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NEUROPHARMACOLOGY

Pharmacological Characterization of a Novel, Potent Adenosine A₁ and A_2A Receptor Dual Antagonist, 5-[5-Amino-3-(4-fluorophenyl)pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854), in Models of Parkinson's Disease and Cognition

Pharmacology Research Laboratories (T.M., K.M., J.Y., Y.M., R.M., H.Y., A.I., N.M.) and Chemistry Research Laboratories (S.A., A.A.), Astellas Pharma Inc., Ibaraki, Japan

Received February 25, 2007; accepted August 6, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Central adenosine A_2A receptor is a promising target for drugs to treat Parkinson's disease (PD), and the central blockade of adenosine A₁ receptor improves cognitive function. In the present study, we investigated the effect of a novel adenosine A₁ and A_2A dual antagonist, 5-[5-amino-3-(4-fluorophenyl) pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854), in animal models of PD and cognition. The binding affinities of ASP5854 for human A₁ and A_2A receptors were 9.03 and 1.76 nM, respectively, with higher specificity and no species differences. ASP5854 also showed antagonistic action on A₁ and A_2A agonist-induced increases of intracellular Ca²⁺ concentration. ASP5854 ameliorated A_2A agonist 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680)- and haloperidol-induced catalepsy in mice, with the minimum effective doses of 0.32 and 0.1 mg/kg, respectively, and it also improved haloperidol-induced catalepsy in rats at doses higher than 0.1 mg/kg. In unilateral 6-hydroxydopamine-lesioned rats, ASP5854 significantly potentiated l-dihydroxyphenylalanine (L-DOPA)-induced rotational behavior at doses higher than 0.032 mg/kg. ASP5854 also significantly restored the striatal dopamine content reduced by 1-metyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment in mice at doses higher than 0.1 mg/kg. Furthermore, in the rat passive avoidance test, ASP5854 significantly reversed the scopolamine-induced memory deficits, whereas the specific adenosine A_2A antagonist 8-((E)-2-(3,4-dimethoxyphenyl)ethenyl)-1,3-diethyl-7-methyl-3,7-dihydro-1H-purine-2,6-dione (KW-6002; istradefylline) did not. Scopolamine- or 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate) (MK-801)-induced impairment of spontaneous alternation in the mouse Y-maze test was ameliorated by ASP5854, whereas KW-6002 did not exert improvement at therapeutically relevant dosages. These results demonstrate that the novel, selective, and orally active dual adenosine A₁ and A_2A receptors antagonist ASP5854 improves motor impairments, is neuroprotective via A_2A antagonism, and also enhances cognitive function through A₁ antagonism.

Adenosine A_2A receptors are specifically localized in the striatum (Svenningsson et al., 1999), where they are coexpressed with dopamine D₂ receptors in the GABAergic striatopallidal neurons; in contrast, there are no A_2A receptors in the neurons projecting from the striatum to the substantia nigra that express D₁ receptors (Ferre et al., 1997). Stimulation of adenosine A_2A receptors decreases the binding affinity of D₂ receptors (Ferré et al., 1991), and it elicits effects opposite to D₂ receptor activation at the level of second-messenger systems and early gene expression (Svenningsson et al., 1999). These data suggest that antagonistic adenosine-dopamine interactions may regulate basal ganglia activity and that they could explain the depressant and stimulating effects of adenosine A_2A receptor agonists and antagonists on motor behavior (Ferre et al., 1997). Parenteral administration and local striatal infusion of adenosine A_2A receptor agonists, such as CGS21680, induce sedation and catalepsy, like dopamine antagonists, and also inhibit the motor-activating effects of dopamine receptor agonists (Svenningsson et al., 1999).

In contrast, adenosine receptor antagonists, including caffeine and related methylxanthines, produce motor stimulant effects, which seem to be related to an action on A_2A, rather than A₁, receptors (Fredholm et al., 1999). The A_2A-selective antagonist KW-6002 (istradefylline) exhibits antiparkinsonian activities in experimental models of the Parkinson's disease (PD) (Kanda et al., 1998; Shiozaki et al., 1999; Koga et al., 2000), and it is developing in clinical stage. More recently, other adenosine A_2A antagonists, SCH-420814 and BIIB014/V2006, also have been developed in clinical stage as antiparkinsonian drugs. These findings support a role for A_2A receptors as neuromodulators of dopaminergic function, and they suggest that they may play an important role in movement disorders such as PD. In addition, antagonism of adenosine A_2A receptors exerts dual actions on motor dysfunction and neurodegenerative processes in animal models of PD (Ikeda et al., 2002). Consistent with its pharmacology, A_2A receptor knockout mice are resistant to both motor impairment and neurochemical changes relevant to neurodegenerative disorders such as PD (Ledent et al., 1997; Chen et al., 2001). Hence, adenosine A_2A receptor antagonists may represent a novel therapeutic approach to pathologies characterized by neurodegenerative events, since they both reverse motor impairment and are neuroprotective.

Adenosine A₁ receptors are expressed throughout the brain, including the hippocampus and prefrontal cortex, which are important areas for cognitive function, and A₁ receptors in the hippocampus are densely concentrated in the CA1 and CA3 regions (Onodera and Kogure, 1988). Adenosine A₁ receptors play an important role in memory formation (Normile and Barraco, 1991; Costenla et al., 1999). In fact, administration of an A₁ agonist before passive avoidance training impaired memory (Normile and Barraco, 1991), and selective adenosine A₁ antagonists enhanced cognition in rodents (Maemoto et al., 2004). Therefore, the blockade of both adenosine A₁ and A_2A receptors might have therapeutic implications for neurodegenerative disease such as PD, Alzheimer's disease, and other neurological diseases. In particular, blockade of both adenosine A₁ and A_2A receptors has potential for the treatment of PD, which presents with both motor disability and cognitive impairment. In fact, theophylline improved both motor and mental impairment scores in a short, open-label study in PD patients (Mally and Stone, 1994).

