European Journal of Pharmacology, 0
1991
ADONIS
203 (1991) 295-297
295
ElsevierSciencePublishersB.V. All rightsreserved 0014-2999/91/$03.50 0014299991007241
EJP 23931
Short communication
ese
io Rachida Bousselmame, Adina Michael-Titus
Unite’ dc Neuropsychopharmacologie
76800 Saint-Etienne
du Rowray,
E&rimentaie~
France and
’
U.R.A. 1170 du C.N.R.S.,
School of Biological Sciences,
’
and Jean Costentin
FacultG de Mkdecine and Pharmacie
QueenMary
Mile End Road, London EI4NS,
de Rouen,
and Westfield College, UniLlersity of London,
U.K.
Received24 June 1991, revised MS received 8 August 1991 accepted 20 August 1991
Acetorphan, an enkephalinase inhibitor, or morphine was injected in mice which had received saline or morphine (32 mg/kg S.C. twice a day on 8 consecutive days) chronically. In the hot-plate test, the analgesia (increase in jump latency) induced by morphine (2 mg/kg i.p.1 or by the CL selective opioid agonist, [D-Ala’,N-Me-Phe4, Gly5-ollenkephalin (DAGO) (1.5, 3 or 6 rig/moose i.c.v.1, was significant in the saline group but was strongly decreased in morphine-pretreated mice. In contrast the analgesic effect of acetorphan (5 mg/kg i.v.1 or of the S selective opioid agonist [D-Pen’,D-Pen”]enkephalin (DPDPE) (0.75, 1.5 or 3 pg/mouse i.c.v.) was similar in both groups. These results suggest that the enkephalins protected by acetorphan act on the 6 receptor site to produce antinociception. Desensitization; CL-Opioid receptors; &Opioid receptors; Acelorphan; Analgesia
1. Introduction Selective inhibitors of enkephalinase, one of the enzymes which degrade the enkephalins, have been shown to induce analgesia in different r;ociception models. Thus, the results obtained with thiorphan (Costentin et al., 1986) or with its systemically active prodrug, acetorphan (Lecomte et al., 1986), suggest that one of the physiological roles of endogenous enkephalins is to modulate nocicep,tion. Several reports have tried to define the type of opioid receptor at which endogenous enkephalins may exert this modulation. Results obtained with enkephalinase inhibitors injected i.c.v. (Chaillet et al., 1984) suggested that, when this route of administration is used, p-opioid receptors are preferentially activated, leading to analgesia. In addition, data obtained with kelatorphan, a mixed peptidase inhibitor which completely protects enkephalins against inactivation, indicate that these endogenous opioids act at 6 sites to induce spinal antinociception (Dickenson et al., 1988). The present report is focused on the analgesic effect of acetorphan, a parenteral’ly active enkephalinase in-
Correspondence to: 3. Costentin. Unit6 de Neuropsychopharmacoloyie Expbimentale, FacultC de MCdecine et Fharmacie. Avenue de I’UniversitC, BP 97. 76603 Saint-Etiennc du Rouvray, France.
hibitor, and on the type of opioid receptor activated after the systemic administration of this inhibitor. The type of receptor involved in the analgesic effect of acetorphan was analysed using a desensitization model described by Gwynn and Domino (1984). The chronic administration of morphine in mice, according to the protocol used by these authors, induces selective tolerance to /.L agonists. Thus, if the administration of acetorphan leads to activation of p receptors, the effect of the inhibitor would be decreased or abolished in morphine-pretreated mice. If, however, S-opioid receptors are activated the effect of acetorphan would not be affected by the selective desensitization of p receptors. The results presented here support ttc Idea of a selective activation of S-opioid receptors.
2. Materials and methods Male Swiss albino mice (CDl, Charles River, 20-22 gi were used throughout. They were housed 30 in a cage (makrolon boxes) with free access to standard semi-synthetic laboratory diet and tap water, under controlled conditions (temperature 22 + 1°C 7:00 a.m.-7:OO p.m. light dark cycle). Experiments were carried out between 10:00 a.m. and 6:00 p.m. The mice were exposed to the nociceptive test only once. The hot plate test was derived from that of Eddy and Leimbach (1953). A plastic cylinder (height = 20 cm, diameter =
to confine the mouse to the heated surface of the plate. The plate was heated to a temperature of 55 + 0.5”C. using a thermoregulatcd water circulating pump. The latency of the jump was detcrmined for each animal. To avoid injury, animals not responding within 740 s were removed from the hot plate. Acetorphan (Laboratoire Bioprojet, Paris, France) was dissolved in distilled water containing 1% dimethylsulfoxyde and .iSc cremophor EL. Morphine hydrohloride (C.P.F. Melun, France) was dissolved in saline. (DAGO) (Ba-Ala’,N-Me-Phe’,Gly”-ollenkephalin them. Bubendorf. Switzerland) was dissolved in diswater. [D-Pen’,D-Pen’]enkephalin (DPDPE) hem, Bubendorf. Switzerland) was dissolved in HCI 0.5 N then the solution was adjusted to pH 7 with NaOH 0.5 N. Drugs were given in a volume of IO ml/kg. The i.c.v. injections were performed according to the method of Haley and McCormick (1057) in a volume of 10 PI/mouse. The effects of chronic treatment with morphine on analgesia induced by morphine or acetorphan were analysed by means of the Newman-Keuls test (fig. I). The effects of chronic treatment with morphine on
Fig 2. Analgesic effect of DAGO or DPDPE in mice treated semichronically with saline or morphine. Saline (IO ~1 per mouse) (Sal) or DAGO (1.5. 3 or h ng per mouse) or DPDPE (0.75, 1.5 or 3 pg per mouse) were administered i.c.v. in mice treated chronically with either saline (0.20 ml/20 g s.c.) ( Sal chronic) or morphine (32 mg/kg s.c.) (MRP chronic) twice a day for X consecutive days, the last injection heing performed IH h before testing.The test injections were given IO min before the hot plate test. The data were analyzed by linear regression analysis. Upper panel: ‘Sal chronic’ group (r 0.83. P < O.tlOl); ‘MRP chronic’ group (r 0.15. P > 0.05). Bartlett’s test indicates that the regression lines of the two groups are not parallel (F(l.52): 2.07, P < 0.001). Lower panel: ‘Sal chronic’ group (r 0.57, P < 0.001 );‘MRP chronic’ group (r 0.81, P < 0.001). Bartlett’s test indicates that the regression lines do not differ (F(l52): 0.48, P > 0.05). M f S.E.M. of seven mice per group.
analgesia induced by increasing doses of DAGO or DPDPE were analysed by linear regression, followed by Bartlett’s test (fig. 2). Sal
URQ
AC5
Sal
URQ
AC5
uw chmnr Fig. 1. Analgesic effect of morphine or acetorphan in mice treated semi-chronically with saline or morphine. Saline (Sal). or morphine 2 mg/kg i.p. (MRPZ) or acetorphan 5 mg/kg i.v. (AC?) were administered in mice treated semi-chronically with saline (0.20 ml/20 g s.c.1 (sal chronic) or with morphine 32 mg/kg s.c.. twice a day for X consecutive days (MRP chronic), the last injection being performed 18 h hefore testing. Acetorphan S mg/kg (AC51 or morphine 2 mg/kg (MRP2) were injected respectively 5 or IS min hefore the hot plate test. The Newman-Keuls test indicates that there is no significant difference when are compared the Sal chronic/Sal, MRP chronic/Sal and MRP chronic/MRPZ groups: there is no significant difference when are compared the Sal chronic/MRPZ, Sal chronic/AC5 and MRP chronic/ACS groups. identified hy an asterisk (*). Conversely there is a significant difference (P < 0.0.5) hetween these two sets of three groups. M f S.E.M. of I I- IS mice per
3. Results
Sal cnmnlc
group.
