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Dopamine D1 Receptor Imaging in the Rodent and Primate Brain Using the Isoquinoline (+)-[11C]A-69024 and Positron Emission Tomography
Dopamine D1 Receptor Imaging in the Rodent and Primate Brain Using the Isoquinoline (þ)-[11C]A-69024 and Positron Emission Tomography LAURENT BESRET,1 FRE´DE´RIC DOLLE´,2 ANNE-SOPHIE HE´RARD,1,3 MARTINE GUILLERMIER,1,3 STE´PHANE DEMPHEL,2 FRANC¸OISE HINNEN,2 CHRISTINE COULON,2 MICHE`LE OTTAVIANI,2 MICHEL BOTTLAENDER,2 PHILIPPE HANTRAYE,1,3 MICHAEL KASSIOU4,5,6 1
CNRS, URA 2210, 4 place du Ge´ne´ral Leclerc, F-91406 Orsay, France
2
CEA, DSV, I2BM, SHFJ, Laboratoire d’Imagerie Mole´culaire Expe´rimentale, F-91406 Orsay, France
3
CEA, DSV, I2BM, Molecular Imaging Research Center, F-92265 Fontenay-aux-Roses, France
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Discipline of Medical Radiation Sciences, University of Sydney, Sydney, NSW 2006, Australia
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Brain and Mind research Institute, University of Sydney, Sydney, NSW 2050, Australia
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School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia
Received 23 May 2007; revised 12 July 2007; accepted 17 July 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21168
ABSTRACT: In vivo pharmacokinetic and brain binding characteristics of (þ)-[11C]A69024, a high-affinity-D1-selective dopamine receptor antagonist, were assessed with micro-PET and b-microprobes in the rat and PET in the baboon. The biodistribution of (þ)-[11C]A-69024 in rats and baboons showed a rapid brain uptake (reaching a maximal value at 5 and 15 min postinjection in rats and baboons, respectively), followed by a slow wash out. The region/cerebellum concentration ratio was characterized by a fourfold higher uptake in striatum and a twofold higher uptake in cortical regions, consistent with in vivo specific binding of the radiotracer in these cerebral regions. Furthermore, this specific (þ)-[11C]A-69024 binding significantly correlated with the reported in vitro distribution of dopamine D1-receptors. Finally, the specific uptake of the tracer in the striatum and cortical regions was completely prevented by either a pretreatment with large doses of nonradioactive ()A-69024 or of the D1-selective antagonist SCH23390, resulting in a similar uptake in the reference region (cerebellum) and in other brain regions. Thus, (þ)-[11C]A-69024 appears to be a specific and enantioselective radioligand to visualize and quantify brain dopamine D1 receptors in vivo using positron emission tomography. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2811–2819, 2008
Keywords:
PET; pharmacokinetics; pharmacodynamics; CNS; receptors
INTRODUCTION
Laurent Besret and Fre´de´ric Dolle´ have contributed equally to this work. Correspondence to: Michael Kassiou (Telephone: 612-93510894; Fax: 612-9351-0652; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 2811–2819 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
Positron emission tomography (PET) is an imaging technique which offers the opportunity to study drug pharmacokinetics and pharmacodynamics in vivo. It allows us to visualize and quantify receptor binding sites in living patients and derive correlates between normal and disease states.
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Several PET radioligands with dopamine D1 receptor antagonistic properties have been developed during the last decade.1–3 Unfortunately, most of these D1-preferential receptor ligands display high in vivo nonspecific binding and/or too fast dissociation kinetics which greatly impede their usefulness as in vivo PET tracers. For example two selective benzazepine D1 antagonists, [11C]SCH23390 and [11C]NNC112, with favorable binding characteristics in vitro have been evaluated in humans in vivo.3,4 However, the pharmacokinetic profile of 11C]SCH23390 is not favorable in vivo displaying a rapid washout from the brain after intravenous injection, a property that greatly limits its ability to quantify D1receptor in regions with intermediate receptor densities, such as cortical regions. Similarly, [11C]NNC112 which was reported as a ligand of choice for dopamine D1 receptor visualization and quantification in various brain regions,3 displays in vivo a high nonspecific binding which limits our ability to assess specific binding in regions like the hippocampus or various cortical areas. As an alternative to these two benzazepine radiotracers, we attempted at developing a structurally unrelated radioligand which would also be a highly selective, high affinity D1-receptor antagonist (Tab. 1) but displaying higher specific binding in vivo.5–7 In vitro, the better D1-selectivity of the (þ) enantiomer of the isoquinoline A-69024 compared to either SCH23390 or other benzazepine derivatives, makes it an interesting candidate for PET analyses.5–8 We therefore undertook the carbon-11 radiosynthesis of each of the enantiomers of A-69024 namely, (þ)[11C]A-69024 and ()-[11C]A-69024 and now report on the pharmacokinetics and in vivo regional brain biodistribution of both [11C]A-69024 enantiomers studied by micro-PET, b-microprobes and conventional PET in rodents and nonhuman primates.
2 - [11 C]methyl - 1,2,3,4 - tetrahydroisoquinoline) and ()-[11C]A-69024 (()-1-(2-bromo-4,5-dimethoxybenzyl)-7-hydroxy-6-methoxy-2-[11C]methyl1,2,3,4-tetrahydroisoquinoline) used the following conditions: (1) trapping at room temperature of the [11C]methyl triflate in 300 mL of acetone containing 1.0 mg of precursor (nor-()-A-69024) and 5 mL of an aqueous 3 M solution of sodium hydroxide (6 equiv.); (2) concentration to dryness of the reaction mixture at 1008C using a nitrogen stream; (3) taking up the crude reaction mixture with 0.5 mL of the HPLC mobile phase; (4) HPLC purification (hexane/isopropanol [60/40 (v:v)]; 3 mL/min) on semipreparative Daicel ChiralCel OD (250 10 mm). Typically, 30–80 mCi (1.11– 2.96 GBq) of (þ)-A-69024 (tR ¼ 9–10 min) and ()A-69024 (tR ¼ 12–13 min) were routinely obtained using these conditions within 25–30 min including HPLC purification with specific radioactivities ranging from 0.7 to 2.5 Ci/mmol (25.9–92.5 GBq/mmol). Formulation of labeled product for i.v. injection was effected as follows: The HPLC-collected fraction containing (þ)- or ()-[11C]A-69024 was concentrated to dryness (using a rotavapor, oil bath temperature: 70–808C), then the residue was taken up in 10 mL of physiological saline containing 10% of ethanol. The radioligand preparation for injection was a clear and colorless solution and its pH was between 5 and 7. The preparation was found to be >95% chemically pure and >99% radiochemically pure, as demonstrated by HPLC analysis (H2O/CH3CN [75/25 (v:v) containing lowUV PIC1 B7 reagent, 20 mL for 1000 mL]; 2.0 mL/ min) on analytical Symmetry-M1 C-18, Waters (50 4.6 mm), free from starting labeling precursor and was shown to be radiochemically stable for at least 60 min. The radiosynthesized (þ)- and ()-[11C]A-69024 also coeluted with an authentic sample of ()-A-69024.
MATERIALS AND METHODS
Biodistribution Experiments
Radiosynthesis of (R)-[11C]A-69024 and ()-[11C]A-69024
Studies were performed on adult male SpragueDawley rats (269 9 g, mean SEM; n ¼ 10, obtained from Harlan, Gannat, France) in compliance with the French law on animal experimentation. Animals were housed in groups of two to four with food and water ad libitum and kept on a 12:12 h light:dark cycle (dark period 07:00 p.m.– 7:00 a.m.) in temperature and humidity-controlled facilities. The animals were decapitated at different times (5, 15, 20, 30, and 45 min) following radioligand injection (50 mCi); brains
Preparation of (þ)-[11C]A-69024 ((þ)-1-(2-bromo4 , 5 - dimethoxybenzyl ) - 7 - hydroxy - 6 - methoxy Table 1. In Vitro Binding Affinities of Selected D1-Receptor Ligands SCH23390 0.14 nM8
NNC112 0.18 nM8
A-69024 þ0.6 nM7, 12.6 nM7
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were rapidly removed and dissected at 48C in order to obtain four cortical and subcortical regions of interest. Samples were weighed and radioactivity was counted with a gamma counter (Cobra autogamma, Perkin-Elmer, Courtaboeuf, France) and expressed as percentage of injected dose/g of tissue (% ID/g). Statistical significance was evaluated using the Dunnett multiple comparisons test. p < 0.05 was considered statistically significant.
b-Microprobe Studies The b-microprobe (Biospace Labs, Paris, France) is an in situ technique involving the insertion of a fine probe into brain tissue in a way very similar to that used for microdialysis and electrophysiological recordings. It functions as an implantable radioactivity counter9,10 that takes advantage of the limited range of positron particles within biological tissues to define a localized detection volume around the implanted probe (2.05 mm around the tip of the probe for carbon-11-labeled radiotracers). The sensitive end of the probe consists of a 1-mm-long and 250–500 mm-diameter plastic scintillating fiber (BCF-12, Bicron, Newbury, OH). This detection tip is fused with a 250– 500 mm-diameter clear optic fiber (BCF-98, Bicron) whose length is adjustable to the depth of the brain structure targeted (e.g., 7 mm in the case of an implantation in the rat striatum). This feature enables one to detect the light exiting the brain. The light is then transmitted through an optic fiber to a photomultiplier (R7400P, Hamamatsu, Hamamatsu City, Japan). The electronic pulses are analyzed by a counting electronic device, and the measured counting rate is displayed online on a computer screen using the LabVIEW software (National Instruments, Austin, TX). The response of the detector was shown to be linear over the whole scale of radioactivity used in this study. The probe sensitivity was experimentally determined to be 0.68 cps/kBq/mL. This sensitivity leads, with a 37-MBq (1 mCi) injected dose, to counting rates on the order of a few hundred counts per second (depending on the specificity of the tracer to its biologic target), allowing us to set the integration time to 1 s. For these b-microprobe experiments, rats (adult male Sprague-Dawley rat, Harlan) were anesthetized by inhalation of isoflurane (5% for induction, then 2.5% during surgery) and their femoral artery and vein catheterized for rapid blood DOI 10.1002/jps
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sampling and injection of drugs, respectively. Body temperature was adjusted by means of a heating pad. The animals were then mounted in a stereotactic frame, and a craniotomy performed to insert two microprobes into one striatum and one cerebellar hemisphere, respectively. The isoflurane-anesthetized rats were then injected with trace amounts of either (þ)-[11C]A-69024 or ()-[11C]A-69024 (675 180 mCi, mean SEM; specific radioactivity ¼ 538 293 Ci/mmol, mean SEM) alone, or 30 min before i.v. pretreatment with 1 mg/kg of either SCH23390 or the nonradioactive racemic ()A-69024. To assess serum pharmacokinetics, arterial blood samples were collected at preestablished intervals (from 0 to 60 min following administration) and centrifuged (3000 rpm; 4 min; 48C); plasmatic radioactivity was counted on 10 mL serum aliquots and expressed as percentage of injected dose per mL (% of ID/mL).
Micro-PET Imaging in the Rat To assess the regional distribution of the (þ)-A69024 enantiomer in the living rat brain, a microPET study was performed on one adult male Sprague-Dawley rat (Harlan), placed under isoflurane (2.5%) anesthesia. To ensure correct positioning of the animal into the apparatus, the animal was placed in a home-made stereotactic frame before i.v. injection with 3.36 mCi of (þ)[11C]A-69024. PET scans were performed using the high-resolution Focus micro-PET (CTI-Siemens, Knoxville, TN), which acquires 95 contiguous planes simultaneously. A transmission scan was performed for attenuation correction. Regions of interest were drawn directly on the micro-PET images, over brain regions corresponding to the striatum, the frontal cortex and the cerebellum.
