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6-[18F]fluoro-A-85380, a novel radioligand for in vivo imaging of central nicotinic acetylcholine receptors
Life Sciences 67 (2000) 463Ð469
Pharmacology letters Accelerated communication
6-[18F]ßuoro-A-85380, a novel radioligand for in vivo imaging of central nicotinic acetylcholine receptors Andrew G. Hortia, Svetlana I. Chefera, Alexey G. Mukhina, Andrei O. Korena, Daniela GŸndischa, Jonathan M. Linksa,b, Varughese Kuriana, Robert F. Dannalsb, Edythe D. Londona,* a
Brain Imaging Center, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA b Divisions of Nuclear Medicine and Radiation Health Sciences, Johns Hopkins Medical Institutions, Baltimore, MD 21205, USA (Submitted June 29, 1999; accepted September 10, 1999; received in Þnal form October 22, 1999)
Abstract A novel positron emission tomography (PET) radiotracer, 6-[18F]ßuoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[18F]ßuoro-A-85380, 6-[18F]FA) was synthesized by a no-carrier-added ßuorination. In vitro 6-[18F]FA bound to nicotinic acetylcholine receptors (nAChRs), with very high afÞnity (Kd 28 pM). In PET studies, 6-[18F]FA speciÞcally labeled central nAChRs in the brain of the Rhesus monkey and demonstrated highest levels of accumulation of radioactivity in brain regions enriched with the a4b2 subtype of nAChR. 6-[18F]FA exhibited a target-to-non-target ratio (estimated as radioactivity in the thalamus to that in the cerebellum) of binding in primate brain similar to that previously determined for a labeled analog of epibatidine, [18F]FPH. In contrast to [18F]FPH, the novel tracer is expected to exhibit substantially less toxicity. Thus, the novel radioligand, 6-[18F]FA, appears to be a suitable candidate for imaging nAChRs in human brain. Published by Elsevier Science Inc. Keywords: Nicotinic acetylcholine receptor; Positron emission tomography (nAChRs); 6-[18F]ßuoro-3-(2(S)azetidinyl-methoxy)pyridine (6-[18F]ßuoro-A-85380, 6-[18F]FA)
Introduction Efforts to develop radiolabeled ligands for a variety of brain receptors and advances in neuroimaging techniques have contributed to the substantial progress in noninvasive imaging
* Corresponding author. Tel.: 410-550-1540; fax: 410-550-1441. E-mail address: [email protected] (E.D. London) 0024-3205/00/$ Ð see front matter Published by Elsevier Science Inc. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 3 5 -4
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of neuroreceptors during the last two decades. Remarkably, unlike the case for several other types of central receptors, imaging of nicotinic acetylcholine receptors (nAChRs) has been limited by the lack of suitable radiotracers. Development of radiotracers for quantitative imaging of nAChRs would be very useful because these receptors, which modulate excitatory neurotransmission, contribute to a variety of brain functions and behavioral states, including learning and memory, attention and anxiety (1). Nicotinic acetylcholine receptors have been implicated in various neurodegenerative disorders, including AlzheimerÕs and ParkinsonÕs diseases, where postmortem brain tissue examination showed profound deÞcits in nAChR concentrations (1, 2). Recent studies have suggested that expression of an abnormal variant of the gene coding for the a7 subtype of nAChRs is associated with the deÞcit in sensory gating that is seen in schizophrenia (3) and that a missense mutation of the a4 subunit of nAChRs can lead to inherited epilepsy (4). These receptors also mediate the action of nicotine, an active ingredient that promotes dependence on tobacco (1). Furthermore, nicotinic agonists show promise as therapeutic agents for several diseases, including ParkinsonÕs disease and TouretteÕs syndrome, and as non-opioid analgesics (1, 5). Imaging nAChRs with [11C]nicotine (6) was hindered due to the high nonspeciÞc binding and rapid egress of the tracer from human brain. Subsequently, the discovery of epibatidine, a highly potent and speciÞc agonist at nAChRs, stimulated the development of its radiolabeled analogs, [3H]epibatidine (7), (6)-exo-2-(2-[18F]ßuoro-5-pyridyl)-7-azabicyclo[2.2.1]heptane ([18F]FPH) (8) and (6)-exo-2-(2-[123I]iodo-5-pyridyl)-7-azabicyclo[2.2.1]heptane ([123I]IPH) (9), as promising nAChR radiotracers for binding assay studies, positron emission tomography (PET), and single photon emission computed tomography (SPECT), respectively. However, epibatidine produces activation of nAChRs of the ganglionic (a3b4) subtype at very low doses (10). Therefore, labeled analogs of epibatidine, [18F]FPH and [123I]IPH, exhibit