Intestinal Inflammation and Morphine Tolerance Alter the Interaction between Morphine and Clonidine on Gastrointestinal Transit in Mice

The protocol was approved by the Institutional Committee of Animal Use and Care. Male Swiss CD-1 mice (20–25 g), housed under controlled standard conditions (12 h light/dark, 22°C temperature, and 66% humidity) were used in the study. Animals had free access to food and water and were acclimated to the housing conditions for at least 1 week before use. All experiments were conducted between 9 am and 3 pm.

Gastrointestinal transit was measured according to procedures used in our laboratory. 13Briefly, food was removed 18 h before the experiment, but animals had free access to water. After 18 h of fasting, a charcoal meal (0.25 ml of a suspension of 10% vegetable charcoal in 5% gum acacia) was administered using a rigid canula introduced through the mouth into the esophagus (orally) in awake animals. In control animals, GIT was evaluated 20 min after charcoal administration, because at this time the marker has traveled approximately 50% of the length of the small intestine, thus permitting the evaluation of the inhibition or increase in GIT (GIT time in the small intestine for control animals was 42–45 min). Animals were killed by cervical dislocation, and the small intestine was separated from the omentum, avoiding stretching. The length of intestine from pyloric sphincter to the ileocecal junction and the distance traveled by the charcoal meal were measured. For each animal, GIT was calculated as the percent of the distance traveled by the charcoal, relative to the total length of the intestine (percent of GIT). The inhibitory effects of drugs on GIT are expressed as a percentage of inhibition of the GIT in a drug-treated animal (test GIT) when compared with the mean GIT measured in a group of vehicle-treated mice (n = 10):

% inhibition =[(vehicle GIT − test GIT)/ (vehicle GIT)]× 100.

Under light ether anesthesia, mice were randomly implanted with 75 mg morphine base or placebo pellets at the nape of the neck. GIT was determined 72 h after pellet implantation. 23 Figure 1shows the experimental design used for the study.

In this study we used a model of intestinal inflammation produced after the intragastric administration of an irritant and cathartic agent (croton oil). Intestinal inflammation was induced by the oral administration of two doses (0.05 ml) of croton oil, 24 h apart, according to the method; control animals received the same volume of saline. In both instances, animals were fasted for 18 h before croton oil or GIT testing (charcoal), except for free access to water. GIT was measured 96 h after the first dose of croton oil. Morphologic changes induced by croton oil have been previously reported by our group 22and were established by optical microscopy. They included a disruption of the mucosa and the infiltration of lymphocytes in the submucosa. In addition, animals treated with croton oil (but not those receiving saline) presented with weight loss (20.8 ± 0.8%) and increased GIT (25.2 ± 2.0%). 22 

Dose–response relationships were first obtained for morphine and clonidine individually, and their median effective doses (ED⁵⁰s) were calculated. In all experiments, six to eight mice were tested per each dose point. Initially, six mice were used for each point; however, when the observed effects in one of the animals deviated from the other five, two additional mice were tested, and the mean value of the eight mice was used for evaluation of the results.

In the present study, ED⁵⁰(a measure of potency) is defined as the dose of a drug or a combination of drugs that produces a 50% inhibition of GIT. Similarly, the ED²⁰, ED⁶⁰, and ED⁸⁰correspond to the doses that produce 20%, 60%, and 80% inhibition, respectively. The term E^max is used to designate the maximal inhibitory effect observed after the administration of a single drug or their combination; actually, the E^maxindicates the maximal effect that can be obtained when increasing the doses of the drugs or their combination. The value is calculated on the basis of the experimental points that form the linear segments of the curves.

Dose–response curves were also obtained with combinations of both drugs. The doses used in fixed potency ratio experiments were multiples of their respective ED⁵⁰s, e.g. , in saline-treated animals, multiples of 1.6 and 0.107 mg/kg of morphine and clonidine, respectively. These mixtures were termed 1:1 combinations (one ED⁵⁰of morphine combined with one ED⁵⁰of clonidine, indicating a 1:1 potency ratio). From the dose–response curves to the combination, the ED⁵⁰and ED⁶⁰ were calculated by linear regression analysis, using the points that formed the linear segments of the curves. Because the dose–response curves of the combination were obtained with a mixture of the two drugs, the results are expressed as total milligrams (resulting from adding up the milligrams of morphine plus the milligrams of clonidine). Similar experiments were also performed in animals that were tolerant to morphine or that had croton oil–induced intestinal inflammation. In each of these cases, different doses were used, but they were always combined at a 1:1 potency ratio (using the ED⁵⁰s obtained under each of the two experimental conditions).

