The prevalence of pain associated with cancer is estimated to be 50%, increasing up to 75% in advanced stages, and pain from bony metastasis is one of the most common and disruptive symptoms.1 As recent advances in both diagnostic and therapeutic techniques have improved the survival time with malignant disease, the prevalence of bony metastasis and the consequent incidence of bone cancer–related pain have increased.2 Although opioids continue to be the mainstay for controlling metastatic bone pain, patients often become refractory to morphine administration and require higher doses, leading to significant side effects such as constipation, sedation, and respiratory depression.3 In a mouse model of bone cancer, the morphine requirement was 10 times that required to relieve inflammatory pain,4 most likely because of the downregulation of the μ-opioid receptor in dorsal root ganglion neurons of tumor-affected animals.5 Additionally, sustained treatment with morphine increased pain, osteolysis, and markers of neuronal damage in dorsal root ganglion cells as well as the expression of proinflammatory cytokines.6 These data suggest a possible detrimental effect of μ-opioid agonists and an urgent requirement for more effective treatments for bone cancer pain.
The κ-opioid receptor has long been established as a functionally differentiated subtype from μ- and δ-opioid receptors. Radioligand studies have demonstrated that there are at least 2 main binding sites, named the κ1 and κ2 receptors.7 Although the antinociception produced by peripherally acting κ1 agonists has been extensively studied in a wide variety of pain models,8–10 including osteosarcoma-induced hyperalgesia,11 relatively little is known regarding the function of κ2 receptors. At the spinal level, the selective κ2 agonist GR89696 displays antihyperalgesic activity in a rat model of inflammatory pain, whereas intrathecal κ1 agonists fail to exhibit analgesia.12,13 Thus, κ1 and κ2 receptors are strongly implicated in pain transmission through peripheral and spinal mechanisms.
An alternative approach for relieving bone cancer pain is through the modulation of inflammatory cytokines. Recent studies have suggested the importance of proinflammatory cytokines such as tumor necrosis factor (TNF)-α14 and interleukin (IL)-1β15 in bone cancer pain. Conversely IL-10, an antiinflammatory cytokine, downregulates TNF-α16 and IL-1β,17 and reduces thermal and mechanical hyperalgesia.
These findings suggest that both κ2-opioid agonists and IL-10 may exhibit antinociceptive activity in bone cancer pain. However, the analgesic effects of these agents have not been examined at the spinal level in a cancer pain model. In this study, we aimed to evaluate the analgesic efficacy of intrathecally administered GR89696 and IL-10 in a rat model of bone cancer pain. We also examined the interaction between the 2 drugs at the spinal level.
These studies were approved by the Institutional Animal Care and Use Committee of Chonnam National University. Experiments were performed using 6-week-old female Sprague-Dawley rats weighing 180 to 200 g each. The rats were housed in groups of 4, with free access to a standard diet and tap water, in a temperature-controlled (22°C ± 0.5°C) room under a 12-hour light/dark cycle. Rats showing a paw withdrawal threshold above 15 g were used in this study.
MRMT-1 rat mammary gland carcinoma cells, provided by Cell Resource Center for Biomedical Research, Tohoku University (Miyagi, Japan), were cultured in RPMI-1640 medium (500 mL; Gibco/Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin sulfate; Gibco). The cells were trypsinized, counted, and diluted to achieve the final concentration for injection, and the diluted cells were maintained on ice until injected.
Induction of Bone Cancer Pain
Syngeneic MRMT-1 rat breast cancer cells (1 × 105) or culture medium (control) was injected into the right tibial medullary cavity of each rat.18 Briefly, the rats were anesthetized with sevoflurane under spontaneous respiration. A 1-cm rostral–caudal incision was made in the skin over the top half of the tibia. Blunt dissection was performed to expose the tibia, ensuring minimal damage to muscles and blood vessels. A round dental bur (1/4 FG, 0.5 mm) was used to access the intramedullary canal, and a 20-μL volume of culture medium or tumor cells was injected into the intramedullary cavity, using a hand-driven, gear-operated syringe pump. The injection site was closed using bone wax, and the skin was closed with 3-0 silk sutures. After surgery, the development of bone cancer pain was evaluated by measuring the mechanical sensitivity of the right paw.
Three days after tumor cell injection, a polyethylene tube (PE-10) was inserted into the subarachnoid space of each rat under sevoflurane anesthesia, as described previously.19 The catheter was inserted through an incision in the atlantooccipital membrane and was advanced caudally by 8.5 cm, to the lumbar enlargement. The external end of the catheter was subcutaneously tunneled to the top of the head and was plugged with a piece of steel wire. The skin was sutured with a 3-0 silk. After intrathecal catheterization, the rats were closely monitored, and those showing a postsurgical neurological deficit (n = 11 of 108 rats) were excluded from the study and immediately euthanized with an overdose of volatile anesthetics.
Assessment of Mechanical Allodynia
To determine the mechanical withdrawal threshold, rats were placed individually in a transparent cage with a wire mesh floor, which allowed full access to the paws. The rats were allowed to adapt to the cage for approximately 15 minutes, until cage exploration and major grooming activities ceased. One of a series of 8 von Frey filaments (Stoelting, Wood Dale, IL) with logarithmically increasing stiffness (0.4, 0.7, 1.2, 2.0, 3.6, 5.5, 8.5, and 15 g) was presented perpendicularly to the plantar surface of a paw. A force just sufficient to cause slight buckling of the filament was applied against the paw and held for approximately 5 seconds. A positive response was assumed when the paw was sharply withdrawn. The 50% withdrawal threshold was determined using the up-down method as previously described.20 When a positive response was not shown even at 15 g of pressure, this value was assigned as a cutoff value. Animals exhibiting mechanical allodynia, i.e., a withdrawal response to the application of a bending force of <5 g (n = 74 of 97 tumor cell–implanted rats), were considered to have bone tumor pain.
The selective κ2 agonist 4-[(3,4-dichlorophenyl)acetyl]-3-(1-pyrrolidinylmethyl)-1-piperazinecarboxylic acid methyl ester fumarate (GR89696) was obtained from Tocris Cookson Ltd. (Bristol, UK) and was dissolved in 0.9% saline. IL-10 (Tocris Cookson Ltd.) was dissolved in distilled water. Intrathecal injection of these drugs was performed using a hand-driven, gear-operated syringe pump. All drugs were delivered in a volume of 10 μL, followed by an additional 10 μL of saline to flush the catheter.
Ten days after the implantation of cancer cells, the rats were allocated to experimental groups receiving intrathecal administration of one of the experimental drugs or control groups administered saline or distilled water accordingly. The rats were tested by an observer blinded to the drug treatments.
Effects of κ2-Opioid Agonist and IL-10
To evaluate the analgesic efficacy of κ2-opioid agonist, saline (n = 5) or GR89696 (30 μg, n = 4; 100 μg, n = 5; 300 μg, n = 5) was injected through the intrathecal catheter. Similarly, distilled water (n = 5) or IL-10 (0.3 μg, n = 6; 1 μg, n = 5; 3 μg, n = 5) was administered intrathecally. Doses of experimental drugs were chosen based on the pilot study and for approximately equal spacing on the log scale. The mechanical withdrawal threshold was measured immediately before (control value) and at 15, 30, 60, 90, 120, 150, and 180 minutes after intrathecal delivery of the experimental drugs. The withdrawal threshold assessed before tumor cell injection was regarded as a baseline threshold.
The characteristics of the drug interaction between GR89696 and IL-10 were analyzed using the isobolographic method.21 The 50% effective dose (ED50) values of GR89696 and IL-10 were obtained from dose-response curves fitted for the first series of experimental data. Next, both GR89696 and IL-10 were coadministered intrathecally at their respective ED50 (n = 5) and fractions (1/2, n = 6; 1/4, n = 5; 1/8, n = 5) of the ED50. The paw withdrawal threshold was then measured using von Frey hairs. From these dose-response data, the ED50 value of the mixture was calculated. An isobologram was constructed by plotting the ED50 values of each drug on the x axis and y axis, respectively. A straight line connecting these 2 points represents the theoretical line of additivity, which indicates the theoretical concentrations of a combination of the 2 drugs predicted to provide the same effect. Additionally, to describe the magnitude of the interaction, a total fraction value was calculated according to the following formula:
Total fraction value = (ED50 of drug 1 with drug 2/ED50 of drug 1 given alone) + (ED50 of drug 2 with drug 1/ED50 of drug 2 given alone)
The fractional value indicates the portion of the single ED50 value that was accounted for by the corresponding ED50 value of the combination. Values near 1 indicate an additive interaction, values >1 imply an antagonistic interaction, and values <1 indicate a synergistic interaction.
To evaluate the behavior effected by the administration of G89696 and IL-10, additional rats receiving the highest dose (n = 5 in each group) were examined at 5, 10, 20, 30, 40, 50, and 60 minutes after intrathecal administration. Motor functions were assessed by examining the righting and place-stepping reflexes. The former was performed by placing the rat horizontally with its back on the table, which normally gives rise to an immediate coordinated twisting of the body to an upright position. The latter was elicited by drawing the dorsum of either hindpaw across the table. Rats normally attempt to place their paws forward, into a position for walking. The pinna and corneal reflexes were assessed as present or absent. Other abnormal behaviors such as serpentine movement or tremors were noted.