untoward effects, such as prolonged catecholamine release at doses approximately 30Ð100 times the mass that would be needed for in vivo imaging (11, 12). A more recently synthesized highly selective nAChR ligand A-85380 [3-(2(S)-azetidinylmethoxy)-pyridine] (10) is equipotent to epibatidine in in vitro binding assays with the human a4b2 subtype of nAChRs. However, it is only 1/100th as potent as epibatidine in activating the a3b4 subtype of nAChRs, which is at least partially responsible for the observed adverse effects of epibatidine (10). Based on these observations, we hypothesized that radiolabeled derivatives of A-85380 would maintain high afÞnity in labeling central nAChRs in vivo and would provide a wider safety margin between an adequate tracer dose and the dose that would produce a biological effect, as compared with epibatidine-based radioligands. Previously, we synthesized a series of halogenated derivatives of A-85380 with high binding afÞnity toward nAChRs and relatively low toxicity (13Ð15) and labeled several of them with isotopes for PET and SPECT imaging (16Ð18). The ßuoro-derivatives of A-85380, 2-ßuoro-3-(2(S)-azetidinylmethoxy)pyridine (2-FA) and 6-ßuoro-3-(2(S)-azetidinylmethoxy) pyridine (6-FA), like FPH, both exhibited very high in vitro binding afÞnities toward nAChRs (respectively, 46 pM and 25 pM (13) vs.37 pM (11)). In in vivo studies, 2-[18F]FA and 6-[18F]FA manifested a very high target-to-non-target ratio of binding and regional distribution in mouse brain matching the distribution of nAChRs (14,15). Unlike FPH, 2-FA and 6-FA demonstrated dramatically lower biological activity in vivo in mice (14,15), identifying the A-85380-based radiotracers as very promising candidates for PET human studies. The
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goal of the present study was to perform PET imaging of nAChRs in monkey brain with 6-[18F]FA in order to investigate its brain distribution, kinetics and speciÞcity toward nAChRs in vivo. Another paper presents our Þndings in PET imaging studies of 2-[18F]FA in monkey brain (19).
Methods Radiochemistry 6-[18F]FA was synthesized by a no-carrier-added nucleophilic radioßuorination through treatment of a dimethylsulfoxide solution of 6-iodo-3-((1-tert-butoxycarbonyl-2(S)-azetidinyl)methoxy)pyridine (13) with anhydrous [KryptoÞx222áK]1[18F]F2 complex at 190oC followed by acidic deprotection of the intermediate compound with trißuoroacetic acid solution in methylene chloride (Fig. 1A), puriÞcation by high-performance liquid chromatography (HPLC), and formulation as a sterile and apyrogenic solution as described previously (15). The radioactive precursors, [18F]ßuoride and [KryptoÞx222áK]1Ð[18F]F2, were produced with a CTI RDS111 negative ion cyclotron and a computer-controlled chemistry module. Receptor binding in vitro Binding assays with 6-[18F]FA were performed as described previously (20). Brießy, P2 membrane fractions (20Ð30 mg protein) from male Fischer-344 rat brain (excluding medulla and cerebellum) were incubated in the presence of 1 Ð 1000 pM 6-[18F]FA in 1 ml of HEPESsalt solution (pH 7.4) at 22 oC for 2 h. Binding was terminated by Þltration through GF/B Þlters pretreated with 1% polyethyleneimine. NonspeciÞc binding was determined in the presence of 500 mM (2)-nicotine. PET studies Two male Rhesus monkeys were used in six PET studies, including Þve control experiments and one displacement assay. The animals were prepared as described previously (19), positioned in a PET scanner (Siemens ECAT EXACT HR1), and a transmission scan was performed for attenuation correction. 6-[18F]FA (4.5Ð14 mCi, corresponding to ,1 nmol/kg) was injected intravenously, followed by PET scanning immediately after the administration. Vital signs, including heart rate, electrocardiogram, oxygen saturation of blood hemoglobin, were monitored using a SpaceLab monitor. A dynamic series of PET data sets were acquired as follows: 5 3 1 min, 5 3 2 min, 5 3 5 min, 8 3 10 min, 10 3 15 min. Each data set consisted of 63 slices over a 15.5 cm axial extent of the brain. The within-plane resolution was approximately 4.5 mm (full width at half maximum) (Fig. 2). The displacement assay was accomplished by subcutaneous injection of 1 mg/kg of cytisine, a high-afÞnity nAChR ligand, 80 min after injection of 6-[18F]FA. Regions of interest (ROIs) were placed in the thalamus, frontal cortex, and cerebellum as previously described (20), and average timeactivity curves (fraction of injected dose normalized to body weight in 1 cm3 of tissue (%ID? kg/cm3 vs. time) were generated (Fig. 1C).
