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Cancer pain is treated using various therapeutic modalities, including non-steroidal anti-inflammatory drugs, opioids, radiation and surgical intervention (Levy, 1996; Cherny, 2000). For advanced cancer pain, opioids are often the mainstay of analgesic therapy (Koshy et al., 1998; Portenoy et al., 1999; Cherny, 2000); however, opioids are often insufficient in treating bone cancer pain (Mercadante and Arcuri, 1998; Portenoy et al., 1999). Recent studies have shown that morphine has different effects on bone cancer pain and inflammatory pain in animal models (Luger et al., 2002; Yamamoto et al., 2008), and that higher doses of morphine are required to obtain significant analgesic effects in a bone cancer pain model than in an inflammatory pain model. In the clinical setting, oxycodone effectively relieves bone cancer pain (Heiskanen and Kalso, 1997; Watson and Babul, 1998; Becker et al., 2000; Gimbel et al., 2003; Watson et al., 2003; Kalso, 2005; Bercovitch and Adunsky, 2006; Silvestri et al., 2008). Previously, we reported that oxycodone inhibited different pain-related behaviours in femur bone cancer (FBC) and nociceptive pain models over a similar dose range, whereas morphine was less effective in the FBC models (Minami et al., 2009). These results suggested that oxycodone had a unique, distinct analgesic profile in bone cancer pain.
Most strong opioids, including morphine and oxycodone, are μ-opioid receptor agonists, and several studies have addressed the pharmacological mechanism underlying the actions of opioids. μ-Opioid receptors are expressed in several brain regions that modulate ascending pain signal transmission from peripheral nociceptive sensory neurons to the CNS, including the spinal cord, periaqueductal grey matter (PAG), lateral thalamus and mediodorsal thalamus (mTH) (Cohen and Melzack, 1985; Basbaum and Jessell, 2000; Narita et al., 2008). The μ-opioid receptor belongs to the large GPCR superfamily (Cox and Weinstock, 1964; Veatch et al., 1964; Pert and Snyder, 1973; Martin et al., 1976; Chen et al., 1993; Min et al., 1994; Narita et al., 2008). Binding of a μ-opioid receptor agonist changes the receptor conformation allosterically, leading to the activation of G-proteins. Since a μ-opioid receptor agonist induces the binding of [35S]-GTPγS to G proteins, measuring [35S]-GTPγS binding to the μ-opioid receptor in pain-related brain regions is valuable for assessing functional changes in this receptor induced by opioid agonists (Lazareno, 1997; Narita et al., 1999).
In this study we investigated the mechanism underlying the unique analgesic profile of oxycodone by comparing it with morphine in a bone cancer pain model. One potential mechanism – differential activation of the μ-opioid receptor by oxycodone and morphine under the bone cancer pain – was observed in this model.
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In this study, we found that μ-opioid receptor activation by oxycodone and morphine was attenuated in the PAG, vPAG, mTH and spinal cord in the FBC model. In addition, the attenuation of μ-opioid receptor activation by oxycodone was less than that of morphine in supraspinal regions, such as the PAG, vPAG and mTH. Therefore, in bone cancer pain, μ-opioid receptor activation in the brain regions related to pain signalling was modified in an agonist-dependent manner. Consistent with this, when the analgesic effects of i.c.v. oxycodone and morphine were examined using three different pain-related behaviours in the FBC model, the overall analgesic potency of oxycodone was greater than that of morphine.
Bone cancer pain is often insufficiently controlled by opioids (Mercadante and Arcuri, 1998; Portenoy et al., 1999). The FBC model is a good animal model for investigating the mechanisms of bone cancer pain (Honore et al., 2000; Minami et al., 2009), because this model mimics some clinical features of human bone cancer pain (Komiya et al., 1999; Pandit-Taskar et al., 2004). For example, pathological changes, such as bone destruction and nerve compression, appear within a few weeks after the implantation of tumour cells (Luger et al., 2002; Minami et al., 2009). Since we have reported that oxycodone has a unique analgesic profile as compared with other opioids in this model (Minami et al., 2009), the mechanism underlying the distinct analgesic effects of oxycodone in bone cancer pain can be investigated.
In the receptor binding assay, FBC mice exhibited a marked decrease in the Bmax of [3H]-DAMGO binding without affecting the Kd in pain-related regions, indicating that the number of μ-opioid receptors on cell membranes was reduced in bone cancer pain. Yamamoto et al. (2008) reported that μ-opioid receptor levels were reduced in the spinal cord and dorsal root ganglion in bone cancer pain model mice. In this study, we also found a reduction in μ-opioid receptors, not only in the spinal site but also in the supraspinal region. The reduction in μ-opioid receptors may result from their phosphorylation and/or internalization with the continuous release of endogenous opioids. It has been reported that pain and electrical stimulation induces the release of endogenous opioids in the brain (Zangen et al., 1998; Zubieta et al., 2001), and the endogenous opioids rectify pain by stimulating the μ-opioid receptor as an internal compensation system. Although we did not measure endogenous opioid release in this model, perhaps such a change in the endogenous opioid level plays a role in the reduction in μ-opioid receptors on cell membranes in pain-related regions.
Our results showed that reduction in the μ-opioid receptor levels depended on the brain regions. When we analysed the relationship between changes in the μ-opioid receptor levels and changes in the GTP activities by oxycodone and morphine, a consistent correlation was not observed. For example, while both the reduction in the number of μ-opioid receptors and that in the opioid-activated GTP level were relatively small at the vTH compared with other regions, the reduction in the μ-opioid receptor level was only 25% in PAG, although the morphine-activated GTP level was more severely decreased (as about 60% reduction) at the PAG. These results showed that the magnitude of the change in the μ-opioid receptor level does not simply reflect the magnitude of the change in the agonist activity level.
One of our main findings was the limited attenuation of μ-opioid receptor activation (measured by GTPγS binding activity) induced by oxycodone as compared with morphine in the pain-related regions in bone cancer pain. There are at least three possibile mechanisms that may explain the agonist-dependent attenuation of μ-opioid receptor activation; (i) a change in the number of μ-opioid receptors; (ii) a change in the μ-opioid receptor binding affinity; and (iii) a change in regulatory mechanism of G-protein activation. It has been reported that agonist activity of a partial agonist tends to be more affected by the change in the number of receptors than that of a full agonist (Cordeaux et al., 2000; McDonald et al., 2003). Since our data and the previous studies by others (Lemberg et al., 2006; Narita et al., 2008) showed that oxycodone is a more nearly partial agonist than morphine, a reduction of μ-opioid receptors in the FBC model mouse would be expected to affect the agonist activity of oxycodone more than that of morphine. However, our results do not corroborate this prediction and, therefore, this possibility is unlikely. The second possibility is that μ-opioid receptor binding affinity was reduced depending on the agonists under the bone cancer pain condition. However, when we examined whether the μ-opioid receptor binding affinities of oxycodone and morphine were affected in the FBC model, no significant change was observed in the displacement levels of [3H]-DAMGO binding by either oxycodone or morphine between the tumour-implanted mice and sham-operated mice. This suggests that changes in μ-opioid receptor binding affinity are an unlikely mechanism for the attenuation of μ-opioid receptor activation. The third possibility is that a regulatory mechanism of GDP-GTP exchange is responsible for the the agonist-dependent modulation in the FBC model. Since we used a non-hydrolysable analogue of GTP ([35S]-GTPγS) in this study, the processes involved in GTP hydrolysation cannot account for the differences in GTP stimulation, and the distinct μ-opioid receptor activation caused by oxycodone and morphine is probably due to the cellular processes following ligand binding to the receptors, before Gα-GDP is exchanged for Gα-GTP. Thus, the differential activation of the μ-opioid receptor by oxycodone and morphine might be due to the mechanism that regulates the binding of GTP to G proteins in the FBC model. Our hypothesis is supported by previous findings showing that the GDP-GTP exchange factor modulates agonist-induced pharmacological effects (Birukova et al., 2006). However, little is known about the importance of the GDP-GTP exchange mechanism in the regulation of GTP activation by opioid agonists; further studies are required to identify the underlying mechanism of the observed agonist-dependent effect.
