Pharmacologically induced weight loss is associated with distinct gut microbiome changes in obese rats.

BACKGROUND: Obesity, metabolic disease and some psychiatric conditions are associated with changes to relative abundance of bacterial species and specific genes in the faecal microbiome. Little is known about the impact of pharmacologically induced weight loss on distinct microbiome species and their respective gene programs in obese individuals. METHODOLOGY: Using shotgun metagenomics, the composition of the microbiome was obtained for two cohorts of obese female Wistar rats (n = 10-12, total of 82) maintained on a high fat diet before and after a 42-day treatment with a panel of four investigatory or approved anti-obesity drugs (tacrolimus/FK506, bupropion, naltrexone and sibutramine), alone or in combination. RESULTS: Only sibutramine treatment induced consistent weight loss and improved glycaemic control in the obese rats. Weight loss was associated with reduced food intake and changes to the faecal microbiome in multiple microbial taxa, genes, and pathways. These include increased ß-diversity, increased relative abundance of multiple Bacteroides species, increased Bacteroides/Firmicutes ratio and changes to abundance of genes and species associated with obesity-induced inflammation, particularly those encoding components of the flagellum and its assembly. CONCLUSIONS: Sibutramine-induced weight loss in obese rats is associated with improved metabolic health, and changes to the faecal microbiome consistent with a reduction in obesity-induced bacterially-driven inflammation.

Using Esterase Selectivity to Determine the In Vivo Duration of Systemic Availability and Abolish Systemic Side Effects of Topical ß-Blockers.

For disorders of the skin, eyes, ears, and respiratory tract, topical drugs, delivered directly to the target organ, are a therapeutic option. Compared with systemic oral therapy, the benefits of topical treatments include a faster onset of action, circumventing the liver first pass drug metabolism, and reducing systemic side effects. Nevertheless, some systemic absorption still occurs for many topical agents resulting in systemic side effects. One way to prevent these would be to develop drugs that are instantly degraded upon entry into the bloodstream by serum esterases. Because topical ß-blockers are used in glaucoma and infantile hemeangioma and cause systemic side effects, the ß-adrenoceptor system was used to test this hypothesis. Purified liver esterase reduced the apparent affinity of esmolol, an ester-containing ß-blocker used in clinical emergencies, for the human ß-adrenoceptors in a concentration and time-dependent manner. However, purified serum esterase had no effect on esmolol. Novel ester-containing ß-blockers were synthesized and several were sensitive to both liver and serum esterases. Despite good in vitro affinity, one such compound, methyl 2-(3-chloro-4-(3-((2-(3-(3-chlorophenyl)ureido)ethyl)amino)-2-hydroxypropoxy)phenyl)acetate, had no effect on heart rate when injected intravenously into rats, even at 10 times the equipotent dose of esmolol and betaxolol that caused short and sustained reductions in heart rate, respectively. Thus, ester-based drugs, sensitive to serum esterases, offer a mechanism for developing topical agents that are truly devoid of systemic side effects. Furthermore, differential susceptibility to liver and serum esterases degradation may also allow the duration of systemic availability for other drugs to be fine-tuned.

Novel selective ß1-adrenoceptor antagonists for concomitant cardiovascular and respiratory disease.

