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NEUROPHARMACOLOGY

LY503430, a Novel -Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid Receptor Potentiator with Functional, Neuroprotective and Neurotrophic Effects in Rodent Models of Parkinson's Disease

Eli Lilly & Co. Ltd., Lilly Research Centre, Surrey, United Kingdom (T.K.M., K.W., C.S.R., M.A.W., C.A.H., D.L., M.J.O.); and Eli Lilly & Co., Lilly Corporate Center, Indianapolis, Indiana (J.L.V., P.B., E.S., M.G., A.M.O., P.S., D.M.Z., E.S.N., D.B.)

Received January 23, 2003; accepted April 28, 2003.

	Abstract

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Glutamate is the major excitatory transmitter in the brain. Recent developments in the molecular biology and pharmacology of the -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors have led to the discovery of selective, potent, and systemically active AMPA receptor potentiators. These molecules enhance synaptic transmission and play important roles in plasticity and cognitive processes. In the present study, we first characterized a novel AMPA receptor potentiator, (R)-4'-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430), on recombinant human GLU_A1–4 and native preparations in vitro and then evaluated the potential neuroprotective effects of the molecule in rodent models of Parkinson's disease. Results indicated that submicromolar concentrations of LY503430 selectively enhanced glutamate-induced calcium influx into human embryonic kidney 293 cells transfected with human GLU_A1, GLU_A2, GLU_A3, or GLU_A4 AMPA receptors. The molecule also potentiated AMPA-mediated responses in native cortical, hippocampal, and substantia nigra neurons. We also report here that LY503430 provided dose-dependent functional and histological protection in animal models of Parkinson's disease. The neurotoxicity after unilateral infusion of 6-hydroxydopamine into either the substantia nigra or the striatum of rats and that after systemic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice were reduced. Interestingly, LY503430 also had neurotrophic actions on functional and histological outcomes when treatment was delayed until well after (6 or 14 days) the lesion was established. LY503430 also produced some increase in brain-derived neurotrophic factor in the substantia nigra and a dose-dependent increases in growth associated protein-43 (GAP-43) expression in the striatum. Therefore, we propose that AMPA receptor potentiators offer the potential of a new disease modifying therapy for Parkinson's disease.

With this in mind, the actions of neurotrophins in Parkinson's disease have recently been evaluated (Bradford et al., 1999). Many investigators have reported that glial derived growth factor (GDNF) promotes dopamine neuron survival (Tomac et al., 1995; Gash et al., 1996; Rosenbald et al., 2000), whereas other studies have shown that brain-derived growth factor (BDNF) can protect against behavioral, biochemical, and immunocytochemical changes after nigrostriatal dopamine lesions (Altar et al., 1992, 1994; Klein et al., 1999). A major drawback of growth factors is their large size and therefore the need to administer these molecules directly into the brain. An alternative and perhaps more clinically relevant approach would be to use a small molecule that could up-regulate neurotrophin expression in the brain. Of direct relevance therefore is the recent report that -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors interact with and signal through the protein tyrosine kinase Lyn (Hayashi et al., 1999). As a result, the mitogen-activated protein kinase pathway is activated and the expression of BDNF is increased (Hayashi et al., 1999).

After the discovery that cyclothiazide and IDRA 21 could potentiate AMPA receptor activity, more potent and selective AMPA receptor potentiators have been discovered (Ornstein et al., 2000; Baumbarger et al., 2001b; Parsons et al., 2002). All of these compounds allosterically regulate AMPA receptor activity at least in part by suppressing the desensitization process of AMPA receptors. As a result, these molecules can enhance calcium flux in HEK293 cells transfected with recombinant human GLU_A1–4 subunits (Miu et al., 2001) and increase AMPA-evoked responses on native neurons in vitro (Baumbarger et al., 2001a,b; Gates et al., 2001) and in vivo (Vandergriff et al., 2001), enhance long-term potentiation, and are active in various cognitive models (Staubli et al., 1994; Hampson et al., 1998a,b; Quirk and Nisenbaum, 2002). In addition, it has also been shown that AMPA potentiators such as CX-546 (Lauterborn et al., 2000), LY392098 (Legutko et al., 2001), and LY404187 (Mackowiak et al., 2002) increase BDNF levels in vitro and in vivo. Based on these observations, we hypothesized that an AMPA potentiator would protect against nigrostriatal degeneration.

In the present study, we have evaluated the effects of a novel AMPA receptor potentiator, LY503430 on glutamate responses in HEK293 cells transfected with human GLU_A1, GLU_A2, GLU_A3, or GLU_A4 AMPA receptor subunits and on glutamate responses in hippocampal, Purkinje, cortical, and substantia nigra neurons in vitro. We also present results showing that this novel AMPA potentiator provided significant neuroprotection in a mouse MPTP and two rat 6-OHDA models of Parkinson's disease. Furthermore, in the unilateral 6-OHDA model, functional and histological protection were maintained when LY503430 treatment was delayed for up to 14 days, suggesting a trophic action. These data suggest that LY503430 could protect and potentially reverse the progression of Parkinson's disease.

	Materials and Methods

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Studies on Recombinant iGlu Receptors Expressed in HEK293 Cells. Ninety-six-well plates containing confluent monolayers of HEK293 cells stably expressing human AMPA receptors were prepared. Cells were incubated in buffer solution (10 mM glucose, 138 mM sodium chloride, 1 mM magnesium chloride, 5 mM potassium chloride, 5 mM calcium chloride, and 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.1–7.3) containing 20 µM Fluo3-AM dye (Molecular Probes, Eugene, OR) for 60 min. Cells were washed in buffer solution and fluorescence measurements were made using a fluorometric imaging plate reader (Molecular Devices Corp., Sunnyvale, CA) that indicated changes in fluorescence upon influx of calcium into cells upon stimulation by glutamate (100 µM) in the presence of cyclothiazide (100 µM) or compound. Compound applications preceded glutamate additions by 5 min, and fluorescent measurements were made immediately before addition of glutamate and 3 min after glutamate addition. Data are expressed as EC₅₀ values determined from maximum responses observed for LY503430 on each cell line in the presence of glutamate (100 µM).