A total of 13 groups of experiments were conducted (table 1). We evaluated dose–response relations for morphine, clonidine, and their 1:1 combination (one ED⁵⁰of morphine plus one ED⁵⁰of clonidine) in the following experimental protocols: (1) naive and (2) morphine-tolerant mice, and (3) saline- and croton oil–treated mice. An additional group of experiments was conducted in tolerant animals in which a complete dose–response curve to morphine was obtained in the presence of a fixed dose of clonidine.

To maintain standardized conditions and minimize errors, control and treated animals were evaluated in parallel on the same day. Animals were randomly assigned to the control or treatment groups, and the technician performing the test (GIT) was blinded as to the treatment administered.

The results are expressed as percent inhibition of GIT. Statistical calculations were performed as described by Tallarida and Murray. 24Because all dose–response curves had a linear segment, effective doses (EDs) were determined by linear regression analysis of this segments, based on at least five different doses (data points) and six to eight mice per dose. Individual slopes of the dose–response curves were compared by Student t  test, according to the test for parallelism described in Tallarida and Murray. 24To evaluate the interaction of morphine and clonidine, we used two methods of analysis: isobolographic analysis and fixed-dose analysis.

Isoboles are graphic representations of the doses of two (or more) drugs used individually or in combination that are needed to produce a specified level of response (e.g. , ED⁵⁰). The doses were obtained from their respective dose–response curves. In constructing an isobole, the dose of each drug that individually produces a given level of response is plotted on the axes of the graph. A diagonal line is then drawn to join the isoeffective doses. The doses of each drug used in combinations producing the same effect are then plotted. Points falling on the diagonal line represent zero interaction (additivity), whereas those located above and below are antagonistic and synergistic, respectively. Mean and SEM values were calculated for all the doses plotted, and the doses expected in the absence of interaction (those on the isobole line) were compared with those obtained experimentally (observed) using a Student t  test. The diagonal noninteraction line of the isobole is described by the equation da/Da + db/Db = 1. In this equation, Da and Db are the doses of the two drugs that individually produce the specified level of response; da and db are the combined doses that also produce that response. The sum of these quotients is the interaction index. An interaction index of 1 indicates additivity (no interaction). If the index differs from 1 an interaction exists: either synergy (index < 1) or antagonism (index > 1). 25,26Using this type of analysis, an interaction can be established only for a given drug ratio and level of effect, and the results cannot be extrapolated to other drug combinations.

In the fixed-dose experiments, we compared a dose–response curve of morphine alone with a dose–response curve to morphine in the presence of a small, fixed dose of clonidine (0.02 mg/kg). This dose was selected because it produces a 10% inhibitory effect that, although small, can be measured and evaluated for statistical purposes. In these experiments, the data was plotted in a log dose–response manner, and the E^max was estimated. In this type of analysis, when the dose–response curve of the combination is shifted to the right, an antagonistic interaction is presumed, whereas a displacement to the left suggests synergy. The drug interaction is statistically corroborated when the sum of the effects produced by each agent alone (the expected effect) is significantly different from the observed effect of the combination. 27Two-factor analysis of variance was used to demonstrate the presence of statistical differences between two treatment groups. We compared the responses obtained with morphine alone with those of morphine in the presence of 0.02 mg/kg of clonidine, and from these data the effects of the drugs, dose, and their interaction were determined. 28In this calculation, a significant effect of the drugs indicates statistical differences between the two treatment groups; a significant effect of the dose indicates that the observed responses vary significantly between different doses tested, and a significant interaction shows that the differences between the treatment groups depend on the dose administered. If all factors (drug, dose, and interaction) are significantly different, the analysis indicates that the effects of the combination are different from additivity, whereas if the interaction is not statistically significant, the effects are considered to be additive. To identify the type of interaction, the dose–response curves obtained with each treatment are also shown. This method is not applicable when dose–response curves are parallel, because it cannot differentiate between parallel–additive and parallel–synergistic or –antagonistic combinations. 29,30 

