Intrathecal cyclooxygenase inhibitor administration attenuates morphine antinociceptive tolerance in rats

C. -S. Wong1,*, M. -M. Hsu1, R. Chou2, Y. -Y. Chou1 and C. -S. Tung3

1Department of Anesthesiology, National Defense Medical Center and Tri-Service General Hospital, Taipei, Taiwan. 2School of Biological Sciences, University of California, Irvine, Irvine, California 92697, USA. 3Department of Physiology and Biophysics, National Defense Medical Center, Taipei, Taiwan

Accepted for publication: May 4, 2000


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several lines of evidence suggest that the N-methyl-D-aspartate receptor (NMDA) and nitric oxide (NO) systems are involved in morphine tolerance. Cyclooxygenase (COX) inhibitors may also play a role in morphine tolerance by interacting with both systems. In the present study, we examined the effects of the COX inhibitors N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide (NS-398, selective COX2 inhibitor) and indomethacin (non-selective COX inhibitor) on the development of antinociceptive tolerance of morphine in a rat spinal model. The antinociceptive effect was determined by the tail-flick test. Tolerance was induced by injection of morphine 50 µg intrathecally (i.t.) twice daily for 5 days. The effects of NS-398 and indomethacin on morphine antinociceptive tolerance were examined after administering these drugs i.t. 10 min before each morphine injection. Neither NS-398 nor indomethacin alone produced an antinociception effect at doses up to 40 µg. NS-398 and indomethacin did not enhance the antinociceptive effect of morphine in naïve and morphine-tolerant rats. However, they shifted the morphine antinociceptive dose–response curve to the left when co-administered with morphine during tolerance induction, and reduced the increase in the ED50 of morphine (dose producing 50% of the maximum response) three- to four-fold. Collectively, these findings and previous studies suggest that COX may be involved in the development of morphine tolerance without directly enhancing its antinociceptive effect.

Br J Anaesth 2000; 85: 747–51

Keywords: enzymes, cyclooxygenase inhibitors; non-steroidal anti-inflammatory drugs; analgesics opioid, morphine


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Non-steroidal anti-inflammatory drugs (NSAIDs) produce their therapeutic effect by inhibiting COX activity, thus reducing the synthesis of prostaglandins (PGs).1 Combinations of NSAIDs with morphine have been used in clinical pain management, particularly in terminal cancer patients.2 The potential advantages of this combination are that analgesia can be maximized while minimizing the adverse effects of morphine. Animal and clinical studies have shown additive or, possibly, synergistic interaction between opioids and NSAIDs.3 4 Interactions between NSAIDs and morphine have also been reported in the visceral nociception and neuropathic pain models.5 6

There is a large body of evidence indicating the involvement of the NMDA receptor and NO systems in opioid tolerance. NMDA antagonists and NO inhibitors have been shown to attenuate or prevent morphine tolerance.7–14 Recently, we further demonstrated that NMDA receptor antagonists inhibit morphine tolerance not only by modulating the binding characteristics of µ-opioid receptors11 but also by partially preventing the constitutive neuronal expression of NO synthase (NOS).14

Mao et al. proposed that opioid tolerance and neuropathic pain syndromes share a common intracellular mechanism; both are expressed as a loss of analgesic effect of opioids.15 In addition, Malmberg and Yaksh reported that NSAIDs could attenuate the hyperalgesia mediated by glutamate receptors.16 Interaction between NMDA- and PG receptor-mediated events during inflammatory nociception has also been reported.17 PGE2 was shown to stimulate the release of NO from rat spinal cord by NMDA receptor activation through EP1 receptors.18 Moreover, cross-communication between the NOS and COX systems has also been demonstrated.19 20 NO interacts directly with COX to enhance its enzymatic activity.19 Inducible NOS activation may increase NO release and subsequently increase PG release, via COX activation.20 These findings imply complicated interactions among NMDA receptors and the NO and COX systems. In the light of these findings, we propose that COX inhibitors modulate antinociceptive tolerance of morphine via interaction with the NMDA–NO system. The present study was designed to examine the effects of the COX-selective inhibitors NS-398 (a selective COX2 inhibitor) and the non-selective COX inhibitor indomethacin on the development of morphine antinociceptive tolerance in a rat spinal model.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The experimental protocol was approved by the Animal Care and Use Committee of the National Defense Medical Center. Male Sprague–Dawley rats (weighing 300–350 g) were anaesthetized with intraperitoneal chloral hydrate (400 mg kg–1), and intrathecal (i.t.) catheters were implanted for drug administration. The rats were then housed in the animal facility at the National Defense Medical Center for a 3-day recovery period. The antinociceptive effects of various drugs were measured by the tail-flick test, as in our previous studies.11 14 A microsyringe (Hamilton, 25 µl) was attached to the i.t. catheter (PE10) for drug administration. Drugs were administered in 5 µl of solution, and drug administration was followed by flushing with 10 µl of saline. Morphine sulphate was purchased from the Narcotics Control Bureau of the Health Department of the Republic of China (Taiwan), and was dissolved in saline. The antinociceptive effects of NS-398 and indomethacin were examined, and the effects of i.t. administration of NS-398 and indomethacin on morphine antinociceptive tolerance were examined by injecting these drugs 10 min before morphine administration. Antinociceptive responses were examined 30 min after morphine injection. The thermal intensity used in the tail-flick test was determined from the mean tail-flick latency (3.0±0.1 s) of 30 rats injected with saline before the study.

In a preliminary study, the effect of 30 µg of NS-398 or indomethacin, alone or with morphine, was not significantly different from that of of 20 µg in the tail-flick test. Therefore, a dose of 20 µg (i.t.) was used for NS-398 and indomethacin in the present study. Tolerance to the antinociceptive effect of morphine was induced by injection of morphine (50 µg, i.t.) twice daily for 5 days. To investigate the effects of COX inhibitors (NS-398 and indomethacin, 20 µg) on morphine tolerance, we calculated the ED50 for morphine antinociception after morphine tolerance had developed. The COX inhibitors were administered 10 min before each morphine injection on each of the 5 days of tolerance induction. The effects of COX inhibitors on the morphine antinociceptive dose–response curve were examined on the first and fifth days of tolerance induction. The tail-flick test was performed daily.

The tail-flick response was converted from a defined latency to the maximum per cent effect (MPE) as follows:


NS-398 and indomethacin (Cayman, MI, USA) were dissolved in dimethyl sulphoxide (DMSO) and saline (1:1). DMSO had no significant effects on antinociception using the tail-flick test.21

The morphine antinociceptive dose–response latency was analysed by computer-assisted linear regression (Cricket Graph 1.32; Islandia, NY, USA). The ED50 was defined as the morphine dose that induced a 50% MPE measured by the tail-flick test and was calculated using the linear regression equations. All data are presented as mean and SEM. The data were subjected to analysis of variance and the Dunnett test, and P values <0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intrathecal administration of NS-398 or indomethacin alone failed to produce any antinociceptive effect (Fig. 1). The maximum antinociceptive effect of morphine was observed on day 1 during the induction of morphine tolerance (Fig. 2). The tail-flick latency for NS-398 or indomethacin co-administered with morphine was higher than that of morphine alone, but the difference was not statistically significant. Morphine antinociceptive tolerance developed by day 3. Morphine maintained an antinociceptive effect when co-administered with NS-398 or indomethacin during tolerance induction; i.e. both NS-398 and indomethacin attenuated morphine antinociceptive tolerance (P<0.01) (Fig. 2). On day 5, maximum tolerance was attained in the rats treated with morphine alone. Neither indomethacin nor NS-398 treatment alone produced any antinociceptive effect during the 5-day test (Fig. 2). As in our previous study14, normal saline injections did not influence tail-flick latency in the rats used as controls (data not shown).



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Fig 1 The thermal antinociceptive effects of intrathecally administered NS-398 and indomethacin in rats, determined by the tail-flick test. Neither drug had any antinociceptive effect. All data points are mean and SEM for at least six rats.

 


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Fig 2 The effects of NS-398 and indomethacin on the development of morphine tolerance. Tolerance was induced by injection of morphine (50 µg, i.t.) twice daily for 5 days. Tail-flick latency was measured on each of these 5 days. The effects of NS-398 or indomethacin on the development of morphine tolerance were tested by administering NS-398 (20 µg, i.t.) or indomethacin (20 µg, i.t.) 10 min before each morphine injection during tolerance induction. The antinociceptive effects of NS-398 (20 µg, i.t.) and indomethacin (20 µg, i.t.) alone were also examined on each of the 5 days. The {diamondsuit} shows the mean tail-flick latency of 30 rats injected with saline before the study. All data points are mean and SEM for at least six rats. *P<0.01 compared with the group injected with morphine alone.

 
The effects of COX inhibitors on the morphine antinociception dose–response curve in morphine-tolerant rats are shown in Fig. 3. On day 1, morphine administration (0.1–5 µg, i.t.) produced a dose-dependent antinociceptive effect, with an ED50 of 0.51 µg (Table 1). Co-administration of a COX inhibitor (NS-398 or indomethacin) did not change the morphine antinociception dose–response curves either before (day 1) or after (day 5) morphine tolerance had developed (Fig. 3). However, when the COX inhibitors were co-administered with each morphine injection during tolerance induction, the dose–response curves of morphine antinociception shifted to the left (Fig. 3). On day 5, the ED50 values were 85.12, 17.38 and 22.19 µg for morphine alone, morphine plus NS-398, and morphine plus indomethacin respectively (Table 1). Furthermore, the mean baseline tail-flick latency of morphine-tolerant rats was lower (0.2–1 s) than that of the saline-injected control rats (data not shown).



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Fig 3 The effects of NS-398 and indomethacin on the dose–response relationship for morphine antinociception, measured by the tail-flick test, before and after morphine tolerance had developed. The dose–response relationship and the effects of NS-398 (20 µg, i.t.; top) and indomethacin (20 µg i.t.; bottom) on this relationship were first examined on day 1. Tolerance was then induced by injection of morphine (50 µg i.t.) twice daily for 5 days. On day 5, the dose–response curve was shifted to the right. Neither NS-398 nor indomethacin affected the curve after tolerance had developed. When NS-398 (20 µg i.t.) or indomethacin (20 µg i.t.) was coinjected with morphine during the induction of tolerance, the morphine dose–response curves were shifted to the left on day 5. All data points are the mean and SEM for at least six rats.

 

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Table 1 Effects of NS-398 and indomethacin on the ED50 values for morphine antinociception after intrathecal injection of morphine twice daily for 5 days. The ED50 values were calculated from the antinociceptive dose–response curves shown in Fig. 3
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study shows that both COX2-selective and non-selective COX inhibitors attenuated morphine antinociceptive tolerance. No potentiation of morphine antinociception was observed when a COX inhibitor was administered either before or after the development of morphine tolerance. These results suggest that COX is involved in the development of morphine tolerance but does not enhance the antinociceptive effect of morphine.

We found that a selective and a non-selective inhibitor of COX attenuated morphine antinociceptive tolerance. This is consistent with a recent study showing that the non-selective COX inhibitors ketorolac and ibuprofen inhibited the development of morphine tolerance.22 The morphine-tolerant rats had lower mean baseline tail-flick latency than the control group, suggesting that thermal hyperalgesia may develop in association with the development of morphine tolerance.10 15 It is interesting that COX2 is not only inducibly expressed after the inflammation process but is also constitutively expressed in the spinal cord of normal rats.23–25 Furthermore, COX2 is distributed in the superficial layer of the dorsal horn and is related to spinal nociceptive processing in the normal condition.25 However, it is not clear which COX isoforms are involved in morphine tolerance in the rat spinal cord. The effects of NS-398, a COX2-selective inhibitor, on morphine tolerance and morphine ED50 values were slightly greater than those of indomethacin, implying that the inhibition of COX2 may play a role in the development of morphine tolerance. Moreover, the present results agree with those of previous reports showing that COX inhibitors did not produce any thermal antinociceptive effects in the tail-flick test.21 26 Although the non-selective COX inhibitor ketorolac has been shown to potentiate the analgesic effects of opioids by modulating the function of the opioid receptor in visceral nociception5, the present results demonstrate that NS-398 and indomethacin did not potentiate morphine antinociception either before or after the development of morphine tolerance. However, these data also confirm that neither COX1 nor COX2 was directly involved in phasic thermal nociceptive transmission in the rat spinal cord.26

Our previous studies have demonstrated that several drugs attenuate morphine tolerance and maintain the antinociceptive efficacy of morphine in a rat spinal model.11 14 Although the non-selective COX inhibitor ketorolac has been shown to potentiate the analgesic effect of opioids by modulating opioid receptor function in visceral nociception,5 in the present study neither NS-398 nor indomethacin potentiated morphine antinociception, either before or after the development of morphine tolerance. We found previously that the NMDA receptor antagonist MK-801 attenuated morphine tolerance by preventing the reduction of high-affinity µ-opioid receptor sites.11 14 In the present study, COX inhibitors were found to attenuate morphine tolerance but did not enhance the antinociceptive effect of morphine. Because NMDA receptor antagonists and COX inhibitors shift the morphine dose–response curve in different directions, our present results suggest that COX inhibitors inhibit PG synthesis and related neurotransmission rather than having a direct inhibitory effect on conformational change of the µ-opioid receptor.

In summary, the present results show that COX inhibitors can attenuate the development of morphine tolerance. However, both the non-selective COX inhibitor indomethacin and COX2 inhibitors failed to produce any analgesic effects or potentiation of morphine antinociception either before or after the development of morphine tolerance. Which isoforms of COX are expressed and what interactions occur among the NMDA, NO and COX systems during morphine tolerance in the spinal cord are worthy of further investigation.


    Acknowledgements
 
This work was supported by grants from the National Science Council (NSC 89–2314-B-016–105) and the Department of Defense (DOD 88–30) of the Republic of China (Taiwan).


    Footnotes
 
* Corresponding author: Department of Anesthesiology, National Defense Medical Center and Tri-Service General Hospital, #8 Section 3, Tingchow Road, Taipei, Taiwan Back


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 Introduction
 Materials and methods
 Results
 Discussion
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