We have recently identified a novel, nonxanthine, adenosine A₁ and A_2A receptor antagonist, ASP5854 (Fig. 1). Here, we characterize the mechanism of action and the pharmacological features of ASP5854 by using in vitro studies and various animal models of PD and cognition, and we compare its effects to the specific A_2A receptor antagonist KW-6002.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Chemical structure of ASP5854.

	Materials and Methods

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Materials
ASP5854 (Fig. 1) and KW-6002 were synthesized at Fujisawa Pharmaceutical Co., Ltd. (now Astellas Pharmaceutical Inc., Tsukuba, Japan). Chinese hamster ovary (CHO) cells were purchased from Biogen (Cambridge, MA). FuGENE 6 Transfection Reagent was purchased from Roche Applied Science (Mannheim, Germany). -Minimum essential medium (MEM) and Opti-MEM I were purchased from Invitrogen (Tokyo, Japan). F-127 was purchased from Invitrogen (Tokyo, Japan). Fluo3-acetoxymethyl ester was purchased from Dojindo (Kumamoto, Japan). N⁶-Cyclopentyladenosine (CPA), CGS21680, (–)-scopolamine hydrobromide trihydrate, and (+)-MK-801 hydrogen maleate were purchased from Sigma-Aldrich (Steinhein, Germany). [³H]8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) and [³H]CGS21680 were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). [³H]N-Ethylcarboxamidoadenosine (NECA) was purchased from GE Healthcare UK Ltd. (Little Chalfont, Buckinghamshire, UK). Haloperidol (Serenace Injection; 5 mg/ml) was purchased from Dainippon Pharmaceutical (Osaka, Japan). Unless otherwise stated, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Binding Affinities for Adenosine Receptors in Various Species
Preparation of Membranes from CHO Cells Expressing the Human Adenosine Receptors. CHO cell lines permanently expressing human adenosine A₁, A_2A, and A₃ were used in this study. CHO cell lines were made according to the method described previously (Chen and Okayama, 1987). These cells were cultured in -MEM supplemented with 10% fetal bovine serum, 50 IU/ml penicillin, and 50 µg/ml streptomycin in a 5% CO₂, 95% atmosphere, and they were harvested and homogenized in 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl₂, 2 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged (30,000g) at 4°C for 20 min, and the pellet was collected and centrifuged again. Then, the pellet was suspended in 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl₂ (assay buffer). This washing process was repeated twice, and the final pellet was stored at –80°C until use.

Preparation of Membranes from Mouse and Rat Brain. Brain synaptosome membranes were prepared from mice and rats. Animals were sacrificed, and the cerebral cortex and basal ganglia used for A₁ and A_2A binding assays, respectively, were removed quickly and homogenized in approximately 15 volumes of 0.36 M sucrose solution. The homogenates were centrifuged (1000g) at 4°C for 10 min, and then the supernatants were centrifuged (17,000g) at 4°C for 20 min. The pellets were suspended in the assay buffer and further centrifuged (30,000g) at 4°C for 10 min. This washing process was repeated twice, and final pellets were stored at –80°C until use.

Radioligand Binding Assay. Radioligand binding experiments for adenosine A₁, A_2A, and A₃ receptors were performed with [³H]DPCPX, [³H]CGS21680, and [³H]NECA, respectively. Membranes were incubated with 4 nM [³H]DPCPX, 15 nM [³H]CGS21680, or 3.2 nM [³H]NECA (32 nM [³H]NECA for KW-6002) in the absence and presence of various concentrations of ASP5854 or KW-6002 in 250 µl of assay buffer containing 1 U/ml adenosine deaminase. The mixture was incubated for 60 min at room temperature, harvested on Whatman GF/C filters (Whatman, Maidstone, UK) presoaked in 0.1% polyethylenimine by a cell harvester (PerkinElmer Inc., Waltham, MA), and washed eight times with 50 mM Tris-HCl buffer, pH 7.4. The radioactivity on the filter was measured by a scintillation counter. All experiments were performed three times in duplicate using 96-well plates. The nonspecific binding was defined as the binding activity in the presence of 10 µM of the corresponding unlabeled ligand, and it was subtracted from the total binding activity for determination of specific binding. K_i values in inhibition studies were calculated with the Prism 3.0 computer program (GraphPad Software Inc., San Diego, CA). To compare the affinity of compounds to A₁ and A_2A receptors, the ratio of K_i values between A₁ and A_2A receptors was calculated.

Functional Assay for Adenosine A₁ and A_2A Receptors in Vitro
Preparation of CHO Cells Expressing the Human Adenosine A₁ and A_2A Receptors. A CHO cell line permanently expressing the human adenosine A₁ or A_2A receptor was cultured in -MEM supplemented with 10% (v/v) fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were grown at 37°C in an environment of 5% CO₂. These cells were seeded on black 96-well assay plates (Falcon; BD Biosciences, Franklin Lakes, NJ) at a density of 15,000 cells/well, and then they were cultured for 24 h.

