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* Toronto Western Research Institute, Toronto, Ontario, Canada; and
Phytopharm Plc, Godmanchester, Cambridgeshire, UK
1Correspondence: Toronto Western Research Institute, MCL 11–419, Toronto, Ontario, M5T 2S8, Canada. E-mail: brotchie{at}uhnres.utoronto.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: BDNF • GDNF • neurorestorative • striatum • Cogane
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Parkinson’s disease is characterized by a progressive and selective loss of nigral dopaminergic neurons, resulting in a pronounced depletion of striatal dopamine. The loss of striatal dopamine and the consequent dysfunction of the nigrostriatal pathway lead to a range of motor symptoms, including resting tremor, rigidity, bradykinesia, and loss of postural reflex (2
, 3)
. As the disease progresses, many patients also develop some cognitive dysfunction, including anxiety, depression, and dementia. In contrast to other neurodegenerative disorders, there are relatively good symptomatic therapies available for the treatment of Parkinson’s disease. These therapies consist predominantly of dopamine replacement and adjuvant surgical therapy to relieve most of the motor symptoms of the disease (2
, 3)
. However, these symptomatic therapies are not themselves without problem; for example, long-term treatment with the dopamine precursor 3,4-dihydroxy-L-phenylalanine (L-DOPA) often leads to the development of debilitating dyskinesias (4)
. Furthermore, at the present time, there is no disease-modifying neuroprotective or neurorestorative therapy available.
There is a strong scientific rationale for developing disease-modifying therapies for Parkinson’s disease that either induce or enhance the signaling of neurotrophic factors, including glial-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). Both GDNF and BDNF and their receptors are expressed both in the region of the cell bodies of dopaminergic neurons in the substantia nigra (5
, 6)
and in the regions targeted by those neurons, including the striatum (7)
. Furthermore, in both rodent and primate models of Parkinson’s disease, intrastriatal or intraventricular injection of GDNF and BDNF in the striatum protects against biochemical, immunocytochemical, and behavioral changes associated with nigrostriatal dopaminergic damage and promotes dopamine neuron survival (8
9
10
11
12
13
14
15
16)
. However, the translation of these findings to the clinic has proved difficult (for reviews see refs. 17
, 18
). Indeed, in a double-blind clinical trial, intraventricular administration of GDNF was found not to be efficacious in reducing the symptoms of Parkinson’s disease (19)
. Although clinical studies (20
, 21)
of intrastriatal delivery of GDNF that were open label and non-placebo controlled showed promise of efficacy, a recent double-blind placebo-controlled study (22)
was unable to demonstrate significant actions of GDNF.
A major difficulty in the development of neurotrophic factor based therapies is the lack of oral bioavailability and subsequent poor delivery to the required target resulting from their large polypeptide structure (15)
. Several approaches to solve this problem have been investigated, ranging from direct infusion of the protein into the brain (20
, 21)
, to viral-vector mediated delivery (23)
, to implantation of cells capable of synthesizing GDNF (24
, 25)
. However, all of these approaches require intracerebral or intracerebroventricular surgical interventions that might not be applicable, desirable, or accessible to a large proportion of the relevant patient population. Alternatively, nonpeptide compounds that are able to readily cross the blood-brain barrier and induce the expression of one or more neurotrophic factors after oral administration represent a promising opportunity for the treatment for Parkinson’s disease.
