Analgesic effect of benzodiazepines and flumazenil

Gen. Pharmac. Vol. 23, No. 4, pp. 739-742, 1992 Printed in Great Britain. All rights reserved

0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd

ANALGESIC EFFECT OF BENZODIAZEPINES AND FLUMAZENIL F. SIERRALTA* and H. F. MIRANDA Department of Pharmacology, Faculty of Medicine, Universidad de Chile, P.O. Box 70,000, Santiago 7, Chile (Received 18 October 1991)

Abstraet--l. In the present work the analgesic effect of benzodiazepines (BZD) and flumazenil (FLU) using the writhing test in mice was studied. 2. Intracerebroventricular administration of BZD exhibited a dose-dependent antinociceptive effect when compared to control value. 3. Intracerebroventricular administration of FLU induced a dose-dependent antinociceptive action that was not antagonized by naloxone (NX). 4. BZD administered as subcutaneous pellets produced an antinociceptive action in the writhing test, when compared to control mice, only at relative high doses and was partially antagonized by naloxone. 5. These findings could be explained assuming that NX and/or FLU have partial agonist properties in a common receptor which mediates the antinociceptive action. 6. The antinociceptive action of BZD could be related to an increased release of adenosine, which by itself has analgesic effects.

INTRODUCTION Benzodiazepines (BZD) were introduced primarily as very useful drugs in the treatment of anxiety with the great advantage over other anxiolytics that BZD did not induce dependence or tolerance. However, with the widespread use of these c o m p o u n d s and the extensive information available on B Z D and other groups o f drugs acting on C N S , the physiological dependence to B Z D is usually associated wth the chronic administration of these drugs. In spite of their multiple actions in different systems, the analgesic effect of B Z D has been barely studied; moreover, B Z D can alter psychomotor performance, a fact which usually is associated with the algesiometric tests. Eddy et al. (1950) demonstrated a poor analgesic effect of B Z D in mice using the hot-plate test. In addition, with the development of specific BZD-receptor antagonists, it was possible to study the withdrawal syndrome in a way similar to the naloxone-morphine syndrome analysis. However, other pharmacological properties of one of the principal antagonists, flumazenil ( F L U ) has not been extensively revised. The purpose o f the present work was to undertake the study o f the analgesic effect of B Z D and F L U using the writhing test in mice.

MATERIALS AND METHODS

Mice of Swiss As/W strain were used for all the experiments, according to the following groups: (A) mice were implanted each week with 1, 2 and 3 tablets of the following *To whom all correspondence should be addressed.

drugs: diazepam (DZP, 10 mg each tablet for 3 weeks), midazolam (7.5 mg each tablet for 2 weeks) or oxazepam (15mR each tablet for 3 weeks). (B) Compounds (DZP, FLU, midazolam) were delivered using a modification of the intracerebroventricular (i.c.v.) injection according to the method described by Haley and McCormick (1957). The animals were lightly anesthetized with etilic ether, an incision was made in the scalp and the bregma located. The injections were made 2 mm caudal and 2 mm lateral to bregma at a depth of 2 mm using a Hamilton microliter syringe with a 26 gauge needle. All i.c.v, injections were made in a volume of 20/~l. (C) Control mice received placebo tablets or i.c.v. saline solution. When the animals were pretreated with naloxone (NX), this drug was injected 30 rain prior to the experimental procedure. Antinociceptive responses were determined according to Hayashi and Takemori (1971). The number of stretches or writhes (arching of the back, development of tension in the abdominal muscles, elongation of the body and extension of the forelimbs) was counted during a 5 rain period starting 5 rain after the i.p. administration of I ml/kg of 0.6% acetic acid. When given by the i.c.v, route, administration of BZD compounds (DZP, FLU, midazolam) or saline solution to mice was made 30 rain prior to testing the animals. The number of animals involved was kept to a minimum, the duration of experiments was as short as possible and the animals were sacrificed immediately after the recording period. Each animal received only one dose of the drug tested. Data are presented as mean values :t: SEM. Significance was assessed by Student's t-test for unpaired data and multiple analysis of variance (ANOVA) followed by Newman-Keuls test when appropriated. Significance was accepted at the 0.05 level. The calculations were made using a modification of the Barlow's statistical software (1983). Diazepam, flumazcnil and midazolam were generously supplied by Roche Laboratories of Chile. Oxazepam was a gift from Wyeth Laboratory, Santiago, Chile and naloxone hydrochloride was purchased from Sigma Chemical Co., St Louis, Mo., U.S.A.

