Screening for adverse gastrointestinal effects represents an important step in the preclinical drug-development process. Constipation is one of the most common gastrointestinal complaints in the United States, resulting in about 2 million doctor visits annually. Furthermore, gastrointestinal complications are commonly reported adverse drug reactions. Many different therapeutic classes interfere with normal gut function, such as blood pressure medications (calcium channel blockers), analgesics (especially narcotics), antidepressants, antacids that contain aluminum and calcium, antiparkinson drugs, antispasmodics, iron supplements, diuretics, and anticonvulsants.

The present study was designed to examine the effects on gut motility of three examples of currently prescribed medications that are commonly associated with constipation: a calcium channel blocker for treatment of high blood pressure (verapamil), a synthetic opiate for treating pain (buprenorphine), and a selective serotonin reuptake inhibitor for treating depression (fluoxetine). We employed an in vitro assay in which segments of guinea pig distal colon are pinned out in an organ bath and test compounds are applied to the specimen via the superfusate. The isolated guinea pig distal colon maintains the intrinsic peristaltic reflex for several hours. The effects of test compounds on fecal pellet propulsion were examined using a state-of-the-art digital imaging system.

Methods

Animals: All experimental procedures involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Committee at Med Associates, Inc. Hartley guinea pigs of either sex (Charles River) weighing approximately 300 grams were used for all experiments. Segments of distal colon were taken from guinea pigs following euthanasia by isoflurane anesthesia and exsanguination. Colon segments were immediately placed in cold oxygenated physiological saline solution (PSS, see below for composition).

Fecal Pellet Propulsion Velocity: Segments of guinea-pig distal colon (5-8 cm long) were pinned out in an organ bath with pins attached to small pieces of mesentery adhering to the wall of the gut at intervals of ~1.5-2 cm. Colonic segments were superfused with oxygenated PSS warmed to ~37°C. A 30-min equilibration period was allowed, during which time the gut developed spontaneous tone and emptied its luminal contents. Following the equilibration period, a dried fecal pellet that had been coated with a synthetic polymer to maintain its structural integrity was placed into the oral end of the colon. The amount of time for the fecal pellet to move 2 to 3 cm in the aboral direction was measured within the center of the colon segment using the Gastrointestinal Motility Monitor system (Figure 1; MED-GIMM, Med Associates, Inc.). A total of three motility trials were conducted in each experiment under baseline conditions, with a five-minute recovery period between each trial. Motility rates from these first three trials were were averaged together to give a measure of baseline motility. After the third baseline trial, a test compound was applied via the superfusate for 10 minutes. At this point, a fecal pellet was inserted into the oral end of the colon, and motility rate was assessed as before. In the presence of the test compound, a total of three motility trials were performed, with 5-minutes between each trial. The average motility rate in the presence of the test compound was expressed as a percentage of the average baseline motility rate in each specimen.

Motility rates are measured by a computer video tracking algorithm that tracks the fecal pellet as it moves down the length of the colon (Figure 2). The distance traveled over time is plotted, and propulsion velocity is indicated (Figure 2).

Solutions, Drugs, and Reagents: PSS was made daily and consisted of (in mM) 119 NaCl, 4.7 KCl, 24 NaHCO₃, 1.2 KH₂PO₄, 2.5 CaCl₂, 1.2 MgSO₄, 11 glucose, and aerated with 95% O₂-5% CO₂ to obtain pH 7.4. Tetrodotoxin (TTX), verapamil, buprenorphine, and fluoxetine were all from Sigma.

Results and Discussion

Repeatability of the Colonic Peristaltic Reflex Measured In Vitro. Fecal pellet propulsion velocity could be measured consistently from one preparation to another, and over the course of several trials within a single specimen (Figure 3). Consistent motility rates could be measured for up to twelve motility trials (Figure 3C). This protocol served as a time control for experiments in which drugs were applied. Pellet propulsion rate averaged ~2 mm/sec for up to 12 trials (Figure 3C).

Pharmacological Neuronal Ablation Paralyzes the Colonic Peristaltic Reflex. In order to verify that the fecal pellet propulsion observed in the present study is attributed to the neuronally-based peristaltic reflex, we treated colon segments with the nerve toxin, tetrodotoxin (TTX; 1 µM), following baseline motility assessment. Under control (baseline) conditions, colons displayed normal pellet propulsive activity (Figure 4). In the presence of TTX, propulsive activity was abolished (Figure 4). Upon washout of TTX, motility could often be restored (Figure 4, Rinse). This finding indicates that the fecal pellet propulsive activity observed in the isolated guinea pig distal colon can be attributed to the neuronally-mediated peristaltic reflex.

Adverse Drug Reactions: Calcium Channel Blockers – Verapamil. Verapamil is a typical example of a calcium channel blocker (CCB) 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. We exposed isolated guinea pig colonic segments to verapamil at concentrations ranging from 5 to 20 µM (Figure 5). Verapamil dose-dependently inhibited gut motility, resulting in complete ablation of peristalsis at 20 µM (Figure 5).

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. We examined the effects of buprenorphine on fecal pellet propulsion in the guinea pig distal colon (Figure 6). Buprenorphine slowed gut motility. In most cases, this was reversible upon washout (Figure 6, Rinse).

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. 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. We found that treating distal colon preparations with fluoxetine (10 µM) slowed motility (Figure 7).

Summary/Conclusions

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 (MED-GIMM) to assess colonic motility, and have begun to validate this system using benchmark compounds.

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.