We present data demonstrating that PYM50028 (Cogane; 5β,20
,25R-spirostan-3β-ol; common name smilagenin; Fig. 1
), an orally active, nonpeptide neurotrophic factor inducer, is able to restore deficits in the dopaminergic system after both 1-methyl-4-phenylpyridinium (MPP+) -induced neuronal damage in mesencephalic neurons in vitro and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) -lesioned mice in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Primary culture of mesencephalic neurons
Rat mesencephalic neurons were cultured as described previously (26
, 27)
. Briefly, the midbrains obtained from 15-day-old rat embryos (Janvier, Le Genest St. Isle, France) were dissected under a microscope. The ventral portion of the mesencephalic flexure, a region of the developing brain rich in dopaminergic neurons, was used for the cell preparations. The midbrain was dissociated by trypsinization for 30 min at 37°C (trypsin: 0.05%; Life Technologies, St. Christophe, France) in the presence of DNase I (0.1 mg/ml; Roche, Meylan, France). The reaction was terminated by addition of Hank’s buffered salt solution containing calcium, magnesium (Life Technologies), and FBS (10%; Life Technologies). The suspension was triturated and centrifuged at room temperature (50 g for 10 min). The pellet of the dissociated cells was resuspended in a medium consisting of Dulbecco’s modified Eagle’s medium (Life Technologies) and F-12 medium (1:1, v/v; Life Technologies) containing B27 (2%, Life Technologies) supplemented with glucose (6 mg/ml; Sigma, Lyon, France) and L-glutamine (1%, Life Technologies). The cells were plated in 60 mm diameter Petri dishes (2x106 cells/dish) previously coated with poly-L-lysine (Sigma) and maintained in a humidified incubator at 37°C in 5% CO2/95% air atmosphere. Near pure neuronal cultures (<0.5% glia) that contained tyrosine hydroxylase (TH) -positive cells (
1–5% of the total number of cells in these cultures) were achieved by maintaining cells in serum-free Neurobasal medium (Life Technologies) supplemented with B27, L-glutamine (1% Life Technologies), BDNF (0.37 nM), and GDNF (0.034 nM).
Prevention of MPP+-induced neuronal loss
To assess the ability of PYM50028 to prevent MPP+-induced neuronal loss in vitro, on day 5 the cultures were washed and placed in fresh medium containing PYM50028 (30 nM), a combination of BDNF (1.85 nM) and GDNF (0.17 nM), or vehicle (DMSO, 0.25%) for 24 h. On day 6, MPP+ (2 µM) was added to the cultures in the presence of PYM50028 (30 nM), a combination of BDNF (1.85 nM) and GDNF (0.17 nM), or vehicle (DMSO, 0.25%) for a further 48 h.
Reversal of MPP+-induced neuronal atrophy and neuronal loss
To assess the ability of PYM50028 to reverse MPP+-induced neuronal atrophy and neuronal loss, on day 5 MPP+ (2 µM) was added to the culture for 24 h. On day 6, the cultures were washed and placed in fresh medium, without MPP+, and containing PYM50028 (0.3 fM to 30 nM), the combination of BDNF (1.85 nM) and GDNF (0.17 nM), or vehicle (DMSO 0.25%) for a further 48 h. Data from three independent studies were normalized to vehicle control, and the EC50 was estimated using nonlinear regression (GraphPad Prism 5.0; GraphPad, San Diego, CA, USA)
Measurement of neuronal atrophy and neuronal loss
Briefly, the cultures were fixed with paraformaldehyde (PFA) in PBS (4%, Sigma). After fixation, the neurons were permeabilized with Triton X-100 (0.05%, Sigma). Neurons were then incubated with anti-TH antibody (Sigma, 1:10,000) at 37°C for 2 h, then washed with PBS, and incubated with goat anti-mouse/Cy3 (Interchim, Montlucon, France) for 2 h at 37°C. The neurons were mounted and examined with fluorescence microscopy. For each condition, 40–230 images were captured with a camera (Coolpix 995; Nikon, Melville, NY, USA) fixed on a microscope (Nikon, objective x40). The number of TH neurons per field and the percentages of TH-positive cells displaying neurites were made using Image-Pro Plus (Biocom, Les Ulis, France). Analysis was conducted by a technician masked to the condition being analyzed.