Transfection of G16 to Human Adenosine A₁ and A_2A CHO Cells. Human-A₁ and -A_2A CHO cells were transfected with G16, a human G protein, at 50 ng/well by means of FuGENE 6 Transfection Reagent and Opti-MEM. These cells were cultured at 37°C in 5% CO₂ for 24 h.

Measurement of Intracellular Ca²⁺ Concentration Using Fluorometric Imaging Plate Reader. Hanks' balanced salt solution, without phenol red, containing 20 mM HEPES and 2.5 mM probenecid was prepared fresh on the day of assay and used as the assay buffer. The dye-loading buffer contained 5 µM Fluo3-acetoxymethyl ester (Ca²⁺-sensitive dye) and 0.05% Pluronic F-127 (Invitrogen). The cells were dye-loaded at 37°C by removing the existing maintenance media and by adding 100 µl of dye-loading buffer to each well. The cells were washed using plate washer (ELx405 Select; Bio-Tek Instruments, Winooski, VT) with the assay buffer, and 100 µl/well was left in each well. Antagonists at 50 µl/well were added 10 s into the fluorescent measurements. After adding drugs, 10 nM CPA (adenosine A₁ receptor agonist) or 10 nM CGS21680 (adenosine A_2A receptor agonist) were added at 50 µl/well. A 96-well plate contained three wells dedicated to the positive control and three wells as a negative control (assay buffer alone). Each experiment was repeated three times, in triplicate at each concentration. All data were normalized to the positive and negative control wells, which were expressed as 100 and 0% signal. The inhibition percentage at each concentration was averaged for three IC₅₀ values determined by the logistic regression method.

Specificity of ASP5854
The affinity of ASP5854 for 70 receptors, eight ion channels, five transporters, and three enzymes was evaluated using radioligand binding assays (performed by Daiichi Pure Chemical Co., Ltd., Ibaraki, Japan). ASP5854 and each of the positive substances were dissolved in dimethyl sulfoxide, and they were diluted serially with the same solvent to prepare the solutions at 100-fold concentrations of the final concentrations (ASP5854, 1 x 10^–5 mol/l; positive substances, 1 x 10^–5 mol/l). These stock solutions were 10-fold diluted with Milli-Q water (Millipore Corporation, Billerica, MA) just before the use to prepare test substance and positive substance solutions.

Duplicate samples of each concentration of ASP5854 and positive substance solutions were assayed once. These assays used standard radioligand binding techniques with tissue membrane preparations. Each inhibition rate was calculated from 100 – binding ratio. Binding ratio was determined by [(B – N)/(B₀ – N)] x 100 (%), where B is radioactivity or fluorescence intensity in the tube for calculation of inhibition rate (individual value), B₀ is radioactivity or fluorescence intensity in the tube for calculation of total reaction (mean value), and N is radioactivity or fluorescence intensity in the tube for calculation of nonspecific reaction (mean value).

CGS21680- or Haloperidol-Induced Catalepsy in Mice
Adult male ddY mice were purchased from Japan SLC Co. Ltd. (Hamamatsu, Japan), and they were used at 7 weeks of age. The number of mice in one group was 14. The mouse forepaws were placed on a horizontal steel bar ( 0.3 cm), elevated 3 cm above the tabletop. The duration during which each mouse maintained position with both forepaws on the bar and both hindpaws on the tabletop was recorded, up to 30 s, as the cataleptic time. Catalepsy was measured 30 min after intracerebroventricular injection of CGS21680 (10 µg/20 µl/head) or intraperitoneal injection of haloperidol (0.32 mg/kg). ASP5854 (0.01–3.2 mg/kg) or KW-6002 (0.01–3.2 mg/kg) was orally administered 1 h before catalepsy measurement. If the catalepsy time reached 30 s in at least in one trial out of two, catalepsy was scored as positive; otherwise, it was scored as negative. The incidence of catalepsy was expressed as the percentage of the mice that were scored as positive versus the total mice in a group. ASP5854 and KW-6002 were suspended in 0.5% methylcellulose, and they were administered in a volume of 10 ml/kg. As the vehicle-treated group, 0.5% methylcellulose was administered instead of test compounds. CGS21680 was dissolved and diluted in saline, and haloperidol was diluted with saline before the experiments.

Haloperidol-Induced Catalepsy in Rats. Adult male Sprague-Dawley rats were purchased from Japan SLC Co. Ltd. (Hamamatsu, Japan), and they were used at 7 weeks of age. The number of rats in one group was 10 to 12. Forepaws of a rat were placed on a horizontal plastic bar ( 0.8 cm) that was wrapped with gauze; the bar was 11 cm above the tabletop. The duration during which each rat maintained this position was recorded up to 120 s as the cataleptic time. All rats were given two trials at intervals of 1 min. Catalepsy was measured 90 min after the i.p. injection of haloperidol (1 mg/kg). ASP5854 (0.01–1 mg/kg) or KW-6002 (0.032–1 mg/kg) was administered orally 60 min before the catalepsy test.

Rotational Behavior in 6-OHDA Lesion Rats
Animals. Thirteen-week-old male Fisher rats were purchased from Japan SLC, Inc. They were subjected to the surgery at 14 weeks of age.

Unilateral 6-Hydroxydopamine Lesion. Rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.), and then they were placed in a stereotaxic frame (Narishige, Tokyo, Japan). 6-OHDA (10 µg/2 µl in saline containing 0.2% ascorbic acid) was stereotaxically injected into the left substantia nigra at the following coordinates (in millimeters) relative to the bregma and the skull surface: A, –4.3; L, 1.7; and V, 7.5. Thirty minutes before 6-OHDA injection, rats received an injection of desipramine hydrochloride (25 mg/kg i.p.) to protect the noradrenergic neurons.

