Disclosures for Prof D L Hay. Research funding: Alder Biopharmaceuticals Inc. Consulting arrangements: Intarcia Therapeutics Inc.

Transcription

1 Disclosures for Prof D L Hay Research funding: Alder Biopharmaceuticals Inc. Consulting arrangements: Intarcia Therapeutics Inc.

2 CGRP & Its Receptors Debbie L Hay University of Auckland, New Zealand

3 There are two forms of calcitonin gene-related peptide (CGRP) CGRP - 37 amino acid neuropeptide 2 forms αcgrp and βcgrp, derived from separate genes αcgrp widely expressed in CNS, PNS βcgrp found in enteric nerves, vasculature, CNS G S V A K N D C T T A P V H T T R C V L F N A G K V L L A F NH 2 Human CGRP N V G S R S G S. G. Amara et al. Science 1985, 229:

4 CGRP: local and systemic mechanisms in cardiovascular regulation F. A. Russell et al. Physiological Reviews 214, 94:

5 Diseases where CGRP may play a role F. A. Russell et al. Physiological Reviews 214, 94:

6 αcgrp in metabolic regulation and lipid usage CGRP knockout mice Walker et al., Endocrinology 151: , 21 Walker et al., Peptides 58:14-19, 214 Danaher et al., Endocrinology 149:154-6, 28

7 CGRP in headache disorders CGRP is found in sensory neurons of the face and head (trigeminal nerve), brain CGRP content is higher in the blood during a migraine attack CGRP infusion into a migraine sufferer can cause an acute migraine attack CGRP also associated with cluster headache Pharmacology & Therapeutics 94: 77-92, 22 Annals Neurology 33: 48-56, 1993

8 CGRP in migraine A Russo Annual Review of Pharmacology & Toxicology 215, 55:

9 CGRP is involved in the pathophysiology of migraine F. A. Russell et al. Physiological Reviews 214, 94:

10 Numerous strategies to block CGRP activity have been developed Anti-CGRP antibodies Chronic/episodic migraine Anti-receptor antibodies Chronic/episodic migraine Receptor antagonists Acute migraine ALL trials have been positive F. A. Russell et al. Physiological Reviews 214, 94:

11 Considerations for optimizing therapeutic strategies for CGRP In a likely new era of migraine therapy against the CGRP axis we need to consider: Responders vs. non-responders vs. super responders? Side-effects with chronic CGRP blockade? Can we achieve even better efficacy? Where are they expressed? How many CGRP receptors are there? CGRP receptors What aspects of CGRP biology do they each control? Which should we block to balance efficacy vs. side-effects? Do current drugs block them all equally?

12 The challenge of identifying CGRP receptors CGRP? Class B GPCR Closely-related to the calcitonin receptor Only responded to CGRP in some cells RDC-1 CXCR7/ACKR3 Calcitonin-like receptor

13 c A M P p r o d u c t i o n ( % o f m a x i m u m ) The first cloned CGRP receptor CLR + RAMP1 RAMP1 was the factor that controlled CLR responsiveness to CGRP RAMP1 CLR h C L R / R A M P 1 McLatchie et al., Nature 393(6683):333-9, R A M P 1 h C L R Hay et al., Brit. J Pharmacol. 14: , L o g [ C G R P ]

14 Major efforts undertaken to develop antagonists against the CGRP receptor for migraine F F 3 C F N O H N O N O N NH Telcagepant N Ubrogepant Tfelt-Hansen, 211 J Headache Pain 12: N N NH 2 N O Br HO N H O Br H N O N O N H N Olcegepant Is this first official CGRP receptor the only one?

15 CGRP shows complex pharmacology in tissues: suggests >1 receptor 1 -fo ld P e rfu s e d v e s s e ls A triu m In te rn a l a n a l s p h in c te r Rat tissues Quirion R, et al., Ann N Y Acad Sci. 657:88-15, 1992 A rte rie s V a s d e fe re n s p A 2 CGRP 8-37 affinity Insights into CGRP 2 receptors: Remi Quirion Ian Marshall David Poyner Patrick Sexton Modified from: Hay, D.L., Poyner, D.R., Quirion, R. Pharmacological Reviews 6(2):143-5, 28

16 CGRP binding sites are widespread in the brain 125 I-CGRP Human (L) Rat (R) Inagaki et al., Brain Research 374(2):287-98, 1986 Sexton et al., Neuroscience 19(4): , 1986

17 There are more CGRP binding sites than CLR accounts for Oliver KR et al., Eur J Neurosci. 14(4):618-28, 21 Oliver KR et al., Brain Res Mol Brain Res. 57(1):149-54, 1998 As all areas of high density 125 I-αCGRP binding were not found to be hybridisation signal positive, this may suggest that there are additional uncharacterised CGRP receptor subtypes yet to be identified in the central nervous system.

18 Cloning and pharmacological characterization of other RAMPcomplexes shows that CLR/RAMP1 is not the only receptor CGRP receptor AM 2 receptor AMY 1 receptor CLR RAMP1 CLR RAMP3 CTR RAMP1 CGRP>adrenomedullin (human, rodent) adrenomedullin>cgrp (rodent) amylin=cgrp (human, rodent) Shared subunits leads to cross-reactivity across receptors

19 c A M P (n M ) c A M P (n M ) CGRP has important differences in receptor pharmacology across species e.g. adrenomedullin receptors CLR RAMP3 CGRP is more potent at AM 2 receptors in rodents Low nm concentrations of CGRP can activate rodent AM 2 receptors h A M 2 ra M 2 m A M 2 c A M P % h A M E m a x h A M h C G R P ra M r C G R P ra M r C G R P lo g [P e p tid e ] (M ) lo g [P e p tid e ] (M ) lo g [P e p tid e ] (M ) Bailey et al., British Journal of Pharmacology 166: , 212 Hay et al., British Journal of Pharmacology 14: , 23

20 c A M P (% M a x im u m ) CGRP is equipotent at the CGRP receptor and AMY 1 receptor C G R P re c e p to r A M Y 1 re c e p to r Human receptor pharmacology summary: CTR RAMP1 CLR RAMP1 Dual amylin/cgrp receptor Current name: AMY 1 receptor Binds: CGRP, amylin Activated by: CGRP, amylin e.g. Tilakaratne et al., 2; Kuwasako et al., 24; Hay et al., 25; Hay et al., lo g [h C G R P ] M Gingell et al., 216 Cell Discovery doi:1.138/celldisc , published online May 17, 216 Name: CGRP receptor Binds: CGRP Activated by: CGRP, AM See:

21 c A M P (% F o rs k o lin ) c A M P (% F o rs k o lin ) c A M P (% F o rs k o lin ) CGRP 8-37, a commonly used tool does not effectively discriminate between CGRP-responsive receptors A M Y 1 r e c e p to r C G R P r e c e p to r A M 2 r e c e p to r n M r A m y + 1 M r C G R P n M r C G R P + 1 M r C G R P n M r C G R P + 1 M r C G R P n M r C G R P + 1 M r C G R P Rat receptors In studies of naturally-expressed CGRP receptors, which receptor is actually present? Data replotted from Bailey et al., British Journal of Pharmacology 166: , 212

22 c A M P ( % ) c A M P ( % ) CGRP 8-37 can sometimes activate CGRP-responsive receptors Human CGRP receptor Human AMY 1 receptor h C G R P r C G R P h C G R P 1 h C G R P r C G R P h C G R P l o g [ P e p t i d e ] ( M ) l o g [ P e p t i d e ] ( M ) Christopher Walker in preparation

23 c A M P ( n M ) - 1 n M C G R P c A M P ( n M ) - 1 n M C G R P Olcegepant can fully inhibit CGRP at both CGRP and AMY 1 receptors 3 Human CGRP receptor 3 Human AMY 1 receptor 2 O l c e g e p a n t CLR RAMP1 2 O l c e g e p a n t CTR RAMP l o g [ A n t a g o n i s t ] ( M ) l o g [ A n t a g o n i s t ] ( M ) In studies of naturally-expressed CGRP receptors, which receptor is actually present? Christopher Walker

24 C R E B ( S e r ) p h o s p h o r y l a t i o n ( % m a x i m u m ) Olcegepant affinity at AMY 1 depends on the signaling pathway measured, changing its selectivity profile hamy 1 receptor CGRP CGRP 1 5 h C G R P + 1 M O l c e g e p a n t AMY 1 CGRP 1 α βγ s AC α βγ s AC 5 [camp] pa [camp] pa l o g [ h C G R P ] ( M ) pcreb pa (3 nm) pcreb pa 2 1. Christopher Walker in preparation response response

25 Migraine investigational drugs, olcegepant and telcagepant, can antagonize both CGRP and AMY 1 receptors Antagonist Signal measured Affinity measure Human CGRP receptor 9.65 ±.26 (5) Olcegepant [camp] pa 2 (.2 nm) Olcegepant Telcagepant Telcagepant pcreb [camp] pcreb pa 2 1. ±.24 (6) (.1 nm) pa 2 9. ±.17 (1) (1 nm) pa ±.4 (5) (2 nm) Human AMY 1 receptor 7.23 ±.14 (4) (59 nm) 8.58 ±.15 (9) (2.6 nm) 7.36 ±.12 (3) (44 nm) 7.69 ±.12 (3) (2 nm) CGRP vs AMY 1 ~26-fold ~26-fold ~4-fold ~1-fold Estimated circulating concentrations at efficacious doses: Olcegepant 2 nm, Telcagepant 4-6 µm (Tfelt-Hansen P, Olesen J. Cephalalgia. 31(6):748-5, 211) Could these and other molecules be acting at both receptors?

26 Is AMY 1 a genuine CGRP receptor in vivo and a potential target for migraine?

27 Mapping CGRP/AMY 1 receptor expression and pharmacology in the trigeminovascular system Pain Cranial vasculature Blood vessel dilation Cell body of a pseudounipolar neuron of the trigeminal nerve Pain Trigeminal ganglia (Pain sensitive neurons) Brainstem

28 Expression of CGRP-responsive receptor components in primary rat trigeminal ganglia neurons Histology Cell bodies and projections Taqman GPCR Array Extensive antibody specificity controls conducted Walker et al., Annals of Clinical and Translational Neurology, 2(6): , 215

29 c A M P ( % M a x i m u m ) c A M P ( % M a x i m u m ) CGRP responses in TG neurons are blocked by olcegepant or an AMY 1 receptor antagonist 1 2 r C G R P 1 2 r C G R P n M O l c e g e p a n t M A C l o g [ r C G R P ] ( M ) l o g [ r C G R P ] ( M ) Additional characterisation (telcagepant, CGRP 8-37 ) consistent with the presence of both CGRP receptors in rat TG neurons Walker et al., Annals of Clinical and Translational Neurology, 2(6): , 215

30 CTR and RAMP1 co-express in rat TG CTR RAMP1 Merged CTR + RAMP1 are coexpressed CLR+RAMP1 co-expression reported e.g.: Eftekhari et al., 21 Neuroscience 169(2):683-96; Miller S et al., Neuroscience S (16)3131-2, 216 Walker et al., Annals of Clinical and Translational Neurology, 2(6): , 215

31 Zoomed image of CTR + RAMP1 co-localization in rat TG Walker et al., Annals of Clinical and Translational Neurology, 2(6): , 215

32 CGRP and AMY 1 receptors are likely to be expressed in discrete cells CTR CLR Merged CTR + CLR are not coexpressed Walker et al., Annals of Clinical and Translational Neurology, 2(6): , 215

33 CTR + RAMP1 are co-expressed in human TG Walker et al., Annals of Clinical and Translational Neurology, 2(6): , 215

34 CTR + RAMP1 are expressed in human brainstem including the spinal trigeminal nucleus (Sp5) Nissl CTR- 9B4 Walker et al., Annals of Clinical and Translational Neurology, 2(6): , human cases yr No neurological disease Bower, R.L. et al,. 216 American Journal of Physiology - Regulatory, Integrative and Comparative Physiology doi: /ajpregu

35 CTR + RAMP1 co-expression in spinal trigeminal tract (sp5) and occasional blood vessels (bv) CTR Walker et al., Annals of Clinical and Translational Neurology, 2(6): , 215

36 A common CGRP binding site between CGRP and AMY 1 receptors? CTR RAMP1 AMY 1 receptor ECD CTR RAMP1 CGRPanalog Common residues for CGRP binding (CLR/CTR): W72/79 F92/99 D94/11 F95/12 W121/128 Y124/131 For AMY 1 see Gingell et al., 216 Cell Discovery doi:1.138/celldisc , published online May 17, 216 For CLR/RAMP1 see: 3N7P Booe et al., 215 Mol Cell 58(6):14-52

37 Summary and considerations for CGRP-targeted therapies (1) CGRP receptor AM 2 receptor AMY 1 receptor CLR RAMP1 CLR RAMP3 CTR RAMP1 CGRP>adrenomedullin (human, rodent) adrenomedullin>cgrp (rodent) amylin=cgrp (human, rodent) CGRP can act at >1 receptor

38 Summary and considerations for CGRP-targeted therapies (2) Is AMY 1 a CGRP or amylin receptor? Could be both, and depend on access by ligand A Russo Annual Review of Pharmacology & Toxicology 215, 55: Neuronal CGRP accessible to AMY 1 receptors within the TG/brainstem/other brain sites Hay, D. L. et al., 215 Pharmacological Reviews 67(3):564-6 Pancreatic amylin accessible to AMY receptors in the circumventricular organs of the brain (AP/NTS)

39 Summary and considerations for CGRP-targeted therapies (3) Greater efficacy could be achieved through pan-cgrp receptor antagonism CGRP has activities beyond the migraine/pain axis Chronic CGRP receptor blockade (receptor antagonists, antibodies) or ligand depletion (CGRP antibodies) could lead to compensatory increases in receptor expression leading to rebound effects on withdrawal

40 Acknowledgements Christopher Walker AMRF Post-doctoral Fellow (LB5 Saturday 12.1) Rebekah Bower Joseph Gingell Lars Edvinsson Sajedeh Eftekhari Chris Salvatore Paul Insel Andy Russo