Search

Parkinson's disease (PD) is a major cause of morbidity and mortality among older individuals. Several researchers have suggested that iron chelators which cross the blood-brain barrier (BBB) might have clinical efficacy in treating PD. Therefore, efforts are made not only in order to improve the effect of L-dopa but also to introduce drugs which provide anti-parkinsonian and neuroprotective effects. In this study, quercetin, a flavonoid, exhibited noticeable neuroprotective effects via iron induced-oxidative stress-dependent apoptotic pathways. Our results suggested that quercetin significantly decreased the catalepsy and exhibited neuroprotective effects in rotenone-induced Parkinson. A model of rotenone-induced Parkinsonism in rats produced the decrease in glutathione, SOD, catalase, and serum iron concentration and the increase in H2O2 and lipid peroxidation activity. Quercetin efficiently halted the deleterious toxic effects of L-dopa, revealing normalization of catalepsy and rotarod score, in addition to amelioration of neurochemical parameters, indicating benefit of both symptomatic and neuroprotective therapies. In silico molecular docking studies have also shown that quercetin could be an ideal potential drug target for aromatic L-amino acid decarboxylase and human catechol-O-methyltransferase. In conclusion, quercetin possesses strong iron-chelating abilities and could be recommended as a disease-modifying therapy when administered in combination with L-dopa, early on in the course of Parkinson's disease.

Dopamine (DA)-replacement therapy utilizing l-DOPA is the gold standard symptomatic treatment for Parkinson's disease (PD). A critical complication of this therapy is the development of l-DOPA-induced dyskinesia (LID). The endogenous opioid peptides, including enkephalins and dynorphin, are co-transmitters of dopaminergic, GABAergic, and glutamatergic transmission in the direct and indirect striatal output pathways disrupted in PD, and alterations in expression levels of these peptides and their precursors have been implicated in LID genesis and expression. We have previously shown that the opioid glycopeptide drug MMP-2200 (a.k.a. Lactomorphin), a glycosylated derivative of Leu-enkephalin mediates potent behavioral effects in two rodent models of striatal DA depletion. In this study, the mixed mu-delta agonist MMP-2200 was investigated in standard preclinical rodent models of PD and of LID to evaluate its effects on abnormal involuntary movements (AIMs). MMP-2200 showed antiparkinsonian activity, while increasing l-DOPA-induced limb, axial, and oral (LAO) AIMs by ∼10%, and had no effect on dopamine receptor 1 (D 1 R)-induced LAO AIMs. In contrast, it markedly reduced dopamine receptor 2 (D 2 R)-like-induced LAO AIMs. The locomotor AIMs were reduced by MMP-2200 in all three conditions. The N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 has previously been shown to be anti-dyskinetic, but only at doses that induce parkinsonism. When MMP-2200 was co-administered with MK-801, MK-801-induced pro-parkinsonian activity was suppressed, while a robust anti-dyskinetic effect remained. In summary, the opioid glycopeptide MMP-2200 reduced AIMs induced by a D 2 R-like agonist, and MMP-2200 modified the effect of MK-801 to result in a potent reduction of l-DOPA-induced AIMs without induction of parkinsonism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study investigated the effects of ombuoside on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced neurotoxicity in PC12 cells. Ombuoside did not affect cell viability at concentrations of up to 50 µM for 24 h, and ombuoside (1, 5, and 10 µM) significantly inhibited L-DOPA-induced (100 and 200 µM) decreases in cell viability. L-DOPA (100 and 200 µM) induced sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) for 6 h, which were significantly decreased by cotreatments with ombuoside (1, 5, and 10 µM). L-DOPA (100 and 200 µM) alone significantly increased c-Jun N-terminal kinase (JNK1/2) phosphorylation for 6 h and cleaved-caspase-3 expression for 24 h, both of which were partially, but significantly, blocked by ombuoside (1, 5, and 10 µM). In addition, ombuoside (1, 5, and 10 µM) significantly restored the L-DOPA-induced (100 and 200 µM) decrease in superoxide dismutase (SOD) activity for 24 h. Taken together, these findings indicate that ombuoside protects against L-DOPA-induced neurotoxicity by inhibiting L-DOPA-induced increases in sustained ERK1/2 and JNK1/2 phosphorylation and caspase-3 expression and L-DOPA-induced decrease in SOD activity in PC12 cells. Thus, ombuoside might represent a novel neuroprotective agent that warrants further study., Competing Interests: The authors declare no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Background and Purpose: We previously showed that nociceptin/orphanin FQ opioid peptide (NOP) receptor agonists attenuate the expression of levodopa-induced dyskinesia in animal models of Parkinson's disease. We now investigate the efficacy of two novel, potent and chemically distinct NOP receptor agonists, AT-390 and AT-403, to improve Parkinsonian disabilities and attenuate dyskinesia development and expression., Experimental Approach: Binding affinity and functional efficacy of AT-390 and AT-403 at the opioid receptors were determined in radioligand displacement assays and in GTPγS binding assays respectively, conducted in CHO cells. Their anti-Parkinsonian activity was evaluated in 6-hydroxydopamine hemi-lesioned rats whereas the anti-dyskinetic properties were assessed in 6-hydroxydopamine hemi-lesioned rats chronically treated with levodopa. The ability of AT-403 to inhibit the D 1 receptor-induced phosphorylation of striatal ERK was investigated., Key Results: AT-390 and AT-403 selectively improved akinesia at low doses and disrupted global motor activity at higher doses. AT-403 palliated dyskinesia expression without causing sedation in a narrow therapeutic window, whereas AT-390 delayed the appearance of abnormal involuntary movements and increased their duration at doses causing sedation. AT-403 did not prevent the priming to levodopa, although it significantly inhibited dyskinesia on the first day of administration. AT-403 reduced the ERK phosphorylation induced by SKF38393 in vitro and by levodopa in vivo., Conclusions and Implications: NOP receptor stimulation can provide significant albeit mild anti-dyskinetic effect at doses not causing sedation. The therapeutic window, however, varies across compounds. AT-403 could be a potent and selective tool to investigate the role of NOP receptors in vivo., (© 2017 The British Pharmacological Society.)

Background: We evaluated the effects of 3-O-methyldopa (3-OMD), a metabolite of L-DOPA which is formed by catechol-O-methyltransferase (COMT), on the uptake, metabolism, and neuroprotective effects of L-DOPA in striatal astrocytes. We examined changes in the numbers of dopaminergic neurons after treatment with L-DOPA and 3-OMD or entacapone, a peripheral COMT inhibitor, using primary cultured mesencephalic neurons and striatal astrocytes., Results: The number of tyrosine hydroxylase-positive dopaminergic neurons was not affected by L-DOPA treatment in mesencephalic neurons alone. However, the increase in viability of dopaminergic neurons in the presence of astrocytes was further enhanced after methyl-L-DOPA treatment (25 µM) in mixed cultured mesencephalic neurons and striatal astrocytes. The neuroprotective effect of 25 µM L-DOPA was almost completely inhibited by simultaneous treatment with 3-OMD (10 or 100 µM), and was enhanced by concomitant treatment with entacapone (0.3 µM). The uptake of L-DOPA into and the release of glutathione from striatal astrocytes after L-DOPA treatment (100 µM) were inhibited by simultaneous exposure to 3-OMD (100 µM)., Conclusions: These data suggest that L-DOPA exerts its neuroprotective effect on dopaminergic neurons via astrocytes and that 3-OMD competes with L-DOPA by acting on target molecule(s) (possibly including glutathione) released from astrocytes. Since some amount of entacapone can cross the blood-brain barrier, this reagent may enhance L-DOPA transportation by inhibiting COMT and increase the astrocyte-mediated neuroprotective effects of L-DOPA on dopaminergic neurons.