Animal Selection. To select successfully denervated animals, all rats were challenged with apomorphine (0.5 mg/kg s.c.) at more than 7 days postsurgery. The 24 animals showing the highest number of contralateral rotations were selected for subsequent experiments. Rotational behaviors were recorded using the Roto-Rat system (MED Associates, East Fairfield, NJ).

Evaluation of Turning Behavior. Selection was performed more than 2 weeks after the 6-OHDA lesion. The 24 apomorphine-challenged rats were challenged with 32 mg/kg L-DOPA plus 8 mg/kg benserazide to induce intense turning behavior. The animals with the top eight rotational responses were tested in subsequent experiments with drugs. L-DOPA at 10 mg/kg plus benserazide at 2.5 mg/kg, which induces modest contralateral turning behavior, was administered at the same time as ASP5854 (0.01–1 mg/kg), or 30 min after the treatment with KW-6002 (0.1–10 mg/kg). Turning was recorded in 10-min intervals over an 180-min period after L-DOPA plus benserazide administration. At least 5 days were allowed to elapse between experiments. ASP5854 and KW-6002 were suspended in 0.5% methylcellulose and orally administered in a volume of 5 ml/kg. L-DOPA (administered with benserazide at the ratio of 4:1) was suspended in 0.5% methylcellulose containing 0.2% ascorbic acid and administered p.o. in a volume of 5 ml/kg.

Contents of Striatal DA and Its Metabolites in MPTP-Treated Mice
Animals. Male C57BL/6 mice (7 or 8 weeks old) were purchased from Charles River Laboratories Japan Co. Ltd. (Yokohama, Japan).

MPTP Treatment. ASP5854 (0.01–0.32 mg/kg) or KW-6002 (0.1–3.2 mg/kg) suspended in 0.5% methylcellulose was administrated twice p.o., 30 min or 1 h before each MPTP injection, in volumes of 10 ml/kg. As the vehicle group, 0.5% methylcellulose was administered instead of the test compounds. Mice received two i.p. injections of MPTP-HCl (20 mg/kg free base) dissolved in saline or saline alone at a 2-h interval (10 ml/kg).

Measurement of Striatal DA, Dihydroxyphenylacetic Acid, and Homovanillic Acid Levels. The contents of DA, DOPAC, and HVA in the striatum were quantified using high-performance liquid chromatography with electrochemical detection. Four days after MPTP treatment, brains were quickly removed, and the striatum was dissected out. Samples were immediately frozen and stored at –80°C until analysis. On the day of the assay, tissue samples were homogenized with 0.1 M perchloric acid and 0.1 mM EDTA-2Na containing 50 ng/ml isoproterenol as an internal standard. After centrifugation, the supernatant, with added sodium acetate, was injected onto a reversed-phase catecholamine column (SC-5ODS; Eicom Co., Ltd., Kyoto, Japan). The mobile phase contained sodium acetate, methanol, and sodium octane sulfate. DA, DOPAC, and HVA contents were normalized to the normal group (100%). Percentage of recovery was calculated relative to control animals (control, 0%; normal, 100%).

View larger version (9K): [in this window] [in a new window]   Fig. 2. Antagonistic effects of ASP5854 for human adenosine A1 and A2A receptors on [Ca2+]i elevation by CPA or CGS216980 in CHO cells expressing the human adenosine A1 receptor or adenosine A2A receptor. [Ca2+]i was measured using fluorometric imaging plate reader. CPA (10 nM; A) or 10 nM CGS21680 (B) was added immediately after ASP5854 addition. ASP5854 dose-dependently inhibited increased [Ca2+]i by CPA and CGS21680 with IC50 values of 59.81 ± 17.27 and 4.21 ± 0.30 nM, respectively. Values are the mean ± S.E.M. of three separate experiments. IC50 values determined by the logistic regression method.

Y-Maze Test
Male ddY mice (Japan SLC Inc.) (5 weeks old) were used for the Y-maze test. Spontaneous alternation behavior was assessed in the Y-maze test (Ukai et al., 1998). The maze was made of gray vinyl chloride. Each arm was 40 cm in length, 13 cm in height, 3 cm wide at the bottom, and 10 cm wide at the top, and converged at an equal angle. Mice were acclimated in the experiment room for 1 h. Each mouse was placed at the end of one arm, and the number of arm entries and alternations were counted for 8 min. Alternation was defined as entries into all three arms on consecutive occasions. The percentage of alternation was calculated as follows: alternation rate (%) = (alternations/total entry – 2) x 100. ASP5854 (0.032–3.2 or 0.032–1 mg/kg) or KW-6002 (0.1–10 or 0.32–10 mg/kg) was administered orally in a volume of 10 ml/kg 50 min before the Y-maze test. Scopolamine (0.5 mg/10 ml/kg i.p.) or MK-801 (0.15 mg/10 ml/kg i.p.) was injected 30 min after administration of the drug.

All animal procedures were carried out as approved by the Animal Care and Use Committee at Fujisawa Pharmaceutical Co., Ltd., or Astellas Pharma Inc.

