Naunyn-Schmiedeberg’s Arch Pharmacol (1999) 359: 17–20

© Springer-Verlag 1999

ORIGINAL ARTICLE

U. Bauer · M. Nakazi · M. Kathmann · M. Göthert E. Schlicker

The stereoselective κ-opioid receptor antagonist Mr 2266 does not exhibit stereoselectivity as an antagonist at the orphan opioid (ORL1) receptor Received: 15 October 1998 / Accepted: 12 November 1998

Abstract Mr 2266 [(-)-(1R,5R,9R)-5,9-diethyl-2-(3-furylmethyl)-2’-hydroxy-6,7-benzomorphan] is an antagonist at κ-opioid receptors and at ORL1 receptors as well. The aim of our study was to examine whether the known stereoselective antagonism of Mr 2266 at κ-opioid receptors also extends to ORL1 receptors. In mouse brain cortex membranes, the binding of the ORL1 receptor agonist [3H]nociceptin was equipotently inhibited by Mr 2266 and its enantiomer Mr 2267 (pKi 4.82 and 5.14, respectively), whereas the binding of the κ-opioid receptor agonist [3H]U-69,593 was inhibited by Mr 2266 more potently (pKi 9.11) than by its enantiomer Mr 2267 (pKi 7.15). In mouse brain cortex slices preincubated with [3H]noradrenaline, the concentration-response curve of nociceptin for inhibition of the electrically evoked overflow of tritium was equipotently shifted to the right by Mr 2266 and Mr 2267 (pA2 5.77 and 5.64, respectively). On the other hand, the inhibitory effect of U-69,593 on the electrically evoked overflow of tritium in guinea-pig brain cortex slices preincubated with [3H]noradrenaline was more potently antagonized by Mr 2266 (pA2 8.81) than by Mr 2267 (pA2 7.15). These data show that the stereoselective antagonism of Mr 2266 at κ-opioid receptors does not extend to ORL1 receptors. Key words κ-opioid receptor · ORL1 receptor · Nociceptin · U-69,593 · Mr 2266 · Mr 2267 · Noradrenaline release · Mouse and guinea-pig brain cortex

Introduction A fourth type of opioid receptors, usually termed orphan opioid or opioid receptor-like1 (ORL1) receptors, has been cloned and the heptadecapeptide nociceptin (also termed U. Bauer · M. Nakazi · M. Kathmann · M. Göthert · E. Schlicker (✉) Institut für Pharmakologie und Toxikologie, Universität Bonn, Reuterstrasse 2b, D-53113 Bonn, Germany e-mail: [email protected], Fax: +49-228-735404

orphanin FQ) has been identified as an endogenous ligand (for review, see Henderson and McKnight 1997; Meunier 1997). ORL1 receptors occur as presynaptic receptors on axon terminals where they cause inhibition of the release of the respective neurotransmitter; this has been shown in the peripheral nervous system and, albeit less frequently, in the CNS as well (for review, see Henderson and McKnight 1997; Meunier 1997). For example we have recently found that nociceptin, via presynaptic ORL1 receptors, inhibits noradrenaline release in the mouse brain cortex (Schlicker et al. 1998). For the identification and characterization of functional ORL1 receptors only a few antagonists are available so far. [Phe1ψ(CH2-NH)Gly2]-nociceptin-(1-13)-NH2 was identified as a selective and moderately potent competitive ORL1 receptor antagonist (Calò et al. 1998; Guerrini et al. 1998) but proved to be a partial or even full agonist in some ORL1-receptor models (see Schlicker et al. 1998 for references). Carbetapentane and rimcazole (Kobayashi et al. 1997) as well as four ligands at classical opioid receptors have been proposed as (unselective) ORL1 receptor antagonists (Dunnill et al. 1996; Nicholson et al. 1998). Among the four opioid receptor ligands, naloxone benzoylhydrazone was identified as a competitive ORL1 receptor antagonist (Dunnill et al. 1996; Nicholson et al. 1998; Schlicker et al. 1998) whereas bremazocine, WIN 44,441 and Mr 2266 behaved like noncompetitive antagonists (Nicholson et al. 1998). Mr 2266 is a stereoselective antagonist at κ-opioid receptors (the enantiomer Mr 2267 being at least ten times less potent than Mr 2266; Jackisch et al. 1986; Smith et al. 1989), and for two reasons we were interested to see whether the stereoselectivity also extends to the ORL1 receptor. First, stereoselective antagonists would be useful tools in experiments dedicated to the identification of ORL1 receptors. Second, stereoselectivity or lack of stereoselectivity might provide further information on common or different structural requirements of the κ and ORL1 receptor proteins with respect to the binding of ligands. We have determined the antagonistic potencies of Mr 2266 and Mr 2267 in our recently described ORL1 receptor model in

