetic Characterization Adult male Sprague Dawley rats Jugular vein catheter Metabolic cages Blood and urine collected Analyzed by LC-MS
- 29. Intravenous Bolus Dose Study
- 31. Development of AT 4 Ligand Molecules Name Structure Activity Angiotensin IV Val-Tyr-Ile-His-Pro-Phe + PNB-0719 Nle -Tyr-Ile-His-Pro-Phe ++ PNB-0718 “ Norleual” Nle -Tyr- Leu- -His-Pro-Phe - - PNB-0405 d-Nle -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH - PNB-0408 Hexanoic acid -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH ++
- 32. Development of AT 4 Ligand Molecules Name Structure Activity Angiotensin IV Val-Tyr-Ile-His-Pro-Phe + PNB-0719 Nle -Tyr-Ile-His-Pro-Phe ++ PNB-0718 “ Norleual” Nle -Tyr- Leu- -His-Pro-Phe - - PNB-0405 d-Nle -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH - PNB-0408 Hexanoic acid -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH ++
- 33. PNB-0405 inhibits HGF-induced migration.
- 34. PNB-0405 inhibits HGF-induced migration.
- 35. PNB-0405 inhibits HGF-induced scattering.
- 36. PNB-0405 inhibits c-Met signaling. c-Met-Gab-1 Association in HEK Cells (Immunoprecipitation)
- 37. PNB-0405 inhibits c-Met signaling. c-Met-Gab-1 Association in HEK Cells (Immunoprecipitation)
- 38. PNB-0405 reduces tumor growth.
- 40. Modeling of Physicochemical Properties Physicochemical Property Predicted Value Interpretation logP 1.45 hydrophobic Fraction Unbound (to plasma proteins) 42.68 ~50% bound to plasma proteins Effective Human Jejunal Permeability 1.53 moderately well absorbed in the gut (oral administration) Average Intestinal Permeability 0.39
- 43. Intravenous Bolus Dose Study
- 46. Subcutaneous Bolus Dose Study
- 48. Angiotensin IV–AT 4 Activity in Cognition
- 51. Development of AT 4 Ligand Molecules Name Structure Activity Angiotensin IV Val-Tyr-Ile-His-Pro-Phe + PNB-0719 Nle -Tyr-Ile-His-Pro-Phe ++ PNB-0718 “ Norleual” Nle -Tyr- Leu- -His-Pro-Phe - - PNB-0405 d-Nle -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH - PNB-0408 Hexanoic acid -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH ++
- 52. Development of AT 4 Ligand Molecules Name Structure Activity Angiotensin IV Val-Tyr-Ile-His-Pro-Phe + PNB-0719 Nle -Tyr-Ile-His-Pro-Phe ++ PNB-0718 “ Norleual” Nle -Tyr- Leu- -His-Pro-Phe - - PNB-0405 d-Nle -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH - PNB-0408 Hexanoic acid -Tyr-Ile- CH-CH-CH-CH-CH-CH-NH 2 OH ++
- 53. PNB-0408 induces c-Met signaling. c-Met-Gab-1 Association in HEK Cells (Immunoprecipitation)
- 54. PNB-0408 induces c-Met signaling. c-Met-Gab-1 Association in HEK Cells (Immunoprecipitation)
- 56. Long-Term Potentiation (LTP) Baseline stimulation (.1Hz) 100 Hz (burst) 5 Hz (1 theta-train) Theta burst stimulation (TBS) tetanus LTP timecourse (% change from baseline) Induction Maintenance 4-trains
- 57. PNB-0408 (“Hex”) induces long-term potentiation (LTP). LTP in Hippocampal Neurons
- 58. Morris Water Maze Task of Spacial Memory Extra-maze cues hidden pedestal 4 - trials per day 120 seconds per trial 20 second on-pedestal rest between trials
- 59. Day 1 Day 2-3 Day 4 Morris Water Maze Representative Swim Patterns 1-2 Trials 3-4 Trials 13+ Trials 5-12 Trials
- 60. PNB-0408 reverses a chemically-induced learning deficit. Rat Performance in the Morris Water Maze
- 61. PNB-0408 improves spacial learning in aged animals. Rat Performance in the Morris Water Maze
- 63. Future Aims for PNB-0408 Determination of physicochemical properties of PNB-0408 by the use of prediction software
- 64. Future Aims for PNB-0408 In-vitro blood stability study
- 69. Questions?
