Evaluation of [11C]RTI-121 as a selective radioligand for PET studies of the dopamine transporter

Nuclear Medicine & Biology, Vol. 23, pp. 377-384, Copyright 0 1996 Elsevier Science Inc.

ISSN 0969s8051/96/$15.00 + 0.00 PII SO969-8051(96)00019-4

1996

ELSEVIER

Evaluation of [l ‘C]RTI-121 as a Selective Radioligand for PET Studies of the Dopamine Transporter Susan P. Hurne,‘” Sajinder K. Luthra,’ David J. Brown, ’ Jolanta Opacku-Jujfry,’ Safiye Oman,’ Sharon Ashworth,’ Ralph Myers,’ Frank Brady,’ F. Ivy Carroll,’ Michael J. Kuhar? and David J. Brooks’ ‘PET METHODOLOGY AND NEUROSCIENCES GROUPS, CYCLOTRON UNIT, MRC CLINICAL SCIENCES CENTRE, ROYAL POSTGRADUATE MEDICAL SCHOOL, HAMMERSMITH HOSPITAL, LONDON, W12 ONN, UK; ‘DEPARTMENT OF ORGANIC AND MEDICINAL CHEMISTRY, RESEARCH TRIANGLE INSTITUTE, RESEARCH TRlANGLE PARK, NC 27709, USA; AND ‘NEUROSCIENCES BRANCH, ADDICTION RESEARCH CENTER, NATIONAL INSTITUTE ON DRUG ABUSE, BALTIMORE, MD 21224, USA

ABSTRACT. The cocaine analogue RTI-121 (3P-(4-iodophenyl)tropane-2@carboxylic acid isopropyl ester), when labeled with carbon-l 1, was evaluated in rats as a potential PET ligand for the dopamine transporter. The compound gave in vipto striatum:cerebellum ratios that were similar to those obtained with the related ligand [’ ‘C]RTI-55 (2@(4-iodophenyl)tropane-2P-carboxylic acid methyl ester) but showed a much greater selectivity for the dopamine compared with the 5-HT uptake site. The results indicate that [’ ‘C]RTIin man. Experimentally, [’ ‘CIRTI12 1 has potential in the 12 1 could be used in preference to [ “C]RTI-55 quantification of dopamine terminal function in rat models of disease, using a combination of autoradiography, postmortem sampling, and in viva tomography. NUCL MED BIOL 23;3:377-384, 1996. KEY

WORDS.

Dopamine

transporter,

RTI-121,

Positron

INTRODUCTION With the aim of achieving a better understanding of the bases of neurological and psychiatric disorders involving the central dopaminergic system, the development of radioligands for either positron emission tomography (PET) or single photon emission tomography (SPET) receives much attention. Among those compounds synthesized or considered for studies of the presynaptic dopamine uptake complex are [“Clnomifensine (1, 38), analogues of GBR designed for either PET, including [18F]GBR 13119 (22) and [“C]NNC 12-0722 (32), or SPET (14), [“C]dl-three-methylphenidate (12), [“Clcocaine (42), and various analogues of cocaine (9). Although there is some evidence from equilibrium binding data that these compounds and dopamine bind in a mutually exclusive way to the dopamine transporter (35), voltammetric methodology suggests a multisubstrate mechanism of dopamine uptake (30). The latter authors postulate that, under normal circumstances, dopamine and Nf bind at different sites on the carrier, followed by Cl- binding; inhibition of uptake by cocaine involves binding of cocaine at the Na+ site following the binding of dopamine. The affinities of cocaine and various cocaine analogues for the dopamine, relative to noradrenaline and 5-HT, uptake sites are listed together with their structures in Boja et al. (4), and early binding studies for a selection of the analogues have been reviewed (3). Although not selective for a single monoamine site, in that it shows almost equal potency in inhibiting dopamine and 5-HT uptake, RTI-55 (3B-(4-iodophenyl)tropane-2B-carboxylic acid methyl ester), also designated B-CIT, has been used in several centres as a SPET ligand for the presynaptic dopamine transporter, when labeled with iodine-123 (8, 24, 26, 40). Differences in anatomical location and kinetics aid in

* Author for correspondence. Accepted 14 January 1996.