The administration of acetljrphan (5 mg/kg i.v.) to saline-pretreated mice induced an increase in jump latency equivalent to that induced by morphine (2 mg/kg i.p.1. In contrast, in mir:e chronically pretreated with morphine (32 mg/kg s.c., twice a day, on 8 consecutive days with the last injection given 18 h before the test), the effect of the enkephalinase inhibitor was unaffected whereas the analgesia induced by morphine was almost completely suppressed (fig. I). Under the same experimental conditions, the p sclcctive opioid agonist, DAGO (1.5, 3 or 6 rig/moose i.c.v.1, or the 6 selective agonist, DPDPE (0.75, I.5 or 3 pg/mousc i.c.v.), induced a significant increase in jump
297
larency in saline-pretreated mice. The effect of DAGO was no longer observed in mice chronically pretreated with morphine, whereas the analgesic effect of DPDPE persisted (fig. 21.
receptcr agonist, DPDPE, and of the enkepha!inase inhibitor, acetorphan. This suggests that endogenous enkephalins, protected from inactivation by acetorphan, act preferentially on the 6 receptor site to induce antinociception.
4. Discussion References The present results showed that the analgesia induced by an enkephalin~se inhibitor administered systemically is unaffected by desensitization of p-opioid receptors. The selective nature of the desensitization was checked by the parallel administration of DAGO and DPDPE, two selective agonists at, respectively, CLand b-opioid receptors (Paterson et ai., 1984). The desensitization method clearly led to tolerance to the effect of CLagonists such as morphine or DAGO but did not modify the analgesia induced by acetorphan or by DPDPE, suggesting that endogenous enkephalins act at 6 sites to modulate nociception in this experimental model. behavioral results obtained with thio~han admdnistered i.c.v. (ChailIet et al., 19841 and recent biochzmical data obtained after the central injection of kelatorphan (Meucci et al., 1989) suggest a preferential tonic occupation of p receptors by endogenous enkephalins. However, it has also been shown that these endogenous peptides may act at S-opioid sites to induce spinal antinociception (Dickenson et al., 19881. These data are not contradictory; enkephalins have been shown to have a higher affinity for the S type of opioid receptors, but their affinity for .p receptors is also compatible with physiolog~cai roles mediated by this subtype of receptors (Paterson et al., 19841. In conclusion, the desensitization of CL-opioid receptors by chronic administration of morphine let unchanged the analgesic effect of the reference S-opioid
Chaiilet, P., A. Coulaud, J.M. Zajac, M.C. FourniC-Zaluski, J. Costentin and B.P. Roques,1984, The p rather thhn the 8 subtype of opioid receptors appears to be involved in enkephalin-induced analgesia, Eur. J. Pharmacol. 101, 83. Costentin, J., A. Vlaiculescu, P. Chaillet, L. Ben Natan, D. Aveaux and J.-C. Schwartz, 1986, Dissociated effects of inhibitors of enkephalin-metabolising peptidases or naloxone on various nocjceptive responses, Eur. J. Pharmacol. 123, 37. Dickenson, A.H., A.F. Sullivan and B.P. Roques, 1988, Evidence that endogenous enkephalins and 6 opioid receptor agonist have a common site of action in spinal antinociception, Eur. J. Pharmacol. 148, 437. Eddy, W.B. and D. Leimbach, 1953, Synthetic analgesics (II): Dithenyfbutenyl and dithenyibutvlamincs, J. Pharmacol. Exp. Ther. 107,385, Gwynn, G.J. and E.F. Domino, f984. Genot~e-dependent behavioral sensitivity to mu vs. kappa opiate agonists. II. Antinociceptive tolerance and physical dependence, J. Pharmacol. Exp. Ther. 231. 312. Haley, T.J. and W.G. McCormick, 1957, Pharmacological effects produced by intra-cerebral injection of drugs in conscious mouse, Et-. J. Pharmaeol. 12, 15. Lecemte, J.M., J. Costentin, A. Vlaiculescu, P. Chaillet. H!, Mar(;aisCollado, C. Llorens-Cartes, M. Leboyer and J.C. Sc!iwartz, 1986. Pharmacological properties of acetorphan, a parr nterally active ‘enkephalinase’ inhibitor, J. Pharmacol. Exp. Ther., 237, 937. Meucci, E., P. Delay-Goyet, B.P. Roques and J.-M. Zajac, 1989, Binding in vivo of selective p and 6 opioid receptor agonists: opioid receptor occupancy by endogenous enkephalins, Eur. J. Pharmacol. 171, 167. Paterson, E-J., L.E. Robson and H.W. Kosterlitz, 1984, Gpioid receptors, in: The Peptides. Opioid Peptides: Biology, Chemistry and Genetics, eds. S. Udenfriend and J. Meienhofer (Academic Press, New York) p. 147.
1991
ADONIS
203 (1991) 295-297
295
ElsevierSciencePublishersB.V. All rightsreserved 0014-2999/91/$03.50 0014299991007241
EJP 23931
Short communication
ese
io Rachida Bousselmame, Adina Michael-Titus
Unite’ dc Neuropsychopharmacologie
76800 Saint-Etienne
du Rowray,
E&rimentaie~
France and
’
U.R.A. 1170 du C.N.R.S.,
School of Biological Sciences,
’
and Jean Costentin
FacultG de Mkdecine and Pharmacie
QueenMary
Mile End Road, London EI4NS,
de Rouen,
and Westfield College, UniLlersity of London,
U.K.
Received24 June 1991, revised MS received 8 August 1991 accepted 20 August 1991
Acetorphan, an enkephalinase inhibitor, or morphine was injected in mice which had received saline or morphine (32 mg/kg S.C. twice a day on 8 consecutive days) chronically. In the hot-plate test, the analgesia (increase in jump latency) induced by morphine (2 mg/kg i.p.1 or by the CL selective opioid agonist, [D-Ala’,N-Me-Phe4, Gly5-ollenkephalin (DAGO) (1.5, 3 or 6 rig/moose i.c.v.1, was significant in the saline group but was strongly decreased in morphine-pretreated mice. In contrast the analgesic effect of acetorphan (5 mg/kg i.v.1 or of the S selective opioid agonist [D-Pen’,D-Pen”]enkephalin (DPDPE) (0.75, 1.5 or 3 pg/mouse i.c.v.) was similar in both groups. These results suggest that the enkephalins protected by acetorphan act on the 6 receptor site to produce antinociception. Desensitization; CL-Opioid receptors; &Opioid receptors; Acelorphan; Analgesia
1. Introduction Selective inhibitors of enkephalinase, one of the enzymes which degrade the enkephalins, have been shown to induce analgesia in different r;ociception models. Thus, the results obtained with thiorphan (Costentin et al., 1986) or with its systemically active prodrug, acetorphan (Lecomte et al., 1986), suggest that one of the physiological roles of endogenous enkephalins is to modulate nocicep,tion. Several reports have tried to define the type of opioid receptor at which endogenous enkephalins may exert this modulation. Results obtained with enkephalinase inhibitors injected i.c.v. (Chaillet et al., 1984) suggested that, when this route of administration is used, p-opioid receptors are preferentially activated, leading to analgesia. In addition, data obtained with kelatorphan, a mixed peptidase inhibitor which completely protects enkephalins against inactivation, indicate that these endogenous opioids act at 6 sites to induce spinal antinociception (Dickenson et al., 1988). The present report is focused on the analgesic effect of acetorphan, a parenteral’ly active enkephalinase in-
Correspondence to: 3. Costentin. Unit6 de Neuropsychopharmacoloyie Expbimentale, FacultC de MCdecine et Fharmacie. Avenue de I’UniversitC, BP 97. 76603 Saint-Etiennc du Rouvray, France.
hibitor, and on the type of opioid receptor activated after the systemic administration of this inhibitor. The type of receptor involved in the analgesic effect of acetorphan was analysed using a desensitization model described by Gwynn and Domino (1984). The chronic administration of morphine in mice, according to the protocol used by these authors, induces selective tolerance to /.L agonists. Thus, if the administration of acetorphan leads to activation of p receptors, the effect of the inhibitor would be decreased or abolished in morphine-pretreated mice. If, however, S-opioid receptors are activated the effect of acetorphan would not be affected by the selective desensitization of p receptors. The results presented here support ttc Idea of a selective activation of S-opioid receptors.
2. Materials and methods Male Swiss albino mice (CDl, Charles River, 20-22 gi were used throughout. They were housed 30 in a cage (makrolon boxes) with free access to standard semi-synthetic laboratory diet and tap water, under controlled conditions (temperature 22 + 1°C 7:00 a.m.-7:OO p.m. light dark cycle). Experiments were carried out between 10:00 a.m. and 6:00 p.m. The mice were exposed to the nociceptive test only once. The hot plate test was derived from that of Eddy and Leimbach (1953). A plastic cylinder (height = 20 cm, diameter =
to confine the mouse to the heated surface of the plate. The plate was heated to a temperature of 55 + 0.5”C. using a thermoregulatcd water circulating pump. The latency of the jump was detcrmined for each animal. To avoid injury, animals not responding within 740 s were removed from the hot plate. Acetorphan (Laboratoire Bioprojet, Paris, France) was dissolved in distilled water containing 1% dimethylsulfoxyde and .iSc cremophor EL. Morphine hydrohloride (C.P.F. Melun, France) was dissolved in saline. (DAGO) (Ba-Ala’,N-Me-Phe’,Gly”-ollenkephalin them. Bubendorf. Switzerland) was dissolved in diswater. [D-Pen’,D-Pen’]enkephalin (DPDPE) hem, Bubendorf. Switzerland) was dissolved in HCI 0.5 N then the solution was adjusted to pH 7 with NaOH 0.5 N. Drugs were given in a volume of IO ml/kg. The i.c.v. injections were performed according to the method of Haley and McCormick (1057) in a volume of 10 PI/mouse. The effects of chronic treatment with morphine on analgesia induced by morphine or acetorphan were analysed by means of the Newman-Keuls test (fig. I). The effects of chronic treatment with morphine on
Fig 2. Analgesic effect of DAGO or DPDPE in mice treated semichronically with saline or morphine. Saline (IO ~1 per mouse) (Sal) or DAGO (1.5. 3 or h ng per mouse) or DPDPE (0.75, 1.5 or 3 pg per mouse) were administered i.c.v. in mice treated chronically with either saline (0.20 ml/20 g s.c.) ( Sal chronic) or morphine (32 mg/kg s.c.) (MRP chronic) twice a day for X consecutive days, the last injection heing performed IH h before testing.The test injections were given IO min before the hot plate test. The data were analyzed by linear regression analysis. Upper panel: ‘Sal chronic’ group (r 0.83. P < O.tlOl); ‘MRP chronic’ group (r 0.15. P > 0.05). Bartlett’s test indicates that the regression lines of the two groups are not parallel (F(l.52): 2.07, P < 0.001). Lower panel: ‘Sal chronic’ group (r 0.57, P < 0.001 );‘MRP chronic’ group (r 0.81, P < 0.001). Bartlett’s test indicates that the regression lines do not differ (F(l52): 0.48, P > 0.05). M f S.E.M. of seven mice per group.
analgesia induced by increasing doses of DAGO or DPDPE were analysed by linear regression, followed by Bartlett’s test (fig. 2). Sal
URQ
AC5
Sal
URQ
AC5
uw chmnr Fig. 1. Analgesic effect of morphine or acetorphan in mice treated semi-chronically with saline or morphine. Saline (Sal). or morphine 2 mg/kg i.p. (MRPZ) or acetorphan 5 mg/kg i.v. (AC?) were administered in mice treated semi-chronically with saline (0.20 ml/20 g s.c.1 (sal chronic) or with morphine 32 mg/kg s.c.. twice a day for X consecutive days (MRP chronic), the last injection being performed 18 h hefore testing. Acetorphan S mg/kg (AC51 or morphine 2 mg/kg (MRP2) were injected respectively 5 or IS min hefore the hot plate test. The Newman-Keuls test indicates that there is no significant difference when are compared the Sal chronic/Sal, MRP chronic/Sal and MRP chronic/MRPZ groups: there is no significant difference when are compared the Sal chronic/MRPZ, Sal chronic/AC5 and MRP chronic/ACS groups. identified hy an asterisk (*). Conversely there is a significant difference (P < 0.0.5) hetween these two sets of three groups. M f S.E.M. of I I- IS mice per
3. Results
Sal cnmnlc
group.
The administration of acetljrphan (5 mg/kg i.v.) to saline-pretreated mice induced an increase in jump latency equivalent to that induced by morphine (2 mg/kg i.p.1. In contrast, in mir:e chronically pretreated with morphine (32 mg/kg s.c., twice a day, on 8 consecutive days with the last injection given 18 h before the test), the effect of the enkephalinase inhibitor was unaffected whereas the analgesia induced by morphine was almost completely suppressed (fig. I). Under the same experimental conditions, the p sclcctive opioid agonist, DAGO (1.5, 3 or 6 rig/moose i.c.v.1, or the 6 selective agonist, DPDPE (0.75, I.5 or 3 pg/mousc i.c.v.), induced a significant increase in jump
297
larency in saline-pretreated mice. The effect of DAGO was no longer observed in mice chronically pretreated with morphine, whereas the analgesic effect of DPDPE persisted (fig. 21.
receptcr agonist, DPDPE, and of the enkepha!inase inhibitor, acetorphan. This suggests that endogenous enkephalins, protected from inactivation by acetorphan, act preferentially on the 6 receptor site to induce antinociception.
