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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Neuroscience Drug Discovery, Pharmaceutical Research Institute, Bristol Myers Squibb Co., Wallingford, Connecticut
Received for publication
October 19, 2004
Accepted
February 1, 2005.
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Abstract |
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). Although the basis for these abnormalities remains to be elucidated, numerous mechanisms have been proposed that involve peripheral or central alteration in cellular motor and sensory systems responsible for normal gastrointestinal motility and perception. For example, maladaptive changes to long-term stress (McEwen, 1998) manifested as inflammation or infection mediated disruption of the mucosal barrier function (Soderholm et al., 2002), sensitization of visceral nociceptors (Bueno and Fioramonti, 2002), aberrant afferent transmission (Gebhart, 1999), or altered processing of sensory information in the brain (Naliboff et al., 2001) have been causally related to abnormal motility and sensation.
Large conductance Ca2+-activated K+ (maxi-K) channels are abundantly present in virtually all excitable cells and are modulated by membrane depolarization and intracellular Ca2+ (Ashcroft, 2000). Their opening in turn regulates cellular excitability by facilitating the K+ efflux-mediated membrane hyperpolarization. Owing to their ubiquitous presence in smooth muscle cells, including the colonic myocytes and an ability to counteract membrane depolarization, maxi-K channels are believed to play a prominent role during the agonist-mediated excitation-contraction sequence (Carl et al., 1996). Conversely, maxi-K channels may not contribute prominently to the resting membrane potential (Ashcroft, 2000). Maxi-K channels have also been identified within the cell bodies of the intrinsic primary afferent neurons of the small intestine and operate to normalize stretch-induced excitation (Kunze et al., 2000). These intrinsic afferents are an integral part of myenteric neuronal networks involved in regulating secretomotor reflexes within the gut (Clerc and Furness, 2004). In the dorsal motor nucleus of the vagus, a principal source for the extrinsic parasympathetic innervation to the gastrointestinal tract, maxi-K channels influence action potential characteristics of the vagal motor neurons (Pedarzani et al., 2000). Lastly, maxi-K channels are amply expressed within the colonic epithelium (Grunnet et al., 1999) where they may influence absorption and secretion. Thus, maxi-K channels may be uniquely positioned to modulate colonic motility, secretory activity, and visceral sensitivity.
In the following study, we tested the effect of BMS-223131 (Fig. 1), a novel and selective small molecule opener of maxi-K channels (Hewawasam et al., 2003), on normal and stress-exacerbated colonic motility and nociception. We hypothesized that maxi-K channels would play an important role in stress-aggravated increase in enteric secretomotor activity and visceral sensitivity and that BMS-223131 would attenuate the stress-aggravated changes, whereas only minimally affecting normal function.
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Materials and Methods |
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25°C) and humidity (55%) and were allowed unlimited access to standard pellet diet and tap water. Experiments were conducted such that on any given day there was an equal representation of animals from all the treatment groups. In experiments where conscious animals were employed, they were acclimated to handling for 3 consecutive days before the start of the experiment. Animals employed in visceral nociception studies were denied of solid food for no more than 14 h before study. Experimental procedures were approved by the Bristol Myers Squibb Institutional Animal Care and Use Committee. 24-h Fecal Output. In some of the following studies, fecal pellet output was used as an index of colonic motility and transit. Animals were randomly assigned to one of the following five treatments: vehicle (PEG-400, 0.5 ml/kg i.p.), BMS-223131 (2, 6, or 20 mg/kg i.p.), or loperamide (10 mg/kg i.p.). After treatment, animals were placed in individual wire-bottomed cages, and fecal output was monitored at 4, 8, 12, and 24 h. Access to food and water was unlimited throughout the experiment.