Since oxycodone and morphine activate μ-opioid receptors differently in pain-related brain regions, the supraspinal administration of oxycodone and morphine leads to distinct analgesic effects in the FBC model. While i.c.v. oxycodone and morphine had equipotent antinociceptive effects in the sham-operated mouse of FBC (C3H/HeN mouse; Table 1) and the naïve mouse (Narita et al., 2008), i.c.v. oxycodone had 5∼7 times greater analgesic potency than morphine in the evaluation of guarding and allodynia-like behaviours in the FBC model. Consequently, the analgesic potencies of oxycodone and morphine are consistent with their profiles of μ-opioid receptor activation in supraspinal sites in the FBC model.
Of the regions examined in this study, the PAG and mTH are thought to play important roles in the analgesic effects of opioids. The PAG is part of the descending analgesic pathway, and morphine suppresses the release of the neurotransmitter GABA from neurons in the PAG (Basbaum and Fields, 1984). In the mTH, the stimulation of μ-opioid receptors by opioids results in an increase of the inwardly rectifying potassium channel current, which hyperpolarizes the cell and changes its firing pattern on the postsynaptic membrane (Brunton and Charpak, 1998). The unique μ-opioid receptor activation profiles in the PAG and mTH with bone cancer pain may result in the distinct in vivo analgesic potency of oxycodone.
A previous study using a neuropathic pain model reported that morphine-induced μ-opioid receptor activation in the PAG, thalamus and spinal cord did not differ significantly between sham-operated and sciatic nerve-ligated mice (Narita et al., 2008). In our study, on the other hand, the Emax of morphine for μ-opioid receptors was attenuated markedly in several regions in the FBC model. These results suggest that the function of μ-opioid receptors is differentially modulated depending on the type of pain.
There are reports that the antinociceptive effect of oxycodone is antagonized by nor-BNI, a κ-opioid receptor antagonist, at a dose that did not affect the antinociceptive effect of morphine in two rat neuropathic pain models (Ross and Smith, 1997; Nielsen et al., 2007). This suggests that oxycodone produces its analgesic effect by acting on the κ-opioid receptor. Conversely, several different groups have reported that oxycodone-induced antinociception is mediated by the μ-opioid receptor, and that oxycodone binds selectively to μ-opioid receptors (Lemberg et al., 2006; Peckham and Traynor, 2006; Narita et al., 2008). There is a possibility that the discrepancies of the receptors responsible for the oxycodone effect might be due to the different routes of administration. Since oxycodone is metabolized to oxymorphone, an active metabolite, by the peripheral administration, the oxycodone-induced pharmacological effect may be the result of both oxycodone and oxymorphone. It has been reported, however, that both oxycodone- and oxymorphone-induced antinociception are mediated by μ-opioid receptors rather than κ-opioid receptors (Lemberg et al., 2006). These studies suggest that the difference in the administration route does not simply account for the different roles of the μ- and κ-opioid receptor in the opioid analgesic effects. In the present study, we showed that increased GTPγS binding by oxycodone and morphine was antagonized by β-FNA, but not by nor-BNI. Our data are consistent with the idea that the pharmacological effects, for example analgesic effects, of both oxycodone and morphine are mediated via the μ-opioid receptor.
Finally, this study showed that the effects of oxycodone and morphine are modulated differently in bone cancer pain, and that μ-opioid receptor activation by oxycodone in brain regions related to pain signalling was attenuated less as compared with the effects of morphine. Consistent with this, the overall analgesic potency of oxycodone was stronger than that of morphine when they were administered i.c.v. in the FBC model. Therefore, the modulation of μ-opioid receptor function under bone cancer pain appears to be one of the mechanisms underlying the unique analgesic profile of oxycodone, and such modulation may determine analgesic efficacy of a particular opioid in chronic pain.