Neuroprotection by safinamide in the 6-hydroxydopamine model of Parkinson's disease.

AIMS: Current therapies in Parkinson's disease mainly treat symptoms rather than provide effective neuroprotection. We examined the effects of safinamide (monoamine oxidase B and sodium channel blocker) on microglial activation and the degeneration of dopaminergic neurons in a rat model of PD in vivo, and on microglia in vitro. METHODS: Rats received unilateral stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle on day 0: The contralateral side served as control. Safinamide or vehicle was delivered from days 0 or 1, for 7 days, via sub-cutaneous mini-pumps. RESULTS: In vehicle-treated rats 6-hydroxydopamine caused a significant increase in the number of activated MHC-II(+) microglia compared with the contralateral side, and only 50% of the dopaminergic neurons survived in the ipsilateral SNc. In contrast, rats treated daily with safinamide 50 and 150 mg/ml (on day 0 or 1) exhibited a significantly reduced number of activated microglia (55% reduction at 150 mg/ml) and a significant protection of dopaminergic neurons (80% of neurons survived) (P < 0.001) compared with vehicle-treated controls. Rasagiline, a monoamine oxidase B inhibitor, and lamotrigine, a sodium channel blocking drug, also protected dopaminergic neurons, indicating that safinamide may act by either or both mechanisms. Safinamide also reduced the activation of microglial cells in response to lipopolysaccharide exposure in vitro. CONCLUSION: Safinamide therapy suppresses microglial activation and protects dopaminergic neurons from degeneration in the 6-hydroxydopamine model of PD, suggesting that the drug not only treats symptoms but also provides neuroprotection.

Safinamide and flecainide protect axons and reduce microglial activation in models of multiple sclerosis.

Axonal degeneration is a major cause of permanent disability in the inflammatory demyelinating disease multiple sclerosis, but no therapies are known to be effective in axonal protection. Sodium channel blocking agents can provide effective protection of axons in the white matter in experimental models of multiple sclerosis, but the mechanism of action (directly on axons or indirectly via immune modulation) remains uncertain. Here we have examined the efficacy of two sodium channel blocking agents to protect white matter axons in two forms of experimental autoimmune encephalomyelitis, a common model of multiple sclerosis. Safinamide is currently in phase III development for use in Parkinson's disease based on its inhibition of monoamine oxidase B, but the drug is also a potent state-dependent inhibitor of sodium channels. Safinamide provided significant protection against neurological deficit and axonal degeneration in experimental autoimmune encephalomyelitis, even when administration was delayed until after the onset of neurological deficit. Protection of axons was associated with a significant reduction in the activation of microglia/macrophages within the central nervous system. To clarify which property of safinamide was likely to be involved in the suppression of the innate immune cells, the action of safinamide on microglia/macrophages was compared with that of the classical sodium channel blocking agent, flecainide, which has no recognized monoamine oxidase B activity, and which has previously been shown to protect the white matter in experimental autoimmune encephalomyelitis. Flecainide was also potent in suppressing microglial activation in experimental autoimmune encephalomyelitis. To distinguish whether the suppression of microglia was an indirect consequence of the reduction in axonal damage, or possibly instrumental in the axonal protection, the action of safinamide was examined in separate experiments in vitro. In cultured primary rat microglial cells activated by lipopolysaccharide, safinamide potently suppressed microglial superoxide production and enhanced the production of the anti-oxidant glutathione. The findings show that safinamide is effective in protecting axons from degeneration in experimental autoimmune encephalomyelitis, and that this effect is likely to involve a direct effect on microglia that can result in a less activated phenotype. Together, this work highlights the potential of safinamide as an effective neuroprotective agent in multiple sclerosis, and implicates microglia in the protective mechanism.

Chemical inducers and transcriptional markers of oligodendrocyte differentiation.

Oligodendrocytes generate and maintain myelin, which is essential for axonal function and protection of the mammalian central nervous system. To advance our molecular understanding of differentiation by these cells, we screened libraries of pharmacologically active compounds and identified inducers of differentiation of Oli-neu, a stable cell line of mouse oligodendrocyte precursors (OPCs). We identified four broad classes of inducers, namely, forskolin/cAMP (protein kinase A activators), steroids (glucocorticoids and retinoic acid), ErbB2 inhibitors, and nucleoside analogs, and confirmed the activity of these compounds on rat primary oligodendrocyte precursors and mixed cortical cultures. We also analyzed transcriptional responses in the chemically induced mouse and rat OPC differentiation processes and compared these with earlier studies. We confirm the view that ErbB2 is a natural signaling component that is required for OPC proliferation, whereas ErbB2 inhibition or genetic knockdown results in OPC differentiation.

Molecular Neuroimaging of the Dopamine Transporter as a Patient Enrichment Biomarker for Clinical Trials for Early Parkinson's Disease.

The Critical Path for Parkinson's (CPP) Imaging Biomarker and Modeling and Simulation working groups aimed to achieve qualification opinion by the European Medicines Agency (EMA) Committee for Medical Products for Human Use (CHMP) for the use of baseline dopamine transporter neuroimaging for patient selection in early Parkinson's disease clinical trials. This paper describes the regulatory science strategy to achieve this goal. CPP is an international consortium of three Parkinson's charities and nine pharmaceutical partners, coordinated by the Critical Path Institute.

The Qualification of an Enrichment Biomarker for Clinical Trials Targeting Early Stages of Parkinson's Disease.

As therapeutic trials target early stages of Parkinson's disease (PD), appropriate patient selection based purely on clinical criteria poses significant challenges. Members of the Critical Path for Parkinson's Consortium formally submitted documentation to the European Medicines Agency (EMA) supporting the use of Dopamine Transporter (DAT) neuroimaging in early PD. Regulatory documents included a comprehensive literature review, a proposed analysis plan of both observational and clinical trial data, and an assessment of biomarker reproducibility and reliability. The research plan included longitudinal analysis of the Parkinson Research Examination of CEP-1347 Trial (PRECEPT) and the Parkinson's Progression Markers Initiative (PPMI) study to estimate the degree of enrichment achieved and impact on future trials in subjects with early motor PD. The presence of reduced striatal DAT binding based on visual reads of single photon emission tomography (SPECT) scans in early motor PD subjects was an independent predictor of faster decline in UPDRS Parts II and III as compared to subjects with scans without evidence of dopaminergic deficit (SWEDD) over 24 months. The EMA issued in 2018 a full Qualification Opinion for the use of DAT as an enrichment biomarker in PD trials targeting subjects with early motor symptoms. Exclusion of SWEDD subjects in future clinical trials targeting early motor PD subjects aims to enrich clinical trial populations with idiopathic PD patients, improve statistical power, and exclude subjects who are unlikely to progress clinically from being exposed to novel test therapeutics.

In vitro and in vivo pharmacological profile of AS057278, a selective d-amino acid oxidase inhibitor with potential anti-psychotic properties.

Non-competitive N-methyl-d-aspartate (NMDA) blockers induce schizophrenic-like behavior in healthy volunteers and exacerbate symptomatology in schizophrenic patients. Hence, a compound able to enhance NMDA neurotransmission by increasing levels of d-serine, an endogenous full agonist at the glycine site of the NMDA receptors, could have anti-psychotic activity. One way to increase d-serine levels is the inhibition of d-amino acid oxidase (DAAO), the enzyme responsible for d-serine oxidation. Indeed AS057278, a potent in vitro (IC(50)=0.91 microM) and ex vivo (ED(50)=2.2-3.95 microM) DAAO inhibitor, was able to increase d-serine fraction in rat cortex and midbrain (10 mg/kg i.v.). AS057278 was able to normalize phencyclidine (PCP)-induced prepulse inhibition after acute (80 mg/kg) and chronic (20 mg/kg b.i.d.) oral administration in mice. Finally, AS057278 after oral chronic treatment (10 mg/kg b.i.d.) was able to normalize PCP-induced hyperlocomotion. These results suggest that AS057278 has the potential to anti-psychotic action toward both cognitive and positive symptoms of schizophrenia.

Targeted disruption of the murine Bin1/Amphiphysin II gene does not disable endocytosis but results in embryonic cardiomyopathy with aberrant myofibril formation.

The mammalian Bin1/Amphiphysin II gene encodes an assortment of alternatively spliced adapter proteins that exhibit markedly divergent expression and subcellular localization profiles. Bin1 proteins have been implicated in a variety of different cellular processes, including endocytosis, actin cytoskeletal organization, transcription, and stress responses. To gain insight into the physiological functions of the Bin1 gene, we have disrupted it by homologous recombination in the mouse. Bin1 loss had no discernible impact on either endocytosis or phagocytosis in mouse embryo-derived fibroblasts and macrophages, respectively. Similarly, actin cytoskeletal organization, proliferation, and apoptosis in embryo fibroblasts were all unaffected by Bin1 loss. In vivo, however, Bin1 loss resulted in perinatal lethality. Bin1 has been reported to affect muscle cell differentiation and T-tubule formation. No striking histological abnormalities were evident in skeletal muscle of Bin1 null embryos, but severe ventricular cardiomyopathy was observed in these embryos. Ultrastructurally, myofibrils in ventricular cardiomyocytes of Bin1 null embryos were severely disorganized. These results define a developmentally critical role for the Bin1 gene in cardiac muscle development.

Safinamide reduces dyskinesias and prolongs L-DOPA antiparkinsonian effect in parkinsonian monkeys.

INTRODUCTION: Safinamide is a compound under investigation for use in the treatment of Parkinson's disease for combination with pharmacological therapy currently available. The objective of this study was to test the effects of safinamide in an animal model of l-DOPA-induced dyskinesias (LID), the MPTP lesioned dyskinetic macaque monkey, in comparison to and in combination with amantadine. METHODS: LID and parkinsonian symptoms were measured in dyskinetic monkeys treated with l-DOPA with and without several dose levels of safinamide, amantadine, and the two in combination. Safinamide plasma levels were monitored during the experiments. RESULTS: Safinamide pre-treatment (3, 10, 20 and 30 mg/kg) dose-dependently reduced LID scores, in two acute and one semi-chronic experiment. Intensity and duration of LID were reduced and inversely correlated with safinamide blood levels. All doses of safinamide tested prolonged the duration of the beneficial antiparkinsonian effect of l-DOPA. Amantadine (5 and 20 mg/kg) reduced LID, but reduced duration of antiparkinsonian response to l-DOPA. When added to amantadine (5 mg/kg), safinamide showed no (3 mg/kg) or modest (20 mg/kg) additional beneficial effects on LID while the combined treatment prevented the reduction of the duration of the l-DOPA antiparkinsonian effect observed with amantadine only. CONCLUSIONS: Safinamide and amantadine reduced LID in this primate model while only safinamide increased the duration of the antiparkinsonian response of l-DOPA, suggesting that safinamide may have effects on LID that are pharmacologically distinct from amantadine, which is in current clinical use for control of LID.

Tremorolytic effects of safinamide in animal models of drug-induced parkinsonian tremor.

Safinamide is an α-aminoamide derivative that is currently in Phase III clinical trial development as an add-on therapy to levodopa or dopamine agonists for patients with Parkinson's disease. Safinamide is a monoamine oxidase B inhibitor with additional non-dopaminergic actions. The present experiments were performed to evaluate the ability of safinamide to attenuate parkinsonian motor impairments using the tremulous jaw movement model, an animal model of parkinsonian tremor. In rats, tremulous jaw movements can be induced with dopamine (DA) antagonists, DA depletion, and cholinomimetics, and can be reversed by various antiparkinsonian drugs, including L-DOPA, DA agonists, anticholinergics and adenosine A2A antagonists. In these present experiments, tremulous jaw movements were induced with the anticholinesterase galantamine (3.0mg/kg IP), the muscarinic agonist pilocarpine (0.5mg/kg IP), and the dopamine D2 antagonist pimozide (1.0mg/kg IP). Safinamide significantly reduced the number of tremulous jaw movements induced by galantamine, pilocarpine, and pimozide, with consistent effects across all three drugs at a dose range of 5.0-10.0mg/kg. The results of this study support the use of safinamide as a treatment for parkinsonian tremor.

The amyloid-beta rise and gamma-secretase inhibitor potency depend on the level of substrate expression.

The amyloid-beta (Abeta) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-beta precursor protein (APP) through consecutive proteolytic cleavages by beta-site APP-cleaving enzyme and gamma-secretase. Unexpectedly gamma-secretase inhibitors can increase the secretion of Abeta peptides under some circumstances. This "Abeta rise" phenomenon, the same inhibitor causing an increase in Abeta at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Abeta rise depends on the beta-secretase-derived C-terminal fragment of APP (betaCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Abeta, known as "p3," formed by alpha-secretase cleavage, did not exhibit a rise. In addition to the Abeta rise, low betaCTF or C99 expression decreased gamma-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, gamma-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Abeta under conditions of low substrate expression. The Abeta rise was also observed in rat brain after dosing with the gamma-secretase inhibitor BMS-299897. The Abeta rise and potency shift are therefore relevant factors in the development of gamma-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Abeta rise, including the "incomplete processing" and endocytic models, are discussed.

Design and synthesis of benzoazepinone-derived cyclic malonamides and aminoamides as potent gamma-secretase inhibitors.

We report the synthesis of benzoazepine-derived cyclic malonamides (2) and aminoamides (3) as gamma-secretase inhibitors for the potential treatment of Alzheimer's disease. The in vitro structure-activity relationships of 2 and 3 along with dog pharmacokinetic results are described.

Synthesis and evaluation of succinoyl-caprolactam gamma-secretase inhibitors.

The synthesis, evaluation, and structure-activity relationships of a series of succinoyl lactam inhibitors of the Alzheimer's disease gamma-secretase are described. Beginning with a screening hit with broad proteinase activity, optimization provided compounds with both high selectivity for inhibition of gamma-secretase and high potency in cellular assays of A beta reduction. The SAR and early in vivo properties of this series of inhibitors will be presented.

Identification and characterization of presenilin-independent Notch signaling.

Presenilin (PS) proteins control the proteolytic cleavage that precedes nuclear access of the Notch intracellular domain. Here we observe that a partial activation of the HES1 promoter can be detected in PS1/PS2 (PS1/2) double null cells using Notch1 Delta E constructs or following Delta 1 stimulation, despite an apparent abolition of the production and nuclear accumulation of the Notch intracellular domain. PS1/2-independent Notch activation is sensitive to Numblike, a physiological inhibitor of Notch. PS1/2-independent Notch signaling is also inhibited by an active gamma-secretase inhibitor in the low micromolar range and is not inhibited by an inactive analogue, similar to PS-dependent Notch signaling. However, experiments using a Notch1-Gal4-VP16 fusion protein indicate that the PS1/2-independent activity does not release Gal4-VP16 and is therefore unlikely to proceed via an intramembranous cleavage. These data reveal that a novel PS1/2-independent mechanism plays a partial role in Notch signal transduction.