In vivo experiment: MPTP-lesioned mice, a model of Parkinson’s disease
Animal husbandry
Five-week-old male C57/bl/6 mice (Charles River, Saint-Constant, QC, Canada) were housed 2–3/cage in a limited-access rodent facility. Animal rooms were maintained at 21 ± 2°C, with lights on from 7 AM to 7 PM. Drinking water and food were supplied ad libitum to each cage. The studies were conducted according to guidelines set by the Canadian Council on Animal Care. A 2-wk period of acclimatization was allowed between delivery of mice and commencement of treatments.
Groups and procedure
Mice were divided into three groups (n=6–8/group). All animals received daily injections of either vehicle (sterile saline, i.p.; group 1) or MPTP (25 mg/kg/day, i.p.; groups 2 and 3) for 5 consecutive days (days 1–5). From day 2 of MPTP treatment onwards, all animals were treated with either PYM50028 (group 3) or vehicle [hydroxylpropylmethylcellulose (HPMC) 0.5% (w/v)/Tween-80 0.2% (v/v); groups 1 and 2] once daily until the end of the study (day 60). On day 60, blood samples were taken by cardiac puncture while the animals were under isofluorane anesthesia for analysis of drug exposure levels. The animals were then killed, and the brains were removed for postmortem analysis of striatal dopamine transporter (DAT) levels and nigral TH-positive cell numbers.
Three additional groups of animals were prepared for quantification of striatal levels of GDNF and BDNF after 60 days of PYM50028 treatment. Animals were administered either vehicle (sterile saline; group 4) or MPTP (25 mg/kg/day, i.p.; groups 5 and 6) once daily for 5 days. In addition, animals were treated once daily with either vehicle [HPMC 0.5% (w/v)/Tween-80 0.2% (v/v), p.o.; groups 4 and 5] or PYM50028 (10 mg/kg, p.o.; group 6) for 60 days.
Two groups of animals (n=9/group) were prepared to demonstrate the level of lesion at day 2, i.e., immediately before PYM50028 treatment initiation. Animals were administered either vehicle (sterile saline; group 7) or MPTP (25 mg/kg/day, i.p.; group 8) once daily. On day 2, animals were killed by cervical dislocation, and the level of DAT binding was assessed.
Two groups of animals (n=9/group) were prepared to demonstrate that PYM50028 did not alter brain levels of MPP+. Animals were administered MPTP (25 mg/kg/day, i.p.) once daily for 5 days. From day 2 of MPTP treatment onwards, all animals were treated with either vehicle [HPMC 0.5% (w/v)/Tween-80 0.2% (v/v); group 9] or PYM50028 (10 mg/kg, p.o.; group 10) once daily. On day 5, 1.5 h after MPTP administration, animals were killed by cervical dislocation, and the brains were removed and prepared for assessment of MPP+ levels.
Tissue preparation
Brains were removed as rapidly as possible and cut in the coronal plane at the level of the cerebral peduncles. The rostral portion of the brain, including the striatum, was immediately frozen in isopentane chilled to –42°C. Cryostat-cut sections (20 µm) containing the striatum were prepared from this fresh-frozen tissue. The caudal portion of the brain, including the mesencephalon, was cut into slices (2 mm) and immersed in PFA (4%). These slices were then cryoprotected by immersion in PFA (4%) in sucrose (10% for 1 day and 30% for 3 days). Cryostat-cut sections (20 µm) containing the substantia nigra were prepared from this postfixed tissue.
Ex vivo measurements
DAT binding: autoradiography
The levels of striatal DAT binding were assessed by [125I]-RTI-121 binding autoradiography in sections prepared from fresh-frozen tissue. Thawed slides were placed in binding buffer (2x15 min, room temperature) containing Tris (50 mM), sodium chloride (NaCl; 120 mM), and potassium chloride (KCl; 5 mM). Sections were then placed in the same buffer containing [125I]-RTI-121 (50 pM, specific activity 2200 Ci/µmol; Perkin-Elmer, Wellesley, MA, USA,) for 120 min at 25°C to determine total binding. Nonspecific binding was defined as that observed in the presence of GBR 12909 (a selective dopamine uptake inhibitor, 100 µM; Tocris Bioscience, Ellisville, MO, USA). All slides were washed (4x15 min) in ice-cold binding buffer, rinsed in ice-cold distilled water, and air dried. Together with [125I]-microscale standards (Amersham, Piscataway, NJ, USA), slides were then apposed to autoradiographic film (Kodak, Rochester, NY, USA) and left for 2 days at 4°C before developing.
Autoradiograms were analyzed using MCID software (Image Research, St. Catherines, ON, Canada). Densiometric analysis of three striata from each animal was carried out by an examiner masked to the treatment given, whereby a reference curve of counts per minute vs. optical density was calculated from 
-emitting [125I] microscale standards and used to quantify the intensity of signal as nanocuries per gram. Background intensity was subtracted from each reading. Data were then expressed as mean ± SE of the signal intensity for each treatment group. Nonspecific binding was calculated in the same way and subtracted from the total binding to give specific binding. Nonspecific binding was found to account for <1% of total binding.
Dopamine neurons: immunohistochemistry
Sections were stained for TH immunoreactivity at the level at which the division of the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) by the medial terminal nucleus of the accessory optic tract clearly distinguishes the cells of the SNc from those of the VTA. Sections were fixed in 4% PFA (15 min) and rehydrated, and endogenous peroxidase activity was quenched with 0.3% H2O2 in distilled water. Slides were then exposed to pepsin (0.2 g Sigma-p-7000 pepsin in 50 ml 0.01 M HCl) for 30 min, and nonspecific binding was blocked with 1.5% normal goat serum (Vectastain rabbit IgG ABC kit; Vector Laboratories, Burlington, ON, Canada). Slides were incubated with polyclonal anti-TH antibody (AB152; Chemicon, Temecula, CA, USA) in PBS for 18 h at room temperature. After overnight incubation, sections were incubated with biotinylated secondary antibody (Vectastain rabbit IgG ABC kit). The horseradish peroxidase (HRP) conjugate (Vectastain rabbit IgG ABC kit) was applied, followed by Chromagen solution (3,3'-diaminobenzidine; SK-4100, Vector). Finally, slides were rinsed, dehydrated, and coverslipped using distyrene plasticizer xylene mountant. Viable TH-positive neurons, defined as those displaying a definite halo, were quantified by the counting of cells in three consecutive sections from each animal by an examiner masked to the treatment.
Assessment of brain MPP+ levels
After dissection, striata were weighed, frozen on dry ice, and stored at –80°C. Samples were assessed for striatal levels of MPP+ using a method adapted from Jackson-Lewis and Przedborski (28)
. At the time of analysis, tissue samples were sonicated on ice in 600 µl of 5% trichloroacetic acid in HPLC-grade water containing 20 µg/ml of 4-phenylpyridine as the internal standard. After centrifugation at 15,000 g for 20 min at 4°C, supernatants were passed through 0.22 µm sterile filters. With the use of a refrigerated autosampler (Varian, Mississauga, ON, Canada), 20 µl of sample was injected onto a C18 reverse phase column (Beckman Ultrasphere; Beckman Instruments, Fullerton, CA, USA). The mobile phase consisted of 90% 50 mM potassium phosphate (adjusted to pH 3.2 with 18 M phosphoric acid) and 10% acetonitrile. The flow rate was 1.5 ml/min. Ultraviolet detector (Varian Prostar) wavelength was set at 295 nm for detection of MPP+. Data were collected and analyzed with reference to a 4-point external calibration curve and processed using chromatography software (Star; Varian). Data were expressed as micrograms of MPP+ per gram of striatal tissue.
GDNF and BDNF quantification by ELISA
The striatum of each animal was dissected and homogenized in 300 µl lysis buffer at 4°C containing 137 mM NaCl, 20 mM Tris, 1% Nonidet P-40, 10% glycerol, 1 mM phenylmethylsulfonylflouride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.5 mM sodium orthovanadate and then centrifuged at 10,000 g for 10 min at 4°C. The supernatants were removed for ELISA analysis, and the protein pellets were retained for protein quantification by the Lowry method. The levels of BDNF and GDNF were determined using commercially available kits (Promega, Madison, WI, USA). Briefly, 96-well plates were coated with either anti-BDNF or anti-GDNF monoclonal antibody (Promega) overnight. Plates were then washed and nonspecific binding was blocked by incubation with block and sample buffer (Promega) for 1 h at room temperature. Plates were then washed, and 100 µl sample supernatants (diluted 1:1 in block and sample buffer) were added. Serial dilutions (0–1000 pg/ml) of both BDNF and GDNF protein standards were carried out in duplicate to produce a standard curve. Plates were then sealed and incubated for either 2 h (BDNF) or 6 h (GDNF) at room temperature. Plates were then washed, polyclonal anti-human BDNF or GDNF in block and sample buffer was added, and plates were incubated for either 2 h at room temperature (BDNF) or overnight at 4°C (GDNF). After incubation, plates were washed and then incubated with anti-IGY HRP-conjugated secondary antibody in block and sample buffer for 1 h at room temperature. Plates were then washed, and TMB One solution (Promega) was added to each well for 10 min. The color change reaction was then terminated by addition of 1 N hydrochloric acid, and absorbance was measured at 450 nm using a plate reader. Data were expressed as picograms per milligram of protein.
Monoamine oxidase A and B assays
Human recombinant monoamine oxidase A and B (MAO-A and MAO-B) from baculovirus-infected insect cells (Gentest 456284; Hi5 cells; BD Biosciences, San Jose, CA, USA) were used. PYM50028 (10, 100, and 1000 nM), vehicle (DMSO, 1%), or R(–)-deprenyl was preincubated with either MAO-A (4.2 µg/ml) or MAO-B (12.5 µg/ml) in KH2PO4 buffer, pH 7.4, at 37°C for 15 min. For both enzymes, the conversion of kynuramine to 4-hydroxyquinoline was assessed. For both assays, the reaction was initiated by the addition of kynuramine (50 µM) and left to run for 60 min. The reaction was then terminated by the addition of NaOH (6 N). The amount of 4-hydroxyquinoline produced was quantified spectrofluorometrically.
Plasma preparation for PYM50028 level analysis
Blood samples (0.8 ml each) were collected 2–4 h after the final administration of PYM50028 by cardiac puncture under isofluorane anesthesia, and the sampling time was recorded (n=8). The blood was transferred into uniquely labeled polypropylene tubes containing EDTA anticoagulant and centrifuged at 4°C for 5 min at 3000 rpm. The supernatant was collected and stored at –80°C. The concentration of PYM50028 in the plasma samples was measured using a liquid chromatographic tandem mass spectrometric (LC-MS/MS) bioanalytical method.
Statistical analysis
All data are expressed as mean ± SE. For the in vitro data, 1–4 cultures were prepared, and 40–90 fields were counted in each culture for each condition. A global analysis of the data was performed using a 1-way ANOVA, followed by Fisher’s paired least significant difference post hoc test. Analysis was performed using StatView version 5.00 for Windows (Adept Scientific, Acton, OH, USA). The in vivo data were analyzed by 1-way ANOVA, followed by Tukey’s post hoc multiple comparison test. Analysis was performed using GraphPad Prism version 4.00 for Windows. Data not falling within 90% confidence limits of the mean were excluded from the statistical analyses. In all cases, significance was accepted as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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45% reduction compared to control (Fig. 2
A). Twenty-four hour pretreatment with PYM50028 (30 nM) significantly prevented the MPP+-induced neuronal damage, with levels remaining at 6.03 ± 0.21 cells/field, equivalent to 96% of control (Fig. 2A
). Similarly, 24 h pretreatment with a combination of BDNF and GDNF (1.85 and 0.17 nM, respectively) significantly prevented the MPP+-induced loss of dopaminergic neurons per field, with levels remaining at 5.98 ± 0.32 cells/field, equivalent to –95% of control (Fig. 2A
).
