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Long-acting agonist and antagonist activities of naltrexamine bivalent ligands in mice
European Journal of Pharmacology, 186 (1990) 285-288 Elsevier
285
EJP 51479
Long-acting agonist and antagonist activities of naltrexamine bivalent ligands in mice 1 A.E. Takemori, C.B. Yim
2, D.L.
Larson 2 and P.S. Portoghese 2
Departments of Pharmacology, Medical School and : Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, U.S.A.
Received 3 May 1990, accepted 26 June 1990
A series of naltrexamine bivalent ligands, compounds with two naltrexamine pharmacophores separated by a spacer which contains a variable number of glycyl units flanking a succinyl group, were synthesized and evaluated in vivo in mice. These compounds possessed long-acting agonist and especially antagonist activities. The bivalent ligands, 2 and 3 displayed antinociceptive activity that lasted > 4 h. Compound 1, a bivalent ligand and 4, the monomer, antagonized the antinociceptive effect of morphine for a week after a single injection i.c.v. The long duration of action may be due to entrapment of these ligands in the central nervous system. These compounds may give future insights into the design of long-acting agonists and antagonists. Naltrexamine bivalent ligands; ~t-Opioid receptor antagonists; Antinociception; Analgesia; (Long acting)
1. Introduction Bivalent ligands, c o m p o u n d s that contain two recognition sites joined through a connecting spacer, have attracted considerable interest as molecular probes because some of them display considerable selectivity for a single opioid receptor type. For example, the triethylenedioxy derivative of fl-naltrexamine ( T E N A ) (Portoghese and Takemori, 1985), binaltorphimine (BNI) and norB N I (Portoghese et al., 1987) are bivalent ligands selective for K opioid receptors. Previously, a series of bivalent ligands with fl-naltrexamine as the
1 This investigation was supported by USPHS grants from the National Institute on Drug Abuse. Studies in this report were carried out in accordance with the Declaration of Helsinki and/or with the guide for the care and use of laboratory animals as adopted and promulgated by the National Institutes of Health. Correspondence to: A.E. Takemori, Department of Pharmacology, 3-249 Millard Hall, University of Minnesota, 435 Delaware St. S.E., Minneapolis, MN 55455 U.S.A.
p h a r m a c o p h o r e and spacers consisting of a central succinyl group with various glycyl units on either side of this group, were examined for their agonist and antagonist properties in smooth muscle preparations (Portoghese et al., 1986). It has been shown in the guinea pig ileal longitudinal muscle preparation (GPI) that the antagonist activity of this series of c o m p o u n d s at opioid receptors is the highest when there are two glycyl groups present on either end of the succinyl group. In this study, we have examined several of these naltrexamine bivalent ligands (fig. 1) in analgesic assays to see whether or not the same antagonist profile exists in vivo.
2. Materials and methods 2.1. Antinociceptive assay
The acetic acid a b d o m i n a l stretching or writhing assay was used (Hayashi and Takemori, 1971). Male Swiss-Webster mice (Bio-lab, White Bear Lake, M N ) weighing between 20 and 25 g were
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
286
2.2. Drugs"
I HO~NH(COCH COMPOLIND 1 2 3
2NH)nCOCH 12 n 0 2
Naltrexamine bivalent ligands were synthesized as described previously (Portoghese et al., 1986) and the structures are depicted in fig. 1. Three of the four compounds studied had two fl-naltrexamine-derived pharmacophores separated by a spacer containing 0 (1), 2 (2) or 4 (3) glycyl units on either side of the succinyl group. The fourth compound (4) studied was a the corresponding monomer that contained an acetylglycylglycyl side chain.
4
3. Results
3.1 Agonist activity of naltrexamine bivalent ligands
O o ~ H
N H(COCH2NH)2COCHa
4 (monomer) Fig. 1. Chemical structures of naltrexamine bivalent ligands.
injected i.p. with 10 m l / k g of 0.6% acetic acid to induce writhing behavior which was characterized by a stretching of the hind limbs accompanied by a constriction of the abdominal muscles. The mice were placed singly into transparent cylindrical containers and the total number of writhes were counted for a 6 min period beginning 5 min after the acetic acid injection. Antinociceptive activity of the drug was expressed as the percentage of inhibition of the number of writhes in a drug administered group compared to the mean number of writhes in a control group. Administration of drugs, either i.c.v. (intracerebroventricular, 5 #1) or s.c. (10 ml/kg), was timed so that the peak effect occurred in the center of the observation period. Minimum of 10 mice were used for each group and three to four doses of drugs were used to generate dose response curves. EDs0 values and their 95% confidence limits were determined by the parallel line assay of Finney (1964).
Compounds 2, 3 and 4 displayed modest antinociceptive activity when administered i.c.v. (table 1) with the monomer (4) having the highest activity. In comparison, morphine administered i.c.v. had an EDs0 of 0.1 (0.03 - 0.4) n m o l / m o u s e which was 16 times more potent than compound 4. Also the antinociceptive effect of these compounds was long lasting with the activities of 2 and 3 still detectable 4 h after administration (table 2). The compound with the shortest spacer (no glycyl units), 1, did not possess antinociceptive activity at the highest dose employed.
3.2. Antagonist activity of naltrexamine bivalent ligands The compounds were administered i.c.v, and when their agonist activity subsided, their antagonist activity against morphine antinocicep-
TABLE 1 Antinociceptive activity of naltrexamine bivalent ligands. Cornpound
EDs0 (nmol i.c.v.)
!
>10
2 3 4
3.87 (3.41-4.40) 5.07 (2.98-11.69) 1.60 (1.05-2.23)
Rel. potency (rain)
Peak effect
0.4 0.3 1.0
120 60 20
287 TABLE 2 Antagonist activity of naltrexamine bivalent ligands. Time after 2 nmol i.c.v.
Morphine EDso (95% confidence limits) mg/kg s.c. 1
2
3
4
None 4h 24 h 3 days 5 days 7 days
0.9 (0.5-1.8) 14.2 (9.7-22.2) 13.3 (9.0-21.4) 5.2 (3.5-8.0) 3.0 (2.0-4.7) 1.4 (1.0-2.2)
b 5.3 (2.9-11.9) 2.2 (1.1-4.5) 1.8 (1.0-4.9)
b
14.8 (10.3-20.6) 12.9 (9.3-18.5) 3.6 (2.5-5.0) 2,1 (1.5-2.9) 1.1 (0.8-1.51
a
-1.6
a Acetic acid writhing assay, b Antinociceptlve activity still detectable.
tion was assessed (table 2). The compound with the shortest spacer, 1, and the monomer, 4, displayed the highest antagonist activity with both compounds raising the EDs0 of morphine by 16fold 4 h after administration of the ligands. The antagonist activity of these two ligands persisted for a week. Compound 2 had about half the antagonist activity as those of 1 and 4 one day after administration of the ligands. Antagonist activity could not be detected with compound 3. Compounds 1 and 4 were investigated further by using the s.c. route of administration. Compound 1 at sublethal doses ( < 50 m g / k g ) , displayed very weak antagonism of morphine antinociception (5-10% inhibition of the effect of ED90 doses of morphine). On the other hand, the monomer, 1 at a dose of 10 m g / k g , raised the EDs0 of morphine 8-fold 4 h after its administration (table 3). The antagonist activity lasted between 3 to 5 days. Compound 1 also possessed antinociceptive activity when administered s.c. that lasted for about 2-3 h and the compound appeared to be a partial agonist by this route of administration.
TABLE 3 Antagonist activity of compound 4 administered s.c. Time after 10 mg/kg of 4 s.c. 0h 4h 18 h 24 h 3 days 5 days
Morphine EDs0 (95% c.1.) mg/kg s.c. 1.0 (0.6-1.6) 8.2 (3.3-21.1) 4.1 (2.7-6.3) 4.1 (3.6-4.5) 1.6 (1.5-1.7) 1.1 (0.9-1.4)
The m a x i m u m inhibition of writhes was about 50% with increasing doses of 1 from 5 to 80 mg/kg.
4. D i s c u s s i o n
The fact that some of the naltrexamine bivalent ligands possessed antinociceptive activity was not surprising because a substituent attached on the 6-amino group confers agonism to the molecule in a number of naltrexamine derivatives, e.g. /3funaltrexamine. This agonism is probably mediated by x opioid receptors because its activity was detected only by the writhing assay, one of the few methods with which activity at x receptors can be detected (Tyers, 1980; Ward and Takemori, 1983). Also, when antagonists of /t opioid receptors possess agonist activity, they usually interact at K opioid receptors to mediate agonism, e.g. nalorphine, levallorphan, /3-funaltrexamine. The antagonist activities of the naltrexamine bivalent ligands in vivo did not parallel those in the GPI. Whereas in the G P I the antagonist activity peaked when there were two glycyl units on either side of the succinyl group (compound 2), in vivo, the greatest antagonist activity resided in the compound with the shortest spacer (no glycyl units, 1) and the m o n o m e r (4) and 2 displayed much less activity. These findings were surprising because in the GPI, the compound with the shortest spacer is more potent in antagonizing x receptor agonists and less potent in antagonizing # receptor agonists (Portoghese et al., 1986). Data from opioid receptor binding assays with these series of compounds
288 does not shed light on the discrepancies between the d a t a in vitro a n d in vivo b e c a u s e the b i n d i n g affinities of all these c o m p o u n d s for o p i o i d receptors are similar ( P o r t o g h e s e et al., 1986). T h e lack of c o r r e l a t i o n b e t w e e n the a n t a g o n i s m of m o r p h i n e in the G P I a n d in vivo m a y merely m e a n that t h e / , r e c e p t o r system in the G P I m a y be f u n c t i o n a l l y different from that in the central nervous system. Alternatively, there m a y be a f u n d a m e n t a l difference b e t w e e n kt agonist a n d a n t a g o n i s t r e c o g n i t i o n sites because agonist b i n d ing and activity in vivo usually correlate well. Evidence for the latter p o s s i b i l i t y has been rep o r t e d in the bt o p i o i d r e c e p t o r system in the G P I (Portoghese a n d T a k e m o r i , 1983). In a d d i t i o n , since an agonist c o m p o n e n t is s u p e r i m p o s e d u p o n an a n t a g o n i s t effect, the net a n t a g o n i s m might be expected to be different from that o b s e r v e d in s m o o t h muscle p r e p a r a t i o n s . This w o u l d also explain the slower onset of a n t a g o n i s m of 2, i.e. the agonist activity c o m p l e t e l y m a s k s the a n t a g o n i s m p r i o r to 24 h. T h e lack of a n t a g o n i s m of 3 at 24 h might also be e x p l a i n e d on this basis in that there is significant sustained agonism. T h e u n e x p e c t e d long d u r a t i o n of action of the agonist a n d p a r t i c u l a r l y the a n t a g o n i s t activity of these n a l t r e x a m i n e derivatives is puzzling. T h e c o m p o u n d s c o n t a i n n o d i s c e r n i b l e s u b s t i t u e n t that w o u l d lead to a n o n - e q u i l i b r i u m a s s o c i a t i o n so that this p o s s i b i l i t y is unlikely. T h e d a t a o b t a i n e d from s.c. a d m i n i s t r a t i o n of c o m p o u n d s 1 a n d 4 m a y suggest that n a l t r e x a m i n e b i v a l e n t l i g a n d s m a y be too b u l k y to be t r a n s p o r t e d easily into the central nervous system. O n the o t h e r hand, once the c o m p o u n d s enter the central nervous system, it m a y b e c o m e very difficult to clear them from the tissues. A l t h o u g h the long d u r a t i o n of action is
u n e x p l a i n e d , the effect is p r o b a b l y due to some sequestration phenomenon, perhaps entrapment by the cells in the central nervous system followed b y slow release. R e g a r d l e s s of the m e c h a n i s m for the long-lasting effect, these c o m p o u n d s m a y give future insights into the design of long-acting agonists a n d a n t a g o n i s t s .
Acknowledgements We thank the capable technical assistance of Joan Naeseth and Mary Schwartz.
References Finney, D.J., 1964, Statistical method in biologic assay, 2nd edn., Hafner Publishing Co., New York. Hayashi, G. and A.E. Takemori, 1971, The type of analgesicreceptor interaction involved in certain analgesic assays, European J. Pharmacol. 16, 63. Portoghese, P.S., D.L. Larson, L.M. Sayre, C.B. Yim, G. Ronsisvalle, S.W. Tam and A.E. Takemori, 1986, Opioid agonist and antagonist bivalent ligands. The relationship between spacer length and selectivity at multiple opioid receptors, J. Med. Chem. 29, 1855. Portoghese, P.S., A.W. Lipkowski and A.E. Takemori, 1987, Binaltorphimine and norbinaltorphimine, potent and selective • opioid receptor antagonists, Life Sci. 40, 1287. Portoghese, P.S. and A.E. Takemori, 1983, Different receptor sites mediate opioid agonism and antagonism, J. Med. Chem. 26, 134l. Portoghese, P.S. and A.E. Takemori, 1985, TENA, a selective kappa opioid receptor antagonist, Life Sci. 36, 801. Tyers, M.B., 1980, A classification of opiate receptors that mediate antinociception in animals, Br. J. Pharmacol. 69, 503. Ward, S.J. and A.E. Takemori, 1983, Relative involvement of mu, kappa and delta receptor mechanisms in opiate-mediated antinociception in mice, J. Pharmacol. Exp. Ther. 224. 525.
285
EJP 51479
Long-acting agonist and antagonist activities of naltrexamine bivalent ligands in mice 1 A.E. Takemori, C.B. Yim
2, D.L.
Larson 2 and P.S. Portoghese 2
Departments of Pharmacology, Medical School and : Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, U.S.A.
Received 3 May 1990, accepted 26 June 1990
A series of naltrexamine bivalent ligands, compounds with two naltrexamine pharmacophores separated by a spacer which contains a variable number of glycyl units flanking a succinyl group, were synthesized and evaluated in vivo in mice. These compounds possessed long-acting agonist and especially antagonist activities. The bivalent ligands, 2 and 3 displayed antinociceptive activity that lasted > 4 h. Compound 1, a bivalent ligand and 4, the monomer, antagonized the antinociceptive effect of morphine for a week after a single injection i.c.v. The long duration of action may be due to entrapment of these ligands in the central nervous system. These compounds may give future insights into the design of long-acting agonists and antagonists. Naltrexamine bivalent ligands; ~t-Opioid receptor antagonists; Antinociception; Analgesia; (Long acting)
1. Introduction Bivalent ligands, c o m p o u n d s that contain two recognition sites joined through a connecting spacer, have attracted considerable interest as molecular probes because some of them display considerable selectivity for a single opioid receptor type. For example, the triethylenedioxy derivative of fl-naltrexamine ( T E N A ) (Portoghese and Takemori, 1985), binaltorphimine (BNI) and norB N I (Portoghese et al., 1987) are bivalent ligands selective for K opioid receptors. Previously, a series of bivalent ligands with fl-naltrexamine as the
1 This investigation was supported by USPHS grants from the National Institute on Drug Abuse. Studies in this report were carried out in accordance with the Declaration of Helsinki and/or with the guide for the care and use of laboratory animals as adopted and promulgated by the National Institutes of Health. Correspondence to: A.E. Takemori, Department of Pharmacology, 3-249 Millard Hall, University of Minnesota, 435 Delaware St. S.E., Minneapolis, MN 55455 U.S.A.
p h a r m a c o p h o r e and spacers consisting of a central succinyl group with various glycyl units on either side of this group, were examined for their agonist and antagonist properties in smooth muscle preparations (Portoghese et al., 1986). It has been shown in the guinea pig ileal longitudinal muscle preparation (GPI) that the antagonist activity of this series of c o m p o u n d s at opioid receptors is the highest when there are two glycyl groups present on either end of the succinyl group. In this study, we have examined several of these naltrexamine bivalent ligands (fig. 1) in analgesic assays to see whether or not the same antagonist profile exists in vivo.
2. Materials and methods 2.1. Antinociceptive assay
The acetic acid a b d o m i n a l stretching or writhing assay was used (Hayashi and Takemori, 1971). Male Swiss-Webster mice (Bio-lab, White Bear Lake, M N ) weighing between 20 and 25 g were
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
286
2.2. Drugs"
I HO~NH(COCH COMPOLIND 1 2 3
2NH)nCOCH 12 n 0 2
Naltrexamine bivalent ligands were synthesized as described previously (Portoghese et al., 1986) and the structures are depicted in fig. 1. Three of the four compounds studied had two fl-naltrexamine-derived pharmacophores separated by a spacer containing 0 (1), 2 (2) or 4 (3) glycyl units on either side of the succinyl group. The fourth compound (4) studied was a the corresponding monomer that contained an acetylglycylglycyl side chain.
4
3. Results
3.1 Agonist activity of naltrexamine bivalent ligands
O o ~ H
N H(COCH2NH)2COCHa
4 (monomer) Fig. 1. Chemical structures of naltrexamine bivalent ligands.
injected i.p. with 10 m l / k g of 0.6% acetic acid to induce writhing behavior which was characterized by a stretching of the hind limbs accompanied by a constriction of the abdominal muscles. The mice were placed singly into transparent cylindrical containers and the total number of writhes were counted for a 6 min period beginning 5 min after the acetic acid injection. Antinociceptive activity of the drug was expressed as the percentage of inhibition of the number of writhes in a drug administered group compared to the mean number of writhes in a control group. Administration of drugs, either i.c.v. (intracerebroventricular, 5 #1) or s.c. (10 ml/kg), was timed so that the peak effect occurred in the center of the observation period. Minimum of 10 mice were used for each group and three to four doses of drugs were used to generate dose response curves. EDs0 values and their 95% confidence limits were determined by the parallel line assay of Finney (1964).
Compounds 2, 3 and 4 displayed modest antinociceptive activity when administered i.c.v. (table 1) with the monomer (4) having the highest activity. In comparison, morphine administered i.c.v. had an EDs0 of 0.1 (0.03 - 0.4) n m o l / m o u s e which was 16 times more potent than compound 4. Also the antinociceptive effect of these compounds was long lasting with the activities of 2 and 3 still detectable 4 h after administration (table 2). The compound with the shortest spacer (no glycyl units), 1, did not possess antinociceptive activity at the highest dose employed.
3.2. Antagonist activity of naltrexamine bivalent ligands The compounds were administered i.c.v, and when their agonist activity subsided, their antagonist activity against morphine antinocicep-
TABLE 1 Antinociceptive activity of naltrexamine bivalent ligands. Cornpound
EDs0 (nmol i.c.v.)
!
