# Presentation: 537665

## 5-HT4 Receptor Physiology and Pharmacodynamic Effects of Tegaserod

**5-HT****4**** Receptor Physiology and Pharmacodynamic Effects of Tegaserod**

**Michael Camilleri, MD**
**Gastroenterology Research Unit****Mayo Clinic Foundation**

**Notes:**
Good morning.  Dr. Hanover, members and guests.  It is my pleasure this morning to present the data referring to the 5HT4 receptor physiology and the effects of tegaserod.  
Next slide, please

## 5-HT4 Receptor Physiology and Pharmacodynamic Effects of Tegaserod

**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**

**Notes:**
My talk will be consisting of two parts.  Initially I will discuss the physiological role of serotonin and the role of its type 4 receptors in GI functions and secondly I will review for you the evidence for the effects of tegaserod, a 5HT4 receptor agonist, and its pharmacodynamic effects in the GI tract.
Next slide, please

## Physiologic Distribution of Serotonin(5-Hydroxytryptamine, 5-HT)

**Physiologic Distribution of Serotonin****(5-Hydroxytryptamine, 5-HT)**

**5% CNS**

**95% GI tract**
**90% EC**** ****s**
**10% Neurons**

**5-Hydroxytryptamine**

**5**

**Notes:**
As you all know, serotonin is a biogenic amine that is predominantly located in the gastrointestinal tracts.  In fact, it has been estimated that 95% of all the serotonin in the body is in the digestive tract.  90% of this is in the enterochromaffin cells in the mucosa, 10% is in the neurons in different plexuses, which we will discuss later.  About 5% of the body serotonin is in the central nervous system. 
Next slide, please

## Role of 5-HT in the GI Tract

**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**

**Notes:**
Serotonin has a number of diverse effects in the gastrointestinal tract. These diverse effects are related to the different serotonin receptor subtypes, the different locations of serotonin reserves, and mechanisms of actions, some of which will be discussing today. With reference to the gastrointestinal tract, there are four main 5HT receptor subtypes - 1, 2, 3, and 4.
 Next slide, please

## Intrinsic Signaling Pathways

**5**

**Serosa**
**Longitudinal muscle**

***Myenteric plexus***

***Submucosal plexus***

**Muscularis mucosae**
**Lamina propria**
**Epithelium**

**Circular muscle**

**Dorsal Root Ganglion**

**Intrinsic Signaling Pathways**

**MOA **

**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**

**Notes:**
This cartoon depicts the different layers of the intestine or stomach.  Here we have, in the mucosa, the epithelial layer with enterocytes as well as enterochromaffin cells depicted with secretory granules from which 5HT can be releasing. There are a number of different nerve populations within the wall of the intestine. Some of these are in the submucosal plexus and serve the function of activating secretion.  Others are intrinsic primary afferent neurons that take the message from the lumen to the myenteric plexus to evoke the peristaltic reflex.  Activation of these neurons therefore, through a series of interneurons results in an excitatory cholinergic transmission here to induce contraction of the circular muscle of the intestine. At the same time as this is happening, the message also goes through the inhibitory nitroergic motor neurons so that the receiving segments can relax and thereby a bolus can move from a contracted segment to a relaxed segment of the intestine. Therefore, activation of 5HT receptors in the gut will result in either contraction through an excitatory cholinergic neuron or a relaxation through an inhibitory nitroergic neuron.
Next slide, please

