Synthesis and in vivo evaluation of novel radiotracers for the in vivo imaging of the norepinephrine transporter

Nuclear Medicine and Biology 30 (2003) 85–92

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Synthesis and in vivo evaluation of novel radiotracers for the in vivo imaging of the norepinephrine transporter Alan A. Wilson*,a, David Patrick Johnsonc, David Mozleyc, Doug Husseya, Nathalie Ginovarta, Jose Nobregab, Armando Garciaa, Jeffery Meyera, Sylvain Houlea a

PET Centre, Centre for Addiction and Mental Health and Department of Psychiatry, University of Toronto, Toronto, Ontario M5T 1R8, Canada b Dept of Neuroimaging, Centre for Addiction and Mental Health and Department of Psychiatry, University of Toronto, Toronto, Ontario M5T 1R8, Canada c Eli Lilly and Co., Indianapolis, Indiana, USA Received 6 May 2002; received in revised form 25 June 2002; accepted 29 June 2002

Abstract The (R,R) and (S,S) enantiomers of 2-[(2-methoxyphenoxy)phenylmethyl]morpholine (MeNER) have been radiolabelled with carbon-11 in good yield and at high specific activity. These radiotracers are close analogues of reboxetine, a potent and selective ligand for the norepinephrine transporter (NET). They were examined as potential ligands for imaging NET in vivo by positron emission tomography (PET). The in vivo brain distribution of both [11C]-labeled enantiomers were evaluated in rats. Following tail-vein injection of the (R,R)-enantiomer regional brain uptake and washout of radioactivity was homogeneous at all time points examined (5-60 min). In contrast, administration of the (S,S)-enantiomer produced a heterogeneous distribution of radioactivity in brain with highest uptake in the hypothalamus, a NET rich region, and lowest uptake in the striatum, a brain region devoid of NET. Hypothalamus to striatum ratios of 2.5 to one were achieved at 60 min post injection of (S,S)-[11C]-MeNER. Pre-injection of the norepinephrine reuptake inhibitors, reboxetine or desipramine, reduced hypothalamus to striatum ratios to near unity while reuptake inhibitors of dopamine and serotonin had no significant effect on binding. In vitro autoradiography studies (rat brain slices) with (S,S)-[11C]-MeNER produced a regional distribution pattern that was consistent with the reported distribution of NET. (S,S)-[11C]-MeNER has the potential to be the first successful PET ligand to image NET. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Carbon-11; Reboxetine; PET; Norepinephrine transporter; Rat; Autoradiography

1. Introduction The norepinephrine transporter (NET) is a 12 membranespanning protein which plays a major role in terminating the physiological effects of norepinephrine in the synaptic cleft [4,17]. Despite recognition that the norepinephrine transporter (NET) is a site of action of many old (e.g. desipramine) and new (e.g. reboxetine) antidepressant drugs in the brain, there have been only a few attempts to develop radiotracers for imaging NET in vivo in the central nervous system, either by positron emission tomography (PET) or single photon emission computed tomography (SPECT). Haka and Kilbourn reported the synthesis of [11C]-nisox-

* Corresponding author. Tel.: ⫹1-416-979-4286; fax: ⫹1-416-9794656. E-mail address: [email protected] (A. Wilson).

etine which demonstrated modest specific binding in mice [10]. Kung et al. synthesized an iodinated derivative of tomoxetine which showed no specific binding in vivo in rat brain and very high lung uptake [7]. The radiosynthesis of [11C]-desipramine has also been reported but no in vivo data was included [22]. Thus there exists a real need for a radiotracer which is capable of the in vivo tomographic imaging NET. Reboxetine is a potent selective inhibitor of norepinephrine re-uptake and has been marketed as an anti-depressant in several countries [28]. Reboxetine (Fig. 1) has two chiral centers and formally exists as a pair of diastereoisomeric enantiomers, with the (R,R)/(S,S) pair being somewhat more potent than the (R,S/(S/R) pair [15,16]. It is marketed as a mixture of the (R,R) and (S,S) enantiomers, with the (S,S) enantiomer being the more potent and selective at NET with a eudismic ratio of 30 with respect to inhibiting

0969-8051/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0969-8051(02)00420-1

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Fig. 1. Structure of the (S,S) enantiomer of reboxetine and its methyl analogue (S,S)-MeNER.

the reuptake of norepinephrine [29]. The original report on the synthesis and activity of reboxetine [15] also noted that its methyl analogue 2-[(2-methoxyphenoxy)phenylmethyl] morpholine, (methylnorethylreboxetine, MeNER, Fig. 1) was three times more potent than reboxetine itself in inhibiting the reuptake of [3H]-norepinephrine in rat synaptosomes. This, together with the relative ease of introducing [11C]-methyl groups into molecules [8], and an anticipated reduced lipophilicity, made (S,S)-[11C]-MeNER a particularly attractive candidate as a radiotracer for the in vivo imaging of NET. We report here the radiosynthesis of both enantiomers of [11C]-MeNER and assess their potential for PET imaging of NET by examining their in vivo and in vitro binding properties in rat brain using regional dissection techniques and autoradiography. 2. Methods Purifications and analyses of radioactive mixtures were performed by high performance liquid chromatogrpahy (HPLC) with an in-line UV (254 nm) detector in series with a NaI crystal radioactivity detector (synthesis and QC) or a Berhhold LB507A radioactivity detector (metabolite analysis) Isolated radiochemical yields were determined with a dose-calibrator (Capintec CRC-712M). THF was freshly distilled under nitrogen from LiAlH4 while DMF was distilled from BaO and stored over 4Å molecular sieves prior to use. (S,S/R,R)-Reboxetine mesylate, (S,S)-reboxetine mesylate, (S,S/R,R)-MeNER mesylate, (S,S)-(2-(morpholin-2-yl-phenyl-methoxy)-phenol, and (R,R)-(2-(morpholin2-yl-phenyl-methoxy)-phenol were supplied by Eli Lilly (Indianapolis, USA); the last two with unassigned absolute con|figuration (vide infra). All other chemicals were obtained from commercial sources. All animal experiments were carried out under humane conditions, with approval from the Animal Care Committee at the CAMH, and in accordance with the guidelines set forth by the Canadian Council on Animal Care. 2.1. Determination of the absolute configuration of precursors (S,S/R,R)-reboxetine mesylate [16](2 mg) or (S,S)-reboxetine mesylate was partitioned between saturated aque-