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Fig. 1. A. Radiosynthesis of 6-[18F]FA. B. Results of a typical binding experiment with 6-[18F]FA in membranes from rat forebrain. The average values from this and two additional experiments were: Kd 28 6 2 pM, Bmax 3.7 6 0.4 pmol/g tissue. C. Regional time-activity curves of 6-[18F]FA. Control: averaged from Þve PET imaging studies. Displacement: one PET imaging study with 1 mg/kg cytisine injected subcutaneously (Thal 5 thalamus, Cx 5 cortex and CB 5 cerebellum). D. Average region/cerebellum ratios vs. time after the radiotracer injection in Þve control PET studies.
Results Radiochemistry 6-[18F]FA was synthesized with a radiochemical yield of up to 21.2% (not-decay-corrected, determined by HPLC). Chemical and radiochemical purity, determined by HPLC, exceeded 95%. The speciÞc radioactivity of the tracer was in the range of 1200Ð5400 mCi/mmol at the
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Fig. 2. PET images of Rhesus monkey brain showing binding of 6-[18F]FA with nAChRs. All images were obtained from a displacement study, except the bottom left one, which was obtained from a control study on the same animal. Top: 80-min scan, beginning 40 min after tracer administration. Bottom, right: displacement of 6-[18F]FA by subcutaneous injection of 1 mg/kg cytisine at 80 min after tracer administration; 60-min scan, beginning 3 h after tracer administration . Bottom, left: 60-min scan, beginning 3 h after tracer administration.
end-of-synthesis. The Þnal product was formulated as a sterile solution in saline. Sterility and apyrogenicity were conÞrmed by conventional methods. In vitro binding assay Saturation studies demonstrated that at a concentration of up to 1 nM, 6-[18F]FA interacted with a single population of binding sites (Fig. 1B). The observed afÞnity (Kd 28 6 2 pM, n53) was consistent with the afÞnity of non-labeled authentic 6-FA at the a4b2 nAChR subtype in rat brain measured previously in competition studies (13). A Bmax value was calculated as 3.7 6 0.4 pmol/g tissue, n53 (153 6 17 fmol/mg protein). Brain regional distribution and kinetics A substantial incorporation of radioactivity into the monkey brain was seen after injection of 6-[18F]FA. The highest accumulation of radioactivity (0.11%ID?kg/cm3 at 55 min) occurred in the thalamus. An intermediate level of radioactivity was found in the frontal cortex (0.08%ID?kg/cm3 at 40 min), and the lowest level of radioactivity was detected in the cerebellum (peak value of 0.07%IDákg/cm3 at 30 min) (Fig. 1C). The initial clearance rate of ra-
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dioactivity from the cerebellum was higher than from any other region studied. The t1/2 values for the clearance from the peak levels of radioactivity were .200, 155, and 50 min for the thalamus, frontal cortex, and cerebellum, respectively. As a result, after injection of the radiotracer, the thalamus/cerebellum and frontal cortex/cerebellum ratios increased steadily over the observation period, reaching values of 4.5 and 2.1, respectively, at 4 h after injection (Fig. 1D). Injection of cytisine reduced regional radioactivities in the thalamus and cortex at 220 min after the tracer administration by the level of radioactivity in the cerebellum (Fig 1C). No displacement of radioactivity from the cerebellum was observed. Administration of 6-[18F]FA at doses of up to 1 nmol/kg did not produce pharmacological responses, such as changes in ECG, in heart rate or in concentration of O2 in blood. Discussion We synthesized a novel PET nAChR radioligand, 6-[18F]FA, and found that it bound to a single population of binding sites in vitro at a concentration range of up to 1 nM. The dissociation constant (Kd) of the receptor-ligand complex agreed well with the inhibition constant (Ki) determined