Stephen D Silberstein, MD

Jefferson Headache Center

Thomas Jefferson University Hospital

Philadelphia, PA

Neuropharmacology 2017

Neuropharmacology

• How do drugs work?

• What are receptors?

• What are second messengers?

• What are monamines?

• What are neuropeptides?

• What are Migraine Drug targets?

Classic Transmitters

• Acetylcholine (ACh), biogenic amines (DA, 5-HT, and NE ), and AA transmitters (glutamate, GABA, glycine)

• Synthesized in nerve terminals; stored in many small synaptic vesicles (SSVs) at high concentrations

• ACh concentration ~100 mM

– Requires high concentration to activate low affinity receptors• Both inotropic and metabotropic

• Energy-dependent reuptake process

– Also can be metabolized

– SSVs localized at active zones

SSVs localized at active zones

Neurotransmitters

Neuropeptides

Neuropeptides

• Neuropeptides synthesized in cell body from precursor protein and inserted into dense core vesicles (DCV)

• Fast axonal transport to nerve terminal over hours

– Low number of DCV not in active zone

• Their neuropeptide concentration low ~ 3 to 10 mM

• Neuropeptides activate high affinity metatrobic receptors at a low concentration

• Are metabolized

– No reuptake process to terminate action

Receptors

• Cell membrane is like a switchboard

– Multiple receptors located on and in plasma membrane

• Receptors have different effectors

1. Linked to excitatory and inhibitory G-proteins

2. Activate Ion channels

3. Act as transporters

Two Classes of Postsynaptic Receptors

• Ionotropic: direct binding to receptor-channel complex

– Postsynaptic Potentials (PSPs) short and fast (classical)

• Fast PSPs duration about 20 ms

• Metabotropic: indirect effect

– Receptors activate G proteins (GPCRs)

– Changes in membrane long-lasting “slow PSPs”

– One reason is that second-messenger systems are slow

Ionotropic: direct binding Metabotropic: indirect effect

to receptor-channel

Drug Action

Signal Transduction

• Agonist binds to receptor

• Agonist-receptor complex catalyzes dissociation of GDP from alpha subunit of G protein

– G binds GTP and dissociates from G-

• Dissociated subunits

– Activates adenyl cyclase

• Cyclic AMP formed

– Activates phosphoinositide-phospholipase (PLC)

• IP3 and DAG formed

• GTP hydrolyzed to GDP by GTPase in G subunit

– G protein reassociates

Presynaptic Autoreceptors

• Mediate retrograde transfer of information by negative feedback mediated by neuronal transmitter

• NE α : two different subtypes: α1 and α2

–Presynaptic autoreceptors α2 subtype.

• α1 - activate phospholipase C, producing ITP and DAG

• α2 - inhibit adenylate cyclase, ↓cyclic AMP levels

• 5-HT1A : Both autoreceptor and Post synaptic heteroceptor

Neurotransmitter Autoreceptor subtype

NE α2A is predominant (also α2B & α2C)

DA D2, short isoform

Acetylcholine M2

5-HT5-HT1A (somatodendritic)

5-HT1D (nerve terminals)

Histamine H3

GABA GABAB

GlutamatemGluR2(−) Negative feedback

mGluR5(+) Positive feedback

Presynaptic Autoreceptors

A) NE crosses synaptic cleft to activate α- or β-adrenoceptors postsynapticallyB) NE acts on presynaptic inhibitory α2A-adrenoceptors to inhibit transmitter release

Presynaptic inhibitory autoreceptors are α2A

Basic Elements Of Neurochemical Transmission

Anterograde Neurotransmission (A) Retrograde Neurotransmission (B)

Serotonin: 5-hydroxytryptamine (5-HT)

• Biogenic amine widely distributed in plant and animal kingdoms

– Vasoconstrictor material in serum identified, crystallized and named by Rapport and Page

– Gives enterochromaffin cells of GI mucosa their unique histochemical property

• In man, 90% in enterochromaffin cells of GI mucosa

– Remainder in platelets and CNS

Thalamus

Raphe nuclei

Hypothalamus

Cerebellum

Serotonergic System in the Brain –Important Role in Migraine Pathology

Serotonergic mechanisms in the migraine brain - a systematic review.Cephalalgia. 2016 Mar 23 [Epub]Deen M, Christensen CE, HougaardA, Hansen HD, Knudsen GM, Ashina M

Low cerebral serotonin level between attacks

Elevated levels duringa migraine attack

Dorsal Horn

5-HT Synthesis

• Made in serotonergic neurons and enterochromaffin cells

– Platelets acquire it from blood

• Tryptophan 5-hydroxylase converts l-tryptophan to 5-

hydroxytryptophan (5-HTP) Rate limiting process

– 5-HTP decarboxylated to 5-HT

– Tryptophan concentration subsaturating

• Exogenous tryptophan raises brain tryptophan levels and increases 5-HT production

– Depends on rate of firing of 5-HT neurons

Serotonin Synthesis

5-HT Metabolism

• 5-HT stored in neuronal vesicles

– Reuptake by serotonin transporter (SERT)

• Action of 5-HT terminated by reuptake by SERT

– SERT inhibitors increase 5-HT synaptic concentration

• Metabolized by MAO to aldehyde

– Dehydrogenated to 5-hydroxyindol acetic acid(5-HIAA)

5-HT: Receptors

Recognizes three distinct molecular structures:

• 13 G protein-coupled receptors (GPCR)

– 5-HT1,2,4,5,6,7,8

• 5-HT1 inhibits cAMP production

• One-Ligand-gated ion channels: 5-HT3

• One-Transporters: SERT (for reuptake)

Serotonin Receptors

5-HT1A,B,D,E,F

5-HT2A,B,C

5-HT3

5-HT4

5-HT5A,5B

5-HT6

5-HT7

ex excitatory}

→ inhibitory → acute migraine Rx

→ excitatory → migraine prophylaxis

→ excitatory → antiemetic

The 5-HT1A Receptor

• First cloned 5-HT receptor

– Pharmacologicaly similar to β-adrenergic receptors

• Somatodendritic autoreceptor (in raphe nuclei)

– Decreases neuronal firing rate and inhibits 5-HT release

– Regulates central serotonergic tone

• Post synaptic heteroceptor on 5-HT and non 5-HT neurons

– Modulates dopamine release

• 5-HT1A activity important in mood, cognition and memory

5-HT1B

5-HT1B

• Presynaptic on trigeminal perivascular

endings

– ↓ SP and CGRP release in trigeminal

nucleus and ganglion

• Expressed on cranial blood vessels

– Mediates vasoconstriction in, meningeal,

and extracranial arteries and coronary

and pulmonary arteries

5-HT1D and 5-HT1F

5-HT1D most common 5-HT receptor in human brain– Expressed on trigeminal neurons

• mRNA in trigeminal ganglia

– Prejunctional : blocks neuropeptide release

5-HT1F

– Expressed on trigeminal neurons

• mRNA in trigeminal ganglia

– Prejunctional : blocks neuropeptide release

5-HT2 Receptors

• All Coupled to Gαq

– Activates phospholipase C →IP3 and DAG →↑calcium

• 5-HT2A most abundant cortical 5-HT receptor

– Involved in mechanism of hallucinogen action

• Agonists (LSD and psilocybin) psychotomimetic

– Positive mental health with 5-HT2A agonists

– May be part of SSRIs benefit

Mechanism of Action of Psychoactive Drugs

Psychoactive Substance Acts on Mechanism of Action

5-MeO-dimethyltryptamine

(DMT)

5-HT1A, 5-HT1B,

5-HT2A, 5-HT2C,

5-HT6

Activation, reuptake

inhibitor

LSD 5-HT2A Activation

Mescaline 5-HT2A Activation

5-HT2B Receptor

• Formerly 5-HT2F receptor

– First cloned and characterized in rat stomach fundus

– Localized in vascular and cardiac tissues, low CNS expression• CNS: septal nuclei, dorsal hypothalamus and medial amygdala

• Activates nitric oxide synthase (NOS),→↑ NO synthesis

– Can lead to cardiac hypertrophy

• Unselective agonist m-chlorophenylpiperazine (trazadone metabolite) induces migraine attacks in migraine sufferers

• Antagonists (methysergide and pizotifen) effective prophylactic treatments

5-HT2C

• Localized almost exclusively in CNS

–Negligible in cardiac and vascular tissues

• Modulates forebrain DA systems

– Selective agonist (lorcaserin) appetite suppressant

• FDA approved

–May have therapeutic potential for drug abuse and addiction.

5-HT3

• Cation-selective ligand-gated ion channel

• Superfamily of Cys-loop receptors

• Induces influx of cations through channel

– Rapid neuronal membrane depolarization

– Presynaptic: modulates DA, GABA, ACh, SP,

and glutamate release

– Postsynaptic: excitatory fast synaptic neurotransmission

Serotonin (5-HT)

Widely distributed throughout body

During migraine attack:

1. Platelet 5-HT decreases

2. Urinary 5-HT increases; 5-HIAA may increase

3. Plasma 5-HT releasing factor appears

4. Changes in plasma 5-HT not of clinical significance in

regulating cerebral arterial tone

Localization of 5HT Receptors in Migraine Pathways

Functional Pharmacology of Triptans

Triptans have high affinity for 5HT1B, 1D and 1F receptors • 5HT1B receptor on blood vessels is vasoconstrictor

• 5HT1D and 1F receptors on trigeminal nerves inhibit pain

367>10,0002201,150Frovatriptan

193127~10,000Naratriptan

25408705119>10,000Rizatriptan

41762120~10,000Zolmitriptan

247~10,000370>10,000Sumatriptan

5HT1F5HT1E5HT1D5HT1B5HT1ADrug

Concentration causing 50% of maximal stimulation (nM)

New Drug Targets

CGRP

NMDA (mGluR2; GluA3)receptor

CGRPreceptor

OR1

ASIC- 3

nNOS

5HT1F

Monoclonal antibodies

δ opioid receptor

Serotonin-5-HT1F Receptor Agonist

• Lasmiditan selective 5-HT1F receptor agonist

– New ditan drug class

– Does not activate 5-HT1B or 5-HT1D receptors

– No vascular effects

• Trigeminal Ganglia Stimulation

– Lasmiditan inhibits PPE

– Lasmiditan inhibits activation of TNC

37

Headache Pain Freedom (mITT population)

*

*

* p < 0.001

Proximal coronary artery

456789

0

50

100

150Sumatriptan

Lasmiditan

Clinically relevant conc.

-Log [Agonist] (M)

Co

ntr

acti

on

(%

100 m

M K

Cl)

Distal coronary artery

456789

0

50

100

150

-Log [Agonist] (M)

Co

ntr

acti

on

(%

100 m

M K

Cl)

Human Coronary Artery – Preliminary Data

Dr. Antoinette Maassen van den Brink

Trigeminal nerve Blood vessel

Speculative Mechanism of Action

Triptans

1B, 1D, 1F

CGRP

Nitric Oxide

GlutamateX 1B

Sensory nerve block Vasoconstrictor

GepantCGRP

Nitric Oxide

Glutamate

X

Selective CGRP receptor blockade Non-vasoconstrictor

Ditan

Sensory nerve block Non-vasoconstrictor

1F

CGRP

Nitric Oxide

GlutamateX

Neuropeptides

• Distinction between neuropeptide and peptide hormone solely based on neuronal synthesis– Synthesized, modified, and degraded by same

enzymes

– Act locally and at distance

• Neuropeptides act both in and out of the CNS– Neuropeptides are often peptide hormones