Levodopa possesses antinociceptive actions against several somatic pain conditions. However, we do not know at this moment whether levodopa is also effective to visceral pain. The present study was therefore performed to clarify whether levodopa is effective to visceral pain and its mechanisms. Visceral sensation was evaluated by colonic distension-induced abdominal withdrawal reflex (AWR) in conscious rats. Subcutaneously (80 mg/rat) or intracisternally (2.5 μg/rat) administered levodopa significantly increased the threshold of colonic distension-induced AWR in conscious rats. The dose difference to induce the antinociceptive action suggests levodopa acts centrally to exert its antinociceptive action against colonic distension. While neither sulpiride, a D2 dopamine receptor antagonist, nor SCH23390, a D1 dopamine receptor antagonist by itself changed the threshold of colonic distension-induced AWR, the intracisternally injected levodopa-induced antinociceptive action was significantly blocked by pretreatment with subcutaneously administered sulpiride but not SCH23390. Treatment with intracisternal SB334867, an orexin 1 receptor antagonist, significantly blocked the subcutaneously administered levodopa-induced antinociceptive action. These results suggest that levodopa acts centrally to induce an antinociceptive action against colonic distension through activation of D2 dopamine receptors and the orexinergic system in the brain., (Copyright © 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.)

The neurotoxicity of levodopa (L-DOPA) on neural stem cells (NSCs) and treatment strategies to protect NSCs from this neurotoxicity remain to be elucidated. Recently, an active form of vitamin D3 has been reported to display neuroprotective properties. Therefore, we investigated the protective effect of 1,25-dyhydroxyvitamin D3 (calcitriol) on L-DOPA-induced NSC injury. We measured cell viability via the cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays and Annexin V/PI staining followed by flow cytometry, cell proliferation using the BrdU and colony-forming unit (CFU) assays, cell differentiation via immunocytochemistry, the levels of free radicals via 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, apoptosis via DAPI and TUNEL staining, and intracellular signaling protein expression via Western blot. Antibody microarrays were also employed to detect changes in the expression of prosurvival- and death-related proteins. Treatment of NSCs with L-DOPA reduced their viability and proliferation. This treatment also increased the levels of free radicals and decreased the expression levels of intracellular signaling proteins that are associated with cell survival. However, simultaneous exposure to calcitriol significantly reduced these effects. The calcitriol-mediated protection against L-DOPA toxicity was blocked by the phosphoinositide 3-kinase (PI3K) inhibitor LY294004. L-DOPA also inhibited the expression of Nestin and Ki-67, and co-treatment with calcitriol alleviated these effects. The expression levels of GFAP, DCX, and Tuj1 were not significantly affected by treatment with L-DOPA or calcitriol. Calcitriol protects against L-DOPA-induced NSC injury by promoting prosurvival signaling, including activation of the PI3K pathway, and reducing oxidative stress.

The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) has been used as an effective drug for treating dopamine depletion-induced Parkinson's disease (PD). However, long-term administration of L-DOPA produces motor complications. L-DOPA has also been found to modify the two key signaling cascades, protein kinase A/dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), in striatal neurons, which are thought to play a pivotal role in forming motor complications. In the present study, we tested the possible effect of a CB1 cannabinoid receptor agonist on L-DOPA-stimulated abnormal behavioral and signaling responses in vivo. Intermittent L-DOPA administration for 3 weeks induced motor fluctuation in a rat model of PD induced by intrastriatal infusion of dopamine-depleting neurotoxin 6-hydroxydopamine (6-OHDA). A single injection of a CB1 cannabinoid receptor agonist WIN-55,212-2 had no effect on L-DOPA-induced motor fluctuation. However, chronic injections of WIN-55,212-2 significantly attenuated abnormal behavioral responses to L-DOPA in 6-OHDA-lesioned rats. Similarly, chronic injections of WIN-55,212-2 influence the L-DOPA-induced alteration of DARPP-32 and ERK1/2 phosphorylation status in striatal neurons. These data provide evidence for the active involvement of CB1 cannabinoid receptors in the regulation of L-DOPA action during PD therapy.

Background: The cosmetics market has rapidly increased over the last years. For example, in 2011 it reached 242.8 billion US dollars, which was a 3.9% increase compared to 2010. There have been many recent trials aimed at finding the functional ingredients for new cosmetics. Gallic acid is a phytochemical derived from various herbs, and has anti-fungal, anti-viral, and antioxidant properties. Although phytochemicals are useful as cosmetic ingredients, they have a number of drawbacks, such as thermal stability, residence time in the skin, and permeability through the dermal layer. To overcome these problems, we considered conjugation of gallic acid with a peptide., Results: We synthesized galloyl-RGD, which represents a conjugate of gallic acid and the peptide RGD, purified it by HPLC and characterized by MALDI-TOF with the aim of using it as a new cosmetic ingredient. Thermal stability of galloyl-RGD was tested at alternating temperatures (consecutive 4°C, 20°C, or 40°C for 8 h each) on days 2, 21, 41, and 61. Galloyl-RGD was relatively safe to HaCaT keratinocytes, as their viability after 48 h incubation with 500 ppm galloyl-RGD was 93.53%. In the group treated with 50 ppm galloyl-RGD, 85.0% of free radicals were removed, whereas 1000 ppm galloyl-RGD suppressed not only L-DOPA formation (43.8%) but also L-DOPA oxidation (54.4%)., Conclusions: Galloyl-RGD is a promising candidate for a cosmetic ingredient.

L-3,4-Dihydroxyphenylalanine (l-DOPA), the gold standard therapy for Parkinson disease (PD), is associated with motor fluctuations and dyskinesias. This study sought to prevent the development of l-DOPA-induced dyskinesias (LID) with the metabotropic glutamate receptor type 5 (mGlu5 receptor) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) in the de novo treatment of monkeys lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a PD model. MPTP-lesioned monkeys were treated once daily for one month with either l-DOPA or l-DOPA + MPEP (10 mg/kg). MPEP (administered 15 min before l-DOPA) plasma concentrations were elevated during all the l-DOPA motor activation and did not accumulate during a month. The antiparkinsonian effect was maintained throughout the treatment period in MPTP-lesioned monkeys treated with l-DOPA + MPEP, while the duration of this effect decreased over time in MPTP-lesioned monkeys treated with l-DOPA alone, suggesting wearing-off. Over the month-long treatment, the mean dyskinesia score increased in l-DOPA-treated monkeys; interestingly, this increase was reduced by overall 72% in the l-DOPA + MPEP group. Mean dyskinesia scores of monkeys correlated inversely with plasma MPEP concentrations. Normal control and saline-treated MPTP-lesioned monkeys were also included for biochemical analyses. All MPTP-lesioned monkeys were extensively and similarly denervated. [(3)H]ABP688 specific binding to mGlu5 receptors increased in the putamen of l-DOPA-treated monkeys compared to control, saline or l-DOPA + MPEP-treated monkeys. Mean dyskinesia scores of MPTP-lesioned monkeys correlated positively with [(3)H]ABP688 specific binding in the putamen. This study showed a beneficial chronic antidyskinetic effect of MPEP in de novol-DOPA-treated MPTP-lesioned monkeys, supporting the therapeutic use of mGlu5 receptor antagonists in PD to prevent LID. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A variety of polyphenol antioxidant compounds derived from natural products have demonstrated neuroprotective activity against neuronal cell death. The objective of this study was to investigate the effect of resveratrol (RESV) and bioflavonoids in attenuating hydrogen peroxide (H(2)O(2))-induced oxidative stress in neuronal cells. H2O2 levels were increased by the addition of L-3,4-dihydroxyphenylalanine (L-DOPA) to cultured dopaminergic SKNSH cells. H(2)O(2) was monitored by peroxyfluor-1, a selective H(2)O(2) optical probe. To examine the neuroprotective effects of RESV and bioflavonoids against L-DOPA, we cotreated RESV, quercetin, or (-) epigallocatechin gallate with L-DOPA and monitored for H(2)O(2) levels. The combination of RESV and L-DOPA was 50% more effective at reducing H(2)O(2) levels than the combination of quercetin or epigallocatechin gallate with L-DOPA. However, the combination of each antioxidant with L-DOPA was effective at preserving cell viability.

Prostaglandin H synthase exerts not only cyclooxygenase activity but also peroxidase activity. The latter activity of the enzyme is thought to couple with oxidation of dopamine to dopamine quinone. Therefore, it has been proposed that cyclooxygenase inhibitors could suppress dopamine quinone formation. In the present study, we examined effects of various cyclooxygenase inhibitors against excess methyl L-3,4-dihydroxyphenylalanine (L-DOPA)-induced quinoprotein (protein-bound quinone) formation and neurotoxicity using dopaminergic CATH.a cells. The treatment with aspirin inhibited excess methyl L-DOPA-induced quinoprotein formation and cell death. However, acetaminophen did not show protective effects, and indomethacin and meloxicam rather aggravated these methyl L-DOPA-induced changes. Aspirin and indomethacin did not affect the level of glutathione that exerts quenching dopamine quinone in dopaminergic cells. In contrast with inhibiting effects of higher dose in the previous reports, relatively lower dose of aspirin that affected methyl L-DOPA-induced quinoprotein formation and cell death failed to prevent cyclooxygenase-induced dopamine chrome generation in cell-free system. Furthermore, aspirin but not acetaminophen or meloxicam showed direct dopamine quinone-scavenging effects in dopamine-semiquinone generating systems. The present results suggest that cyclooxygenase shows little contribution to dopamine oxidation in dopaminergic cells and that protective effects of aspirin against methyl L-DOPA-induced dopamine quinone neurotoxicity are based on its cyclooxygenase-independent property.

The effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Sesamin at concentration ranges of 20-75 μM exhibited a significant increase in intracellular dopamine levels at 24 h: 50 μM sesamin increased dopamine levels to 133% and tyrosine hydroxylase (TH) activity to 128.2% of control levels. Sesamin at 20-100 μM rapidly increased the intracellular levels of cyclic AMP (cAMP) to 158.3%-270.3% of control levels at 30 min. At 50 μM, sesamin combined with L-DOPA (50, 100 and 200 μM) further increased the intracellular dopamine levels for 24 h compared to L-DOPA alone. In the absence or presence of L-DOPA (100 and 200 μM), sesamin (50 μM) increased the phosphorylation of TH, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), as well as the mRNA levels of TH and CREB for 24 h, an effect which was reduced by L-DOPA (100 and 200 μM). In addition, 50 μM sesamin exhibited a protective effect against L-DOPA (100 and 200 μM)-induced cytotoxicity via the inhibition of reactive oxygen species (ROS) production and superoxide dismutase reduction, induction of extracellular signal-regulated kinase (ERK)1/2 and BadSer112 phosphorylation and Bcl-2 expression, and inhibition of cleaved-caspase-3 formation. These results suggested that sesamin enhanced dopamine biosynthesis and L-DOPA-induced increase in dopamine levels by inducing TH activity and TH gene expression, which was mediated by cAMP-PKA-CREB systems. Sesamin also protected against L-DOPA (100-200 μM)-induced cytotoxicity through the suppression of ROS activity via the modulation of ERK1/2, BadSer112, Bcl-2, and caspase-3 pathways in PC12 cells. Therefore, sesamin might serve as an adjuvant phytonutrient for neurodegenerative diseases., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Levodopa (L-dopa) is the most commonly used treatment for alleviating symptoms of Parkinson's disease. However, L-dopa delays gastric emptying, which dampens its absorption. We investigated whether ghrelin prevents L-dopa action on gastric emptying and enhances circulating L-dopa in rats., Methods: Gastric emptying of non-nutrient methylcellulose/phenol red viscous solution was determined in fasted rats treated with orogastric or intraperitoneal (i.p.) L-dopa, or intravenous (i.v.) ghrelin 10 min before orogastric L-dopa. Plasma L-dopa and dopamine levels were determined by high pressure liquid chromatography. Plasma acyl ghrelin levels were assessed by radioimmunoassay. Fos expression in the brain was immunostained after i.v. ghrelin (30 μg kg(-1)) 10 min before i.p. L-dopa., Key Results: Levodopa (5 and 15 mg kg(-1)) decreased significantly gastric emptying by 32% and 62%, respectively, when administered orally, and by 91% and 83% when injected i.p. Ghrelin (30 or 100 μg kg(-1), i.v.) completely prevented L-dopa's (15 mg kg(-1), orogastrically) inhibitory action on gastric emptying and enhanced plasma L-dopa and dopamine levels compared with vehicle 15 min after orogastric L-dopa. Levodopa (5 mg kg(-1)) did not modify plasma acyl ghrelin levels at 30 min, 1, and 2 h after i.v. injection. Levodopa (15 mg kg(-1), i.p.) induced Fos in brain autonomic centers, which was not modified by i.v. ghrelin., Conclusions & Inferences: Ghrelin counteracts L-dopa-induced delayed gastric emptying but not Fos induction in the brain and enhances circulating L-dopa levels. Potential therapeutic benefits of ghrelin agonists in Parkinson's disease patients treated with L-dopa remain to be investigated., (© 2012 Blackwell Publishing Ltd.)

In searching for a new material made from natural resources that could be used as a whitening agent, we focused on the plants used for skin treatment by the native people of East Kalimantan. The methanol extract of the leaves of Eupatorium triplinerve Vahl showed antimelanogenesis activity in a melanin biosynthesis assay. By activity-guided fractionation, 7-methoxycoumarin (1) was isolated as an active compound. The IC50 of 1 on mushroom tyrosinase was 2360 μM (L-tyrosine was used as the substrate) and above 2840 μM (L-DOPA was used as the substrate), respectively. Regarding melanin formation inhibition in B16 melanoma cells, the IC50 of 1 was 1780 μM with 83% cell viability at IC50. Based on these results, we validated that the leaf extract is in line with the traditional use of the Dayak tribe in East Kalimantan., (Copyright © 2012. Published by Elsevier B.V.)

Amantadine is the only drug marketed for treating levodopa-induced dyskinesia. However, its impact on basal ganglia circuitry in the dyskinetic brain, particularly on the activity of striatofugal pathways, has not been evaluated. We therefore used dual probe microdialysis to investigate the effect of amantadine on behavioral and neurochemical changes in the globus pallidus and substantia nigra reticulata of 6-hydroxydopamine hemi-lesioned dyskinetic mice and rats. Levodopa evoked abnormal involuntary movements (AIMs) in dyskinetic mice, and simultaneously elevated GABA release in substantia nigra reticulata (∼3-fold) but not globus pallidus. Glutamate levels were unaffected in both areas. Amantadine (40 mg/kg, i.p.), ineffective alone, attenuated (∼50%) AIMs expression and prevented the GABA rise. Moreover, it unraveled a facilitatory effect of levodopa on pallidal glutamate levels. Levodopa also evoked AIMs expression and a GABA surge (∼2-fold) selectively in the substantia nigra of dyskinetic rats. However, different from mice, glutamate levels rose simultaneously. Amantadine, ineffective alone, attenuated (∼50%) AIMs expression preventing amino acid increase and leaving unaffected pallidal glutamate. Overall, the data provide neurochemical evidence that levodopa-induced dyskinesia is accompanied by activation of the striato-nigral pathway in both mice and rats, and that the anti-dyskinetic effect of amantadine partly relies on the modulation of this pathway., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

L-DOPA-induced dyskinesias or abnormal involuntary movements (AIMs) are a debilitating adverse complication associated with prolonged L-DOPA administration for Parkinson's disease. Few treatments are currently available for dyskinesias. Our recent data showed that nicotine reduced L-DOPA-induced AIMs in parkinsonian animal models. An important question is the nicotinic acetylcholine receptor (nAChR) subtypes through which nicotine exerts this beneficial effect, because such knowledge would allow for the development of drugs that target the relevant receptor population(s). To address this, we used β2 nAChR subunit knockout [β2(-/-)] mice because β2-containing nAChRs are key regulators of nigrostriatal dopaminergic function. All of the mice were lesioned by intracranial injection of 6-hydroxydopamine into the right medial forebrain bundle. Lesioning resulted in a similar degree of nigrostriatal damage and parkinsonism in β2(-/-) and wild-type mice. All of the mice then were injected with L-DOPA (3 mg/kg) plus benserazide (15 mg/kg) once daily for 4 weeks until AIMs were fully developed. L-DOPA-induced AIMs were approximately 40% less in the β2(-/-) mice compared with the wild-type mice. It is interesting to note that nicotine (300 μg/ml in drinking water) reduced L-DOPA-induced AIMs by 40% in wild-type mice but had no effect in β2(-/-) mice with partial nigrostriatal damage. The nicotine-mediated decline in AIMs was much less pronounced in wild-type mice with near-complete degeneration, suggesting that presynaptic nAChRs on dopaminergic terminals have a major influence. These data demonstrate an essential role for β2* nAChRs in the antidyskinetic effect of nicotine and suggest that drugs targeting these subtypes may be useful for the management of L-DOPA-induced dyskinesias in Parkinson's disease.

