Sensory neuron-specific receptor agonist BAM8-22 inhibits the development and expression of tolerance to morphine in rats

Behavioural Brain Research 178 (2007) 154–159

Research report

Sensory neuron-specific receptor agonist BAM8-22 inhibits the development and expression of tolerance to morphine in rats Qiaoyan Cai a , Jianping Jiang a,b , Tingjun Chen a , Yanguo Hong a,b,∗ a

b

College of Life Sciences, Fujian Normal University, People’s Republic of China Provincial Key Laboratory of Developmental Biology and Neuroscience, Fujian Normal University, Fuzhou, Fujian 350007, People’s Republic of China Received 15 October 2006; received in revised form 11 December 2006; accepted 12 December 2006 Available online 17 December 2006

Abstract We observed that intrathecal (i.t.) bovine adrenal medulla 22, an endogenous opioid peptide, partially reverses morphine tolerance. However, its mechanism remains unclear. The present study determined the effects of BAM8-22, a derivative of BAM22 and selective sensory neuron-specific receptor (SNSR) agonist, on the development and maintenance of tolerance to spinal morphine. Intrathecal administration of BAM8-22 at various doses (0.1, 1 and 10 nmol) did not alter withdraw latencies assessed in both paw withdraw and tail flick tests. Co-administration of BAM8-22 (0.1 nmol) every other day, but not daily, with morphine remarkably attenuated the development of morphine tolerance. Pretreatment and cotreatment with BAM8-22 (0.1 nmol) significantly reversed established morphine tolerance. Furthermore, intermittent administration of BAM8-22 with morphine consistently resumed morphine-induced antinociception. However, i.t. BAM8-22 did not alter morphine-induced hyperalgesia. These results suggested that SNSR may be able to modulate the sensitivity of opioid receptor serving as a most probable underlying mechanism for the effects of BAM8-22 on morphine tolerance. This study also demonstrated that intermittent combination of SNSR agonist BAM8-22 with morphine might be better regimen for long-term use of opioids to treat chronic pain. © 2006 Elsevier B.V. All rights reserved. Keywords: Morphine tolerance; Bovine adrenal medulla 8-22 (BAM8-22); Sensory neuron-specific receptor (SNSR); Antinociception; Spinal cord

Although there have been some advances in the therapeutic management of painful conditions, opioid receptor agonists remain the primary drug of choice for the treatment of moderate to severe pain in clinic [1]. This is ascribed to the fact that opioid receptors are most powerful in producing antinociception among all receptors that are involved in pain modulation. However, after being used for certain time, the opioid agonists usually lose their analgesic effectiveness and an increased dose is required to produce the same antinociceptive response (tolerance). Tolerance is a major obstacle to providing adequate pain relief over a long period of time. Efforts have been made to find the agents that reverse opiate tolerance or replace the opioid agonists for treatment of pain [2]. Bovine adrenal medulla 22 docosapeptide (BAM22), a potent opioid agonist [3,4], may possibly be ∗

Corresponding author at: Department of Physiology, College of Life Sciences, Fujian Normal University, Fuzhou, Fujian 350007, People’s Republic of China. Tel.: +86 591 8347 8923; fax: +86 591 8346 5091. E-mail addresses: yanguo [email protected], [email protected] (Y. Hong). 0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.12.014

used to treat pain after the development of opiate tolerance. We have recently demonstrated that BAM22 is not cross-tolerated in morphine-tolerant rats, and that intrathecal (i.t.) administration of BAM22 partially reverses morphine tolerance [5]. However, the mechanism by which BAM22 suppresses the expression of morphine tolerance remains unclear. BAM22 is an endogenous peptide that is derived from proenkephalin A, the precursor of Leu- and Met-enkephalin [6]. In vivo studies show that i.t. BAM22 still prolongs tailflick latency and inhibits formalin-evoked nocifensive behaviors and spinal c-Fos expression in the presence of naloxone [7,8]. This non-opioid biological action of BAM22 might be ascribed to modulate morphine tolerance as opioid receptors have been down-regulated after chronic exposure to morphine. The non-opioid action of BAM22 may be mediated by sensory neuron-specific receptor (SNSR) since the BAM22 peptide, besides opioid receptors [4,9], also binds with high affinity to SNSR which is expressed exclusively in a subset of smalldiameter primary sensory neurons [10] and may be located on the central terminal of primary afferents [11]. This study was

