A new ethyladenine antagonist of adenosine A2A receptors: Behavioral and biochemical characterization as an antiparkinsonian drug
Abstract
Adenosine A2A receptor antagonists have emerged as an attractive non-dopaminergic target in clinical trials aimed at evaluating improvement in motor deficits in Parkinson’s disease (PD). Moreover, preclinical studies suggest that A2A receptor antagonists may slow the course of the underlying neuro- degeneration of dopaminergic neurons. In this study, we evaluated the efficacy of the new adenosine A2A receptor antagonist 8-ethoxy-9-ethyladenine (ANR 94) in parkinsonian models of akinesia and tremor. In addition, induction of the immediate early gene zif-268, and neuroprotective and anti-inflammatory effects of ANR 94 were evaluated. ANR 94 was effective in reversing parkinsonian tremor induced by the administration of tacrine. ANR 94 also counteracted akinesia (stepping test) and sensorimotor deficits (vibrissae-elicited forelimb-placing test), as well as potentiating L-dopa-induced contralateral turning behavior in 6-hydroxydopamine (6-OHDA) lesion model of PD. Potentiation of motor behavior in 6-OHDA-lesioned rats was not associated with increased induction of the immediate early gene zif-268 in the striatum, suggesting that ANR 94 does not induce long-term plastic changes in this structure. Finally, in a subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, ANR 94 protected nigrostriatal dopaminergic neurons from degeneration and counteracted neuroinflammatory processes by contrasting astroglial (glial fibrillary acidic protein, GFAP) and microglial (CD11b) activation. A2A receptor antagonism represents a uniquely realistic opportunity for improving PD treatment, since A2A receptor antagonists offer substantial symptomatic benefits and possibly disease-modifying activity. The characterization of ANR 94 may represent a further therapeutic opportunity for the treatment of PD with this new class of drugs.
1. Introduction
The most widely used and effective therapy in Parkinson’s disease (PD) is treatment with the dopamine precursor L-3,4-dihydroxyphenylalanine (L-dopa). After several years of L-dopa therapy, however, motor complications, including fluctua- tions in motor response and dyskinesias develop (Olanow et al., 2004). Consequently, one of the main targets of research in PD is to identify alternative therapeutic approaches to ameliorate PD symptoms without inducing motor complications.
Preclinical and recent clinical trials have shown that adenosine A2A receptor antagonists increase the therapeutic efficacy of L-dopa without exacerbating L-dopa-associated side effects, suggesting that these drugs could be used as an effective therapy in PD (Grondin et al., 1999; Kanda et al., 2000; Jenner, 2005; Schwarzs- child et al., 2006; Rose et al., 2006, 2007; Hodgson et al., 2009).
Recently, a class of 9-ethyladenine derivatives has been char- acterized as adenosine receptor ligands (Camaioni et al., 1998). Among them, 8-ethoxy-9-ethyladenine (ANR 94) has shown high selectivity and affinity for the human adenosine A2A receptor subtype (Klotz et al., 2003), and high antiparkinsonian activity characterized by its ability to reverse locomotor deficits induced by haloperidol and induce contralateral turning behavior in unilater- ally 6-hydroxydopamine (6-OHDA)-lesioned rats (Pinna et al., 2005). In addition, ANR 94 had the longer-lasting anticataleptic effect and higher efficacy in potentiating the contralateral turning behavior induced by a threshold dose of L-dopa in 6-OHDA-lesioned rats compared with other derivatives in this class (Pinna et al., 2005).
Based on this evidence and the importance of increasing the number of efficacious adenosine A2A receptor antagonists available for clinical trials in PD, we further investigated the behavioral and biochemical antiparkinsonian activities of the new A2A receptor antagonist ANR 94.
To investigate the efficacy of ANR 94, we used the following tests that are able to reveal subtle motor and sensorimotor improvement in models of PD: the tremor test (in the rat tacrine model of PD tremor, in which tremor is induced by acute administration of the acetylcholinesterase inhibitor tacrine) (Salamone et al., 1998; Simola et al., 2004); and in the unilaterally 6-OHDA-lesioned rat model of PD the stepping test (measuring time to initiation of stepping and the number of adjusting steps) and the vibrissae- elicited forelimb-placing test (Olsson et al., 1995; Chang et al., 1999; Schallert et al., 2000; Pinna et al., 2007).
In addition, we assessed the responsiveness of striatal neurons of 6-OHDA-lesioned rats following acute administration of ANR 94 plus a threshold dose of L-dopa compared to a full effective dose of L-dopa by evaluating changes in the mRNA of the immediate early gene zif-268 (a useful tool for studying the responsiveness of striatal neurons). To gain insight into the cell type of striatal neurons expressing zif-268, neurons were double-labelled with enkephalin mRNA in order to differentiate between enkephalin(+) striatopallidal and enkephalin(—) striatonigral neurons.
Finally, on the basis of studies showing the neuroprotective and anti-inflammatory effects of A2A receptor antagonism in different PD experimental models (Chen et al., 2001; Ikeda et al., 2002; Pierri et al., 2005; Carta et al., 2009), we investigated the neuroprotective efficacy of ANR 94 in subchronically 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-treated mice by evaluating the dopa- mine-synthesizing enzyme tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNc) and the involvement of glial activation by studying glial fibrillary acidic protein (GFAP, for astroglia) and CD11b (for microglia) immunoreactivity in the SNc and striatum.
2. Materials and methods
2.1. Animals
2.1.1. Rats experiments
Male SpragueeDawley rats (Charles River, Calco, Italy) weighing 250e400 g were housed in groups of five in polycarbonate cages (33 w, 56 l, 20 h, cm).
2.1.2. Mice experiments
Male C57BL/6J mice, 3 months old (Charles River, Calco, Italy), weighing 25e30 g were housed in groups of 5e6 in polycarbonate cages (24 w, 40 l, 15 h, cm).All animals were allowed to settle under standard conditions (lights on 8.00 a. m.e8.00 p.m, 23 ◦C) with free access to food and water. Behavioral tests were per- formed during the light cycle between 10.00 a.m. and 4.00 p.m. All experiments were conducted in accordance with the guidelines for the care and use of experi- mental animals of the European Communities Directive (86/609/EEC; D.L.,27.1.1992, No. 116).
2.2. Drugs
As previously described (Pinna et al., 2005) the 8-substituted 9-ethyladenine derivative ANR 94 was synthesized starting from commercially available adenine, which was alkylated using ethyl iodide in the presence of potassium carbonate. Both the 9-ethyladenine and its N-7 isomer were obtained, the two isomers were sepa- rated through chromatography and the structure was assigned to each compound using spectrophotometric techniques. The 9-ethyladenine was treated with N-bro- mosuccinimide (NBS) to obtain the compound: 8-bromo-9-ethyladenine (ANR 82; Camaioni et al., 1998); reaction of ANR 82 with ethanol in the presence of sodium hydroxide produced the 8-ethoxy-9-ethyladenine (ANR 94). The synthesis of this compound was reported in Volpini et al. (2009).ANR 94 was dissolved by adding dimethylsulfoxide (DMSO), polyethylene glycol (PEG 400) and water (50:350:600), and vortexing vigorously; the clear solution was injected in a volume of 0.3 ml i.p./100 g body weight of rat or in a volume of 0.1 ml i. p./10 g body weight of mouse.
2.3. Tacrine-induced jaw movements
Rats were divided into two groups and treated with vehicle or ANR 94 (5 mg/kg. i.p.). Tacrine (2.5 mg/kg i.p.) was administered 20 min after vehicle or ANR 94 and the number of tremulous jaw movements and bursts were measured for 60 min.Administration of tacrine induces perioral tremor, which mostly occurs in bursts of jaw movements. Therefore, the number of bursts and jaw movements for burst were recorded. Tremulous jaw movements were defined as vertical deflections of the lower jaw not directed at a particular stimulus (Salamone et al., 1998). Yawns, tongue protrusions, and stereotypies such as grooming were not included in the evaluation.
2.4. 6-OHDA lesion
Rats (275e300 g) were anesthetised with chloral hydrate (Sigma, Milan, Italy) (400 mg/kg i.p.) and infused unilaterally into the left medial forebrain bundle at coordinates A = —2.2, L = +1.5 from bregma, V = —7.8 from dura, according to the atlas of Pellegrino et al. (1979), with 6-OHDA (8 mg/4 ml saline containing 0.05% ascorbic acid) through a stainless steel needle, at a rate of 1 ml/min. All rats were pre- treated with desipramine (10 mg/kg i.p.) 30 min before 6-OHDA to prevent damage to noradrenergic neurons.
2.5. Stepping tests
Tests were carried out as previously described in Pinna et al. (2007). Briefly, two weeks before 6-OHDA lesion, rats were handled by the experimenter for the first 3 days as described below to allow animals to familiarise with the experimenter’s grip. Over the following 2 days the rats were trained to run spontaneously up the ramp (1 m long) towards the home cage.
The fourth week after lesion, rats were randomly divided into three groups and treated with benserazide (6 mg/kg i.p.) plus L-dopa (6 mg/kg i.p.), SCH 58261 (5 mg/ kg i.p.) or ANR 94 (5 mg/kg i.p.).One day after the drug-test, rats were tested without drugs in the same week (fourth week) to evaluate possible spontaneous recovery in stepping and sensori- motor test.
2.5.1. Assessment of step initiation time
The experimenter held the rat with one hand slightly raising the hind part of the rat body above the ramp surface, whilst the other hand impeded movement of the unmonitored forelimb. Initiation time was measured from the moment the free forelimb was placed on the ramp until the rat started to step using the same forelimb.
2.5.2. Assessment of step adjustment
The rat was held in the same position as in the step initiation test with one forelimb touching a table (length of 0.7 m). The rat was moved slowly (0.7 m in 4 s) sideways along the table surface by an experimenter, first in a forward and then in a backward direction. The number of adjusting steps made by the right and left forelimbs in both (forward and backward) directions was counted.
One week before and 2, 3 weeks after the 6-OHDA lesion, rats were tested for 3 days, twice daily, for stepping and adjusting test. Testing sequence was randomly right and left forelimb testing, repeated twice a day. Thus, all rats were tested for obtaining baseline values (pre-lesion test) and re-tested after 6-OHDA lesion to obtain values of lesion-induced deficits. Then, rats were randomly assigned to the three various treatments. Stepping and adjusting tests were performed following drug administration at two different times on the basis of the onset of pharmaco- logical effect of the drugs (first test: 15 min after L-dopa, 45 min after SCH 58261 or 30 min after ANR 94 administration) and full pharmacological effectiveness (second test: 45 min after L-dopa, 90 min after SCH 58261 or 60 min after ANR 94 admin- istration) (Pinna et al., 1996, 2005, 2007). Both stepping and adjusting tests were videotaped for further objective behavioral analysis of animals.
2.6. Vibrissae-elicited forelimb placing
The experimenter held the rat by the torso, allowing forelimbs to hang free, and brushed its vibrissae against the edge of the tabletop to elicit a forelimb placing response from the forelimb on the same side (Schallert et al., 2000; Pinna et al., 2007). Placing was quantified as the percentage of successful placing responses elicited out of 10 subtests. Subtests in which the rat struggled were not counted. Rats were trained on the placing tests two weeks before surgery (10 trials for each forelimb in one day). To facilitate muscle relaxation and eliminate struggling movements, the experimenter made gentle up and down movements in space prior to the assessment of placing response. The vibrissae test was made in the interval between the two stepping tests (vibrissae test: 30 min after L-dopa, 70 min after SCH 58261 and 45 min after ANR 94 administration).