4. Discussion References The present results showed that the analgesia induced by an enkephalin~se inhibitor administered systemically is unaffected by desensitization of p-opioid receptors. The selective nature of the desensitization was checked by the parallel administration of DAGO and DPDPE, two selective agonists at, respectively, CLand b-opioid receptors (Paterson et ai., 1984). The desensitization method clearly led to tolerance to the effect of CLagonists such as morphine or DAGO but did not modify the analgesia induced by acetorphan or by DPDPE, suggesting that endogenous enkephalins act at 6 sites to modulate nociception in this experimental model. behavioral results obtained with thio~han admdnistered i.c.v. (ChailIet et al., 19841 and recent biochzmical data obtained after the central injection of kelatorphan (Meucci et al., 1989) suggest a preferential tonic occupation of p receptors by endogenous enkephalins. However, it has also been shown that these endogenous peptides may act at S-opioid sites to induce spinal antinociception (Dickenson et al., 19881. These data are not contradictory; enkephalins have been shown to have a higher affinity for the S type of opioid receptors, but their affinity for .p receptors is also compatible with physiolog~cai roles mediated by this subtype of receptors (Paterson et al., 19841. In conclusion, the desensitization of CL-opioid receptors by chronic administration of morphine let unchanged the analgesic effect of the reference S-opioid
Chaiilet, P., A. Coulaud, J.M. Zajac, M.C. FourniC-Zaluski, J. Costentin and B.P. Roques,1984, The p rather thhn the 8 subtype of opioid receptors appears to be involved in enkephalin-induced analgesia, Eur. J. Pharmacol. 101, 83. Costentin, J., A. Vlaiculescu, P. Chaillet, L. Ben Natan, D. Aveaux and J.-C. Schwartz, 1986, Dissociated effects of inhibitors of enkephalin-metabolising peptidases or naloxone on various nocjceptive responses, Eur. J. Pharmacol. 123, 37. Dickenson, A.H., A.F. Sullivan and B.P. Roques, 1988, Evidence that endogenous enkephalins and 6 opioid receptor agonist have a common site of action in spinal antinociception, Eur. J. Pharmacol. 148, 437. Eddy, W.B. and D. Leimbach, 1953, Synthetic analgesics (II): Dithenyfbutenyl and dithenyibutvlamincs, J. Pharmacol. Exp. Ther. 107,385, Gwynn, G.J. and E.F. Domino, f984. Genot~e-dependent behavioral sensitivity to mu vs. kappa opiate agonists. II. Antinociceptive tolerance and physical dependence, J. Pharmacol. Exp. Ther. 231. 312. Haley, T.J. and W.G. McCormick, 1957, Pharmacological effects produced by intra-cerebral injection of drugs in conscious mouse, Et-. J. Pharmaeol. 12, 15. Lecemte, J.M., J. Costentin, A. Vlaiculescu, P. Chaillet. H!, Mar(;aisCollado, C. Llorens-Cartes, M. Leboyer and J.C. Sc!iwartz, 1986. Pharmacological properties of acetorphan, a parr nterally active ‘enkephalinase’ inhibitor, J. Pharmacol. Exp. Ther., 237, 937. Meucci, E., P. Delay-Goyet, B.P. Roques and J.-M. Zajac, 1989, Binding in vivo of selective p and 6 opioid receptor agonists: opioid receptor occupancy by endogenous enkephalins, Eur. J. Pharmacol. 171, 167. Paterson, E-J., L.E. Robson and H.W. Kosterlitz, 1984, Gpioid receptors, in: The Peptides. Opioid Peptides: Biology, Chemistry and Genetics, eds. S. Udenfriend and J. Meienhofer (Academic Press, New York) p. 147.
