Beneficial Effects of Trehalose on Striatal Dopaminergic Deficits in Rodent and Primate Models of Synucleinopathy in Parkinson's Disease.

Disease modification in Parkinson's disease (PD) is an unmet medical need. In the current study, we evaluated trehalose, a safe and well-tolerated disaccharide that has previously demonstrated efficacy in rodent models of neurodegenerative diseases, including PD. In a rat model of PD, based on delivery of adeno-associated virus serotype 1/2 containing the mutated human A53T α-synuclein gene (AAV1/2-hourA53T-aSyn) to the substantia nigra (SN), we showed that rats administered trehalose (2.67 g/kg per day, by mouth) for 6 weeks had less forelimb asymmetry (93% reduction) and higher striatal dopamine (54% increase) compared with rats receiving vehicle. In a pharmacokinetic study, we determined that efficacy was associated with plasma C max of 8900 ng/ml and area under the curve from time 0 to infinity (AUC0-inf) of 11,136 hour⋅ng/ml. We then showed, in macaques, that oral administration of trehalose (2.67 g/kg per day) produced plasma exposures of similar magnitude, with plasma C max of 10,918 ng/ml and AUC0-inf of 27,445 hour⋅ng/ml. In a macaque model of PD, also based on delivery of AAV1/2-hourA53T-aSyn to the SN, trehalose (2.67 g/kg per day, by mouth), administered for 142 days, produced higher striatal dopamine (by 39%) and dopamine transporter levels (by 50%), compared with macaques receiving vehicle. In neither model did trehalose treatment prevent loss of tyrosine hydroxylase (TH) positive (TH+ve) cells in the SN or alter α-synuclein levels in the striatum. These studies demonstrated that trehalose reduces striatal dopaminergic deficits in a rodent and macaque model of synucleinopathy in PD. Furthermore, we have determined the pharmacokinetic parameters associated with efficacy, and thus defined exposures to target in future clinical trials.

Pridopidine, a clinic-ready compound, reduces 3,4-dihydroxyphenylalanine-induced dyskinesia in Parkinsonian macaques.

BACKGROUND: Pridopidine, in development for Huntington's disease, may modulate aberrant l-dopa-induced effects including l-dopa-induced dyskinesia (LID). OBJECTIVE: This study investigated whether pridopidine could reduce LID in the MPTP macaque model of Parkinson's disease and characterized the observed behavioral effects in terms of receptor occupancy. METHODS: The pharmacokinetic profile and effects of pridopidine (15-30 mg/kg) on parkinsonism, dyskinesia, and quality of on-time, in combination with l-dopa, were assessed in MPTP macaques with LID. Pridopidine receptor occupancy was estimated using known in vitro binding affinities to σ1 and dopamine D2 receptors, in vivo PET imaging, and pharmacokinetic profiling across different species. RESULTS: Pridopidine produced a dose-dependent reduction in dyskinesia (up to 71%, 30 mg/kg) and decreased the duration of on-time with disabling dyskinesia evoked by l-dopa by 37% (20 mg/kg) and 60% (30 mg/kg). Pridopidine did not compromise the anti-parkinsonian benefit of l-dopa. Plasma exposures following the ineffective dose (15 mg/kg) were associated with full σ1 occupancy (>80%), suggesting that σ1 engagement alone is unlikely to account for the antidyskinetic benefits of pridopidine. Exposures following effective doses (20-30 mg/kg), while providing full σ1 occupancy, provide only modest dopamine D2 occupancy (<40%). However, effective pridopidine doses clearly engage a range of receptors (including adrenergic-α2C , dopamine-D3 , and serotoninergic-5-HT1A sites) to a higher degree than D2 and might contribute to the antidyskinetic actions. CONCLUSIONS: In MPTP macaques, pridopidine produced a significant decrease in LID without compromising the antiparkinsonian benefit of l-dopa. Although the actions of pridopidine were associated with full σ1 occupancy, effective exposures are more likely associated with occupancy of additional, non-sigma receptors. This complex pharmacology may underlie the effectiveness of pridopidine against LID. © 2018 International Parkinson and Movement Disorder Society.

Pharmacokinetic/Pharmacodynamic Correlation Analysis of Amantadine for Levodopa-Induced Dyskinesia.

Dyskinesia is a common motor complication associated with the use of levodopa to treat Parkinson's disease. Numerous animal studies in mice, rats, and nonhuman primates have demonstrated that the N-methyl-d-aspartate antagonist, amantadine, dose dependently reduces levodopa-induced dyskinesia (LID). However, none of these studies characterized the amantadine plasma concentrations required for a therapeutic effect. This study evaluates the pharmacokinetic (PK)/pharmacodynamic (PD) relationship between amantadine plasma concentrations and antidyskinetic efficacy across multiple species to define optimal therapeutic dosing. The PK profile of amantadine was determined in mice, rats, and macaques. Efficacy data from the 6-hydroxydopamine rat and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine macaque model of LID, along with previously published antidyskinetic efficacy data, were used to establish species-specific PK/PD relationships using a direct-effect maximum possible effect model. Results from the PK/PD model were compared with amantadine plasma concentrations and antidyskinetic effect in a phase 2 study in patients with Parkinson's disease treated with ADS-5102, an extended-release amantadine capsule formulation. Outcomes from each of the species evaluated indicate that the EC50 of amantadine for reducing dyskinesia range from 1025 to 1633 ng/ml (1367 ng/ml for an all-species model). These data are consistent with the mean amantadine plasma concentrations observed in patients with Parkinson's disease (∼1500 ng/ml) treated with ADS-5102 at doses that demonstrated a statistically significant reduction in dyskinesia. These results demonstrate that the EC50 of amantadine for reducing dyskinesia is consistent across multiple species and supports a plasma concentration target of ∼1400 ng/ml to achieve therapeutic efficacy.

The pharmacology of L-DOPA-induced dyskinesia in Parkinson's disease.

L-3,4-Dihydroxyphenylalanine (L-DOPA) remains the most effective symptomatic treatment of Parkinson's disease (PD). However, long-term administration of L-DOPA is marred by the emergence of abnormal involuntary movements, i.e., L-DOPA-induced dyskinesia (LID). Years of intensive research have yielded significant progress in the quest to elucidate the mechanisms leading to the development and expression of dyskinesia and maintenance of the dyskinetic state, but the search for a complete understanding is still ongoing. Herein, we summarize the current knowledge of the pharmacology of LID in PD. Specifically, we review evidence gathered from postmortem and pharmacological studies, both preclinical and clinical, and discuss the involvement of dopaminergic and nondopaminergic systems, including glutamatergic, opioid, serotonergic, γ-aminobutyric acid (GABA)-ergic, adenosine, cannabinoid, adrenergic, histaminergic, and cholinergic systems. Moreover, we discuss changes occurring in transcription factors, intracellular signaling, and gene expression in the dyskinetic phenotype. Inasmuch as a multitude of neurotransmitters and receptors play a role in the etiology of dyskinesia, we propose that to optimally alleviate this motor complication, it may be necessary to develop combined treatment approaches that will target simultaneously more than one neurotransmitter system. This could be achieved via three ways as follows: 1) by developing compounds that will interact simultaneously to a multitude of receptors with the required agonist/antagonist effect at each target, 2) by targeting intracellular signaling cascades where the signals mediated by multiple receptors converge, and/or 3) to regulate gene expression in a manner that has effects on signaling by multiple pathways.

Reproducibility of a Parkinsonism-related metabolic brain network in non-human primates: A descriptive pilot study with FDG PET.

BACKGROUND: We have previously defined a parkinsonism-related metabolic brain network in rhesus macaques using a high-resolution research positron emission tomography camera. This brief article reports a descriptive pilot study to assess the reproducibility of network activity and regional glucose metabolism in independent parkinsonian macaques using a clinical positron emission tomography/CT camera. METHODS: [(18)F]fluorodeoxyglucose PET scans were acquired longitudinally over 3 months in three drug-naïve parkinsonian and three healthy control cynomolgus macaques. Group difference and test-retest stability in network activity and regional glucose metabolism were evaluated graphically, using all brain images from these macaques. RESULTS: Comparing the parkinsonian macaques with the controls, network activity was elevated and remained stable over 3 months. Normalized glucose metabolism increased in putamen/globus pallidus and sensorimotor regions but decreased in posterior parietal cortices. CONCLUSIONS: Parkinsonism-related network activity can be reliably quantified in different macaques with a clinical positron emission tomography/CT scanner and is reproducible over a period typically employed in preclinical intervention studies. This measure can be a useful biomarker of disease process or drug effects in primate models of Parkinson's disease.

Pioglitazone may impair L-DOPA anti-parkinsonian efficacy in the MPTP-lesioned macaque: results of a pilot study.

In this pilot study, pioglitazone, an agonist of the peroxisome proliferator-activated receptor gamma (PPAR-γ) used in the treatment of diabetes mellitus, was administered to dyskinetic parkinsonian macaques. Pioglitazone alleviated L-DOPA-induced dyskinesia, but impaired L-DOPA anti-parkinsonian efficacy. These results suggest caution when administering pioglitazone to patients with advanced Parkinson's disease.

L-745,870 reduces the expression of abnormal involuntary movements in the 6-OHDA-lesioned rat.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinson's disease, but chronic administration is complicated by the development of dyskinesia. We have previously demonstrated that the dopamine D4 receptor antagonist L-745,870 reduces the severity of L-DOPA-induced dyskinesia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned macaque without compromising L-DOPA antiparkinsonian benefits. In the current study, we have addressed the effects of L-745,870 on the expression of L-DOPA-induced abnormal involuntary movements (AIMs) in the 6-hydroxydopamine-lesioned rat. Rats were primed with repeated L-DOPA administration, after which acute challenges of L-DOPA/L-745,870 (vehicle, 0.1, 0.3 and 1 mg/kg) were administered, and AIMs were assessed. Rotarod performance and AIMs were assessed. In L-DOPA-primed rats, L-745,870 (1 mg/kg, but not lower doses) alleviated previously established AIMs (by 84%, P<0.001). Whereas rotarod performance was significantly improved by L-DOPA/vehicle treatment, L-DOPA/L-745,870 failed to improve rotarod performance (P>0.05), suggesting that, in contrast to the MPTP-lesioned macaque, L-745,870 reduces L-DOPA antiparkinsonian benefit in the rat model. Overall, these data suggest that L-745,870 may have a narrow therapeutic window as an antidyskinetic agent in advanced Parkinson's disease.

Using artificial intelligence to identify drugs for repurposing to treat l-DOPA-induced dyskinesia.

Repurposing regulatory agency-approved molecules, with proven safety in humans, is an attractive option for developing new treatments for disease. We identified and assessed the efficacy of 3 drugs predicted by an in silico screen as having the potential to treat l-DOPA-induced dyskinesia (LID) in Parkinson's disease. We analysed ∼1.3 million Medline abstracts using natural language processing and ranked 3539 existing drugs based on predicted ability to reduce LID. 3 drugs from the top 5% of the 3539 candidates; lorcaserin, acamprosate and ganaxolone, were prioritized for preclinical testing based on i) having a novel mechanism of action, ii) having not been previously validated for the treatment of LID, iii) being blood-brain-barrier penetrant and orally bioavailable and iv) being clinical trial ready. We assessed the efficacy of acamprosate, ganaxolone and lorcaserin in a rodent model of l-DOPA-induced hyperactivity, with lorcaserin affording a 58% reduction in rotational asymmetry (P < 0.05) compared to vehicle. Acamprosate and ganaxolone failed to demonstrate efficacy. Lorcaserin, a 5HT2C agonist, was then further tested in MPTP lesioned dyskinetic macaques where it afforded an 82% reduction in LID (P < 0.05), unfortunately accompanied by a significant increase in parkinsonian disability. In conclusion, although our data do not support the repurposing of lorcaserin, acamprosate or ganaxolone per se for LID, we demonstrate value of an in silico approach to identify candidate molecules which, in combination with an in vivo screen, can facilitate clinical development decisions. The present study adds to a growing literature in support of this paradigm shifting approach in the repurposing pipeline.

Use of catechol-O-methyltransferase inhibition to minimize L-3,4-dihydroxyphenylalanine-induced dyskinesia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque.

L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia is a complication of dopaminergic treatment in Parkinson's disease. Lowering the L-DOPA dose reduces dyskinesia but also reduces the antiparkinsonian benefit. A therapy that could enhance the antiparkinsonian action of low-dose L-DOPA (LDl) without exacerbating dyskinesia would thus be of considerable therapeutic benefit. This study assessed whether catechol-O-methyltransferase (COMT) inhibition, as an add-on to LDl, might be a means to achieve this goal. Cynomolgus macaques were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Dyskinesia was established by chronic treatment with L-DOPA. Two doses of L-DOPA were identified - high-dose L-DOPA (LDh), which provided good antiparkinsonian benefit but was compromised by disabling dyskinesia, and LDl, which was sub-threshold for providing significant antiparkinsonian benefit, without dyskinesia. LDh and LDl were administered in acute challenges in combination with vehicle and, for LDl, with the COMT inhibitor entacapone (5, 15 and 45 mg/kg). The duration of antiparkinsonian benefit (ON-time), parkinsonism and dyskinesia were determined. The ON-time after LDh was ∼170 min and the ON-time after LDl alone (∼98 min) was not significantly different to vehicle (∼37 min). In combination with LDl, entacapone significantly increased the ON-time (5, 15 and 45 mg/kg being ∼123, ∼148 and ∼180 min, respectively). The ON-time after LDl/entacapone 45 mg/kg was not different to that after LDh. However, whereas the percentage ON-time that was compromised by disabling dyskinesia was ∼56% with LDh, it was only ∼31% with LDl/entacapone 45 mg/kg. In addition to the well-recognized action of COMT inhibition to reduce wearing-OFF, the data presented suggest that COMT inhibition in combination with low doses of L-DOPA has potential as a strategy to alleviate dyskinesia.

Rotigotine polyoxazoline conjugate SER-214 provides robust and sustained antiparkinsonian benefit.

Currently available dopaminergic drugs such as levodopa and dopamine (DA) receptor agonists impart considerable improvement in Parkinson's disease (PD) motor symptoms but often lead to significant motor complications including "wearing-off" and dyskinesia. Such complications are believed to stem from the pulsatile nature of dopaminergic stimulation with these agents. Continuous dopaminergic drug delivery using polyoxazoline (POZ) polymer conjugation may improve motor symptoms, while avoiding development of side effects. The purposes of the current study are to characterize the in vitro and in vivo pharmacokinetics of POZ conjugation of a U.S. Food and Drug Administration (FDA)-approved DA agonist, rotigotine, and to evaluate their effects in an established rat model of PD. After determination of release profiles of several POZ-conjugated constructs ("fast": SER-212; "moderate": SER-213; and "slow": SER-214) using in vitro hydrolysis, normal male Sprague-Dawley rats were used for determination of the pharmacokinetic profile of both acute and chronic exposure. Finally, a separate group of rats was rendered hemiparkinsonian using intracranial 6-hydroxydopamine (6-OHDA) infusions, treated acutely with POZ-rotigotine, and assessed for rotational behavior and antiparkinsonian benefit using the cylinder test. POZ-rotigotine formulations SER-213 and SER-214 led to substantial pharmacokinetic improvement compared to unconjugated rotigotine. In addition, SER-214 led to antiparkinsonian effects in DA-lesioned rats that persisted up to 5 days posttreatment. Repeated weekly dose administration of SER-214 to normal rats for up to 12 weeks demonstrated highly reproducible pharmacokinetic profiles. The continuous dopaminergic stimulation profile afforded by SER-214 could represent a significant advance in the treatment of PD, with potential to be a viable, once-per-week therapy for PD patients.

A novel MDMA analogue, UWA-101, that lacks psychoactivity and cytotoxicity, enhances L-DOPA benefit in parkinsonian primates.

Treatment of Parkinson's disease with dopaminergic agents, such as l-DOPA, is frequently compromised by disabling side effects, particularly dyskinesia and a shortening in duration of antiparkinsonian action. Studies in animal models and anecdotal evidence from a patient with Parkinson's disease show that the illicit drug ecstasy (MDMA) can alleviate these side effects, though with many drawbacks (e.g., psychoactivity). MDMA itself thus has little therapeutic potential. On the basis of known structure-psychoactivity relationships, we designed a series of α-substituted MDMA analogues, one of which, bearing an α-cyclopropyl substituent (UWA-101), enhanced the quality of l-DOPA actions in animal models. Indeed, UWA-101 was more effective than MDMA. Unlike MDMA, UWA-101 did not reduce viability of serotonergic cells, exhibit psychoactive properties, or reduce food intake, and did not substitute for MDMA in drug discrimination assays. UWA-101 displayed a unique receptor/transporter binding profile relative to MDMA, with a >5-fold decrease in affinity for NET and 5-HT(2A) receptors and a 10-fold increase in affinity for DAT. Furthermore, in a functional reuptake assay, UWA-101 inhibited both 5-HT and dopamine reuptake, while having no effect on the reuptake of noradrenaline. UWA-101 is the first selective DAT/SERT inhibitor described with comparable affinities for these two sites. These data identify a new class of therapeutic in Parkinson's disease and highlight the potential benefits of studying illicit drugs that in themselves would never be considered safe for long-term therapy.

Characterization of 3,4-methylenedioxymethamphetamine (MDMA) enantiomers in vitro and in the MPTP-lesioned primate: R-MDMA reduces severity of dyskinesia, whereas S-MDMA extends duration of ON-time.

l-3,4-dihydroxyphenylalanine (l-DOPA) is the most effective treatment for Parkinson's disease, but long-term l-DOPA administration is marred by the emergence of motor complications, namely, dyskinesia and a shortening of antiparkinsonian benefit (wearing-OFF). 3,4-methylenedioxymethamphetamine (MDMA) is unique in that it exerts antidyskinetic effects and may enhance antiparkinsonian actions of l-DOPA. MDMA is composed of two enantiomers with different pharmacological profiles; here, we describe a novel enantiospecific synthesis of the two enantiomers and expand on the previous characterization of their pharmacology. R-MDMA (rectus-MDMA) is relatively selective for 5-HT(2A) receptors, whereas S-MDMA (sinister-MDMA) inhibits both serotonin (SERT) and dopamine transporters (DAT; SERT/DAT ratio of 10 to 1). R- or S-MDMA (1, 3, and 10 mg/kg, s.c.) was administered in combination with l-DOPA (15 mg/kg, s.c.) to six female common marmosets (Callithrix jacchus) rendered parkinsonian by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) administration. Motor disability, including parkinsonism and dyskinesia, and duration of antiparkinsonian benefit (ON-time) were evaluated. After the administration of R-MDMA (3 and 10 mg/kg), the severity of peak-dose dyskinesia was decreased (by 33 and 46%, respectively; p < 0.05); although total ON-time was unchanged (approximately 220 min), the duration of ON-time with disabling dyskinesia was decreased by 90 min when compared to l-DOPA alone (69% reduction; p < 0.05). S-MDMA (1 mg/kg) increased the total ON-time by 88 min compared to l-DOPA alone (34% increase; p < 0.05), though dyskinesia were exacerbated. These data suggest that racemic MDMA exerts simultaneous effects, reducing dyskinesia and extending ON-time, by 5-HT(2A) antagonism and SERT-selective mixed monoamine uptake inhibition, which arise from its R and S enantiomers, respectively.

L-745,870 reduces L-DOPA-induced dyskinesia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson's disease.

L-DOPA-induced dyskinesia remains an unmet challenge in the treatment of Parkinson's disease (PD). Here, we investigate the potential antidyskinetic efficacy of 3-([4-(4-chlorophenyl)piperazin-1-yl]methyl)-1H-pyrrolo[2,3-b]pyridine (L-745,870), a potent and selective dopamine D(4) receptor antagonist with a good toxicology profile and an excellent safety and tolerability record in phase I/II clinical studies, for non-PD indications. Six macaques were rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration. After induction of stable and marked dyskinesia, animals were administered acute challenges of L-745,870 in combination with L-DOPA. To guarantee D(4) selectivity at the doses used in the study, we determined the plasma, cerebrospinal fluid, and brain levels of L-745,870. Coadministration of L-745,870 (1 mg/kg) and L-DOPA significantly reduced the severity of dyskinesia, by up to 59%, in comparison with L-DOPA alone (P < 0.01). L-745,870 had no effect on the duration of antiparkinsonian benefit (ON-time) (P > 0.05). However, L-745,870 (1 mg/kg) significantly increased the duration of ON-time without disabling dyskinesia (+204%; P < 0.001) and decreased duration of ON-time with disabling dyskinesia compared with L-DOPA alone (-56%; P < 0.01). Brain levels of L-745,870 (∼600 ng/g) were within the range at which L-745,870 provides selective D(4) receptor antagonism. Plasma levels were comparable with those demonstrated to be well tolerated in human studies. These data suggest that selective D(4) receptor antagonists represent a potential therapeutic approach for L-DOPA-induced dyskinesia. It is noteworthy that L-745,870 has already undergone significant clinical development, has an excellent profile for a therapeutic candidate, and could be advanced rapidly to phase IIa clinical studies for dyskinesia in PD.

A critique of available scales and presentation of the Non-Human Primate Dyskinesia Rating Scale.

Levodopa-induced dyskinesia (LID) is a major limitation of long-term management of Parkinson's disease. The roadblocks that have hindered the development of new treatments for levodopa-induced dyskinesia were discussed at a meeting organized by the Michael J. Fox Foundation for Parkinson's research (New York, NY, March 2011). Among these, the lack of consensus methodology and clinical applicability for eliciting and rating LID in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys was highlighted as a particular concern. Here we present an update on the practical use of rating scales for evaluating LID in MPTP-lesioned primate models of PD, with a focus on macaques, and present specifics on the Non-Human Primate Dyskinesia Rating Scale.

Increased levels of 5-HT1A receptor binding in ventral visual pathways in Parkinson's disease.

Visual hallucinations are common in advanced Parkinson's disease (PD). The pathophysiology of visual hallucinations may involve enhanced serotonergic neurotransmission. The atypical antipsychotics clozapine and quetiapine, which have affinity for 5-HT(2A) and 5-HT(1A) receptors, are effective against visual hallucinations in PD. 5-HT(2A) receptors are increased in ventral visual pathways in PD patients with visual hallucinations, and we hypothesized that 5-HT(1A) receptors were also involved in visual hallucinations in PD. Autoradiographic binding using [(3) H]-WAY-100,635 and NAN-190 was performed in brain sections from 6 PD patients with visual hallucinations, 6 PD patients without visual hallucinations, and 5 age-matched controls. All PD subjects had been treated with L-dopa. Brain areas studied were the orbitofrontal, inferolateral temporal, and motor cortices, as well as the striatum, globus pallidus, substantia nigra, and thalamus. 5-HT(1A) -binding levels were dramatically increased in the ventral visual pathways of all PD patients compared with controls (0 vs 11 and 0 vs 100 nmol/mg, respectively; both P < .05). There was no significant difference in 5-HT(1A) -binding levels in PD patients with visual hallucinations compared with PD patients without visual hallucinations or with controls in any of the brain areas studied (P > .05). Gross abnormalities in 5-HT(1A) levels in ventral visual areas occurred in all PD patients exposed to L-dopa. However, as there was no difference in 5-HT(1A) -binding levels between hallucinators and nonhallucinators, alterations in 5-HT(1A) receptor levels may not contribute specifically to visual hallucinations in PD. However, the discrete anatomical distribution of rises to the ventral visual areas suggests some role in predisposing to visual hallucinations.

Toward in vivo proof of binding of 18F-labeled inhibitor [18F]TRACK to peripheral tropomyosin receptor kinases.

BACKGROUND: Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are a family of tyrosine kinases primarily expressed in neuronal cells of the brain. Identification of oncogenic alterations in Trk expression as a driver in multiple tumor types has increased interest in their role in human cancers. Recently, first- and second-generation 11C and 18F-labeled Trk inhibitors, e.g., [18F]TRACK, have been developed. The goal of the present study was to analyze the direct interaction of [18F]TRACK with peripheral Trk receptors in vivo to prove its specificity for use as a functional imaging probe. METHODS: In vitro uptake and competition experiments were carried out using the colorectal cancer cell line KM12. Dynamic PET experiments were performed with [18F]TRACK, either alone or in the presence of amitriptyline, an activator of Trk, entrectinib, a Trk inhibitor, or unlabeled reference compound TRACK in KM12 tumor-bearing athymic nude mice as well as B6129SF2/J and corresponding B6;129S2-Ntrk2tm1Bbd/J mice. Western blot and immunohistochemistry experiments were done with KM12 tumors, brown adipose tissue (BAT), and brain tissue samples. RESULTS: Uptake of [18F]TRACK was increasing over time reaching 208 ± 72% radioactivity per mg protein (n = 6/2) after 60 min incubation time. Entrectinib and TRACK competitively blocked [18F]TRACK uptake in vitro (IC50 30.9 ± 3.6 and 29.4 ± 9.4 nM; both n = 6/2). [18F]TRACK showed uptake into KM12 tumors (SUVmean,60 min 0.43 ± 0.03; n = 6). Tumor-to-muscle ratio reached 0.9 (60 min) and 1.2 (120 min). In TrkB expressing BAT, [18F]TRACK uptake reached SUVmean,60 min 1.32 ± 0.08 (n = 7). Activation of Trk through amitriptyline resulted in a significant radioactivity increase of 21% in KM12 tumor (SUVmean,60 min from 0.53 ± 0.01 to 0.43 ± 0.03; n = 6; p < 0.05) and of 21% in BAT (SUVmean,60 min from 1.32 ± 0.08; n = 5 to 1.59 ± 0.07; n = 6; p < 0.05) respectively. Immunohistochemistry showed TrkB > TrkA expression on BAT fat cells, but TrkA > TrkB in whole brain. WB analysis showed sevenfold higher TrkB expression in BAT versus KM12 tumor tissue. CONCLUSION: The present data show that radiotracer [18F]TRACK can target peripheral Trk receptors in human KM12 colon cancer as well as brown adipose tissue as confirmed through in vitro and in vivo blocking experiments. Higher TrkB versus TrkA protein expression was detected in brown adipose tissue of mice confirming a peripheral functional role of brain-derived neurotrophic factor in adipose tissue.

Altered function of glutamatergic cortico-striatal synapses causes output pathway abnormalities in a chronic model of parkinsonism.

OBJECTIVE: In Parkinson's disease, chronic striatal dopamine depletion results in over-activity and under-activity of the indirect and direct striatal output pathways respectively. In this study, we investigated changes in the function of glutamatergic cortico-striatal synapses that contribute to abnormalities in striatal efferents. METHODS: Whole-cell recordings were performed in striatal slices prepared from adult bacterial artificial chromosome mice, chronically lesioned with 6-hydroxydopamine (6-OHDA). Paired pulse facilitation, spontaneous synaptic activity, the ratio of AMPAR to NMDAR-mediated components of excitatory postsynaptic currents, AMPAR and NMDAR kinetics, current-voltage relationship and intrinsic membrane properties were assessed in indirect and direct pathway medium spiny neurons (MSNs), which were identified on the basis of expression of GFP, driven by the promoters of A2A or D1 receptor expression. The trajectory of striatal efferents, with respect to selective targeting of the globus pallidus and substantia nigra was also compared in sham-operated versus 6-OHDA-lesioned mice. RESULTS: Dopamine depletion did not affect the number of pathway specific output neurons or the trajectory of striatal outputs. In sham-operated animals, cortico-striatal synapses of both striatal efferent populations exhibited paired pulse facilitation and similar ratios of AMPAR to NMDAR-mediated components of excitatory postsynaptic currents. Following striatal dopamine depletion, indirect pathway neurons exhibited decreased levels of paired pulse facilitation, enhanced sensitivity to presynaptic stimulation and an increase in the relative contribution of NMDAR to the EPSC but no change in spontaneous synaptic activity. In sham-operated mice, neurons of the direct pathway exhibited lower firing frequency compared to the indirect pathway following current injection. However, in 6-OHDA-lesioned mice, in the direct pathway, firing threshold was reduced, spike frequency adaptation developed and the frequency of spontaneous activity was also reduced. In addition, changes in the properties of NMDAR kinetics suggest that these receptors were desensitised. DISCUSSION: Increased synchronicity between pre and postsynaptic neurons, as indicated by decreased paired pulse facilitation, and increased sensitivity to extracellular stimulation, combined with an increase in the contribution of NMDARs to the EPSC at cortico-striatal synapses, may contribute to the over-activity of indirect pathway neurons in the parkinsonian striatum. In contrast, a decrease in spontaneous activity, postsynaptic desensitisation to excitatory stimuli and spike frequency adaptation of cortico-striatal synapses may underlie under-activity of the direct pathway.

Fatty acid amide hydrolase (FAAH) inhibition reduces L-3,4-dihydroxyphenylalanine-induced hyperactivity in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned non-human primate model of Parkinson's disease.

Dopaminergic therapies remain the most efficacious symptomatic treatments for Parkinson's disease (PD) but are associated with motor complications, including dyskinesia, and nonmotor complications, such as psychosis, impulse control disorders (ICD), and dopamine dysregulation syndrome (DDS). Nondopaminergic neurotransmitter systems, including the endocannabinoid system, are probably critical to the development of these complications. The role of fatty acid amide hydrolase (FAAH) in mediating l-3,4-dihydroxyphenylalanine (L-DOPA)-induced behaviors was explored in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned marmoset model of PD. Pharmacodynamic and locomotor effects of the selective FAAH inhibitor [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (URB597) were assessed via bioanalytical (liquid chromatography-tandem mass spectrometry) and behavioral observation approaches. URB597 (3, 10, 30, or 60 mg/kg p.o.) increased plasma levels of the FAAH substrates N-arachidonoyl ethanolamide (anandamide), N-oleoyl ethanolamide, and N-palmitoyl ethanolamide by 10.3 ± 0.3-, 7.8 ± 0.2-, and 1.8 ± 0.1-fold (mean of URB597 groups ± S.E.M.), respectively, compared with vehicle (all p < 0.001) 4 h after administration. Treatment with L-DOPA (20 mg/kg s.c.) alleviated parkinsonism but elicited dyskinesia, psychosis-like-behaviors and hyperactivity, a potential correlate of ICD and DDS. During the 2 to 4 h after L-DOPA, corresponding to 4 to 6 h after URB597 administration, URB597 reduced total L-DOPA-induced activity and the magnitude of hyperactivity by 32 and 52%, respectively, to levels equivalent to those seen in normal animals. Treatment with URB597 (10 mg/kg p.o.) did not modify the antiparkinsonian actions of L-DOPA or L-DOPA-induced dyskinesia and psychosis. URB597 did not alter plasma L-DOPA levels and was without behavioral effects when administered alone. Inhibition of FAAH may represent a novel approach to reducing L-DOPA-induced side effects, such as ICD and DDS, while maintaining the antiparkinsonian benefits of L-DOPA treatment.

The selective mu-opioid receptor antagonist ADL5510 reduces levodopa-induced dyskinesia without affecting antiparkinsonian action in MPTP-lesioned macaque model of Parkinson's disease.

In Parkinson's disease (PD), dyskinesia develops following long-term treatment with 3,4-dihydroxyphenylalanine (L-dopa). Given the prominent role of the opioid system in basal ganglia function, nonselective opioid receptor antagonists have been tested for antidyskinetic efficacy in the clinic (naltrexone and naloxone), although without success. In the current study, ADL5510, a novel, orally active opioid antagonist with mu opioid receptor selectivity, was examined in L-dopa-treated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) macaques. Antidyskinetic effects were compared with those of naltrexone. Parkinsonian monkeys with established L-dopa-induced dyskinesia (LID) received acute challenges with L-dopa (subcutaneously) in combination with either vehicle, ADL5510 (0.1, 1, 3 or 10 mg/kg by mouth), or naltrexone (1, 3, or 10 mg/kg subcutaneously). Following treatments, behavior was monitored for 6 hours. Parameters assessed were total activity, parkinsonism, and dyskinesia. ADL5510 (1, 3, and 10 mg/kg) reduced activity and LID (chorea and dystonia) without affecting the antiparkinsonian benefits of L-dopa. The antidyskinetic effect of ADL5510 showed a U-shaped dose-response. It was inactive at 0.1 mg/kg, efficacious at 1 and 3 mg/kg (72% and 40% reductions, respectively), and then less effective at 10 mg/kg. The quality of ON time produced by L-dopa was improved, as indicated by a reduction in the percentage of ON time spent experiencing disabling dyskinesia (70% and 61% reductions with 1 and 3 mg/kg, respectively, compared with L-dopa). Naltrexone, in contrast, did not alleviate LID or affect the antiparkinsonian actions of L-dopa. Mu-selective opioid antagonists have the potential to form the basis of novel antidyskinetic therapies for PD.

A novel dopamine D3R agonist SK609 with norepinephrine transporter inhibition promotes improvement in cognitive task performance in rodent and non-human primate models of Parkinson's disease.

Mild cognitive impairment is present in a number of neurodegenerative disorders including Parkinson's disease (PD). Mild cognitive impairment in PD (PD-MCI) often manifests as deficits in executive functioning, attention, and spatial and working memory. Clinical studies have suggested that the development of mild cognitive impairment may be an early symptom of PD and may even precede the onset of motor impairment by several years. Dysfunction in several neurotransmitter systems, including dopamine (DA), norepinephrine (NE), may be involved in PD-MCI, making it difficult to treat pharmacologically. In addition, many agents used to treat motor impairment in PD may exacerbate cognitive impairment. Thus, there is a significant unmet need to develop therapeutics that can treat both motor and cognitive impairments in PD. We have recently developed SK609, a selective, G-protein biased signaling agonist of dopamine D3 receptors. SK609 was successfully used to treat motor impairment and reduce levodopa-induced dyskinesia in a rodent model of PD. Further characterization of SK609 suggested that it is a selective norepinephrine transporter (NET) inhibitor with the ability to increase both DA and NE levels in the prefrontal cortex. Pharmacokinetic analysis of SK609 under systemic administration demonstrated 98% oral bioavailability and high brain distribution in striatum, hippocampus and prefrontal cortex. To evaluate the effects of SK609 on cognitive deficits of potential relevance to PD-MCI, we used unilateral 6-hydroxydopamine (6-OHDA) lesioned rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cynomolgus macaques, with deficits in performance in a sustained attention and an object retrieval task, respectively. SK609 dose dependently improved the performance of 6-OHDA-lesioned rats, with peak performance achieved using a 4 mg/kg dose. This improvement was predominantly due to a significant reduction in the number of misses and false alarm errors, contributing to an increase in sustained attention. In MPTP-lesioned monkeys, this same dose also improved performance in an object retrieval task, significantly reducing cognitive errors (barrier reaches) and motor errors (fine motor dexterity problems). These data demonstrate that SK609 with its unique pharmacological effects on modulating both DA and NE can ameliorate cognitive impairment in PD models and may provide a therapeutic option to treat both motor and cognitive impairment in PD patients.