Midazolam-induced antinociception: possible interaction with morphine response in tail-flick test

Hossein Rastegar PhD**, Mohammad-Amin Janmohammadi PharmD**, Mohammad-Reza Zarrindast PhD*•, Farshad Roushan-Zamir PhD**, Ali-Reza Salimi MD**

*Department of Pharmacology, Tehran University of Medical Sciences, **Department of Pharmacology, Shaheed Beheshti University of Medical Sciences, Tehran, Iran

•Correspondence: M.R. Zarrindast PhD, Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. P.O. Box: 13145-784, Fax: +98-21-6402569, E-mail: zarinmr@ams.ac.ir.

Background – Benzodiazepines have been used with opiates to give better pain relief than opiates alone. However, the interaction between the two groups of drugs is controversial. The aim of this study was to investigate whether midazolam potentiated the antinociceptive effect of morphine.

Methods – Male albino mice were used in this study. Antinociception was measured using the tail-flick test.

Results – Midazolam and morphine caused dose-dependent antinociceptive effects in mice. The combination of midazolam and morphine showed an increase in analgesia. The benzodiazepine receptor antagonist, flumazenil, decreased the response induced by midazolam or midazolam plus morphine but not that of morphine alone. However, the opioid receptor antagonist naloxone, reduced the antinociception induced by morphine, midazolam, or a combination of the two drugs. Methysergide or propranolol increased the analgesic effect of midazolam; ketanserin, phenoxybenzamine and atropine did not.

Conclusion – Midazolam induced antinociception through both benzodiazepine and opioid receptors.

Keywords · flumazenil · mice · midazolam · serotonin receptor antagonists · tail-flick test

Introduction

Receptor sites for benzodiazepines have been shown in the spinal cord, with the highest level within the lamina II of the dorsal horn.1 Benzodiazepine sites appear to be linked to the gamma-aminobutyric acid (GABA)-A receptor complex,2, 3 and benzodiazepines enhance GABA-mediated presynaptic inhibition of primary afferent terminals.4 They interact with the antinociceptive effect of morphine and other opioid drugs.5 – 9 Systemic administration of benzodiazepines induces sedation and muscle relaxation, which may complicate the interpretation of responses to nociceptive stimulation.10 Diazepam and other benzo- diazepines are frequently administered together with opiates to give better pain relief.11 – 13 Benzodiazepines have also been reported to be of some value in the treatment of chronic pain.14 The neurophysiological mechanisms involved in the benzodiazepines effect on nociception have been studied in animal models,5, 7, 15 but the interactions between benzodiazepines and morphine in producing analgesia are controversial. For example, diazepam has been found to decrease,9 increase,16 or have no effect on morphine-induced antinociception. The use of benzodiazepine agonists with opioids has gained wide acceptance among anesthesiologists. Midazolam is an ultra- short-acting benzodiazepine and is used clinically in preoperative medication, as an induction agent for general anesthesia, and for intravenous sedation. It has anxiolytic, hypnotic, anticonvulsant, muscle relaxant, and anterograde amnestic properties characteristic of the benzodiazepines.17, 18 In the present study, the antinociceptive effect of midazolam in the presence or absence of morphine was investigated.

Materials and Methods

Animals

Male NMRI albino mice (25 – 30 g) were used in the study. Animals were housed in plastic cages in an animal room maintained at 23 ± 1○C on a 12-hour dark cycle (light period, 07:00 – 19:00). Food and water were available at all times except during experiments. Each animal was used once only and was sacrificed immediately after the experiment. Seven animals were used in each experiment. All experiments were performed in accordance with institutional animal use guidelines.

Drugs

The chemicals used were: morphine sulphate (MacFarlan Smith Ltd, England), naloxone hydrochloride (Sigma Chemical Co, England), midazolam (Sigma Poole, UK), flumazenil (Ro 15 – 1788; Roche, Switzerland), phenoxybenzamine (SK&F, USA), methysergide (Sandoz, Switzerland), ketanserin (Sigma Chemical Co, USA), atropine (Merck, Germany) and propranolol (Sigma Poole, UK). Morphine and naloxone were dissolved in saline. The drugs were prepared immediately before use and injected at a volume of 10 mL/kg. All drugs were injected intraperitoneally except morphine, which was administered subcutaneously. Antagonist doses and pretreatment times were usually the same as those previously shown to be pharmacologically active.18, 19

Antinociception recording

Antinociception was assessed using the tail-flick test, with the tail-flick apparatus (type 812, Hugo Sachs Elektronic, Germany). The tail-withdrawal latency (sec) was measured before administration of any drug or vehicle. Normal response latencies were usually between 2.5 and 3.0 sec and a 10 sec cut-off was used to prevent tissue damage. The response was tested 15, 30, 45, and 60 minutes after drug administration. Antinociception was quantified as the percentage of maximum possible effect (%MPE) using the method of Keil and DeLander.20 The following formula was used to calculate %MPE: %MPE =

100 x [(test-control latency)/(10 sec control latency)].

