THE EFFECTS OF CANNABIDIOL ON TYPE 2 DIABETES-RELATED VASCULAR PATHOLOGY PRESENTED BY RACHEL DOPART 1 Dokken, 2008

DIABETES FACTS 29.1 million people in USA have diabetes (type 1 and type 2) (CDC, 2015) 29.1 million people in USA have diabetes (type 1 and type 2) (CDC, 2015) Majority of diabetes cases are type 2 Majority of diabetes cases are type 2 Cardiovascular complications are a leading cause of death from type 2 diabetes (Creager, 2003) Cardiovascular complications are a leading cause of death from type 2 diabetes (Creager, 2003) 2

HOW DOES TYPE 2 DIABETES HAPPEN? Blood sugar level is too high Blood sugar level is too high Sugar goes in the intestine, broken into glucose and absorbed in blood stream Sugar goes in the intestine, broken into glucose and absorbed in blood stream Pancreas detects glucose levels, releases insulin to reduce blood glucose. Pancreas detects glucose levels, releases insulin to reduce blood glucose. Insulin binds to receptors on cells, which causes glucose channels to open on cell surface, allowing glucose to enter the cell Insulin binds to receptors on cells, which causes glucose channels to open on cell surface, allowing glucose to enter the cell (Nucleus medical media, 2012) (Nucleus medical media, 2012) 3

HOW DOES TYPE 2 DIABETES HAPPEN? Body’s cells may resist insulin if the receptors are mutated or absent Body’s cells may resist insulin if the receptors are mutated or absent This causes buildup of glucose in bloodstream/ hyperglycemia This causes buildup of glucose in bloodstream/ hyperglycemia More insulin is released More insulin is released Beta cells lose ability to make insulin, blood glucose increases Beta cells lose ability to make insulin, blood glucose increases (Nucleus medical media, 2012) (Nucleus medical media, 2012) 4

DIABETES AND VASOCONSTRICTION NO is a vasodilator Diabetes lowers NO bioavailability (low insulin or insulin resistance) in endothelium Hyperglycemia: acute inhibition of NO production in arteries Signaling cascade from hyperglycemia produces superoxide, reduces NO (Creager, 2003) Decreased NO means that blood vessels will not relax as well in response to shear stress and cytokines. Can cause inefficient exchange of materials between blood and body cells (Dokken, 2008) 5 (Creager, 2003)

THE ENDOCANNABINOID SYSTEM 6 endocannabinoid-system-involved-in-the-progression-of-asd/ (Shire, 1996)

THC AND CANNABIDIOL 7 +and+Epilepsy

THC AND CANNABIDIOL 8 +and+Epilepsy

CANNABIDIOL Found in Cannabis sativa Found in Cannabis sativa Binds to: Binds to: Orphan receptor GPR55 Orphan receptor GPR55 Putative endothelial cannabinoid receptor CBe Putative endothelial cannabinoid receptor CBe Transient receptor potential vanilloid 1 (TRPV1) receptor Transient receptor potential vanilloid 1 (TRPV1) receptor Alpha1-adrenoceptors, mu opioid receptors, 5-HT1A receptors Alpha1-adrenoceptors, mu opioid receptors, 5-HT1A receptors (Stanley, 2015) (Stanley, 2015) 9

CANNABIDIOL AS A POTENTIAL DRUG FOR TREATING BLOOD VESSEL INFLAMMATION CBD comes from cannabis sativa; structurally similar to THC; does not activate CB1 in the brain CBD comes from cannabis sativa; structurally similar to THC; does not activate CB1 in the brain Does not cause psychoactive effects like THC Does not cause psychoactive effects like THC Epidiolex (phase III clinical trials for child epilepsy) Epidiolex (phase III clinical trials for child epilepsy) Orphan status to treat Hypoxic Ischemic Encephalopathy in newborns Orphan status to treat Hypoxic Ischemic Encephalopathy in newborns (Stanley, 2015) (Stanley, 2015) 10 extracts/epidiolex/

PRECLINICAL EVIDENCE FOR THERAPEUTIC POTENTIAL OF CBD Therapeutic towards: Therapeutic towards: Inflammation Inflammation neurodegeneration neurodegeneration diabetes diabetes oxidative stress oxidative stress CBD protects from diabetic retinopathy CBD protects from diabetic retinopathy CBD lowers diabetes-related cardiomyopathy CBD lowers diabetes-related cardiomyopathy (Stanley, 2015) (Stanley, 2015) 11 hcm.html

CANNABINOIDS ALTER ENDOTHELIAL FUNCTION IN THE ZUCKER RAT MODEL OF TYPE 2 DIABETES Christopher P. Stanley, Amanda J. Wheal, Michael D. Randall, Saoirse E. O’Sullivan Christopher P. Stanley, Amanda J. Wheal, Michael D. Randall, Saoirse E. O’Sullivan Cardiovascular Pharmacology Cardiovascular Pharmacology Volume 720, Issues 1-3, 15 November 2013, Pages Volume 720, Issues 1-3, 15 November 2013, Pages of-obesity-part-ii.html

RESULTS 13 (Stanley, 2013)

RESULTS 14 (Stanley, 2013)

RESULTS 15 (Stanley, 2013)

RESULTS: AEA-TREATED AORTAE 16 Stanley, 2013

RESULTS: AEA-TREATED FEMORAL ARTERIES Lean Fatty 17 (Stanley 2013)

RESULTS: AEA-TREATED MESENTERIC ARTERIES Fatty Lean 18 (Stanley, 2013)

CBD-TREATED AORTAE 19 (Stanley, 2013)

CBD-TREATED FEMORAL ARTERIES FattyLean 20 (Stanley, 2013)

CBD-TREATED MESENTERIC ARTERIES 21 Lean Fatty Stanley, 2013

KEY POINTS AEA and CBD increased vasorelaxation in aorta and femoral artery AEA and CBD increased vasorelaxation in aorta and femoral artery AEA did not produce vasorelaxation in mesenteric (small) arteries AEA did not produce vasorelaxation in mesenteric (small) arteries CBD may be better for diabetes because of improved vasorelaxation. CBD may be better for diabetes because of improved vasorelaxation. (Stanley, 2013) (Stanley, 2013) 22

CANNABIDIOL CAUSES ENDOTHELIUM-DEPENDENT VASORELAXATION OF HUMAN MESENTERIC ARTERIES VIA CB1 ACTIVATION Christopher P. Stanley, William H. Hind, Cristina Tufarelli, Saoirse E. O’Sullivan Christopher P. Stanley, William H. Hind, Cristina Tufarelli, Saoirse E. O’Sullivan Cardiovascular research Cardiovascular research June 19, 2015 June 19, , ,

PURPOSE OF THE ARTICLE Characterize the vascular effects that CBD has on human arteries Characterize the vascular effects that CBD has on human arteries Determine the acute effects that CBD has on arteries Determine the acute effects that CBD has on arteries Form a basis for the pharmacology behind the response Form a basis for the pharmacology behind the response (Stanley, 2015) (Stanley, 2015) 24 forums.com/index.php?action=gallery;sa=view;id=13264

METHODS Took mesenteric arteries from 37 colorectal surgery patients Took mesenteric arteries from 37 colorectal surgery patients Arteries were mounted on a Mulvany Halpern myograph Arteries were mounted on a Mulvany Halpern myograph Tension measured w/ isometric force displacement transducers Tension measured w/ isometric force displacement transducers Controls: were potassium or U46619 to contract, and bradykinin to relax Controls: were potassium or U46619 to contract, and bradykinin to relax Concentration-response curves generated for CBD Concentration-response curves generated for CBD CBD added at 5 min intervals, compare response w/ ethanol-treated arteries CBD added at 5 min intervals, compare response w/ ethanol-treated arteries Tested for possible mechanisms of CBD action by using various chemicals to inhibit different pathways (NO, COX, K+ channels, CB1, CB2, Cbe) Tested for possible mechanisms of CBD action by using various chemicals to inhibit different pathways (NO, COX, K+ channels, CB1, CB2, Cbe) (Stanley, 2015) (Stanley, 2015) 25

RESULTS CBD relaxation in mesenteric arteries: CBD relaxation in mesenteric arteries: Acute relaxation, and relaxation over a longer time Acute relaxation, and relaxation over a longer time PPARy antagonist does not affect vasorelaxation (B) PPARy antagonist does not affect vasorelaxation (B) (Stanley, 2015) (Stanley, 2015) 26

RESULTS Concentration response and vasorelaxation over time for a single concentration of CBD (Stanley, 2015) Concentration response and vasorelaxation over time for a single concentration of CBD (Stanley, 2015) 27

RESULTS: REMOVING ENDOTHELIUM FROM THE ARTERIES Significantly reduces relaxation potential Significantly reduces relaxation potential Could suggest that CBD acts in endothelial cells to elicit the vasorelaxant effect Could suggest that CBD acts in endothelial cells to elicit the vasorelaxant effect (Stanley, 2015) (Stanley, 2015) 28

RESULTS: INHIBITING COX WITH INDOMETHACIN No significant results seen No significant results seen CBD likely does not work through COX pathway CBD likely does not work through COX pathway (Stanley, 2015) (Stanley, 2015) 29

RESULTS: ENOS (ENDOTHELIAL NITRIC OXIDE SYNTHASE) ACTIVATION CBD upregulated eNOS phosphorylation CBD upregulated eNOS phosphorylation Potentially important; may indicate that CBD upregulates nitric oxide levels by activating this enzyme Potentially important; may indicate that CBD upregulates nitric oxide levels by activating this enzyme (Stanley, 2015) (Stanley, 2015) 30

RESULTS: ANTAGONIZING CB1 WITH AM251 AND LY Antagonizing CB1 with AM251 significantly reduces vasorelaxation Antagonizing CB1 with AM251 significantly reduces vasorelaxation This may mean that signaling occurs through CB1 This may mean that signaling occurs through CB1 Confirmed by using LY320135, different CB1 antagonist; showed reduction in maximal response to CBD Confirmed by using LY320135, different CB1 antagonist; showed reduction in maximal response to CBD (Stanley, 2015) (Stanley, 2015) 31

RESULTS: ANTAGONIZING CB2 RECEPTOR WITH AM630 Antagonizing CB2 with AM360 had no effect on vasorelaxation Antagonizing CB2 with AM360 had no effect on vasorelaxation It is likely that CB2 receptor is not involved in eliciting the vasorelaxation effects of CBD It is likely that CB2 receptor is not involved in eliciting the vasorelaxation effects of CBD (Stanley, 2015) (Stanley, 2015) 32

RESULTS: DESENSITIZING TRP CHANNELS WITH CAPSAICIN Reduced vasorelaxation caused by CBD Reduced vasorelaxation caused by CBD TRP channels may be important in the pathway for CBD-induced vasorelaxation TRP channels may be important in the pathway for CBD-induced vasorelaxation (Stanley, 2015) (Stanley, 2015) 33

RESULTS: INHIBITING CBE RECEPTOR WITH O-1918 No significant change when antagonizing CBe receptor No significant change when antagonizing CBe receptor Likely not a key player in vasorelaxation of arteries in response to CBD Likely not a key player in vasorelaxation of arteries in response to CBD (Stanley, 2015) (Stanley, 2015) 34

WHAT WE KNOW SO FAR NO EFFECTS ON VASORELAXATION Inhibiting COX Inhibiting CB2 Inhibiting Cbe Inhibiting PPARy HINDERS VASORELAXATION Endothelial cell elimination Inhibiting CB1 Desensitizing TRP channels 35 (Stanley, 2015)

RESULTS: WHERE ARE THE CB1 RECEPTORS? Antagonize CB1 with AM251 Antagonize CB1 with AM251 Remove endothelial cells from arteries Remove endothelial cells from arteries Observing entire concentration response curve Observing entire concentration response curve Denuding and antagonizing with AM251 causes a more significant decrease in vasorelaxation than just AM251 by itself Denuding and antagonizing with AM251 causes a more significant decrease in vasorelaxation than just AM251 by itself May be a significant concentration of CB1 in endothelial cells May be a significant concentration of CB1 in endothelial cells (Stanley, 2015) (Stanley, 2015) 36

RESULTS: VARIABILITY BETWEEN PATIENTS Results ranged from 2-75% vasorelaxation in response to CBD Results ranged from 2-75% vasorelaxation in response to CBD What conditions in the patients account for such a large difference? What conditions in the patients account for such a large difference? Response lower in males Response lower in males No effect: Age, smoking, BMI No effect: Age, smoking, BMI Disease states that lowered vasorelaxation by CBD: Disease states that lowered vasorelaxation by CBD: Type-2 diabetes Type-2 diabetes Hypercholesterolemia Hypercholesterolemia Disease states that had no effect on vasorelaxation by CBD: Disease states that had no effect on vasorelaxation by CBD: Cancer Cancer Heart disease Heart disease High blood pressure’ High blood pressure’ (Stanley, 2015) (Stanley, 2015) 37