ous NaHCO3 (1 mL) and EtOAc (0.3 mL). (S)-␣-Methoxy␣-(trifluoromethyl)phenylacetyl chloride (2 ␮L) was added to the organic layer and the mixture briefly shaken. HPLC analysis showed that the reaction was complete after 2 min. The diastereoisomers were resolved on a Supelco LC-(R)DNBPG 5␮ column (250 x 4.6 mm) with 100/5 (v/v) hexane/ethanol as eluent at 1 mL/min (k’SS 2.9, k’RR 3.5; ␣ ⫽ 1.21). A solution of 0.5 mg 2-(morpholin-2-yl-phenyl-methoxy)-phenol of unknown absolute con|figuration (either SS or RR, vide ante) in DMF (75 ␮L) was treated with tetrabutylammonium hydroxide (3 ␮L, 1N in MeOH) and ethyl iodide (1␮L). After 10 min at ambient temperature, volatiles were removed by bubbling argon gas through the solution. The residue was then derivatised with (S)-␣-methoxy-␣-(trifluoromethyl)phenylacetyl chloride as described above and the product identified as derivatised (S,S)-reboxetine by co-injections with the standard derivatives prepared above. 2.2. (S,S)-[11C]-MeNER [11C]-methylations were carried out inside an HPLC sample loop using our previously described method [25]. Briefly, the normethyl precursor (S,S)-2-(morpholin-2-ylphenyl-methoxy)-phenol, 0.5-0.6 mg) was dissolved in DMF (75 ␮L) and treated with tetrabutylammonium hydroxide solution (1N in MeOH, 3 ␮L). This solution was loaded onto an HPLC injection loop (2 mL) on a Valco™ high performance liquid chromatography (HPLC) injector valve in the load position. [11C]-Iodomethane (produced as previously described [23] was swept into the HPLC loop coated with precursor solution by a stream of N2 gas (8 mL/min) at ambient temperature. Radioactivity trapped on the loop is detected by a proximal radiation detector. When radioactivity peaked in the loop (3-4 min), the flow of N2 was stopped and the reaction allowed to proceed for 4 min at ambient temperature. The contents of the loop were then quantitatively injected onto the HPLC purification column by simply changing the position of the injection valve from the load to the inject position and the reaction mixture purified by semi-prep HPLC; Phenomenex Prodigy C18 10␮ (250 x 10mm, 30% THF:70% H2O ⫹ 0.1N NH4HCO2, 5 mL/min). The desired fraction was collected, evaporated to dryness at 70 °C, and the residue taken up in 10 mL of sterile saline. The saline solution of (S,S)-[11C]-MeNER was passed through a sterile 0.22 ␮m filter into a sterile, pyrogen-free bottle containing aqueous sodium bicarbonate (1 mL, 8.4%). An aliquot (100 ␮L) of the formulated solution was used to establish the chemical and radiochemical purity and specific activity of the final solution by analytical HPLC; Phenomenex Prodigy C18 10␮ (250 x 4.5mm, 30% CH3CN:70% H2O ⫹ 0.1N NH4HCO2, 4 mL/min). Further evidence for the identity of the radiolabelled products were achieved by co-injection with authentic “cold” material using a further two different HPLC columns

A.A. Wilson et al. / Nuclear Medicine and Biology 30 (2003) 85–92

Scheme 1. Radiosynthesis (S,S)-[C-MeNER.

(Alltech C8 Econosil, and Waters C18 Novapak). The optical purity of the radiolabelled product was determined as follows. Two mL of the final formulated solution was added to a vial containing 1 g of NaHCO3 and 0.5 mL of EtOAc. The mixture was shaken vigourously and (S)-␣-methoxy-␣-(trifluoromethyl)phenylacetyl chloride (1 ␮L) added. After two min an aliquot of the organic layer was analyzed by HPLC (Supelco 100/20/2 hexane/dichloroethane/EtOH, 1 mL/min (k’SS 2.76, k’RR 3.39; ␣ ⫽ 1.23). 2.3. (R,R)-[11C]-MeNER This enantiomer was synthesized in an identical manner to the (S,S) enantiomer using the corresponding (R,R)phenolic precursor. 2.3.1. Log P Measurements The partition coefficient of [11C]-MeNER determined between 1-octanol and 0.02 M phosphate buffer at pH 7.4 was measured by a previously described method [27]. 2.3.2. Autoradiography Twenty-micron coronal sections were cut at 0.3 mm intervals from frozen, unfixed rat brains and thaw-mounted onto Fisher Superfrost* slides (Fisher, Canada), dried under vacuum and kept at -80°C until used. All steps of the receptor binding autoradiography procedures were performed in 50-mMTris-HCl buffer, pH 7.4, containing 300 mM NaCl and 5-mM KCl. Slides were brought to room temperature and pre-incubated in buffer for 30 minutes (to remove endogenous ligands). They were then incubated for 10 minutes in 8 nM [11C]-MeNER (547 mCi/␮mol at the start of film exposure). A separate set of slides was incubated for 10 min in 8 nM [11C]-MeNER in the presence of 100 ␮M desipramine in order to define non-specific binding. All slides were then rinsed 3 times for one minute each in ice-cold buffer, and dipped for 10 seconds in ice-cold distilled water. Slides were then quickly dried under cool air and exposed to Kodak Biomax MR film for 24 hr. Films were developed in a Kodak M35A X-omat automated film processor. 2.3.3. Biodistribution in rats Rats (male, Sprague-Dawley, 210-250 g) were kept on a reversed 12-hr light/12-hr dark cycle and allowed food and