Positron Emission Tomography in the Baboon Studies were conducted in accordance with the European convention for animal care (86–406) and the guide for the care and use of laboratory animals adopted by the National Institutes of Health. Two adult baboons (Papio papio), 20.3 1.1 kg body weight, were included in this study. Prior to PET imaging, each animal was submitted to magnetic resonance (MR) imaging examination JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 7, JULY 2008
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on a 1.5 T SIGNA system (General Electric, Milwaukee, WI). Animals were anesthetized by i.m. injection of a mixture of ketamine (15 mg/kg) and xylazine (1.5 mg/kg) and positioned into the magnet unit using a MR compatible headholder. A T1-weighted inversion-recovery sequence in 3D mode and a 256 192 matrix over 124 slices (1.5 mm thick) was used to generate the MR images to be coregistered with the PET images. PET scans were performed in 3D mode using the ECAT EXACT HRþ high-resolution tomograph (CTI-Siemens), which acquires 63 contiguous planes simultaneously. A transmission scan was performed for attenuation correction. Animals were fasted for 12 h and then anesthetized with ketamine (15 mg) followed by a mixture of ketamine/xylazine (15 mg/1.5 mg/kg) followed by isoflurane 1% in a NO2/O2 mixture (3/2, v/v) (Ohmeda ventilator OAV 7710, Ohmeda, Madison, WI). Primates were positioned in a custom-designed stereotaxic headholder aligned with crossed laser beams permanently attached on the tomograph to ensure an exact repositioning of the head in different PET sessions. Three PET scans were performed (6.1 0.8 mCi were injected with a SA of 496 275 Ci/mmol). In one baboon, pharmacokinetics of the radiotracer was followed during 90 min after i.v. injection. One baboon was pretreated with SCH23390 (1 mg/ kg i.v., 20 min before injection of the radiotracer) and the same PET protocol was performed. Alternatively, one baboon was i.v. injected with ()-A69024 (1 mg/kg) 30 min after injection of radiotracer in order to assess the displaceable binding of the radioligand. In all cases, the PET imaging lasted 60 min. During PET acquisition, arterial blood samples were withdrawn from the femoral artery at designated times and plasma radioactivity were counted using a gamma counter (Cobra autogamma, Perkin-Elmer) and expressed as percentage of injected dose per 100 mL of tissue (% ID/100 mL of tissue). For data analysis, regions of interest, selected on MRI slices, were copied on coregistered PET images in order to involve the caudate nucleus, the putamen, the hippocampus, the temporal cortex and the cerebellum. Concentration of radioactivity in the different regions of interest was calculated for each sequential PET scan and plotted versus time. Radioactivity was measured in the selected cerebral structures after correction for carbon11 decay and expressed as pmol/mL of tissue after normalization using the specific radioactivity measured at the time of injection. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 7, JULY 2008
RESULTS AND DISCUSSION Radiolabeling (þ)-A-69024 ((þ)-1-(2-bromo-4,5-dimethoxybenzyl)-7-hydroxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline) and ()-A-69024 (()-1-(2bromo-4,5-dimethoxybenzyl)-7-hydroxy-6-methoxy - 2 - methyl - 1,2, 3,4 - tetrahydroisoquinoline) were labeled with carbon-11 (T1/2: 20.38 min) at the methylamine function using no-carrier-added [11C]methyl triflate as the alkylating agent and the corresponding racemic N-demethylated precursor using minor modifications of published procedures.5 [11C]Methyl triflate was prepared according to a literature procedure from [11C]methyl iodide using silver triflate.11 [11C]Methyl iodide was prepared from [11C]carbon dioxide using the well-known two step protocol, consisting in trapping [11C]CO2 and reduction to [11C]methanol (LiAlH4) followed by iodination using aqueous HI giving [11C]methyl iodide.12 Reaction of [11C]methyl triflate with nor-()-A69024 was performed using standard conditions for the routine radiosynthesis of other N- or O-[11C]methylated radioligands13–17 and gave satisfactory yields for the preparation of ()[11C]A-69024 (12%, decay-corrected and based on starting [11C]carbon dioxide). Chiral HPLC purification gave radiochemically pure (þ)-[11C]A69024 and ()-[11C]A-69024 in 25–30 min.
Biological Evaluation Microdissection studies showed that the highest accumulation of the radioligand, (þ)-[11C]A-69024 occurred in the striatum, compared to all other regions of interest investigated (Fig. 1). Intermediate radioactivity uptake was found in the frontal cortex and the hippocampus, while the lowest (þ)-[11C]A-69024 uptake was found in the cerebellum (virtually devoided of D1 binding sites) and the plasma. Because of the virtual lack of dopamine D1-receptor population in the mammalian cerebellum, this region is often considered the structure of reference to determine nonspecific D1-receptor binding in vivo.18 Accordingly, the calculated region/cerebellum ratio at 30 min postinjection reached a value of 8 and 2 in the striatum and in the cerebral cortex, respectively (not shown). Interestingly, data obtained with the b-microprobe also supported a high selectivity of the radioligand for the dopamine D1-receptors in vivo. Following i.v. DOI 10.1002/jps
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Figure 1. Ex vivo binding profile of (þ)-[11C]A-69024 in various regions of the rat brain and plasma at various time-points after radiotracer i.v. injection. Data are expressed as % of injected dose per gram of tissue (% ID/g: mean SD). The striatum displays the highest brain uptake, followed by intermediate binding in the frontal cortex and finally the lowest binding in the hippocampus, the cerebellum and the plasma.
injection of the radioligand (Fig. 2A), the radioactivity in the striatum increased rapidly to reach a maximum level within 2–3 min postinjection, with a striatum to cerebellum ratio of 3.5. This ratio is notably smaller than the one obtained ex vivo with microdissection experiments. However, this discrepancy can be logically explained by differences in the amount of (þ)-[11C]A-69024 injected for b-microprobe experiments as compared to microdissection suggesting a higher partial occupation of D1 binding sites by the nonradioactive ligand in the b-microprobe study. Alternatively, this underestimation of the striatum/cerebellum ratio in the in vivo condition could result from a contamination from extrastriatal tissue due to the field of view of the probe that goes beyond the limits of the striatum.19 On the other hand, it is well documented that volatile anesthetics, such as isoflurane, interfere with dopamine regulation in the rat striatum,20 a phenomenon that may directly affect the receptor occupancy by the radioligand. Displacement and presaturation studies performed in rats using either the racemic mixture of A-69024 (Fig. 2B and D) or the selective D1receptor antagonist SCH23390 (Fig. 2C and E) indicated that only a displaceable and saturable binding could be evidenced in the striatum with no specific binding observed in any other brain regions containing D1-receptors such as different cortical regions (data not shown). In displacement studies following i.v. injection of either ()-ADOI 10.1002/jps