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the mouse brain (Schlicker et al. 1998) and their affinities in a radioligand binding study on mouse brain cortex membranes, using [3H]nociceptin. For the sake of comparison, the antagonistic potencies of Mr 2266 and Mr 2267 at a functional κ-opioid receptor (in guinea-pig brain cortex slices) and their binding affinities (using the κ-opioid receptor ligand [3H]U-69,593) were determined as well.

Materials and methods Binding studies. Cerebral cortex from male NMRI mice was homogenized in ice-cold Tris-HCl buffer (Tris-HCl 50 mM pH 7.4, EDTA 2 mM, phenylmethylsulfonyl fluoride 100 µM) containing sucrose 0.3 M with a Potter-Elvehjem homogenizer (10 strokes, 1200 rpm) and centrifuged at 1000 g for 10 min (4°C). The supernatant was centrifuged at 35,000 g for 10 min (4°C). The final pellet was resuspended in sucrose-free buffer solution and frozen at –80°C. The binding assay was performed in Tris-HCl buffer in a final volume of 0.5 ml containing 70–100 µg protein. Saturation curves were obtained by using nine concentrations of [3H]U-69,593 (κ-opioid receptor ligand) ranging from 0.1 to 25 nM and eight concentrations of [3H]nociceptin (ORL1 receptor ligand) ranging from 0.05 to 12 nM. The incubation was terminated after 60 min by rapid vacuum filtration through Whatman GF/C filters (pretreated with polyethylenimine 0.3 %) followed by rapid washing of the incubation tubes and filtered three times with 2 ml Tris-HCl buffer. Non-specific binding was determined with Mr 2266 100 µM ([3H]U-69,593 binding) and nociceptin 5 µM ([3H]nociceptin binding). Competition assays were performed as described for saturation studies (see above) with a concentration of 1.0 nM for [3H]U-69,593 and 0.5 nM for [3H]nociceptin binding. Ki values were determined using ten concentrations of Mr 2266 or Mr 2267. Saturation and displacement curves were analyzed using the program Graph Pad Prism (Graph Pad Software, San Diego, Calif., USA). Superfusion studies. Slices (0.3 mm thick, diameter 3 mm) were prepared from cerebral cortex of male NMRI mice, male Wistar rats and male Dunkin-Hartley guinea pigs. The slices were incubated for 60 min with physiological salt solution (PSS; see Schlicker et al. 1998) at 37°C containing [3H]noradrenaline 25 nM. They were then superfused with PSS at 37°C for 110 min at a flow rate of 1 ml/min. The superfusate was collected in 5-min samples. Tritium overflow was evoked by two 2-min periods of electrical field stimulation 40 min (S1) and 90 min (S2) after onset of superfusion. Stimulation parameters were 0.3 Hz, 50 mA and 2 ms. Tritium efflux was calculated as a fraction of tritium content of the slice at the onset of the respective 5-min sample (fractional rate of tritium efflux). The fractional rates in the 5-min collection periods from 55 to 60 (t1) and from 85 to 90 (t2) min of superfusion were determined. In order to quantify the effects of drugs on basal tritium efflux, t1 values (for drugs present in the PSS throughout superfusion) or t2/t1 ratios (for drugs added to the PSS from 62 min of superfusion) were calculated. The stimulation-evoked tritium overflow was calculated as the amount of tritium in excess of the basal tritium efflux. The latter was assumed to decline linearly from the 5-min period before to that 15–20 min after onset of stimulation and was expressed as a percentage of the tritium content in the slices at the onset of the respective stimulation. To quantify drug-induced effects on the stimulated tritium overflow, S1 values (for drugs present throughout superfusion) or S2/S1 ratios (for drugs present from 62 min onward) were determined. Apparent pA2 values for Mr 2266 or Mr 2267 for their antagonism against U-69,593 or nociceptin were calculated according to formula 4 of Furchgott (1972). Statistical analysis. Results are given as means ± SEM of n experiments in triplicate (binding studies) or n experiments (superfusion studies). Student’s t-test was used for comparison of mean values; if more than one experimental series was compared to the same control,

Fig. 1 Effects of Mr 2266 (䊉;䊏) and Mr 2267 (䊊;ⵧ) on the binding of [3H]U-69,593 (䊉;䊊) and [3H]nociceptin (䊏;ⵧ). Membranes were incubated with 1.0 nM [3H]U-69,593 or 0.5 nM [3H]nociceptin and increasing concentrations of the competitor for 60 min. Results, which represent means ± SEM of 6 experiments in triplicate, are given as percent of controls (not shown). For some data points, SEM is contained within the symbol

the Bonferroni correction was applied. In order to evaluate whether the inhibition of [3H]nociceptin and [3H]U-69,593 binding by the drugs under study was better fitted by a one- or a two-site model, the F-test was used. Drugs used. Mr 2266 [(-)-(1R, 5R, 9R)-5,9-diethyl-2-(3-furylmethyl)-2’-hydroxy-6,7-benzomorphan], Mr 2267 [(+)-(1S,5S,9S)5,9-diethyl-2-(3-furylmethyl)-2’-hydroxy-6,7-benzomor-phan; Boehringer Ingelheim, Ingelheim, Germany]; naloxone hydrochloride, U-69,593 [(5α,7α,8β)-(+)-N-(7-[1-pyrrolidinyl]-1-oxa-spiro[4,5] dec-8-yl)benzenacetamide; Sigma, Munich, Germany]; nociceptin (Bachem, Heidelberg, Germany); [leucyl-3H]-nociceptin (specific activity 157–170 Ci/mmol; Amersham, Braunschweig, Germany); R(-)-[ring-2,5,6-3H]-noradrenaline (specific activity 46.8 Ci/mmol), [phenyl-3,4-3H]-U-69,593 (specific activity 39.7 Ci/mmol; NEN, Zaventem, Belgium); desipramine hydrochloride (Ciba-Geigy, Wehr, Germany); rauwolscine hydrochloride (Roth, Karlsruhe, Germany).

Results Binding studies In saturation binding studies on mouse brain cortex membranes, using [3H]U-69,593 and [3H]nociceptin at various concentrations, a dissociation constant (Kd) of 2.15±0.22 nM and 1.35±0.20 nM, respectively, with a maximum number of binding sites (Bmax) of 24±5 fmol/mg protein and 520±40 fmol/mg protein, respectively, was determined (both n=3). Scatchard analysis (not shown) revealed a straight line with a Hill coefficient (nH) of unity for both radioligands. Mr 2266 and less potently Mr 2267 inhibited binding of [3H]U-69,593 whereas the binding of [3H]nociceptin was equipotently inhibited by both enantiomers (Fig. 1; nH values near unity; for pKi values, see Table 1).

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Superfusion studies

Table 1 Affinity and antagonistic potency of Mr 2266 and Mr 2267 at the ORL1 and κ-receptor. Values were derived from the concentration-response curves shown in Fig. 1 (pKi), Fig. 2 (pA2 at ORL1 receptor) and Fig. 3 (pA2 at κ-receptor). Means ± SEM of 6–10 experiments

Basal tritium efflux (expressed as t1 or t2/t1) in control experiments on mouse, rat and guinea-pig brain cortex slices superfused in the presence of desipramine 1 µM and rauwolscine 1 µM was as in our previous study (Schlicker et al. 1998). Basal tritium efflux (t1) was not altered by Mr 2266 or Mr 2267, 0.32 µM each, but increased by Mr 2266 10 µM (by 75±27%, P<0.05) and by Mr 2267 10 µM (by 106±30%, P<0.05). In the experiments with Mr 2266 and Mr 2267, 10 µM each, the t2/t1 ratio was however not altered (not shown). None of the other drugs under study affected basal tritium efflux (t1 or t2/t1). The electrically evoked tritium overflow was expressed as S1 or S2/S1. The S1 value in mouse cortex slices superfused in the presence of desipramine 1 µM and rauwolscine 1 µM was 9.59±1.03% of tissue tritium (n=6). It exceeded (P<0.05) S1 in cortex slices of the rat (4.96±0.62, n=6) and the guinea pig (4.53±0.36, n=10). The S2/S1 values in the various series of control experiments are given in the legends to Figs. 2 and 3. In the experiments on mouse brain cortex slices in which the effect of nociceptin was studied, the superfusion medium routinely contained naloxone 10 µM in addition to desipramine 1 µM plus rauwolscine 1 µM. The electrically evoked tritium overflow (S2/S1) was inhibited by nociceptin in a concentration-dependent manner (Fig. 2). The maximum inhibition, obtained at 1 µM, was 80% (Fig. 2); the pEC50 (concentration causing an inhibition by 40%) was 7.46±0.07. Mr 2266

and Mr 2267, 10 µM each, shifted the concentration-response curve of nociceptin equally to the right without affecting its maximum effect (Fig. 2; for pA2 values, see Table 1). Mr 2266 and Mr 2267 (each in the presence of naloxone) and naloxone itself did not affect the evoked overflow (S1; not shown). In the experiments on mouse, rat and guinea-pig cortex slices in which the effect of U-69,593 was examined, the medium contained desipramine plus rauwolscine but no naloxone. U-69,593 did not affect the evoked overflow (S2/S1) in mouse and rat cortex slices but caused a concen-

Fig. 2 Effect of nociceptin (䉭) on the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]noradrenaline, and antagonism by Mr 2266 (䊏) and Mr 2267 (ⵧ). Slices were superfused with medium containing desipramine 1 µM, rauwolscine 1 µM plus naloxone 10 µM and, when relevant, Mr 2266 10 µM or Mr 2267 10 µM throughout superfusion. Nociceptin was present in the medium from 62 min of superfusion onward. Tritium overflow was evoked twice, after 40 min (S1) and 90 min (S2) of superfusion, and the overflow ratio S2/S1 was determined. Results, which are means ± SEM of 4–9 experiments, are given as a percentage of the S2/S1 values of corresponding controls (no nociceptin; not shown). The S2/S1 values in controls were 1.19±0.07 (䉭), 1.10±0.08 (䊏) and 1.15±0.10 (ⵧ). *P<0.02, **P<0.001, compared to the corresponding controls

