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NEUROPHARMACOLOGY

Dopamine Transporter (DAT) Inhibitors Alleviate Specific Parkinsonian Deficits in Monkeys: Association with DAT Occupancy in Vivo

Department of Psychiatry, Harvard Medical School and New England Primate Research Center, Southborough, Massachusetts (B.K.M., M.A.F., M.G., Z.L., J.B., C.G.); Organix, Inc., Woburn, Massachusetts (P.C.M.); and Department of Nuclear Medicine, Massachusetts General Hospital, Boston, Massachusetts (D.R.E., E.L., A.A.B., A.J.F.)

Received March 27, 2006; accepted August 1, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Viable dopamine neurons in Parkinson's disease express the dopamine transporter (DAT) and release dopamine (DA). We postulated that potent DAT inhibitors, with low affinity for the serotonin transporter (SERT), may elevate endogenously released extracellular dopamine levels to provide therapeutic benefit. The therapeutic potential of eight DAT inhibitors was investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated cynomolgus monkeys (Macaca fascicularis), with efficacy correlated with DAT occupancy as determined by positron emission tomography imaging in striatum. Four potent DAT inhibitors, with relatively high norepinephrine transporter, but low SERT affinities, that occupied the DAT improved activity in parkinsonian monkeys, whereas three high-affinity DAT inhibitors with low DAT occupancy did not. 2-Carbomethoxy-3-(3,4-dichlorophenyl)-7-hydroxy-8-methyl-8-azabicyclo[3.2.1.]octane (O-1163) occupied the DAT but had short-lived pharmacological effects. The benztropine analog difluoropine increased general activity, improved posture, reduced body freeze, and produced sleep disturbances at high doses. (1R)-2-(1-Propanoyl)-3-(4-fluorophenyl)tropane (O-1369) alleviated parkinsonian signs in advanced parkinsonian monkeys, by increasing general activity, improving posture, reducing body freeze, and sedation, but not significantly reducing bradykinesia or increasing locomotor activity. In comparison with the D₂-D₃ DA receptor agonist quinelorane, O-1369 elicited oral/facial dyskinesias, whereas quinelorane did not improve posture or reduce balance and promoted stereotypy. In conclusion, DAT inhibitors with therapeutic potential combine high DAT affinity in vitro and high DAT occupancy of brain striatum in vivo with enduring day-time effects that do not extend into the nighttime. Advanced parkinsonian monkeys (80% DAT loss) respond more effectively to DAT inhibitors than mild parkinsonian monkeys (46% DAT loss). The therapeutic potential of dopamine transport inhibitors for Parkinson's disease warrants preclinical investigation.

The DAT is another candidate target for antiparkinsonian medications, because DAT blockade produces profound increases in extracellular dopamine in normal striatum (Hurd and Ungerstedt, 1989). Conceivably, DAT blockers may also function as neuroprotective agents to prevent ongoing degeneration in Parkinson's disease, because the DAT reportedly is a conduit for entry of neurotoxins into dopamine neurons (Uhl, 1998). Beyond functioning as a carrier for dopamine, DAT may stimulate somatodendritic dopamine release in the substantia nigra (Falkenburger et al., 2001). Accordingly, DAT inhibitors may augment dopamine levels for receptor activation and protect nigral DA neurons from ongoing degeneration by inhibiting DA and neurotoxin sequestration into DA neurons via the DAT.

In 1961, before the discovery of DAT and other monoamine transporters, methylphenidate (now identified as a DAT inhibitor) reduced rigidity and bradykinesia in a small number of PD subjects (Halliday and Nathan, 1961). After the discovery of brain DAT, the antiparkinsonian drug benztropine was found to be an effective inhibitor of dopamine transport, leading to speculation that DAT blockade as well as muscarinic cholinergic receptor antagonism contributed to its anti-parkinsonian properties (Coyle and Snyder, 1969). Subsequently, other DAT inhibitors were shown to produce moderate, slight, or no improvements in motor function (Teychenne et al., 1976; Bedard et al., 1977; Park et al., 1981; Delwaide et al., 1983; Goetz et al., 1984; Frackiewicz et al., 2002; Bara-Jimenez et al., 2004). Clinical interest in DAT inhibitors was heightened with the discovery that methylphenidate potentiated the effects of infused L-dopa, by increasing the percentage of patients' responding and duration of response, while reducing hypotension (Nutt et al., 2004). The authors concluded that residual DAT is functional in PD and a potential target for symptomatic therapy, particularly if combined with L-dopa. Others revisited the hypothesis that DAT inhibitors have therapeutic potential for PD in preclinical models of PD. High-affinity DAT inhibitors (GBR 12909, BTS 74 398, and brasofensine) increased activity and lowered disability scores in MPTP-treated parkinsonian monkeys or in DA-neuron lesioned rats (Hansard et al., 2002a,b, 2004; Pearce et al., 2002; Lane et al., 2005a,b).

In the present study, we investigated properties of DAT inhibitors that would presage therapeutic efficacy for treating motor impairment in parkinsonism. We focused on eight novel, chemically diverse analogs of the phenyltropane CFT, or WIN 35,428, which exhibits high affinity for the DAT (Fig. 1; Table 3; Madras et al., 1989, 1996, 2003; Kaufman and Madras, 1991; Meltzer et al., 1994, 2001). Selection criteria were initially restricted to high DAT affinity and DAT:SERT selectivity, because SERT inhibitors reportedly attenuate DAT inhibitor-mediated improvements in locomotor activity (Hansard et al., 2002a). Compounds were tested in the MPTP model of PD in nonhuman primates, initially by screening with an accelerometer. Lead compounds that significantly increased daytime but not night-time activity were then assessed by observer rating of specific behaviors. We also monitored the effects of a lead DAT inhibitor in subjects, with a range of motor impairments (Goulet and Madras, 2000), to pursue whether a DAT inhibitor would be less ineffective in advanced PD (Bara-Jimenez et al., 2004). To examine the relationship between therapeutic potential of DAT inhibitors and DAT occupancy or DAT loss, we monitored the DAT in striatum by PET imaging with [¹¹C]CFT (Madras et al., 1989, 2001; Morris et al., 1996). High-affinity DAT inhibitors alleviated select parkinsonian symptoms in monkeys, but affinity alone was an insufficient predictor of the therapeutic potential of DAT inhibitors. The results provide guidelines for further assessment of DAT inhibitors as antiparkinsonian medications.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Structure of compounds. Top row, difluoropine, a benztropine analog and O-1369. Middle row, nonamines O-1014, O-1231, and O-1973. Bottom row, 7-hydroxy substituted phenyltropane O-1163, its nonhydrolyzable analog O-2099, and a putative prodrug of O-2099.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Affinities of DAT Inhibitors at DAT, SERT, and Muscarinic Cholinergic Receptors
Tissue Preparation. Brain tissue was harvested from adult male and female cynomolgus monkeys (Macaca fascicularis) euthanized in the course of other studies. Tissue was derived from the brain bank at the New England Primate Research Center (Southborough, MA) and stored at –80°C. The caudate putamen was dissected from coronal sections of brain tissue (1.4 ± 0.4 g; means ± S.E.M.) and homogenized separately. Membranes were prepared as described previously (Madras et al., 1989). In brief, the tissue was homogenized in 10 volumes (w/v) of ice-cold Tris-HCl buffer (50 mM, pH 7.4, at 0–4°C) and centrifuged at 38,700g for 20 min in the cold. The resulting pellet was suspended in 40 volumes of buffer, and the entire wash procedure was repeated twice. The membrane suspension (25 mg, original wet weight of tissue/ml) was diluted to 12 mg/ml in buffer just before assay and dispersed with a Brinkmann Polytron (setting 5; Brinkmann Instruments, Westbury, NY) for 15 s. All experiments were conducted in triplicate, and each experiment was repeated in each of two to four individual tissue preparations. Transport assays were conducted in cell lines expressing the cloned human DAT, NET, or SERT (Madras et al., 2003).

Competition Assays. The potency of novel drugs for the dopamine, norepinephrine, and serotonin transporters was determined in radioligand binding assays by incubating membranes prepared from individual brain specimens with a fixed concentration of [³H]CFT (dopamine transporter, specific activity 80 Ci/mmol), [³H]nisoxetine (norepinephrine transporter, specific activity 74 Ci/mmol), or [³H]citalopram (serotonin transporter, specific activity 82 Ci/mmol) and a range of concentrations of test compound (Madras et al., 1996). Procedures were similar to those described previously (Madras et al., 1996). Novel compounds were dissolved in various ratios of ethanol/HCl, at concentrations previously shown to not interfere with binding assays. The assay tubes received, in Tris-HCl buffer (50 mM, pH 7.4, at 0–4°C; 100 mM NaCl), these constituents at a final assay concentration: test drug or CFT (0.2 ml; 8–15 concentrations ranging from 1 pM to 100 µM), [³H]CFT (0.2 ml; 0.3 nM), membrane preparation (0.2 ml; 4 mg original wet weight of tissue/ml). The 2-h incubation (0–4°C) was initiated by the addition of membranes and terminated by rapid filtration over Whatman GF/B glass fiber filters (Whatman, Maidstone, UK) presoaked for at least 40 min in 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO). The filters were washed twice with 5 ml of 50 mM Tris-HCl buffer, incubated overnight at 0–4°C in scintillation fluor (5 ml; Beckman Ready-Value; Beckman Coulter, Fullerton, CA), and radioactivity was measured by liquid scintillation spectrometry (Beckman 1801). The cpm values were converted to disintegrations per minute following determination of counting efficiency (49–53%) of each vial by external standardization. Total binding was defined as ³H-radioligand bound in the presence of ineffective concentrations of unlabeled drug (1 or 10 pM). Nonspecific binding was defined as ³H-radioligand bound in the presence of an excess drug used to monitor nonspecific binding (for DAT, 30 µM(–)-cocaine). Specific binding was the difference between the two values. In the caudate putamen, total binding of 1 nM [³H]CFT ranged from 1500 to 3500 dpm, and specific binding was approximately 90% of total. Differences in [³H]CFT bound within triplicate samples averaged 3 to 7% of the mean at levels of radioactivity 400 dpm in the caudate putamen. Drug inhibition of [³H]DA uptake was determined in human embryonic kidney 293 cells transfected with the DAT (Madras et al., 2003).

The norepinephrine transporter in thalamus was assayed by similar methods in a 50 mM Tris-HCl buffer, pH 7.4, which contained 300 mM NaCl (Madras et al., 1996). In brief, 0.6 nM [³H]nisoxetine was used to monitor total NET binding. The 16-h incubation at 0–4°C was initiated by addition of tissue and terminated by rapid filtration over Whatman GF/B glass fiber filters presoaked in 0.3% polyethyleneimine for 1 h. Nonspecific binding was defined as [³H]nisoxetine bound in the presence of an excess (10 µM) of desipramine. The serotonin transporter in caudate putamen was assayed using methods similar to those described above. Each tube received in order: test drug or citalopram (0.2 ml; 8–15 concentrations ranging from 1 pM to 100 µM), [³H]citalopram (0.2 ml; 1 nM), membrane preparation (0.2 ml; 4 mg original wet weight of tissue/ml). The 2-h incubation at 0–4°C was terminated by rapid filtration over Whatman GF/B glass fiber filters presoaked with 0.1% polyethylenimine. For the serotonin transporter, total binding (approximately 2000 dpm) was defined as [³H]citalopram bound in the presence of ineffective concentrations of drug (1 or 10 pM), and specific binding (approximately 70% of total binding) was defined by [³H]citalopram bound in the presence of an excess of fluoxetine (1 µM). The muscarinic cholinergic receptor was assayed with [³H]quinuclidinyl benzilate (PerkinElmer Life and Analytical Sciences, Wellesley, MA). Similar to the protocol of the assays described above, assays were conducted in membranes prepared from frontal cortex of cynomolgus monkeys.

The binding potencies of the DAT inhibitors at 30 receptors were screened by the National Institute of Mental Health Psychoactive Drug Screening Program using standard methods. Online protocols are available at http://pdsp.cwru.edu/pdsp.htm. Data were analyzed by Prism (GraphPad Software Inc., San Diego, CA).

Animals
Observations were made on a total of 13 cynomolgus monkeys (two females, 11 males, ages 2–10 years) housed singly at the New England Primate Research Center under a 12-h light/dark cycle. Animal care and treatment were supervised by veterinarians under the guidelines and in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, National Academy Press, Washington, DC, 1996). An animal care protocol was approved by the Harvard Animal Care Committee and was in compliance with the Harvard Medical School animal management program, an institution accredited by the American Association for the Accreditation of Laboratory Animal Care.

Videotape Rating of Behaviors
Animals (M. fascicularis) were placed in a large filming cage (85 x 79 x 88 cm) with food and other enrichment items, with full access to water. Filming was done in the animal housing room to maintain a familiar home environment. A video camera, focused on one Plexiglas cage side panel, was activated. Before drug treatment, baseline behavior for each subject was filmed for 7 h. Vehicle (saline) was injected immediately after the close of the first half-hour session. Drugs were administered via intramuscular injection, and filming continued for 6 h. Videotapes were rated by an observer blinded to the drug treatments. Several monkeys had a prior history of acute drug treatments but had not been treated with any drugs for at least 2 months. Others were placed specifically in this study with no prior exposure to drugs, with the exception of MPTP.

Monkeys were fitted with a jacket containing an inaccessible back pocket, into which an accelerometer was placed. For videotaping, animals were transferred via a transfer box to a large cage fitted with a Plexiglas front window to facilitate videotaping. Taping was conducted from 9:00 AM to obtain baseline activity levels and was continued for as long as 6 h after the last dose of drug. In the videotaping cage, animals had full access to food and water.

MPTP Lesion
Animals were anesthetized with ketamine, an indwelling venous catheter was implanted, and parkinsonism was induced by two or three injections of MPTP (0.6 mg/kg MPTP·HCl i.v., dissolved in saline) within 45 days. Animals were generally able to maintain feeding and drinking behavior. Some animals displayed improvement after the initial decline. The behaviors and activities of the subjects were monitored for 5 to 14 days before initiation of each drug study, and parkinsonian symptoms remained stable before the drug experiments (Goulet and Madras, 2000). Mild and advanced parkinsonism were designated empirically and reflected scores on the parkinsonian rating scale and DAT imaging.

