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Involvement of monoaminergic and peptidergic components in cathinone-induced analgesia
European Journal of Pharmacology, 114 (1985) 231-234
231
Elsevier Short communication
I N V O L V E M E N T OF M O N O A M I N E R G I C AND P E P T I D E R G I C C O M P O N E N T S IN C A T H I N O N E INDUCED ANALGESIA DAVIDE DELLA BELLA *, ANGELO CARENZI, VIVIANA FRIGENI, ANGELO REGGIANI and ANDREA ZAMBON Zambon Farmaceutici Research Laboratories, Via Lillo del Duca 10, 20091 Bresso-Milan, Italy
Received 23 May 1985, accepted 11 June 1985
D. DELLA BELLA, A. CARENZI, V. FRIGENI, A. REGGIANI and A. ZAMBON, Involvement of monoaminergic andpeptidergic components in cathinone-induced analgesia, European J. Pharmacol. 114 (1985) 231-234. Evidence has been obtained suggesting that cathinone-induced analgesia depends upon stimulation of a-adrenoceptors, followed by release of opioid peptides and by activation of serotonergic pathways. This hypothesis is supported by the following. (1) Cathinone potentiated morphine analgesia and the whole effect was antagonized by naloxone whereas onto the cathinone potentiation was counteracted by phenoxybenzamine. (2) Bestatin potentiated cathinoneinduced analgesia and this effect was sensitive to both naloxone and phenoxybenzamine blockade. (3) The analgesic effect of cathinone + bestatin was further potentiated by the serotonin uptake inhibitor citalopram. Cathinone
Analgesia
a-Receptors
Opioid peptides
1. Introduction
Cathinone is a natural compound, isolated from fresh leaves of Catha-edulis, that has psychostimulating and sympathomimetic actions in a number of animal species including man (Halbach 1980, Johanson and Schuster 1981). According to the current literature, cathinone may exert its pharmacological effects by an amphetamine-like action, namely by stimulating the release of central monoamines (Zelger and Carlini 1981, Kalix 1983). In addition, like amphetamines, cathinone may cause a naloxone-dependent antinociceptive effect in mice and rats, suggesting an influence on opioid transmission (Nencini et al., 1984). However, since cathinone does not interact directly with opiate receptors and does not protect endogenous opioid peptides from degradation, its influence on opioid transmission should be, p r e s u m a b l y , a consequence of the activation of central monoaminergic pathways. In order to investigate the mechanism
* To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Bestatin
of these interactions, we decided to test the effects of cathinone after pharmacological treatments affecting opioid function. In particular, morphine was used to stimulate opiate receptors directly and neuropeptidase inhibitors were used to increase the tone of endogenous opioid peptides. The monoaminergic mechanisms underlying cathinone potentiation of opioid-mediated analgesia were also investigated.
2. Materials and methods
Male Swiss albino mice weighing 20-22 g were used. Analgesia was evaluated by the writhing test. Writhing and stretching was induced by i.p. injection of 0.5% acetic acid a few minutes before the test session. The number of movements during the subsequent 20 min was taken as index of pain sensitivity and the analgesic effect of a treatment was estimated as the inhibition of the number of writhings with respect to that in saline-treated mice (% inhibition). All drugs were dissolved in saline and injected subcutaneously (s.c.) unless
232
otherwise indicated. The time of administration of each drug before a test session is indicated in the legend of each figure. Each value is the mean of writhings of 18 mice. Racemic cathinone hydrochloride was synthesized by Zambon Research Laboratories. Morphine was obtained from Sircai. Naloxone was from Endo. Bestatin was a kind gift of Drs. T. Takeuchi and T. Aoyagy of the Institute of Microbial Chemistry of Tokyo. Other drugs and chemicals were from commercial sources.
