Brian J. Eastwood

Analgesic effects of serotonergic, noradrenergic or dual reuptake inhibitors in the carrageenan test in rats: evidence for synergism between serotonergic and noradrenergic reuptake inhibition.

The efficacy of antidepressant drugs with serotonergic, noradrenergic, or dual reuptake inhibition was evaluated in reversing carrageenan-induced thermal hyperalgesia and mechanical allodynia in rats. Duloxetine (1-30mg/kg, i.p.), a balanced serotonergic-noradrenergic reuptake inhibitor (SNRI), was equiefficacious and more potent than the SNRI venlafaxine (3-100mg/kg, i.p.) in reversing both thermal hyperalgesia and mechanical allodynia induced by carrageenan. In addition, the selective noradrenergic reuptake inhibitors (NRIs) thionisoxetine (0.03-10mg/kg, i.p.) and desipramine (1-30mg/kg, i.p.) also produced complete reversals of carrageenan-induced thermal hyperalgesia. However, only thionisoxetine exhibited a greater than 80% reversal of the carrageenan-induced mechanical allodynia. In contrast, the selective serotonergic reuptake inhibitors (SSRIs) paroxetine, sertraline, and fluoxetine (0.3-10mg/kg i.p.) had little or no effect in the carrageenan model. In order to understand whether the observed enhanced effectiveness of the dual SNRIs was due to a possible synergism between serotonergic and noradrenergic reuptake inhibition, the effects of the NRI thionisoxetine alone and in combination with an inactive dose of the SSRI fluoxetine were determined. In the presence of fluoxetine, the potency of thionisoxetine in reversing carrageenan-induced hyperalgesia and allodynia was significantly increased by approximately 100-fold and brain concentrations of thionisoxetine were increased by 1.1- to 5-fold. The present data indicate fluoxetine pharmacodynamically potentiated the analgesic effects of thionisoxetine over and above a metabolic interaction between these two drugs. The present findings thus indicate that, in the carrageenan model, dual serotonergic-noradrenergic reuptake inhibition by dual SNRIs, or SSRI-NRI combinations, produces synergistic analgesic efficacy.

BrainSeq: Neurogenomics to Drive Novel Target Discovery for Neuropsychiatric Disorders

We outline an ambitious project to characterize the genetic and epigenetic regulation of multiple facets of transcription in distinct brain regions across the human lifespan in samples of major neuropsychiatric disorders and controls. Initially focused on schizophrenia and mood disorders, the goal of this consortium is to elucidate the underlying molecular mechanisms of genetic associations with the goal of identifying novel therapeutic targets. The consortium currently consists of seven pharmaceutical companies and a not-for-profit medical research institution working as a precompetitive team to generate and analyze publicly available archival brain genomic data related to neuropsychiatric illness.

Myostatin Neutralization Results in Preservation of Muscle Mass and Strength in Preclinical Models of Tumor Induced Muscle Wasting

Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non–tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass– and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non–tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting. Mol Cancer Ther; 14(7); 1661–70. ©2015 AACR.

Effect of sampling frames on response rates in the WHO MONICA risk factor surveys

Sample surveys are used to investigate occurrence and determinants of diseases in populations. Their reliability is influenced by quality of sampling frame and response rate. We investigated relationship between sampling frame type and response rates and assessed their impact on non-response bias, using data from the WHO MONICA Project, where 37 centres in 20 countries conducted sample surveys, employing the best locally available sampling frame. Sampling frames fell into three categories: Population registers (PR), electoral registers (ER), and health care registers (HR). Response rate (rrs) was factored into components reflecting quality of sampling frame (contact rate cr) and characterizing willingness of sample members to participate (enrolment rate er). The mean quality score for the sampling frames was 92 for PR, 87 for HR and 85 for ER; they contributed on average 23, 20, and 26 to the respective non-response rates. For all frame types and both sexes the lowest quality score occurred in the age group 35–44, suggesting a reduced ability to track migration of a highly mobile population group. The patterns in the age/sex distribution of er indicate at least for males in PR and females in HR a potential for non-response bias. Estimation of non-response bias through an abbreviated questionnaire failed because of low item response. We found that contact rate characterizes sampling frame quality. For all frame types it had a major influence on response rate. It is likely that low er and low cr cause different kind of bias, requiring different measures to minimize their effects.

Advances in the genetics of schizophrenia: toward a network and pathway view for drug discovery.