Small Intestinal Transit. Small intestinal transit was measured according to a widely used procedure (Miller et al., 1981). We compared the effect of BMS-223131 on transit with that of vehicle (PEG-400) and loperamide. Three groups of overnight fasted rats (N = 9–11) received one of the following treatments at time 0: vehicle (1 ml/kg p.o.), BMS-223131 (20 mg/kg), or loperamide (10 mg/kg). After 2 h, a nonabsorbable marker (polystyrene microspheres) (Biopal Inc., Worcester, MA) was injected into the duodenum through a duodenal cannula. Twenty-five minutes later, the rats were euthanized and the distribution of the marker determined; the geometric center (GC) was calculated for each treatment as described elsewhere (Miller et al., 1981).
Stress-Induced Fecal Output. Rats were randomized and treated as described in the 24-h fecal output study. Ninety minutes after treatment, animals were individually placed in cylindrical restrainers (Stoelting, Wood Dale, IL) for a 1-h period while being subjected to intermittent bursts of air (a total of six 1-min long bursts, delivered at 10 psi through a 4-mm diameter tube and directed at the face and the dorsum). Fecal pellet output was recorded immediately after voiding (wet weight) and after desiccation (dry weight; 40°C in a dry heat oven for a period of 24 h); the difference was regarded as the moisture content and expressed as a percentage of wet weight.
5-HT-Induced Diarrhea. Rats were randomized and dosed as above. Ninety minutes later, animals were subcutaneously injected with serotonin solution (5 mg/kg/ml s.c.) and placed in individual wire-bottomed cages. Fecal output and wet and dry weights were recorded as above for 1.5 h.
Distension-Induced Change in Arterial Pressure in Anesthetized Rats. The general experimental procedure has been described in detail elsewhere (Gebhart and Sengupta, 1996). Briefly, overnight fasted rats were anesthetized with pentobarbital (40–50 mg/kg i.p.) and equipped with femoral artery and venous catheters. After evacuating the colonic contents, a 6-cm long lubricated balloon catheter was introduced into the colon and fastened to the base of the tail for making pressure-controlled distensions. Graded distensions (20–70 mm Hg, 30-s duration) were performed before (baseline), after vehicle (control; PEG-400, 0.5 ml/kg i.v.), and after BMS-223131 (1 or 3 mg/kg i.v.). A 5-min interval between distensions allowed for recovery of the arterial pressure. A distribution time of 10 min was allowed after an intravenous injection.
Distension-Induced Abdominal Withdrawal Response in Conscious Rats. Animals were anesthetized with the ultra-short-acting methohexital sodium (40 mg/kg i.p.; Henry Schein, Melville, NY). Following balloon catheterization, they were allowed to recover for up to 90 min as required. Vehicle (0.5 ml/kg PEG-400) or drug (2, 6, or 20 mg/kg BMS-223131) (5 mg/kg i.p. morphine) treatments were made in a blinded fashion. Seventy-five minutes after treatment, the animal was placed on an elevated Plexiglas platform and allowed to acclimate for 15 min. Colorectal distension was made either manually or using a custom-built electronic distender. In either case, the stimulus was presented as an ascending series of rapid (20 mm Hg/s) distensions (10, 30, 60, and 90 mm Hg; 30-s duration) separated by a 3-min interval. Additionally, in some experiments, the threshold for the abdominal response was determined using a slow-ramp (2 mm Hg/s) distension. Distensions were terminated by venting to room air.
Quantification of the Abdominal Withdrawal Response. Abdominal withdrawal response was graded by an observer blinded to treatment or by recording the associated myoelectrical activity (see below). Visual quantification of the abdominal response was made on a scale of 0 to 3 as described previously (Sivarao et al., 2004). To minimize error, an average from three consecutive trials was recorded for each animal. For threshold determination, the pressure at which a clear retraction of the abdomen was noted over three trials and averaged. When no abdominal response was observed, the threshold was recorded as 60 mm Hg.
