Reduced Plasma Levodopa Fluctuations with More Frequent Administration of a Novel Carbidopa/Levodopa Functionally Scored Tablet.

Levodopa/carbidopa remains the gold standard for treating Parkinson disease (PD), but chronic pulsatile administration contributes to motor complications. This Phase 1 study used a new immediate-release (IR) formulation of carbidopa/levodopa 25/100 mg that is functionally scored for easy and precise splitting to evaluate the effects on levodopa plasma variability when smaller doses are taken more frequently. These functionally scored tablets were shown to be bioequivalent to carbidopa/levodopa 25-/100-mg IR generic reference tablets. Twenty-two healthy volunteers received a whole tablet every 4 hours versus half of the tablet every 2 hours. Plasma levodopa fluctuations were significantly reduced with half-tablets dosed every 2 hours, with a 44% reduction in peaks (P < .0001). While drug exposure did not differ, parameters that underlie motor response variations, including mean peak-to-trough difference and variance, were 51% and 56% less, respectively, with more frequent dosing (both P ≤ .0024). Safety and tolerability of both regimens were similar. In conclusion, more frequent administration of half-tablets of the new functionally scored IR formulation safely provided more constant levodopa levels than whole tablets dosed less often. This tablet technology could facilitate the benefits of more physiologic dopamine replenishment in patients with PD, particularly those with reduced manual dexterity.

Bone Marrow-Derived NCS-01 Cells Advance a Novel Cell-Based Therapy for Stroke.

Human mesenchymal stem cells have been explored for their application in cell-based therapies targeting stroke. Identifying cell lines that stand as safe, accessible, and effective for transplantation, while optimizing dosage, timing, and method of delivery remain critical translational steps towards clinical trials. Preclinical studies using bone marrow-derived NCS-01 cells show the cells' ability to confer functional recovery in ischemic stroke. Coculturing primary rat cortical cells or human neural progenitor cells with NCS-01 cells protects against oxygen-glucose deprivation. In the rodent middle cerebral artery occlusion model, intracarotid artery administration of NCS-01 cells demonstrate greater efficacy than other mesenchymal stem cells (MSCs) at improving motor and neurological function, as well as reducing infarct volume and peri-infarct cell loss. NCS-01 cells secrete therapeutic factors, including basic fibroblast growth factor and interleukin-6, while also demonstrating a potentially novel mechanism of extending filopodia towards the site of injury. In this review, we discuss recent preclinical advancements using in vitro and in vivo ischemia models that support the transplantation of NCS-01 in human stroke trials. These results, coupled with the recommendations put forth by the consortium of Stem cell Therapeutics as an Emerging Paradigm for Stroke (STEPS), highlight a framework for conducting preclinical research with the ultimate goal of initiating clinical trials.

Translating intracarotid artery transplantation of bone marrow-derived NCS-01 cells for ischemic stroke: Behavioral and histological readouts and mechanistic insights into stem cell therapy.

The present study used in vitro and in vivo stroke models to demonstrate the safety, efficacy, and mechanism of action of adult human bone marrow-derived NCS-01 cells. Coculture with NCS-01 cells protected primary rat cortical cells or human neural progenitor cells from oxygen glucose deprivation. Adult rats that were subjected to middle cerebral artery occlusion, transiently or permanently, and subsequently received intracarotid artery or intravenous transplants of NCS-01 cells displayed dose-dependent improvements in motor and neurological behaviors, and reductions in infarct area and peri-infarct cell loss, much better than intravenous administration. The optimal dose was 7.5 × 106 cells/mL when delivered via the intracarotid artery within 3 days poststroke, although therapeutic effects persisted even when administered at 1 week after stroke. Compared with other mesenchymal stem cells, NCS-01 cells ameliorated both the structural and functional deficits after stroke through a broad therapeutic window. NCS-01 cells secreted therapeutic molecules, such as basic fibroblast growth factor and interleukin-6, but equally importantly we observed for the first time the formation of filopodia by NCS-01 cells under stroke conditions, characterized by cadherin-positive processes extending from the stem cells toward the ischemic cells. Collectively, the present efficacy readouts and the novel filopodia-mediated mechanism of action provide solid lab-to-clinic evidence supporting the use of NCS-01 cells for treatment of stroke in the clinical setting.