Statistical Analysis
In catalepsy test in mice, statistical analysis between vehicle-treated group and drug-treated groups were calculated with the appearance ratio Dunnett's test. The ED₅₀ values were calculated using the probit method. In the rat catalepsy test and rat passive avoidance test, statistical analysis between vehicle-treated group and drug-treated groups were made using the Kruskal-Wallis test followed by Dunnett's multiple comparisons. In unilateral 6-OHDA-lesioned rat model, analysis of variance, based on randomized block design, followed by Dunnett's multiple comparisons test was used for comparison between the L-DOPA + benserazide (10 + 2.5 mg/kg)-treated group and multiple drug-treated groups. For striatal DA and metabolite content in MPTP-treated mice and the mice Y-maze test, Dunnett's multiple comparison test was used for comparison between the vehicle-treated control group and multiple drug-treated groups.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Adenosine Receptor Binding Affinities and Selectivity of ASP5854. ASP5854 and KW-6002 were evaluated for their potencies in radioligand binding to the human adenosine receptor subtypes. K_i values for inhibiting specific radioligand binding to the three receptor subtypes are summarized in Table 1. These compounds were also tested for their binding affinities to adenosine A₁ and A_2A receptors obtained from mouse and rat brains (Table 1). ASP5854 showed high affinity for human A₁ and A_2A receptors, with K_i values of 9.03 and 1.76 nM, respectively, but they lacked affinity for human A₃ receptors. In contrast, KW-6002 showed lower affinity for human A_2A receptors compared with ASP5854, and no significant affinity for either human A₁ or A₃ receptors. ASP5854 showed similar binding affinity for A₁ and A_2A receptors in humans, rats, and mice.

Functional Assay for Adenosine A₁ and A_2A Receptors in Vitro. In CHO cells stably expressing cloned human adenosine A₁ and A_2A receptors transfected with G₁₆ protein, 10 nM CPA or 10 nM CGS21680 induced increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), but ASP5854 alone did not increase (data not shown). In contrast, CPA-induced [Ca²⁺]_i was concentration-dependently blocked by ASP5854 treatment (Fig. 2A). ASP5854 also concentration-dependently inhibited increased [Ca²⁺]_i by 10 nM CGS21680 (Fig. 2B). The IC₅₀ values of ASP5854 for increased [Ca²⁺]_i by CPA and CGS21680 were 59.81 ± 17.27 and 4.21 ± 0.30 nM, respectively.

Specificity of ASP5854. ASP5854 was screened for interaction against an array of 70 receptors, eight ion channels, five transporters, and three enzymes, including molecular sites that are relevant to antiparkinsonian activity (Table 2). No significant binding to other proteins was found.

CGS21680- or Haloperidol-Induced Catalepsy in Mice. Intracerebroventricular injection of CGS21680 (10 µg/head), an A_2A agonist, induced cataleptic behavior in mice. Oral administration of ASP5854 dose-dependently ameliorated CGS21680-induced catalepsy, with an ED₅₀ value of 0.147 mg/kg and statistical significance at the doses higher than 0.32 mg/kg (Fig. 3A).

View larger version (29K): [in this window] [in a new window]   Fig. 3. Effects of ASP5854 on CGS21680- or haloperidol-induced catalepsy in mice. In CGS21680-induced catalepsy, catalepsy was measured 30 min after intracerebroventricular injection of CGS21680. A, ASP5854 was orally administered 30 min before CGS21680 injection. The column represent the percentage of the mice with catalepsy within the group (n = 14). ASP5854 suppressed CGS21680-induced catalepsy dose-dependently, with an ED50 value of 0.147 mg/kg. In haloperidol-induced catalepsy, catalepsy was measured 30 min after intraperitoneal injection of haloperidol. ASP5854 or KW-6002 was orally administered 30 min before haloperidol injection. Each column represents the percentage of the mice with catalepsy within the group (n = 14). ASP5854 (B) and KW-6002 (C) suppressed haloperidol-induced catalepsy dose-dependently, with ED50 values of 0.066 and 0.092 mg/kg, respectively. *, P < 0.05 and **, P < 0.01, statistically significant compared with the vehicle-treated controls (opened columns) (by the appearance ratio Dunnett's test). ED50 values were calculated by probit method.

Haloperidol-Induced Catalepsy in Rats. Haloperidol (1 mg/kg i.p.) induced catalepsy in rats. ASP5854 reduced the cataleptic duration, with a minimum effective dose of 0.1 mg/kg (Fig. 4A). KW-6002 also inhibited haloperidol-induced catalepsy, although the minimum effective dose was 0.32 mg/kg (Fig. 4B).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Effects of ASP5854 and KW-6002 on haloperidol-induced catalepsy in rats. Catalepsy was measured 90 min after intraperitoneal injection of haloperidol. ASP5854 (A) or KW-6002 (B) was orally administered 30 min following haloperidol injection. Each column represents the mean ± S.E.M. of 10 to 11 rats. *, P < 0.05 and **, P < 0.01, statistically significant compared with the vehicle-treated controls (opened columns) (by Kruskal-Wallis followed by Dunnett's multiple comparison test).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Effects of ASP5854 and KW-6002 on L-DOPA-induced turning behavior in 6-OHDA-lesioned, hemiparkinsonian rats. ASP5854 (A) was administered orally at same time with the oral administration of L-DOPA (administered with benserazide at the ratio of 4:1). KW-6002 (B) was administered 30 min before L-DOPA + benserazide administration. Each column represents the mean ± S.E.M. of total turning counts for 180 min after L-DOPA administration. *, P < 0.05 and **, P < 0.01, statistically significant compared with L-DOPA + benserazide (10 + 2.5 mg/kg) alone controls (opened columns) (by analysis of variance based on randomized block design, followed by Dunnett's multiple comparisons test). ##, P < 0.01, statistically significant compared with L-DOPA + benserazide (10 + 2.5 mg/kg) alone controls (opened columns) (by paired t test).