• > 100 neuropeptides in human brain

“Small protein-like molecules (peptides) used by neurons to communicate with each other” (Wikipedia)

Diverse Neuropeptide Families

http://www.neuropeptides.nl/tabel%20neuropeptides%20linked.htm

Connected to migraine? Other families

CGRP: CGRP (, ), calcitonin, amylin, adrenomedullin

(AM1,2)

Opioids: enkephalins, dynorphin, endorphins, nociceptin

Somatostatin/cortistatin

Glucagon/secretin: PACAP, VIP, glucagon, secretin, GHRH,

GIP

Natriuretic factors: ANF, BNF, CNP

GRP, neuromedins

Vasopressin/ oxytocin Endothelins

CCK/gastrin

F- and Y-amides: NPY, PPY, NPFF Insulins: insulin, IGFs, relaxins

Motilin/ghrelin

Tachykinins: Sub P, neurokinin A, neuropeptide K,

neuropeptide gamma

Galanins

Gonadotropin releasing hormones

Tensins: angiotensin, neurotensin, bradykinin Neuropeptide B/W/S

Neurexophilins

CRH-related: CRH, urocortins, urotensins Cerebellins

Granins: chromogranins, secretogranins

Adipose neuropeptides: leptin, adiponectin, resistins

Family-less: orexins, MCH, TRH, PTHrP,, CART, AGRP,

prolactin, diazepam-binding inhibitor peptide, etc

Features of Neuropeptides

1. Modulate target cells– CNS: enhance/dampen synaptic activity

– Peripheral tissues: neurohormones, many targets, many functions

2. Signal transducers that act at cell-surface receptors– Nearly all act at G protein coupled receptors

– Induce slower modulatory response compared to classic neurotransmitters

3. All processed from precursors– Synthesis to degradation shared features

Neuropeptides: Diffusion & Degradation

• Diffuse from point of release

– Nonsynaptic dispersion

– Slowly removed →long lasting effects

• No reuptake system

– Active at distance despite low concentration

• High receptor affinities

• Inactivated by extracellular proteases– Can create bioactive peptides

Van den Pol (2012)Neuron 76: 98–115

PresynapticNeuron 1

(glutamate)

Neuromodulation: Modulate Activity Of Co-released

Neurotransmitters To Change Synaptic Signaling Strength

http://neuroscience.uth.tmc.edu/s1/chapter14.html

PresynapticNeuron 2

(Neuropeptide)

PresynapticNeuron 3

(Neuropeptide)

PostsynapticNeuron

Neuropeptides in the Periphery

Edvinsson and Uddman,

Brain Research Reviews

48(3):438-56 2005

Parasympathetic nerves:VIP, PACAP, NO

and acetylcholineqSympathetic nerves: NE, ATP and NPY

Sensory fibers:CGRP, SP, NKA, PACAP and NO

New Drug Targets

CGRP

NMDA (mGluR2; GluA3)receptor

CGRPreceptor

OR1

ASIC- 3

nNOS

5HT1F

Monoclonal antibodies

δ opioid receptor

CGRP Receptor Antagonists in Migraine

• CGRP infusion triggers migraine

‒ Levels increase during migraine and

decrease with headache relief

• Triptans and ergots inhibit CGRP

release

• CGRP receptor antagonists:

– Block CGRP in CNS and inhibit pain

transmission

– Not direct vasoconstrictors

Telcagepant: Pain-Free (2 hours)

17.2%

26.9%

30.8%

9.4%

0

5

10

15

20

25

30

35

40

MK-0974

150 mg

MK-0974

300 mg

Zolmitriptan

5 mg

Placebo

Pa

tie

nts

(%

)

* P≤0.010** P≤0.001

*

**

**

Telcagepant: Summary

• Both 300mg and 150mg effective in acute migraine – 2-hour pain freedom and pain relief