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water ad libitum. A previously described method was used to determine the regional brain uptake of radioactivity [24]. Briefly, rats, in a restraining box, received 8-33 MBq of high specific activity radiotracer (1-2 nmole) in 0.3 mL of buffered saline via the tail vein, vasodilated in a warm water bath. Rats were killed by decapitation at various time intervals after radiotracer administration, the brain removed and stored on ice. Brain regions were excised, blotted and weighed and blood collected (from the trunk). Radioactivity in tissues was assayed in an automated gamma counter, back corrected to time of injection, using diluted aliquots of the initial injected dose as standards. Non-specific binding was defined as the %injected dose in the striatum, a brain region containing a negligible density of NET [3,18] and specific binding was defined as %injected dose/gram (brain region) minus %injected dose/gram (striatum). 2.3.4. Blocking studies in rat For pharmacological studies, rats received an injection of a solution (1 mL/kg body wgt., saline or 5% ethanol in saline, pH 4.5, for GBR 12909) of the pharmacological agent while control animals received only saline 15-20 min prior to the radiotracer injection. Animals were killed 60 min post-radiotracer injection and brain regions dissected and counted as described above. 2.3.5. Metabolism studies Rats (male, Sprague-Dawley, 250-300 g) received 200250 MBq of high specific activity (S,S)-[11C]-MeNER (4-5 nmole) in 0.5 mL of buffered saline via the tail vein. Animals were killed at 30 min or 60 min post-injection by decapitation, blood collected from the trunk in a heparinized tube, and the whole brain surgically removed from the skull and stored on ice. Control animals which received no radiotracer were also killed, blood collected, and their brains excised and stored on ice. Two MBq of (S,S)-[11C]-MeNER in 10 ␮L of buffered saline was added to the control brains and control blood only. All brains were homogenized (Polytron, setting 7) in 5 mL of ice-cold 80% aq. ethanol and centrifuged (17,000 rpm, 15 min). The supernatants and pellets were counted for radioactivity, the supernatants were then evaporated to near dryness and reconstituted in 1% aqueous acetonitrile (4 mL) for HPLC analysis. Blood was centrifuged to separate the plasma (2 mL) which was used directly for HPLC analysis. HPLC analysis of plasma and brain extracts were performed by minor modifications of the method described by Hilton [11]. Briefly, samples were loaded onto a 5 mL HPLC injector loop (Valco Texas) and injected onto a small capture column (4.6 x 20 mm) packed in house with OASIS HLB 30␮m, Waters, NJ). The capture column was washed with 1% aqueous acetonitrile (2 mL/min) for four min then back-flushed (30% acetonitile/70% H2O ⫹ 0.1N ammonium formate, pH 4, 2mL/min) onto a Phenomenex 5 ␮

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Fig. 2. Time activity curves in rat brain regions following injection of [11C]-MeNer. A. (S,S)-[11C]-MeNER. B. (R,R)-[11C]-MeNER.

C18Aqua column (150 x 4.6 mm). Both column effluents were monitored by a Berthold LB507A flow radioactivity detector. All radioactivity data was corrected for physical decay and integrated using a PC. Hold-up of radioactivty in the HPLC system was less than 2% of applied radioactivity.

