The 14q32 maternally imprinted locus is a major source of longitudinally stable circulating microRNAs as measured by small RNA sequencing.

Understanding the normal temporal variation of serum molecules is a critical factor for identifying useful candidate biomarkers for the diagnosis and prognosis of chronic disease. Using small RNA sequencing in a longitudinal study of 66 women with no history of cancer, we determined the distribution and dynamics (via intraclass correlation coefficients, ICCs) of the miRNA profile over 3 time points sampled across 2-5 years in the course of the screening trial, UKCTOCS. We were able to define a subset of longitudinally stable miRNAs (ICC >0.75) that were individually discriminating of women who had no cancer over the study period. These miRNAs were dominated by those originating from the C14MC cluster that is subject to maternal imprinting. This assessment was not significantly affected by common confounders such as age, BMI or time to centrifugation nor alternative methods to data normalisation. Our analysis provides important benchmark data supporting the development of miRNA biomarkers for the impact of life-course exposure as well as diagnosis and prognostication of chronic disease.

Colorectal cancer ascertainment through cancer registries, hospital episode statistics, and self-reporting compared to confirmation by clinician: A cohort study nested within the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS).

BACKGROUND: Electronic health records are frequently used for cancer epidemiology. We report on their quality for ascertaining colorectal cancer (CRC) in UK women. METHODS: Population-based, retrospective cohort study nested within the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). Postmenopausal women aged 50-74 who were diagnosed with CRC during 2001-11 following randomisation to the UKCTOCS were identified and their diagnosis confirmed with their treating clinician. The sensitivity and positive predictive value (PPV) of cancer and death registries, hospital episode statistics, and self-reporting were calculated by pairwise comparisons to the treating clinician's confirmation, while specificity and negative predictive value were estimated relative to expected cases. RESULTS: Notification of CRC events were received for 1,085 women as of 24 May 2011. Responses were received from 61% (660/1,085) of clinicians contacted. Nineteen women were excluded (18 no diagnosis date, one diagnosed after cut-off). Of the 641 eligible, 514 had CRC, 24 had a benign polyp, and 103 had neither diagnosis. The sensitivity of cancer registrations at one- and six-years post-diagnosis was 92 (95% CI 90-94) and 99% (97-100), respectively, with a PPV of 95% (95% CI 92/93-97). The sensitivity & PPV of cancer registrations (at one-year post-diagnosis) & hospital episode statistics combined were 98 (96-99) and 92% (89-94), respectively. CONCLUSIONS: Cancer and death registrations in the UK are a reliable resource for CRC ascertainment in women. Hospital episode statistics can supplement delays in cancer registration. Self-reporting seems less reliable.

Heteroalicyclic carboxamidines as inhibitors of inducible nitric oxide synthase; the identification of (2R)-2-pyrrolidinecarboxamidine as a potent and selective haem-co-ordinating inhibitor.

Heteroalicyclic carboxamidines were synthesised and evaluated as inhibitors of nitric oxide synthases. (2R)-2-Pyrrolidinecarboxamidine, in particular, was shown to be a highly potent in vitro (IC(50)=0.12 µM) and selective iNOS inhibitor (>100-fold vs both eNOS and nNOS), with probable binding to the key anchoring glutamate residue and co-ordination to the haem iron.

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.

Temporal dynamics of myelination in the zebrafish spinal cord.

Knowledge of the precise timing of myelination is critical to the success of zebrafish-based in vivo screening strategies for potential remyelination therapies. This study provides a systematic review of the timing of myelination in the zebrafish spinal cord and a critique of techniques by which it may be accurately assessed. The onset of myelination was found to be 3 days postfertilization (d.p.f.); earlier than previously reported. This coincided with the dorsal migration and differentiation of oligodendrocytes and the expression of myelin basic protein (Mbp) transcripts and protein. Our data suggests that immunohistochemistry with zebrafish-specific anti-Mbp from 3 d.p.f. is the optimal histological method for myelin visualization, while quantification of myelination is more reliably achieved by quantitative polymerase chain reaction (qPCR) for mbp from 5 d.p.f.. Transgenic fluorescent lines such as olig2:EGFP can be used to assess oligodendrocyte cell number at 3 d.p.f. and the development of new, more specific lines may enable real time visualization of myelin itself. Quantitative ultrastructural analysis revealed that the myelination of zebrafish axons is regulated according to axonal growth and not absolute axonal size. This study confirms the use of the zebrafish larvae as a versatile and efficient in vivo model of myelination and provides a platform on which future myelination screening studies can be based.