Acute Abdominal Myoelectrical Activity. Fasted rats were anesthetized with methohexital sodium and prepared for myoelectrical recording using a modified method from Basmajian and Stecko (1962) as outlined below. Small (2-cm x 2-cm) areas on the neck, the lower back, and on the right lower abdomen were shaved and prepared with a topical disinfectant. One end of a 14-cm-long Teflon-coated stainless steel wire (0.005 inches i.d., 0.008 inches o.d.; AM Systems, Carlsborg, WA) with both ends bared was introduced through a 23-gauge hypodermic needle such that it protruded from the needle tip by about 0.5 cm. The protruded end was folded back onto the needle shaft, and the needle was introduced into the abdominal rectus (superior to the inguinal ligament). After ensuring that the needle tip was in the abdominal wall, it was carefully withdrawn over the indwelling wire, thus trapping the bared electrode in the muscle belly. Using the shaft of a 21-gauge needle as a guide, the other end of the electrode was tunneled subcutaneously to exit from the animal's back. A silk suture on the skin stabilized the electrode in place. Using a similar technique, a shorter electrode (6 cm) was introduced into the subcutaneous space above the neck and served as a reference. A topical anesthetic (2% lidocaine gel) was applied on the skin around the electrode. The animal was prepared for colorectal distension as outlined above. The entire procedure took less than 20 min, after which the animal was returned to its home cage.
After recovery, the animal was placed on an elevated platform, the hypodermic electrodes were connected to a head stage amplifier probe (see below), and the animal was allowed to acclimate for 15 min after which it was subjected to a first series of graded colorectal distensions. Immediately after, the animal was administered with vehicle (PEG-400, 0.5 ml/kg i.p.) or BMS-223131 (20 mg/kg i.p.) and returned to home cage.
Seventy-five minutes after injection, the animal was again placed on the platform and following a 15-min period of acclimation, subjected to a second series of distensions. Myoelectrical activity was filtered (band pass, 300-8000 Hz) and amplified using a commercially available AC-coupled differential amplifier (ExAmp-20KD; Kation Scientific, Minneapolis, MN), digitized at 1000 Hz (Powerlab; ADInstruments, Colorado Springs, CO), and stored for off-line analysis. Myoelectrical spikes with a typical amplitude of >100 µV were discriminated over a noise threshold of 10 to 20 µV and counted. The difference in myoelectrical activity between the first and second series was regarded as a treatment effect.
Protocol for Chronic Visceral Hypersensitivity. Inflammatory insult to the colon in neonates causes visceral sensitivity lasting into adulthood (Al-Chaer et al., 2000). In a modified protocol, female pups in each litter were assigned to one of two treatments from postnatal day 11 to day 21. Pups from one group (sensitized) received intracolonic injections of 0.2 ml of 5% mustard oil in a peanut oil base, whereas pups from the other group were handled but not injected. After day 21, pups were weaned and group housed according to treatments. Nine weeks later, to determine the effectiveness of the sensitization protocol, a sample (9–10) of these animals along with their untreated cohorts were tested for visceral pain thresholds, response to graded distension, and 24-h basal motility, whereas others were employed in distension-mediated abdominal withdrawal studies as outlined above to study the efficacy of BMS-223131.
Drugs and Treatment. Morphine sulfate, serotonin hydrochloride, and loperamide hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO), whereas BMS-223131 (free base) was obtained from the in-house compound repository. Serotonin and loperamide were dissolved in saline, whereas BMS-223131 was dissolved in PEG-400. Morphine sulfate was solubulized in PEG-400 rather than saline to mask its identity during treatment. Unless otherwise stated, treatments were made by the intraperitoneal route in a volume of 0.5 ml/kg. Investigators grading the abdominal withdrawal response were blinded to treatment. Moreover, all drug treatments in these studies were coded by individuals unconnected to the study, and the code was broken only after the completion of the study.