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PYM50028 reverses MPP+-induced neuronal atrophy in freshly isolated mesencephalic neurons
Under control conditions, 41 ± 3.7% of mesencephalic neurons were found to express neurites. After exposure to MPP+ (2 µM) for 24 h, the number of mesencephalic neurons expressing neurites was significantly reduced to 27 ± 4.5%, representing a reduction of 33% compared with control (Fig. 3
A). Incubation with PYM50028 (0.03 and 30 nM) for 48 h after a 24 h exposure to MPP+ significantly reversed the MPP+-induced decrease in the number of mesencephalic neurons expressing neurites, with levels restored to 41 ± 4.1 and 43 ± 3.9%, respectively, representing 101 and 107% of control (Fig. 3A
). Treatment with a combination of BDNF and GDNF (1.85 and 0.17 nM, respectively) did not significantly reverse the MPP+-induced reduction in the number of mesencephalic neurons expressing neurites, with levels remaining at 25 ± 3.7%, 62% of control (Fig. 3A
).
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PYM50028 reverses MPP+-induced neuronal loss in freshly isolated mesencephalic neurons
Exposure to MPP+ (2 µM) for 24 h significantly decreased the number of mesencephalic neurons per field to 73.9 ± 2.0% of control (Fig. 3B
). Incubation with PYM50028 (0.3 fM to 30 nM) for 48 h after a 24 h exposure to MPP+ significantly reversed the MPP+-induced loss of dopaminergic neurons (Fig. 3B
). The EC50 of PYM50028 in preventing MPP+-induced neuronal damage was 13.4 fM. Similarly, incubation with a combination of BDNF and GDNF (1.85 and 0.17 nM, respectively) significantly reversed the MPP+-induced loss of dopaminergic neurons, with levels remaining at 126.6 ± 3.1% of control (Fig. 3B
).
In vivo experiments
PYM50028 attenuates the loss of striatal DAT binding in MPTP-lesioned mice
After 2 days treatment, the level of specific striatal DAT binding was 24 ± 2.4 nCi/g in vehicle-treated animals and 13.4 ± 1.4 nCi/g in MPTP-treated animals, representing an 45% reduction (group 7 vs. group 8, P<0.01). Thus, at this time point, representative of the point of initiation of treatment with PYM50028, a significant deficit in striatal DAT binding was evident (data not shown). On day 60, in vehicle-treated MPTP-lesioned mice, specific striatal DAT binding was significantly decreased by 68%, as compared with saline-treated control mice (Fig. 4
A; group 2 vs. group 1, P<0.01). In MPTP-treated animals receiving daily oral administration of PYM50028 (initiated on day 2 and continued until day 60; group 3), striatal DAT binding was significantly higher than that of vehicle-treated MPTP-lesioned animals (group 2, P<0.01; Fig. 4
). Indeed, after treatment with PYM50028 in MPTP-lesioned mice (group 3), DAT binding was restored to 94% of, and not significantly different from, that in non-MPTP-lesioned control animals (group 1). The specific [125I]-RTI-121 binding in the striatum was found to represent >99% of the total binding. Sample autoradiograms illustrating both specific and nonspecific striatal DAT binding representative of each treatment group after 60 days treatment are shown in Fig. 4B-D
.
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PYM50028 attenuates the loss of nigral TH-positive cells in MPTP-lesioned mice
On day 60, a significant decrease (42%) in the number of nigral dopaminergic neurons (TH-positive neurons) was apparent in vehicle-treated, MPTP-lesioned mice (group 2) compared with non-MPTP-lesioned mice (group 1, P<0.01; Fig. 5
A). In MPTP-treated animals receiving daily oral administration of PYM50028 (initiated on day 2 and continuing until day 60; group 3), the number of nigral dopaminergic neurons was significantly higher than in MPTP-lesioned animals (group 2, P<0.01; Fig. 5A
). Indeed, in MPTP-treated mice receiving PYM50028 (group 3), the number of nigral dopaminergic neurons was 89% of, and not significantly different from, that in non-MPTP-lesioned control animals (group 1). Representative sections illustrating TH-positive cells at the level of the substantia nigra in each treatment group are shown in Fig. 5B-D
.