The spectacular advance in our understanding of the genetic basis of schizophrenia through genome‐wide association studies has the potential to identify new leads for drug treatment through improved understanding of disease pathophysiology. However, using these genetic associations successfully in drug development and patient stratification requires further target validation, particularly in understanding which gene(s) is causal in the disease, how the risk variants alter gene function and regulation, and how they fit into disease pathways and networks. If researchers consider the disease network as the target, they need to understand which genes should be targeted and in which modality, in order to modulate pathophysiology and obtain a beneficial effect for the patient. In the present article, we review recent genetic findings in schizophrenia and discuss how these might be validated with biology and integrated with epigenetic and transcriptome data to identify targets that lie within disease networks and pathways. This new understanding of disease biology will also facilitate the development of assays that recapitulate specific aspects of the disease using model organisms and cells. These assays can then be used in screening approaches, which manipulate disease networks or pathological processes to generate and test therapeutic strategies.

Investigating the neuroimmunogenic architecture of schizophrenia

The role of the immune system in schizophrenia remains controversial despite numerous studies to date. Most studies have profiled expression of select genes or proteins in peripheral blood, but none have focused on the expression of canonical pathways that mediate overall immune response. The current study used a systematic genetic approach to investigate the role of the immune system in a large sample of post-mortem brain of patients with schizophrenia: RNA sequencing was performed to assess the differential expression of 561 immune genes and 20 immune pathways in dorsolateral prefrontal cortex (DLPFC) (144 schizophrenia and 196 control subjects) and hippocampus (83 schizophrenia and 187 control subjects). The effect of RNA quality on gene expression was found to be highly correlated with the effect of diagnosis even after adjustment for observable RNA quality parameters (i.e. RNA integrity), thus this confounding relationship was statistically controlled using principal components derived from the gene expression matrix. In DLPFC, 23 immune genes were found to be differentially expressed (false discovery rate <0.05), of which seven genes replicated in both directionality and at nominal significance (P<0.05) in an independent post-mortem DLPFC data set (182 schizophrenia and 212 control subjects), although notably at least five of these genes have prominent roles in pathways other than immune function and overall the effect sizes were minimal (fold change <1.1). In the hippocampus, no individual immune genes were identified to be differentially expressed, and in both DLPFC and hippocampus none of the individual immune pathways were relatively differentially expressed. Further, genomic schizophrenia risk profiles scores were not correlated with the expression of individual immune pathways or differentially expressed genes. Overall, past reports claiming a primary pathogenic role of the immune system intrinsic to the brain in schizophrenia could not be confirmed.

Comparative Effects of LY3020371, a Potent and Selective mGlu2/3 Receptor Antagonist, and Ketamine, a Non-Competitive NMDA Receptor Antagonist, in Rodents: Evidence Supporting Use for the Treatment of Depression

The ability of the N-methyl-d-aspartate receptor antagonist ketamine to alleviate symptoms in patients suffering from treatment-resistant depression (TRD) is well documented. In this paper, we directly compare in vivo biologic responses in rodents elicited by a recently discovered metabotropic glutamate (mGlu) 2/3 receptor antagonist 2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY3020371) with those produced by ketamine. Both LY3020371 and ketamine increased the number of spontaneously active dopamine cells in the ventral tegmental area of anesthetized rats, increased O2 in the anterior cingulate cortex, promoted wakefulness, enhanced the efflux of biogenic amines in the prefrontal cortex, and produced antidepressant-related behavioral effects in rodent models. The ability of LY3020371 to produce antidepressant-like effects in the forced-swim assay in rats was associated with cerebrospinal fluid (CSF) drug levels that matched concentrations required for functional antagonist activity in native rat brain tissue preparations. Metabolomic pathway analyses from analytes recovered from rat CSF and hippocampus demonstrated that both LY3020371 and ketamine activated common pathways involving GRIA2 and ADORA1. A diester analog of LY3020371 [bis(((isopropoxycarbonyl)oxy)-methyl) (1S,2R,3S,4S,5R,6R)-2-amino-3-(((3,4-difluorophenyl)thio)methyl)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylate (LY3027788)] was an effective oral prodrug; when given orally, it recapitulated effects of intravenous doses of LY3020371 in the forced-swim and wake-promotion assays, and augmented the antidepressant-like effects of fluoxetine or citalopram without altering plasma or brain levels of these compounds. The broad overlap of biologic responses produced by LY3020371 and ketamine supports the hypothesis that mGlu2/3 receptor blockade might be a novel therapeutic approach for the treatment of TRD patients. LY3020371 and LY3027788 represent molecules that are ready for clinical tests of this hypothesis.

Electroencephalographic, cognitive, and neurochemical effects of LY3130481 (CERC-611), a selective antagonist of TARP-γ8-associated AMPA receptors

&NA; 6‐[(1S)‐1‐[1‐[5‐(2‐hydroxyethoxy)‐2‐pyridyl]pyrazol‐3‐yl]ethyl]‐3H‐1,3‐benzothiazol‐2‐one (LY3130481 or CERC‐611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) &ggr;−8. This molecule has been characterized as a potent and efficacious anticonvulsant in an array of acute and chronic epilepsy models in rodents. The present set of experiments was designed to assess the effects of LY3130481 on the electroencephelogram (EEG), cognitive function, and neurochemical outflow. LY3130481 disrupted food‐maintained responding in rats and spontaneous alternation in a Y‐maze in mice. In rat fear conditioning, LY3130481 caused a deficit in trace (hippocampal‐dependent), but not in delay fear conditioning. Although these effects on cognitive performances were observed, the known cognitive‐impairing anticonvulsant, topiramate, did not always produce deficits under these assay conditions. LY3130481 produced modest increases in wake times in rats. In addition, LY3130481 was able to attenuate some impairing effects of standard antiepileptic drugs. The motor‐impairing effects of the lacosamide were attenuated by LY3130481 as was the decrease in non‐rapid‐eye movement sleep induced by carbamazepine. Evaluation of the effect of LY3130481 on neurotransmitter and metabolite efflux in the rat medial prefrontal cortex, using in vivo microdialysis, revealed significant increases in the pro‐cognitive and wake‐promoting neurotransmitters, histamine and acetylcholine, as well as in serotonin, telemethylhistamine, 5‐HIAA, HVA and MHPG. LY3130481 thus presents a novel behavioral profile that will have to be evaluated in patients to fully appreciate its implications for therapeutics. LY3130481 is currently under clinical development as CERC‐611 as an antiepileptic. HighlightsLY3130481 (CERC‐611) is a selective antagonist of AMPA receptors TARP &ggr;−8 protein.LY3130481 is currently under clinical development as CERC‐611 as an antiepileptic.LY3130481 produced mixed results in rodent cognition assessments.LY3130481 produced modest increases in wake times in rats and increased wake and cognition‐associated neurotransmitters.LY3130481 attenuated some impairing effects of standard antiepileptic drugs.

Preclinical profile of a dopamine D1 potentiator suggests therapeutic utility in neurological and psychiatric disorders

ABSTRACT DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L‐DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinsons disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y‐maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced‐swim test, a model for antidepressant‐like activity. In rhesus monkeys, DETQ increased spontaneous eye‐blink rate, a measure that is known to be depressed in Parkinsons disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinsons disease, Alzheimers disease, cognitive impairment in schizophrenia, and major depressive disorder. HIGHLIGHTSThe dopamine D1 potentiator DETQ was tested in humanized D1 mice and rhesus monkeys.Actions of DETQ were dependent on endogenous dopaminergic tone.DETQ displayed a behavioral profile consistent with central D1 receptor activation.Neurochemical actions of DETQ support potential pro‐cognitive effects.D1 potentiators show promise for Parkinsons disease and other CNS disorders.

Modelling maintenance of wakefulness in rats: comparing potential non-invasive sleep-restriction methods and their effects on sleep and attentional performance.

While several methods have been used to restrict the sleep of experimental animals, it is often unclear whether these different forms of sleep restriction have comparable effects on sleep–wake architecture or functional capacity. The present study compared four models of sleep restriction, using enforced wakefulness by rotation of cylindrical home cages over 11 h in male Wistar rats. These included an electroencephalographic‐driven ‘Biofeedback’ method and three non‐invasive methods where rotation was triggered according to a ‘Constant’, ‘Decreasing’ or random protocol based upon the ‘Weibull’ distribution fit to an archival Biofeedback dataset. Sleep–wake architecture was determined using polysomnography, and functional capacity was assessed immediately post‐restriction with a simple response latency task, as a potential homologue of the human psychomotor vigilance task. All sleep restriction protocols resulted in sleep loss, behavioural task disengagement and rebound sleep, although no model was as effective as real‐time electroencephalographic‐Biofeedback. Decreasing and Weibull protocols produced greater recovery sleep than the Constant protocol, mirrored by comparably poorer simple response latency task performance. Increases in urinary corticosterone levels following Constant and Decreasing protocols suggested that stress levels may differ between protocols. Overall, these results provide insight into the value of choosing a specific sleep restriction protocol, not only from the perspective of animal welfare and the use of less invasive procedures, but also translational validity. A more considered choice of the physiological and functional effects of sleep‐restriction protocols in rodents may improve correspondence with specific types of excessive daytime sleepiness in humans.