ORIGINAL ARTICLE 
Annals of Nuclear Medicine Vol. 14, No. 2, 81-89, 2000 
Further characterization of a CNS adenosine A2a receptor ligand [11C]KF18446 with in vitro autoradiography and in vivo tissue uptake 
Kiichi ISHIWATA,* Nobuo OGI,*,** Junichi SHIMADA,*** Hiromi NONAKA,*** Akira TANAKA,** Fumio SUZUKI*** and Michio SENDA* 
*Positron Medical Center, Tokyo Metropolitan Institute of Gerontology 
**Showa College of Pharmaceutical Sciences 
***Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Company 
PET assessment of the adenosine A2a receptors localized in the striatum offers us a potential new diagnostic tool for neurological disorders. In the present study, we carried out in vitro receptor autoradiography of a newly developed PET ligand [11C]KF18446 ([7-methyl-11C]-(E)-8-(3,4,5-trimethoxystyryl)- 1,3,7-trimethylxanthine) with rat brain sections. [11C]KF18446 showed a high striatum/cortex binding ratio (5.0) and low nonspecific binding (<10%), suggesting that [11C]KF18446 has characteristics comparable or slightly superior to [3H]CGS 21680 or [3H]SCH 58261, which are currently available representative A2a receptor ligands. Scatchard analysis indicated a Kd of 9.8 nM and a Bmax of 170 fmol/mm3 tissue in the striatum and a Kd of 16.4 nM and a Bmax of 33 fmol/mm3 tissue in the cortex. Seven xanthine-type and four nonxanthine-type adenosine receptor ligands with an affinity for the adenosine A2a receptors significantly reduced the in vitro binding of [11C]KF18446 to the brain section. The blocking effects were much stronger in the striatum than in the cortex, but did not necessarily parallel their affinity. On the other hand, four xanthine-type ligands and one nonxanthine-type ligand (SCH 58261) of the 11 ligands studied reduced the in vivo uptake of [11C]KF18446 in mice, but other ligands, including A1-selective and nonselective ligands and three nonxanthine-type A2a-selective antagonists did not. We conclude that [11C]KF18446 is a promising adenosine A2a receptor ligand for PET study. 
Key words: [11C]KF18446, adenosine A2a receptor, striatum, PET 
INTRODUCTION 
ADENOSINE is an endogenous modulator of a number of physiological functions in the central nervous system (CNS) as well as in peripheral organs. Recent advances in molecular biology and pharmacology have demonstrated the presence of at least four subtypes i.e., A1, A2a, A2b, and A3 receptors.1-3 
In the CNS, adenosine A1 receptors which exhibit higher affinity for adenosine and inhibit adenylyl cyclase are present both pre- and postsynaptically in many re-

Received September 22, 1999, revision accepted November 18, 1999. 
For reprint contact: Kiichi Ishiwata, Ph.D., 1-1 Naka-cho, Itabashi, Tokyo 173-0022, JAPAN. 
E-mail: ishiwata@pet.tmig.or.jp 

gions, being rich in the hippocampus, cerebral cortex, thalamic nuclei, the basal ganglia and the cerebellar cortex in animals4-7 and humans.8,9 Adenosine A2a receptors which exhibit lower affinity for adenosine and stimulate adenylyl cyclase are highly enriched in the striatum, nucleus accumbens and olfactory tubercle, in which dopamine D1 and D2 receptors are localized at very high densities.10-12 Recent studies also demonstrated the presence of A2a receptors in the hippocampus and cortex.13-17 Adenosine A2b receptors show a ubiquitous distribution.10-12 By the in situ hybridization technique, adenosine A2a receptor mRNA and dopamine D2 receptor mRNA are found to be mainly expressed in striatopallidal gamma-aminobutyric acid (GABA)-ergic-enkephaline neurons.18,19 The adenosine A2a receptor density is significantly reduced in the striatum of patients with Huntington's chorea with selective degeneration of the striatopallidal neurons, but not in patients with Parkinson's disease with selective degeneration of nigrostriatal dopamine neurons.20 
Recently, adenosine receptors in the CNS have been considered as targets for new drugs for many neurological and psychiatric disorders such as Parkinson's disease and schizophrenia.21-23 In patients with Parkinson's disease, L-dopa treatment is partially effective, and its chronic use over several years may lead to lose efficacy. Therefore, adenosine therapy is expected to be used as an alternative or adjunct therapy.21 In treating schizophrenia, chronic administration of neuroleptics frequently results in the development of severe movement disorders.24 Animal experiments suggest that adenosine A2a receptors are associated with antipsychotic activity of the neuroleptics through interaction with dopamine receptors.25 
In general, development of selective receptor agonists and antagonists is essential for investigating neuroreceptor systems. As for adenosine A2a receptors, a selective A2a receptor agonist [3H]-2-[p-(2-carboxyethyl)phenethyl-amino]-5'-N-ethylcarboxamidoadenosine ([3H]CGS 21680) is currently used as a standard radiolabeled ligand for in vitro studies of adenosine A2a receptors. As selective A2a antagonists, xanthine-type compounds have been used as selective probes, since the first proposal of KF17837 ((E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine) by Shimada et al.26 Nonxanthine-type antagonists have also been proposed,27-29 including 5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo [4,3-e]-1,2,4-triazolo[1,5-c]pyridine (SCH 58261)30 and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol (ZM 241385)31 which have a high affinity and selectivity for the A2a receptors. Their 3H- or 125I-labeled analogs are used as radiolabeled ligands.32-34 
For the purpose of studying the adenosine A2a receptors in humans by positron emission tomography (PET), we have recently prepared [11C]KF17837.35,36 This was preferentially taken up by the striatum in mice, rats and monkeys, but to a smaller extent by the cerebral cortex and cerebellum, suggesting that the selectivity of [11C]KF17837 for the adenosine A2a receptors may not be sufficient as a PET tracer. In the continuing search for a more selective ligand, we found that [7-methyl-11C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([11C]KF18446) showed much more selective affinity and lower nonspecific uptake in vivo than [11C]KF17837.37,38 In a blocking study to characterize the selectivity of [11C]KF18446 in mice, the striatal uptake was blocked by co-injection of any of four high affinity xanthine-type A2a receptor antagonists, but the blocking effect of a nonxanthine-type antagonist SCH 58261 was weaker than that of xanthine-type A2a antagonists.38 Another nonxanthine-type A2a antagonist ZM 241385 did not block the striatal uptake of [11C]KF18446 (unpublished data). Also the uptake of [11C]KF17837 was not significantly decreased in vivo by SCH 58261 or ZM 241385.36 
In the present study, we carried out in vitro receptor autoradiography (ARG) of [11C]KF18446 to characterize the new PET ligand. The blocking effects of various xanthine-type and nonxanthine-type adenosine receptor ligands on the in vitro binding of [11C]KF18446 to the rat brain section are described. We also examined the in vivo blocking effects of the ligands on the brain uptake of [11C]KF18446 in mice by the tissue dissection method. A part of this study was presented at the 6th International Symposium on Purines and Pyrimidines held in May 19-39 24 1998, in Ferrara, Italy.3
MATERIALS AND METHODS 
Materials 
Four xanthine-type adenosine A2a receptor antagonists, KF17837,26 KF18446,38 (E)-1,3-diallyl-7-methyl-8-(3,4,5-trimethoxystyryl)xanthine (KF19631)38 and 8-chlorostyryl-1,3,7-trimethylxanthine (CSC),40 four nonxanthine-type adenosine A2a receptor antagonists, SCH 58261, ZM 241385, 8-chloro-1-phenyl[1,2,4] triazolo[4,3-a]quinoxalin-4-amine (CP-66713)41 and 2-(2-furyl)-5-[2-(morpholino)ethylamino][1,2,4] triazolo [1,5-a] [1,3,5]triazine-7-amine (ZD9255) (Jones, WO 94/14812 1994), and an adenosine A1 antagonist 8-dicyclopropylmethyl-1,3-dipropylxanthine (KF 15372)42 were prepared by Kyowa Hakko Kogyo Company. 3,7-Dimethyl-1-propargylxanthine (DMPX),43 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine (XAC),42 and CGS 21680 were purchased from Research Biochemical, Inc. (Natick, MA, USA). 
[11C]KF18446 was prepared by 11C-methylation of the demethyl compound with [11C]methyl iodide by the known method.37,38 The specific radioactivity was 10-72 TBq/mmol. 
Male Wistar rats (8-9 weeks old) and male ddY mice (7-9 weeks old) were obtained from Tokyo Laboratory Animals Company (Tokyo, Japan). The animal studies were approved by the Animal Care and Use Committee of Tokyo Metropolitan Institute of Gerontology. 
