BJA Advance Access originally published online on November 26, 2008
British Journal of Anaesthesia 2009 102(2):251-258; doi:10.1093/bja/aen347
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SB366791, a TRPV1 antagonist, potentiates analgesic effects of systemic morphine in a murine model of bone cancer pain
Department of Anaesthesiology, Sapporo Medical University School of Medicine, South 1, West 16, Chuo-ku, Sapporo, Hokkaido 060-8543, Japan
* Corresponding author. E-mail: kawamata{at}sapmed.ac.jp
Accepted for publication October 20, 2008.
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Abstract |
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Background: Bone cancer pain has a major impact on the quality of life of cancer patients but is difficult to treat. Therefore, development of a novel strategy for bone cancer pain is needed for improvement of the patient quality of life. In this study, we examined the analgesic effects of the combination of a transient receptor potential vanilloid subfamily 1 (TRPV1) antagonist and morphine on pain-related behaviours in a murine model of bone cancer pain.
Methods: C3H/HeJ mice underwent injection of osteolytic sarcoma cells into the intramedullary space of the femur. The analgesic effects of intraperitoneal morphine and the analgesic effect of a TRPV1 antagonist, SB366791 [N-(3-methoxyphenyl)-4-chlorocinnamide], on bone cancer pain-related behaviours were examined. The analgesic effects of the combination of SB366791 and morphine on bone cancer pain were also examined.
Results: Intraperitoneal morphine significantly reduced the number of spontaneous flinches and improved ambulation only at the highest dose of 10 mg kg–1 whereas weight-bearing was not improved. Intraperitoneal SB366791 at doses of 0.3 and 1.0 mg kg–1, but not at a dose of 0.1 mg kg–1, reduced the number of spontaneous flinches, whereas neither weight-bearing nor ambulation was improved. Addition of a sub-analgesic dose of SB366791 (0.1 mg kg–1) to morphine significantly reduced the number of flinches and improved weight-bearing compared with the effects of morphine alone.
Conclusions: Our findings showed that the combination of morphine and SB366791 has potent analgesic effects on bone cancer pain. The findings of this study may lead to novel strategies for the treatment of bone cancer pain.
Keywords: analgesics opioid; cancer; nerve, transmission; pain
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Introduction |
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As advances in cancer detection and treatment have increased the life expectancy of cancer patients, more attention to improving the patient quality of life is needed. The World Health Organization (WHO) has proposed a structured approach to drug selection for cancer pain, known as the ‘WHO analgesic ladder’. This approach is capable of providing adequate relief to 70–90% of patients.1 2 However, the remaining 10–30% of patients is difficult to treat. One common type of cancer pain that is difficult to treat is bone cancer pain.3 This type of pain is dull and constant, increases with time, and is exacerbated by the use of involved portions of the skeleton. Although opioids are used for the treatment of advanced bone cancer pain, patients often require higher doses of morphine, which produce diverse and disabling side-effects such as sedation, somnolence, and constipation that may diminish quality of life.4 5 Animal studies using a mouse model of bone cancer pain also showed that bone cancer pain is resistant to morphine compared with inflammation pain.6 7 Therefore, development of a novel strategy for bone cancer pain is needed for improvement of the patient quality of life.
Transient receptor potential vanilloid subfamily 1 (TRPV1) is expressed predominantly in unmyelinated neurones and is activated not only by capsaicin but also by noxious heat, protons, and membrane-derived lipids.8 9 Since these stimuli cause pain in vivo, this sensitivity of TRPV1 to multiple types of noxious stimuli might explain the properties of the so-called polymodal nociceptors. In addition, TRPV1 is also present on sensory fibres in mineralized bone and bone marrow.10 Previously, we demonstrated that TRPV1 expression was increased within a distinct subpopulation of dorsal root ganglion neurones in a bone cancer state and that pharmacological block of TRPV1 reduced pain-related behaviours in a murine model of bone cancer pain.11 It has also been reported that the pain-related behaviours observed in this model were attenuated by gene deletion of TRPV1.10 These findings indicate that activation of TRPV1 plays an important role in generation of bone cancer pain. In addition, we recently showed that expression of mu-opioid receptor (MOR) in TRPV1-expressing primary afferent neurones was reduced in a murine model of bone cancer pain.7 This finding could explain attenuation of the analgesic effect of morphine in treatment of bone cancer pain. Therefore, it is possible that the combination of morphine and a TRPV1 antagonist strongly inhibits bone cancer pain compared with the effect of morphine alone.
