Pharmacology and Therapeutics - Research Publications
Now showing items 1-12 of 474
Successes and Challenges: Inhaled Treatment Approaches Using Magnetic Nanoparticles in Cystic Fibrosis
<jats:p>Magnetic nanoparticles have been largely applied to increase the efficacy of antibiotics due to passive accumulation provided by enhancing permeability and retention, which is essential for the treatment of lung infections. Recurring lung infections such as in the life-shortening genetic disease cystic fibrosis (CF) are a major problem. The recent advent of the CF modulator drug ivacaftor, alone or in combination with lumacaftor or tezacaftor, has enabled systemic treatment of the majority of patients. Magnetic nanoparticles (MNPs) show unique properties such as biocompatibility and biodegradability as well as magnetic and heat-medicated characteristics. These properties make them suitable to be used as drug carriers and hyperthermia-based agents. Hyperthermia is a promising approach for the thermal activation therapy of several diseases, including pulmonary diseases. The benefits of delivering CF drugs via inhalation using MNPs as drug carriers afford application of sufficient therapeutic dosages directly to the primary target site, while avoiding potential suboptimal pharmacokinetics/pharmacodynamics and minimizing the risks of systemic toxicity. This review explores the multidisciplinary approach of using MNPs as vehicles of drug delivery. Additionally, we highlight advantages such as increased drug concentration at disease site, minimized drug loss and the possibility of specific cell targeting, while addressing major challenges for this emerging field.</jats:p>
A Reckless Guide to P-values : Local Evidence, Global Errors.
(Springer International Publishing, 2020)
This chapter demystifies P-values, hypothesis tests and significance tests and introduces the concepts of local evidence and global error rates. The local evidence is embodied in this data and concerns the hypotheses of interest for this experiment, whereas the global error rate is a property of the statistical analysis and sampling procedure. It is shown using simple examples that local evidence and global error rates can be, and should be, considered together when making inferences. Power analysis for experimental design for hypothesis testing is explained, along with the more locally focussed expected P-values. Issues relating to multiple testing, HARKing and P-hacking are explained, and it is shown that, in many situations, their effects on local evidence and global error rates are in conflict, a conflict that can always be overcome by a fresh dataset from replication of key experiments. Statistics is complicated, and so is science. There is no singular right way to do either, and universally acceptable compromises may not exist. Statistics offers a wide array of tools for assisting with scientific inference by calibrating uncertainty, but statistical inference is not a substitute for scientific inference. P-values are useful indices of evidence and deserve their place in the statistical toolbox of basic pharmacologists.
Pre-mitotic genome re-organisation bookends the B cell differentiation process
(NATURE RESEARCH, 2021-02-26)
During cellular differentiation chromosome conformation is intricately remodelled to support the lineage-specific transcriptional programs required for initiating and maintaining lineage identity. When these changes occur in relation to cell cycle, division and time in response to cellular activation and differentiation signals has yet to be explored, although it has been proposed to occur during DNA synthesis or after mitosis. Here, we elucidate the chromosome conformational changes in B lymphocytes as they differentiate and expand from a naive, quiescent state into antibody secreting plasma cells. We find gene-regulatory chromosome reorganization in late G1 phase before the first division, and that this configuration is remarkably stable as the cells massively and rapidly clonally expand. A second wave of conformational change occurs as cells terminally differentiate into plasma cells, coincident with increased time in G1 phase. These results provide further explanation for how lymphocyte fate is imprinted prior to the first division. They also suggest that chromosome reconfiguration occurs prior to DNA replication and mitosis, and is linked to a gene expression program that controls the differentiation process required for the generation of immunity.
Probing the correlation between ligand efficacy and conformational diversity at the ?(1A)-adrenoreceptor reveals allosteric coupling of its microswitches
(American Society for Biochemistry and Molecular Biology, 2020-05-22)
G protein–coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.
Iron accumulation in skeletal muscles of old mice is associated with impaired regeneration after ischaemia-reperfusion damage
BACKGROUND: Oxidative stress is implicated in the insidious loss of muscle mass and strength that occurs with age. However, few studies have investigated the role of iron, which is elevated during ageing, in age-related muscle wasting and blunted repair after injury. We hypothesized that iron accumulation leads to membrane lipid peroxidation, muscle wasting, increased susceptibility to injury, and impaired muscle regeneration. METHODS: To examine the role of iron in age-related muscle atrophy, we compared the skeletal muscles of 3-month-old with 22- to 24-month-old 129SvEv FVBM mice. We assessed iron distribution and total elemental iron using laser ablation inductively coupled plasma mass spectrometry and Perls' stain on skeletal muscle cross-sections. In addition, old mice underwent ischaemia-reperfusion (IR) injury (90 min ischaemia), and muscle regeneration was assessed 14 days after injury. Immunoblotting was used to determine lipid peroxidation (4HNE) and iron-related proteins. To determine whether muscle iron content can be altered, old mice were treated with deferiprone (DFP) in the drinking water, and we assessed its effects on muscle regeneration after injury. RESULTS: We observed a significant increase in total elemental iron (+43%, P < 0.05) and lipid peroxidation (4HNE: +76%, P < 0.05) in tibialis anterior muscles of old mice. Iron was further increased after injury (adult: +81%, old: +135%, P < 0.05) and associated with increased lipid peroxidation (+41%, P < 0.05). Administration of DFP did not impact iron or measures of lipid peroxidation in skeletal muscle or modulate muscle mass. Increased muscle iron concentration and lipid peroxidation were associated with less efficient regeneration, evident from the smaller fibres in cross-sections of tibialis anterior muscles (-24%, P < 0.05) and an increased percentage of fibres with centralized nuclei (+4124%, P < 0.05) in muscles of old compared with adult mice. Administration of DFP lowered iron after IR injury (PRE: -32%, P < 0.05 and POST: -41%, P < 0.05), but did not translate to structural improvements. CONCLUSIONS: Muscles from old mice have increased iron levels, which are associated with increased lipid peroxidation, increased susceptibility to IR injury, and impaired muscle regeneration. Our results suggest that iron is involved in effective muscle regeneration, highlighting the importance of iron homeostasis in muscle atrophy and muscle repair.
THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: Overview.
The Concise Guide to PHARMACOLOGY 2017/18 is the third in this series of biennial publications. This version provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13882/full. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are eight areas of focus: G protein-coupled receptors, ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2017, and supersedes data presented in the 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
Chromosomes distribute randomly to, but not within, human neutrophil nuclear lobes
(CELL PRESS, 2021-03-19)
The proximity pattern and radial distribution of chromosome territories within spherical nuclei are random and non-random, respectively. Whether this distribution pattern is conserved in the partitioned or lobed nuclei of polymorphonuclear cells is unclear. Here we use chromosome paint technology to examine the chromosome territories of all 46 chromosomes in hundreds of single human neutrophils - an abundant and famously polymorphonuclear immune cell. By comparing the distribution of chromosomes to randomly shuffled controls and validating with orthogonal chromosome conformation capture technology, we show for the first time that human chromosomes randomly distribute to neutrophil nuclear lobes, while maintaining a non-random radial distribution within these lobes. Furthermore, we demonstrate that chromosome length correlates with three-dimensional volume not only in neutrophils but other human immune cells. This work demonstrates that chromosomes are largely passive passengers during the neutrophil lobing process but are able to subsequently maintain their macro-level organization within lobes.
Copper-ATSM as a Treatment for ALS: Support from Mutant SOD1 Models and Beyond
The blood-brain barrier permeant, copper-containing compound, CuII(atsm), has successfully progressed from fundamental research outcomes in the laboratory through to phase 2/3 clinical assessment in patients with the highly aggressive and fatal neurodegenerative condition of amyotrophic lateral sclerosis (ALS). The most compelling outcomes to date to indicate potential for disease-modification have come from pre-clinical studies utilising mouse models that involve transgenic expression of mutated superoxide dismutase 1 (SOD1). Mutant SOD1 mice provide a very robust mammalian model of ALS with high validity, but mutations in SOD1 account for only a small percentage of ALS cases in the clinic, with the preponderant amount of cases being sporadic and of unknown aetiology. As per other putative drugs for ALS developed and tested primarily in mutant SOD1 mice, this raises important questions about the pertinence of CuII(atsm) to broader clinical translation. This review highlights some of the challenges associated with the clinical translation of new treatment options for ALS. It then provides a brief account of pre-clinical outcomes for CuII(atsm) in SOD1 mouse models of ALS, followed by an outline of additional studies which report positive outcomes for CuII(atsm) when assessed in cell and mouse models of neurodegeneration which do not involve mutant SOD1. Clinical evidence for CuII(atsm) selectively targeting affected regions of the CNS in patients is also presented. Overall, this review summarises the existing evidence which indicates why clinical relevance of CuII(atsm) likely extends beyond the context of cases of ALS caused by mutant SOD1.
TRPA1/NOX in the soma of trigeminal ganglion neurons mediates migraine-related pain of glyceryl trinitrate in mice.
(Oxford University Press (OUP), 2018-08-01)
Glyceryl trinitrate is administered as a provocative test for migraine pain. Glyceryl trinitrate causes prolonged mechanical allodynia in rodents, which temporally correlates with delayed glyceryl trinitrate-evoked migraine attacks in patients. However, the underlying mechanism of the allodynia evoked by glyceryl trinitrate is unknown. The proalgesic transient receptor potential ankyrin 1 (TRPA1) channel, expressed by trigeminal nociceptors, is sensitive to oxidative stress and is targeted by nitric oxide or its by-products. Herein, we explored the role of TRPA1 in glyceryl trinitrate-evoked allodynia. Systemic administration of glyceryl trinitrate elicited in the mouse periorbital area an early and transient vasodilatation and a delayed and prolonged mechanical allodynia. The systemic, intrathecal or local administration of selective enzyme inhibitors revealed that nitric oxide, liberated from the parent drug by aldehyde dehydrogenase 2 (ALDH2), initiates but does not maintain allodynia. The central and the final phases of allodynia were respectively associated with generation of reactive oxygen and carbonyl species within the trigeminal ganglion. Allodynia was absent in TRPA1-deficient mice and was reversed by TRPA1 antagonists. Knockdown of neuronal TRPA1 by intrathecally administered antisense oligonucleotide and selective deletion of TRPA1 from sensory neurons in Advillin-Cre; Trpa1fl/fl mice revealed that nitric oxide-dependent oxidative and carbonylic stress generation is due to TRPA1 stimulation, and resultant NADPH oxidase 1 (NOX1) and NOX2 activation in the soma of trigeminal ganglion neurons. Early periorbital vasodilatation evoked by glyceryl trinitrate was attenuated by ALDH2 inhibition but was unaffected by TRPA1 blockade. Antagonists of the calcitonin gene-related peptide receptor did not affect the vasodilatation but partially inhibited allodynia. Thus, although both periorbital allodynia and vasodilatation evoked by glyceryl trinitrate are initiated by nitric oxide, they are temporally and mechanistically distinct. While vasodilatation is due to a direct nitric oxide action in the vascular smooth muscle, allodynia is a neuronal phenomenon mediated by TRPA1 activation and ensuing oxidative stress. The autocrine pathway, sustained by TRPA1 and NOX1/2 within neuronal cell bodies of trigeminal ganglia, may sensitize meningeal nociceptors and second order trigeminal neurons to elicit periorbital allodynia, and could be of relevance for migraine-like headaches evoked by glyceryl trinitrate in humans.
Delayed Oral LY333013 Rescues Mice from Highly Neurotoxic, Lethal Doses of Papuan Taipan (Oxyuranus scutellatus) Venom
There is an unmet need for economical snakebite therapies with long shelf lives that are effective even with delays in treatment. The orally bioavailable, heat-stable, secretory phospholipase A₂ (sPLA₂) inhibitor, LY333013, demonstrates antidotal characteristics for severe snakebite envenoming in both field and hospital use. A murine model of lethal envenoming by a Papuan taipan (Oxyuranus scutellatus) demonstrates that LY333013, even with delayed oral administration, improves the chances of survival. Furthermore, LY333013 improves the performance of antivenom even after it no longer reverses neurotoxic signs. Our study is the first demonstration that neurotoxicity from presynaptic venom sPLA2S can be treated successfully, even after the window of therapeutic antivenom has closed. These results suggest that sPLA₂ inhibitors have the potential to reduce death and disability and should be considered for the initial and adjunct treatment of snakebite envenoming. The scope and capacity of the sPLA2 inhibitors ability to achieve these endpoints requires further investigation and development efforts.
The potential of P2X7 receptors as a therapeutic target, including inflammation and tumour progression
Seven P2X ion channel nucleotide receptor subtypes have been cloned and characterised. P2X7 receptors (P2X7R) are unusual in that there are extra amino acids in the intracellular C terminus. Low concentrations of ATP open cation channels sometimes leading to cell proliferation, whereas high concentrations of ATP open large pores that release inflammatory cytokines and can lead to apoptotic cell death. Since many diseases involve inflammation and immune responses, and the P2X7R regulates inflammation, there has been recent interest in the pathophysiological roles of P2X7R and the potential of P2X7R antagonists to treat a variety of diseases. These include neurodegenerative diseases, psychiatric disorders, epilepsy and a number of diseases of peripheral organs, including the cardiovascular, airways, kidney, liver, bladder, skin and musculoskeletal. The potential of P2X7R drugs to treat tumour progression is discussed.
Proteolytic activation of the epithelial sodium channel (ENaC) by the cysteine protease cathepsin-S
(SPRINGER HEIDELBERG, 2012-10-01)
Proteolytic processing of the amiloride-sensitive epithelial sodium channel (ENaC) by serine proteases is known to be important for channel activation. Inappropriate ENaC activation by proteases may contribute to the pathophysiology of cystic fibrosis and could be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. We hypothesized that in addition to serine proteases, cathepsin proteases may activate ENaC. Cathepsin proteases belong to the group of cysteine proteases and play a pathophysiological role in inflammatory diseases. Under pathophysiological conditions, cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. The aim of this study was to investigate the effect of purified Cat-S on human ENaC heterologously expressed in Xenopus laevis oocytes and on ENaC-mediated sodium transport in cultured M-1 mouse renal collecting duct cells. We demonstrated that Cat-S activates amiloride-sensitive whole-cell currents in ENaC-expressing oocytes. The stimulatory effect of Cat-S was preserved at pH 5. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating proteolytic channel activation. Mutating two valine residues (V182 and V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S. Pre-incubation of the oocytes with the Cat-S inhibitor morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS) prevented the stimulatory effect of Cat-S on ENaC. In contrast, LHVS had no effect on ENaC activation by the prototypical serine proteases trypsin and chymotrypsin. Cat-S also stimulated ENaC in differentiated renal epithelial cells. These findings demonstrate that the cysteine protease Cat-S can activate ENaC which may be relevant under pathophysiological conditions.