In vitro binding of [11C]KF18446 to rat brain section 
In vitro ARG of [11C]KF18446 was performed by radioluminography as described previously.44 Three sets of 20 um thick adjacent coronal rat brain sections near the bregma were prepared. According to the method of Przedborski et al.,45 two sets of the brain sections were pre-incubated in 50 mM Tris-HCl, pH 7.4, containing 10 mM MgCl2 and 0.2 IU/mL adenosine deaminase for 30 min at room temperature to remove endogenous adenosine, and then the sections were incubated in the same buffer containing 1.54 nM (100 kBq/mL) [11C]KF18446 for 75 min at room temperature. Nonspecific binding was determined by incubating the other set of the sections in the medium, to which cold KF18446 had been added to a final concentration of 20 uM. The assay was stopped by washing the sections with ice-cold 50 mM Tris-HCl (pH 7.4) containing 120 mM NaCl. These procedures were carried out under dim light to prevent isomerization of radioactive and cold KF18446.35,46 Then the brain sections were dried on a hot plate at 60deg.C, and apposed on an imaging plate until the activity decayed out. Regional radioactivity was measured as PSL/mm2 over the regions of interest placed on the striatum and on the cerebral cortex. The specific binding was determined by subtracting the activity under cold KF18446 loading. The radioactivity (cpm) of the other set of sections was measured with a gamma counter for calibration of PSL. The 11C activity was then converted to the concentration of receptor-bound ligand (fmol/mm3 tissue) by using the specific activity of the ligand and efficiency of the gamma counter (cpm/Bq) as previously described.44 
To measure the dissociation constant (Kd) and maximal receptor binding (Bmax), the buffer containing [11C]KF18446 at various concentrations (0.625, 1.25, 2.50, 5.0, 7.5 and 10 nM) of cold KF18446 was prepared, and the 20 um thick brain sections were incubated for 30 min at 37deg.C in each buffer as described above. Nonspecific binding was determined as described above. The concentration of receptor-bound ligand (fmol/mm3 tissue) in the striatum and cortex was measured as described above. The bound ligand (fmol/mm3 tissue) was plotted against the ratio of bound ligand to free ligand (fmol/mm3 tissue/nM), as a Scatchard plot. 
Blocking effects of adenosine receptor ligands on the in vitro binding of [11C]KF18446 to rat brain section 
As described above, the in vitro binding assay was carried out in the same buffer containing 2.0-3.5 nM (100 kBq/mL) [11C]KF18446 with or without one of the following 11 antagonists or an agonist CGS 21680 at 20 uM for 30 min at room temperature. The antagonists used were four xanthine-type adenosine A2a antagonists, KF17837, KF18446, KF19631, and CSC, four nonxanthine-type adenosine A2a antagonists, ZM 241385, SCH 58261, CP-66713 and ZD9255; two nonselective xanthine-type antagonists XAC and DMPX; and a xanthine-type A1 antagonist KF15372. The binding was measured in PSL/mm2, and the ratio of the binding measured with each blocker to that without blockers was calculated. 
In vitro affinity of KF18446 for other neuroreceptors 
The in vitro affinity of KF18446 for other neuroreceptors: adrenergic alpha1 , alpha2 and beta1 ; dopamine D1 and D2; agonist site of GABAA and benzodiazepine site of GABAA; histamine H1 and H2; non-selective muscarine; nicotinic acetylcholine; and serotonin 5-HT1A and 5-HT2 was determined using rat striatal membranes and 10 uM KF18446, as described.47 
Blocking effects of adenosine receptor ligands on the in vivo mouse brain uptake of [11C]KF18446 
[11C]KF18446 (0.54-1.3 MBq/14-72 pmol) with or without one of the 11 antagonists was intravenously injected into mice (33-41 g). The amounts of the co-injected antagonist were 50 nmol/animal for KF17837 and 100 nmol/animal for the others. The mice were killed by cervical dislocation at 15 min after injection. The brain was removed and dissected into the striatum, cerebellum and cerebral cortex. The 11C-radioactivity in the samples was counted in an auto-gamma counter and decay-corrected. After weighing the tissues, the 11C-radioactivity level in tissues was expressed as the percent injected dose per gram tissue (%ID/g). 