AMPA Receptor-Mediated Responses in Acutely Isolated Substantia Nigra and Prefrontal Cortical Neurons. Prefrontal cortical pyramidal neurons or substantia nigra dopamine neurons from young (2–3-week old) male Sprague-Dawley rats (14–22 days old) were acutely isolated from the prefrontal cortex or the midbrain, respectively, using procedures described previously (Baumbarger et al., 2001b). Male Sprague-Dawley rats were deeply anesthetized with methoxyflurane and decapitated. Their brains were removed rapidly from the skull and immersed in a cold (2°C) NaHCO₃-buffered saline solution (126.0 mM NaCl, 3.0 mM KCl, 1.5 mM MgCl₂, 1.25 mM Na₂PO₄, 2.0 mM CaCl₂, 26.0 mM NaHCO₃, and 10.0 mM glucose; pH 7.4, osmolarity 300 ± 5 mOsM/l). The brains were blocked and 400-µm-thick coronal sections were cut through the rostrocaudal extent of the prefrontal cortex or midbrain using a Vibroslice (Campden Instruments, London, England). Slices then were incubated at room temperature (20–22°C) for 0.5 to 6 h in a holding chamber containing the continuously oxygenated (95% O₂, 5% CO₂) NaHCO₃-buffered saline solution. After the incubation period, slices were transferred to a glass Petri dish containing a low-Ca² HEPES-buffered saline solution [140.0 mM NaHOCH₂CH₂SO₃ (Na isethionate), 2.0 mM KCl, 4.0 mM MgCl₂, 0.1 mM CaCl₂, 23.0 mM glucose, and 15.0 mM HEPES; pH 7.4, osmolarity 300 ± 5 mOsM/l and placed under a dissecting microscope. The prefrontal cortex or ventral midbrain from each hemisphere was dissected from the surrounding tissue. The tissue was placed into a holding chamber containing protease type XIV (1 mg/ml; Sigma-Aldrich, St. Louis, MO) dissolved in a HEPES-buffered Hanks' balanced salt solution (HBSS 6136; Sigma-Aldrich) maintained at 37°C and oxygenated (100% O₂), pH 7.4, osmolarity 300 ± 5 mOsM/l. After 30 to 40 min of incubation in the enzyme solution, the cortex was rinsed three times with the low-Ca²⁺ HEPES-buffered saline solution and triturated using two fire-polished Pasteur pipettes having tips of decreasing diameter. Before whole-cell recording, the cell suspension was placed into a 50-mm transparent plastic Petri dish that was mounted onto the stage of an inverted microscope. Prefrontal cortical pyramidal neurons were selected on the basis of their triangular somatic shape, soma size (20–30 µm in diameter), and presence of some apical and basal dendrites. Substantia nigra dopamine neurons were identified on the basis of their large soma size and multipolar morphology.

The whole-cell variant of the patch-clamp technique was used for recording current from acutely isolated prefrontal cortical pyramidal neurons. Electrodes were pulled from borosilicate capillary tubing (Corning 7052; WPI, Sarasota, FL) using a multistage puller (Sutter Instruments Inc., Novato, CA). The electrodes were fire-polished using a Microfuge (Narishige, Tokyo, Japan) before use. The internal electrode filling solution contained 160.0 mM N-methyl-D-glucamine, 4.0 mM MgCl₂, 40.0 mM HEPES, 3.0 mM BAPTA, 12.0 mM phosphocreatine, 2.0 mM Na₂ATP, and 0.2 mM GTP; pH was adjusted to 7.2 with KOH and osmolarity adjusted to 270 to 280 mOsM/l. The extracellular solution contained 140.0 mM Na isethionate, 1.0 mM KCl, 5.0 mM BaCl₂, 1.0 mM MgCl₂, 10.0 mM HEPES, and 0.001 mM tetrodotoxin; pH adjusted to 7.4 with NaOH 1.0 M; osmolarity adjusted to 300 ± 5 mOsM/l with glucose.

Upon placing the recording electrode in the bath, offset potentials were corrected and electrode resistances ranged between 2 and 7 M. Voltage-clamp recordings were made using a 200B amplifier (Axon Instruments, Inc., Foster City, CA). The membrane potential of cells was held at –80 mV. Currents were digitized and monitored with pCLAMP software version 8.0 (Axon Instruments, Inc.) running on a PC Pentium computer. A small amount of constant positive pressure (2–3 cm of H₂O) was applied to the electrodes as they were advanced through the bath. After achieving the whole-cell configuration, series resistance was compensated (70–85%) and monitored periodically. All experiments were conducted at room temperature.

Application of drugs was accomplished using sixteen barrel pipette array made from small diameter (600 µm) glass capillary tubing. Solutions were contained in 10-ml syringes and positioned approximately 12 inches above the recording chamber. Gravity-induced flow of each solution from the syringe to the corresponding barrel was controlled by electronic valves. The pipette array was positioned 100 to 200 µm from the cell before seal formation. The solutions from the drug array were changed (100 ms) by altering the array position with a DC actuator (Newport Inc., Irvine, CA).

Concentration-response profiles for LY503430 and CX-516 were constructed by measuring the peak current amplitude during a 10-s coapplication of compound and AMPA (5 µM), calculating the percentage of increase relative to the AMPA alone response and plotting the data as a function of potentiator concentration. The plotted points then were fit with a logistic equation of the following form: percentage of potentiation = E_max/(1 + ([AMPA potentiator]/EC₅₀))ⁿ, where the maximal percentage of potentiation is relative to the current evoked by glutamate alone, EC₅₀ is the concentration equal to 50% of the maximally effective concentration, and n is the Hill coefficient. The best fit was chosen using the Marquardt-Levenberg algorithm. Average EC₅₀ and E_max values were determined and reported as mean ± S.E.M.

Responses of Hippocampal Neurons to Iontophoretic Application of AMPA and N-Methyl-D-aspartate (NMDA) in Vivo. The principles of the iontophoresis method are well established and described previously (Vandergriff et al., 2001). Briefly, male Sprague-Dawley rats (300–400 g) were anesthetized with chloral hydrate (400 mg/kg i.p.) initially and supplemental doses intermittently to maintain surgical anesthesia. The rats were mounted in a stereotaxic frame and one or two small craniotomies were performed to allow placement of stimulating and recording electrodes. For the hippocampal iontophoretic studies, five-barrel glass microelectrodes (tip diameter of 5–8 µm) were positioned in the CA1 region of the hippocampus. The center recording barrel contained 2 M NaCl, outer barrels contained 5 mM AMPA (in 195 mM NaCl; pH 7.4), 20 mM NMDA (in 180 mM NaCl; pH 7.1), and 2 M NaCl, the latter being used for current balancing. Hippocampal CA1 pyramidal cells were located and identified by their stereotaxic coordinates (bregma, –4.2; lateral, 2.4, 1.8–2.4 mm below pial surface) and characteristic extracellular action potential. Excitation of CA1 neurons was achieved by the alternate and electronically timed electrophoretic ejection of AMPA and NMDA with currents and durations of ejection (normally 0–10 nA and 20–30 s) adjusted to produce near equal and submaximal increases in firing rate. Once stable responses were maintained for a period of at least 10 min, the test drug was administered via a lateral tail vein cannula (Vandergriff et al., 2001). Cumulative dose-response curves to CX-516 and LY503430 were prepared by recording responses to increasing drug doses in logarithmic steps. Raw data and firing data were recorded on a DAT analyzer for subsequent analysis. Changes were calculated as percentage of control firing rates and expressed as the mean ± S.E.M.

MPTP Neurotoxicity in Mice. Male C57Bl6J mice (Harlan UK Ltd., Oxford, UK), weighing 20 to 25 g, were used. They were housed in groups of 10 mice per cage under a 12-h light/12-h dark cycle (lights on 7:00 AM–7:00 PM) with food and water available ad libitum. LY503430 was administered at 0.5 mg/kg s.c for 11 days. On day 8, the animals received 4 x 20 mg/kg MPTP at 2-h intervals.

6-OHDA Studies in Rats. Male Sprague-Dawley rats (Harlan UK Ltd.), weighing 280–320 g, were anesthetized with a gaseous anesthetic consisting of Isoflurane/nitrous oxide/oxygen and were placed on a thermostatically controlled heating blanket and then placed in a Kopf stereotaxic frame.

Substantia Nigra Lesions. Unilateral lesions of the left substantia nigra were made using 4 µg (free base) of 6-OHDA in 1.8 µl of 0.02% ascorbic acid infused stereotaxically into the left SN. Coordinates from bregma according to the atlas of Paxinos and Watson (1986) were AP, –4.8 mm, L, +1.9 mm; V, –8.0 mm (from skull surface at bregma); and toothbar, –3.7 mm. 6-OHDA was infused at a rate of 0.3 µl/min, followed by 2-min equilibration time, with the needle remaining in place (Murray et al., 2002). Sham-operated rats received identical surgery but 1.8 µl of 0.02% ascorbic acid (6-OHDA vehicle) was infused.

Striatal Lesions. Unilateral lesions of the right striatum were made using 10 µg (free base) of 6-OHDA in 2.57 µl of 0.02% ascorbic acid in 0.9% saline infused stereotaxically into the right striatum. Coordinates from bregma were AP, 0.7 mm; L, –2.3 mm; V, –6.0 mm (from skull surface at bregma); and toothbar, –3.3 mm. The infusion was made over a period of 4 min at a rate of 0.643 µl/min, followed by 4-min equilibration time, with the needle remaining in place (Murray et al., 2002).

Drug Studies. For all s.c. studies LY503430 was dissolved in 12.5% -cyclodextrin and sonicated before administration. For all oral studies, LY503430 was dissolved in 1% sodium carboxymethyl-cellulose/0.25% Tween 80/water vehicle.

For nigral lesion studies, LY503430 was administered for either 14 days at 0.1 or 0.5 mg/kg s.c. in initial studies or 10 days at 0.05, 0.1, 0.2, or 0.5 mg/kg p.o to complete full oral dose response starting 1 day after 6-OHDA lesion. Compounds were administered twice daily on weekdays and once a day at weekends. In additional studies, treatment with LY503430 was delayed until 3, 6, or 14 days after 6-OHDA infusion. For studies using striatal lesion model LY503430 was administered for 28 days at 0.5 mg/kg s.c. starting 1 day after 6-OHDA lesion.

Behavioral Assessment Using Rotometers. Twenty-four hours after the final drug treatment the animals were placed in automated rotometers (MED Associates, St. Albans, VT). The apparatus consisted of Perspex bowls where each rat was linked to a harness that had an infrared sensor at the top connected to a computer with ROTORAT software. This software measured the number of contraversive and ipsiversive rotations. The animals were tested for baseline rotations 24 h after the final drug treatment. On the next day, the animals were retested in the presence of apomorphine (0.25 mg/kg s.c.) or amphetamine (5 mg/kg i.p.) to evaluate the effects of drug treatment on stimulant-induced rotations (this is a measure of functional neuroprotection). Data were expressed as asymmetry scores (the difference between the number of contraversive and ipsiversive rotations).

Neurochemical Measurements of Dopamine and Metabolites. The left and right striata were dissected, weighed, and homogenized in 2 volumes of distilled water. A 10-µl aliquot of the homogenate was transferred to a 0.5-ml Eppendorf tube and 20 µl of 1% aqueous trifluoroacetic acid was added, mixed, and spun at 13,000 rpm for 5 min. Two microliters of the supernatant was then assayed by high-performance liquid chromatography with electrochemical detection. All analyses were performed on a Luna 5 C₁₈ column (25 cm x 2 mm) at a flow rate of 200 µl/min. The elution solvent was 88% water/12% acetonitrile containing an overall concentration of 9 g/l sodium dihydrogen phosphate, 200 mg/l EDTA, and 320 mg/l octane sulfonic acid. The pH was adjusted to 4.20 with orthophosphoric acid. Mobile phase was precleaned by passing through a guard cell, controlled via a Coulochem 5100 controller set at 450 mV and situated between the pump and autosampler. Detection was achieved with an Antec electrochemical detector with a cell potential of 750 mV. Data were collected on a Waters Millennium³² chromatography data system. Dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid concentrations in the samples were calculated by comparison with calibration curves constructed from pure reference standards.

Histological Analysis. After behavioral testing, the animals were given an overdose of anesthetic, and the thorax was opened and perfused with 30 ml of saline followed by 30 ml of 10% buffered formalin via the left ventricle or vena cava. The brains were removed, cut twice into 6-mm segments using a rodent brain matrix, processed, and embedded in paraffin wax. Coronal sections (8 µm) were taken on a sledge microtome (Murray et al., 2002).

Tyrosine Hydroxylase, Growth-Associated Protein-43 (GAP-43), and BDNF Immunocytochemistry. Briefly, the sections were deparaffinized and rehydrated and endogenous peroxidase was quenched with 0.3% H₂O₂ for 30 min. The slides were placed in pepsin (0.2 g of Sigma-p-7000 pepsin in 50 ml of 0.01 M HCl) for 30 min, washed, and nonspecific binding was blocked with 1.5% normal goat serum (Vectastain rabbit IgG ABC kit; Vector Laboratories, Burlingame, CA). This was followed by application of the primary rabbit polyclonal anti-tyrosine hydroxylase antibody (AB152 incubated for 18 h at room temperature; Chemicon International, Temecula, CA), the secondary biotinylated antibody (Vectastain rabbit IgG ABC kit for 30 min) and the horseradish peroxidase conjugate (Vectastain rabbit IgG ABC kit for 30 min). Visualization was carried out using 3,3'-diaminobenzidine (Vector SK-4100) as a chromogen. The slides were coverslipped using DPX mountant. Adjacent sections from the oral efficacy studies were stained in a similar way using a primary antibody (AB5220, 1:500 dilution; Chemicon International) to GAP-43 and/or a primary antibody (SC-546 rabbit polyclonal IgG, 1:100 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to BDNF.

Image Analysis. After tyrosine hydroxylase (TH)-immunostaining, the striatal slides were digitized. Using an image analysis system (Optimas 5.2), the areas of the dorsal and ventral striatum of each hemisphere were outlined individually, and the mean gray densities were measured. The staining intensity of each lesioned hemisphere was expressed as a percentage of the respective intact hemisphere from that animal (Murray et al., 2002). Sections of the substantia nigra at –5.00 mm caudal to bregma in the rat (Paxinos and Watson, 1986) and at –3.08 mm caudal to bregma in mouse (Paxinos and Franklin, 1997) were also stained, and the number of intact TH-positive cells in left and right substantia nigra were counted at 25x magnification. The same image analysis system was used to quantify the GAP-43 and BDNF immunoreactivity. The mean gray intensity of the intact and lesioned striatum was calculated and data were then expressed as a percentage change in GAP-43 immunoreactivity between the intact and lesioned hemisphere. The number of BDNF-positive immunoreactive cells per field was counted in the substantia nigra, striatum, hippocampus (CA1, CA3, and dentate gyrus), and in the cortex.

Statistics. Statistical analysis of data were carried out using analysis of variance followed by appropriate post hoc t test using p < 0.05 as the level of significance. All analysis was performed using the statistical analysis package JMP (SAS Institute Inc., Cary, NC).

	Results

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Potency and Selectivity of LY503430
Effects on Recombinant GLU_A1–4 Receptors Expressed in HEK293 Cells. The AMPA potentiator used for this investigation was the biarylpropylsulfonamide LY503430 (Fig. 1). Submicromolar concentrations of LY503430 enhanced glutamate-induced calcium influx into HEK293 cells transfected with human GLU_A1, GLU_A2, GLU_A3, or GLU_A4 AMPA receptors. The potency and efficacy of potentiation by LY503430 were highly dependent on receptor subtype and splice variant. The rank order of potency of LY503430 was GLU_A2 > GLU_A4 > GLU_A1 > GLU_A3 and at all subunits, the compound was considerably more potent on the "flip" splice variants.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Structure and pharmacological profile of the biarylpropylsulfonamide LY503430. EC50 values (nanomolar) for calcium flux at recombinant human AMPA (GLUA1–4 flip and flop) and kainate (GLUK5, GLUK6, and GLUK6/KA2) receptor subtypes expressed in HEK293 cells. Responses on NMDA receptors in cortical and hippocampal neurons and on kainate responses in DRG neurons are also illustrated.

