Abstract The effects of four preslaughter feeding systems on gut microflora, digesta consistency, soiling of the hide with faeces, liveweight loss, dehydration, meat stickiness, and plasma cortisol were examined in 60 Angus steers. The feeding systems were: feeding hay for 48 or 24 hours before transport to slaughter, transporting cattle directly off pasture, and fasting for 24 hours before transport. Cattle were held overnight at the processing plant after 2 hours transport. At slaughter, pasture‐fed cattle were carrying approximately 75 × 109 aerobic bacteria and 86 × 109 facultative anaerobes in their gastro‐inte:5tinal tracts. The number and type of bacteria were strongly influenced by the preslaughter feeding system. Rumen contents of the fasted animals were less acidic and more moist than the other treatments. Fasted animals had more E. coli, Enterobacter, and facultative anaerobes throughout the gut. The 48‐h hay‐fed steers had higher numbers of Enterococci and fewer E. coli. The pasture groups had runnier faeces, and were dirtier after holding overnight at the processing plant. Treatments did not affect dehydration, but urinary sodium was negatively correlated with meat stickiness, suggesting that some individuals were sufficiently dehydrated to influence this meat quality feature. It was concluded that feeding cattle hay for 48 hours before despatch for slaughter provided several advantages over other preslaughter feeding systems.
The discovery of the orexin system represents the single major progress in the sleep field of the last three to four decades. The two orexin peptides and their two receptors play a major role in arousal and sleep/wake cycles. Defects in the orexin system lead to narcolepsy with cataplexy in humans and dogs and can be experimentally reproduced in rodents. At least six orexin receptor antagonists have reached Phase II or Phase III clinical trials in insomnia, five of which are dual orexin receptor antagonists (DORAs) that target both OX1 and OX2 receptors (OX2Rs). All clinically tested DORAs induce and maintain sleep: suvorexant, recently registered in the USA and Japan for insomnia, represents the first hypnotic principle that acts in a completely different manner from the current standard medications. It is clear, however, that in the clinic, all DORAs promote sleep primarily by increasing rapid eye movement (REM) and are almost devoid of effects on slow-wave (SWS) sleep. At present, there is no consensus on whether the sole promotion of REM sleep has a negative impact in patients suffering from insomnia. However, sleep onset REM (SOREM), which has been documented with DORAs, is clearly an undesirable effect, especially for narcoleptic patients and also in fragile populations (e.g. elderly patients) where REM-associated loss of muscle tone may promote an elevated risk of falls. Debate thus remains as to the ideal orexin agent to achieve a balanced increase in REM and non-rapid eye movement (NREM) sleep. Here, we review the evidence that an OX2R antagonist should be at least equivalent, or perhaps superior, to a DORA for the treatment of insomnia. An OX2R antagonist may produce more balanced sleep than a DORA. Rodent sleep experiments show that the OX2R is the primary target of orexin receptor antagonists in sleep modulation. Furthermore, an OX2R antagonist should, in theory, have a lower narcoleptic/cataplexic potential. In the clinic, the situation remains equivocal, since OX2R antagonists are in early stages: MK-1064 has completed Phase I, and MIN202 is currently in clinical Phase II/III trials. However, data from insomnia patients have not yet been released. Promotional material suggests that balanced sleep is indeed induced by MIN-202, whereas in volunteers MK-1064 has been reported to act similarly to DORAs.
Orexin receptor antagonists represent attractive targets for the development of drugs for the treatment of insomnia. Both efficacy and safety are crucial in clinical settings and thorough investigations of pharmacokinetics and pharmacodynamics can predict contributing factors such as duration of action and undesirable effects. To this end, we studied the interactions between various "dual" orexin receptor antagonists and the orexin receptors, OX1R and OX2R, over time using saturation and competition radioligand binding with [(3)H]-BBAC ((S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide). In addition, the kinetics of these compounds were investigated in cells expressing human, mouse and rat OX1R and OX2R using FLIPR® assays for calcium accumulation. We demonstrate that almorexant reaches equilibrium very slowly at OX2R, whereas SB-649868, suvorexant, and filorexant may take hours to reach steady state at both orexin receptors. By contrast, compounds such as BBAC or the selective OX2R antagonist IPSU ((2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one) bind rapidly and reach equilibrium very quickly in binding and/or functional assays. Overall, the "dual" antagonists tested here tend to be rather unselective under non-equilibrium conditions and reach equilibrium very slowly. Once equilibrium is reached, each ligand demonstrates a selectivity profile that is however, distinct from the non-equilibrium condition. The slow kinetics of the "dual" antagonists tested suggest that in vitro receptor occupancy may be longer lasting than would be predicted. This raises questions as to whether pharmacokinetic studies measuring plasma or brain levels of these antagonists are accurate reflections of receptor occupancy in vivo.
This is the first reported case of lymphoproliferative disease presenting with adrenal insufficiency after liver transplantation. A 38-year-old white man was admitted 8 months after transplantation for cryptogenic cirrhosis with fever (38-39 degrees C), chills, cough, and dyspnea. His blood pressure was 100/70 mm Hg, there was pallor of the conjunctiva, and a lymph node was palpable in the left groin. Laboratory analyses revealed the following values: serum sodium concentration (112 mmol/L), potassium (5.4 mmol/L), hemoglobin (7.8 g/L), white blood cell count (7.7 x 10(9)/L), glucose 3.9 (mmol/L), and mildly elevated liver functions. Abdominal ultrasound showed multiple hypoechoic solid-appearing lesions throughout the liver and spleen. Results of a biopsy specimen of the groin node confirmed polymorphic B-cell lymphoma. A negative Epstein- Barr virus screen before transplant became positive. The patient's fever increased to 40 degrees C. He subsequently developed sepsis and later, multiple organ failure. Autopsy confirmed extensive abdominal disease. The adrenal glands had been completely replaced by the tumor. Primary Epstein-Barr virus infection is associated with posttransplant lymphoproliferative disease. Replacement of the adrenal glands with a tumor produces a clinical picture of adrenal insufficiency.
Starting from lead compound 4, the 1,4-oxazine headgroup was optimized to improve potency and brain penetration. Focusing at the 6-position of the 5-amino-1,4-oxazine, the insertion of a Me and a CF3 group delivered an excellent pharmacological profile with a pKa of 7.1 and a very low P-gp efflux ratio enabling high central nervous system (CNS) penetration and exposure. Various synthetic routes to access BACE1 inhibitors bearing a 5-amino-6-methyl-6-(trifluoromethyl)-1,4-oxazine headgroup were investigated. Subsequent optimization of the P3 fragment provided the highly potent N-(3-((3R,6R)-5-amino-3,6-dimethyl-6-(trifluoromethyl)-3,6-dihydro-2H-1,4-oxazin-3-yl)-4-fluorophenyl)-5-cyano-3-methylpicolinamide 54 (NB-360), able to reduce significantly Aβ levels in mice, rats, and dogs in acute and chronic treatment regimens.
Reducing the generation of β-amyloid peptide by inhibition of BACE-1 is currently investigated as a disease-modifying therapy for Alzheimer’s disease. First generations of peptidomimetic BACE-1 inhibitors showed low efficacy in vivo, mainly due to limited exposure in the brain. Recently, compounds with a cyclic amidine scaffold were found that overcome such limitations. We report here the characterization of NB-360, a compound derived from the 1,4-oxazine scaffold, which has excellent potency and brain penetration. NB-360 acutely inhibited the generation of Aβ40 and Aβ42 in APP51/16 mice, rats and dogs. Upon chronic treatment in APP51/16 transgenic mice, NB-360 completely blocked amyloid deposition. Furthermore, NB-360 significantly reduced signs of plaque-related brain inflammation, measured as the number of activated microglia cells and astrocytes. These data demonstrate the a highly attractive combination of drug-like properties in compound NB-360, and further indicate that BACE-1 inhibition, by preventing plaque deposition, may also prevent plaque-associated neuroinflammation
Suvorexant (Belsorma®) is the first orexin receptor antagonist approved by the US FDA (August 2014) for insomnia treatment. Following comprehensive Phase II/III studies, with up to 12 months of treatment in adult and elderly patients, there is little doubt that suvorexant induces and maintains sleep. However, the FDA and sponsor disagreed about effective versus safe doses (November 2012). The FDA considered that 5–15 mg were efficient and probably safe, whereas the sponsors had proposed 15–40 mg. The final approved doses are 5, 10, 15 and 20 mg. The major issues are next-morning somnolence and safety as seen in driving tests, with possible signs of muscle weakness, weird dreams, sleep walking, other nighttime behaviors and suicidal ideation. Despite its limitations, suvorexant's market entry offers a truly novel treatment for insomnia, paving the way for follow-up compounds and opening therapeutic avenues in other disorders for orexin receptor modulating compounds.
Anxiety and depression disorders represent common, serious and growing health problems world-wide. The neurobiological basis of anxiety and depression, however, remains poorly understood. Further, there is a clear need for the development of better treatments for these disorders. Emerging data with genetic and pharmacological tools supports a role for GABAB receptors in both anxiety and depression. GABAB receptors are metabotropic GABA receptors that are comprised of two subunits, GABAB1 and GABAB2, which form heterodimers. The GABAB(1) gene is transcribed into two predominate isoforms, GABAB(1a) and GABAB(1b) which differ in sequence primarily by the inclusion of a pair of sushi domains (or short consensus repeats) in the GABAB(1a) N-terminus. Both isoforms heterodimerize with GABAB2 subunits to form functional receptors. The two GABAB(1) isoforms and the GABAB(2) subunit constitute the majority of the molecular diversity of the GABAB receptor. However, in the absence of any isoform-selective ligands for research, the behavioural function of mammalian GABAB1 receptor isoforms has been inscrutable. Recently mice deficient in GABAB(1a) and GABAB(1b) isoforms were generated. Aspects of anxiety- and depression-related behaviour may be modelled in mice, by using traditional animal models, and by examining specific biological and behavioural components of the human symptomatology, or ‘endophenotypes’. A preliminary aim of this thesis was to determine the utility of GABAB(1) isoform-deficient mice for the dissection of GABAB(1a) and GABAB(1b) isoform-mediated behaviour. The main aim of this thesis was to test the hypothesis, using a combination of traditional and endophenotype murine models, that GABAB(1) receptor isoforms play an important role in the mediation and anxiety and depression-related behaviour.
GABAB(1) Isoforms in GABAB Receptor Function:
Preparatory work in this thesis examined the influence of genetic background on GABAB receptor-mediated responses. Genetic background, in the form of different mouse strains, had a strong, differential effect on the classic responses to GABAB receptor activation; hypothermia and ataxia. This underlined the necessity of including multiple experimental endpoints in the examination of GABAB receptor function in subsequent work with GABAB(1) isoform-deficient mice. Importantly, this study also demonstrated that the BALB/c mouse strain was an appropriate genetic background for carrying the GABAB(1) isoform mutations.
Initial studies with GABAB(1) isoform-deficient mice demonstrated that they were free of gross sensory-motor deficits that may preclude their application in behavioural tasks. Furthermore, GABAB(1a) and GABAB(1b) diverged in their influences on locomotor responses to novelty and circadian activity, although the GABAB receptor agonists baclofen or γ-hydroxybutyrate (GHB) were not specific for either isoform and were unable to discriminate these differences. These findings demonstrated that the GABAB(1) isoforms had differential influences on behaviour. Together these studies demonstrated that the GABAB(1a)-/- and GABAB(1b)-/- mice were applicable for testing the hypothesis that the GABAB(1) isoforms were differentially implicated in anxiety and depression related behaviour.
