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The effects of receptor selective opioid peptides on morphine-induced analgesia
European Journal of Pharmacology, 80 (1982) 427-430
427
Elsevier Biomedical Press Short communication T H E EFFECTS OF R E C E P T O R SELECTIVE O P I O I D P E P T I D E S O N M O R P H I N E - I N D U C E D ANALGESIA RONALD W. BARRETT 1. and JEFFRY L. VAUGHT 2 1 Department of Pharmacology and Toxicology, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, and 2 Department of Biological Research, McNeil Pharmaceuticals, Spring House, Pennsylvania 19477, U.S.A.
Received 30 March 1982, accepted 13 April 1982
R.W. BARRETT and J.L. VAUGHT, The effects of receptor selective opioid peptides on morphine analgesia, European J. Pharmacol. 80 (1982) 427-430. Utilizing the mouse tail-flick assay, four opioid peptides, which have been reported to be selective for either #- or 8-opioid receptors, were examined for their analgesic potency and for their ability to modify morphine-induced analgesia. [D-Ala2,D-LeuS]enkephalin and [D-Ser2,Thr6]leucine-enkephalin, putative 6-receptor selective peptides, produced a potent analgesic response and at subanalgesic doses potentiated morphine-induced analgesia. Morphiceptin and [D-Ala2,ProS]enkephalinamide, putative /~-receptor selective peptides, were similarly found to produce analgesia. However, in contrast to the 8-receptor selective peptides, three p-receptor selective peptides were unable to alter the potency of morphine. Thus, it would appear that the potentiation of morphine analgesia is a unique property of d-receptor selective peptides. Opioid peptides
Morphine
Interaction
Analgesia
1. Introduction Based on previous work from our laboratory and others (Vaught and Takemori, 1979; Vaught et al., 1981; Lee et al., 1980), it appears that the naturally occurring opioid peptide, leucine-enkephalin, has the unique p r o p e r t y of potentiating the antinociceptive potency of morphine. The m e c h a n i s m by which Leu-enkephalin acts to alter the analgesic response to m o r p h i n e is currently unknown, although indirect evidence suggests that it m a y be an opioid receptor mediated process (Vaught et al., 1981). Increasing evidence has supported the subclassification of opioid receptors into morphine-preferring or #-receptors and enkephalin-preferring or 8-receptors (Chang and Cuatrecasas, 1979). In an a t t e m p t to differentiate the possible role of these two types of receptors in the m o d u l a t o r y activity of leucine-enkephalin on morphine-induced analgesia, we have investigated * To whom all correspondence should be addressed: College of Pharmacy, P.O. Box 789, Busch Campus, Rutgers University, Piscataway, New Jersey 08854, U.S.A. 0014-2999/82/0000-0000/$02.75
© 1982 Elsevier Biomedical Press
the activity of peptides which have been recently reported to be selective for/z- or 8-receptors. We n o w report that subanalgesic doses of [D-Ser 2, Thr6]leucine-enkephalin ( D S T L E ) and [D-Ala 2, D-LeuS]enkephalin ( D A D L E ) , putative 8-receptor selective peptides (Fournie-Zaluski et al., 1981; C h a n g and Cuatrecasas, 1979), potentiate morphine-induced analgesia. In contrast, subanalgesic doses of morphiceptin and [ D - A l a 2, ProS]enkephalinamide (DAPA), putative #-receptor selective peptides (Chang et al., 1981; Audigier et al., 1981), do not significantly alter the antinociceptive potency of morphine.
2. Materials and methods The analgesic potency of m o r p h i n e sulfate (Merck and Co., Rahway, N J), morphiceptin and D A D L E (Peninsula Labs, San Carlos, CA), D S T L E (a generous gift from Dr. B.P. Roques), and D A P A (a generous gift from Dr. Y. Audigier) were determined utilizing the tail-flick method as previously described (Vaught and Takemori, 1979).
428 Male Swiss-Webster mice (18-21 g) (Blue Spruce) were used in all experiments. Drugs (dissolved in saline) were administered intracerebroventricularly (i.c.v.) in a volume of 5/xl. Tail-flick latencies were determined at 10, 15 and 20 min after injection. A reaction time for drug-treated mice greater than 3 standard deviations above the mean of the pre-drug latencies for all mice in the group was the criteria for an analgesic response. To examine the effects of the peptides on morphine-induced analgesia, a subanalgesic dose of peptide (approx. ED.01) was simultaneously administered with morphine and tail-flick latencies determined 15 rain later (Lee et al., 1980). A minimum of 3 doses (10 mice/dose) were used in all cases. EDs0, 95% confidence limits and significance of differences in potency were determined according to the method of Litchfield and Wilcoxon (1949).
3. Results For all experiments, potencies were determined at the time of peak analgesic response. Peak analgesia occurred at 15 rain for morphine and DSTLE and 10 min for morphiceptin, D A D L E and DAPA. As seen in table 1, all of the peptides examined produced a pronounced analgesia in mice. D A D L E and D A P A were found to be significantly more potent than morphine while morphi-
TABLE 2 The effect of/~- and 6-receptor selective peptides on morphineinduced analgesia. Drug
EDso (95% confidencelimits) (nmol/mouse)
Morphine + saline + 1.92 nmolmorphiceptin ~ +0.073 nmol DSTLEa +0.0018 nmol DAPA a +0.012 nmol DADLE a
3.61 (1.56- 8.32) 4.16 (2.27- 7.67) 0.90 (0.34- 2.27) b 3.37 (1.06-10.78) 0.80 (0.49- 1.96)b
a This dose of peptide by itself had no effect on tail-flick latencies. b The EDs0 value was significantly less than that of morphine + saline (P<0.05).
ceptin was significantly less potent than morphine. DSTLE did not differ in potency from morphine. N o correlation between reported receptor specificity and analgesic potency is apparent. On the other hand, when subanalgesic doses of the peptides were coadrninistered with morphine, a difference between the purported 6-receptor selective peptides and /~-receptor selective peptides was observed. D A D L E and DSTLE potentiated morphine analgesia while morphiceptin and DAPA had no significant effect on the potency of morphine (table 2).
4. Discussion
TABLE 1 Analgesic potencies of #- and 8-specificpeptides. Drug
EDs0 (95% confidencelimits) (nmol/mouse)
Morphine Morphiceptin DSTLE DADLE DAPA
3.61 (1.56 - 8.32) 19.2 (10.6 -34.5)a 1.22 (0.56 - 2.65) 0.29 (0.16 - 0.53) b 0.052 (0.023- 0.115) b
a The ED~ovalue was significantlygreater than that obtained with morphine (P<0.05). b The EDso value was significantlyless than that obtained with morphine (P<0.05).
The biochemical documentation of ~- and 6opioid receptors in brain membranes (Chang and Cuatrecasas, 1979) has raised the question of whether one or both of these receptors mediate the analgesic action of opiates. Based on correlations of receptor affinities, bioassay profiles and analgesic potencies of a large number of peptide analogues, Audigier et al. (1980) have speculated that when heat is used as the nociceptive stimulus, the analgesia observed after administration of opioids is mediated by ~t-receptor occupancy. However, the results of the analgesic potency determinations of the opioid peptides used in this study would appear to disagree with this hypothesis. D A D L E and DSTLE, which have been
429 reported to be relatively selective for the 6-receptor in binding and bioassay systems, were found to be at least as potent in producing analgesia as morphine and the putative /~-receptor selective peptide morphiceptin. Discounting the involvement of uncontrollable variables (pharmacokinetic factors; intrinsic activity) which might compromise the extrapolation from in vitro determined receptor affinity to analgesic potency, it appears that the analgesic potency of the peptides used in this study is not clearly reflective of/~- or 6-receptor specificity. Although analgesic potency alone does not distinguish between the /~- and 8-peptides employed in this study,, a clear difference is apparent when a subanalgesic dose of the peptides is coadministered with morphine. Only the purported 8-receptor selective peptides were capable of enhancing the antinociceptive potency of morphine. Although there is no method of determining the receptor subtype that these peptides are acting upon in vivo, it seems likely that at low subanalgesic doses, these peptides act preferentially at the receptor for which they have greatest affinity. Because the potentiation of morphine analgesia appears to a be unique property of 8-specific peptides, we propose that although analgesia may be a direct consequence of #-receptor occupancy (Audigier et al., 1980), the 8-receptor may play an indirect modulatory role in analgesia. In support of this modulatory influence of the 8-receptor on a /~-receptor mediated process is the recent biochemical demonstration of an allosteric interaction between/~- and 6-receptors in rat brain membranes (Rothman and Westfall, 1981). Although the proposition of the 6-receptor playing a modulatory role in analgesia must be viewed as tentative due to the previously discussed limitations of utilizing an in vivo method to distinguish receptor function, 6-receptor enhancement of/~-receptor mediated analgesia may explain the unpredicted analgesic potency of the 8-receptor selective peptides. Since the 8-peptides employed in this study are not devoid of /~-receptor affinity, it is entirely possible that they may attain high enough concentrations to bind to the #-receptor and elicit analgesia in this manner. However, we believe that the lack of correlation between the/~-receptor af-
finity and analgesic potency of D A D L E and DST L E may reside in the fact that at analgesic doses, these peptides occupy both /~- and 6-receptors. Thus, the efficacy of a 8-receptor selective peptide at the /~-receptor may be enhanced by another molecule of the peptide occupying a 8-receptor. A more extensive description of this phenomenon will be presented elsewhere (Vaught et al., unpublished results). In summary, we have demonstrated that the enhancement of the antinociceptive potency of morphine appears to be a unique property of 8-receptor selective peptides. This study provides further support for the hypothesis that the 6-opioid receptor may play a modulatory role in analgesic processes.
Acknowledgement The authors wish to express their appreciation to Drs. Audigier and Roques for their generous gifts of peptides. This work was supported by a PMA Foundation Research Starter Grant. R.W.B. is supported by a PMA Foundation Advanced Predoctoral Fellowship.
References Audigier, Y., H. Marzarguil, R. Gout and J. Cros, 1980. Structure-activity relationships of enkephalin analogs at opiate and enkephalin receptors: Correlation with analgesia, European J. Pharmacol. 63, 35. Audigier, Y., H. Marzarguil, J. Roy, J. Morgat and J. Cros, 1981, Binding and metabolism studies with [3H](DAla2,LeuS)enkephalinamide and [3H](D-AlaZ,ProS)enkephalinamide: Evidence for selective interaction of (DAlaZ,ProS)enkephalinamide with k~-receptors, European J. Pharmacol. 74, 273. Chang, K.J. and P. Cuatrecasas, 1979, Multiple opiate receptors: enkephalins and morphine bind to receptors of different specificity,J, Biol. Chem. 254, 2610. Chang, K.J., A. Killian, F. Hazum, P. Cuatrecasas and J.K. Chang, 1981, Morphiceptin (NH4-Thy-Pro-Phe-ProCONH4): A potent and specific agonist for morphine (#) receptors, Science 212 (3), 75. Fournie-Zaluski, M.C., G. Gacel, B. Maigret, S. Premilat and B.P. Roques, 1981, Structural requirements for specific recognition of ~ and 8 opiate receptors, Mol. Pharmacol. 20, 484. Lee, N.M., F. Leybin, J.K. Chang and H.H. Loh, 1980, Opiate and peptide interaction: Effect of enkephalin on morphine analgesia, European J. Pharmacol. 68, 181.
430 Litchfield, J.T. and F. Wilcoxon, 1949, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther. 96, 99. Rothman, R.B. and T.C. Westfall, 1981, Allosteric modulation by leucine enkephalin of [3 H]naloxone binding in rat brain, European J. Pharmacol. 72, 365. Vaught, J.L. and A.E. Takemori, 1979, Differential effects of
leucine and methionine enkephalin on morphine induced analgesia, acute tolerance and dependence, J. Pharmacol. Exp. Ther. 208, 86. Vaught, J.L., T. Kitano and A.E. Takemori, 1981, Interactions of leucine enkephalin and narcotics with opioid receptors, Mol. Pharmacol. 19, 236.
427
Elsevier Biomedical Press Short communication T H E EFFECTS OF R E C E P T O R SELECTIVE O P I O I D P E P T I D E S O N M O R P H I N E - I N D U C E D ANALGESIA RONALD W. BARRETT 1. and JEFFRY L. VAUGHT 2 1 Department of Pharmacology and Toxicology, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, and 2 Department of Biological Research, McNeil Pharmaceuticals, Spring House, Pennsylvania 19477, U.S.A.
Received 30 March 1982, accepted 13 April 1982
R.W. BARRETT and J.L. VAUGHT, The effects of receptor selective opioid peptides on morphine analgesia, European J. Pharmacol. 80 (1982) 427-430. Utilizing the mouse tail-flick assay, four opioid peptides, which have been reported to be selective for either #- or 8-opioid receptors, were examined for their analgesic potency and for their ability to modify morphine-induced analgesia. [D-Ala2,D-LeuS]enkephalin and [D-Ser2,Thr6]leucine-enkephalin, putative 6-receptor selective peptides, produced a potent analgesic response and at subanalgesic doses potentiated morphine-induced analgesia. Morphiceptin and [D-Ala2,ProS]enkephalinamide, putative /~-receptor selective peptides, were similarly found to produce analgesia. However, in contrast to the 8-receptor selective peptides, three p-receptor selective peptides were unable to alter the potency of morphine. Thus, it would appear that the potentiation of morphine analgesia is a unique property of d-receptor selective peptides. Opioid peptides
Morphine
Interaction
Analgesia
1. Introduction Based on previous work from our laboratory and others (Vaught and Takemori, 1979; Vaught et al., 1981; Lee et al., 1980), it appears that the naturally occurring opioid peptide, leucine-enkephalin, has the unique p r o p e r t y of potentiating the antinociceptive potency of morphine. The m e c h a n i s m by which Leu-enkephalin acts to alter the analgesic response to m o r p h i n e is currently unknown, although indirect evidence suggests that it m a y be an opioid receptor mediated process (Vaught et al., 1981). Increasing evidence has supported the subclassification of opioid receptors into morphine-preferring or #-receptors and enkephalin-preferring or 8-receptors (Chang and Cuatrecasas, 1979). In an a t t e m p t to differentiate the possible role of these two types of receptors in the m o d u l a t o r y activity of leucine-enkephalin on morphine-induced analgesia, we have investigated * To whom all correspondence should be addressed: College of Pharmacy, P.O. Box 789, Busch Campus, Rutgers University, Piscataway, New Jersey 08854, U.S.A. 0014-2999/82/0000-0000/$02.75
© 1982 Elsevier Biomedical Press
the activity of peptides which have been recently reported to be selective for/z- or 8-receptors. We n o w report that subanalgesic doses of [D-Ser 2, Thr6]leucine-enkephalin ( D S T L E ) and [D-Ala 2, D-LeuS]enkephalin ( D A D L E ) , putative 8-receptor selective peptides (Fournie-Zaluski et al., 1981; C h a n g and Cuatrecasas, 1979), potentiate morphine-induced analgesia. In contrast, subanalgesic doses of morphiceptin and [ D - A l a 2, ProS]enkephalinamide (DAPA), putative #-receptor selective peptides (Chang et al., 1981; Audigier et al., 1981), do not significantly alter the antinociceptive potency of morphine.
2. Materials and methods The analgesic potency of m o r p h i n e sulfate (Merck and Co., Rahway, N J), morphiceptin and D A D L E (Peninsula Labs, San Carlos, CA), D S T L E (a generous gift from Dr. B.P. Roques), and D A P A (a generous gift from Dr. Y. Audigier) were determined utilizing the tail-flick method as previously described (Vaught and Takemori, 1979).
428 Male Swiss-Webster mice (18-21 g) (Blue Spruce) were used in all experiments. Drugs (dissolved in saline) were administered intracerebroventricularly (i.c.v.) in a volume of 5/xl. Tail-flick latencies were determined at 10, 15 and 20 min after injection. A reaction time for drug-treated mice greater than 3 standard deviations above the mean of the pre-drug latencies for all mice in the group was the criteria for an analgesic response. To examine the effects of the peptides on morphine-induced analgesia, a subanalgesic dose of peptide (approx. ED.01) was simultaneously administered with morphine and tail-flick latencies determined 15 rain later (Lee et al., 1980). A minimum of 3 doses (10 mice/dose) were used in all cases. EDs0, 95% confidence limits and significance of differences in potency were determined according to the method of Litchfield and Wilcoxon (1949).
3. Results For all experiments, potencies were determined at the time of peak analgesic response. Peak analgesia occurred at 15 rain for morphine and DSTLE and 10 min for morphiceptin, D A D L E and DAPA. As seen in table 1, all of the peptides examined produced a pronounced analgesia in mice. D A D L E and D A P A were found to be significantly more potent than morphine while morphi-
TABLE 2 The effect of/~- and 6-receptor selective peptides on morphineinduced analgesia. Drug
EDso (95% confidencelimits) (nmol/mouse)
Morphine + saline + 1.92 nmolmorphiceptin ~ +0.073 nmol DSTLEa +0.0018 nmol DAPA a +0.012 nmol DADLE a
3.61 (1.56- 8.32) 4.16 (2.27- 7.67) 0.90 (0.34- 2.27) b 3.37 (1.06-10.78) 0.80 (0.49- 1.96)b
a This dose of peptide by itself had no effect on tail-flick latencies. b The EDs0 value was significantly less than that of morphine + saline (P<0.05).
ceptin was significantly less potent than morphine. DSTLE did not differ in potency from morphine. N o correlation between reported receptor specificity and analgesic potency is apparent. On the other hand, when subanalgesic doses of the peptides were coadrninistered with morphine, a difference between the purported 6-receptor selective peptides and /~-receptor selective peptides was observed. D A D L E and DSTLE potentiated morphine analgesia while morphiceptin and DAPA had no significant effect on the potency of morphine (table 2).
4. Discussion
TABLE 1 Analgesic potencies of #- and 8-specificpeptides. Drug
EDs0 (95% confidencelimits) (nmol/mouse)
Morphine Morphiceptin DSTLE DADLE DAPA
3.61 (1.56 - 8.32) 19.2 (10.6 -34.5)a 1.22 (0.56 - 2.65) 0.29 (0.16 - 0.53) b 0.052 (0.023- 0.115) b
a The ED~ovalue was significantlygreater than that obtained with morphine (P<0.05). b The EDso value was significantlyless than that obtained with morphine (P<0.05).
The biochemical documentation of ~- and 6opioid receptors in brain membranes (Chang and Cuatrecasas, 1979) has raised the question of whether one or both of these receptors mediate the analgesic action of opiates. Based on correlations of receptor affinities, bioassay profiles and analgesic potencies of a large number of peptide analogues, Audigier et al. (1980) have speculated that when heat is used as the nociceptive stimulus, the analgesia observed after administration of opioids is mediated by ~t-receptor occupancy. However, the results of the analgesic potency determinations of the opioid peptides used in this study would appear to disagree with this hypothesis. D A D L E and DSTLE, which have been
429 reported to be relatively selective for the 6-receptor in binding and bioassay systems, were found to be at least as potent in producing analgesia as morphine and the putative /~-receptor selective peptide morphiceptin. Discounting the involvement of uncontrollable variables (pharmacokinetic factors; intrinsic activity) which might compromise the extrapolation from in vitro determined receptor affinity to analgesic potency, it appears that the analgesic potency of the peptides used in this study is not clearly reflective of/~- or 6-receptor specificity. Although analgesic potency alone does not distinguish between the /~- and 8-peptides employed in this study,, a clear difference is apparent when a subanalgesic dose of the peptides is coadministered with morphine. Only the purported 8-receptor selective peptides were