Fig. 3 Effect of U-69,593 on the electrically evoked tritium overflow from cerebrocortical slices of guinea pig (䉮), rat (䉬) and mouse (X) preincubated with [3H]noradrenaline, and antagonism by Mr 2266 (䊊) and Mr 2267 (䊉) in guinea-pig cortex slices. Slices were superfused with medium containing desipramine 1 µM, rauwolscine 1 µM and, when relevant, Mr 2266 0.32 µM or Mr 2267 0.32 µM. U69,593 was present in the medium from 62 min of superfusion onward. Tritium overflow was evoked twice, after 40 min (S1) and 90 min (S2) of superfusion, and the overflow ratio S2/S1 was determined. Results, which are means ± SEM of 4–9 experiments, are given as a percentage of the S2/S1 values of corresponding controls (no U69,593; not shown). The S2/S1 values in controls were 0.96±0.04 (∇), 0.81±0.04 (䊉), 0.96±0.06 (䊊), 1.10±0.03 (䉬) and 1.07±0.04 (X). *P<0.001, compared to the corresponding controls

Mr 2266 ORL1 κ

pKi pA2a pKi pA2b

4.82 5.77 9.11 8.81

± ± ± ±

0.11 0.16 0.13 0.10

Mr 2267 5.14 5.64 7.15 7.15

± ± ± ±

0.12 0.14 0.20 0.09

a

Based on the concentrations causing an inhibition of the evoked overflow by 40% in the presence and absence of the Mr compound b Based on the concentrations causing an inhibition of the evoked overflow by 50% in the presence and absence of the Mr compound

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tration-dependent inhibition in guinea-pig cortex slices (Fig. 3). The maximum inhibitory effect, reached at 1 µM, was almost 100% (Fig. 3); the pEC50 (concentration causing an inhibition by 50%) was 7.93±0.06. Mr 2266 and Mr 2267, 0.32 µM each, shifted the concentration-response curve of U-69,593 to the right, Mr 2266 being more effective in this respect than Mr 2267 (Fig. 3; for pA2 values, see Table 1). Mr 2266 and Mr 2267 did not affect the evoked overflow (S1) by themselves (results not shown).

Discussion In the present study Mr 2266, which was identified as an antagonist at the ORL1 receptor in the rat vas deferens (Nicholson et al. 1998), acted as an antagonist also at the ORL1 receptor in the mouse brain cortex. Its stereoisomer Mr 2267 behaved as an antagonist as well, exhibiting about the same potency. These data fit well to the binding studies with [3H]nociceptin in which both enantiomers possessed approximately the same affinity. As opposed to the study of Nicholson et al. (1998), Mr 2266 did not depress the maximum effect of nociceptin. A study with higher concentrations of Mr 2266 and Mr 2267 to clarify the type of antagonism (Schild plot) could not be carried out, however. Note that concentrations of both drugs exceeding 10 µM markedly inhibited electrically induced noradrenaline release by themselves (unpublished). Already at 10 µM both drugs increased basal tritium efflux. However, the Mr compounds were present throughout superfusion and nociceptin (which did not affect basal efflux) was added after the first stimulation. Thus, the basic conditions under which the interactions were investigated were identical throughout the experiments (for a more detailed discussion of this problem, see Göthert et al. 1981). For the sake of comparison, we also determined the affinity of the Mr compounds for the κ-opioid receptor, using binding of the ligand [3H]U-69,593, and their antagonistic potency in a functional κ-opioid receptor model. κ-opioid receptor-mediated inhibition of noradrenaline release in the mouse cortex (present study), like in the rat cortex (Werling et al. 1987; present study), was not detectable; for this reason, we studied κ-receptor-mediated inhibition of noradrenaline release in the guinea-pig cortex (shown previously by Werling et al. 1987). Note that for the (U-69,593-sensitive) κ-receptor, species differences between the mouse and the guinea pig do not seem to exist, as shown by virtually identical binding affinities of various ligands at the expressed κ-receptor from either species (for review, see Knapp et al. 1995). In our study, Mr 2266 was, as expected, more potent than Mr 2267, both in the binding (by 2.0 log units) and the functional κ-opioid receptor model (by 1.7 log units). Acknowledgements We wish to thank Mrs. D. Petri for her technical assistance and the companies Boehringer Ingelheim and CibaGeigy for gifts of drugs.

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