– Absence of migraine associated symptoms

• 300 mg efficacy comparable to zolmitriptan 5mg

• Well tolerated: AEs comparable to placebo and lower than zolmitriptan

• But Merck discontinued MK-3207( CGRP receptor antagonist)

– Effective, but some subjects had delayed, asymptomatic liver function abnormalities

New Oral CGRP Antagonists

• Allergan acquired rights to two Merck small molecule oral CGRP receptor antagonists:

– MK-1602 (ubrogepant) for acute treatment

• Phase III study is expected to begin in 2016

– MK-8031 for migraine prevention

• Phase II study of MK-8031 is expected to begin in 2016

– MK-1602 & MK-8031 belong to different chemical series than Telcagepant, and have not shown evidence of liver toxicity

New Drug Targets

CGRP

NMDA (mGluR2; GluA3)receptor

CGRPreceptor

OR1

ASIC- 3

nNOS

5HT1F

Monoclonal antibodies

δ opioid receptor

Pituitary Adenylate Cyclase-activating Polypeptides (PACAP)

• Vasoactive neuropeptide

• Gene: Chromosome 18

• Two forms

– PACP 38 (38 amino acids)

– PACAP 27 (27 amino acids) • Major human type: PACAP38

• Isolated from hypothalamic extract: stimulates cAMP formation

• Belongs to secretin/glucagon/VIP family

– Structurally and functionally similar to VIP

Pituitary Adenylate Cyclase-activating Polypeptides (PACAP)

• PACAP-38 widely distributed in many organs

– In sensory trigeminal neurons and parasympathetic nerve fibers surrounding cerebral vessels

PACAP38 VIP

CNS and PNS

Neurotransmission, neuromodulation, regulation of cicardian

clock, behavioral actions, learning and memory procession

Endocrine system

Regulation of pituitary, adrenal

and pacreatic secretion

Vascular system

Vasodilation

Gastrointestinal system

Regulates lipid/carbohydrate

mebolism, liver cell proliferation

Immune system

Regulates immune reponses

Respiratory system

BronchodilationUrogenital system

Regulates micturation

PACAP Receptors (PAC1, VPAC1, and VPAC2)

• PAC1: PACAP affinity>>VIP affinity

• VPAC1/VPAC2: PACAP affinity = VIP

• PACAP affinity: PAC1 = VPAC1 = VPAC2

• VIP affinity: VPAC1/VPAC2 > PAC1

PACAP

• Vasoactive neuropeptide with pro-nociceptive role inCNS

– Also functions in neuroinflammation and sensitization

• Induces headache and delayed migraine-like attacks

• May be due to activation of PAC1 receptors

• Modulation of dural or extracranial trigeminal nociceptors

• Prolonged dilation of extracranial arteries, activation of perivascular afferents, initiation of inflammatory response

• Mast cell degranulation

• PAC1 antagonists may be useful in migraine treatment

PACAP38 – a role in migraine?PACAP38 induces migraine in 58-73% of migraine patients without aura as shown in two controlled studies using placebo or VIP as comparators

PACAP38 induces sustained dilatation of cranial vessels lasting more than 2 h, despite having a half-life of 3.5 min

Schytz HW et. al. Neurotherapeutics 2010;7:191. Amin FM et al. Brain 2014;137:779.

PAC1 receptor mAb (AMG 301)

• Amgen and Novartis: undergoing Phase 1 clinical trials

• Binds to human PAC1 receptor

– Epitopes different from those of other antibodies against PAC1

• Prevents PACAP from binding to its receptor

– Inhibits vasodilation

– Decreases inflammation

– Modulates pain

• Potential Uses: Migraine, Cluster headache, chronic pain, diabetes mellitus (type II), cardiovascular disorders, and sepsis

“Pain is a more terrible

lord of mankind than

even death itself.”

-Albert Schweitzer