2.7. Assessment of rotational behavior
Rotational behavior was assessed in hemispherical bowls, with sawdust on the floor, in which rats were connected to an automated rotameter system (Panlab s.l., Barcelona, Spain) capable of detecting the number of full (360◦) rotations in both directions (ipsilateral and contralateral to the lesioned hemisphere). Two weeks after 6-OHDA-infusion, rats were primed with an injection of benserazide (15 mg/kg i.p.) plus L-dopa (50 mg/kg i.p.). Rats displaying at least 300 contralateral rotations during the 2 h testing period were included in the study. Rats were placed in the bowls 30 min before drug administration in order to acclimatize and extinguish any spontaneous rotational behavior.
Three days later, rats were administered with vehicle (DMSO/PEG400/H2O i.p.), vehicle + L-dopa (3 mg/kg i.p.), ANR 94 (5 mg/kg i.p.) + L-dopa (3 mg/kg i.p.), vehicle + L-dopa (12 mg/kg i.p.) or with ANR 94 (5 mg/kg i.p.). Vehicle or ANR 94 and L-dopa were administered at the same time. Rats treated with L-dopa were injected with benserazide (6 mg/kg i.p.) 30 min before L-dopa injection. Contralateral rota- tions were measured every 10 min for 1 h.
2.8. In situ hybridization
In situ hybridization studies were carried out in rats sacrificed 1 h after drug or vehicle injection. Rats were killed with CO2 and their brains were rapidly removed, frozen in dry ice-cooled isopentane and stored at —20 ◦C. Cryostat coronal sections (12 mm) of striatum were mounted on glass slides coated with gelatine, and pro- cessed according to Carta et al. (2002, 2005).
Sections were hybridized with [35S]-labelled and digoxigenin-labelled ribonu- cleotide probes complementary to mRNA encoding for zif-268 and enkephalin. Plasmids were linearized with Hind III (zif-268) or EcoRI (enkephalin) restriction enzymes (Promega, Italy). Antisense ribonucleotide probe for zif-268 was generated using T7 RNA polymerase (Promega, Italy) in the presence of [35S]UTP (Perkin Elmer, Italy). Antisense ribonucleotide probes for enkephalin was generated using SP6 RNA polymerase (Promega, Italy) in the presence of [DIG]UTP (Roche Molecular Biochemicals, Italy). Each slide was hybridized with 100 ml of buffer containing 2 × 106 cpm of radioactively labelled probe. Digoxigenin probe (3 ml) was added to each slide for double-labelling experiments in order to label enkephalin(+) stria- topallidal neurons.
Hybridization was carried out at 55 ◦C for 12 h. The next morning, slides were processed for digoxigenin probe visualization (Carta et al., 2002, 2005). Slides were washed (1× SSC, room temperature; RNase A, 20 mg/ml, for 15 min; 4× 20 min in 0.2× SSC at 60 ◦C, brief rinse in water) and incubated in Tris-buffered saline containing 3 % normal goat serum (Sigma, Italy), 0.3 % Triton X-100 and rabbit IgG directed against digoxigenin coupled to alkaline phos- phatase (1:1000; Roche, Italy) for 4 h. Slides were then rinsed 3× 10 min in Tris- buffered saline and placed in a buffer (in mM: Tris HCl, 100; NaCl, 100; MgCl2, 50) containing nitroblue tetrazolium salt (0.34 mg/mL) and 5-bromo-4-chloro-3- indolyl-phosphate toluidinium salt (0.18 mg/mL; Roche, Italy) for 12 h. There- after, slides were rinsed in water, air dried and exposed to X-ray film for 1 week.Then, slides were dipped in a photographic emulsion (LM-1 Amersham, Italy), air dried and allowed to expose in the dark at —20 ◦C. After three weeks slides were developed in Kodak D-19 for 4 min at 18 ◦C, stained with methylgreen and coverslipped.