Effects LY503430 and CX-516 Substantia Nigra Dopamine Neurons and Prefrontal Cortical Neurons in Vitro. The potency and efficacy of LY503430 (0.03–10 µM) and CX-516 (0.3–3.0 mM) on the AMPA-evoked responses of substantia nigra dopamine neurons was evaluated by recording the inward current elicited in response to application of AMPA (5 µM, 10-s duration; holding potential –80 mV) alone and in the presence of each compound. Preliminary experiments demonstrated that this concentration of AMPA was equal to 30% of the maximal response (EC₃₀). The relatively small responses recorded in the presence of AMPA reflect only the desensitized component of the total AMPA response; the peak component is not detectable because of the relatively slow solution switching speed (100 ms) of the actuator used in these experiments. At all concentrations tested, application of LY503430 alone had no effect on the holding current (Fig. 2a). However, when applied in the presence of AMPA, LY503430 potentiated the evoked current in a concentration-dependent manner (Fig. 2, a and b). As previously reported for related biarylpropylsulfonamides (Baumbarger et al., 2001b), the potentiated response displayed a marked time dependence such that a steady-state level was never achieved during the 10-s AMPA stimulus or even if applications were prolonged for up to 120 s. Because of this property, the data were expressed as a percentage of change in peak amplitude from that of the glutamate response alone and plotted as a function of compound concentration. As such, the values for potency and efficacy are estimates. Comparison of the concentration-response profiles for LY503430 and CX-516 revealed that LY503430 was more potent (EC₅₀ = 2.6 ± 0.3 µM) and efficacious (E_max = 181.3 ± 40.0-fold increase; n = 8) than CX-516 (EC₅₀ = >3 mM and E_max = 11.2 ± 2.4-fold increase; n = 9). Similar differences in potency and efficacy of potentiation of prefrontal cortical neurons (Fig. 2c) were found for LY503430 (EC₅₀ = 3.3 ± 0.8 µM and E_max = 86.7 ± 14.3-fold increase; n = 14) and CX-516 (EC₅₀ = 3.7 ± 1.3 mM and E_max = 3.8 ± 1.6-fold increase; n = 6).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Effects of LY503430 and CX-516 on native AMPA receptor activity in vitro and AMPA-mediated responses in vivo. a, concentration-response profile for LY503430 (0.03–10.0 µM) potentiation was assessed by measuring the responses of acutely isolated substantia nigra dopamine neurons to 5 µM AMPA alone and in the presence of potentiator. b and c, plots of the average degree of potentiation by LY503430 (0.03–10.0 µM) and CX-516 (0.3–3.0.0 mM) as a percentage of the 5 µM AMPA response for each concentration of compound tested in acutely isolated substantia nigra dopamine neurons (b) and prefrontal cortical neurons (c). Although both compounds enhanced AMPA-evoked responses in concentration-dependent manner, LY503430 was more potent and efficacious than CX-516 in both cell types. d, plot of the average change in AMPA- and NMDA-evoked discharge of CA1 hippocampal neurons in response to intravenously delivered LY503430 and CX-516. Data points in plots represent mean ± S.E.M.

Cross-Reactivity on Other Glutamate Receptor Subtypes. LY503430 had no discernible effects on kainate-mediated currents in HEK293 cells transfected with GLU_K5, GLU_K6, or GLU_K6/KA2 at concentrations up to 10 µM or on responses of rat cortical and hippocampal neurons to NMDA and to kainate in the presence of GYKI53655 (a selective AMPA antagonist). On rat dorsal root ganglion (DRG) neurons, LY503430 (3 µM) slightly inhibited responses to kainate (Fig. 1). In addition, ligand binding studies indicated that LY503430 did not bind to other neurotransmitter (e.g., adrenergic, nicotinic, muscarinic, serotonergic, and dopaminergic) receptors (data not shown).

Effects of LY503430 on AMPA Responses of Rat Hippocampal Neurons in Vivo. Systemic administration of LY503430 (0.01–10 µg/kg i.v.) enhanced responses of hippocampal neurons to iontophoretic AMPA in a dose-dependent manner (Fig. 2d). There was a smaller increase in responses to NMDA. The dose-response curves for LY503430 are compared with that of CX-516 (Fig. 2d). A dose of 0.1 µg/kg i.v. LY503430 was sufficient to produce a selective increase in hippocampal responses to AMPA, confirming that LY503430 crosses the blood-brain barrier and has central actions.

Neuroprotective Effects of LY503430 in Rodent Models of Parkinson's Disease
Prevention of MPTP-Induced Neurotoxicity in Mice. LY503430, administered for 11 days at 0.5 mg/kg s.c., prevented the loss of tyrosine hydroxylase immunoreactivity (TH-IR) in the striatum (Fig. 3a) and substantia nigra (Fig. 3b) in MPTP-treated mice.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effects of pretreatment for 7 days with LY503430 (0.5 mg/kg s.c.) on the density of TH-immunoreactivity in the striatum (a) and the number of TH-positive intact cells per slide in the substantia nigra at –3.08 mm caudal to bregma (b) after MPTP treatment in mice. LY503430 provided significant protection against the MPTP-induced neurotoxicity in the substantia nigra. n = 6/group.

Functional and Histological Protection after Infusion of 6-OHDA into the Striatum in Rats. In initial studies we found that 10 µg of 6-OHDA infused unilaterally into the striatum produces a slow, partial retrograde degeneration of the cell bodies in the substantia nigra, resulting in an approximate 50% loss in tyrosine hydroxylase-positive cells at 4 weeks and in marked ipsiversive rotations in response to amphetamine (5 mg/kg). Using this model, we found that 28-day treatment with LY503430 (0.5 mg/kg s.c.) attenuated amphetamine-induced ipsiversive rotations (Fig. 4a) and provided significant protection against the loss of TH-positive nigral cell bodies (Fig. 4, b and c).

View larger version (78K): [in this window] [in a new window]   Fig. 4. Effects of LY503430 (0.5 mg/kg s.c. for 28 days starting 1 day after infusion of 6-OHDA into the striatum) on functional outcome (a) and the number of TH-immunoreactive cell bodies per slide in the substantia nigra at 5.00 mm caudal to bregma (b and c). LY503430 attenuated amphetamine (5 mg/kg i.p.)-induced rotational behavior (a), and the number of intact TH-immunoreactive nigral cell bodies was significantly higher in LY503430-treated animals (p < 0.05). Data are based on eight animals per group. **, p < 0.01 versus sham control; +, p < 0.05 versus vehicle control.

It should be pointed out that in these and the subsequent experiments, the LY503430 treatment was stopped 1 to 2 days before the behavioral tests and in separate experiments acute dosing of LY503430 was without effect on turning behavior in previously lesioned control animals.

