Assessment of gait and balance impairment in people with spinocerebellar ataxia using wearable sensors.

OBJECTIVE: To explore the use of wearable sensors for objective measurement of motor impairment in spinocerebellar ataxia (SCA) patients during clinical assessments of gait and balance. METHODS: In total, 14 patients with genetically confirmed SCA (mean age 61.6 ± 8.6 years) and 4 healthy controls (mean age 49.0 ± 16.4 years) were recruited through the Massachusetts General Hospital (MGH) Ataxia Center. Participants donned seven inertial sensors while performing two independent trials of gait and balance assessments from the Scale for the Assessment and Rating of Ataxia (SARA) and Brief Ataxia Rating Scale (BARS2). Univariate analysis was used to identify sensor-derived metrics from wearable sensors that discriminate motor function between the SCA and control groups. Multivariate linear regression models were used to estimate the subjective in-person SARA/BARS2 ratings. Spearman correlation coefficients were used to evaluate the performance of the model. RESULTS: Stride length variability, stride duration, cadence, stance phase, pelvis sway, and turn duration were different between SCA and controls (p < 0.05). Similarly, sway and sway velocity of the ankle, hip, and center of mass differentiated SCA and controls (p < 0.05). Using these features, linear regression models showed moderate-to-strong correlation with clinical scores from the in-person rater during SARA assessments of gait (r = 0.73, p = 0.003) and stance (r = 0.90, p < 0.001) and the BARS2 gait assessment (r = 0.74, p = 0.003). CONCLUSION: This study demonstrates that sensor-derived metrics can potentially be used to estimate the level of motor impairment in patient with SCA quickly and objectively. Thus, digital biomarkers from wearable sensors have the potential to be an integral tool for SCA clinical trials and care.

Adjunctive Lanicemine (AZD6765) in Patients with Major Depressive Disorder and History of Inadequate Response to Antidepressants: A Randomized, Placebo-Controlled Study.

The objective of this study was to investigate the efficacy and safety of adjunctive lanicemine (NMDA channel blocker) in the treatment of major depressive disorder (MDD) over 12 weeks. This phase IIb, randomized, parallel-arm, double-blind, placebo-controlled study was conducted at 49 centers in four countries between December 2011 and August 2013 in 302 patients aged 18-70 years, meeting criteria for single episode or recurrent MDD and with a history of inadequate treatment response. Patients were required to be taking an allowed antidepressant for at least four weeks prior to screening. Patients were randomized equally to receive 15 double-blind intravenous infusions of adjunctive lanicemine 50 mg, lanicemine 100 mg, or saline over a 12-week course, in addition to ongoing antidepressant. The primary efficacy end point was change in Montgomery-Åsberg Depression Rating Scale (MADRS) total score from baseline to week 6. Secondary efficacy outcome variables included change in MADRS score from baseline to week 12, response and remission rates, and changes in Clinical Global Impression scale, Quick Inventory of Depressive Symptomology Self-Report score, and Sheehan Disability Scale score. Of 302 randomized patients, 240 (79.5%) completed treatment. Although lanicemine was generally well tolerated, neither dose was superior to placebo in reducing depressive symptoms on the primary end point or any secondary measures. There was no significant difference between lanicemine and placebo treatment on any outcome measures related to MDD. Post hoc analyses were performed to explore the possible effects of trial design and patient characteristics in accounting for the contrasting results with a previously reported trial.

Concentration-response relationship of the α7 nicotinic acetylcholine receptor agonist FRM-17874 across multiple in vitro and in vivo assays.