previously for non-labeled 6-FA in competition assays with [3H]epibatidine (13). The Bmax value obtained was in good agreement with the density of a4b2 nAChRs in rat brain measured with [3H]-cytisine (21) and 5-[125I]I-A-85380 (17). After administration in monkey, the tracer accumulated substantially in brain, with a pattern of regional distribution consistent with the known distribution of the a4b2 subtype of nAChRs (1) and similar to that of [18F]FPH (22). The high ratios of accumulated radioactivity in nAChR-rich regions vs. nAChR-poor regions (Fig 1D) suggest that the tracer could be used successfully in PET studies. The thalamic/cerebellar ratio of radioactivity from 6-[18F]FA, similar to that from [18F]FPH, is one of the highest reported for tracers to image nAChRs by PET in primate brain (3.3 to 3.8 at 135 min for [18F]FPH (22,23) vs. 3.1 at 135 min for 6-[18F]FA). The dramatic loss of radioactivity from nAChR-rich brain regions (Fig. 1C, 2) after administration of cytisine indicated that 6-[18F]FA bound speciÞcally and reversibly to nAChRs in vivo. Displacement of the tracer from the nAChR-poor cerebellum was negligible, demonstrating that the cerebellum could be used as a measure of non-speciÞc binding. Tracer doses of 6-[18F]FA (about 1 nmol/kg) did not produce any noticeable biological effects (changes in heart rate, electrocardiogram, hemoglobin oxygen saturation) in the monkeys. In a previous study (15), the authentic compound, 6-FA, was found to be substantially less toxic than the ßuoro analog of epibatidine, FPH (11). Collectively, the results obtained in the study suggest that the novel radiotracer, 6-[18F]FA, may be considered as a potentially useful radioligand for imaging nAChRs in humans by PET. References 1. 2. 3. 4.
M.W. HOLLADAY, M.J. DART and J.K. LYNCH, J. Med. Chem. 40 4169Ð4194 (1997). E.D. LONDON, M.J. BALL and S.B. WALLER, Neurochem. Res. 14 745Ð750 (1989). R. FREEDMAN, M. HALL, L.E. ADLER and S. LEONARD, Biol. Psychiatry 38 22Ð33 (1995). O.K. STEINLEIN, J.C. MULLEY, P. PROPPING, R.H. WALLACE, H.A. PHILLIPS, G.R. SUTHERLAND, I.E. SCHEFFER and S.F. BERKOVIC, Nat. Genet. 11 201Ð203 (1995). 5. A.W. BANNON, M.W. DECKER, M.W. HOLLADAY, P. CURZON, D.L. DONNELLY-ROBERT, P.S.
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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
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PUTTFARCKEN, R.S. BITNER, A. DIAZ, A.H. DICKENSON, R.D. PORSOLT, M. WILLIAMS and S.P. ARNERIC, Science 279 77Ð81 (1998). M. MAZIERE, G. BERGER, R. MASSE, D. PLUMMER and D. COMAR, Electrophysiological Effects of Nicotine, A. Remond and C. Izard (eds), 31Ð47, Elsevier, Amsterdam (1979). E.D. LONDON, U. SCHEFFEL, A.S. KIMES and K.J. KELLAR, Eur. J. Pharmacol. 278 R1-R2(1995). A. HORTI, H.T. RAVERT, E.D. LONDON and R.F. DANNALS, J. Labelled Cpd. Radiopharm. 38 355Ð366 (1996). J.L. MUSACHIO, A. HORTI, E.D. LONDON and R.F. DANNALS, J. Labelled Cpd. Radiopharm. 39 39Ð48 (1997). M.A. ABREO, N.H. LIN, D.S. GARVEY, D.E. GUNN, A.M. HETTINGER, J.T. WASICAK, P.A. PAVLIK, Y.C. MARTIN, D.L. DONNELLY-ROBERT, D.J. ANDERSON, J.P. SULLIVAN, M. WILLIAMS, S.P. ARNERIC and M.W. HOLLADAY, J. Med. Chem. 39 817Ð825 (1996). A.G. HORTI, U. SCHEFFEL, A.S. KIMES, J.L. MUSACHIO, H.T. RAVERT, W.B. MATHEWS, Y. ZHAN, P.A. FINLEY, E.D. LONDON and R.F. DANNALS, J. Med. Chem. 41 4199Ð4206 (1998). P.E. MOLINA, Y.S. DING, I. CARROLL, F. LIANG, N.D. VOLKOW, N. PAPPAS, M. KUHAR, N. ABUMRAD, S.J. GATLEY and J.S. FOWLER, Nucl. Med Biol. 24 743Ð747 (1997). A.O. KOREN, A.G. HORTI, A.G. MUKHIN, D. GUNDISCH, A.S. KIMES, R.F. DANNALS and E.D. LONDON, J. Med. Chem. 41 3690Ð3698 (1998). A.G. HORTI, U. SCHEFFEL, A.O. KOREN, H.T. RAVERT, W.B. MATHEWS, J.L. MUSACHIO, P.A. FINLEY, E.D. LONDON and R.F. DANNALS, Nucl. Med. Biol. 25 599Ð603 (1998). U. SCHEFFEL, A.G. HORTI, A.O. KOREN, H.T. RAVERT, J.P. BANTA, P.A. FINLEY, E.D. LONDON, and R.F. DANNALS, Nucl. Med. Biol., in press. A.G. HORTI, A.O. KOREN, H.T. RAVERT, J.L. MUSACHIO, W.B. MATHEWS, E.D. LONDON, and R.F. DANNALS, J. Labelled Cpd. Radiopharm. 41 309Ð318 (1998). A.G. HORTI, A.O. KOREN, K.S. LEE, A.G. MUKHIN, D.B. VAUPEL, A.S. KIMES, M. STRATTON and E.D. LONDON, Nucl. Med. Biol. 26 175Ð182 (1999). A. KOREN, A.G. HORTI, A.G. MUKHIN, D. G†NDISCH, R.F. DANNALS and E.D. LONDON, J. Labelled Cpd. Radiopharm., in press. S.I. CHEFER, A.G. HORTI, A.O. KOREN, D. G†NDISCH, J.M. LINKS, V. KURIAN, R.F. DANNALS, A.G. MUKHIN, and E.D. LONDON, NeuroReport, 10 2715Ð2721(1999). S.I. CHEFER, A.G. HORTI, K.S. LEE, A.O. KOREN, D.W. JONES, J.G. GOREY, J.M. LINKS, A.G. MUKHIN, D.R. WEINBERGER and E.D. LONDON, Life Sci. 63 L355ÐL360(1998). L.A. PABREZA, S. DHAWAN and K.J. KELLAR, Mol. Pharmacol. 39 9Ð12 (1991). V.L. VILLEMAGNE, A. HORTI, U. SCHEFFEL, H.T. RAVERT, P. FINLEY, D.J. CLOUGH, E.D. LONDON, H.N. WAGNER JR and R.F. DANNALS, J. Nucl. Med. 38 1737Ð1741 (1997). Y. DING, J. GATLEY, J.S. FOWLER, N.D. VOLKOW, D. AGGARWAL, J. LOGAN, S.L. DEWEY, F. LIANG, F.I CARROLL and M. KUHAR, Synapse 24 403Ð407 (1996).
Pharmacology letters Accelerated communication
6-[18F]ßuoro-A-85380, a novel radioligand for in vivo imaging of central nicotinic acetylcholine receptors Andrew G. Hortia, Svetlana I. Chefera, Alexey G. Mukhina, Andrei O. Korena, Daniela GŸndischa, Jonathan M. Linksa,b, Varughese Kuriana, Robert F. Dannalsb, Edythe D. Londona,* a
Brain Imaging Center, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA b Divisions of Nuclear Medicine and Radiation Health Sciences, Johns Hopkins Medical Institutions, Baltimore, MD 21205, USA (Submitted June 29, 1999; accepted September 10, 1999; received in Þnal form October 22, 1999)
Abstract A novel positron emission tomography (PET) radiotracer, 6-[18F]ßuoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[18F]ßuoro-A-85380, 6-[18F]FA) was synthesized by a no-carrier-added ßuorination. In vitro 6-[18F]FA bound to nicotinic acetylcholine receptors (nAChRs), with very high afÞnity (Kd 28 pM). In PET studies, 6-[18F]FA speciÞcally labeled central nAChRs in the brain of the Rhesus monkey and demonstrated highest levels of accumulation of radioactivity in brain regions enriched with the a4b2 subtype of nAChR. 6-[18F]FA exhibited a target-to-non-target ratio (estimated as radioactivity in the thalamus to that in the cerebellum) of binding in primate brain similar to that previously determined for a labeled analog of epibatidine, [18F]FPH. In contrast to [18F]FPH, the novel tracer is expected to exhibit substantially less toxicity. Thus, the novel radioligand, 6-[18F]FA, appears to be a suitable candidate for imaging nAChRs in human brain. Published by Elsevier Science Inc. Keywords: Nicotinic acetylcholine receptor; Positron emission tomography (nAChRs); 6-[18F]ßuoro-3-(2(S)azetidinyl-methoxy)pyridine (6-[18F]ßuoro-A-85380, 6-[18F]FA)
Introduction Efforts to develop radiolabeled ligands for a variety of brain receptors and advances in neuroimaging techniques have contributed to the substantial progress in noninvasive imaging
* Corresponding author. Tel.: 410-550-1540; fax: 410-550-1441. E-mail address: [email protected] (E.D. London) 0024-3205/00/$ Ð see front matter Published by Elsevier Science Inc. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 3 5 -4