Donepezil Plus Solifenacin (CPC-201) Treatment for Alzheimer's Disease.

Available cholinergic drugs for treating Alzheimer's disease (AD) provide modest symptomatic benefit. We hypothesized that co-administration of a peripheral anticholinergic to reduce dose-limiting adverse effects (AEs) would enable the safe/tolerable use of higher cholinesterase inhibitor doses and thus improve their antidementia efficacy. A modified single-blind, ascending-dose, phase IIa study of donepezil plus solifenacin (CPC-201) lasting 26 weeks was conducted in 41 patients with probable AD of moderate severity. Entry criteria included the use of donepezil at a dose of 10 mg/day during the preceding 3 months. The primary outcome measure was the maximum tolerated dose (MTD) of donepezil achieved (to protocol limit of 40 mg/day) when administered with the anticholinergic solifenacin 15 mg/day. Secondary measures included assessments of cognitive and global function, as well as of AEs. The mean ± SD donepezil MTD increased to 38 ± 0.74 mg/day (median 40 mg/day; p < 0.001); 88% of the study population safely attained this dose at the end of titration. Markedly reduced donepezil AE frequency, especially gastrointestinal, allowed this dose increase. There were no drug-related serious AEs or clinically significant laboratory abnormalities. At 26 weeks, Alzheimer's Disease Assessment Scale Cognitive Component scores in the efficacy evaluable population improved by 0.35 ± 0.85 points over baseline (p < 0.05), an estimated 2.5 ± 0.84 points above 10 mg/day donepezil and 5.4 ± 0.84 points above historic placebo (both p < 0.05). Clinical Global Impression of Improvement scores improved by 0.94 ± 0.20 to 3.1 ± 0.20 points (p < 0.001). The findings suggest that limiting donepezil AEs by co-administration of solifenacin allows the safe administration of substantially higher cholinesterase inhibitors doses that may augment cognitive and global benefits in patients with AD.

Apathy in neuropsychiatric disease: diagnosis, pathophysiology, and treatment.

Apathy is an increasingly recognized concomitant of a broad range of central nervous system disorders. Nevertheless, its nosology, pathogenesis and therapy remain shrouded in confusion and controversy. As yet, there is little consensus regarding methods for detecting apathy, or distinguishing it from depression, or for assessing its severity. Many now regard the apathy syndrome as primarily reflecting a lack of motivation that compromises emotional, cognitive, and overt behavioral function. Even though under-recognized and under-diagnosed, apathy hardly appears uncommon: current epidemiologic studies suggest over 10 million Americans may be affected. Its reported frequency in various neurologic and psychiatric conditions varies widely, from less than 10 to over 80%, reflecting differences in population characteristics and assessment procedures. Often apathy has been associated with such neurodegenerative disorders as Alzheimer's disease, Parkinson's disease, and fronto-temporal dementia. But it also occurs in those with psychiatric disorders such as schizophrenia and major depression. Clinical, neuropathologic, and neuroimaging observations increasingly suggest that apathy reflects dysfunction of frontal-subcortical circuits, especially those linking the ventromedial prefrontal cortex to related regions in the basal ganglia. Therapeutically, numerous small studies suggest that psychostimulants, dopaminergics, and cholinesterase inhibitors may benefit those manifesting this syndrome. However, no adequately powered, randomized controlled trials have reported success and no medication have ever been approved for this disorder. The accelerating pace of current research nevertheless promises to improve our understanding of apathy and to better address the unmet medical needs of those suffering its consequences.

A 5-HT2A receptor inverse agonist, ACP-103, reduces tremor in a rat model and levodopa-induced dyskinesias in a monkey model.

A potent 5-hydroxytryptamine (5-HT)2A receptor inverse agonist and antagonist, ACP-103 [N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl) carbamide (2R,3R)-dihydroxybutanedioate (2:1, active:salt)], was evaluated for its ability to reduce the primary motor symptom of tremor using tacrine-induced tremulous jaw movements in rats, which is an animal model of parkinsonian tremor. Furthermore, ACP-103 was evaluated for its ability to reduce levodopa-induced dyskinesias in monkeys rendered parkinsonian with MPTP [1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine]. ACP-103 reduced tacrine-induced tremulous jaw movements in rats. In addition, ACP-103 administered in combination with levodopa caused a dose-related reduction in dyskinesias in monkeys. These data suggest that ACP-103 may have the potential to reduce tremor and levodopa-induced dyskinesias in Parkinson's disease.

Nuclear factor-kappaB-dependent cyclin D1 induction and DNA replication associated with N-methyl-D-aspartate receptor-mediated apoptosis in rat striatum.

Cell cycle reentry has been found during apoptosis of postmitotic neurons under certain pathological conditions. To evaluate whether nuclear factor-kappaB (NF-kappaB) activation promotes cell cycle entry and neuronal apoptosis, we studied the relation among NF-kappaB-mediated cyclin induction, bromodeoxyuridine (BrdU) incorporation, and apoptosis initiation in rat striatal neurons following excitotoxic insult. Intrastriatally injected N-methyl-D-aspartate receptor agonist quinolinic acid (QA, 60 nmol) elicited a rise in cyclin D1 mRNA and protein levels (P<0.05). QA-induced NF-kappaB activation occurred in striatal neurons and nonneuronal cells and partially colocalized with elevated cyclin D1 immunoreactivity and TUNEL-positive nuclei. QA triggered DNA replication as evidenced by BrdU incorporation; some striatal BrdU-positive cells were identified as neurons by colocalization with NeuN. Blockade of NF-kappaB nuclear translocation with the recombinant peptide NF-kappaB SN50 attenuated the QA-induced elevation in cyclin D1 and BrdU incorporation. QA-induced internucleosomal DNA fragmentation was blunted by G(1)/S-phase cell cycle inhibitors. These findings suggest that NF-kappaB activation stimulates cyclin D1 expression and triggers DNA replication in striatal neurons. Excitotoxin-induced neuronal apoptosis may thus result from, at least partially, a failed cell cycle attempt.

NF-kappaB contributes to 6-hydroxydopamine-induced apoptosis of nigral dopaminergic neurons through p53.

To evaluate the contribution of NF-kappaB and the NF-kappaB target gene p53 to nigral dopaminergic neuron degeneration in rodent models of Parkinson's disease, time-course of dopaminergic neuron loss as well as changes in the expression of some NF-kappaB-regulated proapoptotic proteins were assayed after unilateral infusion of 6-hydroxydopamine into rat medial forebrain bundle. Substantial loss of tyrosine hydroxylase immunoreactivity in nigral was observed 24 h after 6-hydroxydopamine treatment. The degenerative processes began 12 h after 6-hydroxydopamine administration as evidenced by a positive silver staining. Apoptotic death of dopaminergic neurons was suggested by the appearance of TUNEL-positive nuclei in substantia nigra and internucleosomal DNA fragmentation as detected by agarose gel electrophoresis. NF-kappaB activation in dopaminergic neurons as revealed by immunohistochemistry and electrophoresis mobility shift assay, began at 12 h after 6-hydroxydopamine administration. Levels of c-Myc and p53 immunoreactivities increased after 6-hydroxydopamine treatment, mainly in dopaminergic neurons as indicated by co-localization with tyrosine hydroxylase immunoreactivity. Blockade of NF-kappaB nuclear translocation with recombinant cell-permeable peptide NF-kappaB SN50 inhibited NF-kappaB nuclear translocation and p53 induction. SN50 and the p53 antagonist pifithrin-alpha significantly reduced nigral dopaminergic neuron degeneration. These results suggest that NF-kappaB activation contributes, at least in part, to oxidative stress-induced degeneration of dopaminergic neurons through a NF-kappaB-dependent p53-signaling pathway.

Glutamate release inhibition ineffective in levodopa-induced motor complications.

Reported benefits of various glutamatergic receptor antagonists in Parkinson's disease (PD) prompted an evaluation of the antidyskinetic effect of a putative glutamate release inhibitor in 15 moderately advanced patients. In a 3-week, double-blind, proof-of-concept study, riluzole (200 mg/day) failed to alter parkinsonian or levodopa-induced motor complication severity. Opposing effects of a generalized inhibition of glutamate-mediated synaptic transmission may limit the usefulness of this approach to treat PD.

Susceptibility of striatal neurons to excitotoxic injury correlates with basal levels of Bcl-2 and the induction of P53 and c-Myc immunoreactivity.

The present studies evaluated the potential contribution of Bcl-2, p53, and c-Myc to the differential vulnerability of striatal neurons to the excitotoxin quinolinic acid (QA). In normal rat striatum, Bcl-2 immunoreactivity (Bcl-2-i) was most intense in large aspiny interneurons including choline acetyltransferase positive (CAT+) and parvalbumin positive (PARV+) neurons, but low in a majority of medium-sized neurons. In human brain, intense Bcl-2-i was seen in large striatal neurons but not in medium-sized spiny projection neurons. QA produced degeneration of numerous medium-sized neurons, but not those enriched in Bcl-2-i. Many Bcl-2-i-enriched interneurons including those with CAT+ and PARV+ survived QA injection, while medium-sized neurons labeled for calbindin D-28K (CAL D-28+) did not. In addition, proapoptotic proteins p53-i and c-Myc-i were robustly induced in medium-sized neurons, but not in most large neurons. The selective vulnerability of striatal medium spiny neurons to degeneration in a rodent model of Huntington's disease appears to correlate with their low levels of Bcl-2-i and high levels of induced p53-i and c-Myc-i.

Effects of serotonin 5-HT1A agonist in advanced Parkinson's disease.

Intermittent stimulation of striatal dopaminergic receptors seems to contribute to motor dysfunction in advanced Parkinson's disease (PD). With severe dopaminergic denervation, exogenous levodopa is largely decarboxylated to dopamine in serotonergic terminals. If 5-HT1A autoreceptors regulate dopamine as well as serotonin release, in parkinsonian patients inhibition of striatal serotonergic neuron firing might help maintain more physiological intrasynaptic dopamine concentrations and thus ameliorate motor fluctuations and dyskinesias. To evaluate this hypothesis, effects of a selective 5-HT1A agonist, sarizotan, given orally at 2 and 5 mg twice daily to 18 relatively advanced parkinsonian patients, were compared with baseline placebo function during a 3-week, double-blind, placebo-controlled, proof-of-concept study. Sarizotan alone or with intravenous levodopa had no effect on parkinsonian severity. But at safe and tolerable doses, sarizotan coadministration reduced levodopa-induced dyskinesias and prolonged its antiparkinsonian response (P < or = 0.05). Under the conditions of this study, our findings suggest that 5-HT1A receptor stimulation in levodopa-treated parkinsonian patients can modulate striatal dopaminergic function and that 5-HT1A agonists may be useful as levodopa adjuvants in the treatment of PD.

Neuroprotective effects of prostaglandin A1 in animal models of focal ischemia.

The present study evaluated the neuroprotective potential of prostaglandin A1 (PGA1) in rodent models of focal cerebral ischemia. PGA1 33 nmol reduced infarction volume by about 43% (P < 0.05) when administered intracerebroventricularly before and after transient ischemia in mice. PGA1 16.5-66 nmol diminished infarction volume by 18% to 27% (P < 0.01) when administered immediately following permanent ischemia in rats. PGA1 treatment also significantly ameliorated motor dysfunction after brain ischemia. These results suggest that PGA1 protects neurons from ischemic injury.

Continuous dopaminergic stimulation reduces risk of motor complications in parkinsonian primates.

Levodopa or short-acting dopamine (DA) agonist treatment of advanced parkinsonian patients exposes striatal DA receptors to non-physiologic intermittent stimulation that contributes to the development of dyskinesias and other motor complications. To determine whether continuous dopaminergic stimulation can delay or prevent onset of motor complications, four MPTP-lesioned, levodopa-naive cynomolgus monkeys were implanted subcutaneously with apomorphine containing ethylene vinyl acetate rods. Three other MPTP-lesioned monkeys received daily injections of apomorphine. Animals receiving apomorphine rods showed improved motor function ('ON' state) within 1 day of implantation, and remained continually 'ON' for the duration of treatment (up to 6 months) without developing dyskinesias. Injected animals also showed similar improvement in motor function after each apomorphine injection. However, these primates remained 'ON' for only 90 min and within 7-10 days all developed severe dyskinesias. Implanted monkeys evidenced local irritation, which was alleviated by steroid co-therapy.