L-dopa therapy for Parkinson's disease leads to dyskinesias or abnormal involuntary movement (AIMs) for which there are few treatment options. Our previous data showed that nicotine administration reduced L-dopa-induced AIMs in parkinsonian monkeys and rats. To further understand how nicotine mediates its antidyskinetic action, we investigated the effect of nicotinic receptor (nAChR) agonists in unilateral 6-OHDA-lesioned rats with varying striatal damage. We first tested the drugs in L-dopa-treated rats with a near-complete striatal dopamine lesion (>99%), the standard rodent dyskinesia model. Varenicline, an agonist that interacts with multiple nAChRs, did not significantly reduce L-dopa-induced AIMs, while 5-iodo-A-85380 (A-85380), which acts selectively at α4β2* and α6β2* subtypes, reduced AIMs by 20%. By contrast, both varenicline and A-85380 reduced L-dopa-induced AIMs by 40-50% in rats with a partial striatal dopamine lesion. Neither drug worsened the antiparkinsonian action of L-dopa. The results show that selective nicotinic agonists reduce dyskinesias, and that they are optimally effective in animals with partial striatal dopamine damage. These findings suggest that presynaptic dopamine terminal α4β2* and α6β2* nAChRs are critical for nicotine's antidyskinetic action. The current data have important implications for the use of nicotinic receptor-directed drugs for L-dopa-induced dyskinesias, a debilitating motor complication of dopamine replacement therapy for Parkinson's disease., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The coenzyme tetrahydrofolic acid is the most rapid suicide substrate of tyrosinase that has been characterized to date. A kinetic study of the suicide inactivation process provides the kinetic constants that characterize it: λ(max), the maximum apparent inactivation constant; r, the partition ratio or the number of turnovers made by one enzyme molecule before inactivation; and k(cat) and K(m), the catalytic and Michaelis constants, respectively. From these values, it is possible to establish the ratio λ(max)/K(m), which represents the potency of the inactivation process. Besides acting as a suicide substrate of tyrosinase, tetrahydrofolic acid reduces o-quinones generated by the enzyme in its action on substrates, such as l-tyrosine and l-DOPA (o-dopaquinone), thus inhibiting enzymatic browning.

Neurotoxic effects have been suggested for l-3,4-dihydroxyphenylalanine (L-DOPA), while neuroprotective effects have been proposed for statins. We investigated whether certain statins (simvastatin or pitavastatin) could inhibit L-DOPA neurotoxicity. Neuronally-differentiated PC12 (nPC12) cells were treated with L-DOPA and/or statins for 24h, and their viabilities were measured using a cell counting kit, trypan blue staining, and 4',6-diamidino-2-phenylindole (DAPI) staining. Free radical and specific intracellular signaling protein levels were measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. High concentrations of l-DOPA reduced nPC12 cell viability, but combined treatment with statins restored viability. Treatment with 200 μM L-DOPA increased free radical and hydroxyl radical levels, but combined treatment with 5 μM statins decreased these levels. Survival-related signaling proteins were decreased in nPC12 cells treated with 200 μM L-DOPA, but combined treatment with 5μM statins significantly increased the levels of these proteins. Treatment with 200 μM L-DOPA significantly increased death-related signaling proteins, while combined treatment with 5 μM statins reduced the levels of these proteins. Pretreatment with LY294002, a phosphatidylinositol 3 kinase (PI3K) inhibitor, before combined treatment with statins and L-DOPA almost completely blocked the protective effects of statins. These results indicate that statins reduce L-DOPA neurotoxicity by lowering oxidative stress and by enhancing survival signals and inhibiting death signals via activation of the PI3K pathway., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Long-term motor complications of dopamine replacement, such as L-DOPA-induced dyskinesia (LID) and reduced quality of L-DOPA action, remain obstacles in the treatment of Parkinson's disease. Dysfunctional glutamatergic neurotransmitter systems have been observed in both the untreated parkinsonian and dyskinetic states and represent novel targets for treatment. Here, we assess the pharmacokinetic profile and corresponding pharmacodynamic effects on behavior of the orally active, selective metabotropic glutamate receptor type 5 (mGlu5) antagonist, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) (as the hydrochloride salt) in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned macaque. Six parkinsonian, MPTP-lesioned cynomolgus monkeys, with established LID, were administered acute challenges with MTEP (4.5-36 mg/kg p.o.) or vehicle, either alone or in combination with L-DOPA (33 +/- 1 mg/kg p.o.). Motor activity, parkinsonian disability, and dyskinesia were assessed for a 6-h period. Plasma drug levels were assessed by liquid chromatography-tandem mass spectrometry. MTEP had no antiparkinsonian action as monotherapy. However, administration of L-DOPA in combination with MTEP (36 mg/kg) reduced peak dose LID by 96%. Moreover, although total on-time (duration for which L-DOPA exerted an antiparkinsonian effect) was not significantly reduced, MTEP (36 mg/kg) reduced the duration of on-time with disabling LID by 70% compared with that for L-DOPA alone. These effects were associated with a peak plasma concentration of 20.9 microM and an area under the curve from 0 to 24 h of 136.1 h x microM (36 mg/kg). Although total on-time was not reduced, the peak antiparkinsonian benefit of l-DOPA/MTEP (36 mg/kg) was less than that with L-DOPA alone. Selective mGlu5 inhibitors may have significant potential to ameliorate dyskinesia, but care should be taken to ensure that such effects do not come at the expense of the peak antiparkinsonian benefit of L-DOPA.

l-3,4-dihydroxyphenylalanine methyl ester hydrochloride (l-DOPA) is the gold standard for symptomatic treatment of Parkinson's disease (PD), but long-term therapy is associated with the emergence of abnormal involuntary movements (AIMS) known as l-DOPA-induced dyskinesias (LID). The molecular changes underlying LID are not completely understood. Using the 6-hydroxydopamine-lesioned rat model of PD, we showed that l-DOPA elicits profound alterations in the activity of three LID molecular markers, namely DeltaFosB, dopamine, cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), as well as in phosphorylation levels of the cytoskeletal-associated protein tau. These modifications are triggered by protein kinase A (PKA) activation and intermittent stimulation of dopamine receptors as they are totally prevented by intrastriatal injections of Rp-cAMPS, a PKA inhibitor, or by continuous administration of l-DOPA via subcutaneous mini-pump. Importantly, Rp-cAMPS does not modulate the positive effect of l-DOPA on locomotor deficits and significantly attenuates the emergence of AIMS in 6-hydroxydopamine hydrobromide-lesioned rats. Even if decreased PKA signalling in the striatum may represent a clinical challenge, these data provide novel evidence that PKA activation, through modification of striatal signalling and alterations of cytoskeletal constituents, plays a key role in the manifestation of LID., (Copyright 2009 Elsevier Inc. All rights reserved.)

Aripiprazole is a novel antipsychotic medication characterized by partial agonism at the D2 and 5-HT1A receptors and by antagonism at the 5-HT2A receptor. The aim of the present study was to evaluate, in an open-label pilot study, the effects and safety of very small doses of aripiprazole on L-dopa-induced dyskinesia of a group of PD patients who did not show a significant clinical benefit by pharmacological treatment with amantadine and mirtazapine. Twelve PD patients with peak-dose LID were enrolled in a period of 1 year. Aripiprazole dosage was of 0.625 mg/day. The ten patients who continued taking aripiprazole displayed a significant decrease in the intensity and frequency of dyskinesias in all parts of the body, particularly in trunk movements (AIMS score T(0) = 14.1 +/- 3.6 vs. final score 2.4. +/- 2.6; P = 0.005). Our study suggests that aripiprazole at very low doses is tolerated and could be efficacy in treating LID.

The effects of scoparone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. PC12 cells treated with scoparone at concentrations of 100-200 microM showed a 128-136% increase in dopamine levels over the course of 24 hr. Scoparone significantly increased the secretion of dopamine into the culture medium. Under the same conditions, the activities of tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) were enhanced by treatment with 200 microM scoparone for 6-48 hr, but the activity of TH was regulated for a longer period than that of AADC. The intracellular levels of cyclic AMP and Ca(2+) were increased by treatment with 200 microM scoparone. The levels of TH mRNA and the phosphorylation of cyclic AMP-response element-binding protein (CREB) were also significantly increased by treatment with 200 microM scoparone. In addition, scoparone at a concentration of 200 microM stimulated the activities of cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC), and Ca(2+)/calmodulin kinase II (CaMK II). Finally, pretreatment with 200 microM scoparone reduced the cytotoxicity induced by L-DOPA (20-100 microM) at 24 hr. These results suggest that scoparone enhances dopamine biosynthesis by regulating TH activity and TH gene expression, which is mediated by the PKA, CREB, PKC, and CaMK II pathways, and protects cells from L-DOPA-induced cytotoxicity by inducing cyclic AMP-PKA systems in PC12 cells., ((c) 2009 Wiley-Liss, Inc.)

Rationale: Dyskinesia affects the majority of levodopa-treated parkinsonian patients within 5-10 years of treatment with levodopa. Clinical and preclinical observations suggest that an increase in serotoninergic transmission can contribute to the appearance of dyskinesias. It is thus conceivable that a modulation of synaptic dopamine (DA) levels induced by the inhibition of serotonin (5-HT) release, as a consequence of 5-HT(1A) agonists administration, might alleviate dyskinesias., Objective: Since 5-HT(1A) receptors are expressed in the subthalamic nucleus (STN), the aim of the present study was to assess the effect of the intrasubthalamic administration of sarizotan, a compound with full 5-HT(1A) agonist properties, on levodopa-induced dyskinesias in the 6-hydroxydopamine (6-OHDA) model of parkinsonism., Materials and Methods: Male Sprague-Dawley rats received a unilateral 6-OHDA administration in the nigrostriatal pathway. A test of apomorphine was performed to evaluate dopamine depletion. One week later, a cannula was implanted in the STN. Animals were treated with levodopa (6 mg/kg, i.p., twice at day) for 22 consecutive days. On day 23, several doses (1 ng, 10 ng, or 1 microg) of sarizotan were administered through the cannula to the STN. The higher doses of sarizotan effectively attenuated all levodopa-induced dyskinesias including axial, limb, and orolingual subtypes., Conclusions: These results suggest that the STN is a target structure for the antidyskinetic action of sarizotan and indicate that drug-mediated modulation of STN activity may be an alternative option for the treatment of levodopa-induced dyskinesias in Parkinson's disease.

In the present study, we evaluated the effects of subchronic blockade of mGluR5 by 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) on learning, anxiety and levodopa-induced dyskinesia in rats. In addition, we excluded the possibility that subchronic treatment produced pharmacokinetic changes using brain microdialysis. MTEP (5 mg/kg) impaired spatial learning in a radial maze task and contextual fear conditioning (CFC) when administered acutely, and the same effect was observed following a 4-day pre-treatment regime. Similarly, MTEP (5 mg/kg) exerted anxiolytic-like effects in CFC when given before the test whether administered after acute or sub-chronic treatment. Similarly, in levodopa-induced dyskinesia, sub-chronic (7 subsequent days) treatment with MTEP (5 mg/kg) resulted in a significant reduction in abnormal involuntary movements (AIMs), comparable to single acute administration. The data indicate that tolerance does not develop to the anxiolytic and antidyskinetic effects of mGluR5 antagonist MTEP at least at the used treatment mode and tested doses. However, at least at the doses tested, also no tolerance to the memory impairing effect of MTEP was observed. Depending on the indication and model, the amnesic effects of MTEP should be taken into account as a potential limitation, also after repetitive treatment.

Nicotinic cholinergic receptors play a role in cardiovascular regulation in the lower brain stem. Herein, we present evidence that l-3,4-dihydroxyphenylalanine (DOPA), a putative neurotransmitter in the central nervous system, is involved in the depressor response to microinjection of nicotine into the nucleus tractus solitarii (NTS). Microinjection of nicotine into the medial area of the NTS led to decreases in arterial blood pressure and heart rate in anesthetized rats. Mecamylamine, a nicotinic receptor antagonist, microinjected into NTS, blocked the depressor and bradycardic responses to nicotine. Nicotine-induced depressor and bradycardic responses were blocked by DOPA cyclohexyl ester (DOPA CHE), an antagonist for DOPA. DOPA CHE did not modify the action of carbachol on excitatory postsynaptic potential in rat cortical slices. These results suggest that endogenous DOPA is involved in nicotine-induced depressor responses in the NTS of anesthetized rats.

It is not yet clear how L-dopa, that is the most effective drug for the treatment of Parkinson's disease, enters into the dopaminergic neurons to be transformed into dopamine. It is suggested that L-dopa is mainly transported into cells by a group of L-amino acid carriers named "System L". Since these carriers are selectively inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), we have applied this compound to electrophysiologically recorded dopaminergic neurons of the rat substantia nigra pars compacta to examine the possible modulation of the effects of L-dopa by System L. We have observed that BCH reduced, in a concentration-dependent manner, the membrane hyperpolarization/outward current caused by L-dopa. Interestingly, the actions of dopamine were not changed by this System L inhibitor, suggesting that the reducing effects on L-dopa are not due to a BCH-induced unspecific block of dopamine-mediated events. Therefore, our electrophysiological data that an l-type amino acid carrier, possibly System L, is involved in the transport of L-dopa into dopaminergic neurons.

Adenosine A2A receptors have a unique cellular and regional distribution in the basal ganglia, being particularly concentrated in areas richly innervated by dopamine such as the caudate-putamen and the globus pallidus. Adenosine A2A receptors are selectively located on striatopallidal neurons and are capable of forming functional heteromeric complexes with dopamine D2 and metabotropic glutamate mGlu5 receptors. Based on the unique cellular and regional distribution of this receptor and in line with data showing that A2A receptor antagonists improve motor symptoms in animal models of Parkinson's disease (PD) and in initial clinical trials, A2A receptor antagonists have emerged as an attractive non-dopaminergic target to improve the motor deficits that characterize PD. Experimental data have also shown that A2A receptor antagonists do not induce neuroplasticity phenomena that complicate long-term dopaminergic treatments. The present review provides an updated summary of results reported in the literature concerning the biochemical characteristics and basal ganglia distribution of A2A receptors. We subsequently aim to examine the effects of adenosine A2A antagonists in rodent and primate models of PD and of l-DOPA-induced dyskinesia. Finally, concluding remarks are made on post-mortem human brains and on the translation of adenosine A2A receptor antagonists in the treatment of PD.

Several lines of evidence demonstrate that the striatal enkephalinergic system may be involved in the development of LIDs. Preproenkephalin-B (PPE-B) transcript levels are elevated with LIDs and there are also declines in kappa-opioid and other opioid receptors in different regions of the basal ganglia. If reduced kappa-opioid receptors are linked to LIDs, it is possible that drugs that stimulate this subtype may decrease dyskinesias. We therefore initiated experiments to investigate the effect of kappa-opioid receptor activation on LIDs. We first tested the selective kappa-agonist U50,488 in rats with unilateral lesions of the nigrostriatal pathway. Chronic L-dopa treatment induced abnormal involuntary movements, including axial, orolingual and forelimb dyskinesias contralateral to the lesion. U50,488 administration prior to L-dopa treatment reduced these movements by 70%, suggesting that U50,488 has potential as an anti-dyskinetic treatment. We next tested its effect in a parkinsonian nonhuman primate model, which offers the advantage that parkinsonism and LIDs can clearly be differentiated and that the dyskinesias are similar to those in parkinsonian patients. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys were treated with L-dopa (5 mg/kg p.o.) twice daily for 3 weeks to induce dyskinesias. As in the rodent model, U50,488 (0.1-1.0 mg/kg i.m.) decreased LIDs in a dose-dependent fashion. However, the anti-parkinsonian effect of L-dopa was similarly reduced, and side effects developed, including sedation and vomiting. These data suggest that kappa-opioid agonists such as U50,488 may not be clinically useful antidyskinetic agents because they also reverse the anti-parkinsonian effect of l-dopa.

The synthesis of a series of 4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)methyl-2-arylbenzofuran and 4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)methylbenzofuran-2-carboxamide derivatives as novel alpha(2C)-adrenergic receptor antagonists are described. Their affinity at three different human alpha(2)-adrenergic receptors is reported, and some of these compounds exhibited high affinity for the alpha(2C)-adrenergic receptor with high subtype selectivity. Among them, compound 10e has been found to show the anti-L-dopa-induced dyskinetic activity in marmosets. The structure-activity relationship of these compounds is also discussed.

For medical, pharmacological, and cosmetic reasons, the demand for effective and safe depigmentating agents has increased. In this study, 101 plant extracts (methanol or water extracts) were screened for their inhibitory activities against tyrosinase, (L-3, 4,-dihydroxyphenylalanine) L-DOPA oxidation, and melanin biosynthesis in B16 mouse melanoma cells. Of the extracts examined, 31 showed over 50% inhibition of mushroom tyrosinase at a concentration of 666 microg/Ml, and 11 inhibited L-DOPA auto-oxidation at this concentration. In particular, extracts of Broussonetia kazinoki var. humilis (leaves and stems), Broussonetia papyrifera (leaves and bark), Cornus officinalis (fruit), Rhus javanica (gallnut), and Pinus densiflora (leaves) inhibited both tyrosinase activity and L-DOPA oxidation in a concentration-dependent manner. Seventeen plant extracts that inhibited tyrosinase were further tested for their inhibitory effects on melanogenesis. In B16 mouse melanoma cells, extracts of Acorus gramineus, Capsella bursa-pastoris, Morus bombycis, Perilla frutescens var. crispa, Quercus dentate (bark), Rhus javanica (gallnut), Schizopepon bryoniaefolius, or Sophora flavescens markedly inhibited (>50%) melanin synthesis at 50 microg/Ml. These plants represent a potential source of novel whitening agents for ultraviolet (UV)-sensitive skin.

5-HT1A autoreceptor stimulation can act to attenuate supraphysiological swings in extracellular dopamine levels following long-term levodopa treatment and may be useful in the treatment and prevention of the motor complications. The purpose of this study was to investigate cellular and behavioral effects of 5-HT1A receptor agonist 8-OH-DPAT in a rat model of levodopa-induced motor complications. Two sets of experiments were performed. First, animals were treated with levodopa (50 mg/kg with benserazide 12.5 mg/kg, twice daily), intraperitoneally (i.p.) for 22 days. On day 23, animals received either 8-OH-DPAT (1 mg/kg, i.p.) or 8-OH-DPAT plus WAY-100635 (0.1 mg/kg, i.p) or vehicle with each levodopa dose. In the second set, animals were treated either with levodopa (50 mg/kg, i.p.) plus 8-OH-DPAT (1 mg/kg, i.p.) or levodopa (50 mg/kg, i.p.) plus vehicle, administered twice daily for 22 consecutive days. Our study showed that 8-OH-DPAT plus levodopa both prolonged the duration of the motor response and reduced peak turning. 8-OH-DPAT plus levodopa also decreased the frequency of failures to levodopa. Co-administration of WAY-100635, a 5-HT1A receptor antagonist, with 8-OH-DPAT eliminated the effect of 8-OH-DPAT on motor complications indicating that the observed 8-OH-DPAT responses were probably mediated at the 5-HT1A autoreceptor. Moreover, 8-OH-DPAT plus levodopa significantly reduced hyperphosphorylation of GluR1 at serine 845, which was closely associated with levodopa-induced motor complications.

5-Hydroxytryptamine 1a (5-HT(1a)) receptor agonists, such as sarizotan and tandospirone, are reported to reduce levodopa-induced dyskinesia in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated macaques and in Parkinson's disease without worsening motor disability. However, these compounds are not specific for 5-HT(1a) receptors and also possess dopamine antagonist actions. We now report on the effects of (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin [(R)-(+)-8-OHDPAT], a selective 5-HT(1a) agonist lacking dopaminergic activity, on motor disability and dyskinesia (chorea and dystonia) in levodopa-primed MPTP-treated common marmosets. Administration of (R)-(+)-8-OHDPAT (0.2, 0.6, and 2.0 mg/kg s.c), in conjunction with levodopa/carbidopa (12.5 mg/kg each p.o.) to levodopa-primed animals, dose-dependently reduced levodopa-induced chorea but did not affect dystonic movements. However, (R)-(+)-8-OHDPAT treatment also reduced locomotor activity and the reversal of motor disability. Administration of (R)-(+)-8-OHDPAT alone had no effects of motor behaviors. The effects of (R)-(+)-8-OHDPAT on levodopa-induced motor behaviors were antagonized by the 5-HT(1a) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY-100635) (1.0 mg/kg s.c.). Administration of (R)-(+)-8-OHDPAT (0.6 mg/kg s.c.) also reduced chorea produced by the administration of the D(2)/D(3) dopamine receptor agonist pramipexole (0.06 mg/kg p.o.) to levodopa-primed MPTP-treated animals. However, again the increase in locomotor activity and reversal of motor disability produced by pramipexole were also inhibited. These data suggest that selective 5-HT(1a) agonists do not provide an effective means of suppressing levodopa-induced dyskinesia, except with worsening of parkinsonism.

A series of potent inhibitors of tyrosinase and their structure-activity relationships are described. N-Benzylbenzamide derivatives (1-21) with hydroxyl(s) were synthesized and tested for their tyrosinase inhibitory activity. With this series, compound 15 provided a potent tyrosinase inhibition: it effectively inhibited the oxidation of l-DOPA catalyzed by mushroom tyrosinase with an IC(50) of 2.2microM.

The authors intended to focus the attention of the medical community on the potential therapeutic usefulness of A(2A) adenosine receptors antagonists in the treatment of Parkinson's disease. Basal ganglia express a big amount of A(2A) adenosine receptors, occurring mainly on the external surfaces of neurons located at indirect pathways between the striatum, globus pallidus and substantia nigra. Experiments with the animal models of Parkinson's disease indicate that A(2A) receptors are strongly involved in the regulation of the central movement system. Co-localization of A(2A) and dopaminergic D2 receptors in the striatum creates a milieu for an antagonistic interaction between adenosine and dopamine. The experimental data prove that the best mobility improvement of patients with Parkinson's disease could be achieved with a simultaneous activation of dopaminergic D2 receptors and inhibition of adenosine A(2A) receptors. In animal models of Parkinson's disease, the use of selective antagonists of A(2A) receptors, such as istradefylline, led to the reversibility of movement dysfunction. These compounds might improve the mobility during both monotherapy and co-administration with L-dopa and the dopamine receptor agonists. The use of these antagonists in the combined therapy enables the reduction of the L-dopa doses, as well as reduction of the side effects. In the adjunctive therapy, the A(2A) antagonists might be used in both moderate and advanced stages of Parkinson's disease. The long-lasting administration of the A(2A) receptor antagonists does not decrease patient's response and does not cause side effects typical of the L-dopa therapy. It was proved on different animal models that inhibition of A(2A) receptors not only decreases the movement disturbance, but also reveals the neuroprotective activity, which might slow down or stop the progress of the disease.

