14-P-Methyl-8-oxacyclorphan (BC-3016), a morphinan derivative with high affinity for kappa opioid receptor: comparison with dynorphin-A(1-13)

SIMON LEMAHRE' AND MICHEL DUMOWT Dkparternent de Physiologic et Pkrmacokogie, Facuktt de MCdecine, UmiversitC de Sherbrooke,

Sherbrooke, Quk., Canada J I H 5M4 FRANCOIS JOLICOEUR

Dkprtement de Pgchiatrie, Faculte' de Mtdecine, Universite' de Sherbrooke, Sherbrooke, Qut ., Canada JlU 5M4 A N D

BERNARD BELLEAU Department ojChernistry, McGill University, 886 Sherbrooke St. W., Montre'ak, Qut., CcsmaBk( H2A 2K6

Received November 8, 1985

LEMAPRE, S., M. DUMONT, F. ~OLPCOEUW, and B. BELLEAU. 1986. 14-B-Methyl-8-oxacyelorphan (BC-3016), a morphinaw derivative with high affinity for kappa opioid receptor: comparison with dynorphin-A(1-13). Can. J . Physiol. Phmacol. 64: 787-7 1 1.

14-~-Methyl-8-oxacyc1orpkan (BC-3016) was tested for its ability to depress the electrically evoked contractions of the guinea pig ileum (GPH) and of the mouse vas deferens (MVD) and to compete with the binding of prototype ligands selective for K-, p-, or 6-opioid receptors in membrane preparations of rat brain and guinea pig cerebellum. BC-3016 was a very potent agonist in the GPI and MVB preparations, with of 0.7 a d 3 1 niW, respectively. The activity of levorphanol, a standard alkaloid related to BC-3016, was much lower in Both assays with IDw values of 44 and 86 M , respectively. Conversely, the activity of BC-301.6 was quite comparable to that of dynorphin-A(l-13) in both preparations. In the GPI assay, a putative K-receptor antagonist, MR-2266, was 6.6 and 5.5 times more potent than naloxone in blocking the activity of BC-3016 and dynorphin-A(l-13), respectively. BC-3016 was also very potent in displacing bound [%I~]ethylketocyclazocine ( [ 3 ~ ] ~ ~ ~ ) to membrane preparations sf the guinea pig cerebellum, a brain component containing predominantly K-opioid receptors (Ki of 0.58 nM). Its potency in the displacement of the bound p-ligand, %-labelled b ~ - ~ l a ~ , ~ e ~ h e ~ , ~ l y - ~ ~ ~ ) - e n k e ~ h a l i n (E~HJDAGB), to rat brain homogenates was somewhat lower (Ki of 0.8 niW) but still high when compared with its ability to displace the 8-ligand, 'H-labelled (D-Se?, TIah6)-Leu-enkepidin ( [ 3 ~ ] ~ ~ ~ ~ ~ ) to rat brain homogenates (K, sf 4.45 nM). The affinity of BC-3016 for the opioid receptor was 2.1 -fold higher than that of U-50488M, a selective eopioid ligand. Finally, the relative potency of BC-3016 to bind each one of the receptor types was similar to that of dynorphin-A(1-13). These data indicate that the structural rnoaifications of levorphanol Beading to BC-3016 increases the affinity of the compound for the K-opioid receptor and provides a compound whose profile of activity is similar in some respect to that of endogenous dynorphin-A(l - 13).

LEMAIRE, S., M. DUMONT, F. JOLHCOEUR et B. BELLEAU. 19g6. 14-p-Methyl-8-oxacyclorphan (BC-3016), a morphinan derivative with high affinity for kappa opioid receptor: comparison with dynorphin-A(l - 13). Can. 9. Physiol. Phmacol. 64: 707-7 11.

Un a test6 le BC-3016 (14-p-mCthyl-8-oxacyclorphme) pour sa capacitC de diminuer les contractions CvoquCes CBectriquement de B'illCon de cobaye (IC) et du canal defdrent de ssuris (CDS), ainsi que pour sa capacitd d'entrer en cornpCtition avec la fixation de ligands prototypes sClectifs F u r les rkcepteurs opioides K, p et 6 dans des prCparations de membranes de cerveau de rat et de cervelet de cobaye. Le BC-3016 fut un agoniste trks puissant dans les preparations d'IC et de CDS, avec un IDJo de 0,7 et de 31 M , respectivement. k9activitC du ldvorphanol, un alcalsei'de standard associG au BC-3016, Ctait beaucoup plus faible dans les deux prkparations, avec des valeurs d'IDSO de 4-44 et de 86 niW, respectivement. Inversement, h s les deux prkparations, l'activitd du BC-3016 Chit assez comparable 24 celle de la dynorphine-A(l-13). Dans la prkparation d'IC, un antagsniste putatif dm rkcepteur K, MW-2266, Ctait 6,6 et 5,5 fois plus puissant que le nalsxone pour bloquer I'activitC du BC-3016 et de la dynorphine-A(l- 13), respectivement. Le BC-3816 fut aussi tr&s puissant pour ddplacer la [3~ljethylkdtscyclazocine (I~HIEKC) fixCe aux prkparations de membrane de cervelet de cobaye, une composante du cerveau contenant surtout des rbcepteurs opioides K (Ki de 0.58 nM). Sa capacitC de dCplacer le ligand p RxC, ( ~ - ~ ~ a ~ , ~ e ~ h e ~ , ~ l y - ~ H ~ ) - e n k ~ ~ h a l i n e m q u k au %I ( [ 3 ~ ] ~ ~ ~ ~ ) , aux homoghats du cerveau de rat Chit plus faible (Ki de O,$ nA4) mais toujours grande csemparCe h sa capacit6 de dkplacer le ligand 8, (~-~e?,~hr~)-leu-enkd~haline maquC au 3~ (['HIDSLET), aux homogdnats du cervwu de rat (Ki de 4 4 5 M). L'affinitk du BC-3016 pour le rkcepteur opioyde Ctait 2,1 fois plus haute que celle de 19U-50488H, un ligamd opioide K dlectif. Finalement, la capacitC relative du BC-2016 de fixer chaque type de rkcepteurs Ctait similaire ir celle de la dynorphine-A(l- 13). Ces rksultats indiquent que les modifications stmcturales du lCvorphanol menant au BC-2816 augmentent l'affinitk du compost2 pour le rkcepteur opioyde K et fouhnissent un compost5 dont le profil d'activitk est sirnilaire, d'une certaine rnani&re, ir celui de la dynorphine-A(1- 13) endogbne.

[Traduit par la revue]

Introduction Since the discovery of endogenous opisid peptides (Hughes

et al. 1995; Simantov and Snyder 19761, much interest has focussed on determining the mechanisms of action of opioid compounds at various receptor sites located in the brain, spinal cord, peripheral smooth muscles, and specialized organs (for a

'Resent address: Depmment of Phmacolsgy, School of Medi- cine, University of Ottawa, Ottawa, Canada K1H 8N5.

review see Martin 1976; Copolsv and Helrne 1983). One of the most important findings was the emergence of three different families of endogenous opioid peptides exhibiting relative selectivity for specific opisid receptors: p-endorphin k i n g more specific for the E-receptor, the en4tephdins for the 6- receptor, and dynsrphin-a-neoendorphin for the K-receptor (Hughes et al. 1975; Cox 1982; Fischli et d. 1982). The anti- nocicepkive effects of enkephdins and @-endorphin were dem- onstrated both by intracerebra1 injection o% these peptides (Loh et al. 1996; Chaillet et al. 1984) and by the administration of

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

AC

Q S

ervi

ce/S

eria

ls (

A)

on 1

0/27

/13

For

pers

onal

use

onl

y.

708 CAN. J. PHYSIC&. PHARMACBL. VOL. $4, 1986

specific enkephalin peptidase inhibitors (Chaillet et al. 1983; Foumie-Zaluski et al. 1984). However, even though dynorphin was shown to be the most potent of the opioid peptides in "in vilre" peripheral assays (Goldstein et al. 19791, its analgesic property has remained quite elusive and difficult to demonstrate (Friedman et al. 198 1) except when injected directly into the spin& cord (Rzewlocki et al. 1983; Jhammdas et al. 1985), where the K-type of opioid receptor predominates (Attali et al. H 1982). Moreover, dynorphin has been shown to antagonize HO morphine- and P-endorphin-induced analgesia (Friedmaw et al. 1881).

8 6 - 3 0 1 6 L E V B R P H A N O L A few yeas ago, a novel synthetic opioid alkaloid of the

morphinan class, (14-p-methyl-8-s~acyclorphan~ BC-3026) was synthesized and found ts be devoid of analgesic activity in rats, dogs, and monkeys when standard aversive tkemal stimuli were utilized (Lambert et al. 1978). However, Be-3016 was very potent in inhibiting writhing response in the mouse, an effect which is now thought to be mediated though interaction with K-receptors (Goodman and Pastern& 1984). Furthemore, this synthetic malog was found to antagonize the effects of the p-agonist oxymorphone in the mouse straub tail test (Lambert et d. 1978). Since this overall in vdvo profile of activity appeared quite different from that of its parent morphinan, a detailed study was undertaken to better define its relative affinity for the p,-, ti-, and K-receptors and to compare its profile of activity with those of other opioid dkaloids and dynoqhin-A( 1 - 13).

blahrials and methods 'H-labelled ( ~ - A l a < ~ e ~ h e ~ , ~ ~ l ~ - ~ ~ ~ - e & e ~ h a l i n ( [ 3 ~ ] ~ ~ ~ ~ )

(40Cj/mol; 1 Ci = 3'9 GB ) was obtained from Amershm, England. 9 [~~]e th~ lke tm~e luoc ine ([ HIEKC) (16.4 C 2 m o l ) , and 3~-~abelled (~Se$,~bage)-ku-ernBrepha1in (['HIDSLET) (30.5 Ci lmol) were purchased from New England Nuclear, Boston, MA. Trms-(+)-3,4,dich1om-N-me~y1-N-(2-(1 -pyrro1idiny1)cyclohexyl)- knzeneacetamide methanesulfonate hydrate (U-5W88H), a selective K-opioid ligand (Piercy et al. 19821, was obtained from Upjohn Corn- pmy ( K d m a o o , MI). MR-2266 was generously provided by Dr. W. Merz (Bmfinger Ingelheim). Levorphamol (L-Dromoran) and leval- b q h m tartrate were provided by Hoffman-LaRoche Ltd. (Vaudreuil, QuC.). Dynorphh-A(l -1 3) was synthesized in our laboratory as pre- viously described (Turcotte et al. 1984). The synthesis of BC-3816 (14-~-methyl-8-oxacyclop%shan; Fig. 1 ) was regmad earlier (Lmbert et al. 19'98; MornBrovic a d Lmbert 19'96). Guinea pigs (mde 200-300 g), mice (mde 20-30 g, Swiss Webster), and rats (male 100-200 g) were obtained from the Canadian Breeding F m (St.-Constant, Que.).

Smooth muscle assays The inhibition of electrically induced contractions of the guinea pig

ileum (GPI) and mouse vas deferens (MVD) was measured as reported earlier (Twcotte et al. 1984). Briefly, the animals were sacrificed by a blow s n the neck and their tissues were rapidly dissected and mounted on a 10-waC double-jacketed organ bath in h b s solution (pH 7.4) at 3'9°C. Tensions of 0.15 (vas deferens) and 1.0 g (ileum) were applied and contractions were induced with electrical ~ulsatisns of 60 0. I Hz, 10 ms delay, and 1 rns duration. They were recorded with a force displacement transducer (FT83C) coupled to a Grass polygraph. Dose-response curves were constmeted for all opioid compounds and the concentration causing 564% inhibition (IDs0) obtained from log- probit plots of six graded concentrations, each representing the mean 9 SBM of six different tissues. The potencies of the antagonists were expressed as the equilibrium constant Ke (Kosterlitz and Watt 1968).

Opiate binding assays Guinea pig cerebellum and rat brain homogenates were prepared as

previously described (Turcotte et al. 1984; Rsbson et al. 1984). Briefly, the tissues were homogenized with a glass-Teflon hornoge-

Fig. 1. Structure sf Be-30 16 and levorphanol.

nizer in 10 vol (wlv) of ice-cold 50 mkf Tris-HC1 buffer (pH 7.4). The larger particles were removed by filtration though two layers of cheesecloth and the crude membrane fraction separated by centrifuga- tion at 17 000 X g for 30 min at 4'C in a SorvdB SS34 rotor. Membrane pellets were resuspended in cold THis buffer, incubated at 37°C for 30 f in , and the suspension was centrifuged under the same conditions. The final pellets were resuspended in Tris buffer to yield a concenwa- tim of 10% (wlv) based on the original tissue weight. Binding assays were performed at room temperature (22'C) for 30 min with 2-mL aliquots of the membrane preparation (1 rng protein) in the presence of bacitracin (25 pJQ, captopril (10 @), thiorphan (8.3 and bestatin (30 pM). The labelled ligands used were [ 3 ~ ] ~ ~ ~ (K-Iigand) with the guinea pig cerebellum membrane preparation and [ 3 ~ ] ~ ~ ~ ~ (p-ligand) or %-]labelled (~-~e?,TQ)-~eu-e&e~halin (DSLET) (8- ligand) with the rat brain membrane preparation. Competition experi- ments were performed in the presence of 2 nM of [ 3 ~ ] ~ ~ C or [ 3 ~ ~ ~ ~ ~ ~ ~ or 0.75 sf ['H]DAGO using increasing concentrations (from 10-" to 1 p M) of the unlabelled compound. The incubations were stopped by placing the tubes in an ice-water bath for 15 nain followed by filtration though SFIB M a t m n filters amd four washes with 3-mL fractions of the Tris-HC1 buffer (0°C). The filters were then counted in 10 naL aquasol in a beta counter (Bechan). The specific binding was estimated usin the difference between the counts in the f presence and absence of 10- M levallorphan. For the calculation of the kinetic parmeters of binding of labelled and unlabelled ligands, the equilibrium dissociation constmt (KD) was determined for each triti- ated liganad from saturation curves and the inhibition constant (Ki) calculated using the following equation: Ki -- HC5d(1 + [L]IKD), where b] is. the concentration of the labelled ligand and KD the equilibrium dissociation constant (Cheng and Pasoff 1973).

Smooth muscie responses A comparison between the smooth muscle responses to BC-

30 16, dynorphin-A(l - 13) and levorphanol is presented in Table 1. Ow the GPH preparation, BC-36316, md dynorphin-A( 1 - 13) behaved as potent agonists with IDSo in the nanomolar range (IDso: 0.7 and 0.5 M, respectively). On the other hand, levor- phanol, the standad narcotic analgesic (Fig. I), had only 1 % the potency of BC-30 16 (EDs0 of 4-4. B as compared with 6.7 EM for BC-3016). The difference in the potencies of the two latter compounds in the MVD was less dramatic but BC-3016 was nonetheless 2.8 times more potent than levoqhanol. In both assays, the pattern of activity of Be-3016 followed that of dynoqkn-A(1- 13) but at a somewhat reduced level of potency (1.4 and 1.5 times less potent in the GPI and MVD prepaations, respectively).

Antagonist activity of nalsxone and MR-2256 An important method for the determination of the type of

receptor involved in the activity of an opioid compound in smooth muscle assays consists in the measurement of the p-

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

AC

Q S

ervi

ce/S

eria

ls (

A)

on 1

0/27

/13

For

pers

onal

use

onl

y.

LEMAHRE ET AL. 709

TABLE 1 . Relative inhibitory potencies of Be-3016, levoqhanol, binding amounting to 0.8 and 0.5 nM. Conversely, the affinity dynorghin -A(I - 13) on electrically induced contractions of the guinea of the same two compounds for the &receptor was lower (Ki

pig ileum and mouse vas deferens values for inhibition of [ 3 ~ ] ~ ~ ~ ~ ~ binding of 3.47 and 4.45 nM, respectively). Finally, the selectivity of U-50488M for the

GPI MVD K-receptor was much higher than that of BC-3016 and dynor-

Relative Relative phin-~( l - 13). U-50488k inhibited the binding of ['HI DAGO

Opioid IDS{ potencyb potencyh and [%]DSLET only at high concentrations (Ki of 824 and

compound ( W (%I ( W (%) 12 393 nM, respectively).

BC-3016 0 . 7 k 0 . 2 100 3% + 4 188 HRvorpRmol 44 .123 .0 1 8 6 2 8 36 Dymrphin-A(1-13) 8 .520 .1 148 21 A2 148

Toncentration which gives half maximal response. bRelathe to Be-3016.

TABLE 2. Relative inhibitory potencies of MR-2266 and ndoxone on the activity of BC-3016 and dynorphin -A(l- 13) in the guinea pig

ileum preparation - -

BC-3016 Dynorphin-A(l - 13)

Relative Relative Opioid K potencyb K/ potencyb

antagonist ( W (%I bW (%I

MR-2266 3.1k0.31 100 4 .8k1 .6 188 Ndoxone 20.7k 1.2 15 26.$&2.7 18

The values axe the means 2 SEM of six expstiments. %dative to MW-2266.

tency of known selective opioid antagonists relative to that the test compounds. Naloxone is recognized to possess a high affinity for the p-opioid receptor (Wood et al. 1982) while (-)-(1R,5R,9R)-5,9-diethyl-6,7-benzomorphan (MR-2266) is h o w n to display a high affinity for the K-site (Romer et al. 1980). As shown in Table 2, MR-2266 (K, = 3.1 nM) was 6.6 times more potent than naloxone (K, = 28.7 nM) in antagoniz- ing the activity of BC-34816. The same pattern of activity was observed for the same two compounds against dynorphin-A(1- 13), the K, values being 4.8 and 26.8 nkf for MR-22% and waloxone, respectively. It is worth noting here that the K, value of naloxone against nomorphine, a prototypic p-agonist, has been reported to be quite distinct (3.5 nM, Chavkin et al. 1982) from that found against dynorphin-A(1- 13) (26.8 in Table 2; 21 HEnn in Chavkin et al. 1982).

Selective receptor binding activity There are different ways to determine the selectivity of an

opioid ligand for a specific reeeptor type. One of them involves the use of membrane preparations from the guinea pig cerebel- lum, a tissue known to contain mostly the K-type of opioid receptor (>84%; Robson et al. 1984). Another way calls for the use of selective labelled ligmds such as [ 3 ~ ] ~ ~ G 0 and ['HIDSLET which selectively bind to the p-opioid receptor (Handa et al. 198 1) and 6-opioid receptor (Gacel et al. 1980), respectively. As shown in Table 3, BC-3016, dynorphin-A(1- 13), and U-50488W are all potent displacers of the binding of ['HIEKC to the guinea pig membrane preparation (Ki ranging from 0.1 1 to 1.23 nM). On the other hand, levorphansl was less potent in this assay (Ki = 3.05 nM). In addition, BC-3016 and dynorphin-A(1-13) behaved as potent ligands for the p- receptor, their respective Ki for displacement of [ 3 ~ ] D ~ ~ 0

The existence of multiple opioid receptors was suggested more than 20 years ago when it was discovered that among various opioid dkaloids, some behaved as pure agonists, some as pure antagonists, and others as mixed agonist-antagonists depending on the type of assay used. A more recent classiffication of opioid receptor types was put forward by Martin and his colleagues (Martin et al. 1976) who were able to differentiate between three types of receptors (k, K, and a) according to the behaviousal or physiological responses elicited in the spinal dog. Lord et al. (1977) subsequently suggested the existence of the a-site. The recent development of specific l igads for individual types of opioid receptors and the design of experiments involving protection of specific receptor types against alkylating agents have confirmed the existence of multi- ple opioid receptors although the physiological significance of this receptor multiplicity remains to be clarified (Goldstein and James 1984). Dynorphin is an endogenous opioid peptide dis- playing prominent activity in the guinea pig ileum assay (Gold- stein et al. 1979); however, no clear-cut analgesic activity could be associated with this peptide (Brzewlwki et al. 1983). Its extremely high activity in the guinea pig ileum preparation may be explained by a specific interaction with one or two types of opioid binding sites. Our data, which agree with those of Qui- rion and Pert (198 I), indicate that dynorphin-A(l- 13) has a high affinity for both the K -and preceptors, although some selectivity for the K-receptor can be observed (Ki = 0.1 1 nM, Table 3). Essentially, the same results were obtained with BC- 3016, although its affinity for the K-site was somewhat smaller (Ki = 0.58 M ) .

It is quite interesting to note that the structural modifications of levorphanol leading to BC-30 16 (Fig. 1) bring about a d rma- tic change in the pattern s f activity in the guinea pig ileum preparation as well as in the receptor specific opioid binding assays. Thus, while levorphanol is a g d ligand for the p- receptor, its structural transformation into BC-3016 changes the affinity for the K-site (Table 3). The K-like activity of certain benzomorphans has been attributed to the presence of an oxygen (8) in close proximity (benzylic position) to the nitrogen (N) which would presumably promote binding to an anionic site on the K-receptor (Wood 1984). Analog BC-30 1 6 also incorporates an oxygen atom but at the proximal position 8 of the ring system (Fig. I), a feature which may promote together with the 14-P- methyl group affinity for the K-receptor.

The present data indicate that BC-3816, a novel morphinan analog, bears many similarities in its binding and peripheral activities to those of dynorphin-A-( 1 - 13). A much broader spectrum of biological tests remain to be developed before adopting BC-3016 as a genuine substituent for dynorphin itself. Presently, the fact that these two urnelated structures act simi- larly on the opioid receptors and that they display the same order of potency in two smooth muscle preparations offers a gmd

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

AC

Q S

ervi

ce/S

eria

ls (

A)

on 1

0/27

/13

For

pers

onal

use

onl

y.

7184 CAW. J. BHYSIOL. PHARMACOL. VOL. 64, 1986

TABLE 3. Comparison of the inhibitory effects of BC-3816, dynorphin-A(l-131, and U-50488H on the binding of [ 3 ~ ] ~ ~ ~ (2 to a membrane preparation of guinea pig cerebellum and the binding of [ 3 ~ ] ~ ~ ~ 8 (0.75 M) and [ 3 ~ ] ~ ~ L ~ T (2 nM) to rat brain homogenates

[ 3 ~ ] ~ ~ ~ ['HID AGO [ 3 ~ ] ~ ~ ~ ~ ~

Relative Relative Relative Kia potencyb Ky potencyb Ky potencyb Ratio

Opioid compound (m (%) ow (%I ~:p:8

Values are the mean A SEM of thee sets d duplicate. KD for ['HIEKC, [3H]DAG0, and [3HJDLSET were 1.11, 1.45, and 3.9 M, respectively. bRelative to BC-30 t 6 .

opportunity to specify the structural requirements for synthetic opioids endowed with dynorphin-like activity.

The potential value of exploring the biological activity of BC-3016 and developing related structures is dso supported by the following observations. Firstly, Jhammdas et al. ( 1986) have recently demonstrated that intmthecal injections of dynor- phin-A( 1 - 13) but not morphine ( p-ligand) or U-50488H (K- ligmd) cause a late and long-lasting analgesic effect (24 h or more after injection) that is reversible by naloxone. Therefore? the possible stimulation of a single type of opioid receptor by morphine (p) or U-50488H (K) is not efficient in evoking the biological activity while dynoqhin-A(1- 13), which is a potent ligmd for both p- and K-receptors (Table 3), is a potent effector. It would be of great interest to verify the activity of BC-3016 in such a system. Secondly, the use of opioid alkaloids that mimic the action of dynsrphin-A(1-13) may help to understand its mechanism of action a d clarify its physiological function. For instance, it is interesting to note that the N-substituent in BC- 3016 ( 4 H 2 + ; Fig. 1) is identical to that found in naltrex- one, a potent opioid antagonist. Both Be-3016 and dynorphh- A(1- 13) we potent opioid agonists in the GPI and MVB assays. However, preliminary experiments by F. Jolicoeur and col- laborators (unpublished data) have indicated that BC-3016, while producing no direct antinociceptive effect after intracis- temal injection in the rat, significantly antagonizes the analgesic effects of morphine. This latter property was also reported for dynorphin-A(1-13) (Friedmaw et al. 1981). Finally, it is ex- pected that BC-3016 or related compounds would be more resistant to degradation than the peptide dynorphin-A(1- 13). Therefore, BC-3816 presents biological characteristics that are unique in the rnorphinan class of opioids (high affinity for K-receptor) and of great interest for compkson with endoge- nous dynorphin.

R i s work was supported by research grants from the Fonds de Recherche en SmtC das QuCbec, the Medical Research Goun- ci2 of Canada, and the Canadian Heart Foundation. We wish to express our gratitude to Mrs. Danielle Laurendeau and Lucie Lafrance for secretarial and technical support. Gratitude is also expressed to Dr. Richard Partyka (Bristol-Myers Cs., Syracuse site) for his sustained encouragement md interest and for sup- plies of BC-3016 originally synthesized by Dr. Y. Monkovic.