Pharmacological activation of α7 nicotinic acetylcholine receptors (α7 nAChRs) may improve cognition in schizophrenia and Alzheimer's disease. The present studies describe an integrated pharmacological analysis of the effects of FRM-17874, an analogue of encenicline, on α7 nAChRs in vitro and in behavioral and neurophysiological assays relevant to cognitive function. FRM-17874 demonstrated high affinity binding to human α7 nAChRs, displacing [(3)H]-methyllacaconitine (Ki=4.3nM). In Xenopus laevis oocytes expressing human α7 nAChRs, FRM-17874 acted as an agonist, evoking inward currents with an EC50 of 0.42µM. Lower concentrations of FRM-17874 (0.01-3nM) elicited no detectable current, but primed receptors to respond to sub-maximal concentrations of acetylcholine. FRM-17874 improved novel object recognition in rats, and enhanced memory acquisition and reversal learning in the mouse water T-maze. Neurophysiological correlates of cognitive effects of drug treatment, such as synaptic transmission, long-term potentiation, and hippocampal theta oscillation were also evaluated. Modulation of synaptic transmission and plasticity was observed in rat hippocampal slices at concentrations of 3.2 and 5nM. FRM-17874 showed a dose-dependent facilitation of stimulation-induced hippocampal theta oscillation in mice and rats. The FRM-17874 unbound brain concentration-response relationship for increased theta oscillation power was similar in both species, exhibited a biphasic pattern peaking around 3nM, and overlapped with active doses and exposures observed in cognition assays. In summary, behavioral and neurophysiological assays indicate a bell-shaped effective concentration range and this report represents the first attempt to explain the concentration-response function of α7 nAChR-mediated pro-cognitive effects in terms of receptor pharmacology.

Identification of short-acting κ-opioid receptor antagonists with anxiolytic-like activity.

The κ-opioid receptor plays a central role in mediating the response to stressful life events. Inhibiting κ-opioid receptor signaling is proposed as a mechanism for treating stress-related conditions such as depression and anxiety. Preclinical testing consistently confirms that disruption of κ-opioid signaling is efficacious in animal models of mood disorders. However, concerns about the feasibility of developing antagonists into drugs stem from an unusual pharmacodynamic property of prototypic κ-opioid receptor-selective antagonists; they inhibit receptor signaling for weeks to months after a single dose. Several fundamental questions include - is it possible to identify short-acting antagonists; is long-lasting inhibition necessary for efficacy; and is it safe to develop long-acting antagonists in the clinic. Here, we test representative compounds (AZ-ECPC, AZ-MTAB, and LY-DMPF) from three new chemical series of κ-opioid receptor ligands for long-lasting inhibition. Each compound dose-dependently reversed κ-opioid agonist-induced diuresis. However, unlike the prototypic antagonist, nBNI, which fully inhibited evoked diuresis for at least four weeks, the new compounds showed no inhibition after one week. The two compounds with greater potency and selectivity were tested in prenatally-stressed rats on the elevated plus maze, an exploration-based model of anxiety. Spontaneous exploration of open arms in the elevated plus maze was suppressed by prenatal stress and restored with both compounds. These findings indicate that persistent inhibition is not an inherent property of κ-opioid-selective antagonists and that post-stress dosing with transient inhibitors can be effective in a mood disorder model. This further supports κ-opioid receptor as a promising target for developing novel psychiatric medications.

In vitro metabolism of α7 neuronal nicotinic receptor agonist AZD0328 and enzyme identification for its N-oxide metabolite.

1. AZD0328 was pharmacologically characterized as a α7 neuronal nicotinic receptor agonist intended for treatment of Alzheimer's disease. In vitro AZD0328 cross species metabolite profile and enzyme identification for its N-oxide metabolite were evaluated in this study. 2. AZD0328 was very stable in the human hepatocyte incubation, whereas extensively metabolized in rat, dog and guinea pig hepatocyte incubations. The N-oxidation metabolite (M6) was the only metabolite detected in human hepatocyte incubations, and it also appeared to be the major in vitro metabolic pathway in a number of preclinical species. In addition, N-glucuronide metabolite of AZD0328 was observed in human liver microsomes. 3. Other metabolic pathways in the preclinical species include hydroxylation in azabicyclo octane or furopyridine part of the molecule. Pyridine N-methylation of AZD0328 (M2) was identified as a dog specific metabolite, not observed in human or other preclinical species. 4. Multiple enzymes including CYP2D6, CYP3A4/5, FMO1 and FMO3 catalyzed AZD0328 metabolism. The potential for AZD0328 to be inhibited clinically by co-administered drugs or genetic polymorphism is relative low.