The purpose of this study was to investigate the effect of the catechol-O-methyltransferase (COMT) inhibitor, entacapone, in the reversal and prevention of "wearing-off" phenomena in hemiparkinsonian rats. Catechol-O-methyltransferase (COMT) inhibitors increase the half-life and bioavailability of levodopa, providing more continuous dopamine receptor stimulation. This raises the possibility of using levodopa and a COMT inhibitor not only to treat motor complications, but also to prevent their development. Male Sprague-Dawley rats received a unilateral 6-hydroxydopamine (6-OHDA) administration in the nigrostriatal pathway. Two sets of experiments were performed. First, animals were treated with levodopa (50 mg/kg/day with benserazide 12.5 mg/kg/day, twice daily (b.i.d.), intraperitoneally (i.p.) for 22 days. On day 23, animals received either entacapone (30 mg/kg, i.p.) or vehicle with each levodopa dose. In the second set, animals were treated either with levodopa (50 mg/kg/day, i.p.) plus entacapone (30 mg/kg/day, i.p.) or levodopa (50 mg/kg/day, i.p.) plus vehicle, administered two or three times daily [b.i.d. or thrice daily (t.i.d.), respectively] for 22 consecutive days. Entacapone both reversed and prevented the shortening of the motor response duration that defines "wearing-off" motor fluctuations. Entacapone also decreased the frequency of failures to levodopa. The combination of levodopa and entacapone may reduce the likelihood of motor fluctuation development and may thus become a valuable approach to treat Parkinson disease whenever levodopa is needed.

Neural stem cells (NSCs) are currently considered very hopeful candidates for cell replacement therapy in neurodegenerative pathologies such as Parkinson's disease (PD), but like embryonic neural tissue transplantation, levodopa medication may still be required to improve symptoms even after cell transplantation. The issues of whether levodopa induces cytotoxicity and apoptosis of NSCs following transplantation, as well as the means to prevent these processes from occurring remain to be elucidated. In this study, the possible cytotoxicity of levodopa at different doses on C17.2 neural stem cells and subsequent neuroprotection by pergolide were investigated. The cell viability was determined by the MTT assay. Cell proliferation was assayed by BrdU labeling, while apoptosis was detected by Annexin-V-FLUOS staining and flow cytometry. Levels of p53, Bax, Bcl-2, NFkB, cytochrome c, caspase-3 as well as cleavage of caspase-3 were measured by western blotting. We found levodopa induced a concentration- and time-dependent decrease in cell viability and proliferation. Apoptotic cells were observed at different stages, specifically 12 and 24 h following exposure to levodopa (200 microM). Elevated p53, Bax, cytochrome c, caspase-3 and active fragments of caspase-3 protein were observed in the cells exposed to levodopa. These alterations were partly inhibited by pergolide, a dopamine receptor agonist, while Bcl-2 and NFkB p65 levels remained constant at the various time-points in all the groups examined. These observations indicate that levodopa at high concentrations (> or = 200 microM) was neurotoxic to C17.2 neural stem cells via inhibition of DNA synthesis and cell proliferation. Activation of the mitochondria-dependent pathway and caspase-3 protease may contribute to the mechanism by which levodopa induces apoptosis. Pergolide, an anti-Parkinson drug, has a neuroprotective effect and partly blocks levodopa-induced cytotoxicity.

Sensitization of NMDA receptors containing the NR2B subunit has been increasingly associated with various forms of synaptic plasticity, including those implicated in the pathogenesis of extrapyramidal motor dysfunction. To determine whether activation of NR2B containing receptors contributes to the development and maintenance of levodopa-induced response changes in parkinsonian animals, we evaluated the effects of the selective NR2B antagonist CP-101,606 on these response alterations in unilateral 6-hydroxydopamine (6-OHDA) lesioned rats. Three weeks of twice-daily levodopa treatment decreased the duration of the rotational response to acute levodopa challenge. The response alteration was associated with an increase in GluR1 (S831) phosphorylation in medium spiny neurons of the dorsolateral striatum. Both the attenuated rotational response and augmented GluR1 phosphorylation were decreased by CP-101,606 treatment. These CP-101,606 effects were observed when the compound was administered either at the end of chronic levodopa treatment (ameliorative effect) or together with the twice-daily levodopa treatment for 3 weeks (preventive effect). Furthermore, concurrent administration of CP-101,606 with levodopa potentiated the ability of levodopa challenge to reverse the 6-OHDA lesion-induced contralateral forelimb movement deficit as measured in a drag test. These results suggest that activation of NR2B subunit containing NMDA receptors contributes to both the development and maintenance of levodopa-induced motor response alterations, through a mechanism that involves an increase in GluR1 phosphorylation in striatal spiny neurons.

Objective: Levetiracetam (LEV; Keppra, UCB Pharma) has been shown to reduce established l-3,4 dihydroxyphenylalanine (l-dopa)-induced dyskinesia. This study investigated whether LEV can modify induction of dyskinesia by l-dopa or the process of priming., Methods: Drug-naive MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) -lesioned marmosets were treated for 21 days with l-dopa/LEV or l-dopa alone. Subsequently, the animals were left untreated for 1 week and then both groups were challenged with a single dose of l-dopa alone on day 29. Behavior was assessed by automated activity counts and by post hoc analysis of videotapes using validated rating scales., Results: LEV had no significant effect on the appearance of dyskinesia when administered de novo in combination with l-dopa. However, after a week of drug holiday, the 2 groups exhibited a different response to an acute l-dopa challenge. Thus, animals previously treated with l-dopa alone exhibited a similar level of dyskinesia to that seen on day 21 of the repeated treatment phase of the study. However, animals previously treated with l-dopa/LEV demonstrated significantly reduced dyskinesia compared with day 21 of the repeated treatment phase of the study., Conclusions: LEV does not modify the onset of dyskinesia following de novo treatment with l-dopa. However, concomitant treatment with l-dopa/LEV reduces the level of dyskinesia induced by l-dopa following a drug holiday. Thus, prior treatment with LEV appears to modify the mechanisms responsible for the maintenance of l-dopa-induced dyskinesia.

Alterations of striatal glutamate receptors are believed to be responsible, at least in part, for the pathogenesis of L-dopa-induced dyskinesias (LID). To evaluate whether co-administration of CI-1041, a novel NMDA receptor antagonist selective for the NR1A/NR2B subtype, with L-dopa might prevent the appearance of this side effect, eight de novo parkinsonian monkeys were treated chronically orally with either L-dopa alone or L-dopa plus CI-1041 (n= 4 for each group). After 4 weeks of treatment with L-dopa alone, all four animals developed moderate dyskinesias either choreic or dystonic in nature. CI-1041 co-treatment completely prevented the induction of dyskinesias in three animals and only one monkey developed mild dyskinesias at the end of the fourth week of treatment in the L-dopa + CI-1041 group. The magnitude and duration of the antiparkinsonian action of L-dopa was similar in both groups. These results suggest that selective NMDA receptor antagonism may be interesting for managing LID in Parkinson's disease patients.

Glutamatergic overactivity might be involved in L-dopa-induced motor complications since glutamate antagonists reverse and prevent L-dopa-induced shortening in motor response duration in 6-hydroxydopamine-lesioned (6-OHDA) rats and improve L-dopa-induced dyskinesias in parkinsonian monkeys and in patients with Parkinson's disease (PD). An increase in the subthalamic nucleus (STN) glutamatergic activity is believed to contribute to the pathophysiology of PD. However, the role of STN activity in L-dopa-induced motor complications is not so clear. In this study, the effect of STN lesions on L-dopa-induced motor response complications was investigated in rats with a nigrostriatal pathway lesion induced by 6-OHDA. Animals were injected with 6-OHDA in the medial forebrain bundle and treated with L-dopa or saline for 22 days. On day 16, animals were randomly distributed in groups that underwent surgery in the STN ipsilateral or contralateral to 6-OHDA lesion, or bilateral. Rotational behavior was measured on days 1, 15, and 22. Attenuation of STN activity by contralateral and bilateral, but not ipsilateral, STN lesion reversed the shortening in motor response duration induced by L-dopa. L-dopa administration, but not saline, induced prominent dyskinesias in 6-OHDA-lesioned rats with additional bilateral STN lesions. The results indicate that bilateral lesions of STN potentiate the duration of L-dopa-induced motor response and facilitate chronic L-dopa-induced abnormal involuntary movements in 6-OHDA-lesioned rats. The characteristics of the abnormal involuntary movements observed in these animals are similar to L-dopa-induced dyskinesias in parkinsonian patients and might be useful as an experimental model for the study of L-dopa-induced dyskinesia., (Copyright 2003 Wiley-Liss, Inc.)

