Thomas A. Comery

Acute γ-Secretase Inhibition Improves Contextual Fear Conditioning in the Tg2576 Mouse Model of Alzheimer's Disease

Transgenic mice (Tg2576) overexpressing the Swedish mutation of the human amyloid precursor protein display biochemical, pathological, and behavioral markers consistent with many aspects of Alzheimers disease, including impaired hippocampal function. Impaired, hippocampal-dependent, contextual fear conditioning (CFC) is observed in mice as young as 20 weeks of age. This impairment can be attenuated after treatment before training with the phosphodiesterase-4 inhibitor rolipram (0.1 mg/kg, i.p.). A rolipram-associated improvement is also observed in the littermate controls, suggesting that the effect of rolipram is independent of β-amyloid. Acute treatment before training (but not after training or before testing) with the γ-secretase inhibitor (GSI) N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine-t-butylester (DAPT), at a dose that reduces brain concentrations of β-amyloid (100 mg/kg), attenuates the impairment in 20- to 65-week-old Tg2576 mice. Importantly, DAPT had no effect on performance of control littermates. These data are supportive of a role of β-amyloid in the impairment of CFC in Tg2576 mice. Furthermore, they suggest that acute treatment with GSI may provide improved cognitive functioning as well as disease-modifying effects in Alzheimers disease.

Phosphodiesterase 10A inhibitor activity in preclinical models of the positive, cognitive, and negative symptoms of schizophrenia.

Following several recent reports that suggest that dual cAMP and cGMP phosphodiesterase 10A (PDE10A) inhibitors may present a novel mechanism to treat positive symptoms of schizophrenia, we sought to extend the preclinical characterization of two such compounds, papaverine [1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline] and MP-10 [2-{[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)phenoxy]methyl}quinoline], in a variety of in vivo and in vitro assays. Both of these compounds were active in a range of antipsychotic models, antagonizing apomorphine-induced climbing in mice, inhibiting conditioned avoidance responding in both rats and mice, and blocking N-methyl-d-aspartate antagonist-induced deficits in prepulse inhibition of acoustic startle response in rats, while improving baseline sensory gating in mice, all of which strengthen previously reported observations. These compounds also demonstrated activity in several assays intended to probe negative symptoms and cognitive deficits, two disease domains that are underserved by current treatments, with both compounds showing an ability to increase sociality in BALB/cJ mice in the social approach/social avoidance assay, enhance social odor recognition in mice and, in the case of papaverine, improve novel object recognition in rats. Biochemical characterization of these compounds has shown that PDE10A inhibitors modulate both the dopamine D1-direct and D2-indirect striatal pathways and regulate the phosphorylation status of a panel of glutamate receptor subunits in the striatum. It is striking that PDE10A inhibition increased the phosphorylation of the (±)-α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptor GluR1 subunit at residue serine 845 at the cell surface. Together, our results suggest that PDE10A inhibitors alleviate both dopaminergic and glutamatergic dysfunction thought to underlie schizophrenia, which may contribute to the broad-spectrum efficacy.

Increased Density of Multiple-Head Dendritic Spines on Medium-Sized Spiny Neurons of the Striatum in Rats Reared in a Complex Environment

It has generally been assumed that new synapses added to various brain regions in response to experience are equivalent to those already in existence. Theorists have recently posited that synaptic configurations involving multiple associated contacts may facilitate plastic change. The number of multiple-headed dendritic spines on medium-sized spiny neurons in the rat dorsolateral corpus striatum was determined following rearing in environments differing in complexity. Postweaning rats were either housed as a group in a toy- and object-filled environment or housed individually in standard laboratory cages for 30 days. Dendritic segments of Golgi-Cox impregnated Type I spiny neurons of the complex environment housed rats had approximately 60% more multiple-head spines than those of the individually caged animals. Multiple-head spines may reflect parallel synaptic contacts that modify relative strengths of existing connections or connections with a novel presynaptic terminal that alter the neurons pattern of connections.

ADX47273: A Novel Metabotropic Glutamate Receptor 5 Selective Positive Allosteric Modulator with Preclinical Antipsychotic-Like and Pro-cognitive Activities

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca2+ assays (EC50 = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 μM) and competed for binding of [3H]2-methyl-6-(phenylethynyl)pyridine (Ki = 4.3 μM), but not [3H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.

Begacestat (GSI-953): A Novel, Selective Thiophene Sulfonamide Inhibitor of Amyloid Precursor Protein γ-Secretase for the Treatment of Alzheimer's Disease

The presenilin containing γ-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. γ-Secretase catalyzes the final step in the generation of Aβ40 and Aβ42 peptides from APP. Amyloid β-peptides (Aβ peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimers disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Aβ peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide γ-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Aβ production with low nanomolar potency in cellular and cell-free assays of γ-secretase function, and displaces a tritiated analog of GSI-953 from enriched γ-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Aβ levels, and a reversal of contextual fear-conditioning deficits that are correlated with Aβ load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Aβ levels, confirming pharmacodynamic activity of GSI-953 in humans.

Effects of atomoxetine and methylphenidate on attention and impulsivity in the 5-choice serial reaction time test.

Deficits in attention and response inhibition are apparent across several neurodegenerative and neuropsychiatric disorders for which current pharmacotherapy is inadequate. The 5-choice serial reaction time test (5-CSRTT), which originated from the continuous performance test (CPT) in humans, may serve as a useful translational assay for efficacy in these key behavioral domains. The selective norepinepherine reuptake inhibitor, atomoxetine, represents the first non-stimulant based drug approved for Attention Deficit Hyperactivity Disorder (ADHD) and has replaced methylphenidate (Ritalin) as the first line in pharmacotherapy for the treatment of ADHD. Methylphenidate and atomoxetine have different cortical and sub-cortical neurochemical signatures that could predict differences in cognitive and non-cognitive functions. The present experiments investigated the effects of acute methylphenidate and atomoxetine in male long Evans rats in the 5-choice serial reaction time (5CSRT) test that is hypothesized to serve as a model of vigilance and impulsivity behaviors associated with ADHD. Long Evans rats were trained to perform at 75% correct responses with fewer than 20% missed trials in the 5CSRT test (500 ms stimulus duration, 5 s inter-trial interval (ITI)). By varying the ITI (10, 7, 5, and 4 s) on drug test days, impulsivity (as defined by premature responses) was dramatically increased with a concomitant decrease in attention (percent correct). Subsequently, animals were treated with methylphenidate (2.5 and 5 mg/kg, i.p.) or atomoxetine (0.1, 0.5 and 1 mg/kg, i.p.) using this design. In Experiment 1, treatment with methylphenidate modestly improved overall attention but the highest dose of methylphenidate (5.0 mg/kg) significantly increased impulsivity. In contrast, treatment with atomoxetine induced a marked decrease in impulsivity whilst modestly improving overall attention. Interestingly, no effect was observed on measures of performance (e.g. motivation/sedation) with atomoxetine, whilst moderate hyperactivity (faster overall response latencies; magazine, correct, incorrect) was observed in the methylphenidate group. Those data suggest that the 5CSRT test can be used to differentiate stimulant and non-stimulant pharmacotherapies on measures of impulsivity.

Differential Rearing Alters Spine Density on Medium-Sized Spiny Neurons in the Rat Corpus Striatum: Evidence for Association of Morphological Plasticity with Early Response Gene Expression

Morphological plasticity of medium-sized spiny neurons of the striatum was examined in Long-Evans hooded rats reared in complex or individual cage environments. Rat pups, aged 28-32 days, were housed either individually in standard laboratory cages or as a group in a large toy- and object-filled environment for 30 days. The spine density on dendrites of medium-sized spiny neurons in the dorsolateral striatum was then examined using the Golgi-Cox method. Rats reared in the complex environment displayed an increase of approximately 30% in spine density relative to those reared individually. These results demonstrate experience-dependent changes in neural structure in the striatum and suggest that the mechanisms for information storage in response to experience may be more widespread in the forebrain than previously believed.

Enhanced clearance of Aβ in brain by sustaining the plasmin proteolysis cascade

The amyloid hypothesis states that a variety of neurotoxic β-amyloid (Aβ) species contribute to the pathogenesis of Alzheimers disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Aβ production and clearance. Enzymes responsible for the degradation of Aβ are not well understood, and, thus far, it has not been possible to enhance Aβ catabolism by pharmacological manipulation. We provide evidence that Aβ catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Aβ levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Aβ oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Aβ. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Aβ levels, restore long-term potentiation deficits in hippocampal slices from transgenic Aβ-producing mice, and reverse cognitive deficits in these mice.

Procognitive and Neuroprotective Activity of a Novel α7 Nicotinic Acetylcholine Receptor Agonist for Treatment of Neurodegenerative and Cognitive Disorders

The α7 nicotinic acetylcholine receptor (nAChR) is a promising target for treatment of cognitive dysfunction associated with Alzheimers disease and schizophrenia. Here, we report the pharmacological properties of 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide [SEN12333 (WAY-317538)], a novel selective agonist of α7 nAChR. SEN12333 shows high affinity for the rat α7 receptor expressed in GH4C1 cells (Ki = 260 nM) and acts as full agonist in functional Ca2+ flux studies (EC50 = 1.6 μM). In whole-cell patch-clamp recordings, SEN12333 activated peak currents and maximal total charges similar to acetylcholine (EC50 = 12 μM). The compound did not show agonist activity at other nicotinic receptors tested and acted as a weak antagonist at α3-containing receptors. SEN12333 treatment (3 mg/kg i.p.) improved episodic memory in a novel object recognition task in rats in conditions of spontaneous forgetting as well as cognitive disruptions induced via glutamatergic [5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); MK-801] or cholinergic (scopolamine) mechanisms. This improvement was blocked by the α7-selective antagonist methyllycaconitine, indicating that it is mediated by α7 activation. SEN12333 also prevented a scopolamine-induced deficit in a passive avoidance task. In models targeting other cognitive domains, including attention and perceptual processing, SEN12333 normalized the apomorphine-induced deficit of prepulse inhibition. Neuroprotection of SEN12333 was demonstrated in quisqualate-lesioned animals in which treatment with SEN12333 (3 mg/kg/day i.p.) resulted in a significant protection of choline acetyltransferase-positive neurons in the lesioned hemisphere. Cumulatively, our results demonstrate that the novel α7 nAChR agonist SEN12333 has procognitive and neuroprotective properties, further demonstrating utility of α7 agonists for treatment of neurodegenerative and cognitive disorders.

Discovery of Begacestat, a Notch-1-Sparing γ-Secretase Inhibitor for the Treatment of Alzheimer's Disease

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimers disease.