RESULTS: VARIABILITY BETWEEN PATIENTS CBD response lower: CBD response lower: Beta blockers Beta blockers Hypoglycaemic medication Hypoglycaemic medication Statin Statin CBD response unchanged: CBD response unchanged: ACE inhibitor ACE inhibitor NSAID NSAID (Stanley, 2015) (Stanley, 2015) 38

RESULTS: INTRACELLULAR MECHANISMS OF CBD Reduced phosphorylation in response to CBD doses: (Stanley, 2015) Reduced phosphorylation in response to CBD doses: (Stanley, 2015) 39

RESULTS: INTRACELLULAR MECHANISMS OF CBD Increased phosphorylation in response to CBD dose (Stanley, 2015) Increased phosphorylation in response to CBD dose (Stanley, 2015) 40

RESULTS: ANTAGONIZING CB1 OR TRPV1 Lose ERK1/2 phosphorylation with both treatments Lose ERK1/2 phosphorylation with both treatments Lose Akt phosphorylation when inhibiting CB1 Lose Akt phosphorylation when inhibiting CB1 (Stanley, 2015) (Stanley, 2015) 41

RESULTS: TYPE 2 DIABETIC CONDITIONS Vasorelaxation via CBD was blunted in those with T2DM Vasorelaxation via CBD was blunted in those with T2DM RT-PCR for aortic cells in high glucose and high insulin environments; how do they affect the expression of target sites? RT-PCR for aortic cells in high glucose and high insulin environments; how do they affect the expression of target sites? High insulin or high glucose caused CB2R upregulation, and TRPV1 and CGRPR downregulation. High insulin or high glucose caused CB2R upregulation, and TRPV1 and CGRPR downregulation. (Stanley, 2015) (Stanley, 2015) 42

FUTURE DIRECTIONS Test in a more uniform group of people, or use more subjects for the test: Test in a more uniform group of people, or use more subjects for the test: +: observe human reactions, compare with original findings +: observe human reactions, compare with original findings -: need many people, invasive procedure -: need many people, invasive procedure Test in rat populations again: Test in rat populations again: Specifically test mechanism of CBD vasorelaxation Specifically test mechanism of CBD vasorelaxation Determine drug interactions; why do they happen? Is it due to medication or due to the disease for said medication? Determine drug interactions; why do they happen? Is it due to medication or due to the disease for said medication? What is the endothelial factor that causes CBD-vasorelaxation? What is the endothelial factor that causes CBD-vasorelaxation? How does CBD work with CB1? How does CBD work with CB1? Does it bind directly (it has low affinity for CB1) Does it bind directly (it has low affinity for CB1) Does it prevent degradation of endocannabinoids? Does it prevent degradation of endocannabinoids? How do TRPV1 expression levels change in people with high cholesterol or type 2 diabetes? How do TRPV1 expression levels change in people with high cholesterol or type 2 diabetes? (Stanley, 2015) (Stanley, 2013) (Stanley, 2015) (Stanley, 2013) 43

QUESTIONS? 44

SOURCES “Am I at risk for type 2 diabetes? Taking steps to lover your risk. diabetes-taking-steps-lower-your-risk-diabetes/Pages/index.aspx “Am I at risk for type 2 diabetes? Taking steps to lover your risk. diabetes-taking-steps-lower-your-risk-diabetes/Pages/index.aspx “Am I at risk for type 2 diabetes? Taking steps to lover your risk. diabetes-taking-steps-lower-your-risk-diabetes/Pages/index.aspx “Am I at risk for type 2 diabetes? Taking steps to lover your risk. diabetes-taking-steps-lower-your-risk-diabetes/Pages/index.aspx Creager, Mark A., et al. "Diabetes and vascular disease pathophysiology, clinical consequences, and medical therapy: part I." Circulation (2003): Dokken, Betsy B. "The pathophysiology of cardiovascular disease and diabetes: beyond blood pressure and lipids." Diabetes Spectrum 21.3 (2008): Shire, David, et al. "Structural features of the central cannabinoid CB1 receptor involved in the binding of the specific CB1 antagonist SR A."Journal of Biological Chemistry (1996): Stanley, Christopher P., et al. "Cannabidiol causes endothelium-dependent vasorelaxation of human mesenteric arteries via CB1 activation."Cardiovascular research (2015): “Type 2 diabetes.” Nucleus Medical Media. “Type 2 diabetes.” Nucleus Medical Media. “What is type 2 diabetes?” “What is type 2 diabetes?” 45