Statistical analysis

ANOVA and the Newman-Keuls tests were used to analyze the data. Differences between means with p < 0.05 were considered statistically significant. Each point represents the mean ± SEM of recordings for seven mice.

Results

Effect of morphine or midazolam on tail-flick latency

Subcutaneous (SC) administration of morphine (3, 6, and 9 mg/kg) induced dose-dependent antinociception [F (3, 24) = 79.2, p < 0.0001]. The maximum response was obtained with 9 mg/kg of the drug (Figure 1). Intraperitoneal (IP) injection of the benzodiazepine receptor agonist midazolam (0.03, 0.3, 3, and 6 mg/kg) also induced dose-dependent antinociception [F (4, 30) = 11.9, p < 0.0001]. The maximum response was obtained with 6 mg/kg of the drug (Figure 2).

Figure 1. Antinociceptive effect of different doses of morphine in the tail-flick test. Mice were injected subcutaneously (SC) with saline (5 mL/kg) or morphine (3, 6, or 9 mg/kg). Antinociception was recorded for 60 min after morphine injection. Each point is the mean ± SEM of percentage of maximum possible effect (%MPE) for seven mice. *** p < 0.001 vs saline.

Figure 2. Antinociceptive effect of different doses of midazolam in the tail-flick test. Mice were injected intraperitoneally (IP) with saline (5 mL/kg) or midazolam (0.3, 0.3, 3, or 6 mg/kg). Antinociception was recorded for 60 min after midazolam administration. Each point is the mean ± SEM of %MPE for seven mice. ** p < 0.01, ***p < 0. 001 vs saline.

Effect of midazolam on morphine-induced antinociception

Figure 3 shows the response to the administration of different doses of morphine (3, 6, or 9 mg/kg SC) in the presence or absence of a low dose of midazolam (0.03 mg/kg IP). Two-way ANOVA showed that the combination of morphine with midazolam did not have any effect [F (4, 60) = 0.66, p > 0.05].

Figure 4 shows the response to a combination of a higher dose of midazolam (4 mg/kg IP) with different doses of morphine. However, two-way ANOVA showed no interaction between the two drugs [F (4, 60) = 0.998, p > 0.05].

Figure 3. Antinociceptive effect of different doses of morphine in the presence or absence of lower dose of midazolam in the tail-flick test. Mice were injected with saline (5 mL/kg IP) or midazolam (4 mg/kg IP) 5 min before administration of morphine (3, 6, 9, or 6 mg/kg SC). Antinociception was recorded for 60 min after morphine administration. Each point is the mean ± SEM of %MPE for seven mice.

Figure 4. Antinociceptive effect of different doses of morphine in the presence or absence of higher dose of midazolam in the tail-flick test. Mice were injected with saline (5 mL/kg IP) or midazolam (0.03 mg/kg IP) 5 min before administration of morphine (1, 2, 3, or 6 mg/kg SC). Antinociception was recorded for 60 min after morphine administration. Each point is the mean ± SEM of %MPE for 7 mice. *p < 0.05 vs saline.

Effects of receptor antagonists on antinociception induced by midazolam in the presence or absence of morphine

One-way ANOVA indicates that pretreatment of animals using the benzodiazepine receptor antagonist flumazenil (1 and 2 mg/kg, IP) 15 min before administration of midazolam (6 mg/kg, IP) or 20 min before morphine (4 mg/kg) or prior to the combination of midazolam and morphine reduced the response induced by midazolam [F (2,18) = 10.4, p < 0.001] and midazolam plus morphine [F (2, 18) = 7.4, p < 0.01] but not morphine alone [F (2, 18) = 0.1, p > 0.05]. Flumazenil alone also induced antinociception [F (2, 18) = 5.2, p < 0.05] (Figure 5).