Parkinson's disease is a chronic neurodegenerative disease of the extrapyramidal system associated with dopaminergic neuronal loss in the basal ganglia. However, several other neurotransmitters, such as serotonin, gamma-amino-butyric acid and glutamate, are also related to the symptoms of Parkinson's disease patients and their response to levodopa treatment. The co-expression of cannabinoid and dopamine receptors in the basal ganglia suggests a potential role for endocannabinoids in the control of voluntary movement in Parkinson's disease. In the present study we treated unilaterally 2,4,5-trihydroxyphenethylamine (6-hydroxydopamine)-lesioned rats with the enantiomers of the synthetic cannabinoid 7-hydroxy-delta6-tetrahydrocannabinol 1,1-dimethylheptyl. Treatment with its (-)- (3R, 4R) enantiomer (code-name HU-210), a potent cannabinoid receptor type 1 agonist, reduced the rotations induced by levodopa/carbidopa or apomorphine by 34% and 44%, respectively. In contrast, treatment with the (+)- (3S, 4S) enantiomer (code-name HU-211), an N-methyl-D-aspartate antagonist, as well as the psychotropically inactive cannabis constituent: cannabidiol and its primary metabolite, 7-hydroxy-cannabinol, did not show any reduction of rotational behavior. Our results indicate that activation of the CB1 stimulates the dopaminergic system ipsilaterally to the lesion, and may have implications in the treatment of Parkinson's disease.

Inhibition of catechol-O-methyltransferase (COMT) has protective effects on levodopa (L-DOPA), but not D-DOPA toxicity towards dopamine (DA) neurons in rat primary mesencephalic cultures [Mol. Pharmacol. 57 (2000) 589]. Here, we extend our recent studies to elucidate the mechanisms of these protective effects. Thus, we investigated the effects of all main L-DOPA/DA metabolites on survival of tyrosine hydroxylase immunoreactive (THir) neurons in primary rat mesencephalic cultures. 3-O-Methyldopa, homovanillic acid, dihydroxyphenyl acetate and 3-methoxytyramine had no effects at concentrations up to 300 micro M after 24h, whereas DA was more toxic than L-DOPA with toxicity at concentrations of >or=1 micro M. The coenzyme of COMT, S-adenosyl-L-methionine (SAM), and its demethylated product S-adenosylhomocystein caused no relevant alteration of THir neuron survival or L-DOPA toxicity. In contrast, inhibition of SAM synthesis by selenomethionine showed time- and dose-dependent increase of THir neuron survival, but did not affect L-DOPA toxicity. L-DOPA-induced lipid peroxidation in mesencephalic cultures was not modified by the COMT inhibitor Ro 41-0960 (1 micro M). Increased contamination of the cultures with glial cells attenuated L- and D-DOPA toxicity, but caused significant enhancement of protection by COMT inhibitors against L-DOPA toxicity only. Investigations of L-DOPA uptake in rat striatal cultures using HPLC revealed a significant reduction of extracellular L-DOPA concentrations by Ro 41-0960. Our data confirm that L-DOPA toxicity towards DA neurons is mediated by an autooxidative process, which is attenuated by glial cells. In addition, we demonstrate a second mechanism of L-DOPA toxicity in vitro mediated by a COMT- and glia-dependent pathway, which is blocked by COMT inhibitors, most likely due to enhanced glial uptake of L-DOPA.

Using in situ hybridization, it was found that subchronic treatment with levodopa/benserazide increased preproenkephalin-A and preproenkephalin-B mRNAs in the dopamine-depleted striatum. In order to examine whether dysfunction of the endogenous opioid system may underlie the development of levodopa-induced dyskinesias, the effect of naloxone, an opioid antagonist, on dyskinesias was investigated in two models of parkinsonism in the common marmoset. MPTP-treated monkeys were administered a daily oral dose of levodopa/benserazide which relieved the parkinsonian symptoms but induced severe and reproducible dyskinetic movements. Naloxone (0.1, 0.2 or 0.5 mg/kg) was given subcutaneously (s.c.) during peak-dose dyskinesia, which reduced the dyskinesias significantly using the highest dose, normalized the motor activity, but did not modify the antiparkinson effect. Unilaterally 6-OHDA -lesioned marmosets received apomorphine s.c., which caused a contralateral turning behavior that could be reduced up to 35 percent by concomitant administration of naloxone. Taken together the present results suggest a possible role for the endogenous opioid system in the pathogenesis of levodopa-induced dyskinesia in primates.

In the French West Indies there is an abnormally high frequency of levodopa-resistant parkinsonism, suggested to be caused by consumption of fruit and infusions of tropical plants, especially Annona muricata (corossol, soursop). To determine whether toxic substances from this plant can cause the neuronal degeneration or dysfunction underlying the syndrome, we exposed mesencephalic dopaminergic neurons in culture to the total extract (totum) of alkaloids from Annona muricata root bark and to two of the most abundant subfractions, coreximine and reticuline. After 24 hours, 50% of dopaminergic neurons degenerated with 18 microg/ml totum, 4.3 microg/ml (13 microM) coreximine, or 100 microg/ml (304 microM) reticuline. The effects of the alkaloid totum were not restricted to the population of dopaminergic cells since GABAergic neurons were also affected by the treatment. Nuclei in dying neurons showed DNA condensation or fragmentation, suggesting that neuronal death occurred by apoptosis. Cell death was not excitotoxic and did not require toxin uptake by the dopamine transporter. Neurodegeneration was attenuated by increasing the concentration of glucose in the culture medium, which also reduced the effect of the dopaminergic neurotoxin MPP+, a mitochondrial respiratory chain inhibitor. Toxin withdrawal after short-term exposure arrested cell death. Acute treatment with totum, coreximine, or reticuline reversibly inhibited dopamine uptake by a mechanism that was distinct from that causing neuronal death. GABA uptake was not reduced under the same conditions. This study suggests that alkaloids from A. muricata can modulate the function and the survival of dopaminergic nerve cells in vitro. It is therefore conceivable that repeated consumption could cause the neuronal dysfunction and degeneration underlying the West Indian parkinsonian syndrome., (Copyright 2002 Movement Disorder Society.)

To evaluate the possible involvement of glutamate AMPA receptor-mediated mechanisms in levodopa-induced motor fluctuations, we investigated the effects of LY293558, a competitive AMPA receptor antagonist, on levodopa-induced motor alterations in rats with unilateral 6-OHDA lesion. Acute and chronic administration of LY293558 was studied to evaluate the possible reversion or prevention of these levodopa effects. In the first set of experiments, rats were treated with levodopa (25 mg/kg with benserazide, twice daily, i.p.) for 22 days and on day 23 LY293558 (5 mg/kg, i.p.) was administered immediately before levodopa. In the second set of experiments, rats were treated daily for 22 days with levodopa and LY293558 (5 mg/kg, twice daily, i.p.). In the third set of experiments, the effect of LY293558 (5 mg/kg, i.p.) administration on selective dopamine D-1 (SKF38393, 1.5 mg/kg, s.c.) and D-2 agonist (quinpirole, 0.1 mg/kg, i.p.)-induced rotational behavior after daily levodopa treatment was studied. The duration of the rotational behavior induced by chronic levodopa decreased by 30% after 22 days. Acute administration of LY293558 on day 23 reversed this effect. The group of animals that were chronically treated with levodopa and LY293558 did not show the decrease in this motor response duration. Chronic levodopa treatment attenuated the rotational response to the D-1 agonist SKF38393 and increased the response to the D-2 agonist quinpirole. LY293558 did not reverse the effect of levodopa on rotational behavior induced by the D-1 agonist but significantly reduced the rotational response to the D-2 agonist in levodopa-treated animals by 40%. Our results demonstrate that an AMPA receptor antagonist reverses and prevents levodopa-induced motor alterations in parkinsonian rats and that this effect on motor fluctuations induced by chronic levodopa is probably due to a modulation of the indirect output pathway of the basal ganglia., (Copyright 2001 Wiley-Liss, Inc.)