Drug Testing
Drug treatments were administered in random order. To determine maximal effective doses of the compounds, monkeys received a cumulative dose (spaced 0.5 h apart) of test compound to determine a dose-response function via the accelerometer. After an interval of 2 to 4 weeks, monkeys received single i.m. injections of test compound. At the end of each treatment period, all monkeys were given a minimum 14-day injection-free period. Baseline activity values were determined both by measuring activity in the absence of drug and by monitoring activity after an i.m. injection of saline. All drugs were dissolved in vehicle (minimal concentrations of ethanol and/or HCl in sterile saline), and vehicle was tested to determine whether the composition of the vehicle or injection per se influenced activity. To determine dose-response effects, difluoropine was dissolved in vehicle and administered at noon daily in single doses (0.1–3.0 mg/kg) i.m. after a vehicle injection, and O-1369 (0.1–3 mg/kg) and quinelorane (0.05 mg/kg) were administered similarly.

Behavioral Analyses: Rating Scale
For behavioral analysis, videotapes were scored independently by an observer blinded to the treatment. Inter-rater reliability generally exceeded 90% after a period of training. The rating scale measured the following behaviors: general activity, locomotor activity, bradykinesia, rigidity, posture, imbalance, tremor frequency, body freeze, feeding ability, oral/facial dyskinesia, limb dyskinesia, trunk dyskinesia, stereotypy, sedation, self-grooming, social interaction, penile erection, vomiting, head scanning, and tail rigidity (Table 1). The ratings enabled estimates of parkinsonism signs as well as dyskinesias and other symptoms of dopaminergic drug treatment (stereotypy, self-grooming, penile erection, vomiting, head scanning, and tail rigidity). The ethogram scale and analysis were devised after repeated observation of individual behavioral motifs of normal and MPTP-treated monkeys on videotape (Goulet and Madras, 2000). The intensity and frequency of occurrence of individual behaviors were assessed, and, based on analysis of the behaviors observed, we focused on seven behaviors that were significantly affected by MPTP: general activity, locomotor activity, bradykinesia, body freeze, rigidity, postural abnormalities, and sedation. Nonetheless, all other behaviors reflecting potential therapeutic and side effects were also rated. Oral/facial dyskinesia, stereotypy and penile erection following drug exposure are presented (Table 1). Other behaviors did not show differences in baseline or following drug treatments. Tapes were divided into 30-min segments. Data consist of observational ratings in 2-min blocks for 6 h after drug administration, with each segment scored in 2-min observation periods every 5 min (5, 10, 15, 20, and 25 min) into the session and the average of each segment was used for analysis. Each 30-min time period after drug administration generated five observational scores. Baseline behavior was rated either with no intervention or after saline injections, with an average of 70 baseline observations in each animal before drug administration. Each behavioral motif was given a score of –1, 0, 1, or 2. Behavioral changes assumed to derive from excess dopaminergic activity (e.g., dyskinesias) were assigned scores of –1. Statistical analysis of the data were based on average scores for each 30-min observation period, during treatment, and compared with baseline levels of activity, monitored for four or more sequential days before drug treatment.

Computerized Monitoring of Movement
Motor activity was assessed with an omnidirectional accelerometer (Actiwatch aw4-64K; Mini-Mitter, Sunriver, OR). The animals were sedated with 10 mg/kg ketamine and were fitted to a jacket according to their weight (Lomir, Montreal, QC, Canada). To increase comfort, the jacket was sleeveless and fabricated with an inaccessible pocket (3 x 3 inches) in the lower back. The accelerometer unit was placed in a hard case and inserted in the jacket pocket after a period of 2 to 4 weeks to accommodate to the jacket. The accelerometer was set to record activity continuously every minute for 30- to 45-day periods. Baseline levels of activity were generated for at least 5 days before drug administration, and then activity was compared in predrug and postdrug sessions. The data were analyzed with the software Rhythmwatch (Mini-Mitter), and activity counts were divided into segments as designated and compared with baseline levels of activity.

PET Imaging of the DAT
PET imaging of the DAT in caudate putamen was used for two purposes: to measure the extent of MPTP-induced dopamine neuron degeneration and to determine DAT occupancy by candidate therapeutics. DAT was quantified with the selective DAT probe [¹¹C]CFT (Kaufman and Madras, 1991; Morris et al., 1996; Madras et al., 2001). PET imaging was conducted with monkeys initially anesthetized with ketamine/xylazine (15.0/1.5 mg/kg) and then maintained under general anesthesia with halothane and positioned prone on the imaging bed of a PC 4096 PET camera (GE/Skanditron AB, Uppsala, Sweden). A stereotactic head-holder was used for head immobilization. CFT was demethylated in the C-2 position, and [¹¹C]methyl was inserted by the methyl iodide reaction. After stabilization in the PET camera, 10 mCi of [¹¹C]CFT (specific activity >1500 mCi/µmol) was injected through the venous catheter, and sequential images were acquired in 15-s time frames for the first 2 min and in 1-min frames for 58 min. At the conclusion of each imaging study, the emission and transmission images were reconstructed using a conventional filtered back-projection algorithm to an in-plane resolution of 6-mm full-width half-maximum. All projection data were corrected for nonuniformity of detector response, dead time, random coincidences, and scattered radiation. A sum image was generated by adding all the frames from frame 10 to the end of the study. Regions of interest were drawn on the summed image in the coronal projection as follows; one 4-pixel region was drawn on each caudate putamen on the slice of maximum intensity. For cerebellum, three regions were drawn on cerebellar slices. Time-activity data were produced using the regions of interest on all time frames of the PET data. The same set of regions of interest was used to analyze each scan for an individual subject on the same day. When necessary, new regions were drawn to compensate for repositioning. Binding potential was calculated by published methods (Bonab et al., 1998).

Drugs
Experimental drugs (codesigned by B. K. Madras and P. C. Meltzer) were difluoropine or O-620, O-1369, O-1014, O-1163, O-1231, O-1973, O-2099, and O-2240. Quinelorane was a generous gift from E. Lilly & Co. (Indianapolis, IN). MPTP was purchased from Sigma-Aldrich.

Data Analysis
One-way analysis of variance, nonparametric analysis with Dunnett's comparison with baseline controls was performed, using GraphPad Instat for Windows (GraphPad Software Inc.). Other statistical tests are mentioned in text. Graphs were prepared by the GraphPad Prism 4.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Receptor Binding Profile. Of a wide range of phenyltropane analogs designed collaboratively over the past decade by the author (Fig. 1), we investigated seven high-affinity DAT inhibitors with low affinity for the SERT and one pro-drug (O-2240). With the exception of O-2240, all of the compounds inhibited [³H]CFT binding with affinities less than 40 nM, the majority having less than 10 nM affinities (Table 2). The rank order of potencies of the compounds for inhibiting [³H]CFT binding was O-1163 = O-2099 > O-1369 > O-1231 > O-1014 = difluoropine > O-1973 (Table 2). A similar rank order of potency was obtained for their potencies to block [³H]dopamine transport. None of the compounds displayed nanomolar affinity for the SERT, and the majority were at least 40-fold selective for the DAT compared with the SERT. The compounds displayed less than 1 µM affinity for the NET, with difluoropine, O-1369, O-1163, and O-2099 blocking [³H]norepinephrine transport by the NET with potencies less than 50 nM. Interestingly, high DAT affinity was not predictive of high NET affinity, but nonamines (O-1014, O-1231, and O-1973) generally were less potent at the NET, compared with the DAT. The main focus of the study was two lead compounds, difluoropine, a benztropine analog, and O-1369, an analog of WIN 35,428 (CFT) (Fig. 1; Table 2). Of eight possible isomers previously characterized, difluoropine emerged as the most potent within a series of benztropine analogs (Meltzer et al., 1994), and it was 15 times more potent than its progenitor benztropine (Table 2). In further distinction to benztropine, difluoropine displayed low affinity for the muscarinic cholinergic receptor (Tables 2 and 3). O-1369, which lacks a hydrolyzable ester link in the C-2 position and is considered to be more metabolically stable than its progenitor compound, displayed high affinity (K_i = 15.1 nM) in blocking DAT transport function (Fig. 1; Tables 2 and 3). In a receptor screening array, 10 µM difluoropine displayed some activity at -opiate receptors and inhibited nicotinic cholinergic receptor activity at 1 µM (Table 3). O-1369 displayed low affinity (>10 µM) for a wide range of receptors, but it seemed to enhance 5-hydroxytryptamine 1Db receptor activity at this concentration, possibly indicative of agonist activity (Table 3). The potencies of clinically relevant DAT inhibitors (mazindol, methylphenidate, nomifensine, and bupropion) are shown in Table 2, for comparative purposes.

Pilot Screening of DAT Inhibitors. Pilot screening of compounds was conducted using an accelerometer in parkinsonian monkeys with varying activity levels, measured before and after drug administration. Activity was monitored 1 to 2 weeks preinjection and 3 to 6 days postinjection to monitor onset and duration of effect. Using a range of doses (0.01–3 mg/kg i.m.), we assessed a novel group of DAT inhibitors (O-1014, O-1231, and O-1973), which contain no amine nitrogen in their structure (Madras et al., 1996, 2003). The three nonamines (Fig. 1; Table 2) produced little or no increases in activity of parkinsonian monkeys on the day of, or for 6 days after, administration compared with baseline levels of activity (data not shown).

O-1163, a 7-hydroxylated phenyltropane analog with a hydrolyzable ester in the 2-position, was selected on the basis of high DAT affinity and DAT:SERT selectivity and for comparison with O-2099, the 2-propionyl analog that is not hydrolyzable in this position. O-2099 was compared with O-2240, a putative prodrug of O-2099 that, by virtue of its hydrolyzable carbon chain to mask the 7-hydroxyl group of O-2099, conceivably would provide extended DAT blockade and therapeutic benefit. O-1163 was administered in single doses (0.1–6 mg/kg i.m.) on separate days. O-1163 increased activity during the first hour after administration (Fig. 2), an effect that dissipated at 90 min and did not reoccur during the day or subsequent days.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Pilot study of activity of parkinsonian cynomolgus monkeys (accelerometer) before and after treatment with the candidate DAT inhibitor O-1163 (Fig. 1; Table 2). Parkinsonian monkeys (n = 3) were monitored 24 h continuously at 1-min intervals with an accelerometer. Dose-response effect of O-1163 from 8:30 to 9:30 AM, the only time period in which O-1163 was active.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effects of O-2099 in parkinsonian monkeys. A, top, activity monitoring (Actiwatch accelerometer) of monkeys for 4 days before, on the day of exposure to 0.1 mg/kg O-2099, and the following day. Each bar represents activity (means ± S.E.M.; n = 3) of 16 30-min blocks between 10:00 AM and 6:00 PM, the period of active O-2099 effects. O-2099 significantly increased activity compared with baseline levels (p < 0.02). B, bottom, behavioral effects of 0.1 mg/kg O-2099 after administration at 9:30 AM. Baseline behavior was filmed and scored for 5 days, and drug was administered on day 6. Data are scores recorded for 6 h after drug treatment (means ± S.E.M.; n = 4) of parkinsonian monkeys (*, p < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Effects of 0.3 mg/kg O-2240, a prodrug of O-2099, on activity monitored by an accelerometer in parkinsonian monkeys. Activity is expressed as a percentage of baseline values (100% averaged from 5 days of daily saline injections before drug administration (means ± S.E.M.; n = 6). O-2240 was administered at 8:30 AM, and activity was monitored by an accelerometer. In sequence, each bar shows activity levels of vehicle (baseline), O-2240 (i.m.), the next day (day 1), second dose of O-2240, and seven subsequent days. Top, 12:00 PM–6:00 PM. Middle, 6:00 PM–12:00 AM (midnight); and bottom, 12:00 AM–6:00 AM. No changes were observed from 6:00 AM to 12:00 PM (Dunnett's multiple comparison with control; p < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Effects of difluoropine in cynomolgus monkeys. A, comparison of difluoropine on behaviors exhibited in normal (n = 4) and MPTP-treated cynomolgus monkeys (n = 5), as a function of dose and time, and rated on an ethogram scale (see Table 1). Difluoropine was administered cumulatively (0.1–3.0 mg/kg) at 30-min intervals and after the last dose (3.0 mg/kg), observations were continued at 30-min intervals (f1–f7) for an additional 3.5 h. Difluoropine effects on normal cynomolgus monkeys (left) and in parkinsonian monkeys (right). Ctrl data are baseline levels of activity with no i.m. injections, monitored for 4 to 8 days (means ± S.E.M.). Veh was administered i.m. 30 min before the first dose of difluoropine to determine injection effects. There were no statistically significant injection effects compared with control values. Each data point is based on five observations made during 30-min intervals (means ± S.E.M.) (Dunnett's multiple comparison with control; *, p < 0.05). Bottom right, composite score of those behaviors that significantly improved in parkinsonian monkeys as a function of dose and time (Dunnett's multiple comparison with control; *, p < 0.05). B, side effect profile of difluoropine on various behaviors and sleep fragmentation. Difluoropine was administered i.m. cumulatively at 30-min intervals to MPTP-treated parkinsonian monkeys (n = 5) as described above. Left, effects of difluoropine on oral/facial dyskinesia (O/F) and head scanning were quantified according to the rating scale described in Table 1. Ctrl data are based on observations with no i.m. injection and represent baseline levels of activity (means ± S.E.M.). Saline veh was administered i.m. 30 min before the first dose of difluoropine to determine injection effects, but these effects were not significant. Right, sleep fragmentation in MPTP-treated monkeys produced by 3 to 6 mg/kg difluoropine. Results are means ± S.E.M.; n = 4 (*, p < 0.05; **, p < 0.01).

View larger version (26K): [in this window] [in a new window]   Fig. 6. Comparison of MPTP-induced parkinsonian monkeys with mild (n = 4) and advanced symptoms (n = 4). Parkinsonian monkeys were divided into two groups, based on their composite behavioral scores and on brain imaging of the DAT. A, top left, comparison of behavioral scores of seven behaviors in the mild compared with the advanced group (means ± S.E.M.): general activity (***, p < 0.007), locomotor activity (***, p < 0.005), bradykinesia (***, p < 0.008), rigidity (***, p < 0.006), posture (**, p < 0.007), body freeze (***, p < 0.006), and sedation (*, p < 0.04). B, top right, composite parkinsonian score in the mild compared with the advanced parkinsonian group (p < 0.002). C, bottom right, correlation between the composite parkinsonism score and DAT binding potential (detected by PET imaging), a measure of dopamine neuron degeneration for eight parkinsonian monkeys; each data point represents a single subject (r2 = 0.71; p < 0.01).

The prodrug of O-2099, O-2240, conceivably could provide a more durable response than O-2099 (Fig. 1). O-2240 was administered twice, with the second dose given 2 days later to determine whether this prodrug produced cumulative effects in the course of being metabolized to its active form. After administration at 8:30 AM and monitored by accelerometry, O-2240 did not change activity during the first 3.5 h, but it increased activity levels in the afternoon (12:00 PM–6:00 PM) (Fig. 4A, top). After the second dose on day 3, O-2240 again increased activity, but the increase did not persist on subsequent days during the same time period (Fig. 4A, top). During normal sleep hours, O-2240 increased activity from 6:00 PM to 12:00 AM (Fig. 4, B and C), with the most robust increase detected from 12:00 AM to 6:00 AM on day 2, after the second dose (Fig. 4C, bottom). Based on the modest effects of O-2099 on daytime activity, dose limitations, and heightened activity elicited by O-2240 during the dark cycle, we focused on other high-affinity DAT inhibitors, difluoropine and O-1369.

Effects of Difluoropine on Activity and Behaviors. The benztropine analog difluoropine was selected because of high affinity and low muscarinic cholinergic receptor affinity (Fig. 1; Table 2). Conceivably, these in vitro properties would clarify whether the antiparkinsonian effects of benztropine (difluoropine progenitor) require muscarinic cholinergic receptor blockade (Coyle and Snyder, 1969). Difluoropine effects on motor function and other behaviors were monitored in normal (n = 4) and in MPTP-treated cynomolgus monkeys (n = 5), as illustrated in Fig. 5 (left, normal monkeys; right, MPTP-treated monkeys). Subjects were treated with various doses of difluoropine (0.1–3.0 mg/kg), and activity was rated by an observer (Table 1) and monitored by an accelerometer. Difluoropine was administered cumulatively at 30-min intervals and after the last dose (3.0 mg/kg), observations were continued at 30-min intervals (f1–f7) for an additional 3.5 h. Each data point is based on five observations made during 30-min intervals (means ± S.E.M.). All values were compared with baseline control activities (Ctrl), monitored for 4 to 8 days before injection of the test compound (means ± S.E.M.). Saline vehicle (veh) was administered i.m. 30 min before the first dose of difluoropine to determine injection effects, but no statistically significant injection effects were detected compared with baseline levels. In normal animals, cumulative doses of difluoropine (0.1–3 mg/kg) affected behaviors differently than in parkinsonian monkeys. Difluoropine reduced locomotor activity (not statistically significantly) in accord with the locomotor-reducing effects of cocaine, a DAT inhibitor, in monkeys (Saka et al., 2004). Difluoropine also increased rigidity, but, with high interin-dividual variability, changes were not statistically significant (data not shown). In MPTP-treated parkinsonian monkeys, difluoropine dose-dependently increased general activity (Fig. 5A, top right; *, p < 0.05), reduced severe body freeze (Fig. 5A, right, third from top; **, p < 0.004), and improved posture (Fig. 5A, right, fourth from top). During the 2 h of maximal effectiveness (f4–f7 interval or 3–3.5 h after the last dose), difluoropine significantly improved general activity, posture, and body freeze (Fig. 5A, bottom right; *, p < 0.05). This composite behavioral score was limited to these parameters, because in congruence with other DAT inhibitors (see below), these consistently achieved statistical significance. This composite score is also illustrated because it corresponded temporally to improved activity as monitored by the accelerometer. Accordingly, the latter device may serve as a rapid screening tool for antiparkinsonian drug effects of DAT inhibitors. Difluoropine increased oral/facial dyskinesias (not significant) and head scanning (Fig. 5B, left; *, p < 0.05, **, p < 0.01). At the highest doses tested (3–6 mg/kg), difluoropine promoted sleep fragmentation (*, p < 0.05) on the night after early morning administration (Fig. 5B, right). Sleep fragmentation, an index of restlessness, is calculated as a percentage of minutes spent moving compared with immobility, during the dark cycle (lights out). Based on low muscarinic cholinergic receptor affinity, difluoropine improved motor function independently of muscarinic cholinergic receptor antagonism.

O-1369 in Mild or Advanced Parkinsonism. Because normal animals responded to difluoropine with reduced locomotor activity (Fig. 5A, top left), we postulated that the response of parkinsonian monkeys to DAT inhibitors could reflect the extent of motor impairment before drug exposure. We vigorously tested this postulate with a lead DAT inhibitor, O-1369, in subjects with a range of baseline levels of activity. A cohort of eight MPTP-treated monkeys was divided into two groups, mild (n = 4) and advanced parkinsonism (n = 4), based on observational (Fig. 6, A and B) and PET imaging data (Fig. 6C). The individual parkinsonian scores of seven behaviors distinguished the two groups of mild and advanced, based on general activity (***, p < 0.007), locomotor activity (***, p < 0.005), bradykinesia (***, p < 0.008), rigidity (***, p < 0.006), posture (p < 0.007), body freeze (p < 0.006), and sedation (*, p < 0.04) (Fig. 6A, top left). One group displayed composite scores exceeding 7.0 (range 7.43–10.56, of a maximal possible score of 12) and was designated as "advanced parkinsonism." The other group, with scores of 4 or less (range 0.56–4.01), was designated "mild parkinsonism" (Fig. 6B). The averaged composite score for the mild cohort differed significantly from the advanced cohort (Fig. 6B, top right; p < 0.005). To compare these composite scores with dopamine neuron viability, we conducted PET imaging in each of the subjects. DAT binding potential, detected with the PET ligand [¹¹C]CFT, correlated significantly (r² = 0.71; p < 0.01) with the composite parkinsonism score (Fig. 6C, bottom). Before MPTP administration, baseline DAT binding potential in the mild and advanced groups did not differ: 2.32 ± 0.33 (mild group: means ± S.E.M.; n = 4) and 2.38 ± 0.06 (advanced group: means ± S.E.M.; n = 4). After MPTP administration, DAT binding potential declined to 1.25 ± 0.26 in the mild group (means ± S.E.M.; n = 4) and to 0.45 ± 0.04 in the advanced group (means ± S.E.M.; n = 4) (p < 0.02), equivalent to a loss of 52 and 80% of DAT. The two groups differed from their normal baseline DAT binding potential (mild: p < 0.04; advanced: p < 0.0001). Intriguingly, the 30% lower DAT binding potential of advanced parkinsonian monkeys was associated with a 3-fold increase in parkinsonism scale.

O-1369, a 3-fluorophenyltropane analog of CFT, or WIN 35,428, was selected for scrutiny on the basis of two considerations: first, the 3-boat-conformer is considerably less potent at the SERT and, accordingly, l more DAT:SERT-selective than its 3-diastereomer; and second, the 2-propionyl moiety that replaced the 2-carbomethoxy group is less likely to undergo enzymatic hydrolysis. The behavioral effects and sleep patterns of the potent DAT inhibitor O-1369 were investigated in this larger cohort of parkinsonian monkeys (n = 8), with varying degrees of MPTP-induced parkinsonism. Baseline activity was acquired for five consecutive days, then dose-response effects of O-1369 (0.1, 0.3, 1.0, and 3.0 mg/kg i.m., given between 8:00 and 8:30 AM) on activity of four advanced parkinsonian monkeys were monitored by an accelerometer (Fig. 7A, top, inset). Activity was measured in 1-min units and averaged for 30-min blocks. Results are expressed as percentage of increase over baseline activity levels, for 4.5 h after administration. Subsequently, the effects of a fixed dose of O-1369 (3.0 mg/kg i.m.) on activity of eight subjects with a range of parkinsonian symptoms (mild and advanced parkinsonism) during peak drug response were monitored by an accelerometer (Fig. 7A, top). Data expressed as average activity counts, computed each minute starting at 30 min after drug treatment, were averaged in 30-min blocks, for 3.5 h. O-1369 increased activity in advanced parkinsonian monkeys with low activity levels (1, 3, and 4, but not 2), and in one higher activity animal (6) (means ± S.E.M.; *, p < 0.03, **, p < 0.01; Fig. 7A, top). We subsequently measured the effects of O-1369 (3 mg/kg i.m.) on behavior in the same cohort of eight monkeys displaying mild parkinsonism (Fig. 7B, middle) or advanced parkinsonism (Fig. 7C). In mild parkinsonism (Fig. 7B, middle), none of the behaviors improved significantly with O-1369 (means ± S.E.M.; n = 4). (black bars designating posture, body freeze, and sedation were not visible because behavior was restored to zero for these parameters.) In advanced parkinsonism (Fig. 7C, bottom), O-1369 significantly improved selective, but not all behaviors compared with baseline activity (means ± S.E.M.; n = 4): general activity (**, p < 0.016), posture (*, p < 0.03), body freeze (*, p < 0.03), and sedation (*, p < 0.03) improved, but locomotor activity (N.S.), bradykinesia (N.S.), and rigidity (N.S.) did not (n = 4). The behavioral effects of O-1369 in advanced parkinsonism are shown as a function of time (Fig. 8). The most robust improvements, general activity, posture, body freeze, and sedation, improved within 60 min (*, p < 0.05; **, p < 0.01) and were measurable for 3 h or longer (Fig. 8). Locomotor activity, bradykinesia, and rigidity displayed slight, time-dependent improvements, but changes were not statistically significant (data not shown). In five parkinsonian monkeys with various degrees of impairment, O-1369 increased sleep fragmentation (p < 0.01), at double the dose (6 mg/kg) found to be behaviorally effective (3 mg/kg; Fig. 9), but it did not significantly affect sleep latency or efficiency.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Effects of O-1369 on activity levels in mild and advanced parkinsonian monkeys (n = 8). A, top, activity levels of monkeys treated with O-1369 and monitored by an accelerometer. Baseline activity was acquired for five consecutive days, then O-1369 (3.0 mg/kg i.m.) was administered, and subjects were monitored for 24 h. Data are derived from the day of drug exposure and are expressed as average activity counts, computed each minute, starting at 30 min after drug treatment and averaged in 30-min blocks, for 3.5 h of recording (*, p < 0.03; **, p < 0.01) (means ± S.E.M.). Inset, dose-response effects of O-1369 on activity as measured by an accelerometer. O-1369 was administered i.m. at various doses (0.1, 0.3, 1.0, and 3.0 mg/kg). Activity was measured in 1-min units and averaged for 30 min blocks. Results are expressed as percentage of increase over baseline activity levels, for 4.5 h after O-1369 administration (n = 4; advanced parkinsonian monkeys). B and C, middle and bottom, comparison of mild and advanced parkinsonian monkeys responding to O-1369 during peak drug response (1–3.5 h; see Fig. 8). B, middle, in mild parkinsonism, none of the behaviors improved significantly with O-1369 (3 mg/kg i.m.), (means ± S.E.M.; n = 4). In the mild parkinsonian monkeys (middle; B), posture, body freeze, and sedation in the post-O-1369 state were normal (0), and black bars fell on the x-axis. C, bottom, in advanced parkinsonism monkeys (means ± S.E.M.; n = 4), O-1369 improved several but not all behaviors significantly, compared with baseline activity: general activity (**, p < 0.016), locomotor activity, N.S.; bradykinesia, N.S.; rigidity, N.S.; posture (*, p < 0.026); body freeze (*, p < 0.026); sedation (*, p < 0.03) (n = 4).

View larger version (19K): [in this window] [in a new window]   Fig. 8. Time course of behavioral effects of 3 mg/kg O-1369 in advanced parkinsonian monkeys. O-1369 was administered in the early morning, and behaviors were monitored for the after 6 h (means ± S.E.M.; n = 4, advanced parkinsonism) (*, p < 0.05; **, p < 0.01, Dunnett's post hoc comparison with control values). Data for bradykinesia, imbalance, rigidity, and locomotor activity were not statistically significant compared with control values and are not shown.

View larger version (21K): [in this window] [in a new window]   Fig. 9. Effects of O-1369 on sleep latency, sleep fragmentation, and sleep efficiency, as monitored by an accelerometer in parkinsonian monkeys with different levels of impairment (n = 5). Sleep parameters are acquired during the nighttime period, when lights are turned off (6:00 PM–6:00 AM). Baseline activity (base) was acquired for five days before administration of various doses of O-1369 (i.m.; 8:00 AM–8:30 AM), with each dose of O-1369 administered on a different day. Baseline activity after the last dose was also recorded (post). Sleep fragmentation, defined as sleep restlessness, is a percentage of minutes moving compared with immobility. Each bar is the mean ± S.E.M. of percentage of change from baseline values, as a function of dose. Sleep fragmentation increased at 6 mg/kg, compared with baseline (**, p < 0.01; n = 5). Sleep latency is defined as time required for sleep onset during the dark period, after lights are turned off and was not affected significantly. Sleep efficiency is an index of the amount of time, during the dark period, spent sleeping and this parameter was not affected (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 10. Activity and behavioral monitoring of mild and advanced parkinsonian monkeys (n = 8) before and after treatment with the D2-D3 dopamine receptor agonist quinelorane (0.05 mg/kg). A, top, parkinsonian monkeys with a range of activity levels were monitored continuously by an accelerometer for 5 days, after which quinelorane (0.05 mg/kg i.m.) was administered, and subjects were monitored for five subsequent days. In animals with relatively low levels of activity, quinelorane increased activity (***, p < 0.001), but it did not do so consistently in higher activity animals. B and C, comparison of response to quinelorane in mild and advanced parkinsonian monkeys during peak drug response (1–3.5 h; see Fig. 11). B, middle, in mild parkinsonism, quinelorane (0.05 mg/kg i.m.) did not significantly improve any of the behaviors (means ± S.E.M.; n = 4). C, bottom, quinelorane improved behaviors in advanced parkinsonism (means ± S.E.M.; n = 4), including general activity (*, p < 0.03), locomotor activity (*, p < 0.03), and body freeze (**, p < 0.01), but other behaviors did not improve (bradykinesia, rigidity, posture, and sedation). See Fig. 9 for time course of drug effects.

View larger version (15K): [in this window] [in a new window]   Fig. 11. Time course of behavioral improvements by 0.05 mg/kg quinelorane in advanced parkinsonian monkeys. Quinelorane was administered in the early morning, and behaviors were monitored for the after 6 h (means ± S.E.M.; n = 4, advanced parkinsonism) (*, p < 0.05; **, p < 0.01; Dunnett's post hoc comparison with control values).

View larger version (30K): [in this window] [in a new window]   Fig. 12. PET imaging of [11C]difluoropine. [11C]Difluoropine was administered to cynomolgus monkeys (n = 2). This representative image was acquired in the course of 90 min. Note the robust accumulation of [11C]difluoropine in the striatum.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Accumulating evidence suggests that DAT inhibitors may fill a niche in the expanding list of therapeutic strategies to treat Parkinson's disease (Hansard et al., 2002a,b; Nutt et al., 2004; but see Frackiewicz et al., 2002; Bara-Jimenez et al., 2004). Residual dopamine neurons may produce sufficient dopamine to improve parkinsonian signs or alleviate side effects, if augmented by DAT inhibitors (this study); inhibitors of monoamine oxidase B, which metabolize dopamine; or drugs that promote dopamine release (Pahwa et al., 2006). In the present report, novel DAT inhibitors alleviated selective symptoms of MPTP-induced parkinsonism in cynomolgus monkeys, but they did not induce hyperkinesis or perseverative stereotypies. The convergence of several key parameters was associated with therapeutic potential: high DAT affinity, high DAT occupancy in the striatum, appropriate onset time/duration, minimal side effect profile, and sleep disturbances. High DAT affinity in vitro may be necessary, but it was an inadequate predictor of the therapeutic potential of a DAT inhibitor.

DAT inhibitors alleviated parkinsonian signs, apparently by DAT blockade in residual dopamine neurons. In view of the failure of lead compounds (O-1369, difluoropine) to bind significantly with 32 brain receptors, it is unlikely that other transporters or receptors mediated this positive response. We did not observe the reported blunting effect of NET blockade on DAT inhibitors tested as antiparkinsonian drugs (Hansard et al., 2002a), because difluoropine, O-1369, and O-2099 displayed high NET affinity (and low SERT affinity). NET blockade, however, may promote sympathomimetic side effects. The inability of O-1369 and other effective DAT inhibitors to reduce bradykinesia, rigidity, or tremor paralleled the effects of the DAT inhibitor brasofensine, which did not improve postural or intentional tremor or initiation of movement (Pearce et al., 2002). Higher doses may be required to reduce tremor, rigidity, or bradykinesia, as reported clinically for methylphenidate (Halliday and Nathan, 1961). Alternatively, specific neuronal circuits involved in PD symptoms may be unresponsive to DAT blockade, because they express insufficient DAT or DA. L-dopa, which increases DA levels in excess of endogenous concentrations, improves the full range of motor disabilities in early stages of PD (Mercuri and Bernardi, 2005). Nonetheless, L-dopa is associated with motor complications, possibly related to its short-half-life and tendency to produce pulsatile DA receptor stimulation (Olanow et al., 2004). Unlike normal brain, which maintains a relatively stable level of DA, fluctuating plasma L-dopa concentrations probably generate irregular DA levels. Monoamine oxidase B and DAT inhibitors may provide a stable, consistent supply of DA to enhance the therapeutic benefit of L-dopa. L-dopa combined with the DAT inhibitor methylphenidate enhanced the therapeutic outcome of PD patients in one study (Nutt et al., 2004) but not if L-dopa was combined with DAT inhibitors NS 2330 or brasofensine (Frackiewicz et al., 2002; Bara-Jimenez et al., 2004). In the latter protocol, the long duration of PD (14 ± 5 years), and elderly population (65 ± 12 years) may have precluded a positive outcome. In parkinsonian monkeys, L-dopa synergized with the DAT inhibitor brasofensine (Pearce et al., 2002) but not with BTS 74 398 (Hansard et al., 2004), even though both DAT inhibitors were effective antiparkinsonian agents alone. Synergy between L-dopa and DAT inhibitors may require a temporal convergence of maximal DAT occupancy by DAT inhibitors combined with L-dopa-induced DA synthesis and release.

Our findings underscore the value of PET imaging to identify candidate therapeutics by detecting DAT occupancy of DAT inhibitors. High DAT occupancy within 1 h of administration, but not high DAT affinity, predicted whether a DAT inhibitor would enhance activity in parkinsonian monkeys. Thus, the high-affinity DAT inhibitors O-2099, O-1163, O-1369, and difluoropine occupied DAT in striatum; high-affinity nonamines O-1014, O-1231, and O-1973 (Madras et al., 1996, 2003) neither occupied DAT sites in striatum nor promoted increased activity in parkinsonian monkeys. Because certain nonamines may be especially vulnerable to enzymatic hydrolysis in the C-2 position or peripheral lipophilic interactions that prevent brain accumulation, higher doses may be necessary. PET imaging could have predicted the rapid decline of behavioral efficacy of O-1163, if DAT occupancy had been measured 2 to 3 h after O-1163 injection, with 2-h pretreatment time or using a radiotracer of longer duration (e.g., [¹⁸F]CFT, [¹⁸F]altropane, or possibly [¹⁸F]O-1369). The failure of brasofensine or NS 2330 to improve PD symptoms (Frackiewicz et al., 2002; Bara-Jimenez et al., 2004) may be related to low DAT occupancy at therapeutic doses or low residual DAT and DA levels.

We were particularly attentive to potential side effects of DAT inhibitors. Potent DAT inhibitors may extend enhanced activity into the nighttime period. Difluoropine, but not O-1369, promoted subject-specific disruptions of sleep patterns at therapeutically relevant doses after early morning exposure. The putative prodrug O-2240 uniquely increased activity during sleep periods, particularly from midnight to 6:00 AM, possibly as a consequence of its slow conversion to O-2099 and persistence in brain regions. Although compromised as a candidate for PD treatment, O-2240 may have therapeutic potential for increasing wakefulness during normal sleep hours, similar to modafinil. Dyskinesias and dystonias are common complications of L-dopa or D₂-D₃ DA receptor agonists. DAT inhibitors elicited time-dependent but not statistically significant oral/facial dyskinesias, but no other visible side effects (limb dyskinesias or stereotypies). Other DAT inhibitors (GBR 12909, brasofensine, and BTS 74 398) reportedly did not elicit dyskinesias (Hansard et al., 2002a; Pearce et al., 2002), but the descriptors of dyskinesias in the latter studies (e.g., stereotypy, limb dystonia, chorea, and athetosis) differed from our definitions (Table 1). In contrast to O-1369, the D₂-D₃ dopamine receptor agonist quinelorane improved locomotor activity, but it did not improve posture or sedation and increased stereotypic behaviors in some subjects, a side effect profile mirroring that of L-dopa (Hansard et al., 2002a; Pearce et al., 2002). Abuse liability and reduced appetite are other considerations for potent DAT inhibitors. Although PD patients report blunted responses to the DAT inhibitor methylphenidate (Persico et al., 1998), this class of compounds presents the potential for diversion. Nonetheless, abuse liability in humans cannot be predicted on the basis of in vitro DAT affinities alone or even self-administration paradigms in animals.

At what stage of PD are DAT inhibitors likely to be effective? DAT binding potential, which correlated inversely with severity of parkinsonism, provided an quantifiable measure of parkinsonian severity, as shown in human PD (Fischman et al., 1998). Subjects with low DAT binding potentials (80% loss) responded to O-1369, but those with higher DAT binding potential (465 DAT loss) were not as responsive. Impairment was limited in these subjects, and endogenous compensatory mechanisms conceivably masked the effects of DAT inhibitors. In human PD, DAT binding potential reduction of 80% corresponds to a phase between the onset and end stages of PD. We postulate that subjects with DAT reductions 60 to 80% may fall in a suitable therapeutic range for intervention with DAT inhibitors.

DAT blockade conceivably may provide therapeutic benefits in addition to enhancing and stabilizing extracellular DA levels, reducing the threshold and therapeutic doses of L-dopa, and attenuating the L-dopa on-off syndrome. DAT inhibitors may 1) attenuate access of putative exogenous neurotoxins into DA neurons (Uhl 1998; Fleming et al., 2005); or 2) attenuate purported neurotoxic effects of L-dopa or of dopamine, by blocking DA sequestration into dopamine neurons. Albeit highly controversial, intracellular accumulation of L-dopa and DA with L-dopa treatment is implicated in accelerating neurodegeneration (for review, see Olanow et al., 2004). DAT inhibitors may 3) benefit patients in early stages of Parkinson's disease, because DAT blockade abolishes dendritic release of dopamine and resulting self-inhibition, a process that implicates carrier-mediated release of dopamine (Falkenburger et al., 2001). DAT inhibitors may 4) protect DA neurons by enhancing transport of DA toxins and metabolites into vesicles via the vesicular monoamine transporter-2. DAT inhibitors apparently up-regulate vesicular vesicular monoamine transporter-2 function (Rau et al., 2005) and 5) increase DA diffusion to postsynaptic DA receptors. We postulate that the juxtaposition of the DAT and DA release sites is conceivably misaligned with postsynaptic DA receptors in parkinsonian brains. DAT inhibitors may 6) antagonize the hypotensive or dyskinetic effects of L-dopa, as demonstrated with methylphenidate for hypotension and brasofensine for dyskinesia (Pearce et al., 2002; Nutt et al., 2004) or 7) improve mood, energy, and reduce apathy (Cantello et al., 1989; Chatterjee and Fahn, 2002; Nutt et al., 2004).

In summary, lead DAT inhibitors that alleviate specific motor deficits of MPTP-induced parkinsonism in monkeys display high DAT occupancy in vivo, low nighttime activity, adequate duration of effect, and few side effects. Pharmacokinetic properties, metabolism, toxicity, tolerance alone or in combination with L-dopa/DA receptor agonists, abuse liability, appetite suppression, and cardiovascular effects will further define their therapeutic potential. Nonetheless, the results warrant further preclinical assessment of DAT inhibitors for treating PD.

	Acknowledgements

 
For receptor screening, we thank Drs. Bryan Roth (Case Western Reserve University, Cleveland, OH) and Linda Brady (National Institute of Mental Health, Bethesda, MD). We thank Helen Panas and Ryan Johnson for technical assistance and Jennifer Carter for assistance in manuscript preparation.

	Footnotes

 
This study was supported by National Institutes of Health Grants NS30556 (NINDS), DA11558 and DA06303 (NIDA), and RR00168 (NCRR) and Boston Life Sciences, Inc. (Hopkinton, MA). The receptor-screening program was supported in part by the National Institute of Mental Health Psychoactive Drug Screening Program Grant NO1 MH80005.

This study was presented in abstract form. Madras BK (2001) Occupancy of the dopamine transporter by a transport inhibitor, as measured by PET imaging, is predictive of therapeutic efficacy for parkinsonism. J Nucl Med 42 (Suppl):210P; Madras BK (2002) The therapeutic potential of a dopamine transport (DAT) inhibitor for Parkinson's disease: comparison with a D₂ dopamine agonist in MPTP-treated monkeys. Society for Neuroscience, Washington, DC.

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

doi:10.1124/jpet.106.105312.

ABBREVIATIONS: PD, Parkinson's disease; DAT, dopamine transporter; DA, dopamine; GBR 12909, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; CFT (WIN 35,428), 2-carbomethoxy-3-4-(fluorophenyl)-tropane; SERT, serotonin transporter; NET, norepinephrine transporter; difluoropine or O-620, (S)-(+)-2-carbomethoxy-3-(di-4-fluorophenylmethoxy)tropane; O-1369, (1R)-2-(1-propanoyl)-3-(4-fluorophenyl)tropane; O-1014, 2-carbomethoxy-3-(3,4-dichlorophenyl)-8-oxabicyclo[3.2.1]oct-2-ene; O-1163, 2-carbomethoxy-3-(3,4-dichlorophenyl)-7-hydroxy-8-methyl-8-azabicyclo[3.2.1.]octane; O-1231, 2-carbomethoxy-3-(3,4-dichlorophenyl)bicyclo[3.2.1]oct-2-ene; O-1973, 2-(1-propanoyl)-3-(4-chlorophenyl)-8-oxabicyclo[3.2.1]octane; O-2099, 2-(1-propanoyl)-3-(3,4-dichlorophenyl)-7-hydroxy-8-methyl-8-azabicyclo[3.2.1]octane; O-2240, 2-(1-propanoyl)-3-(3,4-dichlorophenyl)-7-hydroxy-8-methyl-8-azabicyclo[3.2.1]octane laurate; PET, positron emission tomography; Ctrl, control; veh, vehicle; BTS 74 398, (1-[1-(3,4-dichlorophenyl)cyclobutyl]-2-(3-diaminethylaminopropylthio)ethanone monocitrate; NS 2330, tesofensine.

¹ Current affiliation: EnVivo Pharmaceuticals, Watertown, Massachusetts.

² Current affiliation: Laboratory of Cellular Molecular Neurosciences, Rockefeller University, New York, New York.

Address correspondence to: Dr. Bertha K. Madras, Department of Psychiatry, Harvard Medical School, Division of Neurochemistry, New England Primate Research Center, 1 Pine Hill Dr., Southborough, MA 01772-9102. E-mail: bertha_madras{at}hms.harvard.edu

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