High throughput measurement of hERG drug block kinetics using the CiPA dynamic protocol.

The Comprehensive in vitro Proarrhythmic Assay (CiPA) has promoted use of in silico models of drug effects on cardiac repolarization to improve proarrhythmic risk prediction. These models contain a pharmacodynamic component describing drug binding to hERG channels that required in vitro data for kinetics of block, in addition to potency, to constrain them. To date, development and validation has been undertaken using data from manual patch-clamp. The application of this approach at scale requires the development of a high-throughput, automated patch-clamp (APC) implementation. Here, we present a comprehensive analysis of the implementation of the Milnes, or CiPA dynamic protocol, on an APC platform, including quality control and data analysis. Kinetics and potency of block were assessed for bepridil, cisapride, terfenadine and verapamil with data retention/QC pass rate of 21.8% overall, or as high as 50.4% when only appropriate sweep lengths were considered for drugs with faster kinetics. The variability in IC50 and kinetics between manual and APC was comparable to that seen between sites/platforms in previous APC studies of potency. Whilst the experimental success is less than observed in screens of potency alone, it is still significantly greater than manual patch. With the modifications to protocol design, including sweep length, number of repetitions, and leak correction recommended in this study, this protocol can be applied on APC to acquire data comparable to manual patch clamp.

Metabolic and electrolyte abnormalities as risk factors in drug-induced long QT syndrome.

Drug-induced long QT syndrome (diLQTS) is the phenomenon by which the administration of drugs causes prolongation of cardiac repolarisation and leads to an increased risk of the ventricular tachycardia known as torsades de pointes (TdP). In most cases of diLQTS, the primary molecular target is the human ether-à-go-go-related gene protein (hERG) potassium channel, which carries the rapid delayed rectifier current (IKr) in the heart. However, the proarrhythmic risk associated with drugs that block hERG can be modified in patients by a range of environmental- and disease-related factors, such as febrile temperatures, alterations in pH, dyselectrolytaemias such as hypokalaemia and hypomagnesemia and coadministration with other drugs. In this review, we will discuss the clinical occurrence of drug-induced LQTS in the context of these modifying factors as well as the mechanisms by which they contribute to altered hERG potency and proarrhythmic risk.

Pathophysiological metabolic changes associated with disease modify the proarrhythmic risk profile of drugs with potential to prolong repolarisation.

BACKGROUND AND PURPOSE: Hydroxychloroquine, chloroquine and azithromycin are three drugs that were proposed to treat coronavirus disease 2019 (COVID-19). While concern already existed around their proarrhythmic potential, there are little data regarding how altered physiological states encountered in patients such as febrile state, electrolyte imbalances or acidosis might change their risk profiles. EXPERIMENTAL APPROACH: Potency of human ether-à-go-go related gene (hERG) block was measured using high-throughput electrophysiology in the presence of variable environmental factors. These potencies informed simulations to predict population risk profiles. Effects on cardiac repolarisation were verified in human induced pluripotent stem cell-derived cardiomyocytes from multiple individuals. KEY RESULTS: Chloroquine and hydroxychloroquine blocked hERG with IC50 of 1.47 ± 0.07 and 3.78 ± 0.17 µM, respectively, indicating proarrhythmic risk at concentrations effective against severe acute respiratory syndrome-coronovirus-2 (SARS-CoV-2) in vitro. Hypokalaemia and hypermagnesaemia increased potency of chloroquine and hydroxychloroquine, indicating increased proarrhythmic risk. Acidosis significantly reduced potency of all drugs, whereas increased temperature decreased potency of chloroquine and hydroxychloroquine against hERG but increased potency for azithromycin. In silico simulations demonstrated that proarrhythmic risk was increased by female sex, hypokalaemia and heart failure and identified specific genetic backgrounds associated with emergence of arrhythmia. CONCLUSION AND IMPLICATIONS: Our study demonstrates how proarrhythmic risk can be exacerbated by metabolic changes and pre-existing disease. More broadly, the study acts as a blueprint for how high-throughput in vitro screening, combined with in silico simulations, can help guide both preclinical screening and clinical management of patients in relation to drugs with potential to prolong repolarisation.

Exploring How Low Oxygen Post Conditioning Improves Stroke-Induced Cognitive Impairment: A Consideration of Amyloid-Beta Loading and Other Mechanisms.

Cognitive impairment is a common and disruptive outcome for stroke survivors, which is recognized to be notoriously difficult to treat. Previously, we have shown that low oxygen post-conditioning (LOPC) improves motor function and limits secondary neuronal loss in the thalamus after experimental stroke. There is also emerging evidence that LOPC may improve cognitive function post-stroke. In the current study we aimed to explore how exposure to LOPC may improve cognition post-stroke. Experimental stroke was induced using photothrombotic occlusion in adult, male C57BL/6 mice. At 72 h post-stroke animals were randomly assigned to either normal atmospheric air or to one of two low oxygen (11% O2) exposure groups (either 8 or 24 h/day for 14 days). Cognition was assessed during the treatment phase using a touchscreen based paired-associate learning assessment. At the end of treatment (17 days post-stroke) mice were euthanized and tissue was collected for subsequent histology and biochemical analysis. LOPC (both 8 and 24 h) enhanced learning and memory in the 2nd week post-stroke when compared with stroke animals exposed to atmospheric air. Additionally we observed LOPC was associated with lower levels of neuronal loss, the restoration of several vascular deficits, as well as a reduction in the severity of the amyloid-beta (Aß) burden. These findings provide further insight into the pro-cognitive benefits of LOPC.

Visual discrimination impairment after experimental stroke is associated with disturbances in the polarization of the astrocytic aquaporin-4 and increased accumulation of neurotoxic proteins.

Numerous clinical studies have documented the high incidence of cognitive impairment after stroke. However, there is only limited knowledge about the underlying mechanisms. Interestingly, there is emerging evidence suggesting that cognitive function after stroke may be affected due to reduced waste clearance and subsequent accumulation of neurotoxic proteins. To further explore this potential association, we utilised a model of experimental stroke in mice. Specifically, a photothrombotic vascular occlusion targeting motor and sensory parts of the cerebral cortex was induced in young adult mice, and changes in cognition were assessed using a touchscreen platform for pairwise visual discrimination. The results showed that the execution of the visual discrimination task was impaired in mice 10 to 14 days post-stroke compared to sham. Stroke also induced significant neuronal loss within the peri-infarct, thalamus and the CA1 sub-region of the hippocampus. Further, immunohistochemical and protein analyses of the selected brain regions revealed an increased accumulation and aggregation of both amyloid-ß and α-synuclein. These alterations were associated with significant disturbances in the aquaporin-4 protein expression and polarization at the astrocytic end-feet. The results suggest a link between the increased accumulation of neurotoxic proteins and the stroke-induced cognitive impairment. Given that the neurotoxic protein accumulation appeared alongside changes in astrocytic aquaporin-4 distribution, we suggest that the function of the waste clearance pathways in the brain post-stroke may represent a therapeutic target to improve brain recovery.

Growth Hormone Improves Cognitive Function After Experimental Stroke.

BACKGROUND AND PURPOSE: Cognitive impairment is a common outcome for stroke survivors. Growth hormone (GH) could represent a potential therapeutic option as this peptide hormone has been shown to improve cognition in various clinical conditions. In this study, we evaluated the effects of peripheral administration of GH at 48 hours poststroke for 28 days on cognitive function and the underlying mechanisms. METHODS: Experimental stroke was induced by photothrombotic occlusion in young adult mice. We assessed the associative memory cognitive domain using mouse touchscreen platform for paired-associate learning task. We also evaluated neural tissue loss, neurotrophic factors, and markers of neuroplasticity and cerebrovascular remodeling using biochemical and histology analyses. RESULTS: Our results show that GH-treated stroked mice made a significant improvement on the paired-associate learning task relative to non-GH-treated mice at the end of the study. Furthermore, we observed reduction of neural tissue loss in GH-treated stroked mice. We identified that GH treatment resulted in significantly higher levels of neurotrophic factors (IGF-1 [insulin-like growth factor-1] and VEGF [vascular endothelial growth factor]) in both the circulatory and peri-infarct regions. GH treatment in stroked mice not only promoted protein levels and density of presynaptic marker (SYN-1 [synapsin-1]) and marker of myelination (MBP [myelin basic protein]) but also increased the density and area coverage of 2 major vasculature markers (CD31 and collagen-IV), within the peri-infarct region. CONCLUSIONS: These findings provide compelling preclinical evidence for the usage of GH as a potential therapeutic tool in the recovery phase of patients after stroke.

Reconsidering the role of glial cells in chronic stress-induced dopaminergic neurons loss within the substantia nigra? Friend or foe?

Exposure to psychological stress is known to seriously disrupt the operation of the substantia nigra (SN) and may in fact initiate the loss of dopaminergic neurons within the SN. In this study, we aimed to investigate how chronic stress modified the SN in adult male mice. Using a paradigm of repeated restraint stress (an average of 20h per week for 6weeks), we examined changes within the SN using western blotting and immunohistochemistry. We demonstrated that chronic stress was associated with a clear loss of dopaminergic neurons within the SN. The loss of dopaminergic neurons was accompanied by higher levels of oxidative stress damage, indexed by levels of protein carbonylation and strong suppression of both microglial and astrocytic responses. In addition, we demonstrated for the first time, that chronic stress alone enhanced the aggregation of α-synuclein into the insoluble protein fraction. These results indicate that chronic stress triggered loss of dopaminergic neurