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Effect of JTC-801 (nociceptin antagonist) on neuropathic pain in a rat model
Neuroscience Letters 351 (2003) 133–136 www.elsevier.com/locate/neulet
Effect of JTC-801 (nociceptin antagonist) on neuropathic pain in a rat model Hidemichi Suyama*, Masashi Kawamoto, Syafruddin Gaus, Osafumi Yuge Department of Anesthesiology and Critical Care, Division of Clinical Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima, 734-8551, Japan Received 24 February 2003; received in revised form 14 April 2003; accepted 17 April 2003
Abstract JTC-801, a nociceptin antagonist, may alleviate neuropathic pain because nociceptin has been shown to produce pain modulation. We report that JTC-801 alleviates heat-evoked hyperalgesia and investigated the possible protective effect on osteoporosis induced by chronic constriction injury (CCI) in rats. JTC-801 was given orally to rats with CCI at 0% (vehicle), 0.03% (low dose), or 0.06% (high dose) in food. Paw withdrawal latency (PWL) to heat, bone mineral content (BMC) and bone mineral density (BMD) of the whole tibial bone were measured. JTC-801 dose-dependently normalized PWL. Although JTC-801 did not inhibit a CCI-induced decrease in BMC and BMD, it inhibited an increase in the number of osteoclasts in the JTC-801 groups. JTC-801, given orally in food, alleviated heat-evoked hyperalgesia in CCI rats, suggesting that it is useful for the treatment of neuropathic pain. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: JTC-801; Nociceptin antagonist; Neuropathic pain; Rat; Neuropathic osteoporosis
Nociceptin is an endogenous ligand for the opioid receptorlike1 (ORL1) G-protein coupled receptor [11,12]. This ligand and its receptor are localized in various regions of the central nervous system, which are associated with nociception [4]. The pain modulation of nociceptin has been shown in various rat pain models [5,16]. However, most reports regarding nociceptin pain modulation used intrathecal or intracerebroventricular administration [5,16], while only a few have utilized systemic nociceptin [5,9]. JTC-801, N-(4amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl) benzamide monohydrochloride, was recently synthesized as a novel and oral active ORL1 receptor antagonist [13]. It has been demonstrated that JTC-801 antagonizes ORL1 receptor response, and also has efficacious and potent antinociceptive effects in acute pain animal models, not only by intravenous injection but also oral administration [15]. Chronic constriction injury (CCI) of the sciatic nerve produces neuropathic pain as well as osteoporosis in the ipsilateral limbs of test animals [14]. JTC-801, a nociceptin antagonist, may alleviate such neuropathic pain. In the present study, we attempted to confirm that JTC-801 * Corresponding author. Tel.: þ 81-82-257-5267; fax: þ81-82-257-5269. E-mail address: [email protected] (H. Suyama).
alleviates heat-evoked hyperalgesia and investigated the possible protective effect on osteoporosis induced by CCI in rats. All animal protocols were approved by our institutional animal care committee. We prepared 24 male Sprague– Dawley rats with CCI, according to the following protocol. The rats, each weighing between 200 and 300 g and approximately 7 weeks old on the day of surgery, had an experimental nerve injury induced in their right hind limb. They were anesthetized with sodium pentobarbital (50 mg/kg, i.p.; supplemented as necessary). Four loosely constrictive ligatures of 4.0 chromic gut were tied around the right sciatic nerve at the mid-thigh level, as described in detail elsewhere [1]. In each animal, an identical dissection was performed contralaterally, omitting the ligatures (sham operation). The muscle and skin were then closed in layers. Following surgery, the rats were housed individually in cages for at least 5 days. The rats were divided into three groups and given JTC801 (Japan Tobacco Inc., Tokyo) orally at 0% (vehicle), 0.03% (low dose), or 0.06% (high dose) in food from 10 to 38 days after CCI surgery. The thermal nociceptive threshold was measured with a device similar to that used by Hagreaves et al. [10]. Paw
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00502-0
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withdrawal latency (PWL) was measured on both sides, with 10 min intervals between successive trials. Among the 24 rats, 18 showed ipsilateral PWL that was at least 1 standard deviation (SD) shorter than the mean PWL in the controls. PWL to heat was measured every 3 or 4 days after surgery for 38 days, after which the animals were killed for examination of osteopathic changes. Tibial bones were removed and fixed in 3.7% formaldehyde. Bone mineral content (BMC) and bone mineral density (BMD) of the whole tibial bone were measured using a dual energy X-ray absorptiometer (Aloka DCS600EX, Tokyo) 2 days after initiation of fixation. The absorptiometer used enabled measurement of BMC and BMD based on changes in the transmission of monochromatic X-rays at dual low energy levels with a switched pulse method. We used a scan field of 130 £ 150 mm, a scanning speed of 40 mm/s, and a slice thickness of 1 mm. The coefficient of variation, a statistical index expressing measurement precision, was less than 1% in preliminary measurements (data not shown). After measuring BMC and BMD, the fixed tibial bones were then decalcified in 10% EDTA in Tris buffer at pH 7.4 for 3 –4 weeks, after which they were cut into 5 mm sections and stained for tartrate resistant acid phosphate (TRAP) activity to identify osteoclasts, according to Cole’s method [6]. Briefly, Burstone’s complete medium (TRAP solution) for acid phosphate was prepared by dissolving 4 mg of naphthol AS-BI phosphate substrate (Sigma Japan, Tokyo) in 0.25 ml of N,N-dimethyl formamide, followed by the addition of 25 ml of 0.2 M acetate buffer (pH 5.0), 35 mg of Fast Red (Sigma Japan, Tokyo) as the coupling agent, and two drops (60 ml) of 10% MgCl2. Bone sections were incubated at 37 8C for 30 min with TRAP solution. After incubation, the slides were washed for 15 min in running water, allowed to air-dry at 25 8C, and then counterstained with hematoxylin for approximately 10 s. Coverslips were prepared for mounting with Permount (Fisher Scientific Co.,
New Jersey), and the preparations were examined for the presence or absence of reactive products. We counted the number of TRAP-positive multinucleated osteoclasts per unit area (1 mm2) at a position 1 mm from the distal end of the metaphysis. Differences in PWL (DPWL: ipsilateral PWL minus contralateral PWL) among the groups were tested using the Kruskal –Wallis test, followed by a Mann – Whitney U-test. The sequential difference of DPWL in each group was tested using one-way ANOVA, followed by a Bonferroni/Dunn test. Differences in BMC and BMD between ipsi- and contralateral tibiofibula were tested using a Wilcoxon signed-rank test. P , 0:05 was considered statistically significant. All rats in the study showed changes in the appearance of the nerve-injured paw and demonstrated guarding of the paw, as described previously by Bennett and Xie [1]. Characteristically, the toes were ventroflexed and the paw was often held in an elevated position. The rats also tended to contact the medial portion of the ipsilateral hind paw with the glass floor. No such behavioral changes were observed in the contralateral hind paw. No significant differences in the rats’ body weights were observed in all groups throughout the study. At 1 week before surgery, 1 and 10 days after surgery, and 1, 2, 3, and 4 weeks after treatment, the mean ^ SD body weight (g) was 217 ^ 8.3, 289.8 ^ 19.6, 342.8 ^ 26.1, 383.9 ^ 29.7, 408.7 ^ 33.2, 435.4 ^ 39.7, 461.8 ^ 42.4 (control group), 216.4 ^ 3.5, 302.5 ^ 8.6, 355.8 ^ 20.6, 393.9 ^ 18.0, 419.6 ^ 21.1, 443.5 ^ 23.7, 469.9 ^ 29.1 (low dose group), and 216.3 ^ 6.0, 292.3 ^ 21.1, 344.5 ^ 41.5, 380.3 ^ 48.8, 405.4 ^ 59.4, 430.1 ^ 67.1, 452.8 ^ 71.7 (high dose group), respectively. JTC-801 at the high dose (0.06% in food) normalized DPWL 3 days after the start of administration and throughout the feeding period, while that at the low dose (0.03% in food) normalized DPWL 10 days after the start of
Fig. 1. Analgesic effect of JTC-801 on heat-evoked hyperalgesia. †P , 0:01 vs. preparation within a group. *P , 0:01 vs. after surgery within a group. { P , 0:05 vs. vehicle group among groups.
High dose
Low dose
Data values are means ^ SD. Baseline, 1 week before the surgery; POD, postoperative (the surgery) day; PTD, post-treatment days. *P , 0:05, **P , 0:0001 compared with the sham side, by Wilcoxon signed-ranks test.
13.5 ^ 1.8 8.7 ^ 2.2** 12.6 ^ 2.4 10.1 ^ 2.3* 13.9 ^ 2.3 13.1 ^ 2.4 13.9 ^ 2.3 9.8 ^ 2.0** 12.5 ^ 1.6 10.1 ^ 2.3** 13.1 ^ 2.0 12.7 ^ 1.9 13.5 ^ 3.0 9.1 ^ 1.7** 12.6 ^ 1.6 10.7 ^ 2.8** 13.6 ^ 2.3 13.5 ^ 2.2 12.8 ^ 2.3 8.5 ^ 1.5** 12.3 ^ 1.7 10.1 ^ 1.8** 14.9 ^ 2.8 13.6 ^ 2.8* 13.9 ^ 2.2 9.8 ^ 1.6** 13.4 ^ 2.1 10.7 ^ 1.7** 13.7 ^ 2.5 12.4 ^ 2.3* 13.0 ^ 2.7 8.3 ^ 2.5** 12.8 ^ 1.9 9.9 ^ 1.8** 13.4 ^ 2.8 11.5 ^ 2.7** 13.7 ^ 2.0 9.9 ^ 2.1** 13.9 ^ 1.6 9.6 ^ 2.4** 13.4 ^ 1.9 11.9 ^ 1.7** 15.0 ^ 2.1 10.9 ^ 1.8** 13.9 ^ 2.0 9.3 ^ 1.9** 14.5 ^ 1.9 10.3 ^ 1.8** 12.4 ^ 2.2 12.3 ^ 2.0 12.0 ^ 1.4 11.8 ^ 1.5 13.1 ^ 1.9 13.3 ^ 1.8 Vehicle
Sham side CCI side Sham side CCI side Sham side CCI side
POD 7
12.9 ^ 1.8 9.2 ^ 2.1** 14.0 ^ 2.7 8.6 ^ 1.8** 13.2 ^ 1.4 11.6 ^ 1.9**
PTD 24 PTD 21 PTD 17 PTD 14 PTD 10 PTD 7
135
Baseline
PTD 3 Table 1 Data for PWL
administration and throughout the study (Fig. 1). The normalization of DPWL was dose-dependent at the time of measurement (1 – 4 weeks after the treatment) (Fig. 1). DPWL in the vehicle group did not change from 1 week after surgery to the end of this experiment. Table 1 summarizes the PWL data for each group. BMC of the tibial bone on the CCI side was significantly lower than that on the contralateral side in all three groups, with a median (range) reduction of 62 (27 –82), 46 (21 – 64), and 45 (30 –76) mg in the control, low dose, and high dose groups, respectively (Table 2). BMD of the tibial bone on the ipsilateral side was also significantly lower than that on the contralateral side in all three groups, with a median (range) reduction of 15 (7 – 27), 11 (1 – 14), and 9 (4 –19) mg/cm2, respectively (Table 2). The number of osteoclasts was increased in the CCI side compared with the sham side in the vehicle group, however, no significant changes were observed in the number of osteoclasts between the CCI and sham side in the low and high dose groups. Further, the median increase (range) in the number of TRAP-positive multinucleated osteoclasts was 6.2 (2.1 – 18.9) cells/mm2 in the control, 2.3 (2 7.2 – 11.9) cells/mm2 in the low dose, and 3.0 (2 3.6 – 13.9) cells/mm2 in the high dose group (Table 2). The present results are the first known to demonstrate the alleviation of heat-evoked hyperalgesia with an oral nociceptin antagonist, JTC-801, in CCI rats. In sciatic nerve injury model rats, molecular changes (m-opioid receptor, alfa and beta subunits of the sodium channel, galanin) in the spinal cord and dorsal root ganglia have been shown [2,8]. Nociceptin and its receptor appear at the spinal cord level in nociception [16]. During the development of neuropathy due to CCI of the sciatic nerve, two types of ORL1 receptors are expressed in the lumbar spinal cord and L5-L6 dorsal root ganglia [3]. Intracerebroventricular and intrathecal nociceptin inhibit noxious thermal pain, mechanical noxious stimuli, and inflammatory pain [5,16]. Moreover, the anti-nociceptive effect of intrathecal nociceptin in a CCI rat model was reversed by a selective ORL1 receptor antagonist in a previous study [7]. On the other hand, the effect of peripheral (not central) nociceptin is controversial. While intradermal nociceptin evokes clear excitatory responses from the dorsal horn neurons [5], intravenous and intraperitoneal nociceptin attenuated neurogenic inflammation [9]. When given intravenously at dosages of 0.01 mg/kg and above, or orally at 1 mg/kg and above, JTC-801 antagonized nociceptininduced allodynia in mice [15]. Thus, it is suggested that the nociceptin system, including JTC-801, plays an important role in the modulation of nociceptive signals at the peripheral level during a neuropathic pain condition. We previously reported that BMC and BMD of the tibial bone on the injury side in CCI rats were decreased compared with the sham side, along with an increase in the number of TRAP-positive multinucleated osteoclasts [14]. In the present study, BMC and BMD of the tibial bone on the
PTD 28
H. Suyama et al. / Neuroscience Letters 351 (2003) 133–136
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H. Suyama et al. / Neuroscience Letters 351 (2003) 133–136
Table 2 Data for BMC, BMD and osteoclast number
Bone mineral contents (mg) Bone mineral density (mg/cm2) Osteoclast number (/mm2)
Sham side CCI side Sham side CCI side Sham side CCI side
Vehicle group
Low dose group
High dose group
0.332 [0.295–0.382] 0.2915 [0.213–0.319]† 0.124 [0.114–0.129] 0.1065 [0.094–0.122]† 26.5 [18.7–33.0] 36.2 [27.8–45.3]†
0.344 [0.323–0.376] 0.303 [0.277–0.332]† 0.1165 [0.112–0.130] 0.112 [0.099–0.116]† 28.2 [17.6–34.9] 27.1 [21.1–44.4]
0.335 [0.287–0.390] 0.291 [0.246–0.328]† 0.118 [0.116–0.124] 0.1095 [0.102–0.113]† 25.2 [15.7–35.6] 31.8 [16.4–38.6]
Data values are medians, with range in brackets. †P , 0:05 compared with the sham side, by Wilcoxon signed-ranks test.
CCI side were significantly decreased compared with the sham side in all three groups. However, no significant changes were observed in the number of TRAP-positive multinucleated osteoclasts between the CCI and sham sides in either the low or high dose group. We considered that JTC-801 may alleviate osteoporosis that was caused by an increase in the number of TRAP-positive multinucleated osteoclasts in the CCI rats, because it functions to inhibit such an increase. We were unable to find evidence that nociceptin and the nociceptin antagonist are interactive in osteoporosis. If alleviation of heat-evoked hyperalgesia inhibits an increase in the number of TRAP-positive multinucleated osteoclasts in CCI rats, then it is possible that neuropathic osteoporosis is related to heat-evoked hyperalgesia such as neuropathic pain. JTC-801 demonstrated an approximately five-fold higher binding affinity to the human ORL1 receptor than that in rats and has also shown specific high affinity to the human ORL1 receptor as compared to other human opioid receptor subtypes [16]. As a result, JTC-801 may be useful in the treatment of neuropathic pain in humans. In conclusion, JTC-801, given orally in food, alleviated heat-evoked hyperalgesia in CCI rats, suggesting that it is useful for the treatment of neuropathic pain. Although it did not inhibit the decrease of BMC and BMD on the CCI side, JTC-801 inhibited the increase in the number of TRAPpositive multinucleated osteoclasts on the CCI side. Thus, it may be that this ORL1 receptor antagonist is related to osteoporosis induced by CCI.
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Effect of JTC-801 (nociceptin antagonist) on neuropathic pain in a rat model Hidemichi Suyama*, Masashi Kawamoto, Syafruddin Gaus, Osafumi Yuge Department of Anesthesiology and Critical Care, Division of Clinical Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima, 734-8551, Japan Received 24 February 2003; received in revised form 14 April 2003; accepted 17 April 2003
Abstract JTC-801, a nociceptin antagonist, may alleviate neuropathic pain because nociceptin has been shown to produce pain modulation. We report that JTC-801 alleviates heat-evoked hyperalgesia and investigated the possible protective effect on osteoporosis induced by chronic constriction injury (CCI) in rats. JTC-801 was given orally to rats with CCI at 0% (vehicle), 0.03% (low dose), or 0.06% (high dose) in food. Paw withdrawal latency (PWL) to heat, bone mineral content (BMC) and bone mineral density (BMD) of the whole tibial bone were measured. JTC-801 dose-dependently normalized PWL. Although JTC-801 did not inhibit a CCI-induced decrease in BMC and BMD, it inhibited an increase in the number of osteoclasts in the JTC-801 groups. JTC-801, given orally in food, alleviated heat-evoked hyperalgesia in CCI rats, suggesting that it is useful for the treatment of neuropathic pain. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: JTC-801; Nociceptin antagonist; Neuropathic pain; Rat; Neuropathic osteoporosis
Nociceptin is an endogenous ligand for the opioid receptorlike1 (ORL1) G-protein coupled receptor [11,12]. This ligand and its receptor are localized in various regions of the central nervous system, which are associated with nociception [4]. The pain modulation of nociceptin has been shown in various rat pain models [5,16]. However, most reports regarding nociceptin pain modulation used intrathecal or intracerebroventricular administration [5,16], while only a few have utilized systemic nociceptin [5,9]. JTC-801, N-(4amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl) benzamide monohydrochloride, was recently synthesized as a novel and oral active ORL1 receptor antagonist [13]. It has been demonstrated that JTC-801 antagonizes ORL1 receptor response, and also has efficacious and potent antinociceptive effects in acute pain animal models, not only by intravenous injection but also oral administration [15]. Chronic constriction injury (CCI) of the sciatic nerve produces neuropathic pain as well as osteoporosis in the ipsilateral limbs of test animals [14]. JTC-801, a nociceptin antagonist, may alleviate such neuropathic pain. In the present study, we attempted to confirm that JTC-801 * Corresponding author. Tel.: þ 81-82-257-5267; fax: þ81-82-257-5269. E-mail address: [email protected] (H. Suyama).
alleviates heat-evoked hyperalgesia and investigated the possible protective effect on osteoporosis induced by CCI in rats. All animal protocols were approved by our institutional animal care committee. We prepared 24 male Sprague– Dawley rats with CCI, according to the following protocol. The rats, each weighing between 200 and 300 g and approximately 7 weeks old on the day of surgery, had an experimental nerve injury induced in their right hind limb. They were anesthetized with sodium pentobarbital (50 mg/kg, i.p.; supplemented as necessary). Four loosely constrictive ligatures of 4.0 chromic gut were tied around the right sciatic nerve at the mid-thigh level, as described in detail elsewhere [1]. In each animal, an identical dissection was performed contralaterally, omitting the ligatures (sham operation). The muscle and skin were then closed in layers. Following surgery, the rats were housed individually in cages for at least 5 days. The rats were divided into three groups and given JTC801 (Japan Tobacco Inc., Tokyo) orally at 0% (vehicle), 0.03% (low dose), or 0.06% (high dose) in food from 10 to 38 days after CCI surgery. The thermal nociceptive threshold was measured with a device similar to that used by Hagreaves et al. [10]. Paw
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00502-0
134
H. Suyama et al. / Neuroscience Letters 351 (2003) 133–136
withdrawal latency (PWL) was measured on both sides, with 10 min intervals between successive trials. Among the 24 rats, 18 showed ipsilateral PWL that was at least 1 standard deviation (SD) shorter than the mean PWL in the controls. PWL to heat was measured every 3 or 4 days after surgery for 38 days, after which the animals were killed for examination of osteopathic changes. Tibial bones were removed and fixed in 3.7% formaldehyde. Bone mineral content (BMC) and bone mineral density (BMD) of the whole tibial bone were measured using a dual energy X-ray absorptiometer (Aloka DCS600EX, Tokyo) 2 days after initiation of fixation. The absorptiometer used enabled measurement of BMC and BMD based on changes in the transmission of monochromatic X-rays at dual low energy levels with a switched pulse method. We used a scan field of 130 £ 150 mm, a scanning speed of 40 mm/s, and a slice thickness of 1 mm. The coefficient of variation, a statistical index expressing measurement precision, was less than 1% in preliminary measurements (data not shown). After measuring BMC and BMD, the fixed tibial bones were then decalcified in 10% EDTA in Tris buffer at pH 7.4 for 3 –4 weeks, after which they were cut into 5 mm sections and stained for tartrate resistant acid phosphate (TRAP) activity to identify osteoclasts, according to Cole’s method [6]. Briefly, Burstone’s complete medium (TRAP solution) for acid phosphate was prepared by dissolving 4 mg of naphthol AS-BI phosphate substrate (Sigma Japan, Tokyo) in 0.25 ml of N,N-dimethyl formamide, followed by the addition of 25 ml of 0.2 M acetate buffer (pH 5.0), 35 mg of Fast Red (Sigma Japan, Tokyo) as the coupling agent, and two drops (60 ml) of 10% MgCl2. Bone sections were incubated at 37 8C for 30 min with TRAP solution. After incubation, the slides were washed for 15 min in running water, allowed to air-dry at 25 8C, and then counterstained with hematoxylin for approximately 10 s. Coverslips were prepared for mounting with Permount (Fisher Scientific Co.,
New Jersey), and the preparations were examined for the presence or absence of reactive products. We counted the number of TRAP-positive multinucleated osteoclasts per unit area (1 mm2) at a position 1 mm from the distal end of the metaphysis. Differences in PWL (DPWL: ipsilateral PWL minus contralateral PWL) among the groups were tested using the Kruskal –Wallis test, followed by a Mann – Whitney U-test. The sequential difference of DPWL in each group was tested using one-way ANOVA, followed by a Bonferroni/Dunn test. Differences in BMC and BMD between ipsi- and contralateral tibiofibula were tested using a Wilcoxon signed-rank test. P , 0:05 was considered statistically significant. All rats in the study showed changes in the appearance of the nerve-injured paw and demonstrated guarding of the paw, as described previously by Bennett and Xie [1]. Characteristically, the toes were ventroflexed and the paw was often held in an elevated position. The rats also tended to contact the medial portion of the ipsilateral hind paw with the glass floor. No such behavioral changes were observed in the contralateral hind paw. No significant differences in the rats’ body weights were observed in all groups throughout the study. At 1 week before surgery, 1 and 10 days after surgery, and 1, 2, 3, and 4 weeks after treatment, the mean ^ SD body weight (g) was 217 ^ 8.3, 289.8 ^ 19.6, 342.8 ^ 26.1, 383.9 ^ 29.7, 408.7 ^ 33.2, 435.4 ^ 39.7, 461.8 ^ 42.4 (control group), 216.4 ^ 3.5, 302.5 ^ 8.6, 355.8 ^ 20.6, 393.9 ^ 18.0, 419.6 ^ 21.1, 443.5 ^ 23.7, 469.9 ^ 29.1 (low dose group), and 216.3 ^ 6.0, 292.3 ^ 21.1, 344.5 ^ 41.5, 380.3 ^ 48.8, 405.4 ^ 59.4, 430.1 ^ 67.1, 452.8 ^ 71.7 (high dose group), respectively. JTC-801 at the high dose (0.06% in food) normalized DPWL 3 days after the start of administration and throughout the feeding period, while that at the low dose (0.03% in food) normalized DPWL 10 days after the start of
Fig. 1. Analgesic effect of JTC-801 on heat-evoked hyperalgesia. †P , 0:01 vs. preparation within a group. *P , 0:01 vs. after surgery within a group. { P , 0:05 vs. vehicle group among groups.
High dose
Low dose
Data values are means ^ SD. Baseline, 1 week before the surgery; POD, postoperative (the surgery) day; PTD, post-treatment days. *P , 0:05, **P , 0:0001 compared with the sham side, by Wilcoxon signed-ranks test.
13.5 ^ 1.8 8.7 ^ 2.2** 12.6 ^ 2.4 10.1 ^ 2.3* 13.9 ^ 2.3 13.1 ^ 2.4 13.9 ^ 2.3 9.8 ^ 2.0** 12.5 ^ 1.6 10.1 ^ 2.3** 13.1 ^ 2.0 12.7 ^ 1.9 13.5 ^ 3.0 9.1 ^ 1.7** 12.6 ^ 1.6 10.7 ^ 2.8** 13.6 ^ 2.3 13.5 ^ 2.2 12.8 ^ 2.3 8.5 ^ 1.5** 12.3 ^ 1.7 10.1 ^ 1.8** 14.9 ^ 2.8 13.6 ^ 2.8* 13.9 ^ 2.2 9.8 ^ 1.6** 13.4 ^ 2.1 10.7 ^ 1.7** 13.7 ^ 2.5 12.4 ^ 2.3* 13.0 ^ 2.7 8.3 ^ 2.5** 12.8 ^ 1.9 9.9 ^ 1.8** 13.4 ^ 2.8 11.5 ^ 2.7** 13.7 ^ 2.0 9.9 ^ 2.1** 13.9 ^ 1.6 9.6 ^ 2.4** 13.4 ^ 1.9 11.9 ^ 1.7** 15.0 ^ 2.1 10.9 ^ 1.8** 13.9 ^ 2.0 9.3 ^ 1.9** 14.5 ^ 1.9 10.3 ^ 1.8** 12.4 ^ 2.2 12.3 ^ 2.0 12.0 ^ 1.4 11.8 ^ 1.5 13.1 ^ 1.9 13.3 ^ 1.8 Vehicle
Sham side CCI side Sham side CCI side Sham side CCI side
POD 7
12.9 ^ 1.8 9.2 ^ 2.1** 14.0 ^ 2.7 8.6 ^ 1.8** 13.2 ^ 1.4 11.6 ^ 1.9**
PTD 24 PTD 21 PTD 17 PTD 14 PTD 10 PTD 7
135
Baseline
PTD 3 Table 1 Data for PWL
administration and throughout the study (Fig. 1). The normalization of DPWL was dose-dependent at the time of measurement (1 – 4 weeks after the treatment) (Fig. 1). DPWL in the vehicle group did not change from 1 week after surgery to the end of this experiment. Table 1 summarizes the PWL data for each group. BMC of the tibial bone on the CCI side was significantly lower than that on the contralateral side in all three groups, with a median (range) reduction of 62 (27 –82), 46 (21 – 64), and 45 (30 –76) mg in the control, low dose, and high dose groups, respectively (Table 2). BMD of the tibial bone on the ipsilateral side was also significantly lower than that on the contralateral side in all three groups, with a median (range) reduction of 15 (7 – 27), 11 (1 – 14), and 9 (4 –19) mg/cm2, respectively (Table 2). The number of osteoclasts was increased in the CCI side compared with the sham side in the vehicle group, however, no significant changes were observed in the number of osteoclasts between the CCI and sham side in the low and high dose groups. Further, the median increase (range) in the number of TRAP-positive multinucleated osteoclasts was 6.2 (2.1 – 18.9) cells/mm2 in the control, 2.3 (2 7.2 – 11.9) cells/mm2 in the low dose, and 3.0 (2 3.6 – 13.9) cells/mm2 in the high dose group (Table 2). The present results are the first known to demonstrate the alleviation of heat-evoked hyperalgesia with an oral nociceptin antagonist, JTC-801, in CCI rats. In sciatic nerve injury model rats, molecular changes (m-opioid receptor, alfa and beta subunits of the sodium channel, galanin) in the spinal cord and dorsal root ganglia have been shown [2,8]. Nociceptin and its receptor appear at the spinal cord level in nociception [16]. During the development of neuropathy due to CCI of the sciatic nerve, two types of ORL1 receptors are expressed in the lumbar spinal cord and L5-L6 dorsal root ganglia [3]. Intracerebroventricular and intrathecal nociceptin inhibit noxious thermal pain, mechanical noxious stimuli, and inflammatory pain [5,16]. Moreover, the anti-nociceptive effect of intrathecal nociceptin in a CCI rat model was reversed by a selective ORL1 receptor antagonist in a previous study [7]. On the other hand, the effect of peripheral (not central) nociceptin is controversial. While intradermal nociceptin evokes clear excitatory responses from the dorsal horn neurons [5], intravenous and intraperitoneal nociceptin attenuated neurogenic inflammation [9]. When given intravenously at dosages of 0.01 mg/kg and above, or orally at 1 mg/kg and above, JTC-801 antagonized nociceptininduced allodynia in mice [15]. Thus, it is suggested that the nociceptin system, including JTC-801, plays an important role in the modulation of nociceptive signals at the peripheral level during a neuropathic pain condition. We previously reported that BMC and BMD of the tibial bone on the injury side in CCI rats were decreased compared with the sham side, along with an increase in the number of TRAP-positive multinucleated osteoclasts [14]. In the present study, BMC and BMD of the tibial bone on the
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Table 2 Data for BMC, BMD and osteoclast number
Bone mineral contents (mg) Bone mineral density (mg/cm2) Osteoclast number (/mm2)
Sham side CCI side Sham side CCI side Sham side CCI side
Vehicle group
Low dose group
High dose group
0.332 [0.295–0.382] 0.2915 [0.213–0.319]† 0.124 [0.114–0.129] 0.1065 [0.094–0.122]† 26.5 [18.7–33.0] 36.2 [27.8–45.3]†
0.344 [0.323–0.376] 0.303 [0.277–0.332]† 0.1165 [0.112–0.130] 0.112 [0.099–0.116]† 28.2 [17.6–34.9] 27.1 [21.1–44.4]
0.335 [0.287–0.390] 0.291 [0.246–0.328]† 0.118 [0.116–0.124] 0.1095 [0.102–0.113]† 25.2 [15.7–35.6] 31.8 [16.4–38.6]
Data values are medians, with range in brackets. †P , 0:05 compared with the sham side, by Wilcoxon signed-ranks test.
CCI side were significantly decreased compared with the sham side in all three groups. However, no significant changes were observed in the number of TRAP-positive multinucleated osteoclasts between the CCI and sham sides in either the low or high dose group. We considered that JTC-801 may alleviate osteoporosis that was caused by an increase in the number of TRAP-positive multinucleated osteoclasts in the CCI rats, because it functions to inhibit such an increase. We were unable to find evidence that nociceptin and the nociceptin antagonist are interactive in osteoporosis. If alleviation of heat-evoked hyperalgesia inhibits an increase in the number of TRAP-positive multinucleated osteoclasts in CCI rats, then it is possible that neuropathic osteoporosis is related to heat-evoked hyperalgesia such as neuropathic pain. JTC-801 demonstrated an approximately five-fold higher binding affinity to the human ORL1 receptor than that in rats and has also shown specific high affinity to the human ORL1 receptor as compared to other human opioid receptor subtypes [16]. As a result, JTC-801 may be useful in the treatment of neuropathic pain in humans. In conclusion, JTC-801, given orally in food, alleviated heat-evoked hyperalgesia in CCI rats, suggesting that it is useful for the treatment of neuropathic pain. Although it did not inhibit the decrease of BMC and BMD on the CCI side, JTC-801 inhibited the increase in the number of TRAPpositive multinucleated osteoclasts on the CCI side. Thus, it may be that this ORL1 receptor antagonist is related to osteoporosis induced by CCI.
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