Contents of Striatal DA and Its Metabolites in MPTP-Treated Mice. The striatal DA contents of normal and MPTP-treated control mice were 120.76 ± 4.36 and 43.40 ± 2.88 ng/mg protein, respectively. MPTP treatment significantly reduced the striatal DA content to 35.94% compared with normal mice. ASP5854 dose-dependently improved the reduction of striatal DA content by MPTP administration. The percentage of recovery of DA content from the vehicle-treated control level were 7.09, 21.11, 38.78, and 60.41 at doses of 0.01, 0.032, 0.1, and 0.32 mg/kg, respectively, with statistical significance at doses of 0.1 mg/kg and higher (Fig. 6A). The effects of ASP5854 on DOPAC and HVA contents paralleled that on DA. KW-6002 similarly restored the striatal DA content reduced by MPTP treatment in a dose-dependent manner. The percentage of recovery in DA contents at doses of 0.1, 0.32, 1, and 3.2 mg/kg was 22.91, 28.37, 38.79, and 42.45%, respectively. Significant effects were obtained at the doses of 1 and 3.2 mg/kg (Fig. 6B).

View larger version (50K): [in this window] [in a new window]   Fig. 6. Effects of ASP5854 and KW-6002 on MPTP-induced depletion of striatal DA, DOPAC, and HVA content in mice. ASP5854 (A) or KW-6002 (B) was administered twice by p.o. at 2-h intervals. MPTP was intraperitoneally injected 30 min or 1 h after administration of each drug. The striatum was dissected 4 days after drug administration. DA, DOPAC, and HVA were measured by HPLC with electrochemical detection. Data are presented as mean ± S.E.M. of six to eight mice. *, P < 0.05 and **, P < 0.01, statistically significant compared with the vehicle-treated controls (closed columns) (by Dunnett's multiple comparison test). ##, P < 0.01, statistically significant compared with vehicle-treated controls (closed columns) (by Student's t test).

Scopolamine-Induced Memory Deficits in the Rat Passive Avoidance Test. Scopolamine (1 mg/kg) given 30 min before the acquisition trial significantly reduced the latency in the retention trial determined 24 h thereafter (Fig. 7, A and B). Dosing with ASP5854 (0.032–3.2 mg/kg) immediately after the acquisition trial prolonged the latency in the scopolamine-treated rats, and the dose-response curve was bell-shaped, with a statistically significant effect at 0.1 to 3.2 mg/kg (Fig. 7A), and the percentage of rats reaching criteria tended to increase (data not shown). In contrast, KW-6002 at doses of 0.1 to 10 mg/kg had no effect in this model (Fig. 7B).

View larger version (23K): [in this window] [in a new window]   Fig. 7. Effects of ASP5854 and KW-6002 on scopolamine-induced memory deficits in the passive avoidance task in rats. Scopolamine (1 mg/kg) was administered by i.p. 30 min before the acquisition trial. ASP5854 (A) or KW-6002 (B) was administered i.p. immediately after the acquisition trial. Ordinate represents the median retention latencies in a 24-h retention test. *, P < 0.05 and **, P < 0.01; statistically significant compared with scopolamine-alone controls (closed columns) (by Kruskal-Wallis followed by Dunnett's multiple comparisons). ##, P < 0.01 and ###, P < 0.001, statistically significant compared with scopolamine-alone controls (closed columns) (by Wilcoxon's rank sum test). Each column and bar represents the mean ± S.E.M. of 12 to 13 rats.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Effects of ASP5854 and KW-6002 on scopolamine-induced impairment of spontaneous alternation in the Y-maze task in mice. ASP5854 (A) or KW-6002 (B) were administered 50 min before the Y-maze test, and scopolamine was administered 30 min after drug administration. Each value represents the mean ± S.E.M. of 10 mice. *, P < 0.05 and **, P < 0.01, statistically significant compared with scopolamine alone controls (closed columns) (by Dunnett's multiple comparison test). ###, P < 0.001; statistically significant compared with scopolamine alone controls (closed columns) (by Student's t test).

View larger version (15K): [in this window] [in a new window]   Fig. 9. Effects of ASP5854 and KW-6002 on MK-801-induced impairment of spontaneous alternation in Y-maze in mice. ASP5854 (A) or KW-6002 (B) were administered 50 min before the Y-maze test, and MK-801 was administered 30 min after drug administration. Each value represents the mean ± S.E.M. of 10 mice. *, P < 0.05 and ** P < 0.01, statistically significant compared with MK-801 alone controls (closed columns) (by Dunnett's multiple comparison test). ##, P < 0.01, statistically significant compared with MK-801 alone controls (closed columns) (by Student's t test).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The purpose of the present study was to characterize the in vitro and in vivo profiles of the novel adenosine A₁ and A_2A receptor dual antagonist ASP5854, which was recently identified from our optimization screening of pyrazolopyrimidine derivatives. ASP5854 showed potent and selective binding affinity for A₁ and A_2A receptors, with no significant affinity for A₃ receptors, and it did not show species differences. Furthermore, ASP5854 showed no apparent affinity for many other receptors, channels, monoamine transporters, or enzymes, including the adenosine A_2B receptor and phosphodiesterase, which are common targets for other adenosine antagonists that are xanthine derivatives. ASP5854 concentration-dependently inhibited the increase in [Ca²⁺]_i by CPA and CGS21680, with IC₅₀ values of 59.81 and 4.21 nM, respectively. The K_i value of binding affinity for human adenosine A₁ and A_2A receptors are 9.03 and 1.76 nM, respectively, and ratios of the IC₅₀/K_i value were between 2.4 and 6.6. These results clearly demonstrated that ASP5854 is a potent and selective antagonist of the adenosine A₁ and A_2A receptors.

