Sharon Shacham, Ph.D. ICAD, July 29th, 2008 NASDAQ: EPIX

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1 Stimulation of Serotonin Type 4 Receptors Leads to Increases in Alpha-Secreatase Activity and sappalpha: Potential for Disease Modification in Alzheimer's Disease Sharon Shacham, Ph.D. ICAD, July 29th, 2008 NASDAQ: EPIX

2 Metabolism of Amyloid Precursor Protein β α γ Aβ α secretase (non amyloidogenic) β secretase (amylodoigenic) sappα CTFα sappβ CTFβ γ secretase γ secretase p3 CTFγ Aβ CTFγ Schematic representation of APP770 Turner et al,

3 Neuroprotective and Neurotropic Properties of sappα In vitro evidence: In culture cells sappα enhances synaptogenesis, neurite outgrowth, cell survival and cell adhesion. In vivo evidence: Introducing sappα expression construct to APP ko mice eliminates memory deficits i.c.v injection sappα into mice had cognitive enhancing effects Introducing sappα to brains of young/aged mice resulted in an increase of cholinergic, glutamatergic and GABAergic activity and an increase in the number of the number of EGF-responsive progenitor cells i.c.v injection sappα to middle aged rats increased the power spectrum density of low frequency EEG bands in the hippocampus and cortex 3

4 Activation of α-secretase by GPCRs APP processing was shown to be modulated by several GPCRs: Muscarinic receptors: M1/M3 receptor induced activation of α- secretease through PKC and PLA2 dependent mechanism. AF267B, M1 agonist reduced both Aβ and tau pathologies in the hippocampus and cortex of triple ko AD mice. α-secretase ADAM17 was activated to shift APP processing to non amyloidogenic pathway mglur1, P2Y2, HT2C and HT2a were also shown to induce α- secretase activation through PLC pathway PACAP38 and PACAP 27 activates α-secretase through PKC, PI38K and MAPK activation and PKA independent 4

Serotonin 5-HT4 Receptor Expression Prominent sites of expression: Brain high expression in limbic regions, hippocampus, basal ganglia and substantia nigra (5-HT4 a,

5 Serotonin 5-HT4 Receptor Expression Prominent sites of expression: Brain high expression in limbic regions, hippocampus, basal ganglia and substantia nigra (5-HT4 a, b, c and e) Gut - esophagus, duodenum > jejunum > ileum >> colon > rectum (5- HT4 a, b, c and d) Cardiac atria (5-HT4a, b, e and n) Dumuis et al, Curr Drug tar

6 5-HT4 Receptor: Dual Mechanisms of Action Symptomatic Improvement Stimulate Brain Acetylcholine Production Slow Disease Progression Stimulate Alpha- Secretase Pathway 6

7 Serotonin 5-HT4 Receptor in Cognition and Memory Cachard-Chastel et al, Behav Brain Res 2008 Mohler et al, Neuropharm 2007 Dumuis et al, Curr Drug Tar HT4R activation: Increases ACh Release in Hippocampus During Delayed Spontaneous Alternation in Rats Improve working memory in aged rats and dogs Reverse scopolamine memory impairment in rats and AD transgenic mice Have and additive effect with AChE inhibitors in improving memory performance in normal rats and AD transgenic mice 7

8 Serotonin 5-HT4 Receptor and the α- secretase pathway -110 kda Roberts et al, JBC 2001 Mohler et al, Neuropharm

9 Serotonin 5-HT4 Receptor activation is independent of PKC Activation camp dependent activation of sapp secretion PKC is not involved in 5-HT4 induced sappα secretion Roberts et al, FEBS lett

secretion is not affected by PKA inhibitor in CHO cells expressing 5-HT4e The

10 Serotonin 5-HT4 Receptor activation Involves a PKA independent Pathway Through Small GTP Binding Proteins Maillet et al, Nat cell biol HT induced sappα secretion is not affected by PKA inhibitor in CHO cells expressing 5-HT4e The 5-HT4 receptor induces Rac activation. Epac and Rap1 activation regulats Rac activation 10

11 Serotonin 5-HT4 Receptor - link to Aβ pathway? Effect if PRX on Aβ1 40 levels in CHO hht4d cells Prucalopride induced reduction in Aβ levels in Cho hht4d, but not hht4e/g cells Roberts et al, Neurodeg Disease

12 Serotonin 5-HT4 Receptor - link to Aβ pathway? 5-HT4 Receptor activation by RS-67333, inhibited Aβ secretion in Tg cortical neurons 5-HT4 Receptor activation increases survival of Tg cortical neurons Cho et al, Exp neurol

13 Effects of PRX on CSF Aβ Levels in Aged Beagle Dogs Amyloid β 1-42 Amyloid β Abeta 1-40 levels (fmol/ml) (-25%) (-11%) Abeta 1-42 levels (fmol/ml) (-19%) (-11%) Vehicle PRX mg/kg PRX mg/kg 400 Vehicle PRX mg/kg PRX mg/kg * P=0.069; n=6 13

14 Effect of PRX on Hippocampal Brain sappα Levels in Tg2576 Mice Average hippocampal sappα p=0.057 sappα levls (optical density a.u.) Control PRX mg/kg PRX mg/kg PRX mg/kg 16 weeks old at study start, n=10 / group PRX dosed (i.p.) for three weeks produced a dose dependent trend for increase of hippocampal sappα in tg mice 14

15 Effect of PRX on Hippocampal Amyloid β1-40 Levels in Tg2576 Mice 30 Amyloid β (-12%) (-20%) (-30%) Aβ1-40(pmol/g) Vehicle PRX mg/kg PRX mg/kg PRX mg/kg * P=0.04 for treated vs. vehicle (n=10); Model: 3 weeks treatment with PRX i.p. (19 weeks old at study completion) 15

16 Effect of PRX on Hippocampal Amyloid β1-42 Levels in Tg2576 Mice Amyloid β 1-42 (-13%) Aβ1-42 (pmol/g) (-27%) (-20%) Vehicle PRX mg/kg PRX mg/kg PRX mg/kg * P=0.04 for treated vs. vehicle (n=10); Model: 3 weeks treatment with PRX i.p. (19 weeks old at study completion) 16

17 PRX reduces Brain Aβ levels in Aged Beagle Dogs Insoluble Aβ 1-42 Insoluble Aβ (-28%) (-45%) (-55%) n=2 n=3 n= (-70%) 0 Control PRX mg/kg PRX mg/kg 0 Control PRX mg/kg PRX mg/kg Model: Aged dogs, 2 weeks oral dosing of PRX (n=6) 17

18 Conclusions 5-HT4 Receptor induces sappα secretion through activation (and not increase expression) of α-secretase pathway 5-HT4 induced α-secretase activation is camp dependent, but PKC and PKA independent and involves activation of Epac/Rap-1 and Rac. Activation of the 5-HT4 receptor d, but not g/e, isoforms can lead to reduction in Aβ secretion in-vitro Activation of the 5-HT4 receptor can reduce Aβ secretion and increase neuronal cell survival in Tg2576 mice PRX-03140, a potent and selective 5-HT4 receptor agonist in Ph2 clinical trials, increases sappα secretion in Tg2567 mice and reduces Aβ1-40 and Aβ1-42 in Tg2576 and aged dogs in vivo The 5-HT4 Receptor may represent a novel target for the treatment of AD by providing both symptomatic relief of cognitive impairments as well as neuroprotection by increaseing sappα secretion and reducing Aβ generation and toxicity. 18

19 THANK YOU! Prof. Frank Lezoualc h, INSERM Prof. Alaine Dumuis, CNRS Prof. Bill Milgram, Cancog Prof. Kumar Sambamurti, MUSC Dr. Eric Mohler and Prof. Michael Ragozzino Epix 5-HT4 Team: Dr. Yael Marantz Dr. Boaz Inbal Dr. Mercedes Lobera 19

J. Thomas Megerian, MD, PhD Executive Director, Clinical Research March 28, 2008

Results of a Phase 2A Study of a Novel 5HT4 Agonist for the Treatment of Alzheimer s Disease J. Thomas Megerian, MD, PhD Executive Director, Clinical Research March 28, 2008 EPIX: GPCR Focused Drug Discovery