GABAB(1) Isoforms in Endophenotypes of Anxiety and Depression:
Deletion of GABAB(1a) and GABAB(1b) isoforms had profound, differential impacts on the acquisition (GABAB(1a)) and extinction (GABAB(1b)) of aversive memories, as determined in a conditioned taste aversion paradigm. These effects, however, were not accompanied by differences in innate anxiety, as assessed in a comprehensive test battery of unconditioned anxiety tests, including autonomic (stress-induced hyperthermia), active (marble burying) and passive exploratory avoidance (staircase, light-dark box, elevated plus maze, elevated zero maze) behavioural readouts. There was no evidence for a specific influence of either isoform in these tests. This indicated that the GABAB(1) isoforms themselves did not have a defining role in innate anxiety.
GABAB(1a)-/- and GABAB(1b)-/- mice diverged in their cognitive phenotypes. GABAB(1a)-/- mice were impaired in tasks of working spatial and recognition memory, but not in passive avoidance. GABAB(1b)-/- mice were also impaired, to a lesser degree, in a working spatial memory task, but showed preservation of working recognition memory and passive avoidance. Long term recognition memory, however, was also impaired in these mice.
The GABAB(1a) isoform was specifically implicated in depression-related behaviour, as indicated by reduced immobility in a classic test of antidepressant-like behaviour – the forced swim test. This was most probably mediated via the striking interactions of the GABAB(1a) isoform with the serotonergic system, as illustrated in particular by the profound desensitisation of presynaptic 5-HT1A receptors in GABAB(1a)-/- mice. A lack of effect on 5-HT1A receptor expression in GABAB(1a)-/- mice, as indicated by normal 5-HT1A autoradiography densities, suggested an intracellular mechanism for this desensitisation.
Together these studies demonstrated that the GABAB(1) isoforms are functionally important variants of the GABAB receptor, with specific relevance in depression and to aversive learning and memory processes that underlie cognitive symptoms in anxiety disorders.
Abstract Background Tauopathy in the central nervous system (CNS) is a histopathological hallmark of frontotemporal dementia (FTD) and Alzheimer’s disease (AD). Although AD is accompanied by various ocular changes, the effects of tauopathy on the integrity of the cornea, which is densely innervated by the peripheral nervous system and is populated by resident dendritic cells, is still unknown. The aim of this study was to investigate if neuroimmune interactions in the cornea are affected by CNS tauopathy. Methods Corneas from wild type (WT) and transgenic rTg4510 mice that express the P301L tau mutation were examined at 2, 6, 8, and 11 months. Clinical assessment of the anterior segment of the eye was performed using spectral domain optical coherence tomography. The density of the corneal epithelial sensory nerves and the number and field area of resident epithelial dendritic cells were assessed using immunofluorescence. The immunological activation state of corneal and splenic dendritic cells was examined using flow cytometry and compared between the two genotypes at 9 months of age. Results Compared to age-matched WT mice, rTg4510 mice had a significantly lower density of corneal nerve axons at both 8 and 11 months of age. Corneal nerves in rTg4510 mice also displayed a higher percentage of beaded nerve axons and a lower density of epithelial dendritic cells compared to WT mice. From 6 months of age, the size of the corneal dendritic cells was significantly smaller in rTg4510 compared to WT mice. Phenotypic characterization by flow cytometry demonstrated an activated state of dendritic cells (CD86 + and CD45 + CD11b + CD11c + ) in the corneas of rTg4510 compared to WT mice, with no distinct changes in the spleen monocytes/dendritic cells. At 2 months of age, there were no significant differences in the neural or immune structures between the two genotypes. Conclusions Corneal sensory nerves and epithelial dendritic cells were altered in the rTg4510 mouse model of tauopathy, with temporal changes observed with aging. The activation of corneal dendritic cells prior to the gradual loss of neighboring sensory nerves suggests an early involvement of corneal immune cells in tau-associated pathology originating in the CNS.
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