Lipophilicity of adenosine receptor ligands 
Lipophilicity (cLog P) of adenosine receptor ligands was mathematically calculated to evaluate the penetration of the ligands across the blood-brain barrier in vivo.48 
RESULTS 
In vitro binding of [11C]KF18446 to rat brain section 
Figure 1 shows representative in vitro autoradiograms of [11C]KF18446. In the presence of 20 uM KF18446, the binding of [11C]KF18446 to the striatum disappeared. The time course of the [11C]KF18446 binding to the striatum and cerebral cortex increased over 75 min (Fig. 2A). The specific binding was approximately 90% and 60% of the total binding in the striatum and cortex, respectively. The total binding and specific binding in the striatum were 5.0 and 8.2 times, respectively, as high as in the cortex at 45-75 min. 
A saturation binding experiment showed a specific binding of [11C]KF18446 to the striatum and cortex (Fig. 2B). Scatchard analysis (Fig. 2C) indicated a Kd of 9.8 nM and an apparent Bmax of 170 fmol/mm3 tissue in the striatum and a Kd of 16.4 nM and a Bmax of 33 fmol/mm3 tissue in the cortex. 
Blocking effects of adenosine receptor ligands on the in vitro binding of [11C]KF18446 to rat brain section 
The blocking effects of various adenosine receptor ligands on the in vitro binding of [11C]KF18446 are summarized in Table 1. Table 1 also represents the affinity of the ligands for the adenosine A1 and A2a receptors obtained from the literature. All ligands investigated significantly reduced the binding of [11C]KF18446 to the striatum and to the cortex. The blocking effects in general were much larger in the striatum than in the cortex, but the effects did not necessarily parallel their in vitro affinity. KF18446 had the largest blocking effect in both the striatum and cortex. An A1 antagonist KF15372 with a weak affinity for A2a receptors (Ki = 430 nM)41 and a non selective antagonist DPMX (Ki = 8600 nM)43 also had blocking effects. 
Figure 1 shows the in vitro ARG images of [11C]KF18446 blocked by 20 uM CGS 21680. Although KF18446 and CGS 21680 have a similar affinity for adenosine A2a receptors using [3H]CGS 21680 as a radioligand (Table 1), the blockade of striatal binding of [11C]KF18446 by CGS 21680 was incomplete. 
Lipophilicity of adenosine receptor ligands 
Lipophilicity (cLog P) of the 11 adenosine receptor ligands is summarized in Table 1. Among the xanthine-type compounds the highest lipophilicity was found for KF15372, followed by KF17837 and CSC. The cLog P for KF18446 was relatively low. All four nonxanthine-type ligands had lipophilicity ranging from that for KF18446 to that for KF17837. 
In vitro affinity of KF18446 for other neuroreceptors 
We performed an in vitro membrane binding assay of KF18446 for several other neuroreceptors: adrenergic alpha1, alpha2, and beta1; dopamine D1 and D2; agonist site of GABAA and benzodiazepine site of GABAA; histamine H1 and H2; non-selective muscarine; nicotinic acetylcholine; and serotonin 5-HT1A and 5-HT2. As shown in Table 2 affinity of KF18446 for any of these receptors was not detected (<10 uM of Kd values). 
Blocking effects of adenosine receptor ligands on the in vivo mouse brain uptake of [11C]KF18446 
At 15 min after injection of [11C]KF18446 into mice, the uptake of the radioactivity (%ID/g) was 4.39 +- 0.97 in the striatum, 1.55 +- 0.25 in the cortex and l.62 +- 0.24 in the cerebellum (Fig. 3). 
The blocking effects of the various adenosine receptor antagonists on the regional brain uptake of [11C]KF18446 are summarized in Figure 3. The striatal uptake was reduced by each of the four xanthine-type A2a antagonists, and the order of the blocking effect (KF17837 > KF19631 > KF18446 > CSC) paralleled their in vitro affinity. The uptake by the cortex and cerebellum was also slightly reduced by each of these four antagonists in the same way as was observed in the striatal uptake. The blocking effects were much smaller in the cortex and cerebellum than in the striatum. The nonselective antagonists DPMX and XAC and an A1 antagonist KF15372 did not block the uptake of the tracer in any region. Among the four nonxanthine-type adenosine A2a antagonists, SCH 58261 significantly reduce the uptake of [11CKF]18446 in the striatum, but not in the cortex or cerebellum. The other three antagonists did not reduce the uptake in any region. 
DISCUSSION 
From the first proposal of KF17837 as a selective A2a antagonist by Shimada et al.,26 several xanthine-type compounds have been used as selective probes for pharmacological studies of the adenosine A2a receptors.27-29 Recently we prepared 11C-labeled KF17837 and three other xanthine analogs and found that [11C]KF18446 was a promising candidate as an in vivo probe for studying adenosine A2a receptors by PET.35-39 In the present study, we further characterized the new radioligand [11C]KF18446 by in vitro receptor ARG in rats and in vivo tissue uptake in mice. 
A selective A2a receptor agonist, [3H]CGS 21680, is now widely used as a standard ligand for in vitro studies of adenosine A2a receptors, but CGS 21680 does not cross the blood-brain barrier and cannot be used as a PET tracer. The characteristics of [11C]KF18446 were comparable or slightly superior to those of [3H]CGS 21680. In the membrane binding studies, the A2a/A1 selectivity was 270 for KF18446 and 93 for [3H]CGS 21680 (Table 1). By in vitro ARG, nonspecific binding of [11C]KF18446 in the striatum was 9% of the total binding, whereas the corresponding value for [3H]CGS 21680 was estimated to be 19%.47 The total binding ratio of striatum to cortex was 5.0 for [11C]KF18446 and 4.6 for [3H]CGS 21680.13 It was also found that KF18446 scarcely had any affinity for at least 13 other neuroreceptors (Table 2). Recently nonxanthine-type [3H]SCH 58261 is proposed as a selective A2a receptor radioligand. The A2a/A1 selectivity was approximately 800 but 53 with [3H]CGS 21680, and the nonspecific binding was < 15% below a concentration of 3 nM,34 which are not as good as for KF18446. Taken together with a previous in vivo study38 and the present results, [11C]KF18446 is a promissing PET ligand for mapping adenosine A2a receptors. 
The in vitro binding of [11C]KF18446 to the rat striatum was inhibited by all the adenosine receptor ligands used in the present study at 20 uM (Table 1). This is reasonable because each ligand had an affinity for A2a receptors, which was assessed by the Ki value for the binding of [3H]CGS 21680 to A2a receptors on the rat striatal membrane (Table 1), but the blocking effects did not necessarily parallel the affinity. The largest blocking effect was found for KF18446 and SCH 58261 among xanthine and nonxanthine antagonists, respectively. The blocking effect of CGS 21680 was relatively weak among the ligands with affinity of the same magnitude. These findings suggest that those ligands have multiple binding sites: a common binding site regarded as the typical adenosine A2a receptor and their own unknown binding site suggested to be in the cortex and hippocampus.13-17  
On the other hand, the in vivo result showed a striking difference from the in vitro result. The blocking effects of xanthine-type ligands on the in vivo mouse brain uptake of [11C]KF18446 were apparently different from those of nonxanthine-type ligands with the only exception of SCH 58261 (Fig. 3). Although the in vitro and in vivo experiments were carried out in rats and mice, respectively, the species difference is not likely to exist for the adenosine A2a receptors.36,38 The blocking effect of xanthine ligands on the striatal uptake of the tracer paralleled their in vitro affinity: KF17837 > KF19631 > KF18446 > CSC, except for DPMX having a weak affinity and XAC and KF15372 having a high affinity for A1 receptors. 
The discrepancy between in vitro and in vivo findings cannot be explained by the lipophilicity of the ligands (Table 1). A possible explanation is multiple binding sites of the ligands. When the ligands have different binding sites besides the typical adenosine A2a receptors as suggested by in vitro ARG, it is possible that the binding to the undefined sites might hinder the blocking effect on the uptake of [11C]KF18446. Another explanation is that the association rates of the A2a receptor antagonists binding are different for xanthine-type and nonxanthine-type antagonists. In living animals the receptor ligands are delivered into the brain and washed out by the blood flow, so that the receptor-ligand binding is not necessarily kept in a state of equilibrium, which is quite different from the in vitro binding assay. The antagonists with a low association rate may not effectively block the receptor binding of [11C]KF18446. 
The present study suggests the presence of saturable binding sites for [11C]KF18446 in the cortex. Recent in vitro studies also showed that A2a receptors are found in the hippocampus and cortex.13-17 The binding of a standard A2a receptor ligand [3H]CGS 21680 in these tissues may be different from the classical adenosine A2a receptors present in the striatum and from other defined receptor subtypes as well. The Bmax and Kd Values reported are 353 pmol/mg protein and 58 nM in the hippocampus, 264 pmol/mg protein and 58 nM in the cortex and 419 pmol/mg protein and 17 nM in the striatum.15 The cortical binding site of [3H]CGS 21680 was clearly discriminated from the striatal binding site by using another selective adenosine A2a receptor antagonist SCH 58261.49 For [11C]KF18446 we found the specific binding sites with a Kd of 16.4 nM and a Bmax of 33 fmol/mm3 tissue in the cortex, whereas Kd and Bmax values in the striatum were 9.8 nM and 170 fmol/mm3 tissue, respectively. 
As a radioligand for biochemical and pharmacological studies radiolabeled KF18446 has a disadvantage because photoisomerization from a high affinity E-isomer to a low affinity R-isomer easily occurs in the xanthine derivatives with 8-styryl groups.46 Nevertheless, the disadvantage is easily overcome in PET studies, where the radiolabeled ligand is used immediately after preparation.35 We conclude that [11C]KF18446 is a promissing adenosine A2a receptor ligand for PET study. 
ACKNOWLEDGMENTS 
This work was supported by a Grant-in-Aid for Scientific Research (C) 10670883 from the Ministry of Education, Science, Sports and Culture, Japan. The authors also thank Mr. S. Ishii for his assistance in radiosynthesis. 
REFERENCES 
1. Fredholm BB, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, et al. Nomenclature and classification of purinoceptors. Pharmacol Rev 46: 143-156, 1994. 
2. Palmer TM, Stiles GL. Adenosine receptors. Neuropharmacology 34: 683-694. 1995. 
3 Ongini E, Fredholm BB. Pharmacology of adenosine A2A receptors. Trends Pharmacol Sci 17: 364-372, 1996. 
4 Lewis ME, Patel J, Edley SM, Marangos PJ. Autoradiographic visualization of rat brain adenosine receptors using N6-cyclohexyl[3H]adenosine. Eur J Pharmacol 73: 109-110, 1981.
5. Goodman RR, Snyder SH. Autoradiographic localization of adenosine receptors in rat brain using [3H]cyclohexyl-adenosine. J Neurosci 2: 1230-1241, 1982. 
6. Fastbom J, Pazos A, Palacios JM. The distribution of adenosine A1 receptors and 5'-nucleotidase in the brain of some commonly used experimental animals. Neuroscience 22: 813-826, 1987. 
7. Pagonopoulou O, Angelatou F, Kostopoulos G. Effect of pentylentetrazol-induced seizures on A1 adenosine receptor regional density in the mouse brain: a quantitative autoradiographic study. Neuroscience 56: 711-716, 1993. 
8. Fastbom J, Pazos A, Probst A, Palacios JM. Adenosine A1 receptors in the human brain: a quantitative autoradiographic study. Neuroscience 22: 827-839, 1987. 
9. Svenningsson P, Hall H, Sedvall G, Fredholm BB. Distribution of adenosine receptors in the postmortem human brain: an extended autoradiographic study. Synapse 27: 322-335. 1997. 
10. Bruns RF, Lu GH, Pugsley TA. Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. Mol Pharmacol 29: 331-346. 1986. 
11. Jarvis MF, Williams M. Direct autoradiographic localization of adenosine A2 receptors in the rat brain using the A2-selective agonist, [3H]-CGS 21680. Eur J Pharmacol 168: 243-246, 1989. 
12. Parkinson FE, Fredholm BB. Autoradiographic evidence for G-protein coupled A2-receptors in rat neostriatum using [3H]-CGS21680 as a ligand. Naunyn Schmiedebergs Arch Pharmacol 342: 85-89, 1990. 
13. Johansson B, Georgiev V, Parkinson FE, Fredholm BB. The binding of the adenosine A2 receptor selective agonist [3H]CGS 21680 to rat cortex differs from its binding to rat striatum. Eur J Pharmacol 247: 103-110, 1993. 
14 Cunha RA, Johansson B, van der Ploeg I, Sebastiao AM, Ribeiro JA, Fredholm BB. Evidence for functionally important adenosine A2A receptors in the rat hippocampus Brain Res 649: 208-216, 1994. 
15. Cunha RA, Johansson B, Constantino MD. Sebastiao AM, Fredholm BB. Evidence for high-affinity binding sites for the adenosine A2A receptor agonist [3H]CGS 21680 in the rat hippocampus and cerebral cortex that are different from striatal A2A receptors. Naunyn Schmiedebergs Arch Pharmacol 353: 261-271, 1996. 
16. Kirk IP, Richardson PJ. Further characterization of [3H]CGS 21680 binding sites in the rat striatum and cortex. Br J Pharmacol 114: 537-543, 1995. 
17. Johansson B, Fredholm BB. Further characterization of the binding of the adenosine receptor agonist [3H]CGS 21680 to rat brain using autoradiography. Neuropharmacology 34: 393-403, 1995. 
18. Schiffmann SN, Libert F, Vassart G, Vanderhaeghen JJ. Distribution of adenosine A2 receptor mRNA in the human brain Neurosci Lett 130: 177-181, 1991. 
19. Pollack AE, Harrison MB, Wooten GF, Fink JS. Differential localization of A2a adenosine receptor mRNA with D1 and D2 dopamine receptor mRNA in striatal output pathways following a selective lesion of striatonigral neurons Brain Res 631: 161-166, 1993. 
20. Martinez-Mir MI, Probst A, Palacios JM. Adenosine A2 receptors: selective localization in the human basal ganglia and alterations with disease. Neuroscience 42: 697-706, 1991. 
21. Mally J, Stone TW. Potential role of adenosine antagonist therapy in pathological tremor disorders. Pharmacol Ther 72: 243-250, 1996. 
22. Guieu R, Couraud F, Pouget J, Sampieri F, Bechis G, Rochat H. Adenosine and the nervous system: clinical implications Clin Neuropharmacology 19: 459-474, 1996. 
23. Ferre S. Adenosine-dopamine interactions in the ventral striatum. Implications for the treatment of schizophrenia. Psychopharmacology (Berl) 133: 107-120, 1997. 
24. Baldessarini RJ. Drugs and the treatment of psychiatric disorders, In Pharmacological Bases of Therapeuitics (Eds. Goodman GA, Rall TW, Nies AS, Taylor P), Pergamon, New York, pp. 383-435, 1990. 
25. Ferre S. O'Connor WT, Snaprud P, Ungerstedt U, Fuxe K. Antagonistic interaction between adenosine A2A receptors and dopamine D2 receptors in the ventral striopallidal system. Implications for the treatment of schizophrenia. Neuroscience 63: 765-773, 1994. 
26. Shimada J, Suzuki F, Nonaka H, Ishii A, Ichikawa S. (E)-1,3-dialkyl-7-methyl-8-(3,4,5-trimethoxystyryl)xanthines: potent and selective adenosine A2 antagonists. J Med Chem 35: 2342-2345, 1992. 
27. Baraldi PG, Cacciari B, Spalluto G, Borioni A, Viziano M, Dionisotti S, et al. Current development of A2a adenosine receptor antagonists Current Med Chem 2: 707-722, 1995. 
28. Muller CE, Stein B. Adenosine receptor antagonists: structures and potential therapeutic applications. Current Pharmaceut Design 2: 501-530, 1996. 
29. Poulsen SA, Quinn RJ. Adenosine receptors: new opportunities for future drugs. Bioorg Med Chem 6: 619-641, 1998. 
30. Zocchi C, Ongini E, Conti A, Monopoli A, Negretti A, Baraldi, PG, et al. The non-xanthine heterocyclic compound SCH 58261 is a new potent and selective A2A adenosine receptor antagonist. J Pharmacol Exp Ther 276: 398-404, 1996. 
31. Poucher SM, Keddie JR, Singh P, Stoggall SM, Caulkett PW, Jones G, et al. The in vitro pharmacology of ZM 241385, a potent, non-xanthine, A2a selective adenosine receptor antagonist. Br J Pharmacol 115: 1096-1102, 1995. 
32. Palmer TM, Poucher SM, Jacobson KA, Stiles GL. 125I-4-(2-[7-amino-2-{2-furyl} {1,2,4} triazolo {2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol, a high affinity antagonist radioligand selective for the A2a adenosine receptor. Mol Pharmacol 48: 970-974, 1995. 
33. Zocchi C, Ongini E, Ferrara S, Baraldi PG, Dionisotti S. Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist, to rat striatal membranes. Br J Pharmacol 117: 1381-1386, 1996. 