Hinweis der Redaktion
- Ang I binds to nothing Ang II binds to AT1 And AT2 Ang III binds to same – CNS – short acting, probably more like neurotransmitter Ang IV binds to AT4 Ang IV not involved in renin-angiotensin system
- Discovered trying to clone and purify AT1 -- Found a receptor that only binds Ang IV Binding sites widely distributed in terms of tissue, cell type and species. Evolutionarily conserved
- Presence of AT 4 receptors on endothelial cells Importance of endothelial cells in regulating blood vessel growth (angiogenesis) *Numerous pathologies with aberrant blood vessel growth (too much or too little)
- Presence on endothelial cells led to thinking about what role AT4 plays in endothelial cell activity Involvement in angiogenesis which is growth of new blood vessels Angiogenesis requires that endothelial cells proliferate and migrate Lab did series of studies on endothelial cell proliferation and migration Discuss figure Explain Mtt assay
- Number of cells that have migrated across membrane? Stimulated by? Stained with cresyl violet
- AT4 antagonists inhibit proliferation and migration – next step angiogenesis matrigel - an artificial extracellular matrix substitute Area estimated by measuring the distance between the farthest vessels. treat with norleual 10^-10M control receive vehicle measured area of angiogenic sprouting *point out effect is a lot more dramatic when comparing density vs area
- In mice No injections prior to day 13 – rebound effect after injections stopped.
- Gab-1 selective for c-Met.
- Nle – improved affinity for receptor – why tried that? – hydrophobic branched chain hydrophobic straight chain What evidence? – binding studies (slide in spring semenar) Hexanoic acid – acetyl group: OH—CH—CH2—CH2—CH2—CH2—CH2– O Why 6AH? – increase hydrophobicity, stability– amide reduce ability of peptidase to cleave – why six? – approx same length as 3 Aas but w/o side chains
- Not only do AT4 receptor antagonists inhibit endothelial cell growth…
- “ Summary data” from western blots
- +SA Murine breast cancer Elvax pellets
- Before performing pharmacokinetic studies, it is helpful to determine physicochemical properties of molecule
- Electrospray ionization
- Vd suggests PNB-0718 resides predominantly in the central blood compartment.
- Cell type?
- MDCK Why not looking at 0413 instead? – We are but 0405 is prototype molecule.
- Scattering – activity specific to c-Met activation Incubation: 4 days MDCK grown to confluency on round, glass cover slip
- +SA Murine breast cancer Significant difference between control and treated group. Result doesn’t look quite as good as for 0718 but hoped to optimize dosing strategy after PK done.
- Comparison of gut permeability to other orally administered drugs – intermediate.
- Vd suggests PNB-0405 is extensively distributed outside the central blood compartment.
- Summary data from western blots Potentiated HGF effect
- One of the methods we use to measure the cognitive-enhance activity of our compounds is LTP. LTP believed to mimic molecular event underlying learning Help determine if our compounds are directly affecting hippocampal plasticity Hippocampus integrates and amplifies information then sends to neocortex for storage Stimulate CA3 in hippocampus – action potential runs down axons to dendrites of CA1 CA1 stimulation recorded
- If interval between stimulations is far enough apart so that they don’t affect each other – baseline stimulation. Theta burst stimulation – thought to simulate a learning event – observed during active learning event. EPSP – excitatory post synaptic potential
- Work done by Pete and Starla Meighan.
- Note oral administration DW = distilled water