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Cross-tolerance studies distinguish morphine- and metkephamid-induced analgesia
Life Sciences, Vol. 31, pp. 1201-1204 Printed in the U.S.A.
Pergamon Press
CROSS-TOLERANCE STUDIES DISTINGUISH MORPHINE- AND METKEPHAMID-INDUCEDANALGESIA Martin D. Hynes and Robert C. A. Frederickson L i l l y Reseach Laboratories Eli L i l l y and Company Indianapolis, IN 46285 (Received in final form June 14, 1982)
Summary In v i t r o data demonstrate that metkephamid (LY127623), an analog of methionine enkephalin, has a high a f f i n i t y for the a opioid receptor, as well as the u-receptor. Data generated u t i l i z i n g two in vivo measures of receptor s e l e c t i v i t y , furthermore, indicate that ~ p e h a m i d ' s analgesic a c t i v i t y is in part mediated by a opioid receptors. The analgesic a c t i v i t y of metkephamid was investigated in the mouse writhing assay following chronic treatment with morphine, the prototypic u agonist. Mice were treated chronically with increasing doses of morphine or saline and the inhibition of writhing measured in response to an acute injection of morphine or metkephamid. The dose response curve for morphine was shifted to the right 3- to 4-fold following chronic administration of morphine. In contrast, no such s h i f t in the dose response curve for metkephamid was observed in these morphine-tolerant mice. In a further series of tests, a 50 mg/kg dose of naloxazone 20 hr prior to the assessment of morphine or metkephamid analgesia in the mouse hot plate test substantially shifted the doseresponse curve for morphine to the right, while leaving the doseresponse curve for metkephamid unchanged. These results suggest that a-receptor activation contributes to the analgesia produced by metkephamid. Since the structural characterization of the natural opioid-receptor ligands, the enkephalins (1), synthetic efforts have been directed towards production of analogs with useful systemic a c t i v i t y . The naturally occurring ligands for the opioid-receptor are too rapidly degraded to have such a c t i v i t y . Metkephamid is a methionine enkephalin analog s u f f i c i e n t l y resistant to enzymatic degradation to provide analgesia in preclinical pain models (2,3,4) and in the clinical situation (5). Metkephamid has a high a f f i n i t y in vitro for the 6-receptor, which is the receptor site that the naturally occurring enkephalin analogs appear to prefer over the morphine or u site. This work was undertaken to further investigate the in vitro s e l e c t i v i t y of metkephamid and extend this observation to in vivo measures of receptor s e l e c t i v i t y . Two measures of in vivo s e l e c t i v i t y were employed in this investigation, one a cross-tolerance study with the u-agonist morphine and the other an antagonism study with the irreversible antagonist naloxazone (6). Methods Opioid Receptor Bindin 9. Freshly prepared rat brain homogenate was incubated for 20 minutes in t r i p l i c a t e with the test compound, 0.25 nM 3H-naloxone (3H-Nx) (50 Ci/mM,
0024-3205/82/121201-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
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Metkephamid-lnduced Analgesia
Vol. 31, No.s 12 & 13, 1982
New England Nuclear) or 0.75 nM 3H-D-Ala2-D-Leu5-enkephalin (3H-DALA) (31Ci/mM, New England Nuclear). Following incubation, the samples were f i l t e r e d under reduced pressure with Whatman glass fiber f i l t e r s (GF/C) and washed with two 5-ml volumes of cold Tris-HCl buffer. Filters were subsequently placed in v i a l s , and, after adding lO ml of PCS (Phase Combining System, Amersham), counted by liquid s c i n t i l l a t i o n spectrophotometry. Production of Morphine Tolerance. To establish morphine tolerance, CF-I mice (Harlan Industries, Cumberland, IN) were treated daily with subcutaneous doses of morphine. On each day the animals received 4 doses of morphine, totaling 32 mg/kg on day l , 64 mg/kg on day 2, 128 mg/kg on day 3 and 248 mg/kg on day 4. On day 5, 16-20 hours after the last morphine injection, the acute analgesic a c t i v i t y of morphine and metkephamid was determined in the mouse writhing assay. The number of writhes was determined for each of 5 mice observed simultaneously, beginning 5 minutes after the injection of a 0.6 percent solution of acetic acid. Percent inhibition was calculated by comparing the number of writhes for a drug-treated group with the mean number of writhes in the control group. Naloxazone Antagonism. Mice were treated with either saline or naloxazone at a 200, lO0 or 50 mg/kg subcutaneous dose 20 hours prior to the acute assessment of the analgesic effects of morphine or metkephamid in the mouse hot plate test. The hot plate test for analgesia was conducted by means of an apparatus with an e l e c t r i c a l l y heated metal plate thermostatically maintained at 55°C (2). The time in seconds from contact with the plate until a hind paw was licked was recorded, as was the time until an escape jump occurred. The cutoff times were 60 seconds for the hind paw lick response and 240 seconds for the jump response. Results Opiate Receptor A f f i n i t y . I t was previously reported that the pA2 value for naloxone versus metkephamid in the mouse vas deferens was similar to that versus met-enkephalin but different from that versus normorphine (Frederickson et a l . , 1981). This was interpreted as evidence that metkephamid u t i l i z e s the a-receptor rather than the ~-receptor and was confirmed by a ratio of 3-4 for the IC50's on guinea pig ileum versus mouse vas deferens, compared to a ratio of 0.I-0.3 for normorphine. The data in Table l summarize IC50 values for metkephamid and several other opioids for inhibiting the binding of 3H-Nx and 3H-DALA in brain homogenates. In these systems, morphine has s e l e c t i v i t y for 3H-Nx (u-receptor) binding site and DADL has s e l e c t i v i t y for the 3H-DALA (a-receptor) s i t e . Metkephamid, to the contrary, shows high a f f i n i t y for both sites. This contrasts with the apparent a - s e l e c t i v i t y seen with the isolated tissue preparations. Metkephamid, however, is about 17x more potent than morphine on the a-receptor, while only 2.6x more potent on the u-receptor. Cross-Tolerance. The morphine and metkephafnid ~ose-response curves for inhibiting the writhing response in mice treated with chronic saline or morphine are given in Figure I . Morphine produced a dose-dependent analgesic effect in mice treated with chronic saline. This dose-response curve was markedly shifted to the right following chronic morphine. In marked contrast, the metkephamid doseresponse curve was not shifted in mice treated with chronic morphine. Only at the 8 and 16 mg/kg doses of metkephamid was there a significant difference between the groups treated witW cNronic morphine or saline.
Vol. 31, No.s 12 & 13, 1982
Metkephamid-lnduced Analgesia
1203
A f f i n i t y of Metkephamid for Opioid Receptors Labeled In Vitro with ~H-Naloxone or 3H-D-AIa2-D-Leu5 Enkephalin I n h i b i t i o n - o f Stereospecif~c Binding ' ?C-50 ValuesI 3H-D-AIa2-D-Leu5 Enkepha] in
3H-Naloxone Compound Morphine Naloxone MetkeDhamid D-Ala2-D-Leu5 Enkephalin I.
6.5 3.0 2.5
75.0
42.1
1.9
12.3 4.4
The concentration (nM) of each compound i n h i b i t i n g the stereospecific binding by 50 percent.
/
,.