Drugs used in the study were obtained from several sources: morphine sulphate from Alcaiber S.A., Madrid, Spain, and clonidine hydrochloride from Research Biochemicals, Wayuland, MA. All drugs and their combinations were prepared in pyrogen-free water just before use and administered in a final volume of 10 ml/kg. Drugs were injected subcutaneously at the nape of the neck 30 min before the administration of charcoal, and GIT was assessed 20 min later. The time of injection was selected to coincide with the peak effect for gastrointestinal inhibition after subcutaneous administration of the drugs, occurring approximately 45 min after injection. 31–34 

Dose–response relationships to morphine, clonidine, and their 1:1 combination were obtained in naive mice (implanted with a placebo pellet), and the results are shown in figure 2(top). In these animals, calculated ED⁵⁰values for morphine and clonidine were 1.52 ± 0.15 and 0.09 ± 0.008 mg/kg, respectively (table 2); thus, clonidine was approximately 16.8 times more potent than morphine as an inhibitor of GIT. Unlike the dose–response curves for individual drugs, the curve of the morphine–clonidine combination had produced a maximal effect (E^max) of 60%, and then formed a plateau, that is, became asynthotic (parallel to the abscissa). Doses of up to 2.41 mg/kg of the 1:1 combination failed to produce a greater effect. These results coincide with those previously reported by our group in nontreated animals. 16In the present experiments, the dose–response curve for the combination morphine–clonidine in naive animals had an ED⁵⁰value of 0.506 ± 0.084 mg/kg (total milligrams of the combination), of which 0.478 ± 0.08 mg/kg was morphine and 0.028 ± 0.004 mg/kg was clonidine. The linear segments of all of the three curves were parallel, thus permitting the comparison of their potencies.

Dose–response relationships for morphine, clonidine, and their 1:1 combination were also obtained in morphine-tolerant mice (implanted with a morphine pellet), and the results obtained in these animals are shown in the lower panel of figure 2. In tolerant mice, the dose–response curve to morphine was shifted to the right, and the potency of morphine (ED⁵⁰9.73 ± 0.80 mg/kg) decreased approximately 6.4 times when compared with placebo, thus demonstrating the presence of tolerance to morphine. No significant changes in the dose–response curve to clonidine were observed in morphine-tolerant animals (ED⁵⁰0.09 ± 0.006 mg/kg;table 2). The results show the absence of cross-tolerance between clonidine and morphine. When morphine–clonidine combinations (1:1 potency ratio) were administered to tolerant animals, the resulting dose–response curve had an E^max of 67.6%, after which the curve showed a plateau; the response remained unaltered even after doses of 14.6 mg/kg of the combination were administered. The ED⁵⁰of the 1:1 combination in tolerant animals was 0.131 ± 0.09 mg/kg, of which 0.13 ± 0.09 was morphine and 0.0013 ± 0.0005 mg/kg was clonidine. Thus, in tolerant animals, 50% inhibition of GIT was achieved by significantly lower doses of the drug mixture.

When the ED⁵⁰s in naive and tolerant mice were compared, the data showed: (1) a sixfold decrease in the potency of morphine in tolerant mice (a demonstration of tolerance); (2) the absence of cross-tolerance between morphine and clonidine; (3) that the combination regimen was 3.18 times more potent than morphine alone in placebo-treated animals; and (4) that the combination regimen was 74.8 times more potent than morphine alone in tolerant animals.

The isobolographic and interaction index analysis of the morphine:clonidine combination showed that for the linear portion of the curve (i.e. . levels of effect up to the E^max), the interaction was synergistic, both in naive and tolerant mice. However, the interaction became antagonistic when the doses of the combination were further increased (on the plateau). Table 3shows the type of interaction obtained at different levels of effect and that the magnitude of synergy seems to be enhanced in morphine-tolerant animals.

Because the E^max value of the combination was approximately 60% in both naive and tolerant animals, the interaction at the 60% level of response was evaluated using both interaction indexes and isobolographic analysis. Two sets of values were used: the estimated ED⁶⁰from the linear segments of the dose–response curves (fig. 2), and the doses (obtained experimentally) that produced a 60% effect on the plateau, where the dose–response curves of the combination cross the curve of morphine (fig. 2). The experimentally obtained values used in this calculation were 2.41 mg/kg in naive animals, which produced a 60.3 ± 1.9% inhibition, and 14.6 mg/kg in tolerant animals, which produced 59.7 ± 1.7% inhibition. Our results show that at the 60% level of response, the interaction was synergistic with respect to the linear segment of the curve, but it became antagonistic on the plateau. Figure 3graphically represents the isobolographic analysis of the interaction at the 60% effect level in naive and tolerant animals. The isoboles graphically illustrate that the degree of synergy between morphine and clonidine is significantly increased in morphine-tolerant mice (Student t  test, P < 0.05), and that at the 60% level of response the interaction can be synergistic or antagonistic, depending on the doses evaluated.

To further characterize the morphine–clonidine interaction in tolerant mice, we obtained a dose–response curve to morphine in the presence of a fixed dose of clonidine (0.02 mg/kg) that produced approximately a 10% inhibition of GIT (fig. 4). In these experiments, clonidine induced a shift to the left of the dose–response curve of the opioid at morphine doses of 0.6–3 mg/kg. When larger doses of morphine were used (5–60 mg/kg) the dose–response curve reached a plateau. In constructing the curves, the responses were separated into two segments, the first one comprising the linear segment of the dose–response, and a plateau segment starting at 3 mg/kg (the inflection point of the dose–-response curve) and including all of the higher doses tested (up to 60 mg/kg). The dose–response curves obtained in these experiments (fig. 4) were not parallel, and consequently we were able to use a two-factor analysis of variance to establish the presence of an interaction. We compared the dose–response curve of morphine alone to the ascending linear segment of the dose–response curve of the drug combination and to the horizontal line that formed the plateau. Each segment was analyzed separately. Analysis of the ascending segment showed significant effects attributable to the drugs, the dose, and their interaction (P < 0.0001). However, at the plateau, no significant effect attributable to the drugs was observed (P < 0.147), whereas the dose (P < 0.0001) and the interaction (P < 0.0001) remained highly significant. The results demonstrate the presence of an interaction between morphine and clonidine at both segments of the response.

The type of interaction can also be determined by comparing the inhibitory effects of morphine to the effects of morphine plus a fixed dose of clonidine. For each dose, synergy can be established when the sum of the observed effects of each agent individually (at each point tested) is smaller than the effect of the combination. In our experiments, a dose of 0.02 mg/kg clonidine produced a 10.8 ± 1.9% inhibition of GIT. When the effect produced by clonidine alone was added to the effect produced by each dose of morphine alone (from 1 to 3 mg/kg), the observed effects of the combination were always greater than expected (Student t  test, P < 0.01). Antagonism was shown in these experiments by the existence of a plateau in the combination dose–response curve; increasing doses of morphine (from 5 to 60 mg/kg) in the presence of clonidine produced smaller effects than morphine alone (fig. 4). These experiments confirm the results obtained with the isobolographic analysis of the interaction.

Dose–response curves to morphine, clonidine, and morphine:clonidine (1:1 potency ratio) were obtained in mice with intestinal inflammation. Animals receiving oral saline served as control. No significant differences were observed between the curves obtained in placebo-implanted mice (fig. 2) and oral saline controls; thus, the dose–response curve to the combination was linear up to an E^max of 60% and then reached a plateau. Figure 5shows the dose–response curves obtained in controls and during inflammation. In the latter experiments, the dose–response curves to morphine and clonidine individually were each shifted to the left, demonstrating that inflammation increased the potency of the agonists. However, the dose–response curve of the morphine–clonidine combination was shifted to the right and had a similar E^max(80%) as those of the individual drugs. Thus, the presence of inflammation significantly altered the characteristics of the dose–response curve to the morphine–clonidine combination. Table 4shows the potency (ED⁵⁰values) of morphine, clonidine, and their combination in control animals and during inflammation. In controls, the calculated ED⁵⁰values for morphine and clonidine were 1.62 ± 0.16 and 0.107 ± 0.01 mg/kg, respectively, whereas the ED⁵⁰of the morphine–clonidine combination was 0.555 ± 0.075 mg/kg. The presence of inflammation increased the potency of morphine approximately 9.3 times, 22and that of clonidine was increased 7.1 times, whereas the potency of the combination remained unaltered.

The morphine–clonidine interaction was evaluated in the presence of intestinal inflammation and saline controls, using both isobolograms and interaction indexes. In control animals, the interaction was synergistic up to the ED⁶⁰value and became antagonistic when the dose–response curve reached a plateau (fig. 5, top). During inflammation, the analysis of the morphine:clonidine interaction demonstrated antagonism at all levels of effect, and the values of the interaction indexes at the ED⁵⁰are shown in table 4. Figure 6shows the isoboles obtained for the morphine:clonidine interaction in controls and during inflammation, both at the 50% level of response. The results demonstrate that the presence of intestinal inflammation changes the type of interaction between morphine–clonidine when they are administered at a 1:1 potency ratio.

Morphine and clonidine are clinically used in humans, alone or in combination, in the treatment of acute and chronic pain. Although the drugs have independent mechanisms of action, they share several properties such as analgesia, sedation, ileus, constipation, and the development of tolerance after repeated administration. 23,35Beneficial (e.g. , analgesia) and side effects occur simultaneously, and to decrease the latter, morphine and clonidine have been successfully used in combination. When two or more drugs are administered simultaneously, the type of interaction (synergy, antagonism, no interaction) for all pharmacologic effects should be evaluated before the combination can be considered useful.

Intestinal disorders are very frequent in patients treated with opioids. Although the precise incidence and extent of the GIT effects in humans is unknown, studies performed in experimental animal models generate information that can serve as guidelines for human studies. Sensitivity of mice to morphine is quantitatively different than in humans (less than one tenth as potent), but their effects on pain perception and inhibition of GIT are qualitatively similar. 6The gastrointestinal effects of opioids in small mammals (guinea pig, rat, mice) and humans share a common characteristic: they all slow the propulsion of intestinal contents and cause constipation. Several animal models have been successfully used to screen the effects of opioids for their antidiarrheal activity, 7and extrapolations to humans using the transit of a charcoal meal in rodents have been instrumental in the development of gut selective, clinically effective, antidiarrheal opioids. 8Moreover, the same types of opioid receptors, mainly μ and δ, located both at central and peripheral (intestinal) sites mediate the GIT effects of opioids in rodents and humans. 36,37However, the receptors differ considerably in their relative density and distribution in the gut. Thus, the results obtained in animals can be used only as a guideline or to complement clinical studies.

The effects of the combination of morphine and clonidine on GIT in situations such as morphine tolerance or intestinal inflammation have not been investigated. Because these circumstances occur in clinical practice, a preliminary assessment of the effects of the combination using a well-validated animal model could be useful to complement (but not replace) subsequent studies in humans. Similarly, tolerance to the constipating effects of morphine in humans has not been carefully evaluated or quantified. Chronic treatment with morphine produces tolerance to the analgesic effects of the drug. Consequently, increasing doses of opioids are required to obtain adequate analgesia. These patients present with constipation that is probably related to the higher doses of opioids administered. Thus, it is possible that tolerance develops to the constipating effects of morphine at a different rate, and possibly to a lesser degree, than to analgesia. For example, in mice, the development of tolerance to the gastrointestinal effects of opioids has been shown to follow a different pattern than tolerance to other pharmacologic effects such as antinociception. 38In our model, tolerance was manifested by a sixfold decrease in the potency of morphine, thus supporting the results obtained by other investigators in small rodents. 39,40Until the pattern and extent of tolerance to the GIT effects of morphine in humans is fully investigated, we could assume that the human gut behaves in a qualitatively similar manner to other species.

