Design and synthesis of long acting inhaled corticosteroids for the treatment of asthma

In this Letter we present data for a novel series of ICS for the treatment of asthma. 'Inhalation by design' principles have been applied to a series of highly potent steroidal GR agonists, with a focus on optimising the potential therapeutic index in human. Pharmacokinetic properties were tuned with high intrinsic clearance and low oral bioavailability in mind, to minimise systemic exposure and reduce systemically driven adverse events. High CYP mediated clearance as well as glucuronidation were targeted to achieve high intrinsic clearance coupled with multiple routes of clearance to minimise drug-drug interactions. Furthermore, pharmaceutical properties such as stability, crystallinity and solubility were considered to ensure compatibility with a dry powder inhaler. This work culminated in the identification of the clinical candidate 15, which demonstrates preclinically the desired efficacy and safety profiles confirming its potential as an inhaled agent for the treatment of asthma.     

Maternal genome-wide DNA methylation profiling in gestational diabetes shows distinctive disease-associated changes relative to matched healthy pregnancies

Several recent reports have described associations between gestational diabetes (GDM) and changes to the epigenomic landscape where the DNA samples were derived from either cord or placental sources. We employed genome-wide 450K array analysis to determine changes to the epigenome in a unique cohort of maternal blood DNA from 11 pregnant women prior to GDM development relative to matched controls. Hierarchical clustering segregated the samples into 2 distinct clusters comprising GDM and healthy pregnancies. Screening identified 100 CpGs with a mean β-value difference of ≥0.2 between cases and controls. Using stringent criteria, 5 CpGs (within COPS8, PIK3R5, HAAO, CCDC124, and C5orf34 genes) demonstrated potentials to be clinical biomarkers as revealed by differential methylation in 8 of 11 women who developed GDM relative to matched controls. We identified, for the first time, maternal methylation changes prior to the onset of GDM that may prove useful as biomarkers for early therapeutic intervention.     

Disconnected mutants show disruption to the central projections of proprioceptive neurons in Drosophila melanogaster

We used a P[GAL4] enhancer-trap line, C161, in conjunction with the UAS-lacZ reporter construct to visualize the central projections of a defined set of thoracic and abdominal sensory neurons in a disconnected (disco) mutant background. The results show defects in the organization of sensory axons in the larval and adult central nervous system. The defects are indicative of problems with axon growth and development and include (a) poor axon fasciculation, (b) aberrant axon growth, (c) excessive terminal branching, and (d) ectopic innervation. Sensory neuron identity appears to be normal. The defects are comparable to those previously described for larval photoreceptor and adult retinular cells in disco mutants and extend the known effects of this mutation. Reduced larval and adult viability are likely to result from locomotory defects related to the disruption of the sensory system. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 337–347, 1998     

Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.     

Designing rapid onset selective serotonin re-uptake inhibitors. Part 1: Structure-activity relationships of substituted (1S,4S)-4-(3,4-dichlorophenyl)-N-methyl-1,2,3,4-tetrahydro-1-naphthaleneamine

A series of sertraline analogues 4-39 which possess polar groups on the fused tetrahydronaphthalene ring, targeting reduced V(d) as a strategy to reduce T(max) and increase rate of elevation of central 5-HT levels, were prepared. These studies led to the successful identification of 22a, which demonstrated equivalent pharmacology and metabolic stability to 1, but which possessed greatly reduced V(d) leading to significantly shorter T(max), in rat pharmacokinetic studies.     

Circadian timekeeping in Drosophila melanogaster and Mus musculus

The discovery of the period gene mutants in 1971 provided the first evidence that daily rhythms in the sleep-wake cycle of a multicellular organism, the fruit fly Drosophila melanogaster, had an underlying genetic basis. Subsequent research has established that the biological clock mechanism in flies and mammals is strikingly similar and functions as a bimodal switch, simultaneously turning on one set of genes and turning off another set and then reversing the process every 12 h. In this chapter, the current model of the clock mechanism in Drosophila will be presented. This relatively basic model will then be used to outline the general rules that govern how the biological clock operates in mammals.     

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.     

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Background  

Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.