3. Results As pointed out by others (Nencini et al. 1984) and as confirmed in our laboratory (not shown), cathinone caused a dose-dependent inhibition of acetic acid-induced writhing in mice (EDs0 10 mg/kg, s.c.). The effect was sensitive to naloxone blockade and was coupled to increased motor activity (not shown) indicating a simultaneous activation of central peptidergic and monoaminergic mechanisms. A sub-analgesic dose of cathinone (2 mg/kg, s.c.) caused a leftward shift of the dose-response curve of morphine (fig. 1). The whole analgesic effect was completely antagonized by naloxone pretreatment whereas cathinone potentiation but not morphine analgesia, was counteracted by pretreatment with phenoxybenzamine. In contrast the dopamine antagonist haloperidol (0.2 mg/kg, s.c.) did not inhibit either cathinone-induced potentiation or morphine analgesia, thus excluding a specific role of dopamine in mediating the effect of cathinone on pain sensitivity (not shown). In fig. 2A the influence of different doses of the aminopeptidase inhibitor bestatin on the pain threshold of cathinone-pretreated mice is shown. A number of neuropeptidase inhibitors were tested, including thiorphan, but only bestatin was effective to enhance cathinone-induced analgesia. It should be stressed that sub-analgesic doses of cathinone were used and that the intracerebral injection of bestatin did not cause analgesia per se. However when the two drugs were injected together a significant analgesic effect was observed. This effect was fully antagonized by either naloxone pretreatment or
the a-blocker phenoxybenzamine. In fig. 2B the effect of the serotonin uptake inhibitor citalopram (Hyttel 1982) on bestatin enhancement of cathinone-induced analgesia is shown. The cathinone dose was reduced to 1 mg/kg, s.c. to minimize the antinociceptive effect of bestatin + cathinone administration. However, as shown in panel B, the concomitant injection of citalopram (20 mg/kg, s.c.) caused a leftward shift in the dose-response curve for bestatin potentiation. In addition, neither citalopram per se nor its concomitant injection with bestatin or cathinone alone was effective to induce a significant antinociceptive effect (not shown).
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morphine ( m g / k g / s . c . i Fig. 1. Influence of cathinone on morphine-induced analgesia in mice (writhing test). Morphine (0.125-0.25-0.5-1.0 mg/kg, s.c.) was given 15 min before acetic acid; cathinone (2 mg/kg, s.c.) was given 30 min before acetic acid; phenoxybenzamine (2 mg/kg, s.c.) and naloxone (4 mg/kg, s.c.) were given 40 min before acetic acid. Morphine (*); cathinone+morphine (O); phenoxybenzamine+cathinone+morphine (©); naloxone+ cathinone + morphine ( * ).
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bestalin (/U,~/i.c. v./mou se) Fig. 2. Effect of bestatin and citalopram on pain threshold of cathinone-treated mice. (A) Bestatin (5-10-25-50/tg, i.c.v./mouse) was given 2 rain before acetic acid; cathinone (2 m g / k g , s.c.) was given 30 rain before acetic acid; phcno×ybenzamine (2 m g / k g s.c.) and naloxone (4 m g / k g , s.c.) were given 40 rain before acetic acid. Cathinone (*); c a t h i n o n e + b e s t a t i n (*); p h e n o x y b e n z a m i n e + cathinone + bestatin (e); naloxone + cathinone + bestatin (O). (B) Bestatin (5-10-25-50/~g, i.c.v./mouse) was given 2 min before acetic acid; cathinone (1 m g / k g , s.c.) was given 30 min before acetic acid; citalopram (20 m g / k g , s,c.) was given 40 rain before acetic acid. Cathinone (*); cathinone+ bestatin (O); c i t a l o p r a m + c a t h i n o n e + b e s t a t i n (e).
4. Discussion
The present study supports and extends the hypothesis that cathinone, like arfiphetamines, may influence the pain threshold by acting primarily on central monoaminergic pathways. Our results allow the suggestion that cathinone stimulates preferentially the a-adrenergic systems, which in turn activates the endogenous opioid peptide system. The last step should be the activation of serotonergic pathways, which presumably convey the inhibitory signal from supraspinal to spinal structures. The evidence favouring the hypothesis was provided mainly by the following observations. (1) Cathinone potentiation, but not morphine analgesia, was blocked by phenoxybenzamine. Thus the cathinone-mediated release of noradrenaline should stimulate a-receptors and morphine should act distal to this site. (2) Ineffective doses of hestatin together with cathinone caused analgesia but the whole effect was blocked by phenoxybenzamine. Since opioid peptide re-
lease depends on stimulatory events and since bestatin cannot exert its influence on opioid transmission in the absence of the peptides, cathinone-induced stimulation of a-adrenoceptors must cause the release of endogenous opioid peptides. (3) Bestatin + cathinone analgesia, but not that due to bestatin alone or cathinone alone, was potentiated by citalopram. Since citalopram acts on released serotonin, this release should take place after activation of the a-adrenoceptor system by cathinone, and potentiation of the endogenous opioid peptide level by bestatin. It must be pointed out that the hypothesized interactions should take place at the central level. Bestatin effects depend upon the route of injection, since its systemic administration at doses up to 50 mg/kg, i.v. did not cause a significant potentiating effect of cathinone-induced analgesia. The nature of the opioid peptides involved in cathinone action is still obscure. Moreover the stimulation by bestatin coupled to the lack of effect of thiorphan makes it unlikely that penta-
234 p e p t i d e e n k e p h a l i n s m i g h t b e i n v o l v e d in m e d i a t i n g c a t h i n o n e - i n d u c e d effects. I n fact, as p o i n t e d o u t b y S c h w a r t z et al. (1981) a n d as c o n f i r m e d b y o u r studies ( C a r e n z i et al. 1983, R e g g i a n i et al. 1984) t h i o r p h a n , b u t n o t b e s t a t i n s h o u l d b e the m o r e effective i n h i b i t o r of n e u r o p e p t i d a s e activity, by protecting endogenous enkephalins from d e g r a d a t i o n b o t h i n vitro a n d i n vivo. References Carenzi, A., V. Frigeni, A. Reggiani and D. Della Bella, 1983, Effect of inhibition of neuropeptidases on the pain threshold of mice and rats, Neuropharmacology 22 (11), 1315. Halbach, H., 1980, Khat - The problem today, in: Problems of Drug Dependence 1979, Proceedings of the 41st Annual Scientific Meeting, The Committee on Problems of Drug Dependence, Inc., ed. L.S. Harris (DHEW Publication No. (ADM) 80-901, National Institute on Drug Abuse Research Monograph Series No. 27) p. 318. Hyttel, J., 1982, Citalopram-pharmacological profile of a specific serotonin uptake inhibitor with antidepressant activity, in: Progress in Neuro-Psychopharmacology and Bio-
logical Psychiatry (Vol. 6) ed. J. Hyttel (Pergamon Press, Oxford,) p. 277. Johanson, C.E. and C.R. Schuster, 1981, A comparison of the behavioral effects of l- and dl-Cathinone and d-Amphetamine, J. Pharmacol. Exp. Ther. 219 (2), 355. Kalix, P., 1983, Effect of the alkaloid (-)cathinone on the release of radioactivity from rabbit atria prelabelled with [3H]norepinephrine, Life Sci. 32, 801. Nencini, P., A.M. Ahmed, M.C. Anania, M. Moscucci and E. Paroli, 1984, Prolonged analgesia induced by cathinone. The role of stress and opioid and nonopioid mechanisms, Pharmacology 29, 269. Reggiani, A., A. Carenzi, V. Frigeni and D. Della Bella, 1984, Effect of bestatin and thiorphan on [Metg]EnkephalinArg6-PheV-induced analgesia, European J. Pharnlacol. 105, 361. Schwartz, J.C., B. Malfroy and S. de la Baume, 1981, Biological inactivation of enkephalins and the role of enkephalin-dipeptidyl-carboxypeptidase ("enkephalinase") as neuropeptidase, Life Sci. 29, 1715. Zelger, J.L. and E.A. Carlini, 1981, Influence of cathinone (a-aminopropiophenone) and cathine (phenylpropanolamine) on circling behaviour and on the uptake and release of [3H]dopamine in striatal slices of rats, Neuropharmacology 20, 839.
