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Departments of *Medical Pharmacology and Anesthesiology, Rudolf Magnus Institute for Neurosciences, University Medical Centre Utrecht, Utrecht, the Netherlands
Address correspondence and reprint requests to Dr. W. H. Gispen, Department of Medical Pharmacology, Rudolf Magnus Institute for Neurosciences, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands. Address e-mail to w.h.gispen{at}med.uu.nl
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Abstract |
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2-adrenergic agonist tizanidine (50 µg/d). The effects of SHU9119 and MTII are most likely mediated through the MC4 receptor, because this is the only MC-receptor subtype present in the spinal cord. We conclude that the chronic administration of MC4-receptor antagonists might provide a promising tool in the treatment of neuropathic pain. IMPLICATIONS: In this study we demonstrated that continuous intrathecal infusion of the melanocortin-receptor antagonist SHU9119 reduces cold and mechanical allodynia in rats with a chronic constriction injury of the sciatic nerve, a lesion producing neuropathic pain.
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Introduction |
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Extensive research with experimental animal models has led to the discovery of an array of potential new drug targets (2). A possible target in the control of neuropathic pain that has received very little attention is the melanocortin (MC) system. Several lines of research have suggested an involvement of the MC system in nociception. Previous studies have demonstrated hyperalgesia in different tests of acute nociception after intracerebroventricular administration of the MCs -melanocyte-stimulating hormone (-MSH) and adrenocorticotropic hormone (3,4). In addition, the analgesic effects of morphine and ß-endorphin are antagonized by these peptides (5).
At the spinal cord level, the existence of a functional MC system is suggested by the presence of the pro-opio-melanocortin-derived peptides adrenocorticotropic hormone and -MSH and the MC4 receptor (6,7). It is interesting to note that these are all colocalized in the superficial dorsal horn, an area that is important in nociception. Taken together, these findings suggest an involvement of the spinal cord MC system in nociceptive transmission.
We investigated the spinal cord MC system as a new drug target for the control of neuropathic pain. We have shown that a chronic constriction injury (CCI) of the sciatic nerve (8), a condition that causes a syndrome similar to human neuropathic pain, induces an increase in 125I-labeled [Nle4, D-Phe4]-a-MSH binding to the spinal cord, suggesting an increase in MC receptors. Furthermore, acute intrathecal administration of the MC receptor antagonist SHU9119 (directly into the cisterna magna) reduced cold and mechanical allodynia in CCI rats, whereas MC4-selective agonists had the opposite effect (9).
Most drugs used in animal models of neuropathic pain are tested in an acute administration paradigm. Because it is a chronic form of pain, drugs that are suitable for chronic administration and provide long-lasting pain relief are interesting from a clinical point of view. In light of the antiallodynic effect of acutely administered SHU9119, in this study we have investigated the effects of this compound, as well as the MC receptor agonist MTII, on chronic administration.
The effects of these MC-receptor ligands were compared with those of the 2-adrenergic agonist tizanidine, for which the antinociceptive and antiallodynic actions in experimental animals have been well documented (10,11).
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Methods |
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Forty-eight male Wistar rats weighing 350–400 g at the start of the study were used. They were socially housed in groups of two or three on sawdust bedding. The animals were kept on a 12:12-h light/dark cycle, with food and water available ad libitum.
Animals were randomly assigned to different treatment groups. CCI rats were treated with vehicle (n = 10), SHU9119 0.5 µg/d (n = 11), tizanidine hydrochloride 50 µg/d (n = 7), or MTII 0.1 µg/d (n = 10). Sham rats (n = 10) were treated with vehicle.
SHU9119 (cyclo-[Nle4, Asp5, D-Nal(2)7, Lys10]-MSH-[4–10]) was synthesized by using 9-fluorenyl- methoxycarbonyl-based solid phase synthesis as reported elsewhere (13) and purified by using reversed-phase preparative high-pressure liquid chromatography to a purity of ±90%, estimated after analysis by analytical high-pressure liquid chromatography at 215 nm. Molecular weight was confirmed by mass spectrometry performed on a Micromass Quattro (Micromass, Manchester, UK). Tizanidine hydrochloride was purchased from Novartis Pharma AG (Basel, Switzerland). MTII (melanotan-II or cyclo-[Nle4, Asp5, D-Phe7,Lys10] -MSH-[4–10]) was purchased from Bachem Feinchemicalien (Bubendorf, Switzerland).
Drugs were dissolved in saline and continuously administered into the cisterna magna via an Alzet osmotic minipump (type 2002; Charles River, Someren, the Netherlands; pump speed 0.5 µL/h for 14 days).
Before surgery, all animals were anesthetized with a single subcutaneous injection of Hypnorm (Janssen Pharmaceutical Ltd., Beerse, Belgium) containing 0.315 mg/mL fentanyl citrate and 10 mg/mL fluanisone (a butyrophenone), at a dose of 0.3 mL/kg body weight. A CCI was produced by placing four loose ligatures of 4-O chromic catgut (Ethicon Inc., Johnson & Johnson, Somerville, NJ) around the nerve as previously described by Bennett and Xie (8). Subsequently the incision was closed with silk sutures, and the animals were allowed a 2- to 3-day recovery period. For the sham condition, the same procedure was performed except for placement of the ligatures.
Two weeks after the initial surgery, a steel cisterna magna cannula (20 x 0.4 mm) was placed as described by Lankhorst et al. (14). An Alzet osmotic minipump (type 2002; pumping rate, 0.5 µL/h; duration, 14 days), filled with the appropriate solution, was implanted into the right flank and connected to the cannula by subcutaneously tunneled polyethylene tubing (PE 25). The incision in the flank was closed with silk sutures. Because the tube connecting the minipump to the cannula was filled with saline and as a consequence of the length of the tube and pump speed, the drugs were delivered into the cerebrospinal fluid (CSF) starting 3 days after implantation of the pumps. This allowed the animals a 3-day recovery period before testing was initiated. The day on which the drugs were delivered into the CSF is referred to as "Treatment Day 1."
Withdrawal latency to a temperature stimulus was measured by immersing the hind paws on each side into a 10°C or 47.5°C water bath. Upon immersion of the paw, an electronic circuit including a timer was closed. Withdrawal of the paw resulted in a discontinuation of the circuit, which stopped the timer, thus allowing a precise registration of the withdrawal latency time. Cutoff time was set at 10 s to avoid skin damage. The interval of time between consecutive tests was at least 10 min to allow restoration of the original foot temperature.
Before testing, each rat was placed in a plastic testing box with a metal mesh floor and allowed to acclimatize to this environment. Mechanical allodynia was determined by measuring the paw withdrawal threshold after probing of the foot plantar surface with a series of calibrated von Frey filaments (Stoelting, Wood Dale, IL), ranging from 1.08 to 21.09 g. Probing was performed only if the hind paw was in contact with the mesh floor, to correct for paw lifting in response to spontaneous pain. Filaments were applied to the midplantar surface of both feet through the mesh floor until the filament bent and were kept in this position for 6–8 s (15). The smallest force that elicited a foot withdrawal response was considered the threshold stimulus.
Temperature and mechanical stimulation tests were performed the day before the beginning of pharmacologic treatment ("baseline value") and at 2- to 3-day intervals thereafter ("Treatment Days 1–10"). Throughout the experiment, body weight was monitored at regular intervals.
Data are plotted starting from the day before the onset of treatment (baseline) through Treatment Day 10. Withdrawal latencies are expressed as mean ± SEM. Withdrawal thresholds to von Frey stimulation are expressed as median and 25th to 75th percentile range, with values plotted on a logarithmic scale.
Differences in body weight between treatment groups were compared by using a repeated-measures analysis of variance. All other data were analyzed with nonparametric tests. To obtain a linear scale of perceived force in the mechanical stimulation test, withdrawal thresholds were converted to the log of the actual bending force of the filament. Statistical analysis was performed on the transformed data. Differences in baseline values were analyzed with a Mann-Whitney U-test. Overall group differences in mechanical and temperature stimulation tests were analyzed by a Kruskal-Wallis test. The effects of the different drugs were compared with the vehicle-treated CCI group and, in case of a significant difference, with the Sham group by using a Mann-Whitney U-test with Bonferroni’s correction. In the mechanical stimulation test, for the CCI group treated with MTII, thresholds on Treatment Days 1 and 3 were compared with baseline values by using a paired Student’s t-test. Results were considered significant when P < 0.05. For the cold and mechanical stimulation tests, the effect of SHU9119 or tizanidine was quantified as the percentage of maximum possible effect (%MPE), by using the following formula:
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Results |
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All CCI groups developed a cold allodynia of the ligated hind paw, as indicated by a significant reduction in withdrawal latency upon immersion in a 10°C water bath (mean predrug value ± SEM for all CCI groups, 5.5 ± 0.7 s). In the Sham group, none of the animals showed signs of cold allodynia (mean withdrawal latency ± SEM, 9.5 ± 0.3 s). The cutoff latency for the test was 10 s. No signs of cold allodynia developed in the contralateral hind paw of either Sham or CCI animals.
There were no significant differences in baseline responses to heat (47.5°C) stimulation between Sham and CCI animals on either side.
CCI animals displayed a tactile allodynia on the ligated side, as shown by a significant decrease in withdrawal threshold to von Frey stimulation (predrug value ± SEM for all groups of 8.5 [6.2–8.5] g) (median [25th –75th percentile]). Sham animals failed to respond to any filament up to the maximum of 21.09 g, as was the case for the contralateral hind paw of CCI animals. These data are summarized in Figure 1.
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Treatment with MTII (100 ng/d) resulted in a transient, nonsignificant decrease in withdrawal latencies of the ligated hind paw (Fig. 2A).
All treatments were ineffective in causing any significant changes in withdrawal latencies to the cold stimulus on the contralateral side. Also, there were no significant differences in withdrawal latency to a heat stimulus between the vehicle-treated CCI group and any of the other treatment groups on either side (data not shown).
As shown in Figure 2B, treatment with SHU9119 (500 ng/d) increased the withdrawal threshold on the ligated side up to 17.0 [13.2–20.9] and 20.9 [13.2–20.9] g (median [25th –75th percentile]) on Treatment Days 1 and 3; % MPE was 73.0% [26.4%–100%] and 100% [46.6%–100%], respectively. These thresholds were significantly higher than those in the vehicle CCI group (P < 0.05) and did not significantly differ from the maximum threshold observed in the Sham-Operated group.
The administration of tizanidine 50 µg/d resulted in a similar increase in threshold. On Treatment Days 1 and 3, thresholds were 13.2 [13.2–13.2] and 13.2 [13.2–20.9] g, respectively (P < 0.05 versus vehicle CCI group), with corresponding %MPE of 46.6% [24.3%–52.2%] and 46.6% [37.7%–100%] (median [25th–75th percentile]). Only on Treatment Day 3 did this threshold not significantly differ from the threshold of the Sham-Operated group.
On Treatment Day 5, thresholds in both the SHU9119 and tizanidine-treated groups decreased again and were not significantly different from the vehicle CCI group for the remainder of the testing period.
Treatment with MTII 100 ng/d produced a decrease in withdrawal thresholds on Treatment Days 1 and 3 (median [25th –75th percentile] of 4.7 [4.1–5.4] and 5.4 [5.0–5.4]), respectively (Fig. 2B). When compared with the vehicle CCI group, this decrease was not significant because of a slightly higher baseline value in the MTII-treated group. However, within the MTII-treated group, withdrawal thresholds on Treatment Days 1 and 3 were significantly lower than the baseline value.
None of the treatments caused any significant changes in withdrawal threshold on the contralateral side (data not shown).
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Discussion |
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The mechanism through which SHU9119 alleviates allodynia remains to be elucidated. Possibly the endogenous MC-receptor agonist -MSH, released in the dorsal horn (6), exerts a tonic influence on nociception. Consistent with this view is the induction of hyperalgesia upon intracerebroventricular administration of MC-receptor agonists (3,4) and the observed upregulation of spinal cord MC receptors in rats with CCI (9); this could contribute to the increased sensitivity associated with the lesion. We hypothesize that the antiallodynic effects of SHU9119 are caused by a blockade of the endogenous -MSH tonus.
MCs modulate a variety of body functions, including fever, immunity, and body weight homeostasis (17). Body weight homeostasis has received much attention because the MC4 receptor plays a role in disorders of energy balance, such as obesity (18). In rats, MTII and SHU9119 respectively inhibit and stimulate food intake when administered intracerebroventricularly (19). With the use of MCs as possible analgesic drugs, changes in body weight are unwanted side effects. Therefore, in this study, drugs were continuously infused into the cisterna magna, downstream of the cerebroventricular system. The osmotic minipumps we used have a very slow infusion speed (0.5 µL/h), thus allowing a very gradual release of drugs without creating pressure in the direction of the ventricular system. The continuity and speed of drug delivery by the minipumps have been verified earlier in our laboratory (unpublished results). Because at the end of the study, pump reservoirs were empty, it is very unlikely that there was backflow from CSF into the pumps. Thus, in this way drugs were delivered directly in the CSF surrounding the spinal cord, where their proposed site of action, the spinal MC4 receptor, is located. The doses of SHU9119 and MTII we used here (0.5 and 0.1 µg/d, respectively) have been shown in rats to readily affect body weight when administered intracerebroventricularly (19). Here we show that the antiallodynia induced by the chronic administration of the MC-receptor antagonist SHU9119 into the cisterna magna is not accompanied by any changes in body weight, further confirming a spinal site of action.
The magnitude of the antiallodynic effects we find with SHU9119 are comparable to those of tizanidine and were so large that responses were normalized to control (sham) levels. Moreover, the cold antiallodynia corresponds well with that described by Leiphart et al. (10) (more than an 80% decrease in paw withdrawal after 50 µg tizanidine intrathecally). In contrast, they describe a %MPE of only 19% in the paw pinch test, whereas we observed a %MPE of up to 46% in the von Frey test with tizanidine 50 µg/d. This might be explained by the fact that the paw pinch test measures pressure hyperalgesia, whereas the von Frey stimulation that we used measures mechanical allodynia. It is, however, difficult to make full comparisons between tizanidine and SHU9119, because only single doses were tested, and the mechanisms of action (involving the -adrenoreceptors for tizanidine and the MC receptors for SHU9119) were clearly distinct.
In contrast to other groups (8,20), we found no differences in heat sensitivity between CCI and control rats. This discrepancy might result from genetic differences between various rat strains, leading to variations in the predisposition for the development of neuropathic conditions (21) or in sensitivity to noxious stimuli (22). In this study, none of the drugs tested induced changes in the CCI animals’ responses to heat. Also, we have previously demonstrated that injecting a large dose of MTII (500 ng) or SHU9119 (1.5 µg) into the cisterna magna does not affect pain perception in control rats (9). Taken together, these data suggest that both tizanidine and the MCs specifically affect the sensory abnormalities associated with the neuropathic pain state without affecting normal pain perception. Our data confirm previous reports of this selectivity of tizanidine effects in neuropathic pain (10,11).
Despite its potent antinociceptive actions in experimental animals, clinical trials performed with tizanidine had less promising results. In patients with trigeminal neuralgia, the efficacy of tizanidine was inferior to that of carbamazepine (23), and there was a rapid recurrence of painful attacks during tizanidine treatment (24).
Levy et al. (11) have reported that upon chronic intrathecal infusion of tizanidine, after several days rats became tolerant to its analgesic effects. In this study, we observed a similar time course of the effects of tizanidine. Surprisingly, we found that MTII and SHU9119 also had only temporarily effects. Possibly the organism quickly adapts to both a lack of tonic -MSH and a continuous overstimulation of MC receptors, as occur with chronic infusion of SHU9119 and MTII, respectively. These adaptations or development of tolerance might be suppressed by using other dosages of drugs or different drug administration regimens, such as repeated injections at various intervals. Future experiments using these strategies will be helpful in further addressing this question.
However, this rapid decline of effects of the MCs should not exclude MC antagonists from further consideration in the treatment of neuropathic pain in humans, because differences in the speed of the development of tolerance between rats and humans do occur. For instance, tolerance to intrathecal morphine develops quickly in rats in several tests of acute nociception (25), whereas in humans, morphine tolerance can take several months to develop, and morphine can provide long-lasting adequate pain relief in cancer pain (26) and nonmalignant pain (27).
In summary, we demonstrate that chronic intrathecal infusion of the MC-receptor antagonist SHU9119 has profound antiallodynic effects in rats with a CCI of the sciatic nerve. We suggest that these effects are mediated through the spinal cord MC4 receptor. SHU9119 seems to be specifically effective in altering the sensory abnormalities associated with the neuropathic pain state, without affecting normal pain perception. Therefore, we suggest that selective MC4 antagonists might be of value in the treatment of neuropathic pain.
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Acknowledgments |
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