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of neuroreceptors during the last two decades. Remarkably, unlike the case for several other types of central receptors, imaging of nicotinic acetylcholine receptors (nAChRs) has been limited by the lack of suitable radiotracers. Development of radiotracers for quantitative imaging of nAChRs would be very useful because these receptors, which modulate excitatory neurotransmission, contribute to a variety of brain functions and behavioral states, including learning and memory, attention and anxiety (1). Nicotinic acetylcholine receptors have been implicated in various neurodegenerative disorders, including AlzheimerÕs and ParkinsonÕs diseases, where postmortem brain tissue examination showed profound deÞcits in nAChR concentrations (1, 2). Recent studies have suggested that expression of an abnormal variant of the gene coding for the a7 subtype of nAChRs is associated with the deÞcit in sensory gating that is seen in schizophrenia (3) and that a missense mutation of the a4 subunit of nAChRs can lead to inherited epilepsy (4). These receptors also mediate the action of nicotine, an active ingredient that promotes dependence on tobacco (1). Furthermore, nicotinic agonists show promise as therapeutic agents for several diseases, including ParkinsonÕs disease and TouretteÕs syndrome, and as non-opioid analgesics (1, 5). Imaging nAChRs with [11C]nicotine (6) was hindered due to the high nonspeciÞc binding and rapid egress of the tracer from human brain. Subsequently, the discovery of epibatidine, a highly potent and speciÞc agonist at nAChRs, stimulated the development of its radiolabeled analogs, [3H]epibatidine (7), (6)-exo-2-(2-[18F]ßuoro-5-pyridyl)-7-azabicyclo[2.2.1]heptane ([18F]FPH) (8) and (6)-exo-2-(2-[123I]iodo-5-pyridyl)-7-azabicyclo[2.2.1]heptane ([123I]IPH) (9), as promising nAChR radiotracers for binding assay studies, positron emission tomography (PET), and single photon emission computed tomography (SPECT), respectively. However, epibatidine produces activation of nAChRs of the ganglionic (a3b4) subtype at very low doses (10). Therefore, labeled analogs of epibatidine, [18F]FPH and [123I]IPH, exhibit untoward effects, such as prolonged catecholamine release at doses approximately 30Ð100 times the mass that would be needed for in vivo imaging (11, 12). A more recently synthesized highly selective nAChR ligand A-85380 [3-(2(S)-azetidinylmethoxy)-pyridine] (10) is equipotent to epibatidine in in vitro binding assays with the human a4b2 subtype of nAChRs. However, it is only 1/100th as potent as epibatidine in activating the a3b4 subtype of nAChRs, which is at least partially responsible for the observed adverse effects of epibatidine (10). Based on these observations, we hypothesized that radiolabeled derivatives of A-85380 would maintain high afÞnity in labeling central nAChRs in vivo and would provide a wider safety margin between an adequate tracer dose and the dose that would produce a biological effect, as compared with epibatidine-based radioligands. Previously, we synthesized a series of halogenated derivatives of A-85380 with high binding afÞnity toward nAChRs and relatively low toxicity (13Ð15) and labeled several of them with isotopes for PET and SPECT imaging (16Ð18). The ßuoro-derivatives of A-85380, 2-ßuoro-3-(2(S)-azetidinylmethoxy)pyridine (2-FA) and 6-ßuoro-3-(2(S)-azetidinylmethoxy) pyridine (6-FA), like FPH, both exhibited very high in vitro binding afÞnities toward nAChRs (respectively, 46 pM and 25 pM (13) vs.37 pM (11)). In in vivo studies, 2-[18F]FA and 6-[18F]FA manifested a very high target-to-non-target ratio of binding and regional distribution in mouse brain matching the distribution of nAChRs (14,15). Unlike FPH, 2-FA and 6-FA demonstrated dramatically lower biological activity in vivo in mice (14,15), identifying the A-85380-based radiotracers as very promising candidates for PET human studies. The
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goal of the present study was to perform PET imaging of nAChRs in monkey brain with 6-[18F]FA in order to investigate its brain distribution, kinetics and speciÞcity toward nAChRs in vivo. Another paper presents our Þndings in PET imaging studies of 2-[18F]FA in monkey brain (19).
Methods Radiochemistry 6-[18F]FA was synthesized by a no-carrier-added nucleophilic radioßuorination through treatment of a dimethylsulfoxide solution of 6-iodo-3-((1-tert-butoxycarbonyl-2(S)-azetidinyl)methoxy)pyridine (13) with anhydrous [KryptoÞx222áK]1[18F]F2 complex at 190oC followed by acidic deprotection of the intermediate compound with trißuoroacetic acid solution in methylene chloride (Fig. 1A), puriÞcation by high-performance liquid chromatography (HPLC), and formulation as a sterile and apyrogenic solution as described previously (15). The radioactive precursors, [18F]ßuoride and [KryptoÞx222áK]1Ð[18F]F2, were produced with a CTI RDS111 negative ion cyclotron and a computer-controlled chemistry module. Receptor binding in vitro Binding assays with 6-[18F]FA were performed as described previously (20). Brießy, P2 membrane fractions (20Ð30 mg protein) from male Fischer-344 rat brain (excluding medulla and cerebellum) were incubated in the presence of 1 Ð 1000 pM 6-[18F]FA in 1 ml of HEPESsalt solution (pH 7.4) at 22 oC for 2 h. Binding was terminated by Þltration through GF/B Þlters pretreated with 1% polyethyleneimine. NonspeciÞc binding was determined in the presence of 500 mM (2)-nicotine. PET studies Two male Rhesus monkeys were used in six PET studies, including Þve control experiments and one displacement assay. The animals were prepared as described previously (19), positioned in a PET scanner (Siemens ECAT EXACT HR1), and a transmission scan was performed for attenuation correction. 6-[18F]FA (4.5Ð14 mCi, corresponding to ,1 nmol/kg) was injected intravenously, followed by PET scanning immediately after the administration. Vital signs, including heart rate, electrocardiogram, oxygen saturation of blood hemoglobin, were monitored using a SpaceLab monitor. A dynamic series of PET data sets were acquired as follows: 5 3 1 min, 5 3 2 min, 5 3 5 min, 8 3 10 min, 10 3 15 min. Each data set consisted of 63 slices over a 15.5 cm axial extent of the brain. The within-plane resolution was approximately 4.5 mm (full width at half maximum) (Fig. 2). The displacement assay was accomplished by subcutaneous injection of 1 mg/kg of cytisine, a high-afÞnity nAChR ligand, 80 min after injection of 6-[18F]FA. Regions of interest (ROIs) were placed in the thalamus, frontal cortex, and cerebellum as previously described (20), and average timeactivity curves (fraction of injected dose normalized to body weight in 1 cm3 of tissue (%ID? kg/cm3 vs. time) were generated (Fig. 1C).
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Fig. 1. A. Radiosynthesis of 6-[18F]FA. B. Results of a typical binding experiment with 6-[18F]FA in membranes from rat forebrain. The average values from this and two additional experiments were: Kd 28 6 2 pM, Bmax 3.7 6 0.4 pmol/g tissue. C. Regional time-activity curves of 6-[18F]FA. Control: averaged from Þve PET imaging studies. Displacement: one PET imaging study with 1 mg/kg cytisine injected subcutaneously (Thal 5 thalamus, Cx 5 cortex and CB 5 cerebellum). D. Average region/cerebellum ratios vs. time after the radiotracer injection in Þve control PET studies.
Results Radiochemistry 6-[18F]FA was synthesized with a radiochemical yield of up to 21.2% (not-decay-corrected, determined by HPLC). Chemical and radiochemical purity, determined by HPLC, exceeded 95%. The speciÞc radioactivity of the tracer was in the range of 1200Ð5400 mCi/mmol at the
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Fig. 2. PET images of Rhesus monkey brain showing binding of 6-[18F]FA with nAChRs. All images were obtained from a displacement study, except the bottom left one, which was obtained from a control study on the same animal. Top: 80-min scan, beginning 40 min after tracer administration. Bottom, right: displacement of 6-[18F]FA by subcutaneous injection of 1 mg/kg cytisine at 80 min after tracer administration; 60-min scan, beginning 3 h after tracer administration . Bottom, left: 60-min scan, beginning 3 h after tracer administration.
end-of-synthesis. The Þnal product was formulated as a sterile solution in saline. Sterility and apyrogenicity were conÞrmed by conventional methods. In vitro binding assay Saturation studies demonstrated that at a concentration of up to 1 nM, 6-[18F]FA interacted with a single population of binding sites (Fig. 1B). The observed afÞnity (Kd 28 6 2 pM, n53) was consistent with the afÞnity of non-labeled authentic 6-FA at the a4b2 nAChR subtype in rat brain measured previously in competition studies (13). A Bmax value was calculated as 3.7 6 0.4 pmol/g tissue, n53 (153 6 17 fmol/mg protein). Brain regional distribution and kinetics A substantial incorporation of radioactivity into the monkey brain was seen after injection of 6-[18F]FA. The highest accumulation of radioactivity (0.11%ID?kg/cm3 at 55 min) occurred in the thalamus. An intermediate level of radioactivity was found in the frontal cortex (0.08%ID?kg/cm3 at 40 min), and the lowest level of radioactivity was detected in the cerebellum (peak value of 0.07%IDákg/cm3 at 30 min) (Fig. 1C). The initial clearance rate of ra-
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dioactivity from the cerebellum was higher than from any other region studied. The t1/2 values for the clearance from the peak levels of radioactivity were .200, 155, and 50 min for the thalamus, frontal cortex, and cerebellum, respectively. As a result, after injection of the radiotracer, the thalamus/cerebellum and frontal cortex/cerebellum ratios increased steadily over the observation period, reaching values of 4.5 and 2.1, respectively, at 4 h after injection (Fig. 1D). Injection of cytisine reduced regional radioactivities in the thalamus and cortex at 220 min after the tracer administration by the level of radioactivity in the cerebellum (Fig 1C). No displacement of radioactivity from the cerebellum was observed. Administration of 6-[18F]FA at doses of up to 1 nmol/kg did not produce pharmacological responses, such as changes in ECG, in heart rate or in concentration of O2 in blood. Discussion We synthesized a novel PET nAChR radioligand, 6-[18F]FA, and found that it bound to a single population of binding sites in vitro at a concentration range of up to 1 nM. The dissociation constant (Kd) of the receptor-ligand complex agreed well with the inhibition constant (Ki) determined previously for non-labeled 6-FA in competition assays with [3H]epibatidine (13). The Bmax value obtained was in good agreement with the density of a4b2 nAChRs in rat brain measured with [3H]-cytisine (21) and 5-[125I]I-A-85380 (17). After administration in monkey, the tracer accumulated substantially in brain, with a pattern of regional distribution consistent with the known distribution of the a4b2 subtype of nAChRs (1) and similar to that of [18F]FPH (22). The high ratios of accumulated radioactivity in nAChR-rich regions vs. nAChR-poor regions (Fig 1D) suggest that the tracer could be used successfully in PET studies. The thalamic/cerebellar ratio of radioactivity from 6-[18F]FA, similar to that from [18F]FPH, is one of the highest reported for tracers to image nAChRs by PET in primate brain (3.3 to 3.8 at 135 min for [18F]FPH (22,23) vs. 3.1 at 135 min for 6-[18F]FA). The dramatic loss of radioactivity from nAChR-rich brain regions (Fig. 1C, 2) after administration of cytisine indicated that 6-[18F]FA bound speciÞcally and reversibly to nAChRs in vivo. Displacement of the tracer from the nAChR-poor cerebellum was negligible, demonstrating that the cerebellum could be used as a measure of non-speciÞc binding. Tracer doses of 6-[18F]FA (about 1 nmol/kg) did not produce any noticeable biological effects (changes in heart rate, electrocardiogram, hemoglobin oxygen saturation) in the monkeys. In a previous study (15), the authentic compound, 6-FA, was found to be substantially less toxic than the ßuoro analog of epibatidine, FPH (11). Collectively, the results obtained in the study suggest that the novel radiotracer, 6-[18F]FA, may be considered as a potentially useful radioligand for imaging nAChRs in humans by PET. References 1. 2. 3. 4.
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