Functional and Histological Protection after Infusion of 6-OHDA into the Substantia Nigra in Rats. In initial studies, 4 µg of 6-OHDA infused into the substantia nigra produced a loss of cell bodies over the next 4 days and striatal terminals over the next 5 to 6 days, resulting in an 85 to 90% loss in nigra cell bodies, 80 to 90% loss in of TH-IR in the dorsal striatum, and 50 to 60% loss in TH-IR in the ventral striatum. We then carried out a series of experiments to evaluate the effects of LY503430 (0.05, 0.1, 0.2, and 0.5 mg/kg p.o. for 10 days, starting 1 day after 6-OHDA) on functional outcome at 12 days and histological outcome at 13 days after 6-OHDA. Results of the first experiment indicated that both the 0.2 and 0.5 mg/kg oral doses of LY503430 prevented apomorphine-induced rotations (Fig. 5a) and provided significant protection in the dorsal and ventral striatum (Fig. 5, b and c). These effects were accompanied, by only a modest effect on the number of TH-positive cells in the substantia nigra.

View larger version (43K): [in this window] [in a new window]   Fig. 5. Effects of chronic treatment with LY503430 (0.2 and 0.5 mg/kg p.o. for 10 days starting 1 day after infusion of 6-OHDA into the nigra) on rotational behavior (a), tyrosine hydroxylase-immunoreactivity (b and c) in the dorsal and ventral striatum, and number of TH-positive nigral cell bodies (d). Results indicate that both doses of LY503430 provided a significant correction of apomorphine-induced rotational asymmetry (a) and loss of TH staining observed (b) in both dorsal and ventral striatum (c) after unilateral infusion of 6-OHDA into the substantia nigra. In this study, the compound had minimal effects of the number of TH-positive cell bodies in the substantia nigra. In parallel studies, we observed some protection in the nigra after 14 days treatment with 0.5 mg/kg LY503430. Data are based on eight animals per group. ***, p < 0.001 versus baseline rotations or TH, +, p < 0.05; ++, p < 0.01 versus vehicle-treated animals.

Additional experiments (using the same protocol) indicated that 0.1 (Fig. 6, a and b) and 0.05 mg/kg (Fig. 6, c and d) LY503430 attenuated apomorphine-induced rotations and protected against the loss of DAergic terminals in the dorsal striatum to a reduced extent (summarized in Table 1).

View larger version (37K): [in this window] [in a new window]   Fig. 6. Effects of chronic treatment with LY503430 (0.01 and 0.05 mg/kg p.o. for 10 days starting 1 day after infusion of 6-OHDA into the nigra) on rotational behavior (a and b) and tyrosine hydroxylase-immunoreactivity (c and d) in the dorsal and ventral striatum. Results indicate that only the 0.1-mg/kg dose of LY503430 provided a significant correction of apomorphine-induced rotational asymmetry (a), whereas both doses provided some protection against the loss of TH staining observed in the dorsal striatum (c and d) after unilateral infusion of 6-OHDA into the substantia nigra. Data are based on eight animals per group. **, p < 0.01 versus baseline rotations or TH, +, p < 0.05; ++, p < 0.01 versus vehicle-treated animals.

In a separate experiment, we measured levels of dopamine and its metabolites and found that the imbalances caused by nigral infusion of 6-OHDA were reduced by the above treatment with LY503430. Thus, LY503430 at 0.5 mg/kg reduced lesion-induced depletions in dopamine (dopamine levels in the lesioned hemisphere were 9424 ± 1002 in sham-operated animals and 807 ± 220 in vehicle-treated animals, whereas values in LY503430-treated animals were 2775 ± 343). Thus, oral administration of LY503430 provided functional and histological evidence of neuroprotection in a dose-dependent manner. 0

Neurotrophic Effects of LY503430 in Rodent Models of Parkinson's Disease
Effects on of LY503430 on GAP-43 Immunoreactivity in the Striatum. The large functional effect and robust protection of DAergic terminals in the striatum with only minimal protection of cell bodies in the nigra at the highest dose suggested that LY503430 was having a trophic action after infusion of 6-OHDA into the substantia nigra. While assessing TH levels in the striatum, we stained adjacent sections for BDNF and GAP-43. Results indicated that LY503430 provided a dose-dependent increase in GAP-43, but not BDNF, in the lesioned striatum (Fig. 7). We were also not able to detect any changes in BDNF in the striatum or in the hippocampal regions, but did observe an increase in the substantia nigra (Fig. 8). This increase may provide trophic actions on the cell bodies to enhance neurite outgrowth in striatal terminals.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Dose-dependent increase in GAP-43 immunoreactivity in the striatum with LY503430 (0.05, 0.1, 0.2, or 0.5 mg/kg p.o. for 10 days) starting 1 day after unilateral nigral 6-OHDA lesion in the rat. *, p < 0.05; **, p < 0.01 versus vehicle control.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Effects of chronic treatment with LY503430 (0.05 mg/kg s.c. for 14 days starting 1 day after infusion of 6-OHDA into the nigra) on BDNF immunoreactivity in the hipocampal and striatal regions of the brain (a) and the substantia nigra (b). Results indicated that there were similar numbers of BDNF-positive cells in the hippocampal and striatal regions of sham-, vehicle-, and LY503430-treated animals after 14 days treatment, but the LY503430-treated animals had significantly greater numbers of BDNF-positive cells in the substantia nigra. Data are based on eight animals per group. *, p < 0.05 versus vehicle-treated animals.

Effects of Delayed Treatment with LY503430 after Unilateral Nigral Lesion. To distinguish between a neurotrophic and a neuroprotective role, we varied the start of treatment with LY503430 after nigral infusion of 6-OHDA. The level of striatal tyrosine hydroxylase staining was similar whether administration was initiated 1, 3, 6, or 14 days after infusion of 6-OHDA; the data from three such independent experiments are illustrated in Fig. 9. Even, when LY503430 treatment was initiated 1 h before 6-OHDA, there was no improvement over administration at the later time points. Thus, treatment with LY503430 at any time point resulted in 30% TH immunoreactivity in the dorsal striatum, in comparison with 10% in vehicle-treated animals. This apparent neurotrophic effect was also not due simply to up-regulation of TH per se, because there was no effect in control animals treated with LY503430 (Fig. 9) and there were no effects of the compound acutely on turning behavior in lesioned animals. Indeed, in all 195 animals chronically treated with LY503430 there were no overt changes in their behavior.