Q. Cai et al. / Behavioural Brain Research 178 (2007) 154–159

designed to test hypothesis that activation of SNSR modulates the development or/and expression of morphine tolerance. 1. Materials and methods 1.1. Preparation of animals All experiments were performed using male Sprague–Dawley rats, each weighing 220–270 g on the day of surgery. Rats were housed individually in plastic cages with soft bedding at room temperature and maintained on a 12 h light/12 h dark cycle with free access to food and water ad libitum. The following studies were performed under a protocol approved by the Institutional Animal Care and Use Committee. All efforts were made to minimize animal discomfort and to reduce the number of animals used.

1.2. Intrathecal catheter placement For the spinal administration of drugs to the animal, a intrathecal catheter was implanted in the subarachnoid space as described before [12]. Under sodium pentobarbital (50 mg/kg, i.p.) anesthesia, a polyethylene catheter (PE-10 tubing, Stoelting, Wood Dale, IL, USA) was inserted through a small hole made in the atlanto-occipital membrane and threaded 8–8.5 cm down the intrathecal space to the lumbar enlargement level of the spinal cord. The rostral part was sutured to the muscle to immobilize the catheter and the wound was closed in two layers with 4–0 silk. After surgery, the rats were housed individually, had free access to food and water, and were allowed to recover for at least 6 days before habituation and testing. Those with any signs of paralysis, 20% weight loss or catheter occlusion were excluded from the experiment. The proper location of the catheter was confirmed by assessing sensory and motor blockade after i.t. injection of 10 ␮l lidocaine (2%, Shenggong, Shanghai, China). At the end of the study, location of the catheter was examined by postmortem dissection and data obtained from seven animals with a misplaced catheter were excluded from analysis.

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additional 10 ␮l of saline to flush the catheter. Intrathecal administration of any drug or vehicle was performed only once daily. Rats were exposed to morphine (53.2 nmol, i.t.) once daily for six or seven consecutive days. This dose is adequate to produce tolerance within a 7 day time frame [5,17]. Then, 53.2 nmol of morphine-induced effects on nociception were tested and observed for 2 h to assess the ability of various treatments to produce or reverse tolerance.

1.5. Statistical analysis All the behavioral data were normalized to % MPE, expressed as mean ± S.E.M. (standard error of the mean). Data were analyzed by oneway analysis of variance followed by Tukey’s test to detect the changes in the morphine-induced response over time or by two-way ANOVA (treatment group × day) to detect the changes over time between two groups. Pared and un-paired t tests were used to analyze the variance in baseline WL between testing days and in morphine-induced response between groups at certain day point, respectively. P values less than 0.05 were considered significant in each test.

2. Results 2.1. Effect of BAM8-22 on thermal nociception in naive rats To examine the effect of BAM8-22 alone on thermal nociception, various doses of BAM8-22 were administered i.t. followed by WL determinations at 10, 20, 30 and 50 min. In the range of doses tested herein (0.1, 1 and 10 nmol, n = 6 each for tail flick test and n = 5 each for paw withdraw test), BAM8-22 did not alter the noxious heat-induced WL within 50 min after the administration compared with corresponding pretreatment baseline (data not shown).

1.3. Behavioral assessment To minimize intra- and inter-individual variability of behavioral outcome measures, rats were acclimatized to the experimental room and habituated to handling and behavioral testing for 30 min per day for 3 days before behavioral testing was performed. Acute nociceptive sensitivity was assessed using tail flick or paw withdrawal test. Reaction time (tail withdrawal latency or paw withdrawal latency), expressed as WL (withdrawal latency), in the test was determined by immersing the tail [7,11] or paw [13–15] into a slowly stirred water bath at 47.5 ◦ C. To conduct the tests, rats were placed in a custom-made restrainer that held the body without restraining the head, paws and tail [16]. The distal 5 cm of the tail or the hind paw up to the ankle joint dipped into warm water. When a withdrawal response occurred, the stimulus was terminated and the response latency was measured. The water temperature was adjusted to 47.5 ◦ C as this temperature produced an average baseline of WL of 8–10 s in naive rats. Baseline measurements consisted of three trials of tail-flick or paw-withdraw latency at 3-min intertrial intervals. In the absence of a response up to 20 s (cut-off time), the trial was terminated to prevent tissue damage. Investigators were blinded to drugs and doses used. The WL in the pain tests was converted to a percentage of maximum possible effect (% MPE) using following formula: % MPE =

 post-drug latency − baseline latency  cut-off time − baseline latency

× 100%

1.4. Drugs Rats received i.t. injection under conscious condition. BAM8-22 (M.W. 1,971.22) was purchased from Tocris (Bristol, UK) and Huadatianyuan Biological Co. (Shanghai, China). Morphine was obtained from Northeast Pharmaceutical Group (Shenyang, China). All drugs were dissolved in 0.9% saline. The drug or vehicle solution was injected in a volume of 10 ␮l followed by

2.2. Effect of daily or intermittent combination of BAM8-22 and morphine on development of morphine tolerance The effect of daily or intermittent combination of BAM822 with morphine on development of morphine tolerance was determined using tail flick test. To induce tolerance, rats were given 53.2 nmol of morphine (i.t.) once daily. As illustrated in Fig. 1, morphine produced potent antinociception on day 1. The effectiveness of morphine in prolonging the WL gradually decreased from day to day and almost completely disappeared on day 7 (F6,49 = 43.335, P < 0.001, n = 8, Fig. 1A upper panel and B). Baseline WL also significantly decreased on days 5–7 (P < 0.05–0.01 versus day 1, Fig. 1A, lower panel). Repeatedly daily i.t. saline (placebo) for 6 days did not alter the baseline WL. Morphine (53.2 nmol, i.t.) still produced remarkable antinociception in the placebo i.t.administered rats on day 7 (Fig. 1A, n = 7, P > 0.05 versus na¨ıve rats). The antinociceptive effects of 53.2 nmol of morphine peaked at 20–40 min (92 ± 5%) and lasted for approximately 2 h (Fig. 1B). Morphine (53.2 nmol) combined with 0.1 (n = 6) or 10 (n = 8) nmol of BAM8-22 was given i.t. once daily for 7 days and morphine-induced responses were assessed each day. Coadministration of BAM8-22 at the both doses did not prevent the development of morphine tolerance and hyperalgesia. The baseline WL and morphine-induced response in these two groups

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(53.2 nmol, i.t.) still produced remarkable antinociception even following 6-day consecutive administrations and there was significant difference between this and morphine along groups (F5,78 = 4.766, P < 0.001, Fig. 1A). On day 6, morphine-induced antinociception was about 60% MPE and lasted approximately 80 min. Both of amplitude and duration of morphine-induced response were significantly different from those observed in morphine alone group (Fig. 1B). However, intermittent coadministration of BAM8-22 did not prevent baseline from changing. Fig. 1A lower panel illustrates that baseline WL significantly decreased on days 5 and 6 compared with that on day 1. 2.3. Effect of pretreatment or co-treatment with BAM8-22 on established morphine tolerance

Fig. 1. Effects of i.t. daily or intermittent BAM8-22 on the development of morphine tolerance. Morphine (53.2 nmol, i.t., open circle) or saline (10 ␮l, closed circle) was administered from day 1 to day 6 or 7. BAM8-22 was coadministered with morphine daily (open and closed squares) or intermittently (open triangles). The data are illustrated as the mean ± S.E.M. of group. The antinociceptive effect of morphine is defined as % of MPE. (A) Shows saline- or morphine-induced changes in the WL (top panel) and the pretreatment baseline WL (bottom panel). (B) Illustrates time-course curves for morphine-induced responses on day 6 or 7 in the groups of morphine D1-7, saline D1-6/morphine D7 and Intermittent BAM8-22/morphine.  P < 0.05,  P < 0.01 and  P < 0.001 vs. morphine alone group. # P < 0.05 and ## P < 0.01 compared with day 1 in morphine alone group. @ P < 0.05 and @@ P < 0.01 compared with day 1 in group of morphine plus intermittent BAM8-22.  P < 0.001 vs. morphine on day 7 in group of saline D1-6/morphine D7.