Immediate and sustained improvements in working memory after selective stimulation of α7 nicotinic acetylcholine receptors.

BACKGROUND: Nicotine improves cognition in humans and animal models of neuropsychiatric disorders. Here, we sought to establish whether selective stimulation of the neuronal nicotinic α7 receptor could improve spatial working memory in nonhuman primates. METHODS: Beginning with an estimated dose range from rodent studies, the dose of the α7 agonist AZD0328 was titrated for a significant impact on working memory in rhesus macaques after acute administration. After training to stability on the spatial delayed response task, subjects were administered AZD0328 (1.6 ng/kg-.48 mg/kg; intramuscular) or vehicle 30 min before cognitive testing. AZD0328 (1 ng/kg-1.0 µg/kg; intramuscular) was then administered in a repeated, intermittent ascending dose regimen where each dose was given in two bouts for 4 days with a 1-week washout in between bouts, followed by 2-week washout. RESULTS: Acute AZD0328 improved cognitive performance when the dose was titrated down to .0016 and .00048 mg/kg from a cognitively impairing dose of .48 mg/kg. In a subgroup, sustained enhancement of working memory was evident for 1 month or more after acute treatment. Immediate and sustained cognitive enhancement was also found during and after repeated administration of AZD0328 at .001 mg/kg. CONCLUSIONS: These findings demonstrate that extremely low doses of a nicotinic α7 agonist can have profound acute and long-lasting beneficial consequences for cognition, dependent upon the integrity of dorsolateral prefrontal cortex. Thus, the α7 receptor might have a fundamental role in the neural circuitry of working memory and in the synaptic plasticity upon which it might depend.

Linking the cytokine and neurocircuitry hypotheses of depression: a translational framework for discovery and development of novel anti-depressants.

Recent studies suggest a model of depression that links the cytokine hypothesis from the field of psychoneuroimmunology with the neurocircuitry hypothesis derived from burgeoning insight into neurophysiological changes observed in depressed patients. According to the neurocircuitry hypothesis of depression, failure of homeostatic synaptic plasticity in cortical-striatal-limbic nodes of a distributed network of neural circuits involving the sub-genual anterior cingulate cortex is responsible for core symptoms of depression: loss of interest or pleasure (anhedonia) and depressed mood (sadness). According to the cytokine hypothesis of depression, inflammatory cytokines act on neural circuits to evoke the behavioral and physiological changes observed in depression. Synthesis of these hypotheses implicates cytokines released during injury, infection, illness, or psychological stress as a cause of dysregulated synaptic plasticity in cortical-striatal-limbic circuits implicated in depression. These neural circuits process affective and reward-based information for optimal cost-benefit decision-making, a function that may link cytokine-evoked changes in synaptic plasticity to translatable measures of specific behavioral impairments observed in depressed patients. This viewpoint outlines evidence linking the cytokine and neurocircuitry hypotheses of depression to offer a translational model of major depressive disorder suitable for novel drug discovery and development.

Differential coupling of M1 muscarinic and alpha7 nicotinic receptors to inhibition of pemphigus acantholysis.

The mechanisms mediating and regulating assembly and disassembly of intercellular junctions is a subject of intensive research. The IgG autoantibodies produced in patients with the immunoblistering skin disease pemphigus vulgaris (PV) can induce keratinocyte (KC) dyshesion (acantholysis) via mechanisms that involve signaling kinases targeting intercellular adhesion molecules, thus providing a useful model to study the physiologic regulation of KC cohesion. Previous studies showed that activation of Src and protein kinase C are the earliest events in the PV IgG-induced intracellular phosphorylation cascades and that cholinergic agonists are effective for treating patients with pemphigus. In this study, we sought to elucidate the molecular mechanisms allowing cholinergic agonists to inhibit PV IgG-induced acantholysis and phosphorylation of KC adhesion molecules. The extent of acantholysis in KC monolayers correlated closely with the degree of PV IgG-induced phosphorylation of p120- and beta-catenins, with classic isoforms of protein kinase C mediating serine phosphorylation of beta-catenin and Src-tyrosine phosphorylation of p120-catenin. The M(1) muscarinic agonist pilocarpine blocked phosphorylation of both catenins, which could be abolised by the M(1) antagonist MT7. The alpha7 nicotinic agonist AR-R17779 inhibited phosphorylation of P120-cateinin. The alpha7 antagonist methyllycaconitine abolished the effect of AR-R17779. Okadaic acid abrogated protective effects of agonists on phosphorylation of beta-catenin, and pervanadate, on that of p120-catenin. Stimulation of KCs with pilocarpine significantly (p < 0.05) elevated both serine/threonine and tyrosine phosphatase activities in KCs. AR-R17779 both stimulated tyrosine phosphatase and decreased PV IgG-induced Src activity. Methyllycaconitine released Src activity in intact KCs and caused acantholysis. Thus, downstream signaling from M(1) abolished PV IgG-dependent catenin phosphorylation due to activation of both serine/threonine and tyrosine phosphatases, whereas alpha7 action involved both activation of tyrosine phosphatase and inhibition of Src. These findings identified novel paradigm of regulation of signaling kinases associated with cholinergic receptors and provided mechanistic explanation of therapeutic activity of cholinomimetics in PV patients.

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors protect cortical neurons from excitotoxicity.

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, or statins, reduce the incidence of strokes and reduce infarct volume after cerebral ischemia in mice. Excitoxicity caused by overstimulation of glutamate receptors is a major cause of neuronal death after an ischemic brain insult. Experiments presented here explored whether statins protect cultured neurons from excitotoxic death caused by the glutamate receptor agonist NMDA. Treatment with statins preserved NMDA receptor-expressing cortical neurons and potently and substantially reduced lactate dehydrogenase release caused by exposure of embryonic mouse neocortical cultures to NMDA. The rank order of neuroprotective potency was rosuvastatin = simvastatin > atorvastatin = mevastatin > pravastatin, which is similar to the known rank order of potency for inhibition of the HMG-CoA reductase enzyme. Resistance of cultures to NMDA excitotoxicity developed after several days of statin exposure. Neuroprotection by rosuvastatin was coincident with a decrease in cell sterols and occurred with a similar potency as inhibition of cholesterol biosynthesis. Neuroprotection was substantially attenuated by cotreatment with either mevalonate or cholesterol and was mimicked by acute treatment with the cholesterol-extracting agent beta-cyclodextrin, suggesting that neuroprotection was mediated by depletion of a cellular pool of cholesterol because of the inhibition of cholesterol biosynthesis. These results suggest the possibility that, in addition to effects on cerebrovascular function, statins have the potential to render cortical neurons more resistant to NMDA-induced excitotoxic death as a result of changes in cell cholesterol homeostasis.

Microplate gel-filtration method for radioligand-binding assays.

A thin-layer gel-filtration chromatographic method has been developed in a 96-well format to separate free and protein-bound ligand in radioligand-binding assays. The mobile phase in the gel-filtration plate is removed via quick centrifugation before samples are applied. Protein-bound ligand is recovered via centrifugation into another 96-well plate for radioactivity measurements. The method exhibits excellent recovery of protein-ligand complexes and less opportunity for dissociation of the complexes since it eliminates major dilution effects from the mobile phase of a column and from elution steps in conventional gel-filtration chromatography. It offers other advantages: simple, rapid, inexpensive, quantitative, and able to handle a large number of samples as required in drug discovery and clinical settings. This microplate gel-filtration method was optimized in studies of receptor-ligand interactions using estrogen receptors as examples and can be used in other radioligand-binding assays.

Purification, characterization and catalytic properties of human sterol 8-isomerase.

CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2)H(2)O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM)