Evaluating the Therapeutic Potential of the Cannabinoid 2 Receptor in Epilepsy Open Access

Abstract

Epilepsy, characterized by recurrent unprovoked seizures as a result of excessive

synchronous neuronal firing, is one of the most common neurological disorders. Approximately

30% of epilepsy patients do not achieve adequate seizure control with currently available

medications, highlighting the need to identify novel molecular targets for anti-epileptic drug

development. Cannabinoid 2 receptors (CB2Rs) have emerged as a potential therapeutic target in

several neurological disorders, including stroke, traumatic brain injury, and neurodegenerative

disease. CB2R agonists consistently reduce neuroinflammation, attenuate neuron loss, and

improve behavioral outcomes across models of neurological disease. Furthermore, in vitro

studies have identified that neuronally-expressed CB2Rs regulate neuronal excitability in the

hippocampus and cortex, two brain regions which are involved in the generation and propagation

of seizures. Despite accumulating evidence that CB2Rs influence physiological processes that

are relevant to epilepsy, few studies have directly investigated the anti-epileptic potential of

CB2R-targeted therapies. The goal of this dissertation was to evaluate the potential of CB2R

modulation for the treatment of epilepsy. First, we established a direct relationship between

CB2R modulation and seizure phenotypes by demonstrating enhanced seizure susceptibility with

genetic knockout and pharmacological blockade of CB2Rs in mice. We next evaluated whether

pharmacological enhancement of CB2R signaling might confer seizure resistance. Our results

suggest that while the CB2R orthosteric agonist, JWH-133 does not increase seizure resistance,

the novel CB2R positive allosteric modulator, Ec21a, confers robust seizure resistance. We

demonstrate that Ec21a fulfils several clinical requirements including adequate brain uptake, lack

of neurotoxicity, and the ability to maintain protection with repeated dosing. We also found that

Ec21a is similarly seizure protective in a mouse line harboring the human SCN1A R1648H

epilepsy mutation. To further characterize the potential of CB2R-targeted therapies in another

clinically-relevant model, we evaluated the effects of CB2R ligands on a range of pathological

features in a mouse model of mesial temporal lobe epilepsy, a common form of treatmentresistant

epilepsy in adults. Overall, this study provided a comprehensive analysis of the role of

CB2Rs in epilepsy and highlights the therapeutic value of allosteric modulation of CB2Rs as a

potential treatment for refractory forms of epilepsy.

Table of Contents

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION……………………………………………………………… 1

1.1 Overview……………………………………………………………………………………... 2

1.2 Epilepsy………………………………………………………………………………………. 2

1.2.1 Epilepsy etiologies…………………………………………………………………. 3

1.2.2 Treatments for epilepsy…………………………………………………………..… 3

1.2.3 Barriers to effective epilepsy treatment………………………………….................. 4

1.3 Overview of the endocannabinoid system…………...……………………………………….. 5

1.3.1 Distribution of the ECS……………………………………………………………... 6

1.3.2 Cannabinoid receptor signaling pathways………………………………………….. 9

1.3.3 Cannabinoid receptor function…………………………………….…………….... 11

1.3.3.1 Cannabinoid 1 Receptors…………………………………………........... 11

1.3.3.2 Cannabinoid 2 Receptors………………………………………….......... 11

1.4 Cannabinoid-based therapies for neurological disease…………………………………….... 13

1.4.1 Neurodegenerative disease, TBI, and stroke……………………………………… 13

1.4.2 Epilepsy…………………………………………………………………………… 14

1.5 Potential advantages of CB2Rs in epilepsy…………………………………………………. 17

1.6 Summary and goals of dissertation……………………………………………………..…… 20

CHAPTER TWO: REDUCED CANNABINOID 2 RECEPTOR ACTIVITY INCREASES

SUSCEPTIBILITY TO INDUCED SEIZURES IN MICE………………………………….. 26

2.1 Summary………………………………………………………………………………….… 27

2.2 Introduction…………………………………………………………………………………. 28

2.3 Materials and methods………………………………………………………………….….... 30

2.4 Results……………………………………………………………………………………..... 35

2.4.1 Cnr2 mutant mice exhibit increased susceptibility to induced seizures…….…….. 35

2.4.2 The CB2R specific antagonist SR144528 increases seizure susceptibility in WT

mice……………………………………………………………………………….. 38

2.4.3 The CB2R specific agonist JWH-133 does not increases resistance to PTZ-induced

seizures …………………………………………………………………………… 40

2.4.4 Deletion of CB2Rs does not worsen seizure phenotypes in Scn1a mutant

mice………………………………………………………………………...……... 40

2.4.5 Deletion of CB2Rs does not increase spontaneous seizure frequency in Scn1a mutant

mice………………………………………………………………………….……. 45

2.5 Discussion…………………………………………………………………………...……… 45

2.6 Acknowledgements……………………………………………………………….………… 49

CHAPTER THREE: ALLOSTERIC MODULATION OF THE CANNABINOID 2

RECPTOR CONFERS SEIZURE RESISTANCE IN MICE………………………………. 50

3.1 Summary…………………………………………………………………………………… 51

3.2 Introduction…………………………………………………………………………………. 52

3.3 Materials and methods………………………………………………………………….….... 54

3.4 Results……………………………………………………………………………………..... 57

3.4.1 Ec21a pharmacokinetic profile……………………………………..........….…….. 57

3.4.2 Acute effects of Ec32a in CF1 WT mice ……….…….………………….……….. 59

3.4.2.1 Ec21a increases resistance to induced seizures in CF1 mice…….…….. 59

3.4.2.2 CB2Rs, but not CB1Rs, mediate the seizure protection conferred by

Ec21a…………………………………………….……………….......... 60

3.4.2.3 Ec21a does not cause neurotoxicity-induced motor deficits…….……… 61

3.4.3 Ec21a increases seizure resistance in RH mutant mice…………………………… 64

3.4.4 Ec21a-mediated seizure protection is maintained with repeated administration in CF1

mice………………………………………………………………………...……... 64

3.5 Discussion…………………………………………………………………………………... 67

3.6 Acknowledgements……………………………………………………………….………… 71

CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS…………………..……….. 72

4.1 Summary……………………………………………………………….…………………… 73

4.2 Potential mechanisms of CB2R-mediated protection……………………………………….. 74

4.3 Allosteric modulation of CB2Rs as an approach to anti-epileptic therapies……………..…. 77

4.4 Expanding our understanding of the therapeutic potential of CB2Rs in epilepsy………...…. 82

4.5 Overall Conclusions………………………………………………………………...………. 84

REFERENCES………………………………………………………………………………….85

APPENDIX A: ESTABLISHING THE EFFECTS OF CB2R LIGANDS IN A MOUSE

MODEL OF MESIAL TEMPORAL LOBE EPILEPSY……………………………………119

A.1 Introduction……………………………………………………………………………..… 120

A.2 Methods…………………………………………………………………………………… 121

A.3 Results……………………………….……………………………………………………. 122

A.3.1 Establishment of the Pilocarpine Model…………………………………………..124

A.3.2 Effects of CB2R-specific compounds in the pilocarpine model…………………..129

A.4 Summary…………………………………………………………………………………...132

A.5 Acknowledgements ………………………………………………………………………..133

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	cannabinoid 2 receptor epilepsy positive allosteric modulator
Committee Chair / Thesis Advisor	Andrew Escayg, Emory University
Committee Members	Randy Hall, Emory University Stephen Traynelis, Emory University David Weinshenker, Emory University Malu Tansey, Emory University

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