capable of enhancing the antinociceptive potency of morphine. Although there is no method of determining the receptor subtype that these peptides are acting upon in vivo, it seems likely that at low subanalgesic doses, these peptides act preferentially at the receptor for which they have greatest affinity. Because the potentiation of morphine analgesia appears to a be unique property of 8-specific peptides, we propose that although analgesia may be a direct consequence of #-receptor occupancy (Audigier et al., 1980), the 8-receptor may play an indirect modulatory role in analgesia. In support of this modulatory influence of the 8-receptor on a /~-receptor mediated process is the recent biochemical demonstration of an allosteric interaction between/~- and 6-receptors in rat brain membranes (Rothman and Westfall, 1981). Although the proposition of the 6-receptor playing a modulatory role in analgesia must be viewed as tentative due to the previously discussed limitations of utilizing an in vivo method to distinguish receptor function, 6-receptor enhancement of/~-receptor mediated analgesia may explain the unpredicted analgesic potency of the 8-receptor selective peptides. Since the 8-peptides employed in this study are not devoid of /~-receptor affinity, it is entirely possible that they may attain high enough concentrations to bind to the #-receptor and elicit analgesia in this manner. However, we believe that the lack of correlation between the/~-receptor af-
finity and analgesic potency of D A D L E and DST L E may reside in the fact that at analgesic doses, these peptides occupy both /~- and 6-receptors. Thus, the efficacy of a 8-receptor selective peptide at the /~-receptor may be enhanced by another molecule of the peptide occupying a 8-receptor. A more extensive description of this phenomenon will be presented elsewhere (Vaught et al., unpublished results). In summary, we have demonstrated that the enhancement of the antinociceptive potency of morphine appears to be a unique property of 8-receptor selective peptides. This study provides further support for the hypothesis that the 6-opioid receptor may play a modulatory role in analgesic processes.
Acknowledgement The authors wish to express their appreciation to Drs. Audigier and Roques for their generous gifts of peptides. This work was supported by a PMA Foundation Research Starter Grant. R.W.B. is supported by a PMA Foundation Advanced Predoctoral Fellowship.
References Audigier, Y., H. Marzarguil, R. Gout and J. Cros, 1980. Structure-activity relationships of enkephalin analogs at opiate and enkephalin receptors: Correlation with analgesia, European J. Pharmacol. 63, 35. Audigier, Y., H. Marzarguil, J. Roy, J. Morgat and J. Cros, 1981, Binding and metabolism studies with [3H](DAla2,LeuS)enkephalinamide and [3H](D-AlaZ,ProS)enkephalinamide: Evidence for selective interaction of (DAlaZ,ProS)enkephalinamide with k~-receptors, European J. Pharmacol. 74, 273. Chang, K.J. and P. Cuatrecasas, 1979, Multiple opiate receptors: enkephalins and morphine bind to receptors of different specificity,J, Biol. Chem. 254, 2610. Chang, K.J., A. Killian, F. Hazum, P. Cuatrecasas and J.K. Chang, 1981, Morphiceptin (NH4-Thy-Pro-Phe-ProCONH4): A potent and specific agonist for morphine (#) receptors, Science 212 (3), 75. Fournie-Zaluski, M.C., G. Gacel, B. Maigret, S. Premilat and B.P. Roques, 1981, Structural requirements for specific recognition of ~ and 8 opiate receptors, Mol. Pharmacol. 20, 484. Lee, N.M., F. Leybin, J.K. Chang and H.H. Loh, 1980, Opiate and peptide interaction: Effect of enkephalin on morphine analgesia, European J. Pharmacol. 68, 181.
430 Litchfield, J.T. and F. Wilcoxon, 1949, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther. 96, 99. Rothman, R.B. and T.C. Westfall, 1981, Allosteric modulation by leucine enkephalin of [3 H]naloxone binding in rat brain, European J. Pharmacol. 72, 365. Vaught, J.L. and A.E. Takemori, 1979, Differential effects of
leucine and methionine enkephalin on morphine induced analgesia, acute tolerance and dependence, J. Pharmacol. Exp. Ther. 208, 86. Vaught, J.L., T. Kitano and A.E. Takemori, 1981, Interactions of leucine enkephalin and narcotics with opioid receptors, Mol. Pharmacol. 19, 236.