## Extrinsic Signaling Pathways

**5**

**Intrinsic primary**
**afferent neurons**

**Serosa**
**Longitudinal muscle**

***Myenteric plexus***

***Submucosal plexus***

**Muscularis  mucosae**
**Lamina propria**
**Epithelium**

**Circular muscle**

***Enterocytes***

***ECs***

**Inhibitory**
**motor neuron**

**Excitatory**
**motor neuron**

**Extrinsic Signaling Pathways**

**MOA **

**Extrinsic primary**
**afferent neurons**

***ECs***

**Dorsal Root Ganglion**

**CNS**

***Inhibition***

***ECs: Enterochromaffin cells***

**5-HT****4**** receptor**

**Notes:**
Separate from this intrinsic primary afferent neuron, there are longer neurons that project outside the intestine and have their cell body in the dorsal root ganglion. There is evidence that this extrinsic primary afferent pathway is partly dependent upon the 5HT activation.  Data will be summarized for you later in this talk to demonstrate that the use of a 5HT agonist, like tegaserod, can serve to inhibit this extrinsic pathway that send the messages back from the gut to the brain.
Next slide, please

## 5-HT4 Receptor Physiology and Pharmacodynamic Effects of Tegaserod

**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**

**Notes:**
Now in this next section, I plan to review for you the pharmacodynamic effects of tegaserod, a 5HT4 receptor agonist, and it’s affects on the GI tract pertaining to motility and visceral sensitivity. 
Next slide, please

## Tegaserod

**Serotonin**

**5**

**NH****2**

**OH**

**NH**

**N**

**O**

**NH**

**NH**

**NH**

**NH**

**Tegaserod**

**Tegaserod**

**A representative of a new chemical class of compounds, ****aminoguanidine indoles****, designed to act specifically at 5­HT****4**** receptors in the GI tract:**

**Notes:**
Tegaserod is a representative of a new chemical class of compounds, the aminoguanidine indoles, that are designed to act specifically at 5HT4 receptors in the GI tract. Tegaserod is a partial agonist at this 5HT4 receptor.
Next slide, please

## Pharmacologic Profile of Tegaserod

**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**

**Notes:**
Tegaserod displays a high affinity for the human 5HT4 receptor in the nanomolar range.  It serves therefore as a substitute for 5HT and can potently stimulate that intrinsic sensory neuron, which as we showed on the previous slides, is important for triggering the peristaltic reflex.  Again, the EC50 for this effect is in the nanomolar range.  In contrast to this nanomolar sensitivity at the 5HT4 receptor and functional level in the peristaltic reflex, tegaserod has negligible affinity for 5HT3 receptors. At the 5HT3 receptor, you can see that the affinity constant is in the millimolar range, a 1000-fold less, with neither agonist or antagonist properties.
Next slide, please

## Tegaserod Triggers the Peristaltic Reflex

**Tegaserod Triggers the Peristaltic Reflex**

**5**

***Enterocytes***

***ECs***

**5-HT**

**ACh,**
**Substance P**

**VIP,**
**PACAP, NO**

*activation*

**Tegaserod**

**Notes:**
In this slide, we have added some of the important transmitters involved in motor activation.  The ascending contractile response of the peristaltic reflex involves acetylcholine and substance P. The inhibitory response of the peristaltic reflex involves such transmitters as VIP, PACAP and nitric oxide.  5HT and 5HT4 receptors are importantly located on this primary intrinsic afferent.  Therefore it would be expected that the activation of release of 5HT or the extrinsic provision of a 5HT4 agonist, tegaserod, will activate this afferent neuron to induce the peristaltic response. 
Jack Grider and his colleagues have demonstrated in some very elegant studies, the mechanism whereby this action takes place.  Effectively what Grider and colleagues have shown is that tegaserod activates this intrinsic primary afferent neuron which releases excitory and inhibitory transmitters and calcitonin related peptides, a known afferent modulator.  The 5HT4 receptor can be either stimulated endogenously by the release of 5HT from enterochromaffin cells, or exogenously by means of the partial 5HT4 agonist, tegaserod.  In response to this activation, as we’ll see on the next slide, Grider has shown increased levels of the excitatory motor neuron transmitter substance P and the inhibitory transmitter, vasoactive intestinal polypeptide. 
Next slide, please