Data Collection and Analysis. To assess treatment effects, a given variable (fecal output, arterial pressure, or abdominal withdrawal index) was recorded for individual rats and averaged within a treatment group and expressed as mean ± S.E.M. Generally, test groups were compared with their corresponding vehicle-treated group using a one- or two-way analysis of variance (ANOVA) with or without repeated-measures as appropriate, followed by Dunnett's or Bonferroni post-tests for significance. Myoelectrical activity was compared within the same subject before and after treatment. Where applicable, dose dependence was tested using a trend for linearity between the treatments and the response. When applicable, the proportion of the sample responding within each treatment group was compared with that of the vehicle group using a chi square test. Statistical analysis was performed using a commercially available software package (GraphPad Prism version 4.00; GraphPad Software Inc., San Diego, CA). Statistical significance was fixed at P < 0.05.
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Results |
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Effect of BMS-223131 on Small Intestinal Transit. No significant difference was noted between the geometric centers of the vehicle-treated group (GC = 5.8 ± 0.25; N = 9) and the BMS-2231331 (20 mg/kg)-treated group (GC = 5.4 ± 0.20; N = 11). The loperamide (10 mg/kg)-treated group showed a significant reduction in small intestinal transit (GC = 4.0 ± 0.30; N = 11) (P < 0.01; one-way ANOVA followed Dunnett's post-test).
Effect of BMS-223131 on Stress-Induced Fecal Output. An hour of restraint coupled with intermittent bursts of air produced a defecatory response in 11 of 11 vehicle-treated animals and 8 of 10 animals in the groups treated with 2 and 6 mg/kg BMS-223131. In contrast, only 3 of 11 of the 20 mg/kg treated animals defecated in response to stress (chi square test; P = 0.003). One-way ANOVA of the mean fecal output revealed a significant (P < 0.0001) difference in group means, whereas a post-test for linear trend between the group means and treatment revealed a significant correlation (slope, –0.14; r2 = 0.36; P < 0.0001). Moreover, fecal moisture content showed a dose-dependent reduction from a mean of 51.1% (vehicle) to 35.1% (BMS-223131, 20 mg/kg; one-way ANOVA followed by Dunnett's post-test; P < 0.01). None of the loperamide-treated animals (0 of 10) showed a defecatory response. The data are summarized in Fig. 3.
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Effect of BMS-223131 on Serotonin-Induced Diarrhea. In preliminary studies, subcutaneous injection of the enteric neurotransmitter serotonin (1–15 mg/kg) caused watery diarrhea in all animals treated with 5 mg/kg or above. At higher doses (10 and 15 mg/kg s.c.), animals showed a marked hypothermia and lethargy that was not observed at 5 mg/kg (data not shown). Therefore, a 5 mg/kg dose was employed in the current study. All animals treated with vehicle or BMS-223131 responded to serotonin injection. At any dose tested, BMS-223131-treated animals were not significantly different from vehicle-treated animals (one-way ANOVA followed by Dunnett's post-test; Fig. 4). In contrast, although 7 of 8 loperamide-treated animals also responded to the serotonin challenge, the mean fecal output was significantly lower (P < 0.01) compared with the vehicle-treated group. In addition, loperamide significantly reduced the fecal moisture content (P < 0.05).
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Effect of BMS-223131 on Distension-Induced Change in Arterial Pressure in Anesthetized Rats. Transient- and distension-mediated reduction of blood pressure is a validated index of visceral pain (Ness and Gebhart, 1988). As expected, colorectal distension produced a transient and stimulus-dependent reduction in arterial pressure in pentobarbital anesthetized rats. Compared with baseline response, vehicle treatment had no marked effect, whereas BMS-223131 produced a dose-related attenuation in the pseudoaffective response with the 3 mg/kg dose significantly attenuating the response to 70 mm Hg distension (Fig. 5; one-way ANOVA with Dunnett's post-test; P < 0.05).
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Effect of BMS-223131 on Distension-Induced Abdominal Withdrawal Response in Rats. Colorectal distension produced clear behavioral responses in awake animals that included ceasing to move or explore at the initiation of distension, abdominal tightening, and retraction that became more pronounced with increasing intraballoon pressure. Compared with the vehicle-treated group, BMS-223131 showed a dose-related reduction in abdominal withdrawal score (Fig. 6), with the 6 and 20 mg/kg treated animals showing a lower (65 and 68%, respectively) mean response to distensions than the vehicle-treated group (100%); the difference between the groups was significant only at the highest distension of 90 mm Hg (one-way ANOVA followed by Dunnett's post-test; P < 0.05). Morphine-treated animals showed a significantly lower (61%) response to distension at 60 and 90 mm Hg compared with the vehicle-treated animals (Fig. 6).
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A slow ramp distension caused in a progressive sequence abdominal tightening, indentation, and withdrawal, making it feasible to identify the threshold pressure for withdrawal. BMS-223131 produced an increase in the threshold for abdominal withdrawal over the vehicle group at a 20 mg/kg dose (Table 1, top row; one-way ANOVA followed by Dunnett's post-test; P < 0.01); lower doses were not significantly effective. As expected, morphine-treated animals showed a clear elevation in threshold compared with vehicle-treated animals (P < 0.01).
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Effect of BMS-223131 on Distension-Induced Abdominal Myoelectrical Activity. In a design meant to eliminate intersubject variability, a group of animals equipped to monitor abdominal myoelectrical activity was subjected to distensions before and after treatment with BMS-223131 (20 mg/kg). A parallel group treated with vehicle (0.5 ml/kg i.p.) served as a control. The data from the drug-treated group are summarized in Fig. 7 (top panel). Overall, BMS-223131 markedly attenuated the response to distension (two-way ANOVA with repeated measures; P < 0.0001). In contrast, stimulus-response curves before and after vehicle treatment were nearly identical in the control group [two-way ANOVA with repeated measures; P = 0.74; Fig. 7 (bottom panel)].
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Visceral Pain Threshold and Colonic Motility in Mustard Oil-Sensitized Rats and Their Control Cohorts. Abdominal distension threshold was determined in animals injected with intracolonic mustard oil as neonates and in their control cohorts; measurements were made 9 weeks after the last mustard oil treatment. In response to ramped colorectal distension, untreated cohorts showed a mean threshold response of 29.1 ± 3.6 mm Hg, whereas mustard oil-treated animals showed a significantly lower threshold of 17.0 ± 2.5 mm Hg (P = 0.02; unpaired t test; N = 10). Moreover, the distension-response curve in the latter was shifted to the left compared with their control cohorts at all distensions (e.g., 2.4 ± 0.2 versus 1.9 ± 0.3 at 90 mm Hg distension), even though the shift was not statistically significant (P = 0.17; two-way ANOVA with repeated measures). Thus, we were able to confirm a sensitization to distension in these rats. No differences in gross behavior or weight (sensitized, 289 ± 7 g; control cohorts, 283 ± 10 g) were observed between the groups. Although the cumulative 24-h basal fecal output was only marginally higher in the sensitized rats (sensitized, 6.0 ± 1.0 g; control cohorts, 5.6 ± 0.9 g), a significantly higher proportion of sensitized animals showed a defecatory response earlier than their control cohorts (7 of 9 versus 2 of 9 by 8 h; chi square test, P = 0.018).
Effect of BMS-223131 on Distension-Induced Abdominal Withdrawal Response in Neonatally Sensitized Rats. BMS-223131 showed a robust and dose-dependent attenuation of visceral pain response in neonatally sensitized rats (Fig. 8). At the peak distension of 90 mm Hg, all doses of BMS-223131 significantly attenuated the abdominal response (one-way ANOVA followed by Dunnett's post-test). Morphine was highly effective in mitigating the distension-induced nociceptive response (Fig. 8). Visceral pain thresholds were determined in vehicle- and drug-treated groups, and the data are summarized in Table 1 (bottom row). At a threshold of 30.4 ± 4.0 mm Hg, the vehicle-treated group showed a higher distension threshold than untreated sensitized rats (17.0 ± 2.5 mm Hg). This was further increased by BMS-223131 (20 mg/kg) to 42.9 mm Hg. However, this or any other dose was not significantly different from the vehicle (Table 1, bottom row; one-way ANOVA followed by Dunnett's test, P = 0.22). On the other hand, morphine produced a significant elevation (P < 0.01) in the distension threshold.