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69024 (1 mg/kg, Fig. 2B) or SCH23390 (1 mg/kg, Fig. 2C), a rapid decrease in striatal radioactivity was observed, approaching cerebellar levels 10 min later and onwards. These results were essentially confirmed by findings with the pretreatment experiments in which brain uptake values for (þ)-[11C]A-69024 obtained after either SCH23390 (Fig. 2D) or ()-A-69024 (Fig. 2E) administration, showed an essentially uniform tracer distribution across brain regions. Compared to the baseline study, there was a two- to threefold decline in striatal uptake whereas cerebellar radioactive counts remained essentially unaffected. In one study performed with the inactive enantiomer ()-[11C]A-69024, striatal and cerebellar uptake values were superimposable and displayed an essentially similar profile of radioactivity as the one observed in presaturation studies (Fig. 2F), indicating that this radiotracer does not bind specifically to dopamine D1-receptors in vivo. Micro-PET imaging in the rat (Fig. 3A) showed that the radiotracer mainly accumulates into the brain regions highly enriched in D1-receptors like the striatum, to a lesser extend in the frontal cortex regions and to a much lower level in the cerebellum. Time–activity curves revealed a brain uptake profile similar to that obtained with the b-microprobe experiments (Fig. 3B). The striatum to cerebellum ratio was 3.5 from 15 min postinjection and 2 in the frontal cortex. The amount of ligand injected was relatively high (9.91 nmol), nevertheless the quantification showed promising data in terms of regional distribution of the ligand and one could certainly expect an increased specific/nonspecific ratio with higher specific radioactivity. For micro-PET and b-microprobe experiments, all animals were anesthetized with isoflurane which could explain why the brain uptake was lower than that observed with the microdissection procedure obtained in the awake state. PET imaging in the baboon, essentially confirmed results obtained in the rat studies indicating a preferential accumulation of the tracer in caudate nucleus and putamen, intermediate uptake in cortical regions and hippocampus (Fig. 4A) and virtually no retention within the cerebellar region (date not shown). Thus, 5–10 min after injection of (þ)-[11C]A-69024, the uptake was highest in the basal ganglia (caudate nucleus and putamen) with lower binding in cortical areas and hippocampus, the lowest uptake being observed in the cerebellum (Fig. 4B). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 7, JULY 2008
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Figure 2. Time–activity curves of carbon-11 radioactivity derived from b-microprobe recordings in striatal and cerebellar regions after i.v. injection of trace amounts of (þ)[11C]A-69024 in the rat. (A) Control experiment. (B) Displacement experiment with nonradioactive ()-A-69024 (1 mg/kg; i.v.). (C) Displacement experiment with SCH23390 (1 mg/kg; i.v.). (D) Presaturation experiment with nonradioactive ()-A69024 (1 mg/kg; i.v.). (E) Presaturation experiment with SCH23390 (1 mg/kg; i.v.). (F) Time-activity curves of the nonactive enantiomer ()-[11C]A-69024 in the striatum and the cerebellum.
The ROI to cerebellum ratio increase until 40 min p.i. and then remain stable until the end of PET acquisition (90 min). For example, putamen and caudate to cerebellum ratio were 2.5; it reach 1.3 on average in temporal, parietal and occipital cortices; 1.6 in hippocampus and 1.4 in thalamus (data not shown). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 7, JULY 2008
The degree of specific binding for (þ)-[11C]A69024 was demonstrated by a blocking study in which SCH23390 (1 mg/kg, i.v.) was injected 20 min before the radiotracer (Fig. 4C). In this study, the wash out of the radiotracer was very fast and in all brain structures uptake were similar to that of cerebellum. The ROI to DOI 10.1002/jps
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Figure 3. (A) Summed (8–60 min) horizontal (left panel) and coronal (right panel) images obtained from micro-PET acquisition in a normal rat following trace amount injection of (þ)-[11C]A-69024. (B) Time–activity curves in various brain regions of a rat receiving 3.36 mCi of (þ)-[11C]A-69024. Serial images were acquired for 60 min. The data points appearing in the graph are at the midpoint of the interval.
cerebellum ratio never exceeded 1.4 in the striata and 1.1 in the cortical structures. In the displacement study (()-A-69024 1 mg/kg, i.v. injected 30 min after the radioligand), radioactivity levels decreased rapidly in the putamen and the caudate (Fig. 4B) to reach the cerebellar uptake level within 10–15 min postinjection of the cold compound (the ROI to cerebellum ratio, calculated after 40 min, never exceeded 1.2 whatever the brain structure considered). No significant tracer displacement was measured in any other brain region studied. Interestingly in the primate brain, dopaminergic D1-receptors are known to be present in the highest density in the basal ganglia, with intermediate levels in the neocortex and negligible densities in the cerebellum. This regional distribution of D1-receptor densities matches the in vivo distribution of radioactivity observed in the living baboon. In addition, ex vivo experiments have demonstrated that some of the radiotracer binding in the frontal cortex of trace amount-injected animals is displaceable, suggesting the presence of some specific binding of the DOI 10.1002/jps
Figure 4. (A) Coregistered PET and MRI coronal images in the baboon brain obtained after administration of (þ)-[11C]A-69024 (6.25 mCi). (B) Time–activity curves of (þ)-[11C]A-69024 in various brain regions of the baboon. After i.v. administration of nonradioactive ()-A-69024 (1 mg/kg, i.v.) at 30 minutes postradiotracer injection (arrow), a rapid washout (decrease) of the radioactivity can be observed in the putamen and the caudate nucleus, no major changes could be observed in all other brain regions studied. (C) Time–activity curves of (þ)-[11C]A-69024 in the baboon. The solid symbols correspond to the normal (control) brain kinetics of the tracer in the putamen and cerebellum of this primate whereas the open symbols represent the uptake of the tracer observed in the same regions after intravenous presaturation of the D1-receptors using 1 mg/kg of SCH23390. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 7, JULY 2008
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radiotracer in this region of intermediate receptor density. However in the displacement study in the baboon, no displacement could be evidenced in all cortical areas tested. The reason for this is still unclear. However, for this latter experiment one explanation could be derived from an infraoptimal specific activity of the radioligand on the day of the experiment.
CONCLUSION In conclusion, the in vivo binding of (þ)-[11C]A69024 demonstrates: (1) a tracer distribution profile within the brain highly consistent with the known distribution of dopamine D1 receptors, indicating excellent binding selectively in vivo; (2) a high striatum/cerebellum ratio both in rat and baboon, suggesting substantial specific binding relative to nonspecific binding in this region belonging to the basal ganglia; (3) an intermediate specific binding was observed in cortical regions in the nonhuman primate. Accordingly, (þ)-[11C]A69024 appears as a promising tracer to visualize and quantify striatal dopamine D1-receptors by means of positron emission tomography.
ACKNOWLEDGMENTS The authors wish to thank the cyclotron operators Mr. Daniel Gouel, Mr. Christophe Peronne and Mr. Christophe Lecheˆne for performing the irradiations. This work was supported in part by DEST and the French Embassy in Australia under the FAST program (FR040051).
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C, Halldin C, Mauclaire L, Guilloteau D, Mazie`re B, Crouzel C. 2000. Highly efficient synthesis of [11C]PE2I, a selective radioligand for the quantification of the dopamine transporter using PET. J Label Compds Radiopharm 43:997–1004. 16. Dolle´ F, Bramoulle´ Y, Hinnen F, Fowler J. 2002. Efficient synthesis and formulation of (R)-()[11C]deprenyl, a selective radioligand for the quantification of MAO-B activity using PET. J Label Compds Radiopharm 45:803–811. 17. Dolle´ F, Valette H, Demphel S, Coulon C, Ottaviani M, Bottlaender M, Kassiou M. 2004. Radiosynthesis and in vivo evaluation of [11C]Ro-647312: A novel NR1/2B subtype selective NMDA receptor radioligand. J Label Compds Radiopharm 47:911–920.