were not significantly different from those in group treated with morphine alone at any day (data not shown). Morphine (53.2 nmol, i.t.) was administered once daily for 6 consecutive days, and BAM8-22 at a dose of 0.1 nmol was co-administered with morphine on days 1, 3 and 5 (intermittent, n = 8). In this group, the morphine-induced antinociception was only slightly decreased from day to day. Morphine

Effects of pretreatment or co-treatment with BAM8-22 on established morphine tolerance were examined using paw withdraw test. These experiments were designed to determine whether BAM8-22 could mimic BAM22-induced reversal of morphine tolerance [5]. Tolerance was induced by daily injection of morphine (53.2 nmol) for 7 days as previous groups. Then BAM8-22 (0.1 nmol, i.t.) or saline was given on day 8 and the effect of morphine (53.2 nmol, i.t.) was assessed on day 9. Fig. 2 shows that the morphine-induced response was actually not significantly different from baseline on day 7. Following treatment with saline (10 ␮l) on day 8, administration of morphine on day 9 only resulted an increase of 2% MPE in the WL (n = 7, Fig. 2A). However, in the rats that received i.t. BAM8-22 on day 8, morphine produced an increase of 54 ± 7% MPE in prolonging the WL on day 9 (n = 8, Fig. 2A) which was significantly different from that in rats with saline treatment on day 8 (P < 0.001). Potencies of morphine on day 7 and 9 were 11% and 58%, respectively, of the morphine response of naive animals. The effect of morphine lasted for approximately 60 min and was significantly different from that in saline group (P < 0.05–0.001) but less potent than that in morphine-na¨ıve rats (n = 7, P < 0.01–0.001, Fig. 2C). In a separate group that was rendered tolerant to morphine over 7 days, the rats were treated with morphine alone (53.2 nmol) or co-treated with morphine (53.2 nmol) and BAM8-22 (0.1 nmol) on day 8. Fig. 2B illustrates that coadministration of morphine and BAM8-22 (n = 8) resulted in a partial restoration of the morphine-induced response immediately after the administration which was significantly different from that observed in the group with morphine alone on day 8 (n = 8, P < 0.001). The time course and potency of the morphine-induced antinociception following co-administration were similar with that obtained in BAM8-22 pretreatment group (Fig. 2C). As above results showed that both pretreatment and cotreatment with BAM8-22 reversed morphine tolerance, we further determined if intermittent administration of BAM8-22 could mimic BAM22 to restore the potency of morphine in producing antinociception [5]. After rats developed morphine tolerance following 7-day treatments with morphine (53.2 nmol, i.t.), morphine was continuously given (53.2 nmol) on days 8–13

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Fig. 3. Reversal of morphine tolerance by intermittent BAM8-22. Rats were rendered tolerant to morphine by daily treatment with morphine (53.2 nmol, i.t.) for 7 days. Then morphine was continuously administered daily for 6 more days either alone or with BAM8-22 (0.1 nmol) every other day (days 8, 10 and 12). The data are illustrated as the mean ± S.E.M. of seven or eight rats. The antinociceptive effect of morphine is defined as % MPE.  P < 0.001 between the two groups.

3. Discussion

Fig. 2. Reversal effects of pretreatment or co-treatment with BAM8-22 on the established morphine tolerance. Morphine (53.2 nmol) was administered i.t. from day 1 to day 7 to induce tolerance. (A) Shows that BAM8-22 (0.1 nmol) or saline was given i.t. on day 8 and then morphine again on day 9. (B) Illustrates that morphine (53.2 nmol) was given alone or co-administered with BAM822 (0.1 nmol) on day 8. (C) Presents time-course curves for morphine-induced responses on day 7, 8 or 9. The antinociceptive effect of the drugs is defined as % of maximal possible effect. The data are presented as the mean ± S.E.M.  P < 0.001 vs. morphine alone group (open column) in A and B. ++ P < 0.01 and +++ P < 0.001 between groups of BAM8-22 D8/morphine D9 and saline D8/morphine D9. # P < 0.05, ## P < 0.01 and ### P < 0.001 between groups of BAM8-22 plus morphine D8 and morphine D8.

either alone (n = 7) or combined with BAM8-22 every other day (0.1 nmol, on days 8, 10 and 12, n = 8). Fig. 3 illustrates that with intermittent combination with BAM8-22, morphine remarkably increased the WL not only in the same day but also in the following day when BAM8-22 was not given. The potencies of morphine following intermittent combination of BAM8-22 were 40–50% of that observed in morphine-na¨ıve rats. However, intermittent BAM8-22 failed to alter the baseline WL tested before administration of morphine (data not shown).

The salient results of our study are as following. i.t. administration of BAM8-22, a selective agonist at SNSR, did not by itself directly affect acute thermal nociception. Chronic morphine kept producing significant antinociception when it was intermittently co-administered with BAM8-22. Pretreatment or co-treatment with the BAM8-22 peptide also partially restored antinociceptive effects of morphine in morphine-tolerant rats. Furthermore, intermittent administration of BAM8-22 consistently reversed established morphine tolerance. We have demonstrated that prior administration of BAM22 significantly resumed antinociceptive effect of morphine in morphine-tolerant rats [5]. This effect may not be mediated via opioid receptors since chronic morphine desensitized them [18]. It was likely that BAM22-induced reversal of morphine tolerance was due to its non-opioid action. As the BAM22 peptide also binds to SNSR, exhibits non-opioid activities [7,8,10] and does not show cross-tolerance to morphine [5], the reversal effect of BAM22 may be mediated via SNSR. This speculation was tested by using the selective SNSR agonist BAM8-22. BAM822 is a derivative of BAM22 and a synthesized peptide with 15 amino acids. This peptide differs from BAM22 in that it does not contain the classical opioid YGGF motif of BAM22. A study with a broad receptor screen has shown that BAM8-22 is highly selective for SNSR. It binds to SNSR with nanomolar concentration [10]. The BAM8-22 peptide displays longest duration of action compared with other SNSR agonists that have been synthesized to date [11]. Furthermore, in vitro [10] and in vivo [19] studies have demonstrated that BAM8-22 is insensitive to naloxone, a classic antagonist that is widely used in revealing opioid-linked actions. We firstly examined effect of BAM8-22 alone on acute thermal nociception. Surprisingly, we observed that BAM8-22 at doses of 0.1–10 nmol did not alter either paw withdraw latency or tail flick latency within 50 min following drug administration. This observation disagrees with the earlier study showing that BAM8-22 evoked briefly (20 min) and moderately thermal

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hyperalgesia [11]. The reason for the discrepancy was not clear. However, we have also observed that BAM8-22 at a dose of 10 nmol does not alter the nocifensive behaviors in the first phase of the formalin test [19] that was attributed to a direct algogenic effect of formalin on the nociceptors, similar to acute nociception [20,21]. In the present study, daily i.t. injection of morphine produced a progressive decrease in the analgesic response and morphine actually failed to prolong the WL on day 6 or 7. Moreover, animals developed thermal hyperalgesia evidenced by decline of baseline of WT. These results were in agreement with previous studies [22–27] and indicated that tolerance developed to the antinociceptive effect of morphine. It appeared that daily co-administration of BAM8-22 with morphine did not prevent response to i.t. morphine from decreasing as the responses produced by morphine alone and combination of morphine and BAM8-22 were not significantly different each day during 7-day treatments. Failure of daily BAM8-22 to affect morphine tolerance may suggest desensitization of SNSR due to repeated activation of the receptors. This possibility has actually been implied by the previous study showing that daily BAM22 produces a progressive decrease in the analgesic response but morphine still exerts antinociception in BAM22-tolerant rats, suggesting failure of non-opioid mediated mechanism [5]. The phenomenon and its mechanism of potential desensitization of SNSR are being investigated in this laboratory. As BAM22 activates SNSR besides the opioid receptors, desensitization of these two types of receptors could be attributed to the BAM22-induced tolerance. Therefore, we further performed a protocol of intermittent co-administration of BAM8-22 with chronic morphine and observed that morphine still produced significant antinociception even on day 6. The results that intermittent administration of BAM8-22 partially prevented the development of morphine tolerance suggested that activation of SNSR may modulate sensitivity of opioid receptors. It was shown in the present study that activation of SNSR also inhibited the expression of morphine tolerance. We first determined whether BAM8-22 administered 24 h in advance reversed morphine tolerance (see Fig. 2A). This protocol was designed because we wanted to know if BAM8-22 can mimic BAM22 to resume morphine antinociception [5]. We further assessed the effect of coadministration of BAM8-22 on expression of morphine tolerance (see Fig. 2B) since BAM8-22 itself did not affect acute thermal nociception. The results showed that both pretreatment and co-treatment with BAM8-22 significantly resumed potency of morphine as the magnitude of the acquired analgesic tolerance of morphine remarkably decreased as evidenced by a direct comparison of the antinociceptive effects of morphine. The potency of morphine was about 10% of that in morphinena¨ıve animals on day 7 following continuous administration. After co-treatment or pretreatment with BAM8-22, the potency of morphine was increased to approximately 60%. Discontinuous morphine may not be the reason why morphine restored the antinociception as a significant degree of morphine tolerance persists for at least a couple of days [28] which was also shown in the present study. Therefore, the reversal of established mor-

