In Vitro Assay for Assessing Gastrointestinal Side Effects
Gerry Herrera, Ph.D. - Med Associates, Inc.
Screening for adverse gastrointestinal effects represents an important
step in the preclinical drug-development process.
- A 1996 National Health Interview Survey reported that about 3 million Americans suffer from frequent constipation.
- Constipation is one of the most common gastrointestinal complaints in the United States, resulting in about 2 million doctor visits annually.
Constipation afflicts millions of Americans. People try many different remedies to alleviate symptoms, with varying levels of success.
Gastrointestinal Problems Are Commonly Reported Adverse Drug Reactions
- Blood Pressure Medications (Calcium Channel Blockers)
- Pain Medications (Especially Narcotics)
- Antidepressants
- Antacids that Contain Aluminum and Calcium
- Antiparkinson Drugs
- Antispasmodics
- Iron Supplements
- Diuretics
- Anticonvulsants
Many drug classes are associated with constipation. Often times, constipation persists, and patients discontinue treatment because the side effect is burdensome.
This web page focuses on three examples of currently prescribed medications that are commonly associated with constipation: A calcium channel blocker for treatment of high blood pressure, a synthetic opiate for treating pain, and a selective serotonin reuptake inhibitor for treating depression.
The Enteric Nervous System Is Special
The enteric nervous system (ENS) is the branch of the nervous system that is located entirely within the wall of the gastrointestinal tract.
The ENS consists of over 100 MILLION individual neurons. This is roughly the number of neurons in the entire central nervous system, or in both branches of the autonomic nervous system put together.
There are also many neurotransmitter systems in use in the gut. The ENS makes use of cholinergic, adrenergic, purinergic, nitrergic, peptidergic, serotonergic, etc.
The enteric neurons are capable of regulating, controlling, and coordinating the actions of the gut entirely without any extrinsic influence.
An intrinsic reflex circuitry is present that is responsible for mediating the directed movement of gut contents from the oral to aboral end of the gastrointestinal tract.
Intrinsic Reflex Circuits Exist in the Gut
Courtesy of Dr. Gary Mawe, University of Vermont
The animation above shows a cross section of the wall of the gut. The lumen of the gut, where the contents are located, is at the bottom of the slide. The lumen is lined by mucosal epithelial cells. The next layer is the submucosal plexus, a network of enteric nerves. The circular muscle wraps around the circumference of the gut. The myenteric plexus is another network of enteric nerves. The longitudinal muscle is lined up along the long axis of the gut.
A stimulus in the lumen of the gut, for example stretch or distension, results in the release of serotonin (5-HT) by the epithelial cells in the mucosa. The serotonin activates sensory afferent fibers that send projections into the submucosal and myenteric plexus. There are two limbs of the reflex. Some interneurons send projections upstream of the stimulus. These neurons synapse with and activate excitatory motor neurons that release acetylcholine (ACh) and Substance P that trigger smooth muscle contraction. The downstream neurons activate inhibitory motor neurons that release nitric oxide (NO), adenosine triphosphate (ATP), vasoactive intestinal peptide (VIP), and PACAP, for example, that cause relaxation of the smooth muscle.
This contraction of the smooth muscle upstream of the stimulus and relaxation of the smooth muscle downstream of the stimulus results in a pressure gradient that drives luminal contents from the oral end to the aboral end of the gut. This motion is called peristalsis.
In Vitro Assay for Gastrointestinal Motility
- In vitro measurements of GI motility have proven quite useful for elucidating physiological mechanisms and examining pharmacological actions.
- The guinea-pig distal colon has long been used as a model system for such studies.
Gastrointestinal motility can be studied in isolated specimens of GI tissue. Organ bath studies have produced important data clarifying important physiological mechanisms. The peristaltic reflex has been studied in vitro by investigators using a stop watch to measure the amount of time for a fecal pellet to travel a specified distance. This method is labor intensive, requiring the researcher to monitor a single preparation at a time, and also suffers from lack of precision, in that the dependent variable only measures the velocity of pellet propulsion.