34. Fredholm BB, Lindstrom K, Dionisotti S, Ongini E. [3H]SCH 58261, a selective adenosine A2A receptor antagonist, is a useful ligand in autoradiographic studies. J Neurochem 70: 1210-1216, 1998. 
35. Ishiwata K, Noguchi J, Toyama H, Sakiyama Y, Koike N, Ishii S, et al. Synthesis and preliminary evaluation of [11C]KF17837, a selective adenosine A2A antagonist. Appl Radiat Isot 47: 507-511, 1996. 
36. Noguchi J, Ishiwata K, Wakabayashi S, Nariai T, Shumiya S, Ishii S, et al. Evaluation of carbon-11 labeled KF17837: a potential CNS adenosine A2a receptor ligand. J Nucl Med 39: 498-503, 1998. 
37. Ishiwata K, Noguchi N, Sakiyama Y, Shimada J, Ishii S, Sakiyama T, et al. Synthesis and in vivo evaluation of 11C-labeled xanthine derivatives as a PET tracer for mapping CNS and peripheral adenosine A2a receptors. The 12th International Symposium on Radiopharmaceutical Chemistry held in June 15-19, 1997, in Uppsala, Sweden. 
38. Ishiwata K, Noguchi N, Wakabayashi S, Shimada J, Ogi N, Nariai T, et al. Carbon-11 labeled KF18446: a potential CNS adenosine A2a receptor ligand. J Nucl Med 41: 345-354, 2000. 
39. Suzuki F, Ishiwata K. Selective adenosine antagonists for mapping central nervous system adenosine receptors with positron emission tomography: Carbon-11 labeled KF15372 (A1) and KF17837 (A2a). Drug Develop Res 45: 312-323, 1998. 
40. Jacobson KA, Nikodijevic O, Padgett WL, Gallo-Rodriguez C, Maillard M, Daly JW. 8-(3-Chlorostyryl)caffeine (CSC) is a selective A2-adenosine antagonist in vitro and in vivo. FEBS Lett 323: 141-144, 1993. 
41. Sarges R, Howard HR, Browne RG, Lebel LA, Seymour PA, Koe BK. 4-Amino[1,2,4]triazolo[4,3-a]quinoxalines. A novel class of potent adenosine receptor antagonists and potential rapid-onset antidepressants. J Med Chem 33: 2240-2254, 1990. 
42. Suzuki F, Shimada J, Mizumoto H, Karasawa A, Kubo K, Nonaka H, et al. Adenosine A1 antagonists. 2. Structure-activity relationships on diuretic activities and protective effects against acute renal failure. J Med Chem 35: 3066-3075, 1992. 
43. Muller CE, Geis U, Hipp J, Schobert U, Frobenius W, Pawlowski M, et al. Synthesis and structure-activity relationships of 3,7-dimethyl-1-propargylxanthine derivatives, A2A-selective adenosine receptor antagonists. J Med Chem 40: 3496-4405, 1997. 
44. Ishiwata K, Ogi N, Tanaka A, Senda M. Quantitative ex vivo and in vitro receptor autoradiography using 11C-labeled ligands and an imaging plate: a study with a dopamine-D2 like receptor ligand [11C]nemonapride. Nucl Med Biol 26: 291-296, 1999. 
45. Przedborski S, Levivier M, Jiang H, Ferreira M, Jackson-Lewis V, Donaldson D, et al. Dose-dependent lesions of the dopaminergic nigrostriatal pathway induced by intrastriatal injection of 6-hydroxydopamine. Neuroscience 67: 631-647, 1995. 
46. Nonaka Y, Shimada J, Nonaka H, Koike N, Aoki N, Kobayashi H, et al. Photoisomerization of a potent and selective adenosine A2 antagonist, (E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)-7-methylxanthine. J Med Chem 36: 3731-3733, 1993. 
47 Nonaka H, Ichimura M, Takeda M, Nonaka Y, Shimada J, Suzuki F, et al. KF17837 ((E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine), a potent and selective adenosine A2 receptor antagonist. Eur J Pharmacol 267: 335-341. 1994. 
48. Leo AJ. Calculating log Poct from structures. Chem Rev 93: 1281-1308, 1993. 
49. Lindstrom K, Ongini E, Fredholm BB. The selective adenosine A2A receptor antagonist SCH 58261 discriminates between two different binding sites for [3H]-CGS 21680 in the rat brain. Naunyn Schmiedsbergs Arch Pharmacol 354: 539-541, 1996.