## Neurotransmitter Release Induced by Tegaserod is Inhibited by Selective 5-HT4 Antagonist

**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**

**Notes:**
Here are the data from that same paper demonstrating in three different tissues from three different species, the activation of, and release of substance P and VIP.  Note that the exposure of this preparation to a 5HT3 antagonist does not reduce the release of any of these transmitters.  Whereas exposure to a 5HT4 antagonist completely inhibits these responses, suggesting therefore, that the effect of tegaserod to activate the intrinsic primary afferent and excitatory and inhibitory pathways, is related specifically to its 5HT4 effects. Grider obtains a similar response with calcitonin-related peptide.
Next slide

## Tegaserod-Induced Propulsion in Guinea Pig Colon

**1**

**10**

**100**

**1,000**

**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**

**Notes:**
Tegaserod induces propulsion in a dose dependent manner in the guinea pig colon.  Here is an example where the contractile response and the relaxation in the receiving segment of the bolus is activated and the velocity of propulsion is increased with increasing concentrations of this partial 5HT4 agonist.
Next slide, please

##  Tegaserod Stimulates Canine Small Bowel and Colonic Motility After IV Tegaserod

**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**

## Tegaserod Stimulates CanineColonic Motility and Transit

**Distribution of colonic isotope at 30 minutes following IV administration of tegaserod**

**Saline**

**Vehicle**

**0.03**

**0.1**

**0.3**

**0**

**20**

**40**

**60**

**80**

**100**

**Nguyen et al. *****J. Pharmacol. Exp. Ther.***** 1997; 280: 1270-1276**

**5**

**Tegaserod dose  (mg/kg)**

**Colonic contents in region,  % **

**Tegaserod Stimulates Canine****Colonic Motility and Transit**

**Means ± SEM (n = 6)**

**Notes:**
In vivo in dogs, it has been demonstrated that the intestinal transit profile in response to intravenous tegaserod is dramatically altered in a dose dependent manner.  In this slide, you can see that the scintigraphic appearance of the colon is shown here in this question mark-like silhouette.  In the analysis of the data, the orange represents the amount of isotope in the proximal colon, the blue the amount of isotope in the distal colon, and the green in the rectosigmoid portion of the dog.  As you can see in a dose dependent manner within 30 minutes after the exposure to intravenous tegaserod, there is greater aboral movement of isotope towards the rectum and sigmoid.
Next slide, please

## Effect of Tegaserod on Gastrointestinal Transit in Healthy Subjects

**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**

**Degen et al. *****Gastroenterology *****2000; AGA Meeting, abstract**

**Notes:**
What about studies from humans? These are data from Lucas Degen demonstrating the effects of tegaserod on gastrointestinal and colonic transit in healthy volunteers. The data show that the gastric lag time, the time for the initial 10% emptying of a solid meal from the stomach is significantly reduced by tegaserod. The gastric emptying rate, the proportion emptied every minute is significantly increased by tegaserod and the small bowel transit time is significantly reduced.  These data from healthy subjects suggests that there is activation of motor function in both stomach and small intestine.  Degen also showed that the transit through the colon is significantly accelerated in response to oral and intravenous tegaserod.
Next slide, please

## Colonic Filling at 6 HoursEffect of Tegaserod in C-IBS

**Baseline**

**1 week**

**0**

**20**

**40**

**60**

**80**

**100**

**Baseline**

**1 week**

**Tegaserod**

**Placebo**

**Colonic filing, %**

**Prather, et al. *****Gastroenterology*****. 2000.**

**Colonic Filling at 6 Hours****Effect of Tegaserod in C-IBS**

## Effect of Tegaserod on Colonic Transit in C-IBS

**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**

**Notes:**
What about the effects of tegaserod in constipation predominant irritable bowel syndrome?  This slide displays the results of scintiscans of the isotope location in the different parts of the colon 48 hours after tracer ingestion. The data from 12 subjects with C-IBS are summarized in this slide and indicate the significant increase in the proportion of isotope that has moved along the colon and an increase therefore, in the geometric center at 48 hours in response to tegaserod, not to placebo. 
Next slide please.  

