Chapter 1
Ultra-Low-Dose Opioid Antagonists Enhance Opioid Analgesia and Reduce Tolerance Lindsay H. Burns, Todd W. Vanderah, and Hoau-Yan Wang
Abstract Ultra-low-dose opioid antagonists have been shown to enhance opioid analgesia and attenuate the tolerance to analgesic effects normally seen with chronic opioid administration. This chapter reviews the early work with ultralow-dose opioid antagonists starting with electrophysiological recordings of dorsal root ganglion neurons and continuing to antinociception in rodents. These pharmacological findings have not adhered to typical dose response curves and have instead been reported to occur at wide ranges of extremely low doses of several opioid antagonists as well as with the rare opioid agonist. Optimal dose ranges have also been reported to vary with sex and strain of rat. Translation into small clinical studies has been met with varied results, related to variations in dose, route of administration, and antagonist selected. Nevertheless, the clinical studies that have demonstrated enhanced analgesia or opioid sparing effects have utilized opioid antagonist doses in lower dose ranges than the studies that failed to demonstrate efficacy. Furthermore, a large double-blind, placebo- and active-controlled clinical trial demonstrated enhanced opioid analgesia with the extremely low dose of 2 μg naltrexone/patient/day. Preclinical data also extend the effects of ultra-low-dose opioid antagonists to neuropathic pain, which is comparatively resistant to opioid treatment and, interestingly, to cannabinoid analgesia. The mechanism of action has been shown to be the prevention of a chronic opioid-induced mu opioid receptor–G protein coupling switch that is associated with analgesic tolerance and dependence. Finally, recent data shows that this G protein coupling switch is controlled by filamin A and that a high-affinity interaction of naloxone or naltrexone with this scaffolding protein mediates their prevention of the altered coupling.
Keywords: Naloxone; Naltrexone; Neuropathic pain; G protein coupling; Filamin A
L.H. Burns (), T.W. Vanderah, and H.-Y. Wang Pain Therapeutics, Inc., Preclinical Development, 2211 Bridgepointe Parkway, Suite 500, San Mateo, CA 94404 e-mail:
[email protected] R. Dean et al. (eds.), Opiate Receptors and Antagonists, © Humana Press, a part of Springer Science + Business Media, LLC 2009
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Introduction
Opioid analgesics are widely used to manage moderate or severe pain, yet their use is hampered by unwanted side effects, tolerance to their analgesic effects, and physical dependence. The fact that patients often balance analgesia with side effects illustrates the narrow therapeutic window of opioids. Side effects range from nausea, constipation, somnolence, and pruritis to the more serious respiratory depression. Chronic pain patients need analgesia over prolonged or indefinite time periods, yet these patients very often experience a loss of analgesic potency with continued use. Finally, physicians and patients alike are wary of initiating opioid therapy due to the fears or stigma around opioid dependence and even the potential for addiction. As a consequence, the field of pain management is in great need of improved analgesic therapies that provide adequate analgesia for moderate-to-severe pain with fewer adverse effects and minimal potential for tolerance, dependence, and addiction. One such therapy involves the combination of opioid agonists with “ultra-lowdose” opioid antagonists. In contrast to the typical blockade of opioid receptor functions by higher concentrations of opioid antagonists, ultra-low-dose opioid antagonist cotreatment enhances and prolongs opioid analgesia and also prevents or reverses opioid analgesic tolerance. These phenomena have been demonstrated by preclinical data and have now randomized double-blind, controlled clinical trial data. This chapter will review the effects of ultra-low-dose opioid antagonists on analgesia, tolerance, and the hyperalgesia and allodynia in a model of neuropathic pain, while the attenuation of physical dependence and the addictive properties of opioids will be reviewed in Chapater 13. This chapter will also provide a historical overview of studies probing the mechanism of action for these effects from the initial electrophysiology findings to more recent molecular pharmacology data and identification of the high-affinity binding site. These studies indicate that chronic opioid administration causes alterations in the G protein system associated with mu opioid receptors (MORs) and that ultra-low-dose naloxone (and perhaps other opioid antagonists) suppresses these aberrant signaling changes via a picomolar interaction with filamin A.
1.2
Preclinical Evidence of Enhanced Opioid Analgesia and Reduced Tolerance
The history of ultra-low-dose opioid antagonists starts with electrophysiology studies on dorsal root ganglion (DRG) cell cultures. Crain and Shen first observed that while opioid agonists normally inhibit, or shorten, the action potential duration of DRG cells, lower doses of opioid agonists induce an opposite, excitatory effect – a prolongation of the action potential (39, 9). These researchers noted that prolonged exposure to an opioid agonist would also produce this prolongation of action potentials, and they theorized that these excitatory effects of opioids contributed to opioid tolerance (10). This same publication proposed a regula-
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tion by GM1 ganglioside, since exogenous administration of GM1 ganglioside mimicked the prolonged opioid exposure. The hypothesis that excitatory effects of opioids are mediated by opioid receptors coupling to the excitatory G protein Gs rather than the usual inhibitory G proteins, Gi or Go, was suggested by a blockade of the action potential prolongation by administration of cholera toxin-A, an agent that blocks Gs from being activated by receptor stimulation (40). Using their in vitro model of action potential prolongation by low concentrations of morphine, Crain and Shen (11) showed that ultra-low-dose naloxone blocked the action potential prolongation when added at low picomolar concentrations. In the same 1995 publication, Crain and Shen showed that ultra-low-dose naltrexone (10 ng/kg, s.c.) also enhanced and prolonged morphine’s antinociceptive potency in mice. This seminal report also included data demonstrating that cotreatment with ultra-low-dose naltrexone dramatically attenuated morphine withdrawal after an acute high dose of morphine or after a 4-day, twice daily, progressively increasing morphine dosing schedule. Finally, an assessment of tolerance was made in the mice receiving the escalating 4-day morphine treatment, by comparing subsequent antinociception to that of naïve mice. Tolerance was partially prevented by ultra-low-dose naltrexone. A subsequent study by a different lab extended the work of Crain and Shen, demonstrating enhanced morphine antinociception by ultra-low-dose naltrexone administered either systemically or intrathecally to rats (36). This study also demonstrated that coadministration of ultra-low-dose naltrexone (0.05 ng) prevents analgesic tolerance over 7 days of daily intrathecal administration of morphine (15 μg). An even lower intrathecal dose of naltrexone (0.005 ng) as well as i.p. administration of both drugs was less effective. Using rats instead of mice, these data demonstrated enhanced morphine analgesia, a complete prevention of tolerance over a 7-day treatment period, a lower end of an effective ultra-low-dose range by intrathecal administration, and a spinal site of action. Data also showed that established morphine tolerance could be reversed, at least partially. Crain and Shen also followed up their earlier work by demonstrating that both analgesic tolerance and precipitated withdrawal-associated hyperalgesia in mice could be prevented by ultra-low-dose naltrexone combined with either morphine (45) or oxycodone (44). In a further extension of their in vitro data, Crain and Shen demonstrated that acute hyperalgesia elicited by a low dose of an opioid agonist, referred to clinically as “paradoxical hyperalgesia” (27,29), could be reversed by ultra-low-dose naltrexone coadministration, yielding a strong analgesic effect (15). While the early in vitro work primarily utilized naloxone, ultra-low doses of the kappa opioid antagonist nor-binaltorphamine (nor-BNI) and the mu opioid antagonist beta-funaltrexamine (β-FNA) (Crain and Shen, personal communication), as well as the nonselective opioid antagonist diprenorphine (41), have also enhanced the inhibitory effects of opioid agonists in vitro. While most published in vivo work was initially limited to naltrexone, ultra-low-dose naloxone was also recently used in vivo to prevent analgesic tolerance (53; Fig. 1.1). In addition, ultra-low doses of naloxone and nalmefene have demonstrated beneficial effects in clinical studies discussed below. Perhaps more surprisingly, ultra-low doses of the opioid agonists
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Morphine + NLX Morphine (10 mg/kg) NLX (10 ng/kg)
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Fig. 1.1 Co-treatment with ultra-low-dose NLX (10 ng/kg) prevented the antinociceptive tolerance caused by chronic morphine (10 mg/kg, s.c., twice daily for 7 days). Rats treated with morphine + NLX showed stable tail-flick latencies over the week of treatment, while tail-flick latencies of rats receiving morphine alone declined to a level not significantly different from NLX alone. *p < 0.05 and **p < 0.01 for morphine + NLX versus morphine. BLINE predrug baseline, NLX naloxone. Reprinted from Wang et al. (53), with permission from Elsevier
etorphine and dihydroetorphine (41) and of [des-Tyr] fragments of the endogenous opioids dynorphin A-(1–13) and β-endorphin (1–27) (42) also enhance the inhibitory effects of opioid agonists in vitro. Further, ultra-low-dose etorphine enhances morphine’s potency in vivo (43). Together, these findings hinted that these phenomena are not unique to naloxone or naltrexone and are also not dependent on opioid antagonist activity.
1.3
Dose Effects and Dependency on Strain and Sex
Ultra-low-dose opioid antagonist effects do not follow a typical dose-response pattern, but typically span several log units without conforming to bell-shaped curves. A wide range of doses has been used successfully. Although some initial studies tested antagonist:agonist ratios rather that discrete antagonist doses, the much narrower effective dose range for agonists than for ultra-low-dose antagonists in enhancing agonist effects suggests that the antagonist dose is independent of agonist dose. This method was likely initiated for ease of calculating antagonist dose. An examination of the antinociceptive effects of various ratios of naltrexone:oxycodone (1:109, 1:107, and 1:105) combined with a range of oxycodone doses (0.03–3 mg/kg) in male Swiss Webster mice illustrates the wide range of effective doses of naltrexone (Fig. 1.2). Whereas all naltrexone:oxycodone dose ratios examined enhanced the areas under the curve (AUCs) of tailflick latencies
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Oxycodone Doses (mg/kg) Fig. 1.2 Dose-response of ultra-low-dose naltrexone:oxycodone ratios in enhancing oxycodone antinociception in the 52°C hot water immersion tail-flick tests in male Swiss Webster mice. Three different naltrexone:oxycodone ratios (1:109, 1:107, and 1:105) were superimposed onto a dose-response of oxycodone (0.03, 0.1, 0.3, 1, and 3 mg/kg, s.c.). These naltrexone:oxycodone ratios enhanced the effects of all doses of oxycodone, although the 1:109 and 1:107 ratios more potently enhanced the antinociceptive effect of the 3 mg/kg oxycodone dose than did the 1:105 dose ratio. Data are means ± SEM., n = 3. Reprinted from (2) in Recent Developments in Pain Research, with permission from Research Signpost
of the oxycodone doses, the strongest antinociceptive effects were obtained with the 3 and 300 pg/kg naltrexone doses, which appear to enhance the antinociceptive effects of 3 mg/kg oxycodone to a greater extent than a 30 ng/kg dose. While wide antagonist dose ranges have been used effectively, the optimal doses of antagonist vary with both the opioid agonist it is combined with as well as the sex of the animal. The prevention of tolerance and withdrawal-associated hyperalgesia by ultra-low-dose naltrexone was demonstrated at 0.3, 3, 300, and 3,000 ng/kg naltrexone when combined with 3 mg/kg morphine (45) and at 1 pg/kg or 1 ng/kg naltrexone when combined with oxycodone (44). The higher naltrexone doses used in these studies were used in males, suggesting males are less sensitive to ultra-low-dose antagonist effects. Interestingly, although these studies used the same morphine dose for males and females, a tenfold lower dose of oxycodone was administered to males, confirming the previously described increased sensitivity to opioid analgesic effects in male rodents (28). Hence, while male rodents may be more sensitive to opioid analgesia, female rodents appear to be more sensitive to ultra-low-dose naltrexone, suggesting some degree of dissociation between the two. In addition, low doses of oxycodone elicited a greater degree of hyperalgesia in male than in female rats (23), while the reverse was reported with morphineinduced hyperalgesia (22). These findings likely contribute to the variations in
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optimal ultra-low-dose opioid antagonist dose ranges noted both with sex of the animal and the opioid used in combination. In addition to sex differences, the effects of ultra-low-dose naltrexone on morphine antinociception and tolerance can also vary with rat strain (46). While the enhancement of morphine antinociception and attenuation of morphine tolerance was observed in both Sprague–Dawley and Long–Evans rats, naltrexone at the dose range tested (0.1–100 ng/kg) did not enhance morphine antinociception over a range of doses (2.5–7.5 mg/kg) in Fisher 344 or Lewis rats, nor did 10 or 100 ng/kg naltrexone significantly attenuate tolerance in these two strains. Given that pg/kg doses of naltrexone most effectively enhanced oxycodone antinociception in females in the previously mentioned Shen study (44), the question remains whether this even lower range might enhance antinociception in these two rat strains. Nevertheless, clear strain differences exist expanding on the initial report that ultra-low-dose naltrexone enhances morphine antinociception in Swiss Webster mice but antagonizes it in 129/SvEv mice (14). The authors attribute this opposite effect of ultra-low-dose naltrexone in 129/SvEv mice, along with the enhanced potency of morphine and general lack of tolerance in this strain, to its deficiency in GM1 ganglioside. The Terner study (46) also noted strain-specific sex differences. In Sprague– Dawley rats, males and females differed in the dose that most effectively enhanced antinociception, with the best effect in males observed at 10 ng/kg and the highest efficacy in females occurring at the lowest dose tested at 0.1 ng/kg. This greater sensitivity to ultra-low-dose naltrexone in female Sprague–Dawley rats, despite the females being less sensitive to the opioid itself, concurs with the findings by Shen and colleagues in Swiss Webster mice (44, 45). An additional strain-dependent sex difference was revealed in the Terner study; 10 ng/kg naltrexone significantly reversed established morphine tolerance only in female Long–Evans rats. In a separate study examining age and sex effects, morphine antinociception was enhanced by low-dose naltrexone in mature female but not in mature male rats (18–22 weeks) and was negligible in younger rats (21). However, the naltrexone dose range used in that study was again comparatively high (0.002–2 μg/kg), and the effect in mature females was “inversely related to dose.”
1.4
Ultra-Low-Dose Naltrexone Effects on Neuropathic Pain and Cannabinoid Analgesia
Although opioids are not typically very effective in neuropathic pain, recent preclinical data has shown that ultra-low-dose naltrexone enhances the antihyperalgesic and antiallodynic effects of morphine (49) or oxycodone (30) in the L5/L6 spinal nerve ligated rats. The antihyperalgesic effects of intrathecal delivery of these opioids combined with naltrexone at 0.33 ng were complete, though some tolerance developed after 10 days of twice daily administration. Further, while intrathecal morphine did not elicit any antiallodynic effect in this model, its combination with ultra-low-dose naltrexone produced a moderate though transient
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antiallodynic effect. With twice daily oral delivery in this rat model of neuropathic pain, ultra-low-dose naltrexone (0.3–3 μg/kg) combined with oxycodone produced strong antihyperalgesic and antiallodynic effects. Tolerance to these effects was dramatically reduced compared to the more rapid tolerance that developed with oral oxycodone alone. The oral 0.3 μg/kg naltrexone dose combination produced an antihyperalgesic effect that showed no tolerance over this time period. Notably, the enhanced antihypersensitivity effects of the opioids in these studies occurred at slightly higher doses of naltrexone than those shown to enhance antinociception in intact rats. Moreover, these preclinical data suggest that ultra-low-dose naltrexone may augment opioid effects in neuropathic pain and minimize the development of tolerance to them. Further extending the area of research with ultra-low-dose opioid antagonists, a recent study showed ultra-low-dose naltrexone to enhance cannabinoid-induced analgesia (35). This finding may reflect the well-established interaction of the opioid and cannabinoid systems (32), rather than a specific interaction of naltrexone’s binding site on filamin A with cannabinoid receptors. The complete abolition of the analgesic effect of the cannabinoid agonist/naltrexone combination by a cannabinoid antagonist suggests that this analgesia is dependent on the cannabinoid system with any opioid effects upstream. The fact that a high dose of naltrexone only slightly (and nonsignificantly) enhanced here the cannabinoid-induced analgesia suggests that that the potentiation of cannabinoid analgesia by ultra-low-dose naltrexone has some opioid component but does not exclusively rely on opioid receptors.
1.5
Clinical Studies of Ultra-Low-Dose Opioid Antagonist Effects in Analgesia
Clinical experience with ultra-low-dose opioid antagonists has grown from case reports and small clinical studies to blinded, randomized, controlled clinical trials. In contrast to preclinical reports that mostly use naltrexone, clinical studies have reported benefits with ultra-low-doses of naloxone, naltrexone, and also nalmefene. In addition, “ultra-low” doses, dosing schedules, and settings have varied along with results. Most small clinical studies assessed postoperative analgesia and used i.v. (intravenous) administration, while current clinical trials have examined analgesia and dependence and have used oral delivery over 3 weeks or 3 months. While no published clinical studies or clinical trials have been able to assess ultra-low-dose opioid antagonist effects on analgesic tolerance, a case report noted strong analgesia in a severely opioid tolerant diabetic neuropathy patient from ultra-low-dose naltrexone (1 μg orally, b.i.d.) combined with methadone (16). This patient reported a pain intensity reduction from 9 to 3 within 24 h that persisted through a 1-month follow-up. In a postoperative setting, the first clinical study showed that ultra-low-dose naloxone in a continuous i.v. infusion of 0.25 μg/kg/h, but not 1 μg/kg/h, produced an opioid-sparing effect of morphine delivered by patient-controlled analgesia (PCA) following hysterectomy (18). In another study in women undergoing lower
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abdominal surgery, a single dose of 15 or 25 μg nalmefene administered before morphine PCA decreased severity of pain 24 h later and also decreased the need for antiemetics and antipruritics (26). A subsequent study using morphine PCA with or without naloxone at an estimated dose of 0.57 followed by 0.19 μg/kg/h failed to demonstrate enhanced analgesia or opioid-sparing effects (4). Reducing the i.v. naloxone dose tenfold in a later study to an estimated 0.05 followed by 0.006 μg/kg/h, Cepeda again did not demonstrate opioid-sparing effects or enhanced morphine analgesia but reported decreased nausea and pruritis (5). It should be noted that in both studies, PCA doses were increased by 20% when pain intensity exceeded 4 and again at 6 on a 0–10 scale, and a rescue dose 2.5 times the PCA solution was administered when pain intensity reached 6, so accurate assessment of doses is difficult. However, the PCA solution contained 6 μg/cc in the first study and 0.6 μg/cc in the second. A small randomized, double-blind clinical trial of 156 patients presenting to the emergency department with acute, severe pain did not demonstrate any greater analgesic efficacy of a single i.v. bolus of morphine (0.1 mg/kg) combined with one of three different doses of naloxone (0.1, 0.01, or 0.001 ng/kg) compared to the morphine alone (1). In contrast to the only other study that used a single i.v. bolus of an ultra-low-dose opioid antagonist, that is nalmefene administered prior to morphine PCA postoperatively and assessed pain scores 24 h later (26), the present study gave one combined bolus and assessed pain intensity over the next 4 h. Possibly refers to confounding the results of the study were additional doses of morphine or other opioids administered within the 4 h period. While 13 patients in the morphine-only group received additional morphine, 8, 10, and 14 patients in the 0.1, 0.01, and 0.001 ng/ kg naloxone groups required additional morphine, respectively. Although, this data may suggest an opioid-sparing effect at the 0.1 ng/kg naltrexone level these differences were not significant due to the small group sizes. Besides the small group sizes in this study, the diversity of pain conditions and changing rates of pain in patients presenting to the emergency department may have hindered this trial. Nevertheless, this study was unable to demonstrate an effect of ultra-low-dose naloxone in the dose range of 0.001–0.1 ng/kg in augmenting opioid analgesia in acute, severe pain. The first large, randomized, double-blind clinical trial assessed oxycodone and ultra-low-dose naltrexone (Oxytrex™) versus oxycodone alone delivered orally in 350 osteoarthritis patients with pain ≥5 on a 0–10 scale for 3 weeks (7). Naltrexone was formulated with oxycodone at 1 μg per tablet, so that the Oyxtrex b.i.d. dose group received 2 μg/day while the Oxytrex q.i.d. group received 4 μg/day. Both were compared to placebo and oxycodone alone administered q.i.d. in an immediate-release formulation. All active treatment groups received the same daily dose of oxycodone escalating from a starting dose of 10–40 mg/day in the final week. The Oxytrex b.i.d. treatment, with 2 μg/day naltrexone, provided significantly greater analgesia than both placebo and oxycodone by the end of week 2, with the strongest differences seen at the end of week 3. Neither oxycodone itself nor Oxytrex q.i.d., with 4 μg/day naltrexone, separated from placebo in reduction of pain intensity, a result not inconsistent with prior clinical trials of oxycodone at these doses (37, 33). Although this trial included mostly female patients, a sub-analysis by gender illustrated a stronger treatment effect in males, and a stronger separation
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Fig. 1.3 Reduction in pain intensity in males and females. Oxytrex b.i.d. provided the greatest reduction in pain intensity scores in both males and females. At week 3, Oxytrex b.i.d. was significantly better than placebo in males and significantly better than oxycodone in females. Reprinted from Chindalore et al. (7) with permission from American Pain Society
of Oxytrex b.i.d. from placebo in males and a statistical separation of Oxytrex b.i.d. only from oxycodone in females, possibly due to a greater placebo effect in females (Fig. 1.3). Moreover, this first clinical trial demonstrated both enhanced analgesia and the ability to dose b.i.d. without any extended release mechanism, illustrating the prolonged duration of effect seen preclinically. In addition, the enhanced analgesic efficacy of the treatment regimen containing 2 but not 4 μg/patient/day of naltrexone, combined with its ∼20% bioavailability by oral delivery, may shed light on earlier studies using i.v. naloxone at higher doses. A subsequent clinical trial in 750 low-back pain patients showed that Oxytrex b.i.d. provided equivalent analgesia from a significantly lower total average daily dose compared to oxycodone q.i.d. (54). In this trial, patients titrated their dose to a pain score ≤2 or to tolerable side effects up to a maximum of 80 mg/day before being fixed for 12 weeks on their individual doses. Oxytrex b.i.d. also significantly reduced the number of moderate-to-severe events of constipation, somnolence, and pruritis seen with oxycodone alone. To summarize clinical experience with ultra-low-dose opioid antagonists, some studies may have failed due to “ultra-low” doses that are too high, possibly impeding the effect of the opioid by classical receptor antagonism. In addition, differences in efficacy were observed between studies in postoperative or acute pain and chronic pain populations, possibly suggesting greater efficacy in chronic pain, though one demonstrated enhanced efficacy of morphine intra-operatively. Gender differences in sensitivity to ultra-low-dose opioid antagonists have not emerged clinically, though two positive studies were performed in females. Finally, while some positive studies noted enhanced analgesia, others noted opioid-sparing effects or decreased side effects.
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Mechanism of Ultra-Low-Dose Opioid Antagonists
The mechanism of action of ultra-low-dose opioid antagonists in enhancing and prolonging analgesia and preventing tolerance has long been hypothesized as a blockade of excitatory signaling opioid receptors. Mu opioid receptors preferentially couple to pertussis toxin-sensitive G proteins, Gi and Go, to inhibit the adenylyl cyclase/cyclic AMP (cAMP) pathway (31, 8). Since the in vitro excitatory effects of low doses of opioids (the shortening instead of prolongation of the action potential duration) could be pharmacologically blocked by cholera toxin-A, a compound that blocks activation of the “excitatory” G protein Gs by their associated receptors (40), Crain and Shen hypothesized that the excitatory response to opioids, whether from low doses or prolonged exposure, was mediated by MORs coupling to Gs (12). While Gi and Go proteins are known to inhibit adenylyl cyclase and subsequent production of cAMP from ATP, Gs stimulates this enzyme causing the opposite effects on the cell. Based on their electrophysiology work, Crain and Shen also hypothesized that opioid antagonists selectively antagonized such “excitatory opioid receptors” that signal via Gs proteins (13). Much of the controversy over this initial hypothesis arose from earlier research suggesting that observable excitatory effects of opioids were not due to a G protein coupling partner novel to MOR, that is, a switch in G protein coupling, but were simply due to altered signaling of the Gβγ dimer contained in the original Gi or Go proteins (20). The Gβγ dimer of the heterotrimeric G proteins coupling to the MOR are thought to contribute to the analgesic effects of opioids by signaling to ion channels and inhibiting cellular activities by hyperpolarization (24, 38). A Gβγ activation of adenylyl cyclase instead would counteract analgesia as would stimulation of adenylyl cyclase by a Gs protein. Molecular pharmacology work has demonstrated a decrease in MOR Gi and Go coupling concurrent with a striking appearance of Gs coupling in central nervous system (CNS) tissues of rats treated with morphine (10 mg/kg, s.c.) twice daily for 7 days, and an attenuation of these changes by cotreatment with ultra-low-dose naloxone (10 μg/kg, s.c.) (53). A morphine-induced association of MOR with Gs was also demonstrated in Chinese hamster ovary (CHO) cells and in spinal cord (6) by the group who initially demonstrated morphine-induced adenylyl cyclase activation by Gβγ. The development of Gs coupling by MORs may also contribute to the hypersensitivities to thermal and tactile stimuli noted to occur after chronic morphine administration (47, 48). Wang et al. (2005) used a previously established coimmunoprecipitation procedure (50, 25, 55, 56, 3) with antibodies to various Gα proteins to separately isolate each Gα protein along with its associated receptor proteins under basal and morphine-stimulated conditions. Subsequently, the relative amounts of MOR protein associated with each Gα protein were detected via Western blots with a selective antibody directed against an N-terminal epitope of MOR. These relative quantities demonstrated treatment-associated changes in levels of MOR coupling to Gi, Go, and Gs without discernible effects on the expression of either MORs or G proteins (Fig. 1.4). The specificity of antibodies was rigorously demonstrated, and the instatement of Gs coupling with chronic morphine treatment
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MOR immunreactivity Optical Density (Arbitrary Unit)
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+ NLX Fig. 1.4 Densitometric quantifications of Western blots showing that chronic morphine-induced Gs–Mu opioid receptor (MOR) coupling is attenuated by cotreatment with ultra-low-dose naloxone (NLX). MOR protein is detected in immunoprecipitates of Gαi, Gαo and Gαs of spinal cord from rats treated twice daily for 7 days with saline, morphine (10 mg/kg, s.c.), morphine + NLX (10 ng/kg, s.c.) or NLX (10 ng/kg, s.c.). Data are means ± s.e.m. derived from four individual rats in each of the treatment groups that were processed individually (n = 4). Solid bars indicate basal coupling, and hatched bars indicate coupling after receptor stimulation by in vitro morphine.*p < 0.05 versus same Gα protein in vehicle and NLX-treated groups.**p < 0.05 versus same Gα protein in morphine group. Morphine-stimulated coupling was significantly greater (p < 0.01) than basal coupling of each Gα protein within each treatment group. Reprinted from Wang et al. (53), with permission from Elsevier
and its attenuation by cotreatment with ultra-low-dose naloxone were confirmed using tritiated DAMGO instead of the MOR antibody. Wang et al. (53) also examined the interaction of Gβγ with adenylyl cyclase using a similar coimmunoprecipitation procedure using CNS tissues from the same morphine, morphine and naloxone or vehicle-treated rats. Chronic morphine instated an interaction of the MOR-associated Gβγ with adenylyl cyclase types II and IV, and these Gβγ–adenylyl cyclase interactions were similarly attenuated by ultra-low-dose naloxone cotreatment. These treatment-mediated changes in association between Gβγ and adenylyl cyclase type II and IV also occurred without alterations in expression levels of these signaling molecules. We have also demonstrated that the Gβγ that interacts with adenylyl cyclase after chronic morphine treatment originates from a Gs protein that couples to the MOR as opposed to its native G protein (51). Ultra-low-dose opioid antagonists were initially thought to preferentially bind a subset of MORs (13), and a Gs-coupling MOR subpopulation was again recently proposed (6). However, since naloxone prevents MOR–Gs coupling at concentrations well below its affinity for MOR and since our coimmunoprecipitation data showed it to alter the coupling behavior of MOR, we considered proteins that
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interact with MOR and MOR-associated G proteins as the most likely targets, particularly those able to interact with multiple MORs. We recently identified a pentapeptide segment of C-terminal filamin A, a scaffolding protein known to interact with MOR (34), as the high-affinity binding site of ultra-low-dose naloxone in preventing the chronic opioid-induced MOR–Gs coupling (52). Using organotypic striatal slice cultures, we showed that peptide fragments containing the binding site on filamin A abolished the prevention by 10 pM naloxone of both the chronic morphine-induced MOR–Gs coupling and the downstream cAMP excitatory signal. A competition curve showed that naltrexone or naloxone binds filamin A with an affinity of ∼ 4pM, that is ∼200-fold higher than their affinity for MOR (17, 19). This data establishes filamin A as the target for ultra-low-dose naloxone, naltrexone in their prevention of Gs coupling by MOR, and presumably also their prevention of analgesic tolerance and dependence.
1.7
Conclusions
This chapter has provided an overview of ultra-low-dose opioid antagonist research from the initial discovery of preventing opioid excitatory effects observed by electrophysiology in vitro to preclinical findings of enhanced and prolonged analgesia and reduced analgesic tolerance in vivo. The dose–response relationship spans several log units and does not conform to a bell-shaped curve, although dramatic sex and strain effects have been noted within various dose ranges. This chapter reviewed preclinical and clinical studies conducted with a variety of doses and opioid agonist/antagonist combinations in a variety of settings. The recent identification of the high-affinity interaction binding by naloxone or naltrexone to filamin A may partially explain the varied results seen clinically with various opioid antagonists, doses, and routes of administration, on top of the different patient populations, conditions, and endpoints between studies. Further, the observed sex and strain differences could potentially reflect differences in filamin A levels. Although, a few studies have failed to demonstrate benefits of ultra-low-dose opioid antagonists, most of the available evidence, including results of the larger clinical trials, clearly indicates that certain combinations can substantially improve opioid therapy available today. The effects of ultra-low-dose naltrexone on physical dependence and addictive properties of opioids are reviewed in the Chapter 13.
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