emission tomography

distinguishing between the two binding sites (3, 8). Owing to the slow specific uptake and prolonged retention of a high degree of nonspecific signal, however, times of the order of 1 day after radioligand injection are needed to achieve striatum:occipital cortex ratios (representing total:nonspecific counts) of the order of 17 in normal volunteers (19). While this is feasible with the relatively long radioactive half-lives of SPET ligands (for iodine-123, ti,, = 13 h) and a slow in viwo rate of deiodination (33), it makes PET studies using carbon-l 1 labeled RTI55 (ti,* = 20.4 min) difficult to quantify and the specific signals obtained relatively small. Ratios of radioactivity in neocortex:cerebellum of 1.14 (13) and striatum:cerebellum of 3 (16) and 2 (25) have been reported -90 min after radioligand injection. The latter values are comparable to that reported for the equivalent SPET ligand, at a similar time after injection (24). The related compound B-CFT (ZB-carbomethoxy-3B-(4-fluorophenyl)tropane), also designated WIN 35,428, has been used as an alternative ligand for PET, when labeled with carbon-11 (15, 17, 44). Compared with RTI-55, in vitro and in viuo distribution studies suggest that B-CFT is more selective for brain regions containing dopamine terminals, especially at early times after administration (20). In human volunteers, striatum:cerebellum count ratios of the order of 4 have been reported at 90 min after administration of [“C]B-CFI but, as for [“C]RTI-55, this time does not represent an equilibrium state (15). In addition, the relatively high dissociation constant (4.7 nM at the highaffinity site, as reported by Madras et al. (29) for primate striatal membranes), suggests that its in oioo binding could be influenced by endogenous dopamine levels (23). As an alternative ligand, an analogue of RTI-55, namely 3B-(4iodophenyl)tropane-2Bcarboxylic acid isopropyl ester, designated RTI-121, has been identified as combining high affinity and selectivity for the dopamine transporter (4, 6, 41), although recent studies with [1231]RTI-121 have suggested that a high degree of nonspecific binding may limit its usefulness as a SPET ligand (39). In the present study, we

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have compared the in wioo binding characteristics of [“CIRTI-121 with [“C]RTI-55 in rat brain. The aim was to define the in uiefo selectivity and plasma retention of the radiolabeled compounds to evaluate the potential of [’ ‘C]RTI- 12 1 as a PET ligand, not only in man but also in rat using a recently commissioned small-diameter tomograph (2). In the small animal scanner, especially, it was of crucial importance that the ligand was selective for the dopamine transporter since, with the limited resolution of the camera, any binding to 5-HT or noradrenaline uptake sites in regions adjacent to the striatum could influence the final striatal signal obtained because of spillover and partial volume effects. In addition, as with any PET system, unless specific binding to a secondary site can either be accounted for in the final modeling of the data or eliminated by selective blocking, limited dopamine selectivity of the radioligand will lead to an overestimation of remaining dopamine sites in, for example, Parkinson’s disease.

METHODS

Materials Samples of RTI-55, RTI-121 and their Nydesmethyl precursors were prepared as previously reported (5, 10). Citalopram hydrobromide was donated by H. Lundbeck A/S (Copenhagen-Valby, Denmark). GBR12909 dihydrochloride was obtained from Research Biochemicals Inc. (Natick, MA). Chemicals were HPLC grade from BDH Merck Ltd. or were purchased from Sigma (Poole, UK).

Radiochemistry Synthesis of carbon-l 1 labeled RTI-55 was first reported by Mtiller et al. (31). In the present study, RTI-55 and RTI-121 were labeled with carbon-11 by reacting their respective N-desmethyl precursors with [“Cliodomethane routinely prepared from [“Clcarbon dioxide (28). The reaction mixtures were heated to 90°C for 5 min, diluted with water, and injected onto a sample enrichment ClS-column. The column was switched in line with an HPLC column (PL-Bondapak C18, 300 x 7.8 mm i.d.) eluted at 3 mL * min-’ with a mixture of MeCN and KH,PO, (0.07 M) in the proportions [45:55] for RTI-55 or [55:45] for RTI-121, pH adjusted to 7.0 with triethylamine. RTI-55 eluted between lo-11 min and RTI-121 between 12-14 min. The fractions containing the labeled compounds were collected in a flask containing ascorbic acid (100 FL, 5.7 kmol) and the solvent removed by rotary evaporation. The residue was formulated in 10 mL isotonic saline containing ethanol [9.5:0.5]. The pH of the formulated product was 6-6.5. Labeling efficiency was >95% for both compounds, and typical activities at the end of synthesis were 2.6-3.7 GBq. The radiochemical and chemical purities were -98%. Full details of the syntheses, together with their validation, have been reported elsewhere (7).

Biological

Studies

The work was carried out by licensed investigators in accordance with the Home Office’s “Guidance on the Operation of the Animals (Scientific Procedures) Act 1986” (HMSO, Feb., 1990). Adult male Sprague-Dawley rats (mean with SD body weight = 288 + 23 g; Harlan Olac Ltd., Bicester, UK) were used throughout. BIODISTRIBUTION. Catheters were placed in the tail vein and artery of isoflurane-anesthetised rats, which were then allowed to recover for 2 h prior to radioligand injection. The rats were kept under light restraint in Bollman cages, and lo-11 MBq of either [“CIRTI-55 or [“CIRTI-121 was given in 0.20 mL saline (0.9%) via the vein catheter. At time of injection, the specific activity with SD was 27 c 8 (range

22-39) GBq/pmol for [“CIRTI-55 and 39 * 10 (range 26-59) GBq/ pmol for [“CIRTI-121, giving an associated stable content of the order of 1 nmol/kg body weight, for both compounds. At graded times after the injection, blood was collected via the artery catheter and plasma samples obtained by rapid centrifugation. At designated times (between 2 and 120 min) after radioligand injection, rats were killed by overdosing with an IV injection of sodium pentobarbitone, the brain rapidly removed and five regions sampled, namely thalamus, frontal cortex, striatum, hippocampus, and cerebellum. Radioactivity content of blood and brain samples was measured using an LKB Wallac gamma counter with automatic correction for radioactive decay. Data were expressed as: [(MBq/g

tissue or blood)/(MBq

injected)]

* 100.

IN VW0 SELECTIVITY. Selectivity of [“CIRTI-121 for the dopamine transporter was tested by predosing the rats with either citalopram (binding at the 5-HT uptake site [IS]) or GBR 12909 (binding at the dopamine uptake site [21]) and specificity tested by predosing with RTI-121, all at a dose of 2 mg/kg IV given 5 min prior to radioligand injection. In addition, [“C]RTI-121 b‘m d’m g was measured in striata of four older rats (mean with SD body weight 396 ? 17 g), 4-5 months after 6,hydroxydopamine (6-OHDA) 1esioning of the substantia nigra pars compacta, using the method described by Perese et al. (34). Patency of the lesion was confirmed by frequency of turning following i.p. injection of 5 mg/kg amphetamine. PLASMA PROTEIN BINDING. The free fractions of [“C]RTI-55 and [ “ C]RTI-12 1 in plasma were determined using an Amicon Ultrafiltration Unit (Grace & Co., Stonehouse, UK) following incubation of 3 mL blood with 0.37 MBq of either compound, for 10 min at 37°C. Duplicate samples (50 FL) of cell-free plasma were dispensed onto the Amicon unit and centrifuged at 4000 rpm for 15 min. The free fractions were calculated as the ratios of carbon-11 activity in the ultrafiltrate compared with that dispensed onto the unit, after correction for stickiness. METABOLITE ANALYSIS. The fractions of unchanged [l’C]RTI-55 and [“CIRTI-121 in plasma as a function of time after radioligand injection were estimated using a solid-phase extraction (SPE) and reversed-phase HPLC system with on-line radioactivity and UV detection linked to a PC-based integrator, as described previously for other compounds (27). Plasma samples (1 mL) were injected onto the SPE column (octadecyl-derivatizing silica) and eluted using (NH,),HPO, (0.01 M). Duplicate aliquots of plasma and SPE eluent were taken for counting of radioactivity. The retained radioligands and their radiolabeled metabolites were back-eluted from the SPE and HPLC columns using a mixture of KH,PO, (0.07 M), pH 7, and acetonitrile in the proportions [65:35] or [45:55] for RTI-55 and RTI-121, respectively, at a flow rate of 3 mL * min-‘. Retention times were 17 min for [“CIRTI55 and 11 min for [“CJRTI-121. The radioactivity in brain tissue was confirmed as parent compound by similar reversed-phase HPLC analysis of the supematant obtained after homogenization in ice-cold methanol, centrifugation, and rotary evaporation of the residue. Recovery of radioactivity was checked at all stages. PET SCANNING. A small animal tomograph was used to obtain timeradioactivity curves in striatum or cerebellum regions of interest (ROI) following [“C]RTI-121 injection in three rats: control, RTI-121 predosed (2 mg/kg, at zero minus 5 min), or 6-OHDA lesioned. The tomograph, which was designed and built in collaboration with C.T.I. PET Systems (Knoxville, TN), has a diameter of 115 mm and an axial field of view (FOV) of 50 mm (2). With the reconstruction used in the present study, the transaxial resolution is 2.3 mm full width half max-

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of [“CIRTI-121

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In Vi00

imum (FWHM) at the center of the FOV and the axial slice thickness is 4.2 mm FWHM. Isoflurane-anesthetised rats were positioned on a perspex bed with a head holder incorporating perspex ear bars and tooth bar, such that the striata were at the center of the FOV. Radioactivity was given as for the biodistribution studies, and the scanning was begun immediately at injection using a protocol comprising 27 frames of durations: 3 x 5 set, 3 x 15 set, 4 x 60 set, 11 x 300 set, 6 x 600 set, over a total of 2 h. After scanning, the data were reconstructed and the dynamic volumes manipulated and sampled using Analyze software (36). The ROI were chosen on horizontal projections of the Images; sizes were 4 x 8 pixels for right and left striatum and 8 x 6 pixels for cerebellum, where 1 pixel = 0.47 mm.

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RESULTS Following IV injection of either [“CIRTI-55 or [“C]RTI-121, radioactivity cleared rapidly from the peripheral circulation such that by 2 min after injection, radioactivity content per gram of plasma was only 0.07% of that injected. In vitro incubation at 37°C for 10 min indicated a high proportion of plasma protein binding; free fractions in plasma were 0.37 for [“CIRTI-55 and 0.12 for [“CIRTI-121. The plasma clearance curves for the two compounds were indistinguishable, as illustrated in Fig. la. In contrast, radioactivity was retained in whole blood to a greater extent following [“C]RTI-55 (Fig. lb); the blood: plasma ratio for [“CIRTI-55 increased from an initially similar value of 2 for the two compounds to a maximal value of -8 at 20 min, as shown in Fig. lc. Solid-phase extraction and reversed-phase HPLC of plasma samples taken up to 90 min after injection showed that the fractional loss of carbon-l 1 labeled parent compound from the total radioactivity measured was similar for both compounds (Fig. 2). By 90 min, only -50% of plasma radioactivity was associated with the parent compound. Of the remaining radioactivity, the vast majority was in the SPE eluate. At l-2 min after [“C]RTI-55 injection, four additional radioactivity peaks other than that associated with RTI-55 were observed in the back-eluate (retention times of 5, 7, 9, and 13 min compared with 17 min for the parent compound), but these represented only -2% of the total recovered and did not increase with time. For [“CIRTI-121, two additional peaks, corresponding to metabolites, were observed (retention times of 4 and 7.5 min, compared with 11 min for the parent compound) at 5 min after injection. These represented -7% of the total radioactivity and, again, did not increase with time. The radioactivity content of brain tissue at the earliest time of sampling (2 min) was not significantly different following injection of either compound but thereafter differed markedly in all of the tissues sampled. Fig. 3 illustrates data for three of the tissues, namely striatum, thalamus, and cerebellum, representing the range of distributions observed. Despite the much greater accumulation of radioactivity following [“C]RTI-55 injection, compared with [“CJRTI-121, the rate of development and size of specific signal in striatum, expressed as the ratio of striatum:cerebellum counts, was only slightly lower for [’ ‘CIRTI-12 1, as illustrated in Fig. 4a. HPLC analysis of brain samples over the period 40-90 min after injection confirmed that the majority of the radioactive label was associated with the parent compound in both cases. At these times, 96 + 2% (n = 5) of radioactivity was [“CIRTI-55, and 99 + 1% (n = 5) was [“CIRTI-121. For [“CIRTI-55, the additional brain radioactivity had retention times matching three of the additional peaks observed in plasma (i.e., 7, 9, and 13 min). Figure 4b illustrates the thalamus:cerebelIum counts as a function of time after injection of either radioligand. At 2 h, the ratio approached 4 for [“CIRTI-55 but remained at -1.5 for [“CIRTI-121. This indi-

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FIG. 2. Fraction of radioactivity associated with the parent compound in plasma, as a function of time after IV injection of either [“C]RTI-55 (0) or [“C]RTI-121 (0). The radiochemicalpurity at the time of injection was measured by HPLC as 97 * 3% (n = S), for both compounds. Data are individual measurements, l-3 per rat. The time of assay was limited to 90 min because of decreasing count statistics. 0

cation of the greater selectivity of [“C]RTI-121 for the dopamine, relative to the serotonin, transporter was further examined by predosing the rats with either citalopram or GBR-12909. The results are summarized in Fig. 5, as either radioactivity content or ratio of tissueto-cerebellum counts at 90 min after [“C]RTI-121 injection. Compared with the control group (Student’s t test), citalopram predosing had no statistically significant effect on radioactivity content in any tissue. GBR-12909 predosing had a significant effect only in striatum (p c 0.001) where it resulted in a 56% reduction in 90-min content and a reduction in ratio from 4.8 + 0.4 to 2.2 + 0.1. Also included in Fig. 5 is the effect of predosing with RTI-121 itself. Although this resulted in a decrease in radioactivity content in striatum (p < O.OOl), to the extent of that seen with GBR-12909, there was a significant increase in radioactivity content in all other tissues sampled, including cerebellum (p < 0.05 or p < 0.01). The resultant distribution was, however, relatively homogeneous; the tissue:cerebellum ratios ranged from 1.30 f 0.02 in thalamus to 1.45 Y!Z0.05 in the hippocampus. In striatum, the value of 1.44 f 0.05 was significantly less than that observed in the GBR-12909 predosed rats (p < 0.001). Fig. 6 illustrates the 90-min striatum:cerebellum ratios in rats previously lesioned with 6-OHDA to the substantia nigra pars compacta. The ratio was not significantly different from control in the striatum contralateral to the lesion but was reduced to 2.0 + 0.4 in the ipsilateral striatum, a value not significantly different from that observed in the GBR- 12909 predosed rats. Compared with the control group, 6-OHDA lesioning had no significant effect on the tissue:cerebellum ratios for either thalamus, frontal cortex or hippocampus, sampled 90 min after [“CIRTI-121 injection (data not shown). Full time-radioactivity curves obtained from PET scans of three individual rats (control, 6-OHDA lesioned, or RTI-121 predosed) are shown in Fig. 7a-c, respectively. In Fig. 7a, data from right and left striatum were similar and, after the initial delivery and extraction, show the greater retention of [“C]RTI-121 in striatum relative to cerebellum. In Fig. 7b, the curve from the left, ipsilateral striatum in

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blood and brain were markedly different. Most apparent differences were in the selectivity of the compound for the dopamine transporter and the degree of nonspecific binding. The known lack of selectivity of RTI-55 for the dopamine over the 5-HT transporter site (4) was confirmed in the present study, reflected as the specific signal that developed in thalamus as a function of time after injection of [“CIRTI-55 (Fig. 4). Both the extent and the distribution of signal were similar to those previously reported for [‘25C]RTI-55 in mice (11). In addition to the lack of selectivity in brain, the high degree of binding of [“CJRTI-55 to the blood cell fraction (Fig. 1) probably reflects binding to the 5-HT uptake site on platelets (37). Although the rate and the size of development of specific binding in striatum were similar for both [“CIRTI-55 and [“CIRTI-121, the improved selectivity of the latter compound for the dopamine transporter was clearly demonstrated. Predosing with GBR12909 reduced the retention of [“CIRTI-121 only in striatum, and no significant reduction in binding due to citalopram was seen in any 1.0

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the 6-OHDA lesioned rat was initially similar to that from the contralateral striatum but, thereafter, showed less retention of radioactivity with time. As predicted from the 90-min data shown in Fig. 5, the most obvious effect of predosing with RTI-121 (Fig. 7c), in addition to the reduction in specific signal, was an increased retention of radioactivity in all brain tissue, including cerebellum. This increase was associated with an increase in blood and plasma radioactivity; at 90-120 min after radioligand injection, the mean plasma content per gram was 0.15% of that injected, compared with 0.02% in control rats. As in the control rats, only one radioactive peak, that corresponding to RTI-121, was obtained by reversed-phase HPLC analysis of brain homogenate, whether derived from whole brain or cerebellum.

Cortex DISCUSSION Although [“C]RTI-55 and [“C]RTI-121 were cleared similarly in plasma and appeared to be extracted equally into rat brain following IV injection, the subsequent behaviors of the two radioligands in both

FIG. 5. Radioactivity content (a) or tissue:cerebellum ratios (b), 90 min after IV injection of [ “C]RTI-12 1, for the tissues shown. Data are means with SD from the numbers of rats indicated in (a). For predosing, the compounds were given at a dose of 2 mg/kg IV, 5 min prior to the radioligand.

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brain region sampled (Fig. 5). It should be noted, however, that in the GBR-12909 predosed rats the striatum:cerebellum count ratio remained significantly higher than that obtained in the RTI-121 predosed rats. Although it is feasible that this results from the fact that dynamic equilibrium was not achieved within the 90-min assay time, a more probable explanation is that the GBR-12909 binding site is not identical to that/those recognized by cocaine and its analogues (43). The possibility that [“C]RTI-121, at the specific radioactivities used for PET studies, binds to a striatal site other than that associated with the dopamine transporter cannot be eliminated. Recently, a greater degree of nonspecific binding has been reported for [‘2sI]RTI-121 compared with [iZ31]RTI-55 in SPET scans of baboons (39). In the present study, the retention or accumulation of nonspecific binding following [“C]RTI-121 injection in control rats was actually less than that observed with [“CIRTI-55, as illustrated for the cerebellum curves in Fig. 3c. The reason for the differences in nonspecific binding between the two compounds is unclear. It is presumably not related to the plasma profile since, in rats, neither the clearance (Fig. la) nor the rate of metabolism (Fig. 2) of the parent compounds is different. The majority of radiolabeled metabolites were polar; in the case of [’ ‘C]RTI-12 1, none crossed the blood-brain barrier and, following [“CJRTI-55 injection, only -4% of radioactivity in brain was identified as being not parent compound. Nonspecific binding was, however, clearly dependent on the input function since predosing with stable RTI-121 affected plasma counts and caused a marked increase in [“C]RTI-121 retention in all brain tissues sampled, despite a decrease in specific binding in striatum (Figs. 5 and 7). Thus, although the size of the clinical PET signal associated with the dopamine transporter using [’ ‘C]RTI- 12 1 might be justifiably predicted as being of the order of that already obtained with [“C]RTI55 (13, 25), any extrapolation from rat to man with regard to the degree of nonspecific binding is not possible. Using [“CIRTI-55 in man, Farde et al. (13) have not observed the polar radiolabeled-metabolites seen in rats following injection of both [“CIRTI-55 and

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Mid-frame time (minutes after injection) FIG. 7. Dynamic data sets acquired for three anesthetised rats using a prototype small animal PET (2) for ROI corresponding anatomically to either right striatum (0), left striatum (O), or cerebellum (+). Rats were (a) control, (b) lesioned with 6-OHDA to the left substantia nigra pars compacta, 1 month prior to [“CIRTI-121 injection, or (c) predosed with RTI-121 (2 mg/kg IV, 5 min prior to radioligand injection). The scans have been normalized to an injectate of 10 MBq and are decay-corrected to the time of the injection.

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[“ C]RTI-121, but have reported negligible metabolism and a high degree of nonspecific binding. Depending on human metabolism of [’ ‘C]RTI- 12 1, one might expect a similarly high degree of nonspecific binding in [“C]RTI-121 PET scans. However, its improved selectivity, with the added advantage of lower binding in the peripheral circulation (Fig. lb), should justify the preferred use of the isopropyl ester analogue in PET studies. Although dynamic equilibrium was not reached within the 2 h of the study, the ability of [“CIRTI-121 to detect changes in dopamine terminal number in who, within a time period commensurate with PET scanning, is illustrated in Figs. 6 and 7. The data acquired in the small animal tomograph were sufficiently reproducible that [“CIRTI-121 could feasibly be utilized to study changes in dopaminergic terminals in animal models of Parkinson’s disease. It is envisaged that the tomograph (with the advantage that a single animal can be scanned sequentially) will be used to complement in vitro and ex viva autoradiography.

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