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18. Rosa-Neto P, Doudet DJ, Cumming P. 2004. Gradients of dopamine D1- and D2/3-binding sites in the basal ganglia of pig and monkey measured by PET. Neuroimage 22:1076–1083. 19. Ginovart N, Sun W, Wilson AA, Houle S, Kapur S. 2004. Quantitative validation of an intracerebral beta-sensitive microprobe system to determine in vivo drug-induced receptor occupancy using [11C]raclopride in rats. Synapse 52:89–99. 20. Tsukada H, Nishiyama S, Kakiuchi T, Ohba H, Sato K, Harada N, Nakanishi S. 1999. Isoflurane anesthesia enhances the inhibitory effects of cocaine and GBR12909 on dopamine transporter: PET studies in combination with microdialysis in the monkey brain. Brain Res 849:85–96.
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CNRS, URA 2210, 4 place du Ge´ne´ral Leclerc, F-91406 Orsay, France
2
CEA, DSV, I2BM, SHFJ, Laboratoire d’Imagerie Mole´culaire Expe´rimentale, F-91406 Orsay, France
3
CEA, DSV, I2BM, Molecular Imaging Research Center, F-92265 Fontenay-aux-Roses, France
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Discipline of Medical Radiation Sciences, University of Sydney, Sydney, NSW 2006, Australia
5
Brain and Mind research Institute, University of Sydney, Sydney, NSW 2050, Australia
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School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia
Received 23 May 2007; revised 12 July 2007; accepted 17 July 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21168
ABSTRACT: In vivo pharmacokinetic and brain binding characteristics of (þ)-[11C]A69024, a high-affinity-D1-selective dopamine receptor antagonist, were assessed with micro-PET and b-microprobes in the rat and PET in the baboon. The biodistribution of (þ)-[11C]A-69024 in rats and baboons showed a rapid brain uptake (reaching a maximal value at 5 and 15 min postinjection in rats and baboons, respectively), followed by a slow wash out. The region/cerebellum concentration ratio was characterized by a fourfold higher uptake in striatum and a twofold higher uptake in cortical regions, consistent with in vivo specific binding of the radiotracer in these cerebral regions. Furthermore, this specific (þ)-[11C]A-69024 binding significantly correlated with the reported in vitro distribution of dopamine D1-receptors. Finally, the specific uptake of the tracer in the striatum and cortical regions was completely prevented by either a pretreatment with large doses of nonradioactive ()A-69024 or of the D1-selective antagonist SCH23390, resulting in a similar uptake in the reference region (cerebellum) and in other brain regions. Thus, (þ)-[11C]A-69024 appears to be a specific and enantioselective radioligand to visualize and quantify brain dopamine D1 receptors in vivo using positron emission tomography. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2811–2819, 2008
Keywords:
PET; pharmacokinetics; pharmacodynamics; CNS; receptors
INTRODUCTION
Laurent Besret and Fre´de´ric Dolle´ have contributed equally to this work. Correspondence to: Michael Kassiou (Telephone: 612-93510894; Fax: 612-9351-0652; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 2811–2819 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
Positron emission tomography (PET) is an imaging technique which offers the opportunity to study drug pharmacokinetics and pharmacodynamics in vivo. It allows us to visualize and quantify receptor binding sites in living patients and derive correlates between normal and disease states.
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Several PET radioligands with dopamine D1 receptor antagonistic properties have been developed during the last decade.1–3 Unfortunately, most of these D1-preferential receptor ligands display high in vivo nonspecific binding and/or too fast dissociation kinetics which greatly impede their usefulness as in vivo PET tracers. For example two selective benzazepine D1 antagonists, [11C]SCH23390 and [11C]NNC112, with favorable binding characteristics in vitro have been evaluated in humans in vivo.3,4 However, the pharmacokinetic profile of 11C]SCH23390 is not favorable in vivo displaying a rapid washout from the brain after intravenous injection, a property that greatly limits its ability to quantify D1receptor in regions with intermediate receptor densities, such as cortical regions. Similarly, [11C]NNC112 which was reported as a ligand of choice for dopamine D1 receptor visualization and quantification in various brain regions,3 displays in vivo a high nonspecific binding which limits our ability to assess specific binding in regions like the hippocampus or various cortical areas. As an alternative to these two benzazepine radiotracers, we attempted at developing a structurally unrelated radioligand which would also be a highly selective, high affinity D1-receptor antagonist (Tab. 1) but displaying higher specific binding in vivo.5–7 In vitro, the better D1-selectivity of the (þ) enantiomer of the isoquinoline A-69024 compared to either SCH23390 or other benzazepine derivatives, makes it an interesting candidate for PET analyses.5–8 We therefore undertook the carbon-11 radiosynthesis of each of the enantiomers of A-69024 namely, (þ)[11C]A-69024 and ()-[11C]A-69024 and now report on the pharmacokinetics and in vivo regional brain biodistribution of both [11C]A-69024 enantiomers studied by micro-PET, b-microprobes and conventional PET in rodents and nonhuman primates.
2 - [11 C]methyl - 1,2,3,4 - tetrahydroisoquinoline) and ()-[11C]A-69024 (()-1-(2-bromo-4,5-dimethoxybenzyl)-7-hydroxy-6-methoxy-2-[11C]methyl1,2,3,4-tetrahydroisoquinoline) used the following conditions: (1) trapping at room temperature of the [11C]methyl triflate in 300 mL of acetone containing 1.0 mg of precursor (nor-()-A-69024) and 5 mL of an aqueous 3 M solution of sodium hydroxide (6 equiv.); (2) concentration to dryness of the reaction mixture at 1008C using a nitrogen stream; (3) taking up the crude reaction mixture with 0.5 mL of the HPLC mobile phase; (4) HPLC purification (hexane/isopropanol [60/40 (v:v)]; 3 mL/min) on semipreparative Daicel ChiralCel OD (250 10 mm). Typically, 30–80 mCi (1.11– 2.96 GBq) of (þ)-A-69024 (tR ¼ 9–10 min) and ()A-69024 (tR ¼ 12–13 min) were routinely obtained using these conditions within 25–30 min including HPLC purification with specific radioactivities ranging from 0.7 to 2.5 Ci/mmol (25.9–92.5 GBq/mmol). Formulation of labeled product for i.v. injection was effected as follows: The HPLC-collected fraction containing (þ)- or ()-[11C]A-69024 was concentrated to dryness (using a rotavapor, oil bath temperature: 70–808C), then the residue was taken up in 10 mL of physiological saline containing 10% of ethanol. The radioligand preparation for injection was a clear and colorless solution and its pH was between 5 and 7. The preparation was found to be >95% chemically pure and >99% radiochemically pure, as demonstrated by HPLC analysis (H2O/CH3CN [75/25 (v:v) containing lowUV PIC1 B7 reagent, 20 mL for 1000 mL]; 2.0 mL/ min) on analytical Symmetry-M1 C-18, Waters (50 4.6 mm), free from starting labeling precursor and was shown to be radiochemically stable for at least 60 min. The radiosynthesized (þ)- and ()-[11C]A-69024 also coeluted with an authentic sample of ()-A-69024.
MATERIALS AND METHODS
Biodistribution Experiments
Radiosynthesis of (R)-[11C]A-69024 and ()-[11C]A-69024
Studies were performed on adult male SpragueDawley rats (269 9 g, mean SEM; n ¼ 10, obtained from Harlan, Gannat, France) in compliance with the French law on animal experimentation. Animals were housed in groups of two to four with food and water ad libitum and kept on a 12:12 h light:dark cycle (dark period 07:00 p.m.– 7:00 a.m.) in temperature and humidity-controlled facilities. The animals were decapitated at different times (5, 15, 20, 30, and 45 min) following radioligand injection (50 mCi); brains
Preparation of (þ)-[11C]A-69024 ((þ)-1-(2-bromo4 , 5 - dimethoxybenzyl ) - 7 - hydroxy - 6 - methoxy Table 1. In Vitro Binding Affinities of Selected D1-Receptor Ligands SCH23390 0.14 nM8
NNC112 0.18 nM8
A-69024 þ0.6 nM7, 12.6 nM7
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were rapidly removed and dissected at 48C in order to obtain four cortical and subcortical regions of interest. Samples were weighed and radioactivity was counted with a gamma counter (Cobra autogamma, Perkin-Elmer, Courtaboeuf, France) and expressed as percentage of injected dose/g of tissue (% ID/g). Statistical significance was evaluated using the Dunnett multiple comparisons test. p < 0.05 was considered statistically significant.
b-Microprobe Studies The b-microprobe (Biospace Labs, Paris, France) is an in situ technique involving the insertion of a fine probe into brain tissue in a way very similar to that used for microdialysis and electrophysiological recordings. It functions as an implantable radioactivity counter9,10 that takes advantage of the limited range of positron particles within biological tissues to define a localized detection volume around the implanted probe (2.05 mm around the tip of the probe for carbon-11-labeled radiotracers). The sensitive end of the probe consists of a 1-mm-long and 250–500 mm-diameter plastic scintillating fiber (BCF-12, Bicron, Newbury, OH). This detection tip is fused with a 250– 500 mm-diameter clear optic fiber (BCF-98, Bicron) whose length is adjustable to the depth of the brain structure targeted (e.g., 7 mm in the case of an implantation in the rat striatum). This feature enables one to detect the light exiting the brain. The light is then transmitted through an optic fiber to a photomultiplier (R7400P, Hamamatsu, Hamamatsu City, Japan). The electronic pulses are analyzed by a counting electronic device, and the measured counting rate is displayed online on a computer screen using the LabVIEW software (National Instruments, Austin, TX). The response of the detector was shown to be linear over the whole scale of radioactivity used in this study. The probe sensitivity was experimentally determined to be 0.68 cps/kBq/mL. This sensitivity leads, with a 37-MBq (1 mCi) injected dose, to counting rates on the order of a few hundred counts per second (depending on the specificity of the tracer to its biologic target), allowing us to set the integration time to 1 s. For these b-microprobe experiments, rats (adult male Sprague-Dawley rat, Harlan) were anesthetized by inhalation of isoflurane (5% for induction, then 2.5% during surgery) and their femoral artery and vein catheterized for rapid blood DOI 10.1002/jps
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sampling and injection of drugs, respectively. Body temperature was adjusted by means of a heating pad. The animals were then mounted in a stereotactic frame, and a craniotomy performed to insert two microprobes into one striatum and one cerebellar hemisphere, respectively. The isoflurane-anesthetized rats were then injected with trace amounts of either (þ)-[11C]A-69024 or ()-[11C]A-69024 (675 180 mCi, mean SEM; specific radioactivity ¼ 538 293 Ci/mmol, mean SEM) alone, or 30 min before i.v. pretreatment with 1 mg/kg of either SCH23390 or the nonradioactive racemic ()A-69024. To assess serum pharmacokinetics, arterial blood samples were collected at preestablished intervals (from 0 to 60 min following administration) and centrifuged (3000 rpm; 4 min; 48C); plasmatic radioactivity was counted on 10 mL serum aliquots and expressed as percentage of injected dose per mL (% of ID/mL).
Micro-PET Imaging in the Rat To assess the regional distribution of the (þ)-A69024 enantiomer in the living rat brain, a microPET study was performed on one adult male Sprague-Dawley rat (Harlan), placed under isoflurane (2.5%) anesthesia. To ensure correct positioning of the animal into the apparatus, the animal was placed in a home-made stereotactic frame before i.v. injection with 3.36 mCi of (þ)[11C]A-69024. PET scans were performed using the high-resolution Focus micro-PET (CTI-Siemens, Knoxville, TN), which acquires 95 contiguous planes simultaneously. A transmission scan was performed for attenuation correction. Regions of interest were drawn directly on the micro-PET images, over brain regions corresponding to the striatum, the frontal cortex and the cerebellum.
Positron Emission Tomography in the Baboon Studies were conducted in accordance with the European convention for animal care (86–406) and the guide for the care and use of laboratory animals adopted by the National Institutes of Health. Two adult baboons (Papio papio), 20.3 1.1 kg body weight, were included in this study. Prior to PET imaging, each animal was submitted to magnetic resonance (MR) imaging examination JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 7, JULY 2008
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on a 1.5 T SIGNA system (General Electric, Milwaukee, WI). Animals were anesthetized by i.m. injection of a mixture of ketamine (15 mg/kg) and xylazine (1.5 mg/kg) and positioned into the magnet unit using a MR compatible headholder. A T1-weighted inversion-recovery sequence in 3D mode and a 256 192 matrix over 124 slices (1.5 mm thick) was used to generate the MR images to be coregistered with the PET images. PET scans were performed in 3D mode using the ECAT EXACT HRþ high-resolution tomograph (CTI-Siemens), which acquires 63 contiguous planes simultaneously. A transmission scan was performed for attenuation correction. Animals were fasted for 12 h and then anesthetized with ketamine (15 mg) followed by a mixture of ketamine/xylazine (15 mg/1.5 mg/kg) followed by isoflurane 1% in a NO2/O2 mixture (3/2, v/v) (Ohmeda ventilator OAV 7710, Ohmeda, Madison, WI). Primates were positioned in a custom-designed stereotaxic headholder aligned with crossed laser beams permanently attached on the tomograph to ensure an exact repositioning of the head in different PET sessions. Three PET scans were performed (6.1 0.8 mCi were injected with a SA of 496 275 Ci/mmol). In one baboon, pharmacokinetics of the radiotracer was followed during 90 min after i.v. injection. One baboon was pretreated with SCH23390 (1 mg/ kg i.v., 20 min before injection of the radiotracer) and the same PET protocol was performed. Alternatively, one baboon was i.v. injected with ()-A69024 (1 mg/kg) 30 min after injection of radiotracer in order to assess the displaceable binding of the radioligand. In all cases, the PET imaging lasted 60 min. During PET acquisition, arterial blood samples were withdrawn from the femoral artery at designated times and plasma radioactivity were counted using a gamma counter (Cobra autogamma, Perkin-Elmer) and expressed as percentage of injected dose per 100 mL of tissue (% ID/100 mL of tissue). For data analysis, regions of interest, selected on MRI slices, were copied on coregistered PET images in order to involve the caudate nucleus, the putamen, the hippocampus, the temporal cortex and the cerebellum. Concentration of radioactivity in the different regions of interest was calculated for each sequential PET scan and plotted versus time. Radioactivity was measured in the selected cerebral structures after correction for carbon11 decay and expressed as pmol/mL of tissue after normalization using the specific radioactivity measured at the time of injection. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 7, JULY 2008
RESULTS AND DISCUSSION Radiolabeling (þ)-A-69024 ((þ)-1-(2-bromo-4,5-dimethoxybenzyl)-7-hydroxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline) and ()-A-69024 (()-1-(2bromo-4,5-dimethoxybenzyl)-7-hydroxy-6-methoxy - 2 - methyl - 1,2, 3,4 - tetrahydroisoquinoline) were labeled with carbon-11 (T1/2: 20.38 min) at the methylamine function using no-carrier-added [11C]methyl triflate as the alkylating agent and the corresponding racemic N-demethylated precursor using minor modifications of published procedures.5 [11C]Methyl triflate was prepared according to a literature procedure from [11C]methyl iodide using silver triflate.11 [11C]Methyl iodide was prepared from [11C]carbon dioxide using the well-known two step protocol, consisting in trapping [11C]CO2 and reduction to [11C]methanol (LiAlH4) followed by iodination using aqueous HI giving [11C]methyl iodide.12 Reaction of [11C]methyl triflate with nor-()-A69024 was performed using standard conditions for the routine radiosynthesis of other N- or O-[11C]methylated radioligands13–17 and gave satisfactory yields for the preparation of ()[11C]A-69024 (12%, decay-corrected and based on starting [11C]carbon dioxide). Chiral HPLC purification gave radiochemically pure (þ)-[11C]A69024 and ()-[11C]A-69024 in 25–30 min.
Biological Evaluation Microdissection studies showed that the highest accumulation of the radioligand, (þ)-[11C]A-69024 occurred in the striatum, compared to all other regions of interest investigated (Fig. 1). Intermediate radioactivity uptake was found in the frontal cortex and the hippocampus, while the lowest (þ)-[11C]A-69024 uptake was found in the cerebellum (virtually devoided of D1 binding sites) and the plasma. Because of the virtual lack of dopamine D1-receptor population in the mammalian cerebellum, this region is often considered the structure of reference to determine nonspecific D1-receptor binding in vivo.18 Accordingly, the calculated region/cerebellum ratio at 30 min postinjection reached a value of 8 and 2 in the striatum and in the cerebral cortex, respectively (not shown). Interestingly, data obtained with the b-microprobe also supported a high selectivity of the radioligand for the dopamine D1-receptors in vivo. Following i.v. DOI 10.1002/jps
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Figure 1. Ex vivo binding profile of (þ)-[11C]A-69024 in various regions of the rat brain and plasma at various time-points after radiotracer i.v. injection. Data are expressed as % of injected dose per gram of tissue (% ID/g: mean SD). The striatum displays the highest brain uptake, followed by intermediate binding in the frontal cortex and finally the lowest binding in the hippocampus, the cerebellum and the plasma.
injection of the radioligand (Fig. 2A), the radioactivity in the striatum increased rapidly to reach a maximum level within 2–3 min postinjection, with a striatum to cerebellum ratio of 3.5. This ratio is notably smaller than the one obtained ex vivo with microdissection experiments. However, this discrepancy can be logically explained by differences in the amount of (þ)-[11C]A-69024 injected for b-microprobe experiments as compared to microdissection suggesting a higher partial occupation of D1 binding sites by the nonradioactive ligand in the b-microprobe study. Alternatively, this underestimation of the striatum/cerebellum ratio in the in vivo condition could result from a contamination from extrastriatal tissue due to the field of view of the probe that goes beyond the limits of the striatum.19 On the other hand, it is well documented that volatile anesthetics, such as isoflurane, interfere with dopamine regulation in the rat striatum,20 a phenomenon that may directly affect the receptor occupancy by the radioligand. Displacement and presaturation studies performed in rats using either the racemic mixture of A-69024 (Fig. 2B and D) or the selective D1receptor antagonist SCH23390 (Fig. 2C and E) indicated that only a displaceable and saturable binding could be evidenced in the striatum with no specific binding observed in any other brain regions containing D1-receptors such as different cortical regions (data not shown). In displacement studies following i.v. injection of either ()-ADOI 10.1002/jps
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69024 (1 mg/kg, Fig. 2B) or SCH23390 (1 mg/kg, Fig. 2C), a rapid decrease in striatal radioactivity was observed, approaching cerebellar levels 10 min later and onwards. These results were essentially confirmed by findings with the pretreatment experiments in which brain uptake values for (þ)-[11C]A-69024 obtained after either SCH23390 (Fig. 2D) or ()-A-69024 (Fig. 2E) administration, showed an essentially uniform tracer distribution across brain regions. Compared to the baseline study, there was a two- to threefold decline in striatal uptake whereas cerebellar radioactive counts remained essentially unaffected. In one study performed with the inactive enantiomer ()-[11C]A-69024, striatal and cerebellar uptake values were superimposable and displayed an essentially similar profile of radioactivity as the one observed in presaturation studies (Fig. 2F), indicating that this radiotracer does not bind specifically to dopamine D1-receptors in vivo. Micro-PET imaging in the rat (Fig. 3A) showed that the radiotracer mainly accumulates into the brain regions highly enriched in D1-receptors like the striatum, to a lesser extend in the frontal cortex regions and to a much lower level in the cerebellum. Time–activity curves revealed a brain uptake profile similar to that obtained with the b-microprobe experiments (Fig. 3B). The striatum to cerebellum ratio was 3.5 from 15 min postinjection and 2 in the frontal cortex. The amount of ligand injected was relatively high (9.91 nmol), nevertheless the quantification showed promising data in terms of regional distribution of the ligand and one could certainly expect an increased specific/nonspecific ratio with higher specific radioactivity. For micro-PET and b-microprobe experiments, all animals were anesthetized with isoflurane which could explain why the brain uptake was lower than that observed with the microdissection procedure obtained in the awake state. PET imaging in the baboon, essentially confirmed results obtained in the rat studies indicating a preferential accumulation of the tracer in caudate nucleus and putamen, intermediate uptake in cortical regions and hippocampus (Fig. 4A) and virtually no retention within the cerebellar region (date not shown). Thus, 5–10 min after injection of (þ)-[11C]A-69024, the uptake was highest in the basal ganglia (caudate nucleus and putamen) with lower binding in cortical areas and hippocampus, the lowest uptake being observed in the cerebellum (Fig. 4B). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 7, JULY 2008
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Figure 2. Time–activity curves of carbon-11 radioactivity derived from b-microprobe recordings in striatal and cerebellar regions after i.v. injection of trace amounts of (þ)[11C]A-69024 in the rat. (A) Control experiment. (B) Displacement experiment with nonradioactive ()-A-69024 (1 mg/kg; i.v.). (C) Displacement experiment with SCH23390 (1 mg/kg; i.v.). (D) Presaturation experiment with nonradioactive ()-A69024 (1 mg/kg; i.v.). (E) Presaturation experiment with SCH23390 (1 mg/kg; i.v.). (F) Time-activity curves of the nonactive enantiomer ()-[11C]A-69024 in the striatum and the cerebellum.
The ROI to cerebellum ratio increase until 40 min p.i. and then remain stable until the end of PET acquisition (90 min). For example, putamen and caudate to cerebellum ratio were 2.5; it reach 1.3 on average in temporal, parietal and occipital cortices; 1.6 in hippocampus and 1.4 in thalamus (data not shown). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 7, JULY 2008
The degree of specific binding for (þ)-[11C]A69024 was demonstrated by a blocking study in which SCH23390 (1 mg/kg, i.v.) was injected 20 min before the radiotracer (Fig. 4C). In this study, the wash out of the radiotracer was very fast and in all brain structures uptake were similar to that of cerebellum. The ROI to DOI 10.1002/jps
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Figure 3. (A) Summed (8–60 min) horizontal (left panel) and coronal (right panel) images obtained from micro-PET acquisition in a normal rat following trace amount injection of (þ)-[11C]A-69024. (B) Time–activity curves in various brain regions of a rat receiving 3.36 mCi of (þ)-[11C]A-69024. Serial images were acquired for 60 min. The data points appearing in the graph are at the midpoint of the interval.
cerebellum ratio never exceeded 1.4 in the striata and 1.1 in the cortical structures. In the displacement study (()-A-69024 1 mg/kg, i.v. injected 30 min after the radioligand), radioactivity levels decreased rapidly in the putamen and the caudate (Fig. 4B) to reach the cerebellar uptake level within 10–15 min postinjection of the cold compound (the ROI to cerebellum ratio, calculated after 40 min, never exceeded 1.2 whatever the brain structure considered). No significant tracer displacement was measured in any other brain region studied. Interestingly in the primate brain, dopaminergic D1-receptors are known to be present in the highest density in the basal ganglia, with intermediate levels in the neocortex and negligible densities in the cerebellum. This regional distribution of D1-receptor densities matches the in vivo distribution of radioactivity observed in the living baboon. In addition, ex vivo experiments have demonstrated that some of the radiotracer binding in the frontal cortex of trace amount-injected animals is displaceable, suggesting the presence of some specific binding of the DOI 10.1002/jps
Figure 4. (A) Coregistered PET and MRI coronal images in the baboon brain obtained after administration of (þ)-[11C]A-69024 (6.25 mCi). (B) Time–activity curves of (þ)-[11C]A-69024 in various brain regions of the baboon. After i.v. administration of nonradioactive ()-A-69024 (1 mg/kg, i.v.) at 30 minutes postradiotracer injection (arrow), a rapid washout (decrease) of the radioactivity can be observed in the putamen and the caudate nucleus, no major changes could be observed in all other brain regions studied. (C) Time–activity curves of (þ)-[11C]A-69024 in the baboon. The solid symbols correspond to the normal (control) brain kinetics of the tracer in the putamen and cerebellum of this primate whereas the open symbols represent the uptake of the tracer observed in the same regions after intravenous presaturation of the D1-receptors using 1 mg/kg of SCH23390. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 7, JULY 2008
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radiotracer in this region of intermediate receptor density. However in the displacement study in the baboon, no displacement could be evidenced in all cortical areas tested. The reason for this is still unclear. However, for this latter experiment one explanation could be derived from an infraoptimal specific activity of the radioligand on the day of the experiment.
CONCLUSION In conclusion, the in vivo binding of (þ)-[11C]A69024 demonstrates: (1) a tracer distribution profile within the brain highly consistent with the known distribution of dopamine D1 receptors, indicating excellent binding selectively in vivo; (2) a high striatum/cerebellum ratio both in rat and baboon, suggesting substantial specific binding relative to nonspecific binding in this region belonging to the basal ganglia; (3) an intermediate specific binding was observed in cortical regions in the nonhuman primate. Accordingly, (þ)-[11C]A69024 appears as a promising tracer to visualize and quantify striatal dopamine D1-receptors by means of positron emission tomography.
ACKNOWLEDGMENTS The authors wish to thank the cyclotron operators Mr. Daniel Gouel, Mr. Christophe Peronne and Mr. Christophe Lecheˆne for performing the irradiations. This work was supported in part by DEST and the French Embassy in Australia under the FAST program (FR040051).
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