phine tolerance was due to the actual decrease in the magnitude of the acquired analgesic tolerance. SNSR is a newly found receptor. These receptors may be located on the central terminals of primary afferents based on the facts that SNSR mRNA are expressed in the small-diameter neurons in the dorsal root (DRG) and trigeminal ganglia [10] and the radiolabeled C-terminal part of the SNSR agonist ␥2-MSH, ␥2-MSH-6–12, binds with high affinity to membranes derived from rat spinal cord [11]. An in vitro electrophysiological study has shown that activation of SNSR by application of BAM8-22 produces marked inhibition of synaptic responses (presynaptic inhibition) and inhibition of high voltage-activated Ca2+ current [29] that may lead to inhibition of excitatory neurotransmitter release at synapses formed between small-diameter nociceptors and the spinal dorsal horn neurons [30]. However, it is not known whether these events were ascribed for the reversal effects of BAM8-22 on morphine tolerance. Especially, the mechanisms of morphine tolerance are complex and may involve multiple mediations, such as induction of spinal dynorphin expression [31] and protein kinase C [16], activation of spinal NMDA and nonNMDA receptors [16,32], and glutamate release from primary afferent fibers [31], etc. Whatever the mechanisms of morphine tolerance are, abnormal working condition of opioid receptors must be an initial factor and one of key events that occur following chronic exposure to opioids [33]. As opioid receptors and SNSR are both located in the superficial dorsal horn presynaptically [11,34], the restoration of antinociceptive effects of morphine elicited by BAM8-22 probably represents interaction between the opioid receptors and SNSR in the central terminal of primary afferent fibers. Therefore, modulation of sensibility of opioid receptors by SNSR may underlie the reversal of morphine tolerance observed in this study. It would be interesting to determine in the future whether SNSR functions to antagonize the mechanism(s) that is involved in the development or maintenance of morphine tolerance. As opioid receptors are expressed on both presynaptic terminals and the spinal dorsal horn neurons [34–36] and SNSR are only distributed in DRG neurons and, perhaps, on the central terminal of primary afferents, it can be expected that modulation of SNSR only partially inhibited the development and maintenance of morphine tolerance. Inconsistently with NMDA and non-NMDA receptor antagonists [16], BAM8-22 did not prevent and attenuate the development and maintenance of morphine-induced thermal hyperalgesia. This may be ascribed to the fact that the action involved in hyperalgesia occurs in the spinal dorsal horn neurons [37]. The failure of BAM8-22 to reverse hyperalgesia is also one of reasons that this peptide could not completely resume morphine antinociception. The chronic use of opioids is often accompanied by the development of antinociceptive tolerance so that their utilities are greatly limited. The present study demonstrated that spinal administration of the selective SNSR agonist BAM8-22 remarkably attenuated the development of morphine tolerance and resumed morphine antinociception. The results may imply that activation of SNSR modulated desensitization of opioid receptors signaling and resumed their sensitivity. It this is true, it would be perhaps of clinical significance as intermittent combination of SNSR agonist BAM8-22 with morphine might be

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