ATTALI, B., C. GOUARDEES, H. MAZARGUIL, Y. AUDIGIEB, and 9. CROSS. 1982. Differenkid interaction of opiates to multiple "Kappa" binding sites in guinea pig lrambosacrd spinal cord. Life Sci. 31: 1371-1375.

CHAPLLET. P., A. CBULAUD, J. -M. ZAJ AC, M. -C. FOUWHE-ZALWSKI, J. COSTENTPN, and B . P. Roqw~s. 1984. The p rather than the 8 subtype of opioid receptors a p p x s to be involved in enkephalin- induced analgesia. Eur. J. Phmacol. 101: 83-90.

CHAILLET, P., H. MAR$AIS-COLLA~, J. COSTENTIN, C. C. YH, S. DELABAUME, and J. C. SCHWARTZ. 1983. Inhibition of enkephalin metabolism by, a d mtinociceptive activity of, Bestatin, an ami- nopeptidase inhibitor. Eur. J. Phmacol. 86: 329-333.

CHAVMN, C., I. F. JAMES, and A. GOLDSTEIN. 1982. Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science (Washington, B.C., 1979-), 21%: 413-415.

CHENG, Y .-C., md W. H. PRWSQFF. 1973. Relationship between the inhibition constant (Ki) md the concentration of inhibitor which causes 50 per cent inhibition (I&) of an enzymatic reaction. Biochem. Phmacol. 22: 3099-3 102.

C O ~ L O V , D. L., and R. B. HELME. 1983. Enkephalins and endor- phins. Clinical, phmacologicd and therapeutic implications. h g s , 26: 503-519.

Cox, B. M. 1982. Endogenous opioid peptides: a guide to structures and terminology. Life Sci. 3%: 1645- 1658.

RSCHLI, W. A., A. GOLDSTEIN, M. W. HUNKAPIEEER, and L. E. ~ 0 0 ~ . 1982. Isolation and d n s acid sequence analysis sf a 4l330- dalton dynorphin from porcine pituitary. Roc. Natl. Acad. Sci. U.S.A. 78: 5435-5437.

FOU~IE-ZALUSM, M. C., P. CHAHLLET, R. BOUBOUTOU, A. COULAUB, P. CHEROT, G. WAKSMAN, J. COSTENTIN, and B. P. ROQUES. 1984. Analgesic effects of kelatorphan, a new highly ptent inhibitor of multiple enkephalin degrading enzymes. Eur. J . P~XITI~CO~. 102: 525-528.

FWEBMAN, H. PI., M. ]F. EN, J. K. CHANG, N. M. LEE, and H. H. LOH. 898 1. Dynophin: a possible modulatory peptide on morphine or @-endorphin analgesia in mouse. Eur. J . Phmacol. 69: 357- 360.

GACEL, G., M. C. FOURNHE-ZALWSIQI, and B. P. R o q v ~ s . 1980. Tyr-D-Ser-Gly-Phe-Leu-Th, a highly preferential ligand for 6- opiate receptors. FEBS kkt . 118: 245-247.

GOLBSTEIN, A., md'I. F. JAMES. 1984. Multiple opioid receptors criteria for identification and classification. Trends Phmacol. Sci. 5: 503-505.

GOLDSTEIN, A., S. TACHIBANA, E. I. LBWNEY, M. HUNKAPILLER, a d L. HOOD. 1979. Dyaophin-(1-131, an extraordinarily potent opioid peptide. Roc. Natl. Asad. Sci. U.S.A. 76: 6666-6678.

GOODMAN, Re P., md G. W. PASTERNAK. 1984. Multiple opioid receptors. In Analgesics. Edited by M. J. Khar and G. W. Paster- n&. Raven Ress, pp. 69-96.

HANDA, B. M., A. C. LANE, J . A. H. LORD, B. A. MORGAN, M. J. RANCE, and C. F. C. SMITH. 1988. Analogues of p-LPH 61-64 possessing selective agonist activity at the @-opiate receptors. Eur. J. Phmacol. 70: 53 1-540.

HUGHES, J., T. W. SMITH, L. A. KQSTERLITZ, B. A. MORGAN, and B. W. Moms< 1975. Identification of two related penkapepides

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

AC

Q S

ervi

ce/S

eria

ls (

A)

on 1

0/27

/13

For

pers

onal

use

onl

y.

EEMAIIPE ET AL. 71 1

from the brain with potent opiate agonist activity. Nature (London), 258: 577-579.

SHAMANDAS, K., M. SUTAK, and S. LEMAIRE. 1986. Comparative spinal analgesic action of dynorphin-( 1 -$), dynorpkin-(1 - 13) and a kappa receptor agonist U58,488. Can J. Bkmacol. Physiol. 64: 263-268.

KQ~TERLITZ, H. W., and A. J. WATT. 1968. Kinetic parameters of narcotic agonists and antagonists with particular reference to N- allylnoroxyrnorphone (ndoxone) . Br . J . Bkmacol -33: 266-278.

LAMBERT, Y., S. B. DAVIS, and I. MONKOVIC. 1978. Analgesics and narcotic antagonists in the benzomorphan and 8-sxmorphinan series. J. Med. Chem. 21: 423.

LOH, H. N., L.-F. TSENG, E. WEI, and C. H. LI. 1976. P-endorphin is a potent analgesic agent. Proc. Natl. Acad. Sci. U.S .A. 73: 2895- 2898.

LORD, J. A. H., A. A. WATERFIELD, J. HUGHES, and H. W. Ko~TERL~Tz. 1977. Endogenous opioid peptides: multiple agonists and receptors. Nature (London), 267: 495-499.

MARTIN. W. R. 1976. Pharmacology of opioids. Phmacol. Rev. 39: 283-323.

MARTIN, W. R., C. G . EADES, J . A. ~HOMPSON, W. E. HUPPLER, and P. E. GILBERT. 19'76. The effects of morphine- and nalorphine-like drugs in the non-dependent chronic and morphine-dependent spinal dog. J. Phmacol. Exg. Ther. 197: 517-532.

MONKOVIC, I., and Y. L~MBEWT. 1976. 3-14-Substituted-8- oxmorphinm. U.S. Patent No. 3959298. May 25.

RERCY, M. F., R. A. %AHTI, and L. A. SCHRBEDER, F. J. EINSPAHR, and C. BARSUHN. 1982. U-58488H, a pure kappa receptor agonist with spinal analgesic loci in the mouse. Life Sci. 31: 1 19'7- 1280.

PRZEWLWKH, R., 6. T. SHEARMAN, and A. HE^. 1983. Mixed opioid/non opioid effects of dynorphin and dynorphin related pep- tides after their intrathecd injection in rats. Neuropeptides, 3: 233- 246.

Q U ~ O N , R., and C . B. PERT. 11981. Bynorphin: similar relative potencies on F-, 6- and K-opiate receptors. Eur. J . Pharrnacol. 76: 467-468.

ROBSON, L. E., R. W. FOOTE, R. MAURER, and K. W. KOSTERLITZ. 1984. Bpioid binding sites of the K-type in guinea-pig cerebellum. Neuroscience, 12: 62 1-627.

ROMER, D., H. BOSCHER, @. R. WILL, R. MAURER, T. J. PETCHBR, H. B. A. WELL, W. C . C . K. BAKEL, and A. M. AKKERMAN. 1988. Brernazocine, a potent, long-acting opiate kappa-agonis t . Life Sci. 27: 971-973.

SIMANTOV, R, E., md S . H. SNYDER. 1976. Morphine-like peptides in mammalian brain: isolation, structure elucidation and interactions with the opiate receptor. Roc. Natl. Acad. Sci. U. S. A. 73: 251 5- 2519.

TURCSTTE, A., J. M. LALONDE, S. ST-WEWBE, and S. LEMAIRE. 1984. Dynorphin-(1- 13). I. Structure-function relationships of Ala- containing analogs. Int. J. Pept. Protein Wes. 23: 361-367.

WOOD, P. L. 1984. Agonist analgesics: evidence for fi2 and opioid receptor antagonism. h g Dev. Res. 4: 429-435.

Woss, P. L., S. E. CHARLESON, B. LANE, and R. E. HUNIN. 1982. Multiple opiate receptors: differential binding of mu, kappa and delta agonists. Neurophmacology ,28: 12 15- 1220.

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

AC

Q S

ervi

ce/S

eria

ls (

A)

on 1

0/27

/13

For

pers

onal

use

onl

y.