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Effect of propranolol on antinociceptive, tolerance- and dependence-producing properties of morphine in rodents and monkeys
European Journal of Pharmacology, 34 ( 1975) 87--94 ~) North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
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EFFECT OF PROPRANOLOL ON ANTINOCICEPTIVE, TOLERANCEAND DEPENDENCE-PRODUCING PROPERTIES OF MORPHINE IN RODENTS AND MONKEYS* ALAN COWAN and IAN R. MACFARLANE Department of Pharmacology, Pharmaceutical Division, Reckitt and Colman Ltd., Hull HU8 7DS, England
Received 11 April 1975, revised MS received 10 June 1975, accepted 2 July 1975
A. COWAN and I.R. MACFARLANE, Effect of propranolol on antinociceptive, tolerance- and dependenceproducing properties of morphine in rodents and monkeys, European J. Pharmacol. 34 (1975) 87--94. Since an abstinence syndrome may accompany the injection of opioids in addicts pretreated with propranolol the morphine antagonistic properties of this compound were investigated. Racemic propranolol did not significantly affect the antinociceptive EDs 0 of morphine in rodents and neither precipitated abstinence in morphinedependent monkeys nor exacerbated the syndrome in 24 hr withdrawn monkeys. Multiple doses of propranolol did not alter the development of physical dependence on morphine in monkeys. Clinical narcotic antagonism would not be predicted from this profile. Evidence for a possible propranolol--morphine interaction came from studies using the mouse tail flick test. Thus, after 8 injections of pr'opranolol (over 4 days) mice were tolerant to normally effective doses of morphine. Concurrent injections of naloxone antagonised this effect. When propranolol and morphine were administered concurrently the morphine EDs0 (on day 5) was twice that of the group receiving morphine alone. Similar results were obtained with d-propranolol; practolol had a neutral effect. Propranolol
Morphine
Naloxone
Analgesia
1. I n t r o d u c t i o n I t has r e c e n t l y b e e n p r o p o s e d t h a t t h e /3adrenoceptor antagonist, d,l-propranolol, may be useful in t h e t r e a t m e n t o f d e p e n d e n c e on o p i o i d drugs (Grosz, 1972). T h e claim is b a s e d o n t h e f o l l o w i n g o b s e r v a t i o n s . (i) T h e oral a d m i n i s t r a t i o n o f p r o p r a n o l o l t o addicts blocks t h e r e w a r d i n g e f f e c t s o f a s u b s e q u e n t i n j e c t i o n o f h e r o i n ; u n d e r these c i r c u m s t a n c e s , h e r o i n o f t e n increases r a t h e r t h a n diminishes, t h e i n t e n s i t y o f t h e w i t h d r a w a l s y n d r o m e . (ii) P r o p r a n o l o l a t t e n u a t e s t h e residual craving f o r n a r c o t i c s t h a t is usually e x p e r i e n c e d b y exaddicts ( G r o s z , 1 9 7 3 ) .
Physical dependence
Tolerance
T h e s e interesting clinical o b s e r v a t i o n s h a v e p r o m p t e d the p r e s e n t s t u d y in w h i c h t h e influence o f p r o p r a n o l o l o n t y p i c a l m o r p h i n e e f f e c t s - - a n t i n o c i c e p t i o n , t o l e r a n c e and physical d e p e n d e n c e - - has b e e n investigated using mice, rats and m o n k e y s . 2. Materials and m e t h o d s 2.1. A n i m a l s
T h e e x p e r i m e n t s w e r e carried o u t on m a l e albino mice ( M F I / O l a ) , weighing 1 8 - - 2 4 g, o n male S p r a g u e - - D a w l e y rats ( 4 0 - - 6 0 g) and o n rhesus m o n k e y s ( 4 - - 6 kg) o f b o t h sexes. 2. 2. C o m p o u n d s
* This paper was presented, in part, at the 9th Congress of the Collegium Internationale Neuropsychopharmacologicum, in Paris, July 1974.
T h e f o l l o w i n g c o m p o u n d s were dissolved in p h y s i o l o g i c a l saline solution: m o r p h i n e sul-
88
A. COWAN, I.R. MACFARLANE
phate, B.P. (Macfarlan Smith), naloxone hydrochloride (Endo), practolol and d- and d,lpropranolol hydrochlorides (Imperial Chemical Industries). Compounds were injected, at different s.c. sites, in a volume of 10 ml/kg b o d y weight for mice, 5 ml/kg for rats and 0.25 ml/kg for monkeys. The doses quoted refer to the particular salt.
Groups of 10 rats received saline or d,l-propranolol (3, 10 or 30 mg/kg, s.c.) 30 min before saline or one of four doses of morphine (0.10, 0.30, 1 and 3 mg/kg, s.c.). 30 min later squeal thresholds were determined and antinociceptive EDs 0 values were obtained using the method of Litchfield and Wilcoxon (1949).
2.3. Tail flick test
The nociceptive stimulus was water maintained at 55°C. The time taken for each mouse to withdraw its tail from the water was recorded with a stopwatch. Only mice withdrawing their tails within 2 sec were used. Immediately after selection, groups of 10 animals received saline or d,l-propranolol (30 mg/kg, s.c.) either 5 or 30 min before saline or one of four doses of morphine (1,3, 1 0 a n d 30 mg/kg, s.c.}. 30 min later the flick response of each mouse was noted. On the basis of control data, those mice failing to show the flick reflex within 3.5 sec were termed 'non-responders'. Antin0ciceptive EDs 0 values and 95% confidence limits were obtained using the method of Litchfield and Wilcoxon (1949).
5 drug-naive monkeys received the following compounds s.c. at 9.00, 16.00 and 23.00 hr daily for 31 consecutive days: saline + morphine (3 mg/kg) (2 animals); d,l-propranolol (10 mg/kg) + morphine (3 mg/kg) (2 animals) and d,l-propranolol + saline (one animal). The 2 injections were separated by 20 min. The development of physical dependence was monitored by challenging each primate with naloxone (2 mg/kg, s.c.) on day 28, 3 hr after the last dose of drug. Signs of abstinence were noted over 1 hr and the syndrome was classified according to the protocol described by Deneau and Seevers (1964). Physiological saline solution was injected instead of test drugs after day 31 and the animals were observed through a one-way mirror between 9.00--11.00 hr and 14.00--16.00 hr for the following 4 days.
2.4. Tolerance and cross-tolerance tests
2.7. Tests with morphine-dependent monkeys
Groups of 45 mice were injected s.c. at 9.00 and 16.00 hr daily for 4 days with one of the drugs or drug-combinations under study (section 3.3.). On day 5, 17--18 hr after the last injections, the control time for each mouse in the tail flick test was recorded then sub-groups of 15 animals received one of three logarithmically spaced doses of morphine (chosen from 1, 3, 10, 30 and 100 mg/kg, s.c.). 30 min later, the flick responses were again recorded and antinociceptive EDs 0 values for morphine were obtained as described in section 2.3.
8 monkeys, housed in pairs, were selected from a colony of morphine-dependent animals that had received daily injections of the narcotic (6 mg/kg, s.c.) at 9.00, 16.00 and 23.00 hr for 14--16 months, d,l-Propranolol was administered at 12.00 hr in experiments involving non-withdrawn animals (section 3.5.) and at 9.00 hr in experiments involving 24-hr withdrawn animals (section 3.6. ).
2.5. Tail pressure test
3.1. Tail flick test
The procedure used has been previously described (Boura and Fitzgerald, 1966).
When mice were pretreated with either saline or d,l-propranolol (30 mg/kg), 5 min before the
2.6. Direct dependence test
3. Results
I N F L U E N C E O F P R O P R A N O L O L ON M O R P H I N E E F F E C T S
injection of morphine, the antinociceptive EDs 0 values of the narcotic were not significantly different (p > 0.05, Standard assay program, Finney, 1971): 12.5 mg/kg (5.7-27.5, 95% confidence limits) and 9.2 mg/kg (4.8--17.5), respectively. When the pretreatment time was 30 min, there was again no significant difference between the 2 EDs0 values of morphine: 8.0 mg/kg (4.2--15.2) and 8.6 mg/kg (3.4--21.5). By itself, d,l-propranolol (30 mg/kg)produced no antinociceptive effects in either procedure. 3.2. Tail pressure test
The following antinociceptive EDs 0 values were obtained for morphine when rats received either saline or d,l-propranolol (3, 10 or 30 mg/kg), 30 min before the injection of the narcotic: 1.0 mg/kg (0.34--2.9), 1.2 mg/kg (0.71--2.0), 1.3 mg/kg {0.98--1.7) and 0.78 mg/kg ( 0 . 5 2 - t . 2 ) , respectively. These values were not significantly different (p > 0.05). When d,l-propranolol {30 mg/kg) was injected alone, all rats responded within the control pressure range when tested I hr later. 3. 3. Tolerance and cross-tolerance tests
In the first study, 4 groups of mice received s.c. either saline, d,l-propranolol (30 mg/kg), morphine (70 mg/kg) or d,l-propranolol (30 mg/kg) + morphine (70 mg/kg) twice daily for 4 days. With the fourth group, the d,l-propranolol was injected 5 min before the morphine. On day 5, antinociceptive ED50 values for morphine were estimated for each group using the tail flick test. The results are presented in fig. 1. As might be expected, the morphine ED50 values obtained with the groups predosed with saline and morphine, respectively, were significantly different (p < 0.05). Of interest was the finding that the morphine EDs 0 value obtained with mice pretreated with d,l-propranolol was of the same order as that observed with mice receiving the multiple morphine injections. Furthermore, the morphine EDs 0 calculated from the group receiving the
89
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~
dl-Pr~. Morphine (P) (M)
~
P*M
Fig. 1. The effect of various p r e t r e a t m e n t s on the antinociceptive EDs0 of morphine in the mouse tail flick test. 4 groups of 45 mice were injected s.c. at 9.00 and 16.00 hr daily for 4 days with either saline, d,l-propranolol (P, 30 mg/kg), m o r p h i n e (M, 70 mg/kg) or P + M (same doses}; the m o r p h i n e EDs0 values were estimated on day 5, 17--18 hr after the last injections.
drug combination (d,l-propranolol + morphine) was approximately the sum of the EDs 0 values obtained with the d,l-propranolol and morphine groups, respectively. The potency ratios of morphine, relative to the morphine EDs 0 obtained with the saline group, were: d,l-propranolol group, 0.34 {0.18--0.64, 95% confidence limits); morphine group, 0.31 {0.16--0.57) and (d,l-propranolol + morphine) group, 0.16 (0.08--0.28). In the second study, the same experimental design was used, however d,l-propranolol was replaced by d-propranolol (30 mg/kg) which has practically no ~-blocking properties. Similar results (fig. 2) were obtained in that morphine antinociceptive EDs 0 values of the same order were recorded with the d-propranolol and morphine groups, respectively, while the EDs 0 with the (d-propranolol + morphine) group was greater than that of either component group. In this experiment the potency ratios of morphine were: d-propranolol group, 0.34 (0.15--0.76); morphine group, 0.25 (0.11--0.55) and (d-propranolol + morphine) group, 0.18 (0.08--0.41). From subjective impressions, neither d-propranolol nor the race-
90
A. COWAN, I.R. M A C F A R L A N E 80
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Saline
d- Prop. (P)
Morphine (M)
P4 M
Fig. 2. The effect of various pretreatments on the antinociceptive EDs 0 of m o r p h i n e in the mouse tail flick test. 4 groups of 45 mice were injected s.c. at 9.00 and 16.00 hr daily for 4 days with either saline, d-propranolol (P, 30 mg/kg), m o r p h i n e (M, 70 mg/kg) or P + M (same doses); the m o r p h i n e E D s 0 values were estimated on day 5, 17--18 hr after the last injections.
mate attenuated the morphine-induced increase in locomotor activity. Practolol, a ~-adrenoceptor antagonist that does not readily pass the blood--brain barrier, was also examined in the tolerance and crosstolerance experiments. In this case, multiple dosing with practolol (30 mg/kg) did not markedly affect the subsequent morphine antinociceptive ED50 value on day 5 (fig. 3). The potency ratios of morphine were: practolol group, 0.72 (0.36--1.42}; morphine group, 0.30 (0.15--0.62) and (practolol + morphine) group, 0.30 (0.15--0.62). Data from the final study are presented in table 1. It may be noted that, in relation to the experiments described above, much higher doses of morphine were required to produce antinociceptive effects in those mice pre-dosed with morphine. It is of interest that a similar increase in the EDs 0 of morphine was observed in mice pretreated with d,l-propranolol. When naloxone (2 mg/kg, s.c.) was administered to mice 5 min before each of the 8 injections of morphine, the development of tolerance was markedly antagonised as judged by the relatively low morphine EDs 0 value obtained on day 5 (table 1). Naloxone ap-
Saline
Practolol Morphine (P) (M)
P*M
Fig. 3. T h e e f f e c t o f v a r i o u s p r e t r e a t m e n t s o n the anti-
nociceptive ED50 of m o r p h i n e in the mouse tail flick test. 4 groups of 45 mice were injected s.c. at 9.00 and 16.00 hr daily for 4 days with either saline, practolol (P, 30 mg/kg), morphine (M, 70 mg/kg) or P + M (same doses); the m o r p h i n e EDs 0 values were estimated on day 5, 17--18 hr after the last injections.
peared to antagonise the cross-tolerance between d,l-propranolol and morphine since the morphine ED50 obtained on day 5 with the (naloxone + d,l-propranolol) group was approximately one half of that obtained with the group receiving only d,l-propranolol. It should be understood that, irrespective of the nature of the multi-dose schedule, all mice displayed the tail flick reflex within the control time (2.0 sec) when tested immediately prior to morphine administration on day 5. 3.4. Direct dependence test The m o n k e y receiving d,l-propranolol chronically showed no signs of abstinence or unusual behavioural effects after challenge with naloxone or again on abrupt withdrawal of the ~-adrenoceptor antagonist. In contrast, both monkeys receiving morphine chronically and the t w o animals dosed with (d,l-propranolol + morphine) exhibited signs of abstinence after antagonist challenge and on termination of drug administration. Abstinence syndromes of severe intensity were recorded for all 4 monkeys. The behaviour of each primate was characterised by a high incidence of emesis,
INFLUENCE OF PROPRANOLOL ON MORPHINE EFFECTS
91
TABLE 1 The effect of naloxone on cross-tolerance between d,l-propranolol and morphine in mice. 5 groups of 45 mice received test compounds at 9.00 hr and 16.00 hr daily for 4 days. On day 5, antinociceptive EDs0 values for morphine were obtained for each group using the tail flick test. Potency ratios for morphine were calculated using the parallel line assay as described by Finney (1971). Pretreatment (s.c.)
Morphine EDs0 (mg/kg, s.c.) and 95% confidence limits
Potency ratios of morphine and 95% confidence limits
Saline d,l-Propranolol (30 mg/kg) Morphine (70 mg/kg) Naloxone (2 mg/kg) + morphine (70 mg/kg) Naloxone (2 mg/kg) + d,l-propranolol (30 mg/kg)
10 (5-21) 60 (31-116)
1.00 0.18 (0.08-0.38)
62 (43--90) 17 (9--32) 33 (19--56)
0.16 (0.08--0.33) 0.62 (0.28--1.34) 0.35 (0.17--0.81)
fighting, shaking, calling and yawning and by alternating periods of restlessness and reduced l o c o m o t o r activity. Normal behavioural and social patterns were apparent with all 4 monkeys 4 days after abrupt withdrawal of drugs.
3.5. Non-withdrawn morphine-dependent monkeys
after the last dose of narcotic. The withdrawal syndrome was neither augmented nor suppressed, over the following 48 hr, in comparison with 4 saline-injected control monkeys. In contrast to the clinical findings of Grosz (1973), the subsequent injection of narcotic, at the end of the experiment, did not intensify the withdrawal syndrome.
3.5.1. Effect of d,l-propranolol 3 pairs of monkeys each received one of 3 doses of d,l-propranolol (1, 3 or 10 mg/kg, s.c.). No overt behavioural changes were recorded over the following 4 hr or after the following 6 routine injections of morphine.
3.5.2. Effect of d,l-propranolol + naloxone 4 morphine-dependent monkeys received d,l-propranolol (10 mg/kg, s.c.), 3 hr after the morning injection of morphine. 1 hr later each pair of animals was challenged with naloxone (0.5 or 2 mg/kg, s.c.), d,l-Propranolol did not influence the ability of naloxone to precipitate severe abstinence syndromes as judged from parallel observations in 4 monkeys receiving saline and the same doses of narcotic antagonist.
3. 6. Withdrawn morphine-dependent monkeys d,l-Propranolol (10 mg/kg, s.c.) was administered to 4 morphine-dependent monkeys 24 hr
4. Discussion In the present work, single doses of d,1propranolol had no significant effect on the antinociceptive EDs 0 values of morphine in either the hot water version of the mouse tail flick test or the tail pressure test using rats. These observations are consistent with reports of similar interactional studies involving mouse phenylquinone-writhing and hot plate tests (Fennessy and Lee, 1970), the radiant heat version of the mouse tail flick procedure (Dewey et al., 1970), the rat paw pressure test (GSrlitz and Frey, 1972) and the rat hot plate test (Cicero, 1974). In contrast, Heller et al. (1968) and Shah et al. (1974) used the mouse (hot water) and rat (radiant heat) tail flick tests, respectively, and found that similar doses of propranolol significantly antagonised the antinociceptive action of morphine. The reason for these differing results is not known. Conflicting conclusions seem to be a relatively
92
common feature of those studies that involve the administration of test agent and morphine prior to a standard rodent antinociceptive procedure (see Reinhold et al., 1973). From the literature, there is general agreement that the antinociceptive action of morphine is not significantly enhanced by propranolol. Since Cicero et al. (1974b) have recently claimed that the acute administration of phenoxybenzamine or phentolamine increases the reaction time of rats to morphine in the hot plate test, it would appear that ~-, rather than /3-, adrenoceptors are more closely involved in mediating the antinociceptive effect, certainly in this species and situation. At first sight, data from the present multiple-injection experiments point to the possible involvement of /3-adrenoceptors in the antinociceptive action of morphine (fig. 1). More specifically, mice that received d,l-propranolol (30 mg/kg, s.c.) twice daily for 4 days, required surprisingly large doses of morphine on day 5 during estimation of the group antinociceptive EDs 0 value using the tail flick test. In other words, mice that received eight injections of d,l-propranolol became tolerant to normally effective antinociceptive doses of morphine. The morphine EDs 0 value obtained with the d,l-propranololtreated group was, in fact, approximately three times greater than that from the corresponding saline control group and did not differ significantly from that of the group receiving twice daily doses of morphine (70 mg/kg, s.c.). In the interpretation of these results, it is important to appreciate that the antinociceptive test took place 17--18 hr after the last of the multiple injections and at this time, just prior to estimating morphine EDs 0 values, all mice responded to the hot water b y displaying the tail reflex within the control time of 2 sec. Although cross-tolerance between morphine and non-opioid compounds is well known (e.g. methylamphetamine, Pleuvry, 1971), the cross-tolerance described in the present paper is of a less c o m m o n type as one of the agents (d,l-propranolol), on acute administration, does not possess antinociceptive properties in the test situation used.
A. COWAN, I.R. MACFARLANE
It may also be inferred from fig. 1 that the repeated blockade of/3-adrenoceptor sites with d,l-propranolol, just prior to each of the eight injections of morphine, enhances the onset or development of tolerance to the narcotic since the antinociceptive EDs 0 value obtained from this group was twice that from the corresponding group pretreated with morphine alone. The fact that the morphine EDs 0 value for the (d,l-propranolol + morphine) group was approximately the sum of those obtained with the c o m p o n e n t groups may have been fortuitous, however, the possibility that this morphine EDs 0 value was influenced independently by two unrelated processes cannot be discounted. Pertinent to an analysis of this tolerance effect is the report that propranolol interferes with the binding of morphine to subcellular fractions of mouse brain (DeFeudis and Grosz, 1972) while Brunk et al. (1974) state that the metabolism of morphine is uninfluenced by propranolol, at least in normal human subjects. Future work will be directed towards defining the neurochemical consequences of multiple dosing with d,l-propranolol and hence establishing a link with the vast literature that implicates postulated neurotransmitters with agonist actions of morphine (e.g. Way and Shen, 1971). In this c o n t e x t it is of interest that repeated blockade of dopaminergic receptors with haloperidol -- another drug that is claimed to suppress the severity of withdrawal from heroin in addicts (Karkalas and Lal, 1973) -- likewise enhances tolerance to concurrently injected morphine in mice when the hot plate test is used (Eidelberg and Erspamer, 1975). The effects found with d,l-propranolol in the present study were also noted in similar experiments w i t h t h e d-isomer. Since the latter compound shows membrane stabilisation activity rather than /3-blocking activity (Barrett and Cullum, 1968; Jefferson, 1974), it is possible that/3-adrenoceptors are not primarily involved in mediating these effects. Additional experiments are obviously required with a range of /3-adrenoceptor antagonists to clarify the relationship between the extent of (assumed) cen-
INFLUENCE OF PROPRANOLOL ON MORPHINE EFFECTS
tral blockade and the tolerance/cross-tolerance effects. It might be argued that these effects are indeed centrally mediated since multiple dosing with practolol, a ~-adrenoceptor antagonist which does not readily enter the brain (Scales and Cosgrove, 1970), did not markedly affect the subsequent morphine EDs 0 value on day 5 in relation to saline-injected control mice; furthermore, no tolerance enhancement effect was observed when this compound was injected just prior to morphine. The finding that the concurrent administration of naloxone and morphine reduces tolerance development to the antinociceptive effect of the narcotic is in agreement with the data from similar experiments with mice reported by Shen et al. (1970). The fact that naloxone also appeared to antagonise the cross-tolerance between d,l-propranolol and morphine is interesting in light of the suggestion by Charalampous and Askew (1974) that .the ~-adrenergic receptor and the specific opiate receptor (see review by Goldstein, 1974) may be similar in the mouse brain. If this is so then the competition between naloxone and propranolol for mutual binding sites in the CNS may influence, either directly or indirectly, those factors responsible for the cross-tolerance effect. It has to be admitted, however, that this proposition only relates to the mouse at present since Cicero et al. (1974c) found that propranolol has no affect on the binding of 3H-naloxone to brain homogenates obtained from rats. One attraction of using d,l-propranolol, rather than methadone, in the pharmacotherapy of addiction is the supposed negligible physical dependence potential of this compound (Grosz, 1973). At the animal level, signs of abstinence were not observed after naloxone challenge in a rhesus monkey that had been chronically dosed with d,l-propranolol for 28 days or again after abrupt withdrawal of the drug on day 31 (see section 3.4.). It seems clear from the monkey studies that the pre-administration of d,l-propranolol to morphine-dependent animals had no significant influence on the intensity of a subsequent naloxone-induced abstinence syndrome. This finding is in agree-
93
ment with data from recent studies that have involved the precipitation of jumping or 'wet dog' shakes by naloxone in morphine-dependent mice (Chipkin et al., 1974) and rats (Jhamandas et al., 1973; Cicero et al., 1974a), respectively. The monkey studies have also provided information which suggests that d,l-propranolol does not influence the development of dependence on morphine. Moreover, morphine antagonistic properties could not be ascribed to d,l-propranolol since it neither precipitated signs of abstinence in non-withdrawn morphine-dependent monkeys nor exacerbated the syndrome in 24-hr withdrawn morphinedependent animals. Martin et al. {1974) similarly concluded that propranolol ( 5 mg/kg) neither precipitates abstinence nor influences the intensity of withdrawal in morphine-dependent chronic spinal dogs. These laboratory results are surprising when viewed against the reported narcotic blocking action of propranolol in addicts'(Grosz, 1972) and are perhaps more in keeping with the data of Hollister and Prusmack (1974) and Jacob et al. (1975) whose clinical studies clearly (but unfortunately) indicate pessimism for the future use of propranolol as an adjunct to the treatment of withdrawal from opiates.
Acknowledgements It is a pleasure to thank Diane Beilby for expert technical assistance. Generous gifts of naloxone from Endo and practolol and propranolol from ICI are gratefully acknowledged.
References Barrett, A.M. and V.A. Cullum, 1968, The biological properties of the optical isomers of propranolol and their effects on cardiac arrhythmias, Brit. J. Pharmacol. 34, 43. Boura, A.L.A. and A.E. Fitzgerald, 1966, The pharmacology of N-(Cyclopropylmethyl)-19-isopentylnororvinol hydrochloride. A potent and long lasting central depressant, Brit. J. Pharmacol. 26, 307.
94 Brunk, S.F., M. Delle and W.R. Wilson, 1974, Effect of propranolol on morphine metabolism, Clin. Pharmacol. Therap. 16, 1039. Charalampous, K.D. and W.E. Askew, 1974, Effect of non-opiate antagoni.sts on stereospecific binding of tritiated naloxone, Res. Commun. Chem. Pathol. Pharmacol. 8, 615. Chipkin, R.E., W.L. Dewey and L.S. Harris, 1974, The effect of propranolol on the antinociceptive and opiate withdrawal characteristics of morphine in mice, Pharmacologist 16, 204. Cicero, T.J., 1974, Effects of c~-adrenergic blocking agents on narcotic-induced analgesia, Arch. Intern. Pharmacodyn. 208, 5. Cicero, T.J., E.R. Meyer and R.D. Bell, 1974a, Effects of phenoxybenzamine on the narcotic withdrawal syndrome in the rat, Neuropharmacol. 13, 601. Cicero, T.J., E.R. Meyer and B.R. Smithloff, 1974b, ~-Adrenergic blocking agents: antinociceptive activity and enhancement of morphine-induced analgesia, J. Pharmacol. Exptl. Therap. 189, 72. Cicero, T.J., C.E. Wilcox and E.R. Meyer, 1974c, Effect of (~-adrenergic blockers on naloxone-binding in brain, Biochem. Pharmacol. 23, 2349. De Feudis, F.V. and H.J. Grosz, 1972, Effects of propranolol on the binding of 14C-morphine to particulate fractions of mouse brain, Brain Res. 47, 510. Deneau, G.A. and M.H. Seevers, 1964, Drug dependence, in: Evaluation of Drug Activities: Pharmacometrics, Vol. 1, eds. D.R. Laurence and A.L. Bacharach (Academic Press, London) p. 167. Dewey, W.L., L.S. Harris, J.F. Howes and J.A. Nuite, 1970, The effect of various neurohumoral modulators on the activity of morphine and the narcotic antagonists in the tail-flick and phenylquinone tests, J. Pharmacol. Exptl. Therap. 175,435. Eidelberg, E. and R. Erspamer, 1975, Dopaminergic mechanisms of opiate actions in brain, J. Pharmacol. Exptl. Therap. 192, 50. Fennessy, M.R. and J.R. Lee, 1970, Modification of morphine analgesia by drugs affecting adrenergic and tryptaminergic mechanisms, J. Pharm. Pharmacol. 22,930. Finney, D.J., 1971, The comparison of effectiveness, in: Probit Analysis, (Cambridge University Press, Cambridge) p. 100. Goldstein,-A., 1974, Opiate receptors, Life Sci. 14, 615. G6rlitz, B.-D. and H.-H. Frey, 1972, Central monoamines and antinociceptive drug action, European J. Pharmacol. 20, 171. Grosz, H.J., 1972, Narcotic withdrawal symptoms in
A. COWAN, I.R. MACFARLANE heroin users treated with propranolol, Lancet II, 564. Grosz, H.J., 1973, Effect of propranolol on active users of heroin, Lancet II, 612. Heller, B., J.M. Saavedra and E. Fischer, 1968, Influence of adrenergic blocking agents upon morphine and catecholamine analgesic effect, Experientia 24, 804. Hollister, L.E. and J.J. Prusmack, 1974, Propranolol in withdrawal from opiates, Arch. Gen. Psychiat. 31,695. Jacob, R.G., J. Holmstrand, J.H. Eriksson and E. fl.ngg~rd, 1975, Propranolol in the treatment of opiate dependence -- a controlled study, Psychopharmacologia (Berlin) 41, 71. Jefferson, J.W., 1974, /3-Adrenergic receptor blocking drugs in psychiatry, Arch. Gen. Psychiat. 31,681. Jhamandas, K., M. Sutak and S. Bell, 1973, Modification of precipitated morphine withdrawal syndrome by drugs affecting cholinergic mechanisms, European J. Pharmacol. 24,296. Karkalas, J. and H. Lal, 1973, A comparison of haloperidol with methadone in blocking beroin-withdrawal symptoms, Intern. Pharmacopsychiat. 8, 248. Litchfield, J.T. and F. Wilcoxon, 1949, A simplified method of evaluating dose effect experiments, J. Pharmacol. Exptl. Therap. 96, 99. Martin, W.R., C.G. Eades, W.O. Thompson, J.A. Thompson and H.G. Flanary, 1974, Morphine physical dependence in the dog, J. Pharmacol. Exptl. Therap. 189,759. Pleuvry, B.J., 1971, Cross tolerance between methyl* amphetamine and morphine in the mouse, J. Pharm. Pharmacol. 23,969. Reinhold, K., J. Bli/sig and A. Herz, 1973, Changes in brain concentratior of biogenic amines and the antinociceptive effect of morphine in rats, Naunyn Schmiedeb. Arch. Pharmacol. 278, 69. Scales, B. and M.B. Cosgrove, 1970, The metabolism and distribution of the selective adrenergic /3blocking agent, practolol, J. Pharmacol. Exptl. Therap. 175,338. Shah, U.H., M.N. Jindal, V.K. Patel and V.V. Kelkar, 1974, Central actions of some ~-adrenoceptor blocking agents, Arzneim. Forsch. 24, 1581. Shen, F.-H., H.H. Loh and E.L. Way, 1970, Brain serotonin turnover in morphine tolerant and dependent mice, J. Pharmacol. Exptl. Therap. 175, 427. Way, E.L. and F.-H. Shen, 1971, Catecholamines and 5-hydroxytryptamine, in: Narcotic Drugs, Biochemical Pharmacology, ed. D.H. Clouet (Plenum Press, New York) p. 229.
87
EFFECT OF PROPRANOLOL ON ANTINOCICEPTIVE, TOLERANCEAND DEPENDENCE-PRODUCING PROPERTIES OF MORPHINE IN RODENTS AND MONKEYS* ALAN COWAN and IAN R. MACFARLANE Department of Pharmacology, Pharmaceutical Division, Reckitt and Colman Ltd., Hull HU8 7DS, England
Received 11 April 1975, revised MS received 10 June 1975, accepted 2 July 1975
A. COWAN and I.R. MACFARLANE, Effect of propranolol on antinociceptive, tolerance- and dependenceproducing properties of morphine in rodents and monkeys, European J. Pharmacol. 34 (1975) 87--94. Since an abstinence syndrome may accompany the injection of opioids in addicts pretreated with propranolol the morphine antagonistic properties of this compound were investigated. Racemic propranolol did not significantly affect the antinociceptive EDs 0 of morphine in rodents and neither precipitated abstinence in morphinedependent monkeys nor exacerbated the syndrome in 24 hr withdrawn monkeys. Multiple doses of propranolol did not alter the development of physical dependence on morphine in monkeys. Clinical narcotic antagonism would not be predicted from this profile. Evidence for a possible propranolol--morphine interaction came from studies using the mouse tail flick test. Thus, after 8 injections of pr'opranolol (over 4 days) mice were tolerant to normally effective doses of morphine. Concurrent injections of naloxone antagonised this effect. When propranolol and morphine were administered concurrently the morphine EDs0 (on day 5) was twice that of the group receiving morphine alone. Similar results were obtained with d-propranolol; practolol had a neutral effect. Propranolol
Morphine
Naloxone
Analgesia
1. I n t r o d u c t i o n I t has r e c e n t l y b e e n p r o p o s e d t h a t t h e /3adrenoceptor antagonist, d,l-propranolol, may be useful in t h e t r e a t m e n t o f d e p e n d e n c e on o p i o i d drugs (Grosz, 1972). T h e claim is b a s e d o n t h e f o l l o w i n g o b s e r v a t i o n s . (i) T h e oral a d m i n i s t r a t i o n o f p r o p r a n o l o l t o addicts blocks t h e r e w a r d i n g e f f e c t s o f a s u b s e q u e n t i n j e c t i o n o f h e r o i n ; u n d e r these c i r c u m s t a n c e s , h e r o i n o f t e n increases r a t h e r t h a n diminishes, t h e i n t e n s i t y o f t h e w i t h d r a w a l s y n d r o m e . (ii) P r o p r a n o l o l a t t e n u a t e s t h e residual craving f o r n a r c o t i c s t h a t is usually e x p e r i e n c e d b y exaddicts ( G r o s z , 1 9 7 3 ) .
Physical dependence
Tolerance
T h e s e interesting clinical o b s e r v a t i o n s h a v e p r o m p t e d the p r e s e n t s t u d y in w h i c h t h e influence o f p r o p r a n o l o l o n t y p i c a l m o r p h i n e e f f e c t s - - a n t i n o c i c e p t i o n , t o l e r a n c e and physical d e p e n d e n c e - - has b e e n investigated using mice, rats and m o n k e y s . 2. Materials and m e t h o d s 2.1. A n i m a l s
T h e e x p e r i m e n t s w e r e carried o u t on m a l e albino mice ( M F I / O l a ) , weighing 1 8 - - 2 4 g, o n male S p r a g u e - - D a w l e y rats ( 4 0 - - 6 0 g) and o n rhesus m o n k e y s ( 4 - - 6 kg) o f b o t h sexes. 2. 2. C o m p o u n d s
* This paper was presented, in part, at the 9th Congress of the Collegium Internationale Neuropsychopharmacologicum, in Paris, July 1974.
T h e f o l l o w i n g c o m p o u n d s were dissolved in p h y s i o l o g i c a l saline solution: m o r p h i n e sul-
88
A. COWAN, I.R. MACFARLANE
phate, B.P. (Macfarlan Smith), naloxone hydrochloride (Endo), practolol and d- and d,lpropranolol hydrochlorides (Imperial Chemical Industries). Compounds were injected, at different s.c. sites, in a volume of 10 ml/kg b o d y weight for mice, 5 ml/kg for rats and 0.25 ml/kg for monkeys. The doses quoted refer to the particular salt.
Groups of 10 rats received saline or d,l-propranolol (3, 10 or 30 mg/kg, s.c.) 30 min before saline or one of four doses of morphine (0.10, 0.30, 1 and 3 mg/kg, s.c.). 30 min later squeal thresholds were determined and antinociceptive EDs 0 values were obtained using the method of Litchfield and Wilcoxon (1949).
2.3. Tail flick test
The nociceptive stimulus was water maintained at 55°C. The time taken for each mouse to withdraw its tail from the water was recorded with a stopwatch. Only mice withdrawing their tails within 2 sec were used. Immediately after selection, groups of 10 animals received saline or d,l-propranolol (30 mg/kg, s.c.) either 5 or 30 min before saline or one of four doses of morphine (1,3, 1 0 a n d 30 mg/kg, s.c.}. 30 min later the flick response of each mouse was noted. On the basis of control data, those mice failing to show the flick reflex within 3.5 sec were termed 'non-responders'. Antin0ciceptive EDs 0 values and 95% confidence limits were obtained using the method of Litchfield and Wilcoxon (1949).
5 drug-naive monkeys received the following compounds s.c. at 9.00, 16.00 and 23.00 hr daily for 31 consecutive days: saline + morphine (3 mg/kg) (2 animals); d,l-propranolol (10 mg/kg) + morphine (3 mg/kg) (2 animals) and d,l-propranolol + saline (one animal). The 2 injections were separated by 20 min. The development of physical dependence was monitored by challenging each primate with naloxone (2 mg/kg, s.c.) on day 28, 3 hr after the last dose of drug. Signs of abstinence were noted over 1 hr and the syndrome was classified according to the protocol described by Deneau and Seevers (1964). Physiological saline solution was injected instead of test drugs after day 31 and the animals were observed through a one-way mirror between 9.00--11.00 hr and 14.00--16.00 hr for the following 4 days.
2.4. Tolerance and cross-tolerance tests
2.7. Tests with morphine-dependent monkeys
Groups of 45 mice were injected s.c. at 9.00 and 16.00 hr daily for 4 days with one of the drugs or drug-combinations under study (section 3.3.). On day 5, 17--18 hr after the last injections, the control time for each mouse in the tail flick test was recorded then sub-groups of 15 animals received one of three logarithmically spaced doses of morphine (chosen from 1, 3, 10, 30 and 100 mg/kg, s.c.). 30 min later, the flick responses were again recorded and antinociceptive EDs 0 values for morphine were obtained as described in section 2.3.
8 monkeys, housed in pairs, were selected from a colony of morphine-dependent animals that had received daily injections of the narcotic (6 mg/kg, s.c.) at 9.00, 16.00 and 23.00 hr for 14--16 months, d,l-Propranolol was administered at 12.00 hr in experiments involving non-withdrawn animals (section 3.5.) and at 9.00 hr in experiments involving 24-hr withdrawn animals (section 3.6. ).
2.5. Tail pressure test
3.1. Tail flick test
The procedure used has been previously described (Boura and Fitzgerald, 1966).
When mice were pretreated with either saline or d,l-propranolol (30 mg/kg), 5 min before the
2.6. Direct dependence test
3. Results
I N F L U E N C E O F P R O P R A N O L O L ON M O R P H I N E E F F E C T S
injection of morphine, the antinociceptive EDs 0 values of the narcotic were not significantly different (p > 0.05, Standard assay program, Finney, 1971): 12.5 mg/kg (5.7-27.5, 95% confidence limits) and 9.2 mg/kg (4.8--17.5), respectively. When the pretreatment time was 30 min, there was again no significant difference between the 2 EDs0 values of morphine: 8.0 mg/kg (4.2--15.2) and 8.6 mg/kg (3.4--21.5). By itself, d,l-propranolol (30 mg/kg)produced no antinociceptive effects in either procedure. 3.2. Tail pressure test
The following antinociceptive EDs 0 values were obtained for morphine when rats received either saline or d,l-propranolol (3, 10 or 30 mg/kg), 30 min before the injection of the narcotic: 1.0 mg/kg (0.34--2.9), 1.2 mg/kg (0.71--2.0), 1.3 mg/kg {0.98--1.7) and 0.78 mg/kg ( 0 . 5 2 - t . 2 ) , respectively. These values were not significantly different (p > 0.05). When d,l-propranolol {30 mg/kg) was injected alone, all rats responded within the control pressure range when tested I hr later. 3. 3. Tolerance and cross-tolerance tests
In the first study, 4 groups of mice received s.c. either saline, d,l-propranolol (30 mg/kg), morphine (70 mg/kg) or d,l-propranolol (30 mg/kg) + morphine (70 mg/kg) twice daily for 4 days. With the fourth group, the d,l-propranolol was injected 5 min before the morphine. On day 5, antinociceptive ED50 values for morphine were estimated for each group using the tail flick test. The results are presented in fig. 1. As might be expected, the morphine ED50 values obtained with the groups predosed with saline and morphine, respectively, were significantly different (p < 0.05). Of interest was the finding that the morphine EDs 0 value obtained with mice pretreated with d,l-propranolol was of the same order as that observed with mice receiving the multiple morphine injections. Furthermore, the morphine EDs 0 calculated from the group receiving the
89
~--~40
4. o
20.
4'
~
5
~
~flne
~
dl-Pr~. Morphine (P) (M)
~
P*M
Fig. 1. The effect of various p r e t r e a t m e n t s on the antinociceptive EDs0 of morphine in the mouse tail flick test. 4 groups of 45 mice were injected s.c. at 9.00 and 16.00 hr daily for 4 days with either saline, d,l-propranolol (P, 30 mg/kg), m o r p h i n e (M, 70 mg/kg) or P + M (same doses}; the m o r p h i n e EDs0 values were estimated on day 5, 17--18 hr after the last injections.
drug combination (d,l-propranolol + morphine) was approximately the sum of the EDs 0 values obtained with the d,l-propranolol and morphine groups, respectively. The potency ratios of morphine, relative to the morphine EDs 0 obtained with the saline group, were: d,l-propranolol group, 0.34 {0.18--0.64, 95% confidence limits); morphine group, 0.31 {0.16--0.57) and (d,l-propranolol + morphine) group, 0.16 (0.08--0.28). In the second study, the same experimental design was used, however d,l-propranolol was replaced by d-propranolol (30 mg/kg) which has practically no ~-blocking properties. Similar results (fig. 2) were obtained in that morphine antinociceptive EDs 0 values of the same order were recorded with the d-propranolol and morphine groups, respectively, while the EDs 0 with the (d-propranolol + morphine) group was greater than that of either component group. In this experiment the potency ratios of morphine were: d-propranolol group, 0.34 (0.15--0.76); morphine group, 0.25 (0.11--0.55) and (d-propranolol + morphine) group, 0.18 (0.08--0.41). From subjective impressions, neither d-propranolol nor the race-
90
A. COWAN, I.R. M A C F A R L A N E 80
40
,,40
o~
E
u~
E 2O
o
Saline
d- Prop. (P)
Morphine (M)
P4 M
Fig. 2. The effect of various pretreatments on the antinociceptive EDs 0 of m o r p h i n e in the mouse tail flick test. 4 groups of 45 mice were injected s.c. at 9.00 and 16.00 hr daily for 4 days with either saline, d-propranolol (P, 30 mg/kg), m o r p h i n e (M, 70 mg/kg) or P + M (same doses); the m o r p h i n e E D s 0 values were estimated on day 5, 17--18 hr after the last injections.
mate attenuated the morphine-induced increase in locomotor activity. Practolol, a ~-adrenoceptor antagonist that does not readily pass the blood--brain barrier, was also examined in the tolerance and crosstolerance experiments. In this case, multiple dosing with practolol (30 mg/kg) did not markedly affect the subsequent morphine antinociceptive ED50 value on day 5 (fig. 3). The potency ratios of morphine were: practolol group, 0.72 (0.36--1.42}; morphine group, 0.30 (0.15--0.62) and (practolol + morphine) group, 0.30 (0.15--0.62). Data from the final study are presented in table 1. It may be noted that, in relation to the experiments described above, much higher doses of morphine were required to produce antinociceptive effects in those mice pre-dosed with morphine. It is of interest that a similar increase in the EDs 0 of morphine was observed in mice pretreated with d,l-propranolol. When naloxone (2 mg/kg, s.c.) was administered to mice 5 min before each of the 8 injections of morphine, the development of tolerance was markedly antagonised as judged by the relatively low morphine EDs 0 value obtained on day 5 (table 1). Naloxone ap-
Saline
Practolol Morphine (P) (M)
P*M
Fig. 3. T h e e f f e c t o f v a r i o u s p r e t r e a t m e n t s o n the anti-
nociceptive ED50 of m o r p h i n e in the mouse tail flick test. 4 groups of 45 mice were injected s.c. at 9.00 and 16.00 hr daily for 4 days with either saline, practolol (P, 30 mg/kg), morphine (M, 70 mg/kg) or P + M (same doses); the m o r p h i n e EDs 0 values were estimated on day 5, 17--18 hr after the last injections.
peared to antagonise the cross-tolerance between d,l-propranolol and morphine since the morphine ED50 obtained on day 5 with the (naloxone + d,l-propranolol) group was approximately one half of that obtained with the group receiving only d,l-propranolol. It should be understood that, irrespective of the nature of the multi-dose schedule, all mice displayed the tail flick reflex within the control time (2.0 sec) when tested immediately prior to morphine administration on day 5. 3.4. Direct dependence test The m o n k e y receiving d,l-propranolol chronically showed no signs of abstinence or unusual behavioural effects after challenge with naloxone or again on abrupt withdrawal of the ~-adrenoceptor antagonist. In contrast, both monkeys receiving morphine chronically and the t w o animals dosed with (d,l-propranolol + morphine) exhibited signs of abstinence after antagonist challenge and on termination of drug administration. Abstinence syndromes of severe intensity were recorded for all 4 monkeys. The behaviour of each primate was characterised by a high incidence of emesis,
INFLUENCE OF PROPRANOLOL ON MORPHINE EFFECTS
91
TABLE 1 The effect of naloxone on cross-tolerance between d,l-propranolol and morphine in mice. 5 groups of 45 mice received test compounds at 9.00 hr and 16.00 hr daily for 4 days. On day 5, antinociceptive EDs0 values for morphine were obtained for each group using the tail flick test. Potency ratios for morphine were calculated using the parallel line assay as described by Finney (1971). Pretreatment (s.c.)
Morphine EDs0 (mg/kg, s.c.) and 95% confidence limits
Potency ratios of morphine and 95% confidence limits
Saline d,l-Propranolol (30 mg/kg) Morphine (70 mg/kg) Naloxone (2 mg/kg) + morphine (70 mg/kg) Naloxone (2 mg/kg) + d,l-propranolol (30 mg/kg)
10 (5-21) 60 (31-116)
1.00 0.18 (0.08-0.38)
62 (43--90) 17 (9--32) 33 (19--56)
0.16 (0.08--0.33) 0.62 (0.28--1.34) 0.35 (0.17--0.81)
fighting, shaking, calling and yawning and by alternating periods of restlessness and reduced l o c o m o t o r activity. Normal behavioural and social patterns were apparent with all 4 monkeys 4 days after abrupt withdrawal of drugs.
3.5. Non-withdrawn morphine-dependent monkeys
after the last dose of narcotic. The withdrawal syndrome was neither augmented nor suppressed, over the following 48 hr, in comparison with 4 saline-injected control monkeys. In contrast to the clinical findings of Grosz (1973), the subsequent injection of narcotic, at the end of the experiment, did not intensify the withdrawal syndrome.
3.5.1. Effect of d,l-propranolol 3 pairs of monkeys each received one of 3 doses of d,l-propranolol (1, 3 or 10 mg/kg, s.c.). No overt behavioural changes were recorded over the following 4 hr or after the following 6 routine injections of morphine.
3.5.2. Effect of d,l-propranolol + naloxone 4 morphine-dependent monkeys received d,l-propranolol (10 mg/kg, s.c.), 3 hr after the morning injection of morphine. 1 hr later each pair of animals was challenged with naloxone (0.5 or 2 mg/kg, s.c.), d,l-Propranolol did not influence the ability of naloxone to precipitate severe abstinence syndromes as judged from parallel observations in 4 monkeys receiving saline and the same doses of narcotic antagonist.
3. 6. Withdrawn morphine-dependent monkeys d,l-Propranolol (10 mg/kg, s.c.) was administered to 4 morphine-dependent monkeys 24 hr
4. Discussion In the present work, single doses of d,1propranolol had no significant effect on the antinociceptive EDs 0 values of morphine in either the hot water version of the mouse tail flick test or the tail pressure test using rats. These observations are consistent with reports of similar interactional studies involving mouse phenylquinone-writhing and hot plate tests (Fennessy and Lee, 1970), the radiant heat version of the mouse tail flick procedure (Dewey et al., 1970), the rat paw pressure test (GSrlitz and Frey, 1972) and the rat hot plate test (Cicero, 1974). In contrast, Heller et al. (1968) and Shah et al. (1974) used the mouse (hot water) and rat (radiant heat) tail flick tests, respectively, and found that similar doses of propranolol significantly antagonised the antinociceptive action of morphine. The reason for these differing results is not known. Conflicting conclusions seem to be a relatively
92
common feature of those studies that involve the administration of test agent and morphine prior to a standard rodent antinociceptive procedure (see Reinhold et al., 1973). From the literature, there is general agreement that the antinociceptive action of morphine is not significantly enhanced by propranolol. Since Cicero et al. (1974b) have recently claimed that the acute administration of phenoxybenzamine or phentolamine increases the reaction time of rats to morphine in the hot plate test, it would appear that ~-, rather than /3-, adrenoceptors are more closely involved in mediating the antinociceptive effect, certainly in this species and situation. At first sight, data from the present multiple-injection experiments point to the possible involvement of /3-adrenoceptors in the antinociceptive action of morphine (fig. 1). More specifically, mice that received d,l-propranolol (30 mg/kg, s.c.) twice daily for 4 days, required surprisingly large doses of morphine on day 5 during estimation of the group antinociceptive EDs 0 value using the tail flick test. In other words, mice that received eight injections of d,l-propranolol became tolerant to normally effective antinociceptive doses of morphine. The morphine EDs 0 value obtained with the d,l-propranololtreated group was, in fact, approximately three times greater than that from the corresponding saline control group and did not differ significantly from that of the group receiving twice daily doses of morphine (70 mg/kg, s.c.). In the interpretation of these results, it is important to appreciate that the antinociceptive test took place 17--18 hr after the last of the multiple injections and at this time, just prior to estimating morphine EDs 0 values, all mice responded to the hot water b y displaying the tail reflex within the control time of 2 sec. Although cross-tolerance between morphine and non-opioid compounds is well known (e.g. methylamphetamine, Pleuvry, 1971), the cross-tolerance described in the present paper is of a less c o m m o n type as one of the agents (d,l-propranolol), on acute administration, does not possess antinociceptive properties in the test situation used.
A. COWAN, I.R. MACFARLANE
It may also be inferred from fig. 1 that the repeated blockade of/3-adrenoceptor sites with d,l-propranolol, just prior to each of the eight injections of morphine, enhances the onset or development of tolerance to the narcotic since the antinociceptive EDs 0 value obtained from this group was twice that from the corresponding group pretreated with morphine alone. The fact that the morphine EDs 0 value for the (d,l-propranolol + morphine) group was approximately the sum of those obtained with the c o m p o n e n t groups may have been fortuitous, however, the possibility that this morphine EDs 0 value was influenced independently by two unrelated processes cannot be discounted. Pertinent to an analysis of this tolerance effect is the report that propranolol interferes with the binding of morphine to subcellular fractions of mouse brain (DeFeudis and Grosz, 1972) while Brunk et al. (1974) state that the metabolism of morphine is uninfluenced by propranolol, at least in normal human subjects. Future work will be directed towards defining the neurochemical consequences of multiple dosing with d,l-propranolol and hence establishing a link with the vast literature that implicates postulated neurotransmitters with agonist actions of morphine (e.g. Way and Shen, 1971). In this c o n t e x t it is of interest that repeated blockade of dopaminergic receptors with haloperidol -- another drug that is claimed to suppress the severity of withdrawal from heroin in addicts (Karkalas and Lal, 1973) -- likewise enhances tolerance to concurrently injected morphine in mice when the hot plate test is used (Eidelberg and Erspamer, 1975). The effects found with d,l-propranolol in the present study were also noted in similar experiments w i t h t h e d-isomer. Since the latter compound shows membrane stabilisation activity rather than /3-blocking activity (Barrett and Cullum, 1968; Jefferson, 1974), it is possible that/3-adrenoceptors are not primarily involved in mediating these effects. Additional experiments are obviously required with a range of /3-adrenoceptor antagonists to clarify the relationship between the extent of (assumed) cen-
INFLUENCE OF PROPRANOLOL ON MORPHINE EFFECTS
tral blockade and the tolerance/cross-tolerance effects. It might be argued that these effects are indeed centrally mediated since multiple dosing with practolol, a ~-adrenoceptor antagonist which does not readily enter the brain (Scales and Cosgrove, 1970), did not markedly affect the subsequent morphine EDs 0 value on day 5 in relation to saline-injected control mice; furthermore, no tolerance enhancement effect was observed when this compound was injected just prior to morphine. The finding that the concurrent administration of naloxone and morphine reduces tolerance development to the antinociceptive effect of the narcotic is in agreement with the data from similar experiments with mice reported by Shen et al. (1970). The fact that naloxone also appeared to antagonise the cross-tolerance between d,l-propranolol and morphine is interesting in light of the suggestion by Charalampous and Askew (1974) that .the ~-adrenergic receptor and the specific opiate receptor (see review by Goldstein, 1974) may be similar in the mouse brain. If this is so then the competition between naloxone and propranolol for mutual binding sites in the CNS may influence, either directly or indirectly, those factors responsible for the cross-tolerance effect. It has to be admitted, however, that this proposition only relates to the mouse at present since Cicero et al. (1974c) found that propranolol has no affect on the binding of 3H-naloxone to brain homogenates obtained from rats. One attraction of using d,l-propranolol, rather than methadone, in the pharmacotherapy of addiction is the supposed negligible physical dependence potential of this compound (Grosz, 1973). At the animal level, signs of abstinence were not observed after naloxone challenge in a rhesus monkey that had been chronically dosed with d,l-propranolol for 28 days or again after abrupt withdrawal of the drug on day 31 (see section 3.4.). It seems clear from the monkey studies that the pre-administration of d,l-propranolol to morphine-dependent animals had no significant influence on the intensity of a subsequent naloxone-induced abstinence syndrome. This finding is in agree-
93
ment with data from recent studies that have involved the precipitation of jumping or 'wet dog' shakes by naloxone in morphine-dependent mice (Chipkin et al., 1974) and rats (Jhamandas et al., 1973; Cicero et al., 1974a), respectively. The monkey studies have also provided information which suggests that d,l-propranolol does not influence the development of dependence on morphine. Moreover, morphine antagonistic properties could not be ascribed to d,l-propranolol since it neither precipitated signs of abstinence in non-withdrawn morphine-dependent monkeys nor exacerbated the syndrome in 24-hr withdrawn morphinedependent animals. Martin et al. {1974) similarly concluded that propranolol ( 5 mg/kg) neither precipitates abstinence nor influences the intensity of withdrawal in morphine-dependent chronic spinal dogs. These laboratory results are surprising when viewed against the reported narcotic blocking action of propranolol in addicts'(Grosz, 1972) and are perhaps more in keeping with the data of Hollister and Prusmack (1974) and Jacob et al. (1975) whose clinical studies clearly (but unfortunately) indicate pessimism for the future use of propranolol as an adjunct to the treatment of withdrawal from opiates.
Acknowledgements It is a pleasure to thank Diane Beilby for expert technical assistance. Generous gifts of naloxone from Endo and practolol and propranolol from ICI are gratefully acknowledged.
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