A Drug from the Cannabis Field Appears to Protect From Seizure Related Brain Damage Long C 1, Quizon PMP 1, Romine H 1, Kelly W 1, Naidoo V 1, Ruiz S 1, Suggs S 1, Elliott L 1, Ellerbe J 1, Makriyannis A 2, and Bahr BA 1,2 1 Biotechnology Research and Training Center, The University of North Carolina at Pembroke, NC, USA 2 Center for Drug Discovery, Northeastern University, Boston, MA, USA Excitotoxic events such as seizures have been shown to cause brain damage, decreasing neurons and astrocytes—a component of the Blood Brain Barrier (BBB)—their ability to properly recover. Two endogenous compounds from the cannabinoid system, endocannabinoids anandamide (AEA) and 2- arachidonoylglycerol (2-AG), have been linked with on- demand response in protecting against excitotoxic injuries. An excitotoxin, kanic acid (KA), injected (i.p) is used to mimic seizure related events, and has been observed to raise AEA levels in the brain. In order to modulate the endocannabinoid response, various classes of the AM drug (e.g. AM6642, AM5206, AM6701) have been shown to inhibit the AEA deactivating enzyme fatty acid amide hydrolase (FAAH). KA caused calpain- mediated spectrin breakdown, declines in synaptic markers, and disruption of neuronal integrity in cultured hippocampal slices. The pre- and postsynaptic proteins were protected by the FAAH inhibitor to levels comparable to those found in healthy control brains. Using also, Glial Fibrillary Acidic Protein (GFAP) and Brainspecific Angiogenesis Inhibitor 1 (BAI1) as astrocyte markers, it was shown that KA also seemed to reduce and inhibited astrocyte macrophages their ability to play their role in neuroprotection. FAAH inhibition with AM5206 protected against the neurodegenerative and astrocyte degredation cascade assessed in the slice models various times postinsult. In vivo, KA administration induced seizures and the same neurodegenerative events exhibited in vitro. BAI1 activation and GFAP markers appeared to demonstrate positive effects showing the protective effects of AM5206 on not only neuron, but astrocytes as well. This data supports the idea that endocannabinoids are released and converge on pro-survival pathways that prevent excitotoxic progression for both neurons and astrocytes. 1.Various classes of the AM drug have been shown to inhibits FAAH and MAGL to protect against cytoskeletal breakdown in vivo. 2.Dual blockade of endocannabinoid inactivation mechanisms may therefore be an ideal target for neuroprotective modulation of cannabinergic signaling to offset neuropathological events including excitotoxic brain damage. 3.AM5206 not only appears to have a neuroprotective effect, but also appears to have a astrocyte protective effect. Conclusions Summary An alternative for medical marijuana is needed due to cannabinoid side effects and broad action in the brain Mechanism of Action of  9 -THC: CB1 Localization Herkenham, M., et. al., PNAS : Breakdown of endogenous cannabinoids can be blocked by selective inhibitors (e.g. 6642) to enhance the internal repair system that responds to pathogenic events 3.Seizures were induced in a rat model and AM6642 caused a dose dependent reduction in seizure severity 4. AM5206 provides synaptic and cellular protection when administered 2-5 h post-insult. 5. KA initiates a robust integrin response that is protected against with FAAH inhibitors. It also affects astrocyte markers that are also protected with other class of FAAH inhibitors FIGURE 1. Dual FAAH and MAGL inhibitor AM6642 reduces severity of kainic acid (KA) induced excitotoxicity in vivo. Animals were injected i.p. with 9 mg/kg KA immediately followed by either vehicle (n=15), 1 mg/kg AM6642 (n=7), or 7-8 mg/kg AM6642 (n=8). No-insult control rats received only one vehicle injection (n=16). (A) Seizures were scored by blinded observers within a 4 h period. Mean 2 nd h seizure scores post-injection are shown (± SEM). ANOVA, P<0.0001; post hoc test compared to insult-only data: ***P< References Naidoo V, Karanian DA, Vadivel SK, et al. Equipotent inhibition of fatty acid amide hydrolase and monoacylglycerol lipase - dual targets of the endocannabinoid system to protect against seizure pathology. Neurotherapeutics. 2012;9(4): Hwang J, Adamson C, Butler D, Janero DR, Makriyannis A, Bahr BA. Enhancement of endocannabinoid signaling by fatty acid amide hydrolase inhibition: a neuroprotective therapeutic modality. Life Sci. 2010;86(15- 16): Naidoo V, Nikas SP, Karanian DA, et al. A new generation fatty acid amide hydrolase inhibitor protects against kainate-induced excitotoxicity. J Mol Neurosci. 2011;43(3): Karanian DA, Bahr BA. Cannabinoid drugs and enhancement of endocannabinoid responses: strategies for a wide array of disease states. Curr Mol Med. 2006;6(6): Karanian DA, Karim SL, Wood JT, et al. Endocannabinoid enhancement protects against kainic acid-induced seizures and associated brain damage. J Pharmacol Exp Ther. 2007;322(3): (A) Figure 2. AM5206 provides synaptic and cellular protection when administered 2-5 h post-insult. Animals received either vehicle, or 8 mg/kg AM5206 immediately or 2-5 h following KA administration. At 48 h post-KA-injection, hippocampal tissue for immunoblotting and histology was rapidly dissected from these animals and from no insult control rats. The postsynaptic marker GluR1, presynaptic marker synapsin II, and actin were assessed on single immunoblots Immunoblots show that administration of the FAAH inhibitor protects against KA-induced synaptic damage (GluR1, synapsin II). Note that actin remained unchanged across treatment groups. Hippocampal tissue fixed for histology was paraffin embedded, sectioned, and stained with hematoxylin and eosin. Photomicrographs of the CA1 field show that the excitotoxin-induced neuronal loss and pyknotic changes were ameliorated by the FAAH inhibitor injected immediately or 2-5 h following KA administration. sp, stratum pyramidale; sr, stratum radiatum. Size bar: 45 mm Acknowledgements: This work was supported by grant #5R25GM from the NIGMS (National Institute of General Medical Sciences) supporting the UNCP RISE Program. t Actin Load Control Actin Load Control NT KA KA+ Only AM5206 A. Top Left: Integrated optical density units of β1 integrin band in rat hippocampus homogenates. Between KA Only and KA+FAAH Inhibitor P< Top Right: Western blot of rat hippocampus with no treatment, KA only and KA plus FAAH inhibitor stained for integrin β1. Actin to show load control Middle left: Integrated optical density units of integrin β1 in rat neocortex homogenates. Middle Right: Westen blot of integrin β1 in rat neocortex homogenates. Actin to show load control. Bottom Left: Using imageJ software to determine the optical density of the GFAP marker P<0.05. Bottom Right: Western blot of hippocampus with no treatment, KA only, and KA+AM5206 stained for BAI1, GFAP, and Actin for load control. GFAPGFAP GFAPGFAP GFAPGFAP GFAP, astrocyte marker P<0.05 ANOVA P<0.006