Our goal was to develop a semi-automated method for GI motility measurements that would allow for increased throughput, as well as provide more detailed data.
Objectives
Allow for increased throughput over fully manual methods
Provide more detailed data regarding the peristaltic reflex using fecal pellet propulsion velocity as an index of motility.
Gastrointestinal
Motility Monitor
Shown to the right is a schematic of the Gastrointestinal Motility Monitor (GIMM) system). The gut segment is pinned out in an organ bath and superfused with warmed, oxygenated physiological saline solution. The specimen is illuminated from underneath, and a digital video camera captures images and sends the video data to a PC for analysis.
In Vitro Gastrointestinal Motility Monitor: Software Interface
The software interface allows the video image to be spatially calibrated. The fecal pellet is introduced into the oral end of the gut, and the peristaltic reflex is activated. As the pellet is propelled distally, the video camera saves the video and the software tracks the fecal pellet (outlined in red) and provides a graph of the distance traveled over time. This gives a point-by-point readout of motility. The slope of the distance-time graph is equivalent to the average propulsion velocity. Furthermore, the raw data can be examined to determine if the motility rate was consistent (a linear graph), or changes over time (a curved graph).
Pharmacological Neuronal Ablation Paralyzes the Colonic Peristaltic Reflex
Shown below are some video clips of motility trials from a single specimen of guinea-pig distal colon. They are played back here at 4X normal speed. The clip on the left is a control trial, showing propulsion of the fecal pellet from the oral to aboral end of the colon under baseline conditions. Notice the smooth, coordinated movement of the fecal pellet. It is moving at a rate of about 1.5 mm/sec. This is a typical example of the guinea-pig distal colon. Baseline motility rates average around 1.5 to 2.5 mm/sec.
Control |
Tetrodotoxin 1 µM |
In the clip on the right, the same gut is shown after treatment with 1µM tetrodotoxin (TTX), which inhibits enteric neuronal activity by blocking voltage-dependent sodium channels on the nerve axons. Notice that the fecal pellet is not propelled, but that the gut remains contracting. This illustrates the fact that the GI smooth muscle has an intrinsic myogenic contraction, but that efferent nerve activity is required for coordinating, directing, and controlling those contractions to result in directed movement of the gut contents.
The figure to the right shows the raw data from the clips shown above. The black line is the motility data from the control trial. Following TTX, pellet propulsion is abolished. Upon washout of TTX, motility is restored (Rinse).
Repeatability of the Colonic Peristaltic Reflex Measured In Vitro
The figure above is an example showing the reproducibility of this preparation. Panel A shows raw motility data from a series of trials recorded in a single specimen of the guinea-pig distal colon over the course of an experiment. Subsequent trials were performed (a total of 12 trials) with at least three minutes between each trial. Notice that the motility rate remains constant over time. Panel B shows the summary data from a total of 8 such experiments. The motility rate remains fairly constant, around 2 mm/sec, for up to 12 trials.
We can use examples of clinically available compounds that are commonly associated with constipation to validate the use of this GI motility monitoring system as a preclinical screening tool for adverse GI reactions. The first example compound is from the calcium channel blockers for treating hypertension.
Adverse Drug Reactions: Calcium Channel Blockers - Verapamil
- Verapamil is a phenylalkylamine class calcium channel blocker (CCB) approved for treating hypertension in the US.
- The major mechanism of therapeutic action is inhibition of calcium entry into the smooth muscle cells lining the resistance blood vessels.
Verapamil is a typical example of a calcium channel blocker for treating high blood pressure. The main sites of therapeutic action of verapamil, and all the calcium channel blockers, are the vascular smooth muscle cells that line the resistance vasculature. Voltage-dependent calcium channels (VDCC) are the main source of calcium entry into the smooth muscle cells. When calcium levels are high, the muscle contracts, and blood pressure rises. Verapamil blocks the entry of calcium into the vascular smooth muscle cell, decreasing calcium into the cell, leading to relaxation and lowering of blood pressure.
- CCBs also exert a potent inhibition of calcium channels in the smooth muscle that lines the gastrointestinal tract.