## Summary : Motor Effects of Tegaserod

**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**

**Notes:**
So in summary from a motor standpoint, I have shown you that tegaserod mimics the physiological response to serotonin which is released from the enterochromaffin cells and it triggers the peristaltic reflex. Tegaserod stimulates propagative motility in animals and humans and it can exert motor effects to stimulate activity in both the upper and the lower gastrointestinal tract. 
Next slide, please

## 5-HT4 Receptor Physiology and Pharmacodynamic Effects of Tegaserod

**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**

**Notes:**
What about it’s effects on visceral sensitivity? Here I’m going to be summarizing two sets of data.  One of which relates to the function of nerves that are teased out of those arising from the colon and rectum of a cat in an in vitro study and the second study pertains to the reflex, abdominal contractions that are induced in an experimental animal model of IBS during the distention of the colon. 
Next slide please

## 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)**

## Effect of Tegaserod on Visceral Sensitivity in Cats

**	****(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**

**Notes:**
This is the feline model in which a stimulus response is induced and a single afferent nerve from the dorsal root of the S2 sacral 2 dermatome is being recorded. These tracings are pulses that are being perceived by that single S2 afferent nerve.  As you can see as the balloon pressure within a rectal segment is increased, there is an increase in the firing of these afferent nerves. You will notice that there is a saturation of the firing possibilities in that afferent nerve at about 65mm Hg pressure within that balloon.  You’ll also notice as indicated by this arrow that the process is almost saturable with just submaximal stimulation occurring about 50mm Hg. What we’re going to see on the next slide is a dose related effect of tegaserod during stimulation with this submaximal pressure increase within the bowel.
Next slide please

## Dose-Dependent Inhibition by Tegaserod of Afferent Nerve Activity to Rectal Distension in Cats

**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**

**	****0****	****0.3****	****0.6****	****0.9****	****1.2****	****1.5**
**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**

**Notes:**
In all of these experiments, the afferent nerve firing rate has been measured during that submaximal distention at 50mm Hg and the data have also been normalized to exclude the firing rate observed with vehicle that is used to dissolve the drug.  In a dose dependent manner we can see that this firing rate is reduced in response to tegaserod and we can also see that the effective threshold dose, the dose that first has a significant effect from the 100% control is at 0.3 mg/kg.
Next slide, please

## Tegaserod Inhibits Abdominal Contractions in Response to Colorectal Distension in Awake Rats

**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.**

**Notes:**
This set of studies that was presented in a couple of weeks ago, demonstrate that in response to colorectal distention, abdominal contractions occur. This is a model for IBS in an experimental animal that has been used extensively and is well represented in the literature.  Notice that in response to control and vehicle, as the pressure within that balloon is increased, so also there is an increase in the number of contractions observed as part of this visceral somatic pain reflex.  However, tegaserod treatment significantly reduces the number of contractions in response to those same colorectal distentions.
Next slide please.  

## 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**

**Notes:**
So to summarize the sensory effects of tegaserod, tegaserod reduces visceral afferent firing in cats. Tegaserod inhibits visceral nociception in rats. This inhibition occurs without any change in colorectal compliance suggesting in fact that this is an affect on the afferent sensory fibers, not on the wall of the intestine itself.  And finally these effects are presumed to be due to stimulation of 5HT4 receptors.
Next slide please. 

## 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**

**Notes:**
In summary, Mr. Chairman, ladies and gentlemen, serotonin and its 5HT4 receptors are involved in motor, and sensory processes in the gastrointestinal tract. Tegaserod, a partial 5HT4 receptor agonist, appears to have a therapeutic potential to influence small bowel and colonic motor and sensory functions which are involved in the pathophysiology of constipation predominant irritable bowel syndrome.  It’s pharmacological properties suggest that tegaserod may influence the sensory motor dysfunctions and symptoms of constipation predominant irritable bowel syndrome. 
I thank you for your attention.
No additional slides