Current and Future Perspectives in Psychiatric Drug Discovery.

On March 12, 2009, the Society for Medicines Research held a 1-day meeting entitled Current and Future Perspectives in Psychiatric Drug Discovery at GlaxoSmithKline, Harlow, U.K. The meeting, organized by Wendy Alderton, Eric Karran and Simon Ward, brought together experts from industry and academia to review the current challenges in the treatment of depression, schizophrenia and addiction. Recent advances in the development of novel agents for the treatment of these diseases were also presented.

Zebrafish: An in vivo model for the study of neurological diseases.

As the population ages, there is a growing need for effective therapies for the treatment of neurological diseases. A limited number of therapeutics are currently available to improve cognitive function and research is limited by the need for in vivo models. Zebrafish have recently become a focus of neurobehavioral studies since larvae display neuropathological and behavioral phenotypes that are quantifiable and relate to those seen in man. Due to the small size of Zebrafish larvae, assays can be undertaken in 96 well plates and as the larvae can live in as little as 200 mul of fluid, only a few milligrams of compound are needed for screening. Thus in vivo analysis of the effects of compounds can be undertaken at much earlier stages in the drug discovery process. This review will look at the utility of the zebrafish in the study of neurological diseases and its role in improving the throughput of candidate compounds in in vivo screens.

The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer .

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.

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.

GW274150 and GW273629 are potent and highly selective inhibitors of inducible nitric oxide synthase in vitro and in vivo.

1 GW274150 ([2-[(1-iminoethyl) amino]ethyl]-L-homocysteine) and GW273629 (3-[[2-[(1-iminoethyl)amino]ethyl]sulphonyl]-L-alanine) are potent, time-dependent, highly selective inhibitors of human inducible nitric oxide synthase (iNOS) vs endothelial NOS (eNOS) (>100-fold) or neuronal NOS (nNOS) (>80-fold). GW274150 and GW273629 are arginine competitive, NADPH-dependent inhibitors of human iNOS with steady state K(d) values of <40 and <90 nM, respectively. 2 GW274150 and GW273629 inhibited intracellular iNOS in J774 cells in a time-dependent manner, reaching IC(50) values of 0.2+/-0.04 and 1.3+/-0.16 microM, respectively. They were also acutely selective in intact rat tissues: GW274150 was >260-fold and 219-fold selective for iNOS against eNOS and nNOS, respectively, while GW273629 was >150-fold and 365-fold selective for iNOS against eNOS and nNOS, respectively. 3 The pharmacokinetic profile of GW274150 was biphasic in healthy rats and mice with a terminal half-life of approximately 6 h. That of GW273629 was also biphasic in rats, producing a terminal half-life of approximately 3 h. In mice however, elimination of GW273629 appeared monophasic and more rapid (approximately 10 min). Both compounds show a high oral bioavailability (>90%) in rats and mice. 4 GW274150 was effective in inhibiting LPS-induced plasma NO(x) levels in mice with an ED(50) of 3.2+/-0.7 mg kg(-1) after 14 h intraperitoneally (i.p.) and 3.8+/-1.5 mg kg(-1) after 14 h when administered orally. GW273629 showed shorter-lived effects on plasma NO(x) and an ED(50) of 9+/-2 mg kg(-1) after 2 h when administered i.p. 5 The effects of GW274150 and GW273629 in vivo were consistent with high selectivity for iNOS, as these inhibitors were of low potency against nNOS in the rat cerebellum and did not cause significant effects on blood pressure in instrumented mice.