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Discussion |
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), was evaluated in a battery of tests that examined its efficacy against basal and stress-induced fecal pellet output (an index of colonic motility), small intestinal motility, and distension-induced visceral pain. BMS-223131, although only marginally affecting the basal colonic motility, markedly attenuated the stress-induced increase in fecal output and moisture content. Small intestinal transit was unaffected. Furthermore, BMS-223131 dose dependently attenuated autonomic and behavioral responses to colorectal distension in normal as well as chronically hypersensitive rats.
The chronic condition of IBS manifests with symptoms centered around the lower digestive tract: altered bowel function (diarrhea, constipation, or a mix of the two) in conjunction with abdominal pain (Horwitz and Fisher, 2001). A significant number of IBS sufferers attribute acute episodes of worsening symptoms to stressful life events (Drossman et al., 1988). Stress susceptibility appears to be especially marked among IBS patients when compared with healthy cohorts or patients suffering from other lower bowel disorders (Kumar and Wingate, 1985). Even among the healthy, acute psychological or physical stress evokes myoelectrical spiking, strong propulsive and nonpropulsive contractions, and pain (Almy and Tulin, 1947; Schang et al., 1988). In rodents, restraint stress stimulates colonic myoelectrical spiking (Gue et al., 1991), accelerates transit (Williams et al., 1987), disrupts the mucosal barrier (Saunders et al., 2002), and increases mucous secretion (Castagliuolo et al., 1998), outcomes that are mediated in part by activation of myenteric neurons and the release of inflammatory mediators (Groot et al., 2000; Soderholm and Perdue, 2001). Defecation per se in response to an unfamiliar environment is a validated index of stress in rodents (Hall, 1934). It is in this overall context that the effect of BMS-223131 on baseline and stress-induced changes in motility and secretion were evaluated.
BMS-223131 has been shown to be an effective opener of iberiotoxin-sensitive maxi-K channels expressed in Xenopus oocytes (Hewawasam et al., 2003). Additionally, BMS-223131 was found to have little affinity (<50% inhibition at 10 µM) at 70 known neurotransmitter receptors and ion channels including voltage-dependent or ATP-sensitive potassium channels (data not shown). Notably, BMS-223131 showed negligible interaction at opiate receptors, producing <20% inhibition of radioligand binding at µ, 
, or 
receptors (unpublished data). Thus, the primary pharmacological effects of BMS-223131 are likely mediated by maxi-K channels. This conclusion is further supported by a recent report that characterized the mechanism of action of close structural analogs of BMS-223131 in smooth muscle strips (Malysz et al., 2004). Unfortunately, due to the current unavailability of a safe in vivo blocker of maxi-K channels, we were unable to directly confirm the selectivity of the in vivo effects of BMS-223131.
BMS-223131 showed a marked effect against stress-induced colonic motility, although the 24-h basal output was only slightly reduced. BMS-223131 also significantly reduced the fecal moisture content. Small intestinal transit was unaffected. The ability to normalize stress-induced fecal output and secretion while leaving the basal intestinal motility largely unaffected may be desirable since a consistent inhibition of motility could lead to constipation and other complications. Indeed, loperamide whose prolonged use can cause severe constipation was extremely effective in reducing basal as well as stress-induced intestinal motility. On the other hand, a modest and selective reduction in basal colonic motility may be of special help to IBS patients who suffer increased postprandial colonic contractions and a resultant fecal urgency (Camilleri and Ford, 1998).