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PYM50028 increases striatal levels of both GDNF and BDNF in MPTP-lesioned mice
There was no significant difference in striatal levels of GDNF between normal and MPTP-lesioned animals treated with vehicle (group 4 vs. group 5; Fig. 6
A). However, after 60 days of treatment with PYM50028 (10 mg/kg, p.o.; group 6), striatal GDNF levels were significantly increased by 297 ± 80 and 372 ± 96%, as compared with non-MPTP-lesioned control mice (group 4) and vehicle-treated, MPTP-lesioned mice (group 5, P<0.001 and P<0.01, respectively). Similarly, there was no significant difference in striatal levels of BDNF between normal and MPTP-lesioned animals treated with vehicle (Fig. 6B
). However, after 60 days treatment with PYM50028 (10 mg/kg, p.o.), striatal BDNF levels were significantly increased by 511 ± 122 and 164 ± 53%, as compared with non-MPTP-lesioned mice and MPTP-lesioned mice, respectively (both P<0.01).
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PYM50028 does not inhibit MOA-A and MOA-B
PYM50028 (10, 100, and 1000 nM) did not significantly alter the activity of either MAO-B or MAO-A. The activity of MAO-B in the presence of PYM50028 (10, 100, and 1000 nM) was 96, 97, and 100% of control (vehicle DMSO, 1%), respectively. Similarly, the activity of MAO-A in the presence of PYM50028 (10, 100, and 1000 nM) was 99, 98, and 97% of control (vehicle DMSO, 1%), respectively. R(–)-deprenyl (selegiline), an MAO-B inhibitor, and clogyline, a MAO-A inhibitor, were used as reference controls and inhibited MAO-B and MAO-A with an IC50 of 5.26 and 0.288 nM, respectively.
PYM50028 does not alter brain levels of MPP+ after MPTP administration in vivo
After 5 days of MPTP treatment, the brain concentration of MPP+, as detected by HPLC, was not significantly different in mice treated with vehicle or PYM50028 (10 mg/kg, p.o.; 5.4±1.7 µg/g tissue compared with 5.2±0.4 µg/g tissue, respectively, P>0.05, t test).
Plasma PYM50028 levels
The plasma concentration of PYM50028, as detected by LC-MS/MS, on day 60, in PYM50028-treated, MPTP-treated mice (group 3) 2–4 h after last oral administration was 482.9 ± 36 ng/ml.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies in vitro have shown that both BDNF and GDNF can protect dopaminergic neurons against a range of insults (including MPP+ and 6-hydroxydopamine), increase markers of dopaminergic function, and increase neuritic complexity (29
30
31
32)
. In the current study, in mesencephalic cultures, the combination of BDNF and GDNF produced similar actions, preventing loss of dopaminergic cells, if given with MPP+, and restoring the same parameters if given after MPP+. The concentrations of GDNF and BDNF used here, 0.17 and 1.85 nM, respectively, are equivalent to 5 and 50 ng/ml; these are broadly equivalent to concentrations used to demonstrate the positive effects of these agents in similar cellular systems (1–50 ng/ml for GDNF and 25–50 ng/ml for BDNF; refs. 27
, 29
, 30
, 32
33
34
). Other concentrations of GDNF and BDNF (3 and 30 ng/ml and 4 and 40 ng/ml) were also tested in this system, although the effects of these concentrations were less robust and more variable in our hands. Here we show for the first time that these effects of GDNF and BDNF can be mimicked by PYM50028. Furthermore, PYM50028 was extremely potent in providing these actions, with an EC50 of 13.4 fM. The conditions used to maintain the mesencephalic cultures preferentially promote the survival of neurons, and the cultures can be considered "near-pure" neuronal cultures. TH-positive cells comprised 5% of the total number of cells in these cultures in line with previous reports (27)
. The mixed nature of the neuronal population of the cultures precludes any definitive conclusion to be drawn with respect to whether the effects of PYM50028 result from a direct action on those cells or via an indirect interaction involving one or more other cells types. Further studies will be required to define in detail the mechanisms of the action of PYM50028 in exerting its actions.
