Neuropharmacology 2017

Stephen D Silberstein, MD Jefferson Headache Center Thomas Jefferson University Hospital Philadelphia, PA 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 Presynaptic Autoreceptors

Neurotransmitter Autoreceptor subtype

NE α2A is predominant (also α2B & α2C)

DA D2, short isoform

Acetylcholine M2

5-HT1A (somatodendritic) 5-HT 5-HT1D (nerve terminals)

Histamine H3

GABA GABAB

mGluR2(−) Negative feedback Glutamate mGluR5(+) Positive feedback Basic Elements Of Neurochemical Transmission

Anterograde Neurotransmission (A) Retrograde Neurotransmission (B)

A) NE crosses synaptic cleft to activate α- or β-adrenoceptors postsynaptically

B) NE acts on presynaptic inhibitory α2A-adrenoceptors to inhibit transmitter release Presynaptic inhibitory autoreceptors are α2A 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 Serotonergic System in the Brain – Important Role in Migraine Pathology

Low cerebral serotonin level between attacks

Elevated levels during a migraine attack

Thalamus Serotonergic mechanisms in the migraine brain - a systematic Hypothalamus review. Raphe Cerebellum Cephalalgia. 2016 Mar 23 [Epub] nuclei Deen M, Christensen CE, Hougaard Dorsal Horn A, Hansen HD, Knudsen GM, Ashina M 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 → inhibitory → acute migraine Rx 5-HT2A,B,C → excitatory → migraine prophylaxis 5-HT3 → excitatory → antiemetic

5-HT4 5-HT 5A,5B ex excitatory 5-HT6 } 5-HT7 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 5-HT1A, 5-HT1B, Activation, reuptake (DMT) 5-HT2A, 5-HT2C, inhibitor 5-HT6

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

Concentration causing 50% of maximal stimulation (nM)

Drug 5HT1A 5HT1B 5HT1D 5HT1E 5HT1F

Sumatriptan >10,000 70 3 ~10,000 247

Zolmitriptan ~10,000 20 1 62 417

Rizatriptan >10,000 119 5 870 2540

Naratriptan ~10,000 7 2 31 19

Frovatriptan 1,150 20 2 >10,000 367

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 New Drug Targets

δ opioid receptor NMDA (mGluR2; GluA3) receptor Monoclonal antibodies CGRP

ASIC- 3

OR1

5HT1F CGRP receptor nNOS 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 Headache Pain Freedom (mITT population)

* *

* p < 0.001 37 Human Coronary Artery – Preliminary Data

Proximal coronary artery Distal coronary artery 150 150 Sumatriptan Lasmiditan 100 Clinically relevant conc. 100

50 50

0 0 Contraction (%Contraction KCl) mM 100 Contraction (% 100 mM KCl) (% mM Contraction 100 9 8 7 6 5 4 9 8 7 6 5 4 -Log [Agonist] (M) -Log [Agonist] (M) Dr. Antoinette Maassen van den Brink Speculative Mechanism of Action Trigeminal nerve Blood vessel CGRP Triptans Nitric Oxide 1B, 1D, 1F GlutamateX 1B Sensory nerve block Vasoconstrictor

CGRPX Gepant Nitric Oxide Glutamate

Selective CGRP receptor blockade Non-vasoconstrictor

CGRP Ditan Nitric Oxide 1F XGlutamate Sensory nerve block Non-vasoconstrictor Neuropeptides

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

• 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 Diverse Neuropeptide Families

Connected to migraine? Other families

CGRP: CGRP (, ), calcitonin, amylin, adrenomedullin Opioids: enkephalins, dynorphin, endorphins, nociceptin (AM1,2) Somatostatin/cortistatin

Glucagon/secretin: PACAP, VIP, glucagon, secretin, GHRH, Natriuretic factors: ANF, BNF, CNP GIP GRP, neuromedins Vasopressin/ oxytocin Endothelins CCK/gastrin

F- and Y-amides: NPY, PPY, NPFF Insulins: insulin, IGFs, relaxins Motilin/ghrelin Galanins Tachykinins: Sub P, neurokinin A, neuropeptide K, neuropeptide gamma 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

http://www.neuropeptides.nl/tabel%20neuropeptides%20linked.htm 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 Van den Pol (2012) – Can create bioactive peptides Neuron 76: 98–115 Neuromodulation: Modulate Activity Of Co-released Neurotransmitters To Change Synaptic Signaling Strength

Presynaptic Postsynaptic Neuron 1 Neuron (glutamate)

Presynaptic Neuron 2 (Neuropeptide)

Presynaptic Neuron 3 (Neuropeptide)

http://neuroscience.uth.tmc.edu/s1/chapter14.html Neuropeptides in the Periphery

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

Parasympathetic nerves: VIP, PACAP, NO qSympathetic nerves: and acetylcholine NE, ATP and NPY

Edvinsson and Uddman, Brain Research Reviews 48(3):438-56 2005 New Drug Targets

δ opioid receptor NMDA (mGluR2; GluA3) receptor Monoclonal antibodies CGRP

ASIC- 3

OR1

5HT1F CGRP receptor nNOS 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)

40 **

35 ** 30 30.8% 25 26.9% * 20

15 17.2% Patients (%) Patients 10 9.4% 5 0 MK-0974 MK-0974 Zolmitriptan Placebo 150 mg 300 mg 5 mg

* 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

δ opioid receptor NMDA (mGluR2; GluA3) receptor Monoclonal antibodies CGRP

ASIC- 3

OR1

5HT1F CGRP receptor nNOS 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

CNS and PNS Neurotransmission, neuromodulation, regulation of cicardian clock, behavioral actions, learning and memory procession

Endocrine system Gastrointestinal system Regulation of pituitary, adrenal Regulates lipid/carbohydrate and pacreatic secretion mebolism, liver cell proliferation

Immune system Vascular system Regulates immune reponses Vasodilation

Urogenital system Respiratory system Regulates micturation PACAP38 VIP Bronchodilation 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