Many behavioral, biochemical, and functional studies have indicated an antagonistic interaction between adenosine A_2A receptors and dopamine D₂ receptors in the striatum (Svenningsson et al., 1999). Modulation of adenosine A_2A receptors profoundly influences motor function (Ferre et al., 1997). In fact, the i.c.v. injection of CGS21680, an A_2A agonist, induced catalepsy in mice, as do dopamine D₂ antagonists such as haloperidol (Shiozaki et al., 1999). We also found that i.c.v. injection of CGS21680 and i.p. injection of haloperidol induced catalepsy in mice and rats, and ASP5854 dose-dependently reversed the catalepsy in this study. Interestingly, ED₅₀ values obtained from CGS21680- and haloperidol-induced catalepsy in mice were comparable, suggesting that A_2A antagonism of ASP5854 mediated the anticataleptic actions in both models.

Contralateral turning behavior in rats subjected to unilateral 6-OHDA lesions of the dopaminergic nigrostriatal pathway is another well established animal model of PD (Ungerstedt, 1971). In this model, a selective A_2A antagonist reportedly potentiated the contralateral turning behavior induced by direct dopamine agonists (Koga et al., 2000), implying that adenosine A_2A antagonists might be beneficial for the treatment of PD. In fact, KW-6002 shortened the wearing-off time in PD patients in clinical trials (Hauser et al., 2003). In the present study, ASP5854 potentiated the rotational behavior induced by L-DOPA without inducing stereotyped behavior on its own. Drugs capable of selectively reversing the reduction in the duration of L-DOPA antiparkinsonian action without augmenting its response intensity have significant therapeutic potential. Therefore, although further studies in nonhuman primates are warranted, the present findings suggest that ASP5854 may be useful as adjunct therapy in the treatment of advanced PD.

The pathogenesis of PD involves degeneration of striatal neurons that project to the external pallidum (indirect pathway) leading to overactivity, whereas the activity of striatal neurons projecting directly to the basal ganglia output nuclei (direct pathway) is reduced (Kase, 2001). Because adenosine A_2A receptors are located on striatal projection neurons (Svenningsson et al., 1999), especially within the indirect pathway, ASP5854 may work by blocking adenosine A_2A receptors on striatal neurons projecting to the external pallidum. This assumption implies a selective suppressive action of ASP5854 on the indirect pathway. The density of A₁ receptors is moderately higher in the motor regions of the brain, such as the cerebellum and motor cortex, compared with A_2A receptors (Rivkees et al., 1995). In the striatum, A₁ receptors are weakly expressed in approximately 40% of striatal neurons (Rivkees et al., 1995), and they are present presynaptically on glutamate, dopamine, and acetylcholine afferents to medium-sized neurons (Fredholm and Dunwiddie, 1988; Flagmeyer et al., 1997). A₁ receptors antagonistically and specifically modulate the binding and functional characteristics of dopamine D₁ receptors, much like the A_2A-D₂ antagonistic interaction (Ferre et al., 1997), suggesting that adenosine A₁ antagonism could improve motor impairment. In fact, adenosine A₁ antagonist potentiated the motor effects induced by D₁ agonist in rats (Popoli et al., 1996). However, our specific adenosine A₁ antagonist, FR194921, did not improve motor impairment in the same PD models used here (data not shown). Another A₁ antagonist, DPCPX, alone did not improve motor impairment in a nonhuman primate PD model (Kanda et al., 1998). Therefore, the antiparkinsonian effect of ASP5854 is primarily mediated by the adenosine A_2A antagonism. Nevertheless, it is tempting to speculate that the A₁ antagonism of ASP5854 could potentiate the effects of dopamine D₁ or nonselective dopamine agonists, especially since ASP5854 was more potent in the 6-OHDA-lesion model than other PD models, although it warrants the further investigation to address this hypothesis.

Although the pathological hallmark in PD patients is progressive dopaminergic neurodegeneration in the basal ganglia, none of the currently existing dopaminergic-based drugs affect the neurodegeneration that occurs in the basal ganglia in PD. There is an urgent need for a neuroprotective approach that may delay disease progression. MPTP-induced dopaminergic neuronal cell death model provides a useful test for neuroprotective drugs (Iwashita et al., 2004). ASP5854 improved the striatal dopamine depletion produced by MPTP administration. ASP5854 did not bind MAO-B, which oxidizes MPTP to the active toxin MPP⁺, or the dopamine transporter, which transports MPP⁺ into the cell body of dopaminergic neurons (Table 2). ASP5854 also did not significantly affect MPP⁺ concentrations in plasma and brain following the injection of MPTP (data not shown). Chen et al. (2001) demonstrated that MPTP toxicity was reduced by administration of adenosine A_2A receptor antagonists, and in genetically A_2A knockout mice. Ikeda et al. (2002) showed that A_2A receptor antagonists, but not adenosine A₁ antagonists, protected against dopaminergic neurodegeneration induced by MPTP and 6-OHDA. Our study also suggests that blockade of the A_2A receptor could result in neuroprotection; however, further immunohistochemical and mechanistic studies are required.

