Adenosine: Key to Blood Brain Barrier Regulation Health Science Symposium ( 5 /6/14)

1. Adenosine: Key to Blood Brain Barrier Regulation Health Science Symposium (5/6/14) Special thanks to the Organizers for the Invitation Margaret Bynoe, Ph.D. Associate Professor, Director of Graduate Studies Cornell University, College of Veterinary Medicine Ithaca, NY Adenosine

2. Adenosine is a purine nucleoside: regulates inflammation, immune cell migration CD73 Four adenosine receptors: A1 A2A A2B A3 CD39 Ado 10 Sec Extracellular adenosine regulates the blood- brain barrier (BBB)

3. What is the blood brain barrier (BBB)? The BBB: is a semi-permeable barrier that consists of a single layer of endothelial cells that line cerebral vasculature Blood vessel Astrocytes WEB OF VESSELS: The brain is replete with blood vessels—some 500 miles of them—. By Megan Scudellari | November 1, 2013 By Marta Toran The blood to brain barrier refers to a lack of direct communication between the blood and peripheral tissues and the brain

4. Adenosine receptors (ARs) are expressed on BBB cells • Ecto-enzymes, CD39 and CD73 are expressed on BBB cells CD73 and CD39 expression on mouse brain endothelial cells A1 and A2A ARs are expressed on mouse brain endothelial cells CD39 CD73 4

5. The Scientific Problem The blood brain barrier (BBB) hinders the delivery of therapeutic drugs into the central nervous system (CNS). The Challenge To safely and effectively modulate the BBB to permit the entry of therapeutic drugs into the CNS.

6. Modulation of adenosine receptor signaling alters blood brain barrier permeability DMSO Mice treated with 10 mg/kg FITC-dextran and DMSO or adenosine receptor modulator for 1 hr. NECA: (general AR agonist) SCH58261: (A2AAR antagonist)

7. AR facilitates anti-beta amyloid antibody entry into the brain of Alzheimer’s transgenic mice Lexiscanvs NECA in Rats • FDA-approved A2A agonist is more potent than NECA

8. 30 60 90 Adenosine receptors are highly expressed on primary human brain endothelial cells 150 Control Relative TEER Changes (%) LEX 1uM 130 NECA 1uM VEGF 110 S1P 90 A1 AR 70 50 0 10 20 30 Minutes post - treatment How do we Test whether Adenosine Receptor Signaling operates the human Blood Brain Barrier? A2A AR 8 Control ** 6 Trans-endothelial electrical resistance An in vitro human BBB Concentration Relative FITC-Dextran T cell 4 * * * * * GAPDH Endothelial Cell 2 * Tight Junction Molecule 0 HBMVEC HCMEC-D3 AR Agonist Porous membrane

9. Could Activation of A2A Adenosine Receptor permeabilize human BBB to Gemcitabineextravasation and kill Glioblastoma cells Lexiscan or NECA Gemcitabine Brain endothelial cell monolayer Porous membrane Glioblastoma cells Incubation of receiver chamber for 4 days at 37 °C 15 5 30 60 Removal of donor chamber (Minutes post treatment)

10. A2A AR activation permeabilized primary human brain endothelial cell barrier to Gemcitabine passage

11. Modifications often result in loss of drug activity Approaches to CNS drug delivery technologies • Pro-Drug Current approaches are either -too invasive -painful -result in permanent brain damage -loss of drug efficacy • Lipidization • Drug Modification to Cross BBB • Fusion Protein Modulation of BBB • CNS Delivery • Collodial • Endogenous Control • Invasive • Costly • Non-patient friendly • High probability of causing permanent brain damage • Disruption • BBB Modification/ Bypass • Mechanical pump • Intercranial • injection

12. Approaches to CNS drug delivery technologies The use of adenosine signaling to modulate BBB permeability has many advantages over other approaches to deliver therapeutics to the CNS: 1) It makes use of an endogenous mechanism for BBB control 2) It has the potential for precise time dependent control of BBB permeability 3) The process is reversible 4) ARs are located directly on BBB endothelial cells 5) Over 50 commercial reagents for ARs are available; some FDA-approved 6) Use of in vivo and in vitro model systems, there is great potential to gain molecular mechanistic understanding; can lead to other targets Adenosine • Endogenous Control • CNS Delivery Modulation of BBB Disadvantages of adenosine as a facilitator of BBB permeability: -drug-drug interaction -multifunctional properties of adenosine

13. Conclusion/s We propose that blood brain barrier permeability is mediated by A2A AR signaling • The BBB, mediated by the A2A receptor operates as a DOOR - “blood brain door”. Local adenosine concentration is the “key”. • Differential modulation of adenosine receptor signaling represents a “tunable” system that can be exploited for therapeutic purposes. Opening the BBB to therapeutics: Closing the BBB to damaging inflammatory cells: Alzheimer’s disease Parkinson’s disease Cancers of the CNS Neurological manifestation of HIV-AIDS Mental disorders Multiple sclerosis

14. Work Presented today by Past and Present members of the lab: Dr. Aaron Carman Dr. Jeff Mills Dr. Dogeun Kim (DVM) Cindy Meuller Special thanks to Philip Owhfor many encouraging conversation