Serotonin-induced diarrhea was essentially unchecked by BMS-223131. The fecal output of the control group was nearly twice that of the control group in the stress model. We speculate that the 5-HT-induced propulsive and secretory stimuli were too robust to permit adequate suppression by BMS-223131, but not by the high efficacy of loperamide. It may be recalled that when the defecatory stimulus was more modest, as in the case of stress, a high dose of maxi-K opener was nearly as effective as loperamide.
Chronic abdominal pain in the absence of organic disease is arguably the most troubling condition for IBS patients. Numerous clinical studies have demonstrated an increased sensitivity to luminal distension (Ritchie, 1973; Whitehead et al., 1990). Possible explanations include an emotions-driven increase in bowel contractility (Almy and Tulin, 1947), auto-coid sensitization of the visceral afferents in the peripheral nervous system (Bueno et al., 1997) and the spinal cord (Mayer and Gebhart, 1994), and altered processing of visceral inputs by the brain (Naliboff et al., 2001).
The visceral analgesic property of BMS-223131 was first demonstrated by testing its effect on the distension-sensitive change in blood pressure, a bulbospinal reflex activated by noxious stimuli (Ness and Gebhart, 1988). To confirm, we tested the compound's ability to ameliorate distension-induced abdominal withdrawal in fully conscious rats equipped with colorectal balloons. BMS-223131 demonstrated a dose-dependent attenuation of response while elevating the pain threshold significantly at the highest dose. In a confirmatory test that insulated treatment effect from intersubject variability, BMS-223131 attenuated the abdominal myoelectrical activity at 30, 60, and 90 mm Hg, compared with before treatment. In contrast, vehicle-treated animals showed a nearly superimposable response before and after treatment. Lastly, in animals rendered chronically hypersensitive to distension, a condition that attempts to mimic hyperalgesia often seen in IBS patients, BMS-223131 was efficacious at all doses tested. Thus, compared with naive animals, BMS-223131 was even more potent and efficacious in sensitized animals. In a parallel observation, morphine's efficacy and potency also increased in comparison to naive animals. For example, five of nine morphine-treated sensitized rats had distension thresholds greater than 60 mm Hg compared with two of seven of the morphine-treated naive rats.
Treatment effect on distension threshold of sensitized rats was more complex; whereas morphine was highly effective in increasing the pain threshold, BMS-223131 was not. This was puzzling since both morphine and BMS-223131 were markedly effective in reducing the distension-mediated response in sensitized rats. A closer look at the data revealed that the distension threshold (42.9 mm Hg) of the high dose-treated sensitized rats was virtually identical to the high dose-treated naive rats (41.3 mm Hg). However, the vehicle-treated, neonatally sensitized animals showed a higher threshold (30.4 ± 4.0 mm Hg) than the previously tested group of sensitized animals. We speculated that it is either a true variability of response or, more likely, due to the development of some coping mechanism (i.e., to handling stress and treatment in the sensitized rats). This may also explain the somewhat lower abdominal withdrawal scores in these animals. Unfortunately, we were unable to investigate this further.
The constitutive and regulatory role of maxi-K channels within the gastrointestinal tract is just beginning to be understood (Carl et al., 1996). Although maxi-K channels are virtually ubiquitous in the gastrointestinal tract, their dependence on membrane depolarization and elevated Ca2+ make it difficult to conceive a tonic role for them, except in specialized tissues. Instead, throughout the largely phasic gastrointestinal tract, they may operate episodically as part of a negative feedback by opening during agonist-driven membrane depolarization. Indeed, charybdotoxin, another peptide blocker of maxi-K channels, did not affect the electrical or mechanical activity of colonic circular smooth muscle strips in vitro (Carl et al., 1995). However, in the presence of acetylcholine, it increased slow wave duration and amplitude and potentiated acetylcholine-induced contractions. Maxi-K channels were also shown to modulate the excitability of intrinsic primary afferents within the guinea pig enteric nervous system that provide vital feedback to stretch-activated efferent motor neurons and limit distension-induced excitability (Kunze et al., 2000). Consistent with the view that maxi-K channels may play a more significant role in phasic tissue, BMS-223131 did not significantly alter mean arterial pressure (30 mg/kg i.v.; unpublished data) even though maxi-K channels are found in the vascular system.