SB366791 [N-(3-methoxyphenyl)-4-chlorocinnamide] is a more selective and in vivo also a more potent TRPV1 antagonist than a commonly used TRPV1 antagonist capsazepine12 and has been widely used as a selective TRPV1 antagonist in pain research.13–16 In this study, we examined the analgesic effects of morphine and SB366791 on pain-related behaviours in a murine model of bone cancer pain. We also examined analgesic interaction of SB366791 with morphine in bone cancer pain.
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Animals
The present experiments were approved by the Sapporo Medical University Animal Care Committee and were in accordance with the ethical guidelines of the National Institutes of Health. All efforts were made to minimize animal suffering and the number of animals that were used. Experiments were conducted in 103 adult male C3H/HeJ mice weighing 20–25 g (Japan SLC, Hamamatsu, Japan). The mice were housed in a temperature-controlled [21 (1)°C] room with a 12 h light/dark cycle and given free access to food and water. The animals were humanely killed with intraperitoneal injection of pentobarbital (100 mg kg–1) after completion of the experiments.
Drug preparation
SB366791 (BIOMOL International, Plymouth Meeting, PA, USA) was dissolved in 100% dimethyl sulphoxide (DMSO) and diluted in physiological saline. Morphine was dissolved in physiological saline.
Bone cancer model
Murine sarcoma cells (NCTC 2472; ATCC, Rockville, MD, USA) were maintained in NCTC 135 media containing 10% horse sera (HyClone, Logan, UT, USA) and passaged weekly according to ATCC recommendations. Implantation of sarcoma cells was performed as previously described.17 Briefly, mice were anaesthetized with isoflurane (3% in 100% oxygen), and a superficial incision was made in the skin overlying the left patella. The patellar ligament was then cut, exposing the condyles of the distal femur. A 0.5 mm depression was then made using a half-round burr in a pneumatic dental high-speed handpiece to facilitate mechanical retention of the amalgam plug. Then either 20 µl of 
-minimum essential medium (sham-implanted mice) or 20 µl of medium containing 1x105 sarcoma cells (sarcoma-implanted mice) was injected using a 29 gauge needle and a 0.25 ml syringe. To prevent leakage of cells outside the bone, the injection site was closed with dental-grade amalgam, followed by copious irrigation with filtered water. The wound was then closed. To diminish postoperative pain, the animals received intraperitoneal morphine (10 mg kg–1) after wound closure.
Analysis of bone cancer pain
Behavioural testing was performed at day 14 after sarcoma implantation, based on results of studies showing that the tumour occupies 
20% of the intramedullary space at day 6 after implantation and increases to 90% by day 14 after implantation18 19 and that sarcoma implantation-induced pain-related behaviours first appear at day 10 after implantation and continue to escalate until severe impairment at days 17–21 after implantation, when fracture of the affected femur occurs.20
Behavioural assessments included observation of limb use during spontaneous ambulation, quantification of spontaneous flinches, and observation of weight-bearing during spontaneous standing as described previously.7 11 Observation of limb use during spontaneous ambulation and quantification of spontaneous flinches were used for assessment of movement-evoked pain and ongoing pain, respectively. In addition to these behaviours, weight-bearing during spontaneous standing was used as an indicator of movement-evoked pain. Limb use during spontaneous ambulation was assigned scores on a scale of 0–4: 0, complete lack of limb use; 1, relative lack of use of the limb in locomotor activity; 2, limping and guarding behaviour; 3, substantial limping; and 4, normal use. The number of spontaneous flinches was recorded during a 2 min observation period. Flinches were defined as holding of the hindpaw aloft while not ambulatory. In addition, weight-bearing during standing was scored on a scale of 0–3: 0, no weight-bearing (the hindpaw on the injected side always being lifted and never touching the floor); 1, touch weight-bearing (the hindpaw on the injected side being lifted from the floor but occasionally touching the floor); 2, partial weight-bearing (partial support of body weight with the hind limb on the injected side); and 3, full weight-bearing (full support of 100% of body weight with both hind limbs).
Before starting behavioural assessment, mice were placed in a clear plastic box and allowed to acclimatize for 30 min. After recording basal values of behavioural assessment, drugs were administered. Morphine was dissolved in normal saline, and mice received intraperitoneal morphine (1, 3, or 10 mg kg–1 in 200 µl solution) or vehicle alone (200 µl normal saline). SB366791 was also intraperitoneally administered at doses of 0.1, 0.3, and 1.0 mg kg–1 (in 200 µl solution). In the SB366791 study, 50% DMSO (200 µl) was used as a vehicle. To examine the interaction between morphine and SB366791, mice received the solution including morphine and SB366791. Behavioural assessment was performed 0.5, 1, 1.5, and 2 h after drug administration. The observer was blinded to the type of treatment.
Statistical analysis
The scores for limb use and weight-bearing are expressed as medians with first and third quartiles, and 10th and 90th percentiles. Data for area under the time–effect curve (AUC) and the number of flinches are expressed as means (SD). AUC of analgesia was measured by deducting the area under the time–response curve of the vehicle from the area under the time–response curve of drug treatments. AUCs in the presence of SB366791 were compared with those in the absence of SB366791 using the Mann–Whitney U-test. The scores for limb use, weight-bearing, and the number of flinches were compared with the basal values using the Kruskal–Wallis test followed by Bonferroni’s test. P<0.05 was considered to be statistically significant. We assumed an SD of 2.3 for the number of flinches (taken from our preliminary study), an of 0.05, and a β of 0.2. Power analysis showed that 10 animals were necessary to detect a difference of three in the number of flinches.
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Pain-related behaviour in sarcoma-implanted mice
The sham-implanted mice exhibited no detectable pain-related behaviour at day 14 after implantation (n=6). We used 87 mice with sarcoma implantation in the drug treatment experiments (32 mice in the experiments using morphine treatment, 33 mice in the experiments using SB366791 treatment, and 32 mice in the experiments using the combination treatment). Although mice exhibited no detectable pain-related behaviour before sarcoma implantation, they exhibited spontaneous flinches [12 (1) flinches over a 2 min observation period] and impaired limb use during spontaneous ambulation (limb use score, 2) at day 14 after implantation. The sarcoma implantation also resulted in impaired weight-bearing (weight-bearing score, 1).
Analgesic effects of intraperitoneal morphine on bone cancer-related pain behaviour
Intraperitoneal morphine at doses of 1 and 3 mg kg–1 did not significantly alter any of the three pain-related behaviours (Fig. 1A–C). On the other hand, 10 mg kg–1 of morphine slightly but significantly (P<0.05) improved limb use score (3, median) and reduced the number of spontaneous flinches at 0.5 h after administration compared with those before administration [from 13 (2) to 9 (2)] (Fig. 1A and C). These effects of morphine disappeared 1 h after administration. Morphine 10 mg kg–1 did not improve the weight-bearing score (Fig. 1B). Intraperitoneal vehicle did not affect pain-related behaviour (data not shown). Because doses of more than 30 mg kg–1 of morphine produced hypoactivity and sedation which interfered with assessment of behaviour, we used doses of 1, 3, and 10 mg kg–1 of morphine in a behavioural study.
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Analgesic effects of oral SB366791 on bone cancer-related pain behaviour
Intraperitoneal SB366791 at doses of 0.3 and 1.0, but not at a dose of 0.1 mg kg–1, reduced the number of flinches (Fig. 2C). SB366791 at a dose of 0.3 mg kg–1 slightly but significantly (P<0.05) decreased the number of flinches at 0.5 h after administration compared with that before administration [from 12 (2) to 9 (2)]. SB366791 at a dose of 1.0 mg kg–1 also significantly (P<0.05) decreased the number of flinches at 0.5 and 1 h after administration compared with that before administration [from 12 (2) to 7 (2) and 8 (2), respectively]. However, intraperitoneal SB366791 at all doses used in this study did not have any effects on the scores of weight-bearing and ambulation throughout the observation period (Fig. 2A and B). The vehicle also did not affect pain-related behaviour (data not shown).
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Analgesic effects of combination of intraperitoneal morphine and oral SB366791 on bone cancer-related pain behaviour
Next, we examined the analgesic interaction of morphine with SB366791 at a dose of 0.1 mg kg–1, which was ineffective for inhibiting bone cancer pain-related behaviour. Figure 3 shows the time course of analgesic effects of the combination of morphine and SB366791. The combination of SB366791 and morphine at all three doses significantly (P<0.05) reduced the number of flinches (Fig. 3C). Duration of the effect of morphine at the dose of 10 mg kg–1 was prolonged by the addition of SB366791 compared with that of morphine alone (also see Fig. 1C). Weight-bearing was also significantly (P<0.05) improved by the combination of SB366791 and morphine at doses of 3 and 10 mg kg–1 (Figs 1C and 3C). Improvement in ambulation was observed at 0.5 h after administration of the combination of SB366791 and morphine at a dose of 10 mg kg–1 (Fig. 3A). However, morphine (10 mg kg–1) alone also improved ambulation as shown in Figure 1A. Next, AUCs were analysed in order to compare the effect of the combination with the effect of morphine alone. Figure 4 and Table 1 show the data for AUCs. Addition of SB366791 did not significantly increase AUC of morphine at a dose of 1.0 mg kg–1 for any of the three behaviours. AUCs of combinations of SB366791 and morphine at doses of 3.0 and 10.0 mg kg–1 for flinches and weight-bearing, but not ambulation, were significantly (P<0.05) larger than those of morphine alone. These results indicate that a sub-analgesic dose of SB366791 potentiates the effects of morphine at doses of 3.0 and 10.0 mg kg–1 on flinches and weight-bearing.
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Discussion |
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In the present study, we investigated the analgesic effects of a TRPV1 antagonist, SB366791, and the analgesic interaction of SB366791 with morphine in pain-related behaviours in a murine model of bone cancer pain. Our major findings are as follows: (i) the TRPV1 antagonist SB366791 reduced the number of flinches but did not improve ambulation and weight-bearing and (ii) a sub-analgesic dose of SB366791 potentiates the effects of morphine at doses of 3.0 and 10.0 mg kg–1 on flinches and weight-bearing but not ambulation. These findings may lead to the development of a novel therapeutic strategy for bone cancer pain.
TRPV1 antagonist SB366791 and bone cancer pain-related behaviour
Previous studies showed that gene deletion of TRPV1 or other TRPV1 antagonists, JN-17203212 and I-RTX, reduced both ongoing pain and movement-evoked pain in a murine model of bone cancer pain.10 11 These studies indicate that TRPV1 activation is involved in generation of both ongoing pain and movement-evoked pain. On the other hand, in the present study, SB366791 reduced the number of flinches but did not improve ambulation and weight-bearing, suggesting that SB366791 effectively reduces ongoing pain but not movement-evoked pain. It is unclear why these antagonists have different analgesic effects on bone cancer pain. There may be some different pharmacological profiles among these TRPV1 antagonists. For example, JN-17203212 and I-RTX are potent antagonists of TRPV1 activation by either capsaicin or proton, whereas SB366791 is an antagonist of capsaicin, but not proton, activation.21–23 Such different pharmacological profiles may contribute to different analgesic actions for bone cancer pain among these TRPV1 antagonists. In addition, the dose of SB366791 may be involved in different analgesic actions. In the present study, we used 0.1, 0.3, and 1.0 mg kg–1 of SB366791. It has been reported that 0.5 mg kg–1 of intraperitoneal SB366791 completely inhibited topical application of capsaicin-induced change of blood flow and partially inhibited capsaicin-induced acute nociception.12
Interaction of SB366791 with morphine
In the present study, intraperitoneal morphine was relatively ineffective in inhibiting bone cancer pain-related behaviour, as was previously reported.24 Several mechanisms are thought to be involved in the relative resistance of bone cancer pain to morphine. At day 14 after sarcoma implantation, when we assessed bone cancer pain-related behaviour, it has been reported that the tumour occupies 90% of the intramedullary space and that some dorsal root ganglion neurones appear to express ATF3, which is a marker of nerve injury.19 Therefore, peripheral nerves within the intramedullary space would be injured. Peripheral nerve injury alters trafficking of MOR to the membrane, expression of MOR, expression of the regulator of the G protein signalling family of proteins, and expression of anti-opioid peptides in the spinal cord,25 leading to opioid resistance of pain. Actually, our recent study showed down-regulation of MOR in primary afferent neurones.7 In the present study, the combination of morphine and SB366791 had more potent analgesic effects on flinches and weight-bearing than did morphine alone, suggesting that the combination reduced both ongoing pain and certain movement-evoked pain. Possible mechanisms of the analgesic interaction between SB366791 and morphine are as follows. First, change in the pharmacokinetics of each drug may occur when they are administered in combination. Secondly, the difference between the sites of action of morphine and SB366791 might contribute to the potent analgesic interaction because SB366791 affects points different from those affected by morphine along a common nociceptive pathway. Since MOR expression was decreased in TRPV1-positive primary afferent neurones in this bone cancer pain model, resulting in attenuation of the analgesic effect of morphine, it appears that inhibition of TRPV1 rescues decreased morphine-induced analgesia.
Clinical implication
Bone cancer pain is often debilitating, difficult to treat, and insufficiently relieved.3 Pain originating from bone cancer is commonly divided into two categories: ongoing pain and breakthrough or incident pain.4 26 Although opioids are often used for the treatment of advanced bone cancer pain, the efficacy of potent opioids is minimal for this type of pain.27 Accordingly, patients generally require higher doses of morphine, with associated side-effects.4 5 SB366791 may be useful for an adjuvant analgesic to morphine for clinical bone cancer pain management, although further study on side-effects is needed.
In conclusion, our findings showed that the combination of morphine and SB366791 has potent analgesic effects on bone cancer pain. The findings of this study may lead to novel strategies for the treatment of bone cancer pain.
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Support was provided by a Grant-in-Aid for Scientific Research (19791074 and 19791076) to T.K. and Y.N., respectively.
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This article is accompanied by Editorial II. 
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