Scopolamine, a centrally active anticholinergic agent, and MK-801, a noncompetitive NMDA antagonist, are commonly used in the study of learning and memory. Pretreatment of scopolamine 30 min before training impaired retention performance in the rat passive avoidance test, in which animals acquire memory related to aversive stimulus, consistent with earlier observations (Matsuoka et al., 1992). In the Y-maze test, scopolamine and MK-801 induced spontaneous alternation memory impairments in mice, which is consistent with previous findings (Fraser et al., 1997; Ukai et al., 1998). ASP5854 ameliorated the scopolamine-induced impairment of memory in the rat passive avoidance test and the scopolamine- and MK-801-induced impairment of memory in the mouse Y-maze test, with comparable potency to PD models. Although these animal models used here do not perfectly mirror the cognitive deficits associated with PD and further studies will be required, the present findings suggest that ASP5854 could improve cognition in patients with memory disturbances.

A₁ activation depresses cholinergic, noradrenergic, and GABAergic transmission (Phillis and Kostopoulos, 1975; Hollins and Stone, 1980). The cholinergic system is important in learning and memory processes, and cholinesterase inhibitors are useful treatments for Alzheimer's disease (Seltzer, 2006). Thus, blockade of adenosine A₁ receptors may indirectly stimulate cholinergic neurotransmission and thereby enhance cognition. The selective adenosine A₁ antagonist KFM-19 enhanced acetylcholine release and synaptic transmission in rat hippocampal slices and improved learning performance in aged and nucleus basalis magnocellularis-lesioned rats (Schingnitz et al., 1991). FR194921, a selective and potent A₁ antagonist, also improved scopolamine-induced memory deficits in the rat passive avoidance test (Maemoto et al., 2004). These findings suggest that the adenosine A₁ antagonist activity of ASP5854 mediates its cognitive function improvements in the context of cholinergic dysfunction. Endogenous acetylcholine regulates the induction of long-term potentiation (Ovsepian et al., 2004), an underlying event in learning and memory (Malenka et al., 1989). The activation of NMDA receptors also has key role in the induction of LTP, and a competitive NMDA receptor antagonist interfered with spatial learning in the adult rat by blockade of LTP (Morris et al., 1986). Adenosine A₁ receptor activations play a role in the development of LTP, particularly in the CA1 region (Costenla et al., 1999). Thus, the present study demonstrates, for the first time, that adenosine A₁ antagonism leads to improvement of the memory deficits associated with hypoglutamatergic state.

Role for adenosine A_2A receptors in memory has been controversial. Although there is a report showing that genetic deletion of the receptor exhibits improvement in mnemonic process (Wang et al., 2006), the effect of selective adenosine A_2A antagonists on memory disruption is not clear. Although both A_2A agonist and antagonist improve MK-801-induced memory disruption in the mice Y-maze (Fraser et al., 1997), improvement in this model in our hands did not require A_2A antagonism. Specifically, KW-6002 did not show any effect in the rat passive avoidance test. Furthermore, KW-6002 required a 10-fold higher dose in the Y-maze study than in the PD models including catalepsy in mice and rats and the turning behavior in 6-OHDA-lesioned rats. KW-6002 has no binding affinity for human adenosine A₁ receptors but moderate affinity for A₁ receptors in rodents (Table 1), being consistent with previous results by other (Shimada et al., 1997). Thus, central adenosine A₁ receptors may be more useful targets for treatment of cognitive impairment than adenosine A_2A receptors. It was suggested that ASP5854 might improve both cholinergic and glutamatergic memory disruption via potent adenosine A₁ antagonistic activity. Although adenosine A₁ antagonism can influence the cardiovascular system (Hayes, 2003), ASP5854 with higher doses did not affect cardiovascular function in rodent and dog toxicity studies (data not shown).

In conclusion, the novel adenosine A₁ and A_2A dual antagonist ASP5854 might improve both motor impairment and neuronal degeneration via A_2A receptor blockade, and cognitive impairments via A₁ receptor blockade, and could represent a novel class of agents for PD and dementia.

	Acknowledgements

 
We thank Dr. Raymond D. Price for providing helpful comments during preparation of this manuscript.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.121962.

ABBREVIATIONS: CGS21680, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine; NECA, N-ethylcarboxamidoadenosine; KW-6002, 7-methyl-3,7-dihydro-1H-purine-2,6-dione; PD, Parkinson's disease; ASP5854, 5-[5-amino-3-(4-fluorophenyl)pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one; CHO, Chinese hamster ovary; MEM, minimum essential medium; CPA, N⁶-cyclopentyladenosine; MK-801, 5H-dibenzo-[a,d]cyclohepten-5,10-imine (dizocilpine maleate); DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; NECA, N-ethylcarboxamidoadenosine; 6-OHDA, 6-hydroxydopamine; DA, dopamine; MPTP, 1-metyl-4-phenyl-1,2,3,6-tetrahydropyridine; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; NMDA, N-methyl-D-aspartate; LTP, long-term potentiation; MAO, monoamine oxidase; GBR 12909, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine; U-69593, (+)-(5,7,8)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide; SCH-420814, 2-(2-furyl)-7-(2-{4-[4-(2-methoxyethoxy)phenyl]piperazin-1-yl}ethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine; BIIB014/V2006, 2-isopropyl-4-(1,3-thiazol-2-yl)thieno[3,2-d]pyrimidine; FR194921, 2-(1-methyl-4-piperidinyl)-6-(2-phenylpyrazolo[1,5-]pyridin-3-yl)-3(2H)-pyridazinone; KFM-19, 8-(3-oxocyclopentyl)-1,3-dipropyl-7H-purine-2,6-dione.

Address correspondence to: Dr. Takuma Mihara, Neuroscience Discovery Research, Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki, 305-8585, Japan. E-mail: takuma.mihara{at}jp.astellas.com

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