Acute stress-induced changes within the gastrointestinal tract have been extensively studied and include increased colonic motility (Monnikes et al., 1992; Tache et al., 1993), disruption of mucosal homeostasis (Soderholm and Perdue, 2001), and sensitization of afferent terminals (Bueno et al., 1997; Gue et al., 1997), changes that are at least partly mediated by myenteric neurons and by proinflammatory mediators (see Soderholm and Perdue, 2001). Since maxi-K channels have been demonstrated to be abundant in both the apical and the basolateral colonic epithelium (Grunnet et al., 1999), it may be speculated that their opening may oppose agonist-mediated disruption in the epithelial barrier function and help restore the balance between absorptive and secretive processes. Additionally, a decrease in propulsive activity leading to an increased transit time also encourages net absorption. Based on the abundant presence of the maxi-K channels in the enteric neuromuscular apparatus and consistent with our preliminary findings with in vitro jejunal strips where maxi-K openers reduce spontaneous contractility (data not shown), it is reasonable to speculate that a significant portion of the effects of BMS-223131 may result from directly affecting the enteric secretomotor mechanisms.
Stress has been shown to cause abdominal pain in normal as well as IBS sufferers (Drossman et al., 1988). The neurological basis for this is yet to be completely elucidated, although enhanced activity of the lumbar splanchnic afferents in response to distension has been suggested (Lembo et al., 1994). In animals rendered chronically hypersensitive to visceral stimulus, there too appears to be an enhanced sensitivity of pelvic afferents to luminal distension as well as enhanced firing of the lumbosacral neurons (Su et al., 1997; Al-Chaer et al., 2000). Maxi-K channel openers may reduce this by making the action potentials shorter and less frequent. Additionally, distension-sensitive intrinsic primary afferents within the enteric nervous system may be sensitized by stress-mediated release of neurotransmitters or autocoids (Clerc et al., 2002). Since it is known that they express maxi-K channels (Kunze et al., 2000), it may be speculated that these neurons would be a target for BMS-223131 as well. Even though BMS-223131 permeates only poorly across the blood brain barrier (brain to plasma ratio is 0.17; L. Pajor, unpublished data), we cannot rule out the possible involvement of central maxi-K+ channels in the observed visceral effects. However, we think that the peripheral afferents may be a ready target. Although the exact basis for the efficacy of BMS-223131 against visceral pain remains to be elucidated, we speculate that the cellular excitation invoked by distension and long-term colonic sensitization was dampened by BMS-223131, leading to an overall reduction in distension-mediated sensation.
In conclusion, the current study presents the first functional evidence for maxi-K openers as a novel therapeutic class useful in normalizing dysfunctional motility and visceral sensitivity. Moreover, these effects seem to be relevant in mitigating the symptoms of IBS.
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Footnotes |
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ABBREVIATIONS: IBS, irritable bowel syndrome; maxi-K, large conductance Ca2+-activated potassium; BMS-223131, 4-(5-chloro-2-hydroxyphenyl)-3-(2-hydroxyethyl)-6-(trifluoromethyl)-quinolin-2(1H)-one; PEG, polyethylene glycol; GC, geometric center; 5-HT, serotonin or 5-hydroxytryptamine; ANOVA, analysis of variance.
Address correspondence to: Dr. Digavalli V. Sivarao, Neuroscience Drug Discovery (3CD-422), Bristol Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06067. E-mail: siva.digavalli{at}bms.com
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References |
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