1. Non-invasive Vagus Nerve Stimulation for Treatment of Bronchoconstriction, Migraine and Epilepsy Bruce J. Simon, Ph.D. VP Research ElectroCore, LLC

2. Company Overview
ElectroCore® is a healthcare company focused on neuromodulation therapies.
The Company’s first three commercial products are for the treatment of:
Human respiratory disorders, including asthma, exercise-induced bronchoconstriction (EIB), and COPD (AlphaCore®)
Primary headache in humans, including migraine and cluster headache (GammaCore®)
Canine epilepsy (the GammaCore VET™)
The Company is currently in the early commercial phase within the EU and other key countries for these first two products (IDE phase in the United States), and the early commercial phase in the US for the animal health indication.
There are several additional therapies in pre-clinical development, including ones for depression and anxiety, epilepsy, neuropathic pain, IBS and Alzheimer’s disease. 2

3. Vagus Nerve Stimulation
The vagus nerve has long been known to play an important role in the autonomic nervous system
Cyberonics pioneered the use of electrical stimulation of the vagus nerve for the treatment of epilepsy (FDA approved in 1997) and depression (FDA approved in 2005) using an implantable device which required a surgical procedure
Physicians have attempted the treatment of a wide variety of other conditions using the Cyberonics implantable device, including:
Anxiety – George, et al., Brain Stimulation ,
Alzheimer’s Disease – Sjogren, et al J Clin Psychiatry, 63:11
Migraine – Mauskop, et al., Cephalalgia, 2005, 25, 82-86
Fibromyalgia – Lange, et al., Pain Medicine 2011, 12:
Stroke – Mravec, Auton Neurosci, 2010 Dec 8;158(1-2):8-12.
Obesity – Pardo, et al., Int J Obesity (Lond) Nov; 31(11):
We have developed a non-invasive way to stimulate the cervical vagus nerve and shown that 2 minutes of stimulation can have an effect that lasts up to 8 hours in animal and clinical models of bronchonstriction, migraine and epilepsy

4. ElectroCore Development History
ElectroCore initially developed a VNS signal that could block a histamine-induced airway constriction in guinea pig with direct to vagus stimulation. The effect was mediated by stimulation of low threshold, afferent A fibers and not the efferent, high threshold C fibers which cause classic vagally- mediated bronchoconstriction and bradycardia
The signal was shown to be effective in treating asthmatic patients reporting to the ED with bronchospasm using a percutaneous electrode placed near the vagus nerve under ultrasound guidance
A non-invasive stimulation technology (nVNS) was developed which could get a therapeutic signal to the vagus nerve without stimulation of painful nociceptors. A 2 minute treatment produced a benefit that lasted 4-8 hours
The signal was tested in a second ED asthma study and shown to be safe and effective
Treated asthma patients with migraines reported a significant decrease in migraine pain within minutes of treatment
US IDE acute migraine study showed rapid pain relief with 2 hour results comparable to triptans with no side effects. An EU study of cluster patients showed a 60% reduction in frequency and pain
In a rat chronic migraine model, a 2 min treatment reversed allodynia by inhibiting glutamate release in the trigeminal nucleus caudalis (TNC)
The mechanisms of action in migraine and bronchoconstriction may involve up regulation of inhibitory neurotransmitters (e.g. serotonin, GABA, NE) in the TNC and nucleus ambiguus, respectively
We are currently in RCT US IDE trials for cluster headache, chronic migraine and COPD, and RCT European trials for cluster headache, chronic migraine, ED and home COPD treatment, EIB and IBS
Preliminary treatment of seizures in dogs in an open US study are encouraging.

5. Rationale for Treating Bronchoconstriction with Electrical Stimulation
Asthma affects over 30 million people in the United States each year and accounts for over 2 million ED visits, 1/2 million hospitalizations, and over 4 thousand deaths each year
A large component of bronchoconstriction is due to a neurally mediated smooth muscle contraction
The dominant neural pathways for controlling airway caliber are thought to utilize oppositional controls in the parasympathetic nervous system where cholinergic nerves mediate contraction and inhibitory, non-adrenergic, non-cholinergic (iNANC) nerves mediate relaxation
It was hypothesized that an electrical signal could be developed that would:
Block efferent vagal action potentials causing bronchoconstriction
Direct vagus nerve stimulation was optimized in a histamine-induced bronchoconstirction guinea pig model 5

6. Bronchoconstriction: Histamine Challenge
Dr. Charles Emala (Columbia Univ.), had developed a guinea pig model in which he used electricity to cause bronchoconstriction. Using the same model, direct to nerve vagal stimulation with a wholly different signal was found to relieve bronchoconstriction in a histamine model in guinea pigs1 Tying off the nerve below stimulation did not block the effect, while tying off above did Propranolol abolished the effect
Control
Control
Stimulation
Stimulation
Airway Pressure (cm H2O)
1. Hoffman, et al., Low voltage vagal nerve stimulation reduces bronchoconstriction in guinea pigs through catecholamine release, Neuromodulation, 2012 May 2. doi: /j x.

7. Initial Human Studies – Airway Disease
Percutaneous Delivery Study
25 patient pilot study conducted in 5 emergency departments
Patients failed 1 hour standard of care (SOC)
Non-Significant Risk (NSR) Study
76 matched control patients
Patients failed 1 hour of SOC
FEV1 7

8. Is There a Better Way to Stimulate the Vagus Nerve?
Many forms of energy have been shown to depolarize nerves:
Light
Heat
Mechanical
Ultrasound
Trans esophageal electrical stimulation*
Electric field induction by a time-varying vector potential*
Electric field induction by a time-varying magnetic field*
Transcutaneous electric field*
*These devices were built and tested

9. Trans Esophageal Stimulation Using an Inflatable
Balloon Electrode Attached to a Pediatric Nasogastric Tube
The vagus nerve runs between the esophagus and the trachea
Worked but some patients had a tendency to gag when the balloon was inflated!

10. Transcranial Magnetic Stimulation Device
Used for Peripheral Nerve Stimulation and Treating Depression

11. GammaCore and AlphaCore
ElectroCore developed several important breakthroughs in vagus nerve modulation:
A unique electrode configuration that produces a uniform electric field across the surface of the electrode
A novel signal waveform that minimizes stimulation of skin pain nociceptors to allow for a stronger signal that can reach the vagus nerve
Discovered that a 2 minute treatment can produce CNS effects that last up to 8 hours
Selectively stimulates low threshold, mylinated A afferent nerve fibers, and not the higher threshold efferent C fibers responsible for bradycardia and bronchoconstriction
GammaCore
AlphaCore 11

12. Comparison of nVNS with Percutaneous Stimulation
Device Tested in an ED pilot study in S. Africa: Asthma patients who failed SOC were treated with AlphaCore for 90 sec. and followed for 60 min. (Data compared with previous ED study) 12

13. Spontaneous Headache Relief
From the South African ED Asthma Pilot Study:
“Subject is a 58 year-old female enrolled in the study and stimulated with the AlphaCore™ on October 10, 2011 according to study protocol. The subject presented to the emergency room with a previously diagnosed respiratory infection, bronchospasms and headache. The subject underwent stimulation and exhibited marked improvement in FEV1 and shortness of breath. The subject also reported spontaneous relief of her headache following stimulation”.
From the US IDE Office-based Asthma Pilot Study:
Subsequent literature review revealed five publications showing reduction in frequency and intensity of headaches with implanted VNS 13

14. Acute Migraine – US Pilot Study
30 episodic migraineurs were enrolled and instructed to treat 4 migraines as soon as their migraine intensity reach moderate (2) or severe (3), but no later than 20 minutes after initial onset of pain
Data from 26 patients (79 migraines): 28% reached no pain by 2 hours and 58% had mild or no pain, comparable to triptans 14

15. Treatment of Cluster Headache with GammaCore
Data from the UK Open Label Cluster Headache trial (n=14) demonstrated greater than 60% improvement in frequency and severity of attacks1. Seven patients reported a significant or total reduction in their abortive treatments.
Reduction in Severity
Reduction in Frequency
1Nesbitt et al. (2012). Non-invasive Vagus Nerve Stimulation for the Treatment of Cluster Headache:
A Case Series. EHMTIC, London

16. Mechanism of Action Bronchoconstriction16

17. Parasympathetic MOA Summary
Parasympathetic Role: Afferent vagal A fiber activation, without simultaneous C fiber activation, causes an increase in neuronal output from the nTS, which in turn stimulates
The locus coeruleus1,2,4, resulting in elevated levels of norepinephrine5
The dorsal raphe nucleus1,2 resulting in elevated levels of serotonin1,2,6
The periaqueductal gray7, resulting in an increase in GABA3,7
All of which directly suppress activity in the Airway-related Vagal Preganglionic Neurons (AVPNs) in the Nucleus Ambiguus
1Manta S, Optimization of vagus nerve stimulation parameters using the firing activity of serotonin neurons in the rat dorsal raphe. Eur Neuropsychopharmacol (4):
2Dorr and Debonnel. Effect of Vagus Nerve Stimulation on Serotonergic and Noradrenergic Transmission.  J Pharmacol Exp Ther. 318(2):
3Ben-Menachem et al., Effects of vagus nerve stimulation on amino acids and other metabolites in the anaesthetized rat Epilepsy Research 20:221–227
4Groves et al Recordings from the rat locus coeruleus during acute vagal nerve stimulation in the anaesthetized rat. Neurosci. Lett., 379, 174–179.
5Hassert et al. The Effects of Peripheral Vagal Nerve Stimulation at a Memory-Modulating Intensity on Norepinephrine Output in the Basolateral Amygdala.  Behav Neurosci 118:79– Hammond Neurochemical effects of vagus nerve stimulation in humans. Brain Research 583:300– Prabha and Martin, Respir Physiol Neurobiol (3): 213–222 17 17

18. Parasympathetic Control of Airways
Airway-related Vagal
Preganglionic Neurons
Raphe
Nucleus
Locus
Coeruleus
Periaqueductal
Gray
Nucleus Tractus
Solitarius
Unmylenated
C Fibers
Mylenated A
Fibers
Airway-related
Postganglionic Neurons
Afferent Vagus Fibers
Efferent Vagus Fibers
Airway Smooth Muscle

19. Noxious Stimulus Airway-related Vagal Preganglionic Neurons Raphe
Nucleus
Locus
Coeruleus
Periaqueductal
Gray
Nucleus Tractus
Solitarius
Unmylenated
C Fibers
Mylenated A
Fibers
Airway-related
Postganglionic Neurons
Noxious Stimulus
Afferent Vagus Fibers
Efferent Vagus Fibers
Airway Smooth Muscle

20. Increase in AVPN Firing
Glutamate (+)
Airway-related Vagal
Preganglionic Neurons
Raphe
Nucleus
Locus
Coeruleus
Periaqueductal
Gray
Nucleus Tractus
Solitarius
Unmylenated
C Fibers
Mylenated A
Fibers
Vagal
Postganglionic Neurons
Noxious Stimulus
Afferent Vagus Fibers
Efferent Vagus Fibers
Airway Smooth Muscle Contraction

21. AlphaCore Stimulation
Airway-related Vagal
Preganglionic Neurons
Raphe
Nucleus
Locus
Coeruleus
Periaqueductal
Gray
Nucleus Tractus
Solitarius
Unmylenated
C Fibers
Mylenated A
Fibers
Airway-related
Postganglionic Neurons
Afferent Vagus Fibers
AlphaCore Stimulation
Efferent Vagus Fibers
Airway Smooth Muscle

22. X X X Decrease in AVPN Firing AlphaCore Stimulation Serotonin (-)
Airway-related Vagal
Preganglionic Neurons
Serotonin (-)
(-) GABA
(-) Norepinephrine X
Raphe
Nucleus
Locus
Coeruleus
Periaqueductal
Gray
Nucleus Tractus
Solitarius
Unmylenated
C Fibers
Mylenated A
Fibers
Airway-related
Postganglionic Neurons
Afferent Vagus Fibers
AlphaCore Stimulation X
Efferent Vagus Fibers
Airway Smooth Muscle Relaxation

23. Conclusions
AlphaCore provides a non-invasive, painless signal to the vagus nerve which relieves acute bronchoconstriction and congestion in asthma and COPD patients within seconds of treatment.
Works when -agonists have failed
Can replace albuterol as an asthma/COPD rescue medication
Clinically significant increases in WOB, PEF and FEV1
Patients may leave ED sooner
Decreased use of drugs, reduced morbidity
Decreased hospital admissions
The MOA may involve the inhibition of cholinergic outflow from AVPNs to airway smooth muscle and mucus glands by release of GABA, serotonin and norepinephrine from vagal afferent activation of the periaqueductal gray, raphe nucleus and locus coeruleus, respectively.

24. Mechanism of Action Primary Headache24

25. Rat Chronic Migraine Model
Methods:
A rat model was developed to mimic the hypersensitivity seen in migraine patients1
An inflammatory soup (histamine, serotonin, bradykinin and PGE2) was infused onto the dura of awake rats 3x/week for 4 weeks until a long lasting increase in trigeminal sensitivity was obtained
Trigeminal sensitivity was measured by a decrease in periorbital pressure thresholds as determined by von Frey monofilament testing
Periorbital thresholds were measured before and after 1 minute of stimulation with the GammaCore signal at 5 min, 30 min, 1.5, 2.5, 3.5 and 24 hr. post stimulation
Thresholds were compared with untreated animals and Control animals that had not been stimulated
In a second study, animals were injected with glyceryl trinitrate (GTN) an NO donor that causes headaches in migraineurs, and levels of glutamate were measured in the trigeminal nucleus caudalis (TNC).
1 Episodic dural stimulation in awake rats: A model for recurrent headache. Oshinsky et al Headache 47(7):
Cyberonics had never studied any mechanism of action studies on migraine and we want to understand how it works, Our literature search found someone with a classic migraine model to demonstrate possible mechanisms.
Rat Model that mimics symptoms of migrainers – allodynia (sensitivity to touch). Administer an inflammatory soup to rates (similar to the chemical released in a migraine) the rats become sensitive to stimuli.
Rats that have increased allodynia, have a lower pain threshold (around 2 grams of pressure) vs. normal rats (around 10 grams of pressure)
We used nVNS on the sensitized rats for 1 minute and measured trigemincal sensitivity at 1, 5, 30, 1.5, 2.5, 3.5 and 24 hr. post stimulation.