The gastrointestinal effects of α²-adrenergic agonists, such as clonidine, and the presence of α²-adrenergic receptors in the intestine of small rodents have been clearly demonstrated. 23,41However, in humans, the intestinal effects of systemic clonidine are poorly characterized, although several studies demonstrate the constipating/antidiarrheal effects of the drug in humans. 9–12 

The results of the present study show that the antitransit effects of the combination of morphine and clonidine can be synergistic or antagonistic according to the level of effect of the drugs combined. After defining the interaction between morphine and clonidine on GIT, we wanted to determine if the combination would behave in a similar manner in two distinct conditions: previous exposure to opioids (tolerance) and intestinal inflammation. Chronic administration of morphine induces tolerance to the antinociceptive and antitransit effects of morphine. Clonidine has been reported to delay and reduce the development of tolerance to the analgesic effects of morphine, 20although the mechanisms and clinical relevance of these findings are not well established. In the present study we investigated the effects of a 1:1 (potency ratio) morphine–clonidine combination in morphine-tolerant animals to establish the possible benefits of drug combinations on the inhibition of GIT. The drug ratio used in this study (1:1) was selected because preliminary studies revealed a decrease on the inhibition of GIT with increasing doses of the combination. Such antagonism was considered particularly relevant because it could reflect a dissociation of the beneficial and adverse effects of the drugs involved. In control conditions, our results showed that clonidine was approximately 17 times more potent than morphine as an inhibitor of transit. The dose–response curve to the combination was linear but had an E^max of 60%. The decrease in the E^maxof the combination suggests (1) a decrease in receptor affinity or density, 42(2) a partial antagonism revealed when the drugs are used in combination, and/or (3) a common pathway in the intracellular signal transduction mechanisms that mediate the antitransit response. 43 

The results in tolerant animals showed that no cross-tolerance existed between morphine and clonidine. In these animals, the dose–response curve of the combination was shifted to the left and also had an E^maxof 60%. When the doses of the combination were increased, the dose–response curve developed a plateau. The E^maxand the plateau were similar in naive and tolerant animals, showing that morphine tolerance did not alter the response when high doses of the combination were used (fig. 2). The analysis of the interaction showed that synergy was significantly enhanced in tolerant animals.

Changes in the type of interaction (from synergistic to additive) between morphine and clonidine administered intrathecally have been reported in tolerant mice for antinociception. 18In this report, linear dose–response curves for the morphine–clonidine combination (1:1) with E^max values of 80% or higher were obtained. These results, together with our findings, suggest that morphine and clonidine (1:1 potency ratio) interact in a different manner when producing spinal antinociception and inhibition of GIT (a combination of central and peripheral-intestinal effects). Tolerance converts synergy to additivity for the antinociceptive effects, while the synergistic interaction for the antitransit action (< a 60% response) is significantly enhanced. The different interaction between morphine and clonidine for the antinociceptive and antitransit effects in tolerant mice could be related to the different routes of administration and/or to other methodologic variables. However, our results support those of other investigators when evaluating the antinociceptive effects of morphine and clonidine combined in morphine-tolerant mice, after spinal administration. 44 

The effects of the combination of morphine and clonidine were also evaluated in a model of intestinal inflammation. In this model, peripheral opioid and α²-adrenergic receptors have been reported to be “sensitized” or “up-regulated” according to the type and duration of the inflammatory process. 21,22Our results show that during inflammation, the potencies of morphine and clonidine increased nine and seven times, respectively. However, the potency of the combination remained unaltered, and the dose–response curve of the combination was linear up to an E^max value > 80%. This observation would be consistent with an increase in the number of “functional” receptors. Further analysis of the data showed that during inflammation, the interaction between morphine and clonidine was transformed from synergistic to antagonistic at all levels of response. These data suggest that even in the presence of a higher “density” of receptors, both agonists could interact and compete with limited levels (“pools”) of common membrane/intracellular substrates required for signal transduction.

In conclusion, our results show that interaction between morphine and clonidine on GIT is altered during morphine tolerance and in the presence of intestinal inflammation. The effect of the combination is increased during morphine tolerance, where it becomes highly synergistic; however, in the presence of inflammation, the synergistic interaction is converted to antagonistic. The antagonism between morphine and clonidine in both experimental conditions could be explained by a common pathway in the intracellular signal transduction mechanisms that mediate the antitransit effects of both drugs.

The authors thank Sergi Leanez for excellent technical assistance.