The data are presented as means ± SD of the mechanical withdrawal threshold in grams or percentage of maximal possible effect (%MPE). The difference in withdrawal threshold between the cancer cell–implanted and sham groups was analyzed using an unpaired t test. For constructing the dose-response curve, the 50% paw withdrawal threshold was converted to a percentage of the %MPE:
%MPE = (Postdrug threshold − tumor-implanted baseline threshold)/(Cutoff threshold − tumor-implanted baseline threshold) × 100
The dose-response data were analyzed using 1-way analysis of variance with the Dunnett T3 post hoc test. The dose-response (%MPE) data were fitted using least-squares linear regression on log[dose] data, and ED50 with a 95% confidence interval (CI) was calculated as previously described.22 The difference between the theoretical ED50 and experimental ED50 was analyzed by comparing the 95% CIs as described by Tallarida.22 A P value <0.05 was considered to indicate statistical significance.
Bone Destruction and Development of Mechanical Allodynia in Cancer Cell–Implanted Rats
The progression of bone destruction was comparable to previous reports.23,24 Intramedullary injection of cancer cells induced endosteal destruction by day 10 postinjection, and further deterioration with full-thickness cortical bone loss by day 14 after inoculation.
Behaviorally, the paw withdrawal threshold of tumor-implanted rats (n = 6) was significantly reduced compared with that of the media-injected sham group (n = 6) by 7 days after cancer cell injection (Fig. 1). By postinjection day 10, the mechanical withdrawal threshold decreased to 4.15 ± 0.98 g, and this reduced threshold persisted for 3 to 4 days. The withdrawal threshold of the sham group did not differ significantly from the baseline value.
After intrathecal injection of GR89696 and/or IL-10, righting and place-stepping reflexes were not impaired. Pinna and corneal reflexes were also preserved. There was no apparent abnormal behavior after the administration of the experimental drugs.
Effects of Intrathecal κ-Opioid Receptor Agonist and IL-10
Intrathecal GR89696 and IL-10 significantly increased the paw withdrawal threshold of the cancer cell–injected rats, in a dose-dependent manner (Figs. 2 and 3). The highest doses of both drugs almost completely restored the withdrawal threshold to the normal preinjured level. The ED50 values (95% CI) of intrathecal GR89696 and IL-10 were 50.78 μg (31.80–80.07 μg) and 0.83 μg (0.59–1.15 μg), respectively.
Intrathecal coadministration of GR89696 and IL-10 dose dependently decreased the mechanical withdrawal threshold of the ipsilateral paw. Isobolographic analysis revealed a synergistic interaction between intrathecal GR89696 and IL-10 (Fig. 4), and the total fraction value was 0.11. The ED50 (95% CI) of the experimental mixture and the theoretical additive value was 2.71 μg (1.17–6.29 μg) and 25.80 μg (19.12–32.49 μg), respectively; because the 95% CIs did not overlap, P < 0.05 according to the description by Tallarida.22
In this study, intrathecal GR89696 and IL-10 effectively reduced bone cancer pain behavior, in a dose-dependent manner. In addition, interaction analysis revealed a synergism between the 2 drugs. These results raise the intriguing possibility of κ2-opioid receptor agonists and IL-10 as a new therapeutic approach for bone cancer pain management.
Despite radioligand studies demonstrating a heterogeneity of κ-binding sites,25 only the κ1 receptor has been cloned from humans and rodents, and the definition of the κ2 receptor remains an operational label based on binding and agonist/antagonist profiles.26 Thus, κ2-opioid receptor binding refers to the naloxone-sensitive bremazocine binding that remains when μ, δ, and κ1 receptors have been blocked by selective ligands.27 Because only 1 κ receptor has been cloned, it has been suggested that the distinction between the 2 subtypes may represent posttranslational modification of 1 gene product, heterogeneous interactions between different receptor systems, or altered affinity states of the same receptor.28,29 Nonetheless, the uncertainty regarding the molecular nature of the κ2 receptor does not devalue the potential pharmacological significance of the site for the treatment of chronic pain. In the current study, activation of the spinal κ2 receptor by GR89696 resulted in the suppression of bone cancer pain, consistent with previous reports describing inflammatory pain.12 Furthermore, κ2 receptor binding sites have been demonstrated to be relatively abundant in the spinal cord of both rats and humans.30 Interestingly, unlike μ-opioid receptor agonists, κ receptor agonists do not inhibit intestinal transit or induce euphoria, addiction, or respiratory depression.7 Therefore, intrathecal administration of selective κ2 receptor agonists may be effective in the treatment of bone cancer pain.
The mechanism by which spinal κ2 receptors result in antinociception is not entirely understood. Caudle et al.27 demonstrated that GR89696 inhibited N-methyl-D-aspartate (NMDA) receptor–mediated synaptic currents in the CA3 region of the guinea pig hippocampus, and this inhibition was antagonized by naloxone, suggesting that κ2 receptors function through the inhibition of NMDA receptor function.
There has been increasing interest regarding the role of inflammatory cytokines in bone cancer–related pain. TNF-α, IL-1, and IL-6 protein levels are upregulated at the tumor site and in the spinal cord of murine models of bone cancer–induced pain.31,32 Tactile allodynia and spontaneous pain in female rats with tibia tumors were attenuated by the suppression of TNF-α and IL-1β after the administration of a p38 mitogen-activated protein kinase inhibitor.33 Nevertheless, at the spinal level, the role of inflammatory cytokines for bone cancer pain is controversial. Systemic administration of selective cytokine antagonists has been shown to block mechanical hyperalgesia, whereas intrathecal IL-1 antagonists failed to show antinociception, indicating a peripheral mechanism of proinflammatory cytokines in bone cancer pain.31,32 However, the administration of a single selective proinflammatory cytokine antagonist is not likely to be effective, because several cytokines act together in these conditions. In contrast, IL-10 may suppress the production and function of all proinflammatory cytokines.34 Thus, intrathecal administration of IL-10 in the present study could have effectively inhibited bone cancer–related pain. These results are consistent with previous studies in which intrathecal IL-10 reversed intrathecal dynorphin-induced allodynia,35 peri-sciatic phospholipase A2–induced allodynia,36 and chronic constriction injury–induced allodynia and hyperalgesia.37 Moreover, IL-10 not only prevents the development of pathological pain but also can reverse established pathological pain states.37
This study demonstrates a synergistic interaction between intrathecal GR89696 and IL-10. These results may provide a theoretical basis for the development of a multitargeted drug strategy to augment antinociception while preventing or reducing the untoward side effects of each individual drug, thus enhancing analgesic efficacy. However, the mechanism of these interactions could not be established from the results of the current study. Persistent NMDA-dependent signals are, at least in part, responsible for glial fibrillary acidic protein upregulation, which is critical for morphological changes of astrocytes, thereby producing proinflammatory cytokines such as IL-1β.38 In addition, the synaptic mechanisms of central sensitization by proinflammatory cytokines are associated with enhanced NMDA-induced current.39 Therefore, the intrathecal κ2 receptor agonist may suppress the production and function of proinflammatory cytokines through the inhibition of NMDA-mediated synaptic currents, thus enhancing the analgesic action of IL-10 in the spinal cord.
Although μ-opioid agonists are recommended as the mainstay of pharmacotherapy for moderate or severe cancer-related pain, some patients develop intolerable side effects before achieving adequate pain relief.3 Currently, neither κ2 receptor agonist nor IL-10 for spinal administration is available in clinics. However, in the future, these agents may be considered as effective alternatives to μ-opioid agonists in the treatment of intractable cancer pain.
In conclusion, intrathecal administration of the κ2-opioid receptor agonist GR8969 and IL-10 attenuated bone cancer–induced pain in a synergistic manner in the spinal cord, suggesting the therapeutic use of κ2-opioid receptor agonists and IL-10 to relieve bone cancer–related pain at the spinal level. Moreover, the combination of the 2 agents may provide additional benefits for the management of bone cancer pain.
Name: Woong Mo Kim, MD.
Contribution: This author helped conduct the study, analyze the data, and write the manuscript.
Attestation: Woong Mo Kim has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Cheol Won Jeong, MD.
Contribution: This author helped conduct the study.
Attestation: Cheol Won Jeong has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Seong Heon Lee, MD.
Contribution: This author helped conduct the study.
Attestation: Seong Heon Lee has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Yeo Ok Kim, MD, PhD.
Contribution: This author helped conduct the study.
Attestation: Yeo Ok Kim has seen the original study data, reviewed the analysis of the data, approved the final manuscript.
Name: Jin Hua Cui, MD.
Contribution: This author helped conduct the study.
Attestation: Jin Hua Cui has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Myung Ha Yoon, MD, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Myung Ha Yoon has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Tony L. Yaksh, PhD.
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© 2011 International Anesthesia Research Society
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