231
Elsevier Short communication
I N V O L V E M E N T OF M O N O A M I N E R G I C AND P E P T I D E R G I C C O M P O N E N T S IN C A T H I N O N E INDUCED ANALGESIA DAVIDE DELLA BELLA *, ANGELO CARENZI, VIVIANA FRIGENI, ANGELO REGGIANI and ANDREA ZAMBON Zambon Farmaceutici Research Laboratories, Via Lillo del Duca 10, 20091 Bresso-Milan, Italy
Received 23 May 1985, accepted 11 June 1985
D. DELLA BELLA, A. CARENZI, V. FRIGENI, A. REGGIANI and A. ZAMBON, Involvement of monoaminergic andpeptidergic components in cathinone-induced analgesia, European J. Pharmacol. 114 (1985) 231-234. Evidence has been obtained suggesting that cathinone-induced analgesia depends upon stimulation of a-adrenoceptors, followed by release of opioid peptides and by activation of serotonergic pathways. This hypothesis is supported by the following. (1) Cathinone potentiated morphine analgesia and the whole effect was antagonized by naloxone whereas onto the cathinone potentiation was counteracted by phenoxybenzamine. (2) Bestatin potentiated cathinoneinduced analgesia and this effect was sensitive to both naloxone and phenoxybenzamine blockade. (3) The analgesic effect of cathinone + bestatin was further potentiated by the serotonin uptake inhibitor citalopram. Cathinone
Analgesia
a-Receptors
Opioid peptides
1. Introduction
Cathinone is a natural compound, isolated from fresh leaves of Catha-edulis, that has psychostimulating and sympathomimetic actions in a number of animal species including man (Halbach 1980, Johanson and Schuster 1981). According to the current literature, cathinone may exert its pharmacological effects by an amphetamine-like action, namely by stimulating the release of central monoamines (Zelger and Carlini 1981, Kalix 1983). In addition, like amphetamines, cathinone may cause a naloxone-dependent antinociceptive effect in mice and rats, suggesting an influence on opioid transmission (Nencini et al., 1984). However, since cathinone does not interact directly with opiate receptors and does not protect endogenous opioid peptides from degradation, its influence on opioid transmission should be, p r e s u m a b l y , a consequence of the activation of central monoaminergic pathways. In order to investigate the mechanism
* To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Bestatin
of these interactions, we decided to test the effects of cathinone after pharmacological treatments affecting opioid function. In particular, morphine was used to stimulate opiate receptors directly and neuropeptidase inhibitors were used to increase the tone of endogenous opioid peptides. The monoaminergic mechanisms underlying cathinone potentiation of opioid-mediated analgesia were also investigated.
2. Materials and methods
Male Swiss albino mice weighing 20-22 g were used. Analgesia was evaluated by the writhing test. Writhing and stretching was induced by i.p. injection of 0.5% acetic acid a few minutes before the test session. The number of movements during the subsequent 20 min was taken as index of pain sensitivity and the analgesic effect of a treatment was estimated as the inhibition of the number of writhings with respect to that in saline-treated mice (% inhibition). All drugs were dissolved in saline and injected subcutaneously (s.c.) unless
232
otherwise indicated. The time of administration of each drug before a test session is indicated in the legend of each figure. Each value is the mean of writhings of 18 mice. Racemic cathinone hydrochloride was synthesized by Zambon Research Laboratories. Morphine was obtained from Sircai. Naloxone was from Endo. Bestatin was a kind gift of Drs. T. Takeuchi and T. Aoyagy of the Institute of Microbial Chemistry of Tokyo. Other drugs and chemicals were from commercial sources.
3. Results As pointed out by others (Nencini et al. 1984) and as confirmed in our laboratory (not shown), cathinone caused a dose-dependent inhibition of acetic acid-induced writhing in mice (EDs0 10 mg/kg, s.c.). The effect was sensitive to naloxone blockade and was coupled to increased motor activity (not shown) indicating a simultaneous activation of central peptidergic and monoaminergic mechanisms. A sub-analgesic dose of cathinone (2 mg/kg, s.c.) caused a leftward shift of the dose-response curve of morphine (fig. 1). The whole analgesic effect was completely antagonized by naloxone pretreatment whereas cathinone potentiation but not morphine analgesia, was counteracted by pretreatment with phenoxybenzamine. In contrast the dopamine antagonist haloperidol (0.2 mg/kg, s.c.) did not inhibit either cathinone-induced potentiation or morphine analgesia, thus excluding a specific role of dopamine in mediating the effect of cathinone on pain sensitivity (not shown). In fig. 2A the influence of different doses of the aminopeptidase inhibitor bestatin on the pain threshold of cathinone-pretreated mice is shown. A number of neuropeptidase inhibitors were tested, including thiorphan, but only bestatin was effective to enhance cathinone-induced analgesia. It should be stressed that sub-analgesic doses of cathinone were used and that the intracerebral injection of bestatin did not cause analgesia per se. However when the two drugs were injected together a significant analgesic effect was observed. This effect was fully antagonized by either naloxone pretreatment or
the a-blocker phenoxybenzamine. In fig. 2B the effect of the serotonin uptake inhibitor citalopram (Hyttel 1982) on bestatin enhancement of cathinone-induced analgesia is shown. The cathinone dose was reduced to 1 mg/kg, s.c. to minimize the antinociceptive effect of bestatin + cathinone administration. However, as shown in panel B, the concomitant injection of citalopram (20 mg/kg, s.c.) caused a leftward shift in the dose-response curve for bestatin potentiation. In addition, neither citalopram per se nor its concomitant injection with bestatin or cathinone alone was effective to induce a significant antinociceptive effect (not shown).
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morphine ( m g / k g / s . c . i Fig. 1. Influence of cathinone on morphine-induced analgesia in mice (writhing test). Morphine (0.125-0.25-0.5-1.0 mg/kg, s.c.) was given 15 min before acetic acid; cathinone (2 mg/kg, s.c.) was given 30 min before acetic acid; phenoxybenzamine (2 mg/kg, s.c.) and naloxone (4 mg/kg, s.c.) were given 40 min before acetic acid. Morphine (*); cathinone+morphine (O); phenoxybenzamine+cathinone+morphine (©); naloxone+ cathinone + morphine ( * ).
233 (A)
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bestalin (/U,~/i.c. v./mou se) Fig. 2. Effect of bestatin and citalopram on pain threshold of cathinone-treated mice. (A) Bestatin (5-10-25-50/tg, i.c.v./mouse) was given 2 rain before acetic acid; cathinone (2 m g / k g , s.c.) was given 30 rain before acetic acid; phcno×ybenzamine (2 m g / k g s.c.) and naloxone (4 m g / k g , s.c.) were given 40 rain before acetic acid. Cathinone (*); c a t h i n o n e + b e s t a t i n (*); p h e n o x y b e n z a m i n e + cathinone + bestatin (e); naloxone + cathinone + bestatin (O). (B) Bestatin (5-10-25-50/~g, i.c.v./mouse) was given 2 min before acetic acid; cathinone (1 m g / k g , s.c.) was given 30 min before acetic acid; citalopram (20 m g / k g , s,c.) was given 40 rain before acetic acid. Cathinone (*); cathinone+ bestatin (O); c i t a l o p r a m + c a t h i n o n e + b e s t a t i n (e).
4. Discussion
The present study supports and extends the hypothesis that cathinone, like arfiphetamines, may influence the pain threshold by acting primarily on central monoaminergic pathways. Our results allow the suggestion that cathinone stimulates preferentially the a-adrenergic systems, which in turn activates the endogenous opioid peptide system. The last step should be the activation of serotonergic pathways, which presumably convey the inhibitory signal from supraspinal to spinal structures. The evidence favouring the hypothesis was provided mainly by the following observations. (1) Cathinone potentiation, but not morphine analgesia, was blocked by phenoxybenzamine. Thus the cathinone-mediated release of noradrenaline should stimulate a-receptors and morphine should act distal to this site. (2) Ineffective doses of hestatin together with cathinone caused analgesia but the whole effect was blocked by phenoxybenzamine. Since opioid peptide re-
lease depends on stimulatory events and since bestatin cannot exert its influence on opioid transmission in the absence of the peptides, cathinone-induced stimulation of a-adrenoceptors must cause the release of endogenous opioid peptides. (3) Bestatin + cathinone analgesia, but not that due to bestatin alone or cathinone alone, was potentiated by citalopram. Since citalopram acts on released serotonin, this release should take place after activation of the a-adrenoceptor system by cathinone, and potentiation of the endogenous opioid peptide level by bestatin. It must be pointed out that the hypothesized interactions should take place at the central level. Bestatin effects depend upon the route of injection, since its systemic administration at doses up to 50 mg/kg, i.v. did not cause a significant potentiating effect of cathinone-induced analgesia. The nature of the opioid peptides involved in cathinone action is still obscure. Moreover the stimulation by bestatin coupled to the lack of effect of thiorphan makes it unlikely that penta-
234 p e p t i d e e n k e p h a l i n s m i g h t b e i n v o l v e d in m e d i a t i n g c a t h i n o n e - i n d u c e d effects. I n fact, as p o i n t e d o u t b y S c h w a r t z et al. (1981) a n d as c o n f i r m e d b y o u r studies ( C a r e n z i et al. 1983, R e g g i a n i et al. 1984) t h i o r p h a n , b u t n o t b e s t a t i n s h o u l d b e the m o r e effective i n h i b i t o r of n e u r o p e p t i d a s e activity, by protecting endogenous enkephalins from d e g r a d a t i o n b o t h i n vitro a n d i n vivo. References Carenzi, A., V. Frigeni, A. Reggiani and D. Della Bella, 1983, Effect of inhibition of neuropeptidases on the pain threshold of mice and rats, Neuropharmacology 22 (11), 1315. Halbach, H., 1980, Khat - The problem today, in: Problems of Drug Dependence 1979, Proceedings of the 41st Annual Scientific Meeting, The Committee on Problems of Drug Dependence, Inc., ed. L.S. Harris (DHEW Publication No. (ADM) 80-901, National Institute on Drug Abuse Research Monograph Series No. 27) p. 318. Hyttel, J., 1982, Citalopram-pharmacological profile of a specific serotonin uptake inhibitor with antidepressant activity, in: Progress in Neuro-Psychopharmacology and Bio-
logical Psychiatry (Vol. 6) ed. J. Hyttel (Pergamon Press, Oxford,) p. 277. Johanson, C.E. and C.R. Schuster, 1981, A comparison of the behavioral effects of l- and dl-Cathinone and d-Amphetamine, J. Pharmacol. Exp. Ther. 219 (2), 355. Kalix, P., 1983, Effect of the alkaloid (-)cathinone on the release of radioactivity from rabbit atria prelabelled with [3H]norepinephrine, Life Sci. 32, 801. Nencini, P., A.M. Ahmed, M.C. Anania, M. Moscucci and E. Paroli, 1984, Prolonged analgesia induced by cathinone. The role of stress and opioid and nonopioid mechanisms, Pharmacology 29, 269. Reggiani, A., A. Carenzi, V. Frigeni and D. Della Bella, 1984, Effect of bestatin and thiorphan on [Metg]EnkephalinArg6-PheV-induced analgesia, European J. Pharnlacol. 105, 361. Schwartz, J.C., B. Malfroy and S. de la Baume, 1981, Biological inactivation of enkephalins and the role of enkephalin-dipeptidyl-carboxypeptidase ("enkephalinase") as neuropeptidase, Life Sci. 29, 1715. Zelger, J.L. and E.A. Carlini, 1981, Influence of cathinone (a-aminopropiophenone) and cathine (phenylpropanolamine) on circling behaviour and on the uptake and release of [3H]dopamine in striatal slices of rats, Neuropharmacology 20, 839.