~-'~
,,
ii
f
/
;
--m ' ' ~
I ~ I oc..o.,os....+
//~ _fllV
/
Acute Morphine • Chronic Morphine + Acute Morphine I'1 Chron'm Salin e + .. Acute Metkephamid
/ ~
~/4_~" C I ~ / mm
/-
,/
/ •~" ~ ~ " ~ tAcute' Metkephamid ~
d I
0.13
I
I
0.5 2 Dose (mglkg)
l
i
8
32
FIG. l Inhibition of writhing by morphine and metkephamid in morphine-tolerant mice and saline-treated mice. Naloxazone Antagonism. Administration of naloxazone (50 mg/kg s.c.) 20 hours prior to the determination of the morphine dose-response curve in the mouse hot plate assay
1204
Metkephamid-induced
Analgesia
Vol. 31, No.s 12 & 13, 1982
produced a substantial shift to the right in comparison to mice pretreated with saline. The morphine ED50 value was increased by 3- to 4-fold after pretreatment with naloxazone. No such shift in the dose-response curve for metkephamid was seen after pretreatment with naloxazone. Discussion In v i t r o data generated on mouse vas deferens and guinea pig ileum indicated that metkephamid has a degree of s e l e c t i v i t y for the a-receptor, and i t has been proposed that metkephamid produces analgesia by an action on 6-receptors (2). The general belief, however, is that analgesia is mediated via u-receptor activation (6,7,8). Indeed, the present binding studies indicate that metkephamid has high a f f i n i t y for both the u and 6 sites. Metkephamid, however, has considerably higher a f f i n i t y for the a-receptor than does morphine, while their a f f i n i t i e s for the u-receptor are similar. Cross-tolerance studies were initiated to compare the analgesic mechanisms of morphine and metkephamid. These studies demonstrated that the analgesic dose-response curve for metkephamid was not shifted to the right as was the curve for morphine in mice treated chronically with morphine. Only at the higher end of the dose-response curve was there some reduction in the analgesic a c t i v i t y of metkephamid in the morphine-tolerant mice. We interpret this to indicate that a 6-receptor-mediated mechanism contributes to the analgesia produced by metkephamid, although a u mechanism appears to become mnre prominent at the higher doses. This is consistent with the in vitro data showing a f f i n i t y for both receptor types. Indeed, some tolerance-7-n 6-receptors would not be unexpected in animals treated with high doses of morphine since this opiate has some a f f i n i t y for this receptor as well. This emphasizes the importance of careful dose ranging in such experiments to distinguish receptor types since we have as yet no pharmacological tools with absolute s p e c i f i c i t y for the ~- or a-receptor. Further support for a-mediated analgesia by metkephamid was provided by studies with naloxazone, since at the proper dose i t was able to selectively antagonize morphine-induced analgesia. Metkephamid's unique a b i l i t y to produce analgesia by action at the a-receptor may explain the novel c l i n i c a l p r o f i l e reported with this compound (2,5). Acknowledgments. The authors gratefully acknowledge the generous g i f t s of naloxazone from Dr. Gavril W. Pasternak and naloxone from Endo Laboratories. References 1. J. HUGHES, T. W. SMITH, H. W. KOSTERLITZ, L. A. FOTHERGILL, B. A. MORGAN AND H. R. MORRIS, Nature 258 577-579 (1975). 2. R. C. A. FREDERICKSON, E. L. SMITHWICK AND D. P. HENRY, Neuropeptides and Neural Transmission, pp. 227-235, eds. C. Ajmone-Marsan and W. Z. Traczyk, Raven Press, New York (1980). 3. R. C. A. FREDERICKSON, E. L. SMITHWICK, R. SHUMANAND K. G. BEMIS, Science 211 603-605 (1981). 4. R. C. A. FREDERICKSON, C. J. PARLI, G. D. DE VANE, R. R. RUFFOLO, E. L. SMITHWICK AND M. D. HYNES, National Institute on Dru9 Abuse Research Monograph, ed. L. S. Harris (in press) (1982). 5. J. F. CALIMLIM, W. M. WARDELL, K. SRIWATANAKUL, L. LASAGNAAND C. COX, Lancet (in press) (1982). 6. G. W. PASTERNAK, S. R. CHILDERSAND S. H. SNYDER, Science 208 514-516 (1980).
7. G. GACEL, M.-C. ZALUSKI, [. FELLION AND B. P. ROQUES, J. Med. Chem. 24 ll19-l124 (1981). 8. J. L. VAUGHT, R. B. ROTHMANAND T. C. WESTFALL, Life Sci. 30 1443-1455 (1982).
Pergamon Press
CROSS-TOLERANCE STUDIES DISTINGUISH MORPHINE- AND METKEPHAMID-INDUCEDANALGESIA Martin D. Hynes and Robert C. A. Frederickson L i l l y Reseach Laboratories Eli L i l l y and Company Indianapolis, IN 46285 (Received in final form June 14, 1982)
Summary In v i t r o data demonstrate that metkephamid (LY127623), an analog of methionine enkephalin, has a high a f f i n i t y for the a opioid receptor, as well as the u-receptor. Data generated u t i l i z i n g two in vivo measures of receptor s e l e c t i v i t y , furthermore, indicate that ~ p e h a m i d ' s analgesic a c t i v i t y is in part mediated by a opioid receptors. The analgesic a c t i v i t y of metkephamid was investigated in the mouse writhing assay following chronic treatment with morphine, the prototypic u agonist. Mice were treated chronically with increasing doses of morphine or saline and the inhibition of writhing measured in response to an acute injection of morphine or metkephamid. The dose response curve for morphine was shifted to the right 3- to 4-fold following chronic administration of morphine. In contrast, no such s h i f t in the dose response curve for metkephamid was observed in these morphine-tolerant mice. In a further series of tests, a 50 mg/kg dose of naloxazone 20 hr prior to the assessment of morphine or metkephamid analgesia in the mouse hot plate test substantially shifted the doseresponse curve for morphine to the right, while leaving the doseresponse curve for metkephamid unchanged. These results suggest that a-receptor activation contributes to the analgesia produced by metkephamid. Since the structural characterization of the natural opioid-receptor ligands, the enkephalins (1), synthetic efforts have been directed towards production of analogs with useful systemic a c t i v i t y . The naturally occurring ligands for the opioid-receptor are too rapidly degraded to have such a c t i v i t y . Metkephamid is a methionine enkephalin analog s u f f i c i e n t l y resistant to enzymatic degradation to provide analgesia in preclinical pain models (2,3,4) and in the clinical situation (5). Metkephamid has a high a f f i n i t y in vitro for the 6-receptor, which is the receptor site that the naturally occurring enkephalin analogs appear to prefer over the morphine or u site. This work was undertaken to further investigate the in vitro s e l e c t i v i t y of metkephamid and extend this observation to in vivo measures of receptor s e l e c t i v i t y . Two measures of in vivo s e l e c t i v i t y were employed in this investigation, one a cross-tolerance study with the u-agonist morphine and the other an antagonism study with the irreversible antagonist naloxazone (6). Methods Opioid Receptor Bindin 9. Freshly prepared rat brain homogenate was incubated for 20 minutes in t r i p l i c a t e with the test compound, 0.25 nM 3H-naloxone (3H-Nx) (50 Ci/mM,
0024-3205/82/121201-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1202
Metkephamid-lnduced Analgesia
Vol. 31, No.s 12 & 13, 1982
New England Nuclear) or 0.75 nM 3H-D-Ala2-D-Leu5-enkephalin (3H-DALA) (31Ci/mM, New England Nuclear). Following incubation, the samples were f i l t e r e d under reduced pressure with Whatman glass fiber f i l t e r s (GF/C) and washed with two 5-ml volumes of cold Tris-HCl buffer. Filters were subsequently placed in v i a l s , and, after adding lO ml of PCS (Phase Combining System, Amersham), counted by liquid s c i n t i l l a t i o n spectrophotometry. Production of Morphine Tolerance. To establish morphine tolerance, CF-I mice (Harlan Industries, Cumberland, IN) were treated daily with subcutaneous doses of morphine. On each day the animals received 4 doses of morphine, totaling 32 mg/kg on day l , 64 mg/kg on day 2, 128 mg/kg on day 3 and 248 mg/kg on day 4. On day 5, 16-20 hours after the last morphine injection, the acute analgesic a c t i v i t y of morphine and metkephamid was determined in the mouse writhing assay. The number of writhes was determined for each of 5 mice observed simultaneously, beginning 5 minutes after the injection of a 0.6 percent solution of acetic acid. Percent inhibition was calculated by comparing the number of writhes for a drug-treated group with the mean number of writhes in the control group. Naloxazone Antagonism. Mice were treated with either saline or naloxazone at a 200, lO0 or 50 mg/kg subcutaneous dose 20 hours prior to the acute assessment of the analgesic effects of morphine or metkephamid in the mouse hot plate test. The hot plate test for analgesia was conducted by means of an apparatus with an e l e c t r i c a l l y heated metal plate thermostatically maintained at 55°C (2). The time in seconds from contact with the plate until a hind paw was licked was recorded, as was the time until an escape jump occurred. The cutoff times were 60 seconds for the hind paw lick response and 240 seconds for the jump response. Results Opiate Receptor A f f i n i t y . I t was previously reported that the pA2 value for naloxone versus metkephamid in the mouse vas deferens was similar to that versus met-enkephalin but different from that versus normorphine (Frederickson et a l . , 1981). This was interpreted as evidence that metkephamid u t i l i z e s the a-receptor rather than the ~-receptor and was confirmed by a ratio of 3-4 for the IC50's on guinea pig ileum versus mouse vas deferens, compared to a ratio of 0.I-0.3 for normorphine. The data in Table l summarize IC50 values for metkephamid and several other opioids for inhibiting the binding of 3H-Nx and 3H-DALA in brain homogenates. In these systems, morphine has s e l e c t i v i t y for 3H-Nx (u-receptor) binding site and DADL has s e l e c t i v i t y for the 3H-DALA (a-receptor) s i t e . Metkephamid, to the contrary, shows high a f f i n i t y for both sites. This contrasts with the apparent a - s e l e c t i v i t y seen with the isolated tissue preparations. Metkephamid, however, is about 17x more potent than morphine on the a-receptor, while only 2.6x more potent on the u-receptor. Cross-Tolerance. The morphine and metkephafnid ~ose-response curves for inhibiting the writhing response in mice treated with chronic saline or morphine are given in Figure I . Morphine produced a dose-dependent analgesic effect in mice treated with chronic saline. This dose-response curve was markedly shifted to the right following chronic morphine. In marked contrast, the metkephamid doseresponse curve was not shifted in mice treated with chronic morphine. Only at the 8 and 16 mg/kg doses of metkephamid was there a significant difference between the groups treated witW cNronic morphine or saline.
Vol. 31, No.s 12 & 13, 1982
Metkephamid-lnduced Analgesia
1203
A f f i n i t y of Metkephamid for Opioid Receptors Labeled In Vitro with ~H-Naloxone or 3H-D-AIa2-D-Leu5 Enkephalin I n h i b i t i o n - o f Stereospecif~c Binding ' ?C-50 ValuesI 3H-D-AIa2-D-Leu5 Enkepha] in
3H-Naloxone Compound Morphine Naloxone MetkeDhamid D-Ala2-D-Leu5 Enkephalin I.
6.5 3.0 2.5
75.0
42.1
1.9
12.3 4.4
The concentration (nM) of each compound i n h i b i t i n g the stereospecific binding by 50 percent.
/
,.
~-'~
,,
ii
f
/
;
--m ' ' ~
I ~ I oc..o.,os....+
//~ _fllV
/
Acute Morphine • Chronic Morphine + Acute Morphine I'1 Chron'm Salin e + .. Acute Metkephamid
/ ~
~/4_~" C I ~ / mm
/-
,/
/ •~" ~ ~ " ~ tAcute' Metkephamid ~
d I
0.13
I
I
0.5 2 Dose (mglkg)
l
i
8
32
FIG. l Inhibition of writhing by morphine and metkephamid in morphine-tolerant mice and saline-treated mice. Naloxazone Antagonism. Administration of naloxazone (50 mg/kg s.c.) 20 hours prior to the determination of the morphine dose-response curve in the mouse hot plate assay
1204
Metkephamid-induced
Analgesia
Vol. 31, No.s 12 & 13, 1982
produced a substantial shift to the right in comparison to mice pretreated with saline. The morphine ED50 value was increased by 3- to 4-fold after pretreatment with naloxazone. No such shift in the dose-response curve for metkephamid was seen after pretreatment with naloxazone. Discussion In v i t r o data generated on mouse vas deferens and guinea pig ileum indicated that metkephamid has a degree of s e l e c t i v i t y for the a-receptor, and i t has been proposed that metkephamid produces analgesia by an action on 6-receptors (2). The general belief, however, is that analgesia is mediated via u-receptor activation (6,7,8). Indeed, the present binding studies indicate that metkephamid has high a f f i n i t y for both the u and 6 sites. Metkephamid, however, has considerably higher a f f i n i t y for the a-receptor than does morphine, while their a f f i n i t i e s for the u-receptor are similar. Cross-tolerance studies were initiated to compare the analgesic mechanisms of morphine and metkephamid. These studies demonstrated that the analgesic dose-response curve for metkephamid was not shifted to the right as was the curve for morphine in mice treated chronically with morphine. Only at the higher end of the dose-response curve was there some reduction in the analgesic a c t i v i t y of metkephamid in the morphine-tolerant mice. We interpret this to indicate that a 6-receptor-mediated mechanism contributes to the analgesia produced by metkephamid, although a u mechanism appears to become mnre prominent at the higher doses. This is consistent with the in vitro data showing a f f i n i t y for both receptor types. Indeed, some tolerance-7-n 6-receptors would not be unexpected in animals treated with high doses of morphine since this opiate has some a f f i n i t y for this receptor as well. This emphasizes the importance of careful dose ranging in such experiments to distinguish receptor types since we have as yet no pharmacological tools with absolute s p e c i f i c i t y for the ~- or a-receptor. Further support for a-mediated analgesia by metkephamid was provided by studies with naloxazone, since at the proper dose i t was able to selectively antagonize morphine-induced analgesia. Metkephamid's unique a b i l i t y to produce analgesia by action at the a-receptor may explain the novel c l i n i c a l p r o f i l e reported with this compound (2,5). Acknowledgments. The authors gratefully acknowledge the generous g i f t s of naloxazone from Dr. Gavril W. Pasternak and naloxone from Endo Laboratories. References 1. J. HUGHES, T. W. SMITH, H. W. KOSTERLITZ, L. A. FOTHERGILL, B. A. MORGAN AND H. R. MORRIS, Nature 258 577-579 (1975). 2. R. C. A. FREDERICKSON, E. L. SMITHWICK AND D. P. HENRY, Neuropeptides and Neural Transmission, pp. 227-235, eds. C. Ajmone-Marsan and W. Z. Traczyk, Raven Press, New York (1980). 3. R. C. A. FREDERICKSON, E. L. SMITHWICK, R. SHUMANAND K. G. BEMIS, Science 211 603-605 (1981). 4. R. C. A. FREDERICKSON, C. J. PARLI, G. D. DE VANE, R. R. RUFFOLO, E. L. SMITHWICK AND M. D. HYNES, National Institute on Dru9 Abuse Research Monograph, ed. L. S. Harris (in press) (1982). 5. J. F. CALIMLIM, W. M. WARDELL, K. SRIWATANAKUL, L. LASAGNAAND C. COX, Lancet (in press) (1982). 6. G. W. PASTERNAK, S. R. CHILDERSAND S. H. SNYDER, Science 208 514-516 (1980).
7. G. GACEL, M.-C. ZALUSKI, [. FELLION AND B. P. ROQUES, J. Med. Chem. 24 ll19-l124 (1981). 8. J. L. VAUGHT, R. B. ROTHMANAND T. C. WESTFALL, Life Sci. 30 1443-1455 (1982).