- The gut is unable to move contents along as efficiently when the smooth muscle is partly relaxed with CCBs.
- Constipation is one of the most frequently reported side-effect of CCB therapy, and often requires cessation of treatment.
Inhibition of GI Smooth Muscle Contractility by Verapamil
The above schematic of a GI smooth muscle cell illustrates the importance of voltage dependent calcium channels (VDCC) in mediating contraction of these cells. Just as in vascular smooth muscle, these channels are sensitive to inhibition by verapamil.
Slowing of Peristalsis Induced by the Calcium Channel Blocker Verapamil
The figure to the left shows motility data from an experiment on an isolated segment of guinea-pig distal colon. Motility was recorded under control conditions (baseline, black line). Increasing concentrations of verapamil caused a progressive decrease in motility rate. Motility was completely inhibited by a concentration of verapamil (20 µM) that would completely relax the GI smooth muscle.
Analgesics are also commonly associated with constipation.
Adverse Drug Reactions: Narcotic Analgesic: Opiate Agonist - Buprenorphine
- Buprenorphine is a synthetic morphine analog commonly used for addiction therapy, cancer pain management, and post-surgical analgesia.
- In addition to the central effects of opiates, the enteric nervous system is particularly susceptible to being impacted by exogenous opiates.
Patients taking buprenorphine for post-surgical analgesia often suffer from constipation.
Opiate Agonists
By inhibiting synaptic communication in the enteric nervous system, opiates such as buprenorphine interfere with the efficiency of the peristaltic reflex. Opiates have a presynaptic inhibitory effect on transmitter release from interneurons and motor neurons.
Courtesy of Dr. Gary Mawe, University of Vermont
The figure to the left shows the effect of 2 µM buprenorphine on colonic motility. Buprenorphine reduced motility by ~25%. This effect was reversible upon buprenorphine washout.
The final example compound we've investigated so far is from the antidepressant class.
Adverse Drug Reactions: Antidepressant: Serotonin Reuptake Inhibitor - Fluoxetine
- Fluoxetine is one of the better tolerated antidepressants, with adverse effect profiles generally superior to those of the tricyclic amine antidepressants.
- However, constipation may still be a problem for some patients.
Fluoxetine is a selective serotonin reuptake inhibitor (SSRI). It inhibits uptake of serotonin by the serotonin transporter.
Serotonin Reuptake Inhibitors
In the presence of Fluoxetine, serotonin that is released in response to stretch of the GI wall accumulates. Increased persistent levels of serotonin result in desensitization of the serotonin receptors on the sensory nerves that initiate the peristaltic reflex, effectively inhibiting initiation of peristalsis.
Courtesy of Dr. Gary Mawe, University of Vermont
Slowing of Peristalsis Induced by the Selective Serotonin Reuptake Inhibitor Fluoxetine
Below are video clips showing a single segment of guinea-pig distal colon before and after fluoxetine treatment. The clips are played at 4X normal speed. You can see that motility is significantly reduced in the presence of fluoxetine.
Control |
Fluoxetine 10 µM |
Slowing of Peristalsis Induced by the Selective Serotonin
Reuptake Inhibitor Fluoxetine
The figure to the left shows the raw data from the above video clips. The profound reduction in response to fluoxetine is evident.
Summary
- GI ailments, constipation in particular, are commonly experienced by patients taking a wide range of medications.
- Often times patients must discontinue drug therapy due to the GI side-effects.
- We have successfully used a PC-interfaced digital video imaging system to assess colonic motility, and have begun to validate this system using benchmark compounds.
Conclusions
- Assessment of gastrointestinal side effects is an important stage of the preclinical drug development process.
- The Gastrointestinal Motility Monitor system can be effectively used to screen pharmaceutical compounds for potential interactions with the gut. It replaces the need to have a technician watch over a preparation with a stopwatch and ruler. Multiple preparations can be run simultaneously, with all data being recorded by the computer.
- Data obtained in this system provide an informative view of the peristaltic reflex, yielding cumulative distance vs. time recordings for fecal pellets being propelled by the gut.
Acknowledgements
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