National variation in the composition of rheumatology multidisciplinary teams: a cross-sectional study.

The objective of this study is to describe the composition of multidisciplinary teams (MDT) working within rheumatology departments across the UK. All rheumatology departments in the United Kingdom (UK) were invited to participate in a national electronic survey between February 2014 and April 2015 as a part of a national audit for the management of rheumatoid and early inflammatory arthritis commissioned by Healthcare Quality Improvement Partnership. Rheumatology departments were asked to report their MDT composition; defined as a rheumatologist (consultant or specialist trainee), specialist nurse, occupational therapist physiotherapist, and podiatrist. The data were collected as Whole Time Equivalent (WTE) of each professional group at each department adjusted to 100,000 population. The data were grouped according to British Society for Rheumatology regions to study regional variations. The survey was completed by 164/167 departments (98% response rate). All departments reported an MDT comprising a rheumatologist (consultant or specialist trainee) and almost all included a specialist nurse but only 28 (17%) of the departments had MDTs comprising all the professional groups. There was a high degree of regional variation in the provision of Allied Health Professionals (physiotherapists, occupational therapists, and podiatrists) in the UK. MDT care is recommended for the management of inflammatory arthritis, but few UK rheumatology departments have a full complement of healthcare professionals within their MDT. There is a high degree of regional variation in the composition and staffing levels of the rheumatology MDT across the UK; the impact of which warrants further investigation.

Drug reprofiling using zebrafish identifies novel compounds with potential pro-myelination effects.

Treatment of the autoimmune demyelinating disease multiple sclerosis (MS) requires therapies that both limit and repair damage. While several immunomodulatory treatments exist to limit damage there are currently no treatments that promote the regenerative process of remyelination. A rapid way of screening potential pro-remyelination compounds is therefore required. The use of larval zebrafish in a drug reprofiling screen allows rapid in vivo screening and has been used successfully in the past as an efficient way of identifying new indications for existing drugs. A novel screening platform for potential pro-myelination compounds was developed using zebrafish larvae. Two percent of compounds screened from reprofiling libraries altered oligodendrocyte lineage cell recruitment and/or proliferation, as measured by the numbers of dorsally migrated spinal cord olig2(+) cells. Selective screening identified three compounds that altered levels of myelination, as measured by whole larvae myelin basic protein (mbp) transcript levels; the src family kinase inhibitor PP2, a biogenic amine and a thioxanthene. As well as many previously unrecognised compounds, identified compounds included those with previously known effects on myelin and/or the oligodendrocyte lineage, such as a PPAR agonist, steroid hormones and src family kinase inhibitors. As well as providing methods for further assessment of potentially beneficial compounds, this screen has highlighted 25 targets that are able to alter oligodendrocyte lineage cell recruitment or proliferation and/or mbp transcript levels in vivo and are worthy of further investigation for their potential effects on remyelination.

Decreased BDNF levels are a major contributor to the embryonic phenotype of huntingtin knockdown zebrafish.

Huntington's disease (HD) is an autosomal dominant, neurodegenerative condition caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract in the protein huntingtin. Genetic and transgenic studies suggest that the mutation causes disease predominantly via gain-of-function mechanisms. However, loss of normal huntingtin function resulting from the polyglutamine expansion might also contribute to the pathogenesis of HD. Here, we have studied the effects of huntingtin knockdown in zebrafish using morpholino antisense oligonucleotides, as its huntingtin orthologue has 70% amino acid identity with the human protein. Reduced huntingtin levels did not impact on gastrulation and early development, but caused massive apoptosis of neuronal cells by 24 hpf. This was accompanied by impaired neuronal development, resulting in small eyes and heads and enlargement of brain ventricles. Older huntingtin knockdown fish developed lower jaw abnormalities with most branchial arches missing. Molecular analysis revealed that BDNF expression was reduced by approximately 50%. Reduction of BDNF levels by injection of a BDNF morpholino resulted in phenotypes very similar to those seen in huntingtin knockdown zebrafish. The phenotypes of both huntingtin- and BDNF-knockdown zebrafish showed significant rescue when treated with exogenous BDNF protein. This underscores the physiological importance of huntingtin as a regulator of BDNF production and suggests that loss of BDNF is a major cause of the developmental abnormalities seen with huntingtin knockdown in zebrafish. Increasing BDNF expression may represent a useful strategy for Huntington's disease treatment.