Effect of monoamine reuptake inhibitor NS 2330 in advanced Parkinson's disease.

Dopamine reuptake blockers, by enhancing and stabilizing intrasynaptic transmitter levels, could help palliate motor dysfunction in Parkinson's disease. This randomized, double-blind, placebo-controlled study compared the acute effects of the monoamine uptake inhibitor NS 2330 to those of placebo in 9 relatively advanced parkinsonian patients. At the dose administered, no change in parkinsonian scores was found when NS 2330 was given alone or with levodopa. Moreover, NS 2330 coadministration did not appear to alter dyskinesia severity or the duration of the antiparkinsonian response to levodopa. The drug was well tolerated. Under the conditions of this study, the present results failed to support the usefulness of dopamine reuptake inhibition in the treatment of advanced Parkinson's disease.

Striatal plasticity and extrapyramidal motor dysfunction.

Knowledge of molecular events contributing to motor dysfunction in Parkinson's disease has advanced rapidly during the past decade. Studies in animal models as well as in patients afflicted by this disorder suggest that the nonphysiologic stimulation of striatal dopamine receptors, first as a result of dopaminergic denervation and later as a consequence of the intermittent high-intensity stimulation produced by standard therapeutic regimens, leads to plastic changes in striatal medium spiny neurons. The clinical appearance of parkinsonism and subsequently of motor response complications is associated with the aberrant activation of signaling cascades within medium spiny neurons that modify the phosphorylation state of their ionotropic glutamatergic receptors. Resultant NMDA and AMPA receptor sensitization augments cortical excitatory input to these spiny efferent neurons, thus altering striatal output in ways that compromise motor function. These findings have already yielded new insight into mechanisms subserving motor memory and synaptic integration as well as accelerated development of novel approaches to the improved treatment of motor disability.

Mapping of rat brain using the Synuclein-1 monoclonal antibody reveals somatodendritic expression of alpha-synuclein in populations of neurons homologous to those vulnerable to Lewy body formation in human synucleopathies.

The neuronal protein alpha-synuclein has been implicated in the pathogenesis of Parkinson disease and other neurodegenerative diseases. Although many studies report that alpha-synuclein expression is restricted to neuronal presynaptic terminals, this protein aggregates in Lewy bodies in somata that are typically distant from their axon terminals. Few studies have addressed this paradox and there has been no compelling explanation proposed for the apparent discrepancy between the locus of neuronal alpha-synuclein expression and the loci of Lewy bodies in the majority of Parkinson disease cases. We explored this issue by extensively characterizing the monoclonal antibody Synuclein-1 (Syn-1) and using this highly selective antibody to map the distribution of alpha-synuclein throughout rat brain and in human substantia nigra (SN). Additionally, alpha-synuclein expression in rat SN detected by 2 polyclonal antibodies against alpha-synuclein was compared with that detected by the Syn-1 antibody. In contrast with many previous reports, alpha-synuclein was detected by Syn-1 in neuronal somata and dendrites in restricted brain regions, as well as more ubiquitously in axons and terminals. The strongest alpha-synuclein neuronal expression in rat was found in brainstem and cortical regions that are homologous to regions prone to Lewy body formation in humans. The Syn-1 antibody labeled abundant somatodendritic alpha-synuclein in both rat and human SN, a major locus of Lewy body formation and neurodegeneration in Parkinson disease. By contrast, very few immunoreactive somata in the rat SN were labeled by the 2 polyclonal antibodies. We explore possible explanations for the differences in conflicting reports of patterns of alpha-synuclein expression in brain, including differences among antibodies.

Striatal glutamatergic mechanisms and extrapyramidal movement disorders.

The nonphysiologic stimulation of striatal dopaminergic receptors, as a result of disease- or drug-related denervation or intermittent excitation, triggers adaptive responses in the basal ganglia which contribute to the appearance of parkinsonian symptoms and later to the dyskinesias and other alterations in motor response associated with dopaminergic therapy. Current evidence suggests that these altered responses involve activation of signal transduction cascades in striatal medium spiny neurons linking dopaminergic to coexpressed ionotropic glutamatergic receptors of the N-methyl-D-aspartate (NMDA) and Alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) classes. These intraneuronal signaling pathways appear capable of modifying the phosphorylation state of NMDA and AMPA receptor subunits; resultant sensitization enhances cortical glutamatergic input which in turn modifies striatal output in ways that compromise motor behavior. The regulation of these spiny neuron glutamate receptors can also be affected by the activation state of coexpressed nondopaminergic receptors as well as by changes associated with Huntington's disease. These observations lend new insight into molecular mechanisms contributing to the integration of synaptic inputs to spiny neurons. They also suggest novel approaches to the pharmacotherapy of extrapyramidal motor dysfunction.

Complications of a trophic xenotransplant approach in parkinsonian monkeys.

Various restorative cell transplantation strategies have been investigated to substitute for lost dopamine (DA) neurons or to enhance DA synthesis in Parkinson's disease. Intracerebral implantation of engineered cells encapsulated in a semipermeable polymer membrane constitutes one way to deliver bioactive substances unable to cross the blood-brain barrier while avoiding the need for long-term immunosuppression. Glial cell line-derived neurotrophic factor (GDNF) has shown trophic effects on DA neurons but effective and sustained delivery within the brain parenchyma remains problematic. The long-term efficacy and late complications of a xenotransplant approach utilizing GDNF-expressing encapsulated baby hamster kidney (BHK) cells were examined. Each of five MPTP-lesioned parkinsonian cynomolgus monkeys received five devices containing active or inert cells grafted bilaterally in the striatum in a two-stage procedure 9 months apart and animals were sacrificed 4 months later for analyses. No definite motor benefit was observed, DA levels were comparable between GDNF- and control cell-implanted striata, and tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra showed no consistent recovery. Cell viability and GDNF synthesis in the explanted devices were negligible. The brain tissue surrounding all implants showed an intense immune reaction with prominent "foreign body" inflammatory infiltrates. Membrane biophysics, the cell type used, and the extended period of time the devices remained in situ may have contributed to the negative outcome and should be addressed in future investigations using this approach.

Cyclic AMP responsive element binding protein phosphorylation and persistent expression of levodopa-induced response alterations in unilateral nigrostriatal 6-OHDA lesioned rats.

Activation of cAMP responsive element binding protein (CREB) has been increasingly implicated in the formation and maintenance of long-term memory. To elucidate molecular mechanisms that underlie the persisting alterations in motor response occurring with levodopa (L-dopa) treatment of parkinsonian patients, we evaluated the time course of these changes in relation to the activation of striatal CREB in 6-hydroxydopamine (6-OHDA) lesioned animals. Three weeks of twice-daily L-dopa treatment reduced the duration of the rotational response to acute L-dopa challenge in hemiparkinsonian rats, which lasted about 5 weeks after withdrawal of chronic L-dopa therapy. This shortened response duration, resembling human wearing-off fluctuations, was associated with a marked increase in Ser-133 phosphorylated CREB (pCREB) immunoreactivity in medium spiny neurons in dorsolateral striatum in response to acute dopaminomimetic challenge. Intermittent treatment with the D1 receptor-preferring agonist SKF 38393, but not the D2 receptor-preferring agonist quinpirole, produced a similar rise in CREB phosphorylation. The time course of changes in CREB phosphorylation correlated with the time course of changes in motor behavior after cessation of chronic L-dopa therapy. Both the altered motor response duration and the degree of CREB phosphorylation were attenuated by the intrastriatal administration of CREB antisense or protein kinase A inhibitor Rp-cAMPS. The results suggest that region-specific Ser-133 CREB phosphorylation in D1 receptor containing spiny neurons contributes to the persistence of the motor response alterations produced by intermittent stimulation of striatal dopaminergic receptors.