>10
2 3 4
3.87 (3.41-4.40) 5.07 (2.98-11.69) 1.60 (1.05-2.23)
Rel. potency (rain)
Peak effect
0.4 0.3 1.0
120 60 20
287 TABLE 2 Antagonist activity of naltrexamine bivalent ligands. Time after 2 nmol i.c.v.
Morphine EDso (95% confidence limits) mg/kg s.c. 1
2
3
4
None 4h 24 h 3 days 5 days 7 days
0.9 (0.5-1.8) 14.2 (9.7-22.2) 13.3 (9.0-21.4) 5.2 (3.5-8.0) 3.0 (2.0-4.7) 1.4 (1.0-2.2)
b 5.3 (2.9-11.9) 2.2 (1.1-4.5) 1.8 (1.0-4.9)
b
14.8 (10.3-20.6) 12.9 (9.3-18.5) 3.6 (2.5-5.0) 2,1 (1.5-2.9) 1.1 (0.8-1.51
a
-1.6
a Acetic acid writhing assay, b Antinociceptlve activity still detectable.
tion was assessed (table 2). The compound with the shortest spacer, 1, and the monomer, 4, displayed the highest antagonist activity with both compounds raising the EDs0 of morphine by 16fold 4 h after administration of the ligands. The antagonist activity of these two ligands persisted for a week. Compound 2 had about half the antagonist activity as those of 1 and 4 one day after administration of the ligands. Antagonist activity could not be detected with compound 3. Compounds 1 and 4 were investigated further by using the s.c. route of administration. Compound 1 at sublethal doses ( < 50 m g / k g ) , displayed very weak antagonism of morphine antinociception (5-10% inhibition of the effect of ED90 doses of morphine). On the other hand, the monomer, 1 at a dose of 10 m g / k g , raised the EDs0 of morphine 8-fold 4 h after its administration (table 3). The antagonist activity lasted between 3 to 5 days. Compound 1 also possessed antinociceptive activity when administered s.c. that lasted for about 2-3 h and the compound appeared to be a partial agonist by this route of administration.
TABLE 3 Antagonist activity of compound 4 administered s.c. Time after 10 mg/kg of 4 s.c. 0h 4h 18 h 24 h 3 days 5 days
Morphine EDs0 (95% c.1.) mg/kg s.c. 1.0 (0.6-1.6) 8.2 (3.3-21.1) 4.1 (2.7-6.3) 4.1 (3.6-4.5) 1.6 (1.5-1.7) 1.1 (0.9-1.4)
The m a x i m u m inhibition of writhes was about 50% with increasing doses of 1 from 5 to 80 mg/kg.
4. D i s c u s s i o n
The fact that some of the naltrexamine bivalent ligands possessed antinociceptive activity was not surprising because a substituent attached on the 6-amino group confers agonism to the molecule in a number of naltrexamine derivatives, e.g. /3funaltrexamine. This agonism is probably mediated by x opioid receptors because its activity was detected only by the writhing assay, one of the few methods with which activity at x receptors can be detected (Tyers, 1980; Ward and Takemori, 1983). Also, when antagonists of /t opioid receptors possess agonist activity, they usually interact at K opioid receptors to mediate agonism, e.g. nalorphine, levallorphan, /3-funaltrexamine. The antagonist activities of the naltrexamine bivalent ligands in vivo did not parallel those in the GPI. Whereas in the G P I the antagonist activity peaked when there were two glycyl units on either side of the succinyl group (compound 2), in vivo, the greatest antagonist activity resided in the compound with the shortest spacer (no glycyl units, 1) and the m o n o m e r (4) and 2 displayed much less activity. These findings were surprising because in the GPI, the compound with the shortest spacer is more potent in antagonizing x receptor agonists and less potent in antagonizing # receptor agonists (Portoghese et al., 1986). Data from opioid receptor binding assays with these series of compounds
288 does not shed light on the discrepancies between the d a t a in vitro a n d in vivo b e c a u s e the b i n d i n g affinities of all these c o m p o u n d s for o p i o i d receptors are similar ( P o r t o g h e s e et al., 1986). T h e lack of c o r r e l a t i o n b e t w e e n the a n t a g o n i s m of m o r p h i n e in the G P I a n d in vivo m a y merely m e a n that t h e / , r e c e p t o r system in the G P I m a y be f u n c t i o n a l l y different from that in the central nervous system. Alternatively, there m a y be a f u n d a m e n t a l difference b e t w e e n kt agonist a n d a n t a g o n i s t r e c o g n i t i o n sites because agonist b i n d ing and activity in vivo usually correlate well. Evidence for the latter p o s s i b i l i t y has been rep o r t e d in the bt o p i o i d r e c e p t o r system in the G P I (Portoghese a n d T a k e m o r i , 1983). In a d d i t i o n , since an agonist c o m p o n e n t is s u p e r i m p o s e d u p o n an a n t a g o n i s t effect, the net a n t a g o n i s m might be expected to be different from that o b s e r v e d in s m o o t h muscle p r e p a r a t i o n s . This w o u l d also explain the slower onset of a n t a g o n i s m of 2, i.e. the agonist activity c o m p l e t e l y m a s k s the a n t a g o n i s m p r i o r to 24 h. T h e lack of a n t a g o n i s m of 3 at 24 h might also be e x p l a i n e d on this basis in that there is significant sustained agonism. T h e u n e x p e c t e d long d u r a t i o n of action of the agonist a n d p a r t i c u l a r l y the a n t a g o n i s t activity of these n a l t r e x a m i n e derivatives is puzzling. T h e c o m p o u n d s c o n t a i n n o d i s c e r n i b l e s u b s t i t u e n t that w o u l d lead to a n o n - e q u i l i b r i u m a s s o c i a t i o n so that this p o s s i b i l i t y is unlikely. T h e d a t a o b t a i n e d from s.c. a d m i n i s t r a t i o n of c o m p o u n d s 1 a n d 4 m a y suggest that n a l t r e x a m i n e b i v a l e n t l i g a n d s m a y be too b u l k y to be t r a n s p o r t e d easily into the central nervous system. O n the o t h e r hand, once the c o m p o u n d s enter the central nervous system, it m a y b e c o m e very difficult to clear them from the tissues. A l t h o u g h the long d u r a t i o n of action is
u n e x p l a i n e d , the effect is p r o b a b l y due to some sequestration phenomenon, perhaps entrapment by the cells in the central nervous system followed b y slow release. R e g a r d l e s s of the m e c h a n i s m for the long-lasting effect, these c o m p o u n d s m a y give future insights into the design of long-acting agonists a n d a n t a g o n i s t s .
Acknowledgements We thank the capable technical assistance of Joan Naeseth and Mary Schwartz.
References Finney, D.J., 1964, Statistical method in biologic assay, 2nd edn., Hafner Publishing Co., New York. Hayashi, G. and A.E. Takemori, 1971, The type of analgesicreceptor interaction involved in certain analgesic assays, European J. Pharmacol. 16, 63. Portoghese, P.S., D.L. Larson, L.M. Sayre, C.B. Yim, G. Ronsisvalle, S.W. Tam and A.E. Takemori, 1986, Opioid agonist and antagonist bivalent ligands. The relationship between spacer length and selectivity at multiple opioid receptors, J. Med. Chem. 29, 1855. Portoghese, P.S., A.W. Lipkowski and A.E. Takemori, 1987, Binaltorphimine and norbinaltorphimine, potent and selective • opioid receptor antagonists, Life Sci. 40, 1287. Portoghese, P.S. and A.E. Takemori, 1983, Different receptor sites mediate opioid agonism and antagonism, J. Med. Chem. 26, 134l. Portoghese, P.S. and A.E. Takemori, 1985, TENA, a selective kappa opioid receptor antagonist, Life Sci. 36, 801. Tyers, M.B., 1980, A classification of opiate receptors that mediate antinociception in animals, Br. J. Pharmacol. 69, 503. Ward, S.J. and A.E. Takemori, 1983, Relative involvement of mu, kappa and delta receptor mechanisms in opiate-mediated antinociception in mice, J. Pharmacol. Exp. Ther. 224. 525.