ß-Blockers reduce mortality and improve symptoms in people with heart disease; however, current clinically available ß-blockers have poor selectivity for the cardiac ß1-adrenoceptor (AR) over the lung ß2-AR. Unwanted ß2-blockade risks causing life-threatening bronchospasm and reduced efficacy of ß2-agonist emergency rescue therapy. Thus, current life-prolonging ß-blockers are contraindicated in patients with both heart disease and asthma. Here, we describe NDD-713 and -825, novel highly ß1-selective neutral antagonists with good pharmaceutical properties that can potentially overcome this limitation. Radioligand binding studies and functional assays that use human receptors expressed in Chinese hamster ovary cells demonstrate that NDD-713 and -825 have nanomolar ß1-AR affinity >500-fold ß1-AR vs ß2-AR selectivity and no agonism. Studies in conscious rats demonstrate that these antagonists are orally bioavailable and cause pronounced ß1-mediated reduction of heart rate while showing no effect on ß2-mediated hindquarters vasodilatation. These compounds also have good disposition properties and show no adverse toxicologic effects. They potentially offer a truly cardioselective ß-blocker therapy for the large number of patients with heart and respiratory or peripheral vascular comorbidities.-Baker, J. G., Gardiner, S. M., Woolard, J., Fromont, C., Jadhav, G. P., Mistry, S. N., Thompson, K. S. J., Kellam, B., Hill, S. J., Fischer, P. M. Novel selective ß1-adrenoceptor antagonists for concomitant cardiovascular and respiratory disease.

Profiling of molecular interactions in real time using acoustic detection.

Acoustic sensors that exploit resonating quartz crystals to directly detect the binding of an analyte to a receptor are finding increasing utility in the quantification of clinically relevant analytes. We have developed a novel acoustic detection technology, which we term resonant acoustic profiling (RAP). This technology builds on the fundamental basics of the "quartz crystal microbalance" or "QCM" with several key additional features including two- or four-channel automated sample delivery, in-line referencing and microfluidic sensor 'cassettes' that are pre-coated with easy-to-use surface chemistries. Example applications are described for the quantification of myoglobin concentration and its interaction kinetics, and for the ranking of enzyme-cofactor specificities.

Quantification of small molecule-receptor affinities and kinetics by acoustic profiling.

The label-free RAPid 4 system that exploits resonant acoustic profiling (RAP) from Akubio (Cambridge, UK) was used to determine the affinity and kinetics for several different small molecule-receptor interactions. This was achieved by attaching the target receptor to the surface of quartz crystal resonators through a variety of specific coupling chemistries, followed by application of a small-molecular-weight ligand to the receptor via a microfluidic flow-based delivery system. Rank order of binding was determined for very weak interactions such as cofactor binding to glucose dehydrogenase. Moderate interaction affinities and binding kinetics could be determined for biotin binding to a specific antibody, and also for several low-molecular-weight sulfonamide analogues binding to human carbonic anhydrase isoform II. The equilibrium binding constants were in general agreement with the values obtained by kinetic analysis of the data, as well as with previously published values obtained using surface plasmon resonance, stopped flow fluorescence, and isothermal titration calorimetry.

Direct quantification of analyte concentration by resonant acoustic profiling.

BACKGROUND: Acoustic sensors that exploit resonating quartz crystals directly detect the binding of an analyte to a receptor. Applications include detection of bacteria, viruses, and oligonucleotides and measurement of myoglobin, interleukin 1beta (IL-1beta), and enzyme cofactors. METHODS: Resonant Acoustic Profiling was combined with a microfluidic lateral flow device incorporating an internal reference control, stable linker chemistry, and immobilized receptors on a disposable sensor "chip". Analyte concentrations were determined by analyzing the rate of binding of the analyte to an appropriate receptor. RESULTS: The specificity and affinity of antibody-antigen and enzyme-cofactor interactions were determined without labeling of the receptor or the analyte. We measured protein concentrations (recombinant human IL-1beta and recombinant human myoglobin) and quantified binding of cofactors (NADP+ and NAD+) to the enzyme glucose dehydrogenase. Lower limits of detection were approximately 1 nmol/L (17 ng/mL) for both IL-1beta and human myoglobin. The equilibrium binding constant for NADP+ binding to glucose dehydrogenase was 2.8 mmol/L. CONCLUSIONS: Resonant Acoustic Profiling detects analytes in a relatively simple receptor-binding assay in <10 min. Potential applications include real-time immunoassays and biomarker detection. Combination of this technology platform with existing technologies for concentration and presentation of analytes may lead to simple, label-free, high-sensitivity methodologies for reagent and assay validation in clinical chemistry and, ultimately, for real-time in vitro diagnostics.

Development of a generic dual-reporter gene assay for screening G-protein-coupled receptors.

Multiple assay formats have been developed for the pharmacological characterization of G-protein-coupled receptors (GPCRs) and for screening orphan receptors. However, the increased pace of target identification and the rapid expansion of compound libraries present the need to develop novel assay formats capable of screening multiple GPCRs simultaneously. To address this need, the authors have developed a generic dual-reporter gene assay that can detect ligand activity at 2 GPCRs within the same assay. Two stable HEK293 cell lines were generated expressing either a firefly (Photinus) luciferase gene under the control of multiple cAMP-response elements (CREs) or a Renilla luciferase gene under the control of multiple 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs). Coseeded reporter cells were used to assess ligand binding activity at both Galphas-and Galphaq-coupled receptors. By selectively coexpressing receptors with a chimeric G-protein, agonist activity was assessed at Galphai/o-coupled receptors in combination with either Galphas-or Galphaq-coupled receptors. The dual-reporter gene assay was shown to be capable of simultaneously performing duplexed screens for a variety of agonist and/or antagonist combinations. The data generated from the duplexed reporter assays were pharmacologically relevant, and Z' factor analysis indicated the suitability of both agonist and antagonist screens for use in high-throughput screening.

Increased 5-HT2A receptor expression and function following central glucocorticoid receptor knockdown in vivo.

Central glucocorticoid receptor function may be reduced in depression. In vivo modelling of glucocorticoid receptor underfunctionality would assist in understanding its role in depressive illness. The role of glucocorticoid receptors in modulating 5-HT(2A) receptor expression and function in the central nervous system (CNS) is presently unclear, but 5-HT(2A) receptor function also appears altered in depression. With the aid of RNAse H accessibility mapping, we have developed a 21-mer antisense oligodeoxynucleotide (5'-TAAAAACAGGCTTCTGATCCT-3', termed GRAS-5) that showed 56% reduction in glucocorticoid receptor mRNA and 80% down-regulation in glucocorticoid receptor protein in rat C6 glioma cells. Sustained delivery to rat cerebral ventricles in slow release biodegradable polymer microspheres produced a marked decrease in glucocorticoid receptor mRNA and protein in hypothalamus (by 39% and 80%, respectively) and frontal cortex (by 26% and 67%, respectively) 5 days after a single injection, with parallel significant up-regulation of 5-HT(2A) receptor mRNA expression (13%) and binding (21%) in frontal cortex. 5-HT(2A) receptor function, determined by DOI-head-shakes, showed a 55% increase. These findings suggest that central 5-HT(2A) receptors are, directly or indirectly, under tonic inhibitory control by glucocorticoid receptor.

Migration of neurons between ganglia in the metamorphosing insect nervous system.

Migration of neurons over long distances occurs during the development of the adult central nervous system of the sphinx moth Manduca sexta, and the turnip moth Agrotis segetum. From each of the suboesophageal and three thoracic ganglia, bilaterally-paired clusters of immature neurons and associated glial cells migrate posteriorly along the interganglionic connectives, to enter the next posterior ganglion. The first sign of migration is observed at the onset of metamorphosis, when posterio-lateral cell clusters gradually separate from the cortex of neuronal cell bodies and enter the connectives. Cell clusters migrate posteriorly along the connective to reach the next ganglion over the first three days (approximately 15%) of pupal development. During migration, each cell cluster is completely enveloped by a single giant glial cell spanning the entire length of the connective between two adjacent ganglia. Intracellular cobalt staining reveals that each migrating neuron has an ovoid cell body and an extremely long leading process which extends as far as the next posterior ganglion; this is not a common morphology for migrating neurons that have been described in vertebrates. Once the cells arrive at the anterior cortex of the next ganglion, they rapidly intermingle with the surrounding neurons and so we were unable to determine the fate of the migrating neurons at their final location.