Loss of PINK1 function affects development and results in neurodegeneration in zebrafish.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder in the Western world. PTEN (phosphatase/tensin homolog on chromosome 10)-induced putative kinase 1 (PINK1), a putative kinase that is mutated in autosomal recessive forms of PD, is also implicated in sporadic cases of the disease. Although the mutations appear to result in a loss of function, the roles of this protein and the pathways involved in PINK1 PD are poorly understood. Here, we generated a vertebrate model of PINK1 insufficiency using morpholino oligonucleotide knockdown in zebrafish (Danio rerio). PINK1 knockdown results in a severe developmental phenotype that is rescued by wild-type human PINK1 mRNA. Morphants display a moderate decrease in the numbers of central dopaminergic neurons and alterations of mitochondrial function, including increases in caspase-3 activity and reactive oxygen species (ROS) levels. When the morphants were exposed to several drugs with antioxidant properties, ROS levels were normalized and the associated phenotype improved. In addition, GSK3beta-related mechanisms can account for some of the effects of PINK1 knockdown, as morphant fish show elevated GSK3beta activity and their phenotype is partially abrogated by GSK3beta inhibitors, such as LiCl and SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)1H-pyrrole-2,5-dione]. This provides new insights into the biology of PINK1 and a possible therapeutic avenue for further investigation.

Zebrafish based assays for the assessment of cardiac, visual and gut function--potential safety screens for early drug discovery.

INTRODUCTION: Safety pharmacology is integral to the non-clinical safety assessment of new chemical entities prior to first administration to humans. The zebrafish is a well established model organism that has been shown to be relevant to the study of human diseases. The potential role of zebrafish in safety pharmacology was evaluated using reference compounds in three models assessing cardiac, visual and intestinal function. METHODS: Compound toxicity was first established in zebrafish to determine the non toxic concentration of a blinded set of 16 compounds. In the cardiac assay, zebrafish larvae at 3 days post fertilisation (d.p.f.) were exposed to compounds for 3 h before measurement of the atrial and ventricular rates. To investigate visual function, the optomotor response was assessed in 8 d.p.f. larvae following a 5 day compound exposure. In the intestinal function assay, the number of gut contractions was measured in 7 d.p.f. larvae after a 1 h compound exposure. Finally, compound uptake was determined for 9 of the 16 compounds to measure the concentration of compound absorbed by the zebrafish larvae. RESULTS: Seven compounds out of nine produced an expected effect that was statistically significant in the cardiac and visual functions assays. In the gut contraction assay, six out of ten compounds showed a statistically significant effect that was also the expected result whilst two displayed anticipated but non-significant effects. The compound uptake method was used to determine larval tissue concentrations and allowed the identification of false negatives when compound was poorly absorbed into the zebrafish. DISCUSSION: Overall, results generated in three zebrafish larvae assays demonstrated a good correlation between the effects of compounds in zebrafish and the data available from other in vivo models or known clinical adverse effects. These results suggest that for the cardiac, intestinal and visual function, zebrafish assays have the potential to predict adverse drug effects and supports their possible role in early safety assessment of novel compounds.

Non-associative learning in larval zebrafish.

Habituation, where a response is reduced when exposed to a continuous stimulus is one of the simplest forms of non-associative learning and has been shown in a number of organisms from sea slugs to rodents. However, very little has been reported in the zebrafish, a model that is gaining popularity for high-throughput compound screens. Furthermore, since most of the studies involving learning and memory in zebrafish have been conducted in adults, we sought to determine if zebrafish larvae could display non-associative learning and whether it could be modulated by compounds identified in previous rodent studies. We demonstrated that zebrafish larvae (7 days post fertilization) exhibit iterative reduction in a startle response to a series of acoustic stimuli. Furthermore, this reduction satisfied criteria for habituation: spontaneous recovery, more rapid reductions in startle to shorter intertrial intervals and dishabituation. We then investigated the pathways mediating this behavior using established compounds in learning and memory. Administration of rolipram (PDE4 inhibitor), donepezil (acetylcholinesterase inhibitor), and memantine (N-methyl-D-aspartic acid (NMDA) receptor antagonist) all increased the acoustic startle response and decreased habituation in the larvae, similar to previous rodent studies. Further studies demonstrated that NMDA blocked the memantine response and the effect of donepezil was blocked by mecamylamine but not atropine suggesting that the donepezil response was mediated by nicotinic rather than muscarinic receptors. Zebrafish larvae possess numerous advantages for medium to high-throughput screening; the model described herein therefore offers the potential to screen for additional compounds for further study on cognition function.

Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants.

The search for novel anticonvulsants requires appropriate model systems in which to test hypotheses through focused compound screening or genetic manipulation, or conduct black box screening of large numbers of compounds or potential genetic modifiers. Many models are currently in existence that subserve particular roles in achieving these aims, but all have their limitations. Zebrafish have been suggested as an additional model of epilepsy, but their optimum role is unclear. They are more amenable to high throughput analysis, but are more genetically removed from humans than rodents. We therefore sought to develop assay methodology applicable to medium/high throughput screening using an automated tracking system to measure the amount of movement induced by exposure to the proconvulsant, pentylene tetrazole (PTZ). We then used this system to explore how many known anti-epileptic drugs (AEDs) would be detected when running such a screen. We were able to detect suppression of PTZ-induced excessive movements with 13 out of 14 standard AEDs. A parallel sedation and toxicity screen suggested these effects were due to direct anti-epileptic effect, although non-specific effects cannot be fully excluded. These results suggest zebrafish may be a useful high throughput primary screen to pick up potential novel AEDs.

Identification and optimisation of a series of substituted 5-pyridin-2-yl-thiophene-2-hydroxamic acids as potent histone deacetylase (HDAC) inhibitors.

Further investigation of a series of thienyl-based hydroxamic acids that included ADS100380 and ADS102550 led to the identification of the 5-pyridin-2-yl-thiophene-2-hydroxamic acid 3c, which possessed modest HDAC inhibitory activity. Substitution at the 5- and 6-positions of the pyridyl ring of compound 3c provided compounds 5a-g, 7a, b, 9, and 13a. Compound 5b demonstrated improved potency, in vitro DMPK profile, and rat oral bioavailability, compared to ADS102550. Functionalisation of the pendent phenyl group of compounds 5b, 5e and 13a provided analogues that possessed excellent enzyme inhibition and anti-proliferative activity.

Identification and optimisation of a series of substituted 5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids as potent histone deacetylase (HDAC) inhibitors.

Optimisation of ADS100380, a sub-micromolar HDAC inhibitor identified using a virtual screening approach, led to a series of substituted 5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids (6a-i), that possessed significant HDAC inhibitory activity. Subsequent functionalisation of the pendent phenyl group of compounds 6f and 6g provided analogues 6j-w with further enhanced enzyme and anti-proliferative activity. Compound 6j demonstrated efficacy in a mouse xenograft experiment.

Quinazoline and benzimidazole MCH-1R antagonists.

We have modified the previously reported 2-aminoquinoline 1 to provide two novel series of MCH-1R antagonists. Representative compounds from the quinazoline and benzimidazole series have been shown to be potent and selective, with promising in vitro eADME profiles.

A virtual screening approach to finding novel and potent antagonists at the melanin-concentrating hormone 1 receptor.

Melanin-concentrating hormone (MCH) has been known to be an appetite-stimulating peptide for a number of years. However, it is only recently that MCH has been discovered to be the natural ligand for a previously "orphan" G-protein-coupled receptor, now designated MCH-1R. This receptor has been shown to mediate the effects of MCH on appetite and body weight, and consequently, drug discovery programs have begun to exploit this information in the search for MCH-1R antagonists for the treatment of obesity. In this paper, we report the rapid discovery of multiple, structurally distinct series of MCH-1R antagonists using a variety of virtual screening techniques. The most potent of these compounds (12) demonstrated an IC(50) value of 55 nM in the primary screen and exhibited antagonist properties in a functional cellular assay measuring Ca(2+) release. More potent compounds were identified by follow-up searches around the initial hit. A proposed binding mode for compound 12 in a homology model of the MCH-1R is also presented.

Structure-activity relationships of a novel series of melanin-concentrating hormone (MCH) receptor antagonists.

A new series of 2-aminoquinolines has been identified as antagonists of the melanin concentrating hormone receptor (MCH-1R). Syntheses and structure-activity relationships are described leading to a compound having low nanomolar activity against the receptor and demonstrating functional antagonism. Studies also showed that some of the compounds were selective against a range of other G protein-coupled receptors.