1. CMOA 1 5 5-HT 4 Receptor Physiology and Pharmacodynamic Effects of Tegaserod Michael Camilleri, MD Gastroenterology Research Unit Mayo Clinic Foundation Michael Camilleri, MD Gastroenterology Research Unit Mayo Clinic Foundation

2. CMOA 2 5 5-HT 4 Receptor Physiology and Pharmacodynamic Effects of Tegaserod  Physiologic role of serotonin (5-HT) and its type-4 receptors (5-HT 4 ) in GI functions  Tegaserod, a partial 5-HT 4 receptor agonist, and its pharmacodynamic effects on the GI tract  Physiologic role of serotonin (5-HT) and its type-4 receptors (5-HT 4 ) in GI functions  Tegaserod, a partial 5-HT 4 receptor agonist, and its pharmacodynamic effects on the GI tract

3. CMOA 3 Physiologic Distribution of Serotonin (5-Hydroxytryptamine, 5-HT) 5% CNS 95% GI tract  90% EC s  10% Neurons 95% GI tract  90% EC s  10% Neurons 5-Hydroxytryptamine NH 2 OH NH 5

4. CMOA 4 5 Role of 5-HT in the GI Tract  Four main 5-HT receptor subtypes identified in the human GI tract : 5-HT 1, 5-HT 2, 5-HT 3, and 5-HT 4 –Diverse effects due to different receptor subtypes  5-HT 3 and 5-HT 4 receptors involved in multiple GI functions: motor, sensory, secretory  Location of 5-HT 4 receptors: neurons, EC cells, enterocytes, smooth muscle cells  Four main 5-HT receptor subtypes identified in the human GI tract : 5-HT 1, 5-HT 2, 5-HT 3, and 5-HT 4 –Diverse effects due to different receptor subtypes  5-HT 3 and 5-HT 4 receptors involved in multiple GI functions: motor, sensory, secretory  Location of 5-HT 4 receptors: neurons, EC cells, enterocytes, smooth muscle cells

5. Inhibition 5 Serosa Longitudinal muscle Serosa Longitudinal muscle Myenteric plexus Submucosal plexus Muscularis mucosae Lamina propria Epithelium Muscularis mucosae Lamina propria Epithelium Circular muscle Dorsal Root Ganglion Intrinsic Signaling Pathways MOA 5 Intrinsic primary afferent neurons Intrinsic primary afferent neurons Enterocytes ECs Extrinsic primary afferent neurons ECs CNS Inhibitory motor neuron Excitatory motor neuron 5-HT Activation ECs: Enterochromaffin cells 5-HT 4 receptor

6. 5 Intrinsic primary afferent neurons Serosa Longitudinal muscle Serosa Longitudinal muscle Myenteric plexus Submucosal plexus Muscularis mucosae Lamina propria Epithelium Muscularis mucosae Lamina propria Epithelium Circular muscle Enterocytes ECs Inhibitory motor neuron Excitatory motor neuron Extrinsic Signaling Pathways MOA 6 Extrinsic primary afferent neurons Extrinsic primary afferent neurons ECs Dorsal Root Ganglion CNS Inhibition ECs: Enterochromaffin cells 5-HT 4 receptor

7. CMOA 7 5 5-HT 4 Receptor Physiology and Pharmacodynamic Effects of Tegaserod  Physiologic role of serotonin (5-HT) and its type-4 receptors (5-HT 4 ) in GI functions  Tegaserod, a partial 5-HT 4 receptor agonist, and its pharmacodynamic effects –Effects on motility –Effects on visceral sensitivity  Physiologic role of serotonin (5-HT) and its type-4 receptors (5-HT 4 ) in GI functions  Tegaserod, a partial 5-HT 4 receptor agonist, and its pharmacodynamic effects –Effects on motility –Effects on visceral sensitivity

8. CMOA 8 Serotonin 5 NH 2 OH NH N O Tegaserod A representative of a new chemical class of compounds, aminoguanidine indoles, designed to act specifically at 5­HT 4 receptors in the GI tract:

9. CMOA 9 5 Pharmacologic Profile of Tegaserod  Tegaserod, a partial agonist, displays high affinity for human 5-HT 4 receptors (K B = 18 nM)  Tegaserod mimics action of 5-HT and potently stimulates intrinsic sensory neurons triggering the peristaltic reflex (EC 50  5 nM)  Tegaserod has negligible affinity for 5-HT 3 receptors (K B = 7,200 nM); therefore, it is devoid of relevant 5-HT 3 receptor antagonism  Tegaserod, a partial agonist, displays high affinity for human 5-HT 4 receptors (K B = 18 nM)  Tegaserod mimics action of 5-HT and potently stimulates intrinsic sensory neurons triggering the peristaltic reflex (EC 50  5 nM)  Tegaserod has negligible affinity for 5-HT 3 receptors (K B = 7,200 nM); therefore, it is devoid of relevant 5-HT 3 receptor antagonism

10. CMOA 10 Tegaserod Triggers the Peristaltic Reflex 5 Enterocytes ECs 5-HT ACh, Substance P ACh, Substance P VIP, PACAP, NO VIP, PACAP, NO Tegaserod

11. CMOA 11 5 Neurotransmitter Release Induced by Tegaserod is Inhibited by Selective 5-HT 4 Antagonist Means ± SE (n = 3 - 8) Grider et al. Gastroenterology 1998; 115: 370-380  fmol / 100 mg · min Human jejunum Guinea pig colon Rat colon 8 7 6 5 4 3 2 1 0 Substance P  fmol / 100 mg · min 7 6 5 4 3 2 1 0 Human jejunum Guinea pig colon Rat colon VIP 8 Tegaserod alone + 5-HT 3 antagonist + 5-HT 4 antagonist

12. CMOA 12 1101001,000 0 80 100 120 140 160 180 200 % of control Tegaserod concentration (nM) Jin et al. J. Pharmacol. Exp. Ther. 1999; 288: 93-97 5 Tegaserod-Induced Propulsion in Guinea Pig Colon Means ± SEM (n = 3 - 4) Velocity of propulsion

13. CMOA 13 50 mm Hg Colon Jejunum Ileum 0 90 Tegaserod Minutes Nguyen et al. JPET. 1997;280:1270-1276. 5 Tegaserod Stimulates Canine Small Bowel and Colonic Motility After IV Tegaserod

14. CMOA 14 Distribution of colonic isotope at 30 minutes following IV administration of tegaserod SalineVehicle0.030.10.3 0 20 40 60 80 100 Nguyen et al. J. Pharmacol. Exp. Ther. 1997; 280: 1270-1276 5 RectosigmoidProximalDistal Tegaserod dose (mg/kg) Colonic contents in region, % Tegaserod Stimulates Canine Colonic Motility and Transit Means ± SEM (n = 6)

15. CMOA 15 Effect of Tegaserod on Gastrointestinal Transit in Healthy Subjects Means ± SEM (n = 12) *P <.05 vs placebo, † P <.005 vs placebo Percent of Control -60 -40 -20 0 20 40 60 Gastric emptying rate Small bowel transit time * * † † Tegaserod 0.6 mg IV Tegaserod 6 mg PO Degen et al. Gastroenterology 2000; AGA Meeting, abstract

16. CMOA 16 Baseline1 week 0 20 40 60 80 100 Baseline1 week Tegaserod Placebo Colonic filing, % Prather, et al. Gastroenterology. 2000. Colonic Filling at 6 Hours Effect of Tegaserod in C-IBS

17. CMOA 17 Effect of Tegaserod on Colonic Transit in C-IBS Placebo Tegaserod (2 mg b.i.d.) 0 1 2 3 4 5 Stool Prather et al. Gastroenterology 2000; 118: 463-468 5 * Means ± S.E.M. (n = 12) *P <.05 vs baseline Colonic localization of radiotracer

18. CMOA 18 5 Summary : Motor Effects of Tegaserod  Tegaserod mimics the physiologic response to serotonin released from enterochromaffin cells, triggering the peristaltic reflex  Tegaserod promotes motility throughout the GI tract in animals and humans  Tegaserod mimics the physiologic response to serotonin released from enterochromaffin cells, triggering the peristaltic reflex  Tegaserod promotes motility throughout the GI tract in animals and humans

19. CMOA 19 5 5-HT 4 Receptor Physiology and Pharmacodynamic Effects of Tegaserod  Physiologic role of serotonin (5-HT) and its type-4 receptors (5-HT 4 ) in GI functions  Tegaserod, a partial 5-HT 4 receptor agonist, and its pharmacodynamic effects –Effects on motility –Effects on visceral sensitivity  Physiologic role of serotonin (5-HT) and its type-4 receptors (5-HT 4 ) in GI functions  Tegaserod, a partial 5-HT 4 receptor agonist, and its pharmacodynamic effects –Effects on motility –Effects on visceral sensitivity

20. CMOA 20 Visceral Sensitivity End-Points for Preclinical Studies in Small Animals  Single afferent fiber recordings  Pseudo-affective measurements: –Animal behavior, eg, withdrawal, writhing –Abdominal contractions –Drop in arterial BP (under anesthesia)  Single afferent fiber recordings  Pseudo-affective measurements: –Animal behavior, eg, withdrawal, writhing –Abdominal contractions –Drop in arterial BP (under anesthesia)

21. CMOA 21 Static stimulus- response curve (single afferent unit in dorsal root S 2 ) Submaximal firing rates at 50 mm Hg Static stimulus- response curve (single afferent unit in dorsal root S 2 ) Submaximal firing rates at 50 mm Hg Ruehl et al. Neurogastroenterol. Mot. 1998; 10: 219-222 Effect of Tegaserod on Visceral Sensitivity in Cats 5 1 ms

22. CMOA 22 Schikowski et al. Gastroenterology 1999; 116: A643 5 Dose-Dependent Inhibition by Tegaserod of Afferent Nerve Activity to Rectal Distension in Cats 100 80 60 40 20 0 00.30.60.91.21.5 Tegaserod dose (mg/kg IV) EC 50 = 0.7 mg/kg Effect inhibited by 5-HT 4 antagonist * * * * * Means ± SD (n = 9) *P <.05 % of vehicle control Firing rate at 50 mm Hg distension

23. CMOA 23 Tegaserod Inhibits Abdominal Contractions in Response to Colorectal Distension in Awake Rats * * 5 Mean ± SEM (n = 7) * P <.05 versus vehicle Coelho, et al. Gastroenterology. 2000;118:A835.

24. CMOA 24 Summary : Sensory Effects of Tegaserod During colorectal distension:  Tegaserod reduces visceral afferent firing in cats via stimulation of 5-HT 4 receptors  Tegaserod inhibits visceral discomfort / pain in rats During colorectal distension:  Tegaserod reduces visceral afferent firing in cats via stimulation of 5-HT 4 receptors  Tegaserod inhibits visceral discomfort / pain in rats

25. CMOA 25 Summary  Serotonin and its 5-HT 4 receptors are involved in motor, secretory, and sensory processes in the GI tract  Tegaserod, a partial 5-HT 4 receptor agonist, stimulates GI motor functions, and inhibits visceral sensitivity  Tegaserod may influence the sensorimotor dysfunctions and symptoms of constipation- predominant IBS  Serotonin and its 5-HT 4 receptors are involved in motor, secretory, and sensory processes in the GI tract  Tegaserod, a partial 5-HT 4 receptor agonist, stimulates GI motor functions, and inhibits visceral sensitivity  Tegaserod may influence the sensorimotor dysfunctions and symptoms of constipation- predominant IBS