N1′-fluoroethyl-naltrindole (BU97001) and N1′-fluoroethyl-(14-formylamino)-naltrindole (BU97018) potential δ-opioid receptor PET ligands

Nuclear Medicine and Biology 29 (2002) 455– 462

N1⬘-fluoroethyl-naltrindole (BU97001) and N1⬘-fluoroethyl-(14formylamino)-naltrindole (BU97018) potential ␦-opioid receptor PET ligands Robin J. Tyackea, Emma S.J. Robinsona, Rebecca Schnabela, John W. Lewisb, Stephen M. Husbandsc, David J. Nutta, Alan L. Hudsona,* a

Psychopharmacology Unit, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK b School of Chemistry, University of Bristol, Bristol, BS8 1TD, UK c Department of Pharmacy and Pharmacology, University of Bath, Bath, UK Received 12 February 2002; accepted 14 February 2002

Abstract The properties of two prospective positron emission tomography (PET) ligands for the ␦-opioid receptor, N1⬘-fluoroethyl-naltrindole (BU97001) and N1⬘-fluoroethyl-(14-formylamino)-naltrindole (BU97018) were investigated. Both were antagonists in the mouse vas deferens, and showed high affinity and selectivity, 1.81 nM and 3.09 nM respectively. [3H]BU97001 binding to rat whole brain was also of high affinity, KD of 0.42 nM of and BMAX of 59.95 fmol mg of protein-1. In autoradiographic studies, it was found to bind to brain areas previously shown to be associated with the ␦-opioid receptor and good correlations were found to exist with naltrindole and DPDPE. BU97018 and especially BU97001 appear to show good potential as ␦-opioid receptor PET ligands with the incorporation of 18F. © 2002 Elsevier Science Inc. All rights reserved. Keywords: ␦-Opioid receptor; N1⬘-fluoroethyl-naltrindole (BU97001); N1⬘-fluoroethyl-(14-formylamino)-naltrindole (BU97018); Naltrindole; Positron emission tomography; Rat brain; Mouse vas deferens; Autoradiography

1. Introduction There is increasing evidence that the opioid receptors play an important role in a number of disease states, for example addiction [7,13], Parkinson’s, Huntington’s, Alzheimer’s [4] and seizure disorders [11] as well as their more traditional role in analgesia. However, little is known about the role that the individual opioid receptors mediate in these conditions. Morphine and morphine based analogues are highly effective analgesics, albeit with some undesirable side effects (depression of respiration, tolerance/dependence, effects on mood). These compounds tend to have their highest affinity for the ␮-opioid receptor, though there is increasing evidence that ␬-opioid and ␦-opioid agonists may have potential as analgesics but without the associated * Corresponding author. Tel.: ⫹44-0-117-925-3066; fax: ⫹44-0-117928-9700. E-mail address: [email protected] (A.L. Hudson).

problems. Similarly, in addiction, the precise roles the individual opioid receptors play in analgesia are also largely unknown. A better understanding of the roles the individual opioid receptors play would lead to the development of better treatments for addiction and the development of improved analgesics. A major reason for the scarcity of knowledge in these areas is the lack of adequate pharmacological tools especially for work in humans. A number of radioligands are currently available for use in positron emission tomography (PET) with which to image opioid receptors in human brain. These are the ␮-opioid receptor ligand [11C]carfentanil, the non selective opioid receptor ligand [11C]diprenorphine, the mixed ␮ and ␬-opioid receptor ligand [18F]cyclofoxy, and the ␦-opioid receptor ligand [11C]methyl naltrindole [12]. Our group has recently reported the synthesis of a number of N-fluoroethyl substituted naltrindole analogues as potential [18F]-␦-opioid PET ligands that would, in theory, demonstrate better temporal qualities than those displayed by

0969-8051/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 2 ) 0 0 3 0 0 - 1