Both GDNF (8
, 15
, 35
36
37)
and, to a lesser extent, BDNF (38
39
40)
have also been shown to protect against or restore dopaminergic loss in preclinical models of Parkinson’s disease. Although the clinical significance of the potential neuroprotective and neurorestorative effects of both BDNF and GDNF is undisputed, their large polypeptide structure and consequent poor oral bioavailability represents a major challenge to be overcome in their clinical application. Many studies have attempted to solve this issue using a variety of delivery methods, including the transplantation of immortalized neural progenitor cells (39
, 40)
, intrastriatal grafting of genetically modified fibroblasts (41)
, direct intrathecal administration of the protein (38)
, and the use of lentiviral vectors (36
, 37)
. However, to date, the translation of these findings to the clinic has proven difficult (reviewed in refs. 17
, 18
). Although there are currently several promising delivery systems in development that may reduce a number of the problems associated with these invasive delivery techniques, a further limitation may emerge as the complex trophic requirements of neurons potentially limit the efficacy achieved by a single factor. Furthermore, the amount of neurotrophic factor required and the duration of the treatment to achieve clinical benefits are not yet fully understood.
PYM50028 provides an alternative, clinically applicable, approach to enhance neurotrophic factor function in Parkinson’s disease. Our study demonstrates that PYM50028 mimics the effects of a combination of GDNF and BDNF in vitro. In addition, it shows that oral administration of PYM50028 is able to elevate the level of GDNF and BDNF in the striatum of MPTP-lesioned mice and provide encouraging evidence to support an action to restore loss of markers of both dopaminergic cell body and terminal function, i.e., nigral TH cells and striatal DAT. It should be noted that these studies did not use full stereological analysis of TH cell number nor did they assess dopamine levels. Thus, although we can suggest that the actions of PYM50028 are not confined to one pole of the dopaminergic neuron, further studies will be required to define the full extent of the protection and restoration afforded by PYM50028.
The mechanism by which PYM50028 might elevate GDNF and BDNF levels is not fully understood. However, PYM50028 did not bind to any of the 168 targets assessed using SpectrumScreen (MDS Pharma Services, Taipei, Taiwan; data not shown). In the absence of any identified binding site, the mechanism of action that leads to elevation of GDNF and BDNF remains to be determined.
Importantly, and in contrast to several studies in which single neurotrophic factors have been delivered to a single brain site, our study shows that oral administration of PYM50028 attenuates damage to both the cell body and terminal regions of dopaminergic neurons of the nigrostriatal pathway. Thus, PYM50028 administration reduces both the loss of striatal DAT binding and nigral TH-positive neurons observed in MPTP-lesioned mice. Furthermore, the demonstration of an effect, despite a delayed initiation of PYM50028 therapy (2 days after commencement of MPTP administration, when a significant loss of striatal DAT was already present), may indicate that PYM50028 is able to reverse the early neuronal damage observed in this model. Additional studies will be required to confirm the restorative effect of a further delayed treatment initiation.
In conclusion, the present study demonstrates that PYM50028 has both neuroprotective and neurorestorative effects in vitro and in vivo. Thus, PYM50028 may have the potential not only to delay the progression of Parkinson’s disease but also to reverse existing neurodegenerative damage. In concert, these data suggest that PYM50028 is a promising candidate for the treatment of Parkinson’s disease.
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ACKNOWLEDGMENTS |
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Received for publication October 26, 2007. Accepted for publication February 21, 2008.
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