View larger version (33K): [in this window] [in a new window]   Fig. 9. Effects of delayed treatment with LY503430 (0.5 mg/kg s.c. for 14 days) on TH-immunoreactivity in the dorsal striatum after a unilateral nigral 6-OHDA lesion in the rat. These data are based on three independent experiments and plotted together using the same axis. The data indicate that LY503430 provided similar effects on the density of TH-immunoreactivity in the dorsal striatum whether administration was initiated 1, 3, 6, or 14 days after 6-OHDA. ***, p < 0.01 versus sham control; +, p < 0.05; ++, p < 0.01 versus vehicle control. n = 8/group.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we have demonstrated that a novel, potent, selective, and systemically active AMPA receptor potentiator provided neuroprotective actions in three rodent models of Parkinson's disease. The compound also increased GAP-43 expression in the lesioned striatum and reduced the 6-OHDA-induced effects on turning behavior and on striatal DAergic innervation. LY503430 is a new class of compound with potential utility for providing neuroprotection and neuronal repair in Parkinson's disease.

LY503430 as a Selective Potentiator of AMPA Receptors. This novel biarylpropylsulfonamide selectively potentiated responses of human recombinant and rat native AMPA receptors. The results indicate that LY503430 is more selective for the flip splice variants and in particular for hGLU_A2 with an EC₅₀ value of 33 nM, whereas the EC₅₀ value was 2.25 µM on the "flop" variant. AMPA receptor currents in isolated substantia nigra, striatal, hippocampal, and prefrontal cortical neurons were also potentiated with EC₅₀ values of 100 nM to 7 µM, illustrating the heterogeneity across central neurons. In contrast, functional NMDA, GLU_K5, and GLU_K6, GLU_K6/KA2 receptor activities in hippocampal, cortical, DRG, and transfected HEK293 cells, as described previously with other AMPA potentiators (Baumbarger et al., 2001a,b; Gates et al., 2001) were not affected by concentrations of 10 to 100 µM LY503430. In addition, LY503430 showed no binding affinity at 18 other different neurotransmitter receptors (D. Calligaro, unpublished observations).

Systemic administration of LY503430 (0.01–10 µg/kg i.v.) also enhanced responses of hippocampal neurons to AMPA in a dose-dependent manner, suggesting reasonable blood-brain barrier permeation. The small increase in NMDA responses is likely to be due to enhancement of tonic AMPA receptor activity, causing depolarization and relieving the Mg²⁺ brake of NMDA receptor channels, as shown previously for other AMPA potentiators (Vandergriff et al., 2001). For comparison, the equivalent dose of CX-516 required to produce a 20% increase in AMPA-mediated responses was 1000 µg/kg i.v.

Neuroprotective Actions of LY503430. The exact mechanisms of Lewy body formation and cell death in PD remains to be elucidated, but evidence suggests that it is a combination of oxidative stress, genetic (Scott et al., 1997), environmental factors (Tanner and Langston, 1990) and malfunction of ubiquitin-proteosome systems (McNaught et al., 2001). Familial PD is associated with mutations in Parkin or -synuclein genes, but it is interesting that -synuclein-overexpressing mice have inclusion bodies, but no cell death (Masliah et al., 2000). Other recent studies have shown that the mitochondrial Complex I inhibitor rotenone can produce a slow progressive degeneration of dopaminergic neurons, with inclusion bodies in some rats (Betarbet et al., 2000). In the present study, we have used well established models with MPTP and 6-OHDA that produce cell loss and permanent dopamine depletion in vivo (Flint Beal, 2001). Our results indicated that LY503430 could protect whether we used systemic MPTP in mice, or infusion of 6-OHDA in the rat either into the cell body region, which produces a relatively rapid lesion or into the striatum, which produces a slow partial lesion of the nigra. We counted the number of intact nigral cells at one stereotaxic level and further studies using stereological methods are required to provide conclusive evidence for neuroprotection. However, in most cases, we used both rotational behavior and TH-immunoreactivity in the same animals to demonstrate the improvement in outcome, which were also confirmed by measurement of dopamine and its metabolites in a separate experiment.

Many investigators have reported that antioxidants, nitric oxide synthase inhibitors, anti-inflammatory agents, nicotine, immunophilins, and related molecules can provide protection in these models (Korczyn and Nussbaum, 2002; O'Neill and Siemers, 2002). Our in-house comparisons indicate, however, that LY503430 provides superior protection to all these other pharmacological interventions. We have not evaluated central administration of growth factors, but, for example, GDNF is effective in both nigral and striatal 6-OHDA lesion models (Altar et al., 1992, 1994; Rosenbald et al., 2000). In addition, in the current studies we observed an increased level of BDNF immunoreactivity in the substantia nigra after LY503430 treatment in the 6-OHDA-lesioned animals.

Neurotrophic Actions of LY503430. The finding that, after LY503430 treatment, there was a greater increase in tyrosine hydroxylase staining in the terminal region relative to the cell body region in the nigro-striatal pathway, led us to propose a neurotrophic mode of action. Indeed, in this protocol we found an up-regulation of the neurotrophic agent GAP-43 in the lesioned animals treated with LY503430. It is well established that GAP-43 is implicated in formation of new synapses and neurite outgrowth (Benowitz and Routtenberg, 1997; Namgung and Routtenberg, 2000). For example, transgenic animals overexpressing GAP-43 exhibit increased sprouting and repair, and GAP-43 is up-regulated in the penumbra after ischemic brain lesions.

The improvement in both functional and histological outcomes, even when treatment was delayed until after the cell death process, provides direct evidence of a neurotrophic effect. Because LY503430 has little effect on the number of surviving nigral neurons but does increase the tyrosine hydroxylase staining in the striatum, the improvement is largely confined to the terminal regions, which is consistent with enhanced sprouting. The parallel improvement in the behavioral measure suggests that this increase in tyrosine hydroxylase staining represents functional dopaminergic synapse formation. Furthermore, because the degree of improvement is similar whether the treatment is started immediately before, during, or after the cell death process, this neurotrophic effect seems to be the dominant feature of the drug's action. In support of this, treatment with LY503430 from days 1 to 5 postlesioning was ineffective, suggesting that whatever the mechanism of action it takes several days to activate the effect. This is consistent with the downstream activation by AMPA receptors of one or more signaling pathways (e.g., Lyn kinase and mitogen-activated protein kinase). Thus, we hypothesize that in this way LY503430 has trophic actions via the remaining nigral cell bodies and/or their striatal terminals to enhance DAergic sprouting.

Mode of Action of LY503430. The exact mechanism of neuroprotection is not clear, but one mechanism may be an up-regulation of trophic factors, in particular BDNF. In 1999, Hayashi et al. (1999) reported that AMPA receptor potentiators can increase BDNF via the tyrosine kinase Lyn, which is physically associated with the AMPA receptor. Further studies have demonstrated that AMPA potentiators can increase BDNF expression in both cerebellar granule and hippocampal neurons (Legutko et al., 2001) and in astrocytic cultures (V. Lakics, M. J. O'Neil, unpublished data) in vitro. In addition, subchronic treatment with LY404187 was reported to increase tyrosine kinase B and BDNF mRNA in the rat hippocampus in vivo (Mackowiak et al., 2002). We did not observe any increases in BDNF using immunocytochemical methods in striatal slices at the end of the current efficacy studies. The lack of changes in the striatum may be because 1) the BDNF levels are too low here, 2) immunocytochemistry is not of sufficient sensitivity, 3) the 12- to 14-day endpoints are too late and we missed the effect, 4) the BDNF is increased in other brain areas that can alter sprouting in the straitum. In agreement with this, we did observe an increase in BDNF levels in the substantia nigra in one study after chronic 0.5 mg/kg LY503430 treatment, suggesting it may play some role. It is also clear that both BDNF and GDNF can protect dopaminergic neurons in vitro and in vivo (Rosenbald et al., 2000), and BDNF plays a key role in synaptic plasticity (Kovalchuk et al., 2002; Messaoudi et al., 2002). We also observed a dose-dependent increase in GAP-43 in both the intact and lesioned striatum. The magnitude of the increase was larger in the lesioned striatum, suggesting that a combination of lesion and AMPA receptor potentiator is required to robustly increase GAP-43. Manipulation of GAP-43 has profound effects on neurite outgrowth in cell culture systems and agents that increase GAP-43 in rat cerebral cortex accelerate functional recovery in vivo. It seems likely that LY503430 signaling through the AMPA receptor can increase trophic factors (such as BDNF) and enhance sprouting of striatal terminals (in this case, increased GAP-43).

Concluding Remarks. In conclusion, we have demonstrated that LY503430 is a potent potentiator of recombinant human and rat native AMPA receptors in vitro and can also increase AMPA-evoked responses in vivo. The present results provide the first evidence that AMPA receptor potentiators can provide functional and histological protection in rodent models of PD. In addition, these effects were maintained after delayed administration and accompanied by an increase in GAP-43 expression in the striatum provided tantalizing evidence for neurotrophic action. AMPA potentiators have been shown to be effective in animal models of depression (Li et al., 2001; Skolnick et al., 2001), rodent models of cognition (Staubli et al., 1994; Hampson et al., 1998a,b; Quirk and Nisenbaum, 2002), and in some memory tests in aged humans (Lynch et al., 1997) and hence may be of additional benefit in PD patients by treating these concurrent symptoms. These results suggest that LY503430 or another related AMPA potentiator would be a suitable molecule to advance as a clinical candidate to reduce or halt and potentially reverse the degeneration observed in human Parkinson's disease.

	Acknowledgements

 
We thank David Caligaro for valuable help profiling LY503430 across neurotransmitter receptor subtypes, Sandra Woodhouse for help with dopamine and metabolite measurements, and Roger Moore for statistical assistance.

	Footnotes

 
DOI: 10.1124/jpet.103.049445.

ABBREVIATIONS: PD, Parkinson's disease; SN, substantia nigra; GDNF, glial derived growth factor; BDNF, brain derived neurotrophic factor; AMPA, -amino-3-hydroxy-5-methylisoxazole-4-propionic acid; HEK, human embryonic kidney; 6-OHDA, 6-hydroxydopamine; NMDA, N-methyl-D-aspartate; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; GAP-43, growth associated protein-43; CX-516, 1-(quinoxalin-6-ylcarbonyl)piperidine; TH, tyrosine hydroxylase; DRG, dorsal root ganglion; TH-IR, tyrosine hydroxylase-immunoreactivity; DA, dopamine; IDRA-21, 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiacine-S,S-dioxide; CX-546, 1-(1,4-benzodioxan-6-ylcarbonyl)piperdine; LY503430, (R)-4'-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide; LY392098, R,S-N-2-(4-(3-thienyl)phenyl)propyl-2-propanesulfonamide; LY404187, R,S-N-2-(4-(4-cyanophenyl)phenyl)propyl-2-propanesulfonamide.

¹ Current address: Developmental Biology Unit, Institute of Child Health, University of London, London, WC1N 1EH, UK.

² Current address: Department of Biology and Biochemistry, University of Bath, University of Bath, Bath BA2 7AY, UK.

³ Current address: ReNeuron, Guildford, 10 Nugent Rd., Surrey Research Park, Guildford, Surrey, GU2 7AF, UK.

⁴ Current address: DOV Pharmaceuticals, Inc., 433 Hackensack Ave., Hackensack NJ 07601.

Address correspondence to: Dr. Michael J. O'Neill, Eli Lilly & Co. Ltd., Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK. E-mail: oneill_michael_j{at}lilly.com

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