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[11C]methyl naltrindole due to the longer half life of 18F (t1/2 ⫽ 109.6 min) compared with that of 11C (t1/2 ⫽ 20.4 min). Pre-clinical studies, competition binding analysis demonstrated that the best of these were BU97001 and BU97018, which showed selectivity and affinity for the ␦-opioid receptor [2]. Naltrindole is a selective and high affinity antagonist for the ␦-opioid receptor in a wide variety of tissues; mouse and rat vas deferens, guinea pig brain and ileum and the rat brain [17,20]. The aim of the present study was to evaluate these two N-fluoroethyl derivatives of naltrindole, for their functional characteristics (mouse vas deferentia), at the ␦-opioid receptor subtype. The most promising compound was then tritiated and further characterized, saturation radioligand binding analysis and in vitro autoradiography, to determine its ability to specifically bind to the ␦-opioid receptor in rat brain.

2. Methods 2.1. Tissue preparation and bioassay Mice (T/O, male, 20 –30 g) were stunned by a blow to the head and killed by cervical dislocation. Their vas deferentia were dissected out and the connective tissue removed and placed in Mg2⫹-free Krebs solution on ice (118 mM NaCl, 29 mM NaHCO3, 4.7 mM KCl, 1.2 mM KH2PO4, 11.1 mM glucose, 2.6 mM CaCl2 and gassed with 95% O2 and 5% CO2 [6]). The vas deferentia were suspended between two platinum electrodes in an organ bath of 10 ml capacity containing Mg2⫹-free Krebs solution. A constant temperature of 35°C was maintained using a Techne Circulator and the tissue was gassed with 95% O2 and 5% CO2 throughout. An initial tension of 0.5 g was applied to the vas deferentia and the tissue was stimulated with paired shocks with a 10s delay between pulses of 0.5 ms, delivered at a rate of 50 Hz, modified from the method of Ronai et al. [18]. The electrically induced contractions were detected by a mechanoelectrical transducer and displayed on a potentiometric recorder. Prior to all experiments the tissue was allowed to stabilize for approximately 30 min, until the contractions were constant. Throughout the stabilization period the preparation was washed with Mg2⫹free Krebs at regular intervals. Cumulative dose response curves, for the inhibition of electrically induced contractions, were constructed for the selective opioid receptor agonists DPDPE (␦), DAMGO (␮) and U69593 (␬). The effect of a single concentration of test compound on these dose response curves was determined. The tissue was incubated with the test compound for 5 min prior to the construction of the second cumulative dose response curve. The agonists and test compounds were added directly to the Mg2⫹-free Krebs solution bathing the tissue.

2.2. Saturation binding studies Crude P2 brain membranes were prepared as follows, all procedures were carried out a 4°C unless otherwise stated, rat brains (male, Wistar, 250 –300 g) were homogenized in 10 vols of ice cold buffer (50 mM TRIS HCl, 1 mM MgCl2 and 320 mM sucrose, pH 7.4). The homogenate was centrifuged (1000 g for 10 min) and the precipitate discarded. The supernatant was centrifuged a second time (32,000 g for 20 min) and the supernatant discarded, with the remaining precipitate making up the crude P2 membrane preparation. This was washed twice in excess buffer (50 mM TRIS HCl, 1 mM MgCl2) at room temperature, 30 ml were added, the precipitate re-suspended and centrifuged (32,000 g for 20 min). The washed membrane preparations were stored at ⫺70°C until use. Prior to use they were thawed and washed twice (as above). Membrane aliquots (400 ␮l, 0.2– 0.3 mg protein) were incubated with 9 concentrations of [3H]BU97001 over the range 0.01–3 nM in the absence or presence of 10 ␮M naloxone, total and non-specific binding respectively, in a final volume of 500 ␮l. Each incubation was performed in triplicate, at room temperature and allowed to reach equilibrium (1 hr). Bound and free radioactivity were separated by vacuum assisted rapid filtration through pre-soaked (0.5% polyethleneimine) glass-fiber filters (Whatman GF/B) using a Brandel M-24 cell harvester. The filters were then washed twice with 5 ml of ice-cold buffer and the membrane bound radioactivity remaining on the filters was determined by liquid scintillation counting (Emulsifier-safe, Packard; Wallac 1409 ␤-counter). Protein content was determined throughout using the method of Bradford [1], bovine serum albumin was used to construct the standard curves. 2.3. [3H]BU97001 autoradiography Rats (male, Wistar, 250 g) were anesthetized using sodium pentobarbital (60 mg kg-1 i.p.) and perfused (intracardiac) with ice-cold phosphate (10 mM) buffered saline (pH 7.4, 1 ml g-1). Brains were carefully removed and rapidly frozen in isopentane cooled on dry ice. Sections of frozen brain were cut (12 ␮m thick; transverse or parasagittal in the plane of the atlas of Paxinos and Watson, 1986) using a cryostat (2800 Frigocut, Reichert-Jung, Germany) at ⫺16°C to ⫺20°C and thaw mounted on to gelatin coated glass microscope slides. These were stored at –70°C until use. Sections were thawed and pre-washed for 15 min at room temperature (21–25°C) in buffer (50 mM TRIS HCl, 1 mM MgCl2) and dried under a stream of cool air. Sections were incubated for 90 min room temperature, in 200 ␮l of buffer containing [3H]BU97001 (1 nM), with or without 10 ␮M naloxone, to measure total or non-specific binding respectively. Incubations were terminated by aspirating off the free ligand and the sections washed by three 10 min rinses in ice cold buffer, followed by a dip rinse in ice cold distilled water. The sections were dried rapidly under a

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stream of cool air and apposed to [3H] Hyperfilm (Amersham, UK) with [3H] microscale standards (Amersham, UK), in X-ray cassettes, at room temperature for 6 –10 weeks. The films were developed in Ilford High Contrast developer for 3– 4 min, immersed in an acetic acid (0.01%) stop bath for 20 sec, fixed with Ilford Hypam fixer plus Ilford rapid hardener for 6 – 8 min, washed for 20 min in distilled water with wetting agent to prevent spotting and allowed to air dry. The films were then quantified by computer assisted densitometry (MICD 5) and the values converted to fmol [3H]ligand mg-1 wet tissue equivalent using calibrated [3H] microscale standards. For each brain section or region analyzed the area was outlined using the tools provided (MCID 5) and an average densitometric determination made. The value from each section or region, from each animal, was then expressed as the mean ⫾ S.D., (n ⫽ 3). 2.4. Analysis of data Data were analyzed with iterative non-linear regression curve fitting procedures supplied with GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego California USA). Each experiment was analyzed independently and the EC50 of the agonist in the presence and absence of the test compound was used to calculate the dose ratio. This in turn was used to calculate the equilibrium dissociation constant (Ke) using the “single dose method” of Osterlitz and Watt [14]. Values are given as the mean ⫾ S.D. for 3 to 4 separate experiments. Similarly each individual saturation binding assay was analyzed independently and the KD and BMAX determined and the resulting values given as the mean ⫾ S.D. for 5 separate experiments. Correlation analysis was performed using Pearson Product Moment Correlation, SigmaStat for Windows ver. 2.03, the correlation was determined to be significant if P ⬍ 0.05. 2.5. Drugs and chemicals BU97001, BU97018 and naltrindole were synthesized by Prof. John W. Lewis’s group, University of Bristol as previously described [2]. [3H]BU97001 (specific activity, 1.2 TBq mmol-1) was custom synthesized by SmithKline Beecham Pharmaceuticals, King of Prussia, PA 194060939, USA, from a di-brominated precursor, BU98009, also prepared by Prof. John W. Lewis’s group. Naloxone, naloxonazine and nor-BNI were obtained from Tocris Cookson Ltd, UK. DPDPE, DAMGO and U69593 were obtained from Sigma-Aldrich Co. Ltd, Poole, UK.

3. Results 3.1. Bioassay BU97001, BU97018 and naltrindole were tested against the ␦-opioid receptor antagonist DPDPE in the isolated

Fig. 1. Cumulative agonist dose response curves for the inhibition of electrically induced contractions of mouse vas deferens, by the ␦-opioid agonist DPDPE in the presence and absence of the antagonists, naltrindole (50 nM, panel a) BU97001 (100 nM, panel b) and BU97018 (100 nM, panel c). Data were analyzed as described in text; each value represents the mean ⫾ S.D. (vertical bars) from 3 or 4 experiments.

mouse vas deferens preparation, to determine their functional characteristics. Fig. 1 shows the agonist dose response curves in the absence and presence of 50 nM naltrindole (panel (a)) and 100 nM BU97001 and BU97018 (panels (b) and (c) respectively). Naltrindole, the ␦-opioid antagonist, had no inhibitory effect on the electrically induced contractions of the mouse vas deferens alone, but antagonised the effect of DPDPE, shifting the dose response curve to the right, Ke values were determined (Table 1), using the single dose method [14]. Naltrindole also antagonised the effects of DAMGO and U69593, shifting the dose

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Table 1 Equilibrium dissociation constant (Ke, nM) and apparent selectivity for the compounds antagonising the effect of DPDPE, DAMGO and U69593 on the electrically induced contractions of mouse vas deferens

Table 2 List of brain regions their abbreviations and values for the specific binding of [3H]BU97001 Brain region

Abbreviation

Specific binding (fmol mg tissue⫺1)

Frontal cortex Olfactory bulb External plexiform layer Internal granular layer Anterior olfactory nucleus Nucleus accumbens Claustrum Olfactory tubercle Corpus callosum Caudate putamen Cingulate cortex Lateral ventricular Globus pallidus Piriform cortex Internal capsule Cortex Laminae 1–3 Laminae 4 Laminae 5–6 Retrosplenial granular cortex Hippocampus (entire) Dentate gyrus Mediodorsal thalamic nucleus Dorsomedial hypothalamic nucleus Hypothalamus Cerebellum 4th ventricular Thalamus

Fr2

31.60 ⫾ 10.78

Epi Igr AO Acb Cl Tu fmi CPu Cg LV GP Pir ic

60.01 ⫾ 10.24 39.62 ⫾ 14.11 27.67 ⫾ 13.12 25.69 ⫾ 11.27 30.65 ⫾ 13.22 39.43 ⫾ 12.08 9.81 ⫾ 2.36 26.51 ⫾ 2.19 22.89 ⫾ 1.07 18.79 ⫾ 2.92 7.95 ⫾ 7.04 21.64 ⫾ 17.73 6.19 ⫾ 3.54

Lam1-3 Lam4 Lam5-6 Rsg Hippo DG MDM DMD Hyp Cere 4V Thal

24.48 ⫾ 12.61 19.11 ⫾ 3.66 20.95 ⫾ 3.39 5.54 ⫾ 4.54 9.53 ⫾ 1.91 11.94 ⫾ 9.78 7.14 ⫾ 6.46 9.04 ⫾ 5.66 8.03 ⫾ 5.66 10.03 ⫾ 15.06 9.20 ⫾ 8.62 7.94 ⫾ 4.00

Opioid receptor type

Naltrindole BU97001 BU97018

␦ Ke (nM)

␮ Ke (nM)

␬ Ke (nM)

5.22 ⫾ 0.96 1.81 ⫾ 1.57 3.09 ⫾ 3.31

46.0 ⫾ 39.2 47.6 ⫾ 5.8 84.1 ⫾ 18.4

209.0 ⫾ 91.5 160.5 ⫾ 103.7 20.6 ⫾ 17.1

Selectivity

␮/␦

␬ /␦

9 26 27

40 89 7

Data were analysed as described in text; each value represents the mean ⫾ S.D. from 3 or 4 experiments.

response curves to the right, but with a lower affinity. BU97001 antagonised the effect of the agonists at all three opioid receptors, but showed the greatest affinity at the ␦-opioid receptor. At this receptor the effect of DPDPE was inhibited with an affinity (Ke) of 1.81 ⫾ 1.57 nM. At the other opioid receptors BU97001 inhibited the agonists with lower affinities DAMGO, 47.6 ⫾ 5.8 nM, and U69593, 160.5 ⫾ 103.7 nM. BU97018 also antagonised the effect of all three agonists and again showed the greatest affinity at the ␦-opioid receptor Ke ⫽ 3.09 ⫾ 3.31 nM. At the other opioid receptors it inhibited the agonists with much lower affinities (see Table 1). BU97001 also appeared to inhibit the electrically induced contractions of the mouse vas deferens when added on its own. It caused full inhibition with an EC50 of 2.3 ␮M. However this action could not be blocked by 100 nM of known selective opioid antagonists naltrindole (␦), naloxonazine (␮) and nor-BNI (␬) (data not shown), indicating that it was not an agonist at any of the opioid receptor types. 3.2. Saturation binding [3H]BU97001 binding to rat whole brain membranes was saturable and of high affinity, over the concentration range 0.1–3 nM. Fig. 2 shows the specific binding of [3H]BU97001 over this concentration range and represents

Data analysed as described in the text and expressed as the means ⫾ S.D. for 3– 4 animals.

the cumulative results from five separate experiments. Analysis of these data using iterative non-linear curve fitting indicated that the [3H]BU97001 binding was best fitted to a single population of sites and the values for the KD and BMAX were determined to be 0.42 ⫾ 0.23 nM and 59.95 ⫾ 23.64 fmol mg of protein-1 respectively. 3.3. Autoradiography

Fig. 2. Saturation binding analysis of [3H]BU97001 to whole rat brain membranes. Data represent the mean ⫾ S.D. (vertical bars) of five separate experiments performed in triplicate.

Quantitative analyses of the autoradiographs from rat brain sections obtained from three rats are summarized in Table 2. The highest specific binding of [3H]BU97001 was seen in the areas of the forebrain. There was uniformly high binding in the frontal cortex, the anterior olfactory nucleus, the nucleus accumbens and the claustrum (approximately 25 to 31 fmol mg of tissue-1). The highest levels of specific binding were seen in the external plexiform layer (60.01 ⫾ 10.24 fmol mg of tissue-1) and the internal granular layer (39.62 ⫾ 14.11 fmol mg of tissue-1) of the olfactory bulb and the olfactory tubercle (39.43 ⫾ 12.08 fmol mg of tissue-1). Caudal to these areas the specific binding in the brain regions was low (less than 10 fmol mg of tissue-1). Exceptions to this were the caudate putamen (26.51 ⫾ 2.19

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Fig. 3. Representative autoradiographs of the total binding of [3H]BU97001 to rat brain sections. Panels (a)–(e) show the binding at different regions of the brain relative to bregma, (a) is 5.20 mm from bregma, (b) is 2.70 mm from bregma, (c) is 1.60 mm from bregma, (d) is ⫺0.92 mm from bregma and (e) is ⫺2.80 mm from bregma. Measurements were determined from the rat brain atlas of Paxinos and Watson (1986). Fr-Frontal cortex, Epi-External plexiform layer of the Olfactory bulb, fmi-corpus callosum, Tu-Olfactory tubercle, Cg-Cingulate cortex, CPu-Caudate putamen, LV-Lateral ventricular, GP-Globus pallidus, Hippo-Hippocampus, Thal-Thalamus and Hyp-Hypothalamus. Scale bar represents 5mm.

fmol mg of tissue-1), the cingulate cortex (22.89 ⫾ 1.07 fmol mg of tissue-1), the lateral ventricular (18.79 ⫾ 2.92 fmol mg of tissue-1) and the cortical laminae (19 to 24 fmol mg of tissue-1). Fig. 3 shows representative autoradiographs of the binding of the total and non-specific binding of [3H]BU97001 to different brain regions, moving caudally from panel (a) to panel (e). The regions of the brain were determined relative to bregma using the rat brain atlas of Paxinos and Watson [15]. Bregma is an anatomical reference point on the skull where the coronal and sagittal suture lines meet. 4. Discussion and conclusions Our group recently reported the synthesis and initial evaluation of a number of ␦-opioid ligands with the goal of

producing a suitable PET ligand [2]. The two compounds from that study which showed the best affinity and selectivity in competition binding, BU97001 and BU97018, were taken forward and investigated for their functional characteristic in the isolated tissue preparation of the mouse vas deferens. The more selective of these compounds, BU97001, was tritiated at this time and its in vitro binding characterized. Both compounds, BU97001 and BU97018, showed antagonism of the electrically induced contraction of the mouse vas deferens by shifting the agonist dose response curves to the right for all of the different agonists tested. BU97001 antagonised the action of DPDPE with high affinity, but showed lower affinity in its antagonism of DAMGO and U69593 responses showing around 26 fold

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selectivity for the ␦-opioid receptor over the ␮-opioid receptor and 89 fold selectivity for the ␦-opioid receptor over the ␬-opioid receptor. BU97018 also antagonised the effect of DPDPE and the other opioid agonist DAMGO and U69593 and similarly to BU97001 it demonstrated the best antagonism towards the ␦-opioid receptor agonist DPDPE. Its selectivity for the ␦-opioid receptor over the ␮-opioid receptor, 27 fold, was comparable with that of BU97001. However, the affinity and selectivity for the ␦-opioid receptor over the ␬-opioid receptor were lower for BU97001. The values obtained for BU97001 and BU97018, not only being comparable with each other, also showed good comparisons with the known ␦-opioid antagonist naltrindole (see Table 1). This was not unexpected, as it is the parent compound of BU97001 and structurally very similar to BU97018. The fact that the additions of the fluoroethyl groups had only a small effect on affinity or selectivity is advantageous for the future addition of the PET nucleotide 18F. On its own BU97001 also displayed agonist properties in the mouse vas deferens, by inhibiting the electrically induced contractions, albeit with low potency (EC50 of 2.3 ␮M). This effect could not be blocked by 100 nM of the selective opioid antagonists naltrindole (␦), naloxonazine (␮) or nor-BNI (␬) indicating that this effect was not being mediated via opioid receptors. Neither naltrindole nor BU97018, though both structurally very similar, showed any agonist like properties in this system. The mechanism through which BU97001 has its agonist effect is presently unknown. The total number of binding sites labeled by [3H]BU97001 (BMAX ⫽ 59.9 ⫾ 23.6 fmol mg of protein-1) was very similar to those previously reported for the parent compound naltrindole and the selective ␦-opioid ligand DPDPE 63.4 ⫾ 2.0 fmol mg of protein-1 [20] and 79.9 fmol mg of protein-1 [5] respectively in rat brain. The affinity shown by [3H]BU97001 was high (0.42 ⫾ 0.23 nM) and was in good agreement with its Ki at the ␦-opioid receptor (0.24 nM) as determined by the inhibition of [3H]DPDPE binding [2]. It showed a lower affinity than its parent compound naltrindole (37 pM [20]; 0.25 nM [3], but this was not unexpected as the addition of the fluoroethyl group has been previously shown to reduce the affinity and selectivity (as above and [2]). The autoradiographic analysis of the specific binding of [3H]BU97001 to brain sections showed binding to the areas previously shown to be labeled by both the parent compound naltrindole [3] as well as the selective ␦-opioid receptor ligand DPDPE [5]. Highest levels of binding were observed in regions such as the external plexiform layer and the internal granular layer of the olfactory bulb, the olfactory tuderacle, the nucleus accumbens, the claustrum and the caudate putamen. This is shown graphically in Fig. 4. Panel (a) shows the correlation of the autoradiographical binding and distribution of [3H]BU97001 with [3H]naltrindole (values taken from [3]), r ⫽ 0.737 and P ⫽ 0.00003. While panel (b) shows the correlation between

[3H]BU97001 and the highly selective ␦-opioid receptor ligand [3H]DPDPE (values taken from [5]), r ⫽ 0.77 and P ⫽ 0.005. There are some discrepancies between the autoradiography of these ligands compared with [3H]BU97001 the main one was that there was a moderate level of specific binding in the lateral ventricle and it appeared that the [3H]BU97001 was binding to the choroid plexus which neither DPDPE nor naltrindole were reported to do. An area of concern with this ligand was that its non specific binding in both the autoradiography and saturation binding was higher than that reported for [3H]naltrindole [3,20] or [3H]DPDPE [5]. This higher non-specific binding is probably due to the increased lipophilicity conferred on the BU97001 by the fluoroethyl group. Also, it should be noted that in these investigations no BSA was added to help reduce the level of non-specific binding as was in those reports of the binding of [3H]naltrindole [3,20] or [3H]DPDPE [5]. The opioid PET ligands currently available are not without their problems, [11C]diprenorphine is a non-selective antagonist and [11C]carfentanil and [11C]buprenorphine are agonist and partial agonist respectively, which can give rise to undesirable side effects [16]. At present the only “purpose built” opioid receptor PET ligand is the ␦-opioid receptor N1⬘-([11C]methyl)naltrindole [8] which has been used to image and quantify [19] ␦-opioid receptors in the human brain. Both BU97001 and BU97018 appear to be comparable with, if not slightly better, than N-methyl-naltrindole in our hands [2]. Additionally as they use the more stable radioisotope 18F (t1/2 109.6 min) and not 11C (t1/2 20.4 min) they should have better temporal properties. A preliminary account of this work was presented to the 1st European Opioid Conference, Guildford, UK in April 1997. Subsequent to this Mathews et al., have disclosed their data on N1⬘-fluoroethyl-naltrindole showing that they also find it has high affinity and selectivity in rat brain homogenates in vitro (Ki ⫽ 93pM) [9] and that N1⬘-[18F]fluoroethyl-naltrindole shows specific binding to ␦-opioid receptors in mouse brain [10]. The aim of the program of work was to produce a ligand that was easily amenable to labeling with a suitable PET radioligand, in this case 18F, which had good affinity and selectivity for the ␦-opioid receptor and was an antagonist to ensure it had limited biological action. BU97001 appears to fit these criteria well. It has sub nanomolar affinity in saturation analysis as well as in competition binding where it also demonstrates reasonable selectivity [2]. This affinity and selectivity is also seen in the mouse vas deferens. The correlations between DPDPE and naltrindole with [3H]BU97001 autoradiography are very good indicating that it is binding to the same sites. The fluoroethyl moiety can readily be introduced into the molecule as a final step. Although the initial binding data [2] indicated that its selectivity was not one hundred percent ideal as a PET ligand, it certainly warranted further investigation. However, taking into account the saturation, functional and in vitro autoradiographic data, as well as the limitations of the currently

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Fig. 4. Panel A shows the correlation of the autoradiographical binding and distribution of [3H]BU97001 with [3H]naltrindole (values taken from Drower et al. (1993)), r ⫽ 0.737 and P ⫽ 0.00003. Panel B shows the correlation between [3H]BU97001 and the highly selective ␦-opioid receptor ligand [3H]DPDPE (values taken from Gulya et al. (1986)), r ⫽ 0.77 and P ⫽ 0.005 (Pearson Product Moment Correlation, SigmaStat for Windows ver. 2.03). The numbered squares refer to different brain areas; 1-retrosplenial granular cortex, 2-internal capsule, 3-globus pallidus, 4-hypothalamus, 5-hippocampus (entire), 6-dentate gyrus, 7-thalamus, 8-piriform cortex, 9-laminae 4, 10-anterior olfactory nucleus, 11-claustrum, 12-frontal cortex, 13-laminae 5– 6, 14-laminae 1–3, 15-internal granular layer, 16-olfactory tubercle, 17-caudate putamen, 18-nucleus accumbens, 19-external plexiform layer.

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available opioid ligands, we conclude that this ligand shows potential as a possible ␦-opioid receptor PET ligand. Although only BU97001 was tritiated at this time the similarity it shares with BU97018 both in the competition binding studies [2] and the mouse vas deferens suggest that BU97018 too shows potential as another ␦-opioid receptor PET ligand.

[9]

[10]

Acknowledgments [11]

This work was supported by an MRC ROPA award, number G9513279. [12]

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