26. Results
Stimulation for 2 min with nVNS increases the trigeminal threshold to that of naïve animals. The effect lasts for 3.5 hrs.
Treatment with the NO donor, glyceryl trinitrate (GTN) increases glutamate levels in sensitized rats (8x) and naïve rats (2x) and causes a further 10-fold decrease in trigeminal thresholds in sensitized rats.
nVNS inhibits the increase in sensitized rats, reducing glutamate levels to that of naïve rats.
Ongoing studies will measure levels of the inhibitory neurotransmitters, serotonin, NE and GABA in the TNC as a possible mechanism for the glutamate inhibition

27. Parasympathetic Control of Migraine Pain

28. GammaCore Stimulation
Perception of Pain
Is Blocked
Cortex X
Thalamus X
(-) Glutamate
Migraine Pain
Generators
Trigeminal Nucleus
Caudalis
Serotonin (+)
(+) GABA
(+) Norepinephrine
Raphe
Nucleus
Locus
Coeruleus
Periaqueductal
Gray
Stimulation of afferent vagal fibers may upregulate the release of norepinephrine from the nerves of the locus coeruleus throughout their projections.
Nucleus Tractus
Solitarius
GammaCore Stimulation
Afferent Vagus Fibers

29. Non-Invasive Vagus Nerve Stimulation
Provides rapid relief of bronchoconstriction in asthma and COPD patients with 2 minutes of treatment
Decreases the frequency and severity of cluster, acute, chronic migraine and other primary headache disorders
May be effective in treating other CNS disorders including anxiety, Alzheimer’s disease, neuropathic pain, depression, IBS, stroke and obesity
May normalize the underlying autonomic CNS dysfunction in these disease states by modulating the expression of excitatory and inhibitory neurotransmitter levels in the brain

30. Future Research
fMRI
fMRI
EEG
GSR, HRV,
Evoked Potential

31. Thank You

32. Probability of Successful Headache Treatment in those Patients that Treated Four Headaches (65%)
P=.63 for these 52 headaches

33. Responder/Non-Responder Model
We therefore attempted to model the data assuming two groups of patients, responders who have a high probability of successful headache treatment and non-responders with low success probability.
The probability of successfully treating k headaches out of n is given by:
P(k,n) = n!/(k! . (n-k)!) . (NR . PRk . (1-PR)n-k + NN . PNk . (1-PN)n-k )
Where:
NR = Fraction of patients who are responders
NN = Fraction of patients who are non-responders = 1-NR
PR = Responder success probability
PN = Non-responder success probability

34. Probability of Successful Headache Treatment
65% Responders: Probability of success = .92
35% Non-responders: Probability of success = .10

35. Responders and Non-responders

36. Non-Responders? Why might some patients be responders and others not?
Biological differences in response to vagal stimulation
Differences in underlying pathology of the migraine
Active signal not getting to the vagus nerve due to anatomical differences (e.g. deeper vagus in heavier patients), or insufficient stimulation amplitude
A subset of patients (6 with 21 headaches) reported AE’s related to muscle stimulation, suggesting an amplitude sufficient to provide therapy.
These subjects had a 90% success rate.
Can we improve the success rate with better patient training on the correct use of the device?

37. Is Vagus Nerve Stimulation Safe?
Stimulation of vagal efferent fibers has long been known to cause bradycardia and bronchoconstriction
A key breakthrough by ElectroCore was the discovery of a way to stimulate only the low threshold afferent vagal fibers and NOT the high threshold efferent fibers 37 37

38. High Voltage but NOT Low Voltage Induces Bradycardia
Stimulation is effective at .5 – 1 V
3 Volts
10 Volts
Time (s)
Time (s)
Blood Pressure Tracings

39. High Voltage but NOT Low Voltage Induces Bronchoconstriction
Airway Pressure (cm H20)
2 Volt
6 Volt
10 Volt
16 Volt
Time (min)

40. nVNS Has no Effect on Blood Pressure or Heart Rate in Humans