2.9. MPTP-protocol
Mice were treated with vehicle, MPTP (20 mg/kg i.p.) once a day for 4 days or ANR 94 (0.5 mg/kg i.p.) twice a day plus MPTP (20 mg/kg i.p.) once a day for 4 days. Mice treated with ANR 94 plus MPTP received ANR 94 the day before MPTP treat- ment, 30 min before and 12 h after MPTP treatment. ANR 94 was also administered after MPTP treatment discontinuation, every day, once a day, until sacrifice, which occurred 3 days after MPTP treatment. Injections of ANR 94 during MPTP treatment were made at 8:00 a.m. and 8:00 p.m.
2.9.1. Immunohistochemistry
Brains of mice transcardially perfused with paraformaldehyde were cut in coronal sections at level of striatum and SNc with a vibratome. Sections were incubated for 1 h with 5% normal goat serum as blocking agent, then overnight in primary antibodies (polyclonal rabbit anti-TH, 1:1000, Biomol; monoclonal mouse anti-GFAP, 1:400, Sigma; monoclonal rat anti-mouse CD11b, 1:1000, Serotec). Subsequently sections were incubated in biotinylated secondary antibodies (goat anti-rabbit IgG for TH, goat anti-mouse IgG for GFAP and goat anti-rat IgG for CD11b, all purchased from Vector) and visualized by standard avidinebiotinehorseradish peroxidase technique (ABC, Vector), using 3,3′-diaminobenzidine (Sigma) as chro- mogen. Sections were mounted on gelatine-coated slides, dehydrated and coverslipped.Adjacent SNc sections were stained with cresyl violet, in order to evaluate cell loss in this area. The entire SNc, was captured at 10× magnification and immuno- stained sections of one dorsolateral (DL) and one ventromedial portion of striatum were captured at 20× magnification.
2.10. Data analysis and statistics
2.10.1. Statistic of behavioral studies
Mean S.E.M of the number of jaw movements and bursts and of the six subtests of stepping and adjusting step tests performed without drug and of each time test with drug were calculated. For vibrissae test, mean S.E.M of placing was expressed as the percentage of successful placing responses elicited out of 10 subtests for each rat. Mean S.E.M. of the number of contralateral turns performed were calculated throughout a 1 h period.
Significance between groups was evaluated by Student’s t-test followed by NewmaneKeuls or by one or two-way ANOVA followed by Tukey’s HSD post hoc test. Results were considered significant at p < 0.05. 2.10.2. Analysis and statistics of autoradiograms and double-labelling sections Quantitative analysis of labelling was performed using the image analysis program Scion Image (Carta et al., 2005). The average grey value from white matter was subtracted from each autoradiographic section to correct for background labelling. Zif-268 mRNA levels were measured in the DL portion of the lesioned and unlesioned striatum. For double-labelling studies the selected areas were digitized at 400× through a Zeiss microscope, under combined epiluminescence and low trans- mitted light. About one hundred and fifty neurons from the DL portion of the lesioned striatum were analysed. Silver grains as white dots were counted automatically above each cell with an image analyser (Zeiss KS 300) and a frequency distribution of silver grains per neuron in enkephalin(+) striato- pallidal and enkephalin(—) striatonigral neurons was obtained. In order to confirm the result obtained with the first analysis, a second analysis on enkephalin(—) striatonigral neurons stained with methylgreen was done and silver grains as black dots were counted. Analysis on enkephalin(—) striatonigral neurons showed the same result.Data were statistically compared with two-way ANOVA, followed by Tukey's HSD post hoc test for comparison between individual groups. Results were considered significant at p < 0.05. 