3. Results HPLC conditions were not found which allowed a separation of the (S,S) from the (R,R) enantiomers of reboxetine or MeNER. However the diastereoisomeric amides formed upon reaction with (S)-␣-methoxy-␣-(trifluoromethyl)phenylacetyl chloride were well resolved using a chiral phase HPLC column. This allowed the assignation of the absolute con|figuration of the phenolic precursors by a sequence of O-ethylation and N-acylation, followed by a comparison of the HPLC properties of the diastereoisomeric amides with the amide derived from authentic (S,S)-reboxetine [16]. Radiosynthesis of both enantiomers of [11C]-MeNER (Scheme 1) proceeded smoothly using [11C]-iodomethane,

the corresponding phenolic precursor, and our previously describe “Loop” method [25]. Radiochemical yields were 25-40% (uncorrected, based on [11C]-iodomethane trapped) in a synthesis time of 25 min (from end-of bombardment). Final products were sterile, pyrogen-free and radiochemically pure (⬎ 98%) by TLC and HPLC and contained less than 0.02 ␮g/mL of normethyl precursor. A typical bombardment of 10 ␮Ahr resulted in 100 mCi of final, formulated product with specific activities of 35-65 GBq/␮mole at end-of-synthesis. Optical purities were ⬎97% and the log P (pH 7.4) for [11C]-MeNER was 2.35 (⫾ 0.06, n⫽12). Upon tail vein injection of S,S-[11C]-MeNER in rats moderate brain uptake was observed (0.53% injected dose/ organ at 5 min post-injection) with a slow washout of radioactivity in NET rich regions such as hypothalamus and cortex and a faster washout of activity in the striatum, a region lacking in NET [3,18]. Hypothalamus to striatum ratios of 2.5 to one were reached at 60 min post injection (Fig. 2A). Rank order of region to striatum ratios was hypothalamus ⬎ hippocampus ⬇ cortex ⬇ thalamus ⬇ brain stem ⬎ cerebellum ⬎ striatum. In contrast, no regional retention of radioactivity was found upon injection of the enantiomeric radiotracer R,R-[11C]-MeNER (Fig. 2B).

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Fig. 3. Effects of pre-treatment of rats with various drugs on (S,S)-[11C]-MeNER binding in rat brain regions. Rats were treated with drug or saline (Tail-vein) 15-20 min prior to administration of the radiotracer and killed 60 min post radiotracer injection.

Instead, a relatively homogeneous distribution, with similar washout rates, was found in all examined brain regions. Pre-treatment of rats with the norepinephrine reuptake inhibitors reboxetine [28] or desipramine [12], followed by injection of S,S-[11C]-MeNER, resulted in no specific retention of radioactivity in hypothalamus or other brain regions compared to striatum (Fig. 3.) However pre-treatment of rats with DASB (a serotonin reuptake inhibitor [26]), L-deprenyl (a monoamine oxidase type B inhibitor [9]) or GBR 15909 (a dopamine reuptake inhibitor [21]) had no significant effect on the regional distribution of radioactivity following injection of S,S-[11C]-MeNER compared with saline injected controls (Fig. 3). HPLC analysis of rat plasma showed that S,S-[11C]MeNER was quickly metabolized with only 50% unchanged radiotracer present at 30 min post-injection and 20% after 60 min. Radioactive metabolites were all hydrophilic. HPLC analysis of whole rat brain extracts showed that greater than 95% of radioactivity in the brain was unchanged S,S-[11C]-MeNER. In vitro incubation of slide-mounted rat brain sections with 10 nM [11C]-MeNER for 10 min produced a regional distribution pattern that was remarkably similar to patterns previously reported in autoradiographic assays of [3H]nisoxetine in rat brain [2,20]. As illustrated in Fig. 4, this included high levels in the anteroventral nucleus of the thalamus, the bed nucleus of the stria terminalis, the paraventricular nucleus of the hypothalamus, and the locus co-