Pretreatment of animals with the opioid receptor antagonist naloxone (1 and 2 mg/kg, IP) 15 min before the administration of midazolam (6 mg/kg, IP), 20 min before morphine (4 mg/kg) or prior to midazolam plus morphine reduced the response induced by midazolam [F (2, 18) = 12.25, p < 0.001], morphine [F (2, 18) = 16.3, p < 0.001] and midazolam plus morphine [F (2, 18) = 18.3, p < 0.001]. Naloxone alone did not induce any response [F (2, 18) = 0.26, p > 0.05] (Figure 6).

Figure 5. Change in antinociceptive effect of morphine, midazolam, or morphine plus midazolam due to flumazenil injection 15 min before midazolam (6 mg/kg or saline IP) or 20 min prior to morphine (4 mg/kg or saline IP) administration. Antinociception was recorded for 60 min after morphine/midazolam administration. Each point is the mean ± SEM of %MPE for seven mice. *p < 0.05, **p < 0.001, ***p < 0.01 vs saline.

Figure 6. Effect of naloxone on antinociceptive effect of morphine, midazolam, or morphine plus midazolam. Naloxone was injected 15 min before midazolam (6 mg/kg or saline IP) or 20 min prior to morphine (4 mg/kg or saline SC) administration. Antinociception was recorded for 60 min after morphine administration. Each point is the mean ± SEM of %MPE for seven mice. **p < 0.01, ***p < 0.001 vs saline.

Effect of serotonergic, cholinergic and adrenoceptor antagonists on midazolam-induced response

One-way ANOVA indicates that when the animals were treated with different doses of ketanserin (1 and 2 mg/kg) [F (2, 18) = 2.4, p > 0.05], atropine (5 and 10 mg/kg) [F (2, 18) = 0.16, p > 0.05] 15 min, or phenoxybenzamine (5 and 10 mg/kg) [F (2, 18) = 0.72, p > 0.05] 60 min before midazolam administration (6 mg/kg), the antinociception induced by midazolam was unchanged. While one-way ANOVA indicated that administration of methysergide (1 and 2 mg/kg) [F (2, 18) = 13.6, p < 0.05] or propranolol (5 and 10 mg/kg) [F (2, 18) = 3.6, p < 0.05] 15 min before midazolam administration increased the midazolam response (Table), two-way ANOVA did not show any interactions between propranolol and midazolam [F (2, 36) = 0.21, p > 0.05]. Two-way ANOVA showed an interaction between methysergide and midazolam response [F (2, 36) = 4.54, p < 0.05].

Table. Effects of different receptor antagonists on midazolam-induced antinociception in the tail-flick test. Mice were treated intraperitoneally (IP) with saline (5 mL/kg), or atropine (5 and 10 mg/kg) 15 min, methysergide (1 and 2 mg/kg) 15 min, propranolol (5 and 10 mg/kg) 15 min, ketanserin (1 and 2 mg/kg) 15 min or phenoxybenzamine (phenoxy; 5 and 10 mg/kg) 60 min prior to midazolam (6 mg/kg) administration. Antinociception was recorded for 60 min after morphine injection. Each point is the mean ± SEM of %MEP for 7 mice.
 

%MPE of mean ± SEM
 

Saline

Midazolam

Saline

4.5 ± 0.7

16.9 ± 0.9

Atropine 5 mg

5.6 ± 0.6

16.5 ± 0.8

Atropine 10 mg

4.7 ± 0.6

17.2 ± 0.9

Saline

4.5 ± 0.7

16.8 ± 1.5

Methysergide 1 mg

5.3 ± 0.6

22 ± 1.2†

Methysergide 2 mg

7.6 ± 0.5*

26 ± 1‡

Saline

4.5 ± 0.7

16.4 ± 0.8

Propranolol 5 mg

5.7 ± 0.7

17.8 ± 0.6

Propranolol 10 mg

6.2 ± 0.6

19 ± 0.5

Saline

4.5 ± 0.7

17.4 ± 0.4

Ketanserin 1 mg

5.2 ± 0.4

18.7 ± 0.9

Ketanserin 2 mg

6.4 ± 0.5

20.2 ± 1.2

Saline

4.5 ± 0.7

17.5 ± 0.8

Phenoxybenzamine 5 mg

6.7 ± 0.3

16.8 ± 0.7

Phenoxybenzamine 10 mg

8.3 ± 0.7*

16.2 ± 0.8

MPE = maximum possible effect; *p < 0.05 different from respective saline control groups. †p < 0.05, ‡p < 0.01 different from midazolam control groups.

Discussion

The present study investigated the antinociceptive response to midazolam in the presence or absence of morphine using the tail-flick test. Different doses of midazolam, a benzodiazepine receptor agonist,19 induced antinociception dose dependently. Flumazenil, a benzodiazepine receptor antagonist,8 reduced the midazolam-induced effect. Therefore, the benzodiazepine receptor mechanism is apparently involved in the response induced by midazolam. These results agree with those obtained by others21, 22 indicating that benzodiazepines can elicit antinociception. Contradictory results show that systemic administration of benzodiazepine agonists in animals can decrease8, 9 or increase23 antinociception induced by systemically administered morphine.

It has been proposed that when both drugs are administered at the supraspinal level, midazolam decreases the antinociceptive potency and duration of action of morphine. Thus, different mechanisms may be involved in antinociception at the levels of spinal cord and in the brain.24 Our results showed that systemic administration of a combination of midazolam and morphine increased the response to low doses of morphine. However, two-way ANOVA did not indicate any potentiation. The antinociception induced by morphine was not reduced by a benzodiazepine receptor antagonist, indicating that the response induced by midazolam, but not morphine, was mediated through a benzodiazepine receptor mechanism. When animals were treated with the opioid receptor antagonist naloxone, the response induced by either midazolam, morphine, or a combination of the two drugs was reduced. This indicates that the effect of midazolam may, at least partly, be mediated through an opioid receptor mechanism. Whether these different effects of midazolam and naloxone are the result of different responses to the drugs at spinal and supraspinal levels needs to be further investigated. The results may be consistent with those of others demonstrating that the opioid system may be involved in some responses to benzodiazepines, such as the anxiolytic effect.25 Considerable evidence now exists for the involvement of the GABA-A receptor in benzodiazepine effects.26 – 28 For example, there is an association between the GABA and serotonergic systems.29 Whether the analgesic effect of midazolam is partly due to such associations remains to be clarified. Our results also showed that methysergide, a serotonin (5-HT) receptor antagonist, increased the response induced by midazolam. However, the 5-HT2 receptor antagonist ketanserin30 did not alter the effect of midazolam in the present study, which indicates that at least the 5-HT2 receptor mechanism is not involved. Other receptor antagonists, atropine and phenoxybenzamine, did not alter the midazolam effect. While the highest dose of propranolol tended to increase midazolam antinociception, they did not affect the midazolam response statistically significant. Thus, the involvement of cholinergic and adrenoceptor mechanisms can be excluded.

In conclusion, there are clinical reports indicating that the benzodiazepines are able to potentiate opioid response in gastrointestinal examination,11 postoperative analgesia,12 dental procedures,13 acute muscle spasm and lancinating neuropathic pain.14 However, study did not show any such a response to these combinations.

References

  1. Keller AF, Coull JA, Chery N, et al. Region-specific developmental specialization of GABA-glycine cosynapses in laminas I-II of the rat spinal dorsal horn. J Neurosci. 2001; 21: 7871 – 80.

  2. Sigel E, Buhr A. The benzodiazepine binding site of GABA-A receptors. Trends Pharmacol Sci. 1997; 18: 425 – 9.

  3. Smith GB, Olsen RW. Functional domains of GABA-A receptors. Trends Pharmacol Sci. 1995; 16: 162 – 8.

  4. Persohn E, Malherbe P, Richards JG. In situ hybridization histochemistry reveals a diversity of GABA-A receptor subunit mRNAs in neurons of the rat spinal cord and dorsal root ganglia. Neuroscience. 1991; 42: 497 – 507.

  5. Pick CG. Strain differences in mice antinociception: relationship between alprazolam and opioid receptor subtypes. Eur Neuropsychopharmacol. 1996; 6:
    201 – 5.

  6. Luger TJ, Hayashi T, Weiss CG, et al. The spinal potentiating effect and the supraspinal inhibitory effect of midazolam on opioid-induced analgesia in rats. Eur J Pharmacol. 1995; 275: 153 – 62.

  7. Luger TJ, Lorenz IH, Grabner-Weiss C, et al. Effect of the NMDA-antagonist, MK 801, on benzodiazepine-opioid interactions at the spinal and supraspinal level in rats. Br J Pharmacol. 1995; 114: 1097 – 103.

  8. Luger TJ, Hayashi T, Lorenz IH, et al. Mechanisms of the influence of midazolam on morphine antinociception at spinal and supraspinal levels in rats. Eur J Pharmacol. 1994; 271: 421 – 31.

  9. Rosland JH, Hole K. Benzodiazepine-induced antagonism of opioid antinociception may be abolished by spinalization or blockade of the benzodiazepine receptor. Pharmacol Biochem Behav. 1990; 37: 505 – 9.

  10. Rosland JH, Hole K. 1, 4-Benzodiazepines antagonize opiate-induced antinociception in mice. Anesth Analg. 1990; 71: 242 – 8.

  11. Paspatis GA, Manolaraki M, Xirouchakis G, et al. Synergistic sedation with midazolam and propofol versus midazolam and pethidine in colonoscopies: a prospective, randomized study. Am J Gastroenterol. 2002; 97: 1963 – 7.

  12. Nathan JE, Vargas KG. Oral midazolam with and without meperidine for management of the difficult young pediatric dental patient: a retrospective study. Pediatr Dent. 2002; 24: 129 – 38.

  13. Weinbroum AA, Weisenberg M, Rudick V, et al. Flumazenil potentiation of postoperative morphine analgesia. Clin J Pain. 2000; 16: 193 – 9.

  14. Reddy S, Patt RB. The benzodiazepines as adjuvant analgesics. J Pain Symptom Manage. 1994; 9:
    510 – 4.

  15. Nishiyama T, Hanaoka K. The synergistic interaction between midazolam and clonidine in spinally-mediated analgesia in two different pain models of rats. Anesth Analg. 2001; 93: 1025 – 31.

  16. Wala EP, Sloan JW, Jing X, et al. The effects of diazepam dependence and withdrawal on morphine-induced antinociception and changes in locomotion in male and female rats. Pharmacol Biochem Behav. 2001; 69: 475 – 84.

  17. Woods JH, Katz JL, Winger G. Benzodiazepines: use, abuse, and consequences. Pharmacol Rev. 1992; 44: 151 – 347.

  18. Zarrindast MR, Hodjati MR, Pejhan A, et al. Bupropion induces sniffing: a possible dopaminergic mechanism. Eur Neuropsychopharmacol. 1996; 6: 299 – 303.

  19. Rattan AK, Sribanditmongkol P. Effect of morphine-induced catalepsy, lethality, and analgesia by a benzodiazepine receptor agonist midazolam in the rat. Pharmacol Biochem Behav. 1994; 48: 357 – 61.

  20. Keil GJ, Delander GE. Time-dependent antinociceptive interactions between opioids and nucleotide transport inhibitors. J Pharmacol Exp Ther. 1995; 274: 1387 – 92.

  21. Yanez A, Sabbe MB, Stevens CW, et al. Interaction of midazolam and morphine in the spinal cord of the rat. Neuropharmacology. 1990; 29: 359 – 64.

  22. Pick CG. Antinociceptive interaction between alprazolam and opioids. Brain Res Bull. 1997; 42: 239 – 43.

  23. Wynn RL, Bergman SA, Rudo FG, et al. GABAmimetics diminish antinociception of meperidine under conditions which enhance other opioid μ-agonists. Arch Int Pharmacodyn Ther. 1990; 304: 136 – 46.

  24. Luger TJ, Hayashi T, Weiss CG, et al. The spinal potentiating effect and the supraspinal inhibitory effect of midazolam on opioid-induced analgesia in rats. Eur J Pharmacol. 1995; 275: 153 – 62.

  25. Tsuda M, Suzuki T, Misawa M, et al. Involvement of the opioid system in the anxiolytic effect of diazepam in mice. Eur J Pharmacol. 1996; 307:
    7 – 14.

  26. Sigel E. Mapping of the benzodiazepine recognition site on GABA-A receptors. Curr Top Med Chem. 2002; 2: 833 – 9.

  27. Nemmani KV, Ramarao P. Role of benzodiazepine-GABA-A receptor complex in attenuation of U-50,488H-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin in mice. Life Sci. 2002; 70: 1727 – 40.

  28. Lelas S, Rowlett JK, Spealman RD, et al. Role of GABA-A/benzodiazepine receptors containing alpha 1 and alpha 5 subunits in the discriminative stimulus effects of triazolam in squirrel monkeys. Psychopharmacology (Berl). 2002; 161: 180 – 8.

  29. Feng J, Cai X, Zhao J, et al. Serotonin receptors modulate GABA-A receptor channels through activation of anchored protein kinase C in prefrontal cortical neurons. J Neurosci. 2001; 21: 6502 – 11.

  30. Sawynok J, Zarrindast MR, Reid AR, et al. Adenosine A3 receptor activation produces nociceptive behaviour and edema by release of histamine and 5-hydroxytryptamine. Eur J Pharmacol. 1997; 338: 1 – 7.

AIM Home | Table of Contents