2.10.3. Analysis of neuroprotection study For each animal, three sections from the SNc and striatum were analysed for each protein evaluated in the study.Mean number of TH- or GFAP-positive cells, obtained from each experi- mental group, was first normalized as respect to the vehicle, values from different levels were averaged thereafter. For CD11b analysis, images were digitized in a grey scale, and CD11b immunostaining evaluated with the analysis program Scion Image. A threshold, the value of which was set above the mean value S.E.M. of the background, was applied for background-correction. Inside each frame, the area occupied by grey values above the threshold was auto- matically calculated. For each level of SNc or striatum, the obtained value was first normalized as respect to vehicle, values from different levels were averaged thereafter.In all experiments, significance of differences between groups for each param- eter was evaluated by one-way ANOVA followed by Tukey's post hoc test. Results were considered significant at p < 0.05. 3. Results 3.1. Effect of ANR 94 on tacrine-induced jaw movements The effects of ANR 94 (5 mg/kg i.p.) on tacrine-induced tremu- lous jaw movements are shown in Fig. 1. The results reveal that pre- treatment with ANR 94 (5 mg/kg i.p.) was associated with a significantly lower number of tremulous jaw movements induced by acute administration of tacrine (2.5 mg/kg i.p.) (p < 0.05 vs vehicle) (Fig. 1A). Moreover, ANR 94 (5 mg/kg) pre-treatment was associated with a significantly lower number of bursts induced by tacrine (p < 0.05 vs vehicle) (Fig. 1B). Fig. 1. Effect of ANR 94 (5 mg/kg i.p.) pre-treatment on tremulous jaw movements and jaw bursts induced in rats by tacrine (2.5 mg/kg i.p.). Results are mean SEM of jaw movements (A) and mean SEM of jaw bursts (B) recorded for 60 min after tacrine administration. ANR 94 significantly reduced tremulous jaw movements and jaw bursts compared to vehicle. *p < 0.05 vs vehicle; N = 6. Statistical significance was determined by Student's t-test followed by the NewmaneKeuls post hoc test. 3.2. Effect of ANR 94 in the stepping test In the stepping test, unilaterally 6-OHDA-lesioned rats displayed significantly impaired motor performance of the right forelimb contralateral to the 6-OHDA lesion compared to the pre-lesion test. Latency of step initiation progressively increased in the right fore- limb, contralateral to the lesion (p < 0.0001 vs pre-6-OHDA lesion), from 2 to 3 weeks post-lesion (p < 0.0001 vs pre-6-OHDA lesion), and remained stable the 4th week after the 6-OHDA lesion (Fig. 2). No significant impairment was observed in the left forelimb and so these results are not reported here. The efficacy of ANR 94 in the stepping test was compared to that of L-dopa and the well-characterized A2A receptor antagonist SCH 58261. The dose of L-dopa used was, according to Pinna et al. (2007), subthreshold for induction of rotation that could have otherwise interfered with the results of the tests. The doses of the A2A receptor antagonists SCH 58261 and ANR 94 were chosen on the basis of previous studies in which these compounds were evaluated for their pharmacological efficacy on turning behavior in 6-OHDA-lesioned rats (Pinna et al., 1996, 2005) and on antagonism of haloperidol-induced catalepsy (Pinna et al., 2005). 5. Conclusions The present results suggest that administration of the A2A receptor antagonist ANR 94 improves parkinsonian initiation movement deficits, akinesia/hypokinesia, gait deficits, sensori- motor impairments and tremor even without the combined administration with L-dopa. These findings predict that ANR 94, similarly to other proven A2A receptor antagonists evaluated in clinical trials, may be effective in PD patients. In addition, the ability of ANR 94 to produce moderate activation of zif-268 mRNA compared to a full dose of L-dopa suggests that ANR 94 would be effective as a symptomatic treatment without provoking long-term plastic changes.Finally, the neuroprotective and anti-inflammatory effects of ANR 94 offer a unique opportunity to counteract neuro- degeneration, one of the most intriguing aspects of PD pathology.