eruleus and subcoeruleus area. Coincubation with 100 ␮M desipramine abolished binding in all cases (Fig. 4, panel E). 4. Discussion The radiosynthesis of both enantiomers of [11C]-MENER was rapid and effective. Selective methylation of the phenoxide anion was achieved without protection of the morpholine nitrogen. A reaction time of 4.5 min at ambient temperature was sufficient to ensure ⬎90% conversion of [11C]-iodomethane into product. Curiously, under the employed reverse-phase HPLC conditions using THF as the organic co-solvent, the more lipophilic product eluted before the precursor, enabling a fast and efficient separation (Fig. 5). With acetonitrile as co-solvent, the expected order of elution was observed, i.e. precursor before product. It has been suggested that the “LOOP” technique of performing [11C]-methylations suffers from the disadvantage of HPLC column degradation due to the injection of 2 mL of nitrogen with the reaction mixture onto the column [13]. Such concerns are unfounded. The HPLC column used to produce the chromatograms in Fig. 5 had been used for over two hundred such injections. The marked differences in regional brain uptake of the two carbon-11 labeled strongly suggests that the preferential binding in NET rich regions such as the hypothalamus and thalamus compared with the NET poor striatal region is a reflection of binding of (S,S)-[11C]-MeNER to NET. This

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Fig. 4. Coronal rat brain sections incubated with 8 nM [11C]-MeNER. Abbreviations: CPu; caudate-putamen nucleus; BNSt: Bed nucleus of the stria terminalis; PVN: paraventricular nucleus of the hypothalamus; DR: Dorsal raphe´ nucleus; PPN: Pedunculopontine nucleus; LC: locus coeruleus; SC: Subcoeruleus area. Non-specific binding (panel E) was defined in the presence of 100 ␮M desipramine.

hypothesis is strengthened by the results of drug blocking studies (Fig. 3). Only the NET selective drugs, reboxetine and desipramine, inhibit the preferential regional brain uptake of radioactivity while DASB and GBR 12909, selective drugs for the serotonin transporter and dopamine transporter respectively, have no significant effect on the distribution of radioactivity. Further evidence for (S,S)[11C]-MeNER binding to NET is provided by the pattern of binding obtained from the in vitro autoradiography studies (Fig. 4). A necessary requirement for an imaging radiotracer of central nervous system receptors is a lack of radiolabelled metabolites in the brain. The presence of such radioactive species can reduce signal to noise ratios and, more significantly, can obfuscate the quantification of the imaging process. Only polar metabolites were found in rat plasma by HPLC analysis, following tail-vein injection of (S,S)-[11C]MeNER. Such hydrophilic metabolites are not likely to cross the blood brain barrier, at least by a passive diffusion mechanism. Direct examination of rat brain extracts showed only minor amounts of radioactive metabolites (⬍ 5%), most of which could be accounted for by the contribution from the brain vascular compartment. Unless a species spe-

cific metabolic pathway is followed, radioactive metabolites should not confound the use of (S,S)-[11C]-MeNER in imaging the living human brain using PET. Perhaps one reason why it has proved to be so difficult to develop a radiotracer for in vivo imaging of NET is the low amount of receptor protein in the brain compared with other successfully imaged systems such as DAT, and SERT. Estimates of the Bmax of NET in rat brain regions vary enormously in the literature, [1–3,5,6,18 –20,30]. Nevertheless, it is probable that levels of NET are substantially lower than e.g. SERT or DAT. Brunswick et al found that NET Bmax values in e.g. hypothalamus were at least four fold lower than SERT Bmax values in the same region and 17 fold lower than DAT Bmax in the striatum [5]. Thus in vivo imaging of NET by tomographic techniques represents a significant challenge. (S,S)-[11C]-MeNER has demonstrated promising in vivo pharmacokinetics, pharmacology, and metabolism for a potential in vivo NET radiotracer. To determine whether this ligand provides sufficient signal and contrast to succeed in imaging NET in humans by PET will require human imaging trials. While this work has focused on the imaging of NET in the central nervous system, it should be noted that

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Fig. 5. Chromatograms from the purification of (S,S)-[11C]-MeNER. A. Radioactivity. B. Mass (UV at 254 nM). See text for HPLC condition.

(S,S)-[11C]-MeNER may also be worth evaluating as a radiotracer to image cardiac sympathetic innervation [14].

Acknowledgments The authors wish to thank Ruiping Guo, Lisa Richardson, Kevin Chung, and Roger Raymond for their technical assistance. This work was financially supported in part by Eli Lilly (Canada).

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