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Fig. I. Antinociceptive effect induced by the i.c.v, administration of diazepam in the writhing test. Values are expressed as mean + SEM of control (n = 13), DZP 2 mg, 1 mg, lO0~ug (n = I0 each), DZP I mg + NX I mg (n = 12) and DZP 100~ug + NX I mg. *P < 0.05 against control.

Fig. 3. Antinociceptive effect induced by the i.c.v, administration of midazolam in the writhing test. Values are expressed as mean + SEM of control (n = 13), midazolam 2 m g (n = 10) and midazolam 2 0 0 p g (n = I0). *P <0.05 against control.

RESULTS

The i.c.v, administration of FLU induced a dosedependent antinociceptive action in this study, since the number of writhings were significantly decreased by all the doses used. In addition, NX was unable to antagonize the antinociceptive effect of 2 mg/kg of FLU (see Fig. 2.) Midazolam administered i.c.v, also produced a dose-dependent antinociceptive effect, since the number of writhings induced by the administration of acetic acid was significantly decreased with 2 mg/kg

Antinociceptive effects of BZD and related drugs administered i.c.v. DZP exhibited a dose-dependent antinociceptive effect in the w r i t h i n g test o f mice w h e n c o m p a r e d to c o n t r o l value, u s i n g the t-test, as c a n be seen in Fig. 1. H o w e v e r , N X w a s u n a b l e to a n t a g o n i z e the n u m b e r o f w r i t h i n g s i n d u c e d by ! m g / k g o f D Z P b u t w a s effective in b l o c k i n g t h e a n t i n o c i c e p t i v e a c t i o n o f 100 m g / k g o f D Z P , see Fig. 1.

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Fig. 2. Antinociceptive effect induced by the i.c.v, administration of flumazenil in the writhing test. Values are expressed as mean 4- SEM of control (n = 13), FLU 2 rag, 600/ag, 300/tg (n = l0 each) and F L U 2 m g + N X lmg (n = 12). *P < 0.05 against control.

Diozepom (rag} Fig. 4. Antinociceptive effect of subcutaneous implantation of diazepam pellets in the writhing test. Values are expressed as mean + SEM of control (n = 13), DZP I0 mg (n = 10), DZP 20mg (n = 12) and DZP 30mg (n = 12). *P <0.05 against control.

741

Analgesia of benzodiazepines and flumazenil

doses of the drug (30 and 45 mg/kg) were devoid of antinociceptive effects when compared to control by Student's t-test. See Fig. 5. The pretreatment of mice with subcutaneous pellets of midazolam (7.5 and 15 mg/kg), for 7 and i 5 days respectively, was not able to produce antinociceptive actions in the writhing test (see Fig. 6).

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Fig. 5. Antinociceptive effect of subcutaneous implantation of oxazepam pellets in the writhing test. Values are expressed as mean _+SEM of control (n = 13), oxazepam 15rag (n = 10), oxazepam 30mg (n = 10) and oxazepam 45 rag. *P < 0.05 against control. but not with 200/ag/kg of midazolam, data shown in Fig. 3.

Antinociceptive effects of BZD administered as subcutaneous pellets DZP administered as subcutaneous pellets produced an antinociceptive action in the writhing test, when compared to control mice, only at a dose of 30 mg/kg (t-test), as can be seen in Fig. 4. The administration of oxazepam in pellets induced an antinociceptive action only at 15 mg/kg; higher [~']

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DISCUSSION The results obtained in the present study demonstrated that BZD, as well as the antagonist of BZD, flumazenil, were able to induce antinociceptive action in mice, using the writhing test as an algesiometric assay. The antinociceptive action of BZD in the writhing test is not due to a spasmolytic effect of BZD, since this property has not been described for this type of drugs (Bellantuono et al., 1980) and also was not antagonized by atropine 0.1-1 mg/kg (data not shown). This property of antinociception is common to BZDs of long action as diazepam, short action as oxazepam, ultrashort action (hypnotic) as midazolam and to their antagonist FLU. The effect described is dose-dependent, usually demonstrable with relative high doses of the drugs and is partially related with opioid-receptors, since the antinociceptive action of diazepam, in low doses (100 pg/kg, i.c.v.) was antagonized by NX and at higher doses (1 mg/kg i.c.v.) was not antagonized. The BZD-induced antinociceptive effect was also obtained when the drugs were administered by subcutaneous implantation, with the exception of midazolam which was not able to produce antinociceptive effects with all the doses used. In relation with the oxazepam administration by pellets, the antinociceptive effect was obtained only at a dose of 15 rag. The pretreatment with diazepam produced antinociceptive action in the writhing test at a dose of 30 rag. The i.c.v, administration of FLU, a known antagonist of BZD, produced a dose-dependent antinociceptive effect that was not antagonized by pretreatment of the animals with NX. This finding could be explained assuming that NX and/or F L U have partial agonist properties in a common receptor which mediates the antinociceptive action. The partial agonist properties of NX have been described in some actions displayed by delta-opioid receptors (Koren and Maurice, 1989). Additionally, the partial agonist effect of FLU has been demonstrated in several behavioral paradigms. It seems that the anxiogenicanxiolytic actions of FLU are due to a blocking interaction of this compound on adenosine receptors, since FLU-anxiogenic induced effect is a consequence of the blockade and the anxiolytic action is due to up-regulation induced by FLU at adenosine receptors to overcome the antagonism (Phillis and O'Regan, 1988). Among the different substances that produce analgesia, adenosine is one of them. Adenosine analogs have antinociceptive action in the hot-plate test and in the writhing test, which is blocked by adenosine antagonists, whereas other antagonists such as NX are ineffective (Dunwiddie, 1985). Furthermore, biochemical and behavioral evidence has suggested that the action of opioids and related

742

F. SIERRALTAand H. F. MIRANDA

drugs may be mediated in part via increased release of purines in the brain since methylxanthines can antagonize the analgesic effects of morphine. Thus, it has been suggested that the ability of methylxanthines to antagonize the antinociceptive effect of opioids is not due to an interaction with opioid receptors, nor to an inhibition of the opioid response (facilitation of purine release), but rather to an antagonism of the effects of adenosine (Dunwiddie, 1985; Sawynok et al., 1989). Taking into consideration the above facts, a similar mechanism is proposed for the action of BZD, which could be related to an increased release of adenosine. Acknowledgements--The authors wish to express their gratitude to Roche Laboratories and Wyeth Laboratories of Chile and to Dr G. Pinardi for helpful suggestions during the elaboration of this manuscript. REFERENCES

Barlow R. B. (1983) Biodata Handling with Microcomputers. Elsevier, Amsterdam.

Bellantuono C., Reggi V., Tognoni G. and Garattini S. (1980) Benzodiazepines: clinical pharmacology and therapeutics use. Drugs 19, 195-219. Dunwiddie T. (1985) The physiological role of adenosine in the central nervous system. Int. Rev. Neurobiol. 27, 63-139. Eddy N. B., Touchberry C. F. and Liebcrman J. E. 0950) Synthetic analgesic. I. Methadone isomers and derivatives. J. Pharmac. exp. Ther. 98, 121-137. Haley T. J. and McCormick W. G. 0957) Pharmacological effect produced by intracerebral injection of drugs in the conscious mouse. Br. J. Pharmac. 12, 12-17. Hayashi G. and Takemori A. E. (1971) The type of analgesic-receptor interaction involved in certain analgesic assays. Fur. J. Pharmac. 16, 63. Koren G. and Maurice L. 0989) Pediatric uses of opioids. Pediat. Clin. N. Am. 36, 1141-1156. Phillis J. W. and O'Regan M. H. (1988) The role of adenosine in the central actions of the benzodiazepines. Prog. Neuropsychopharmac. biol. Psychiat. 12, 389404.

Sawynok J., SweeneyM. I. and White T. D. (1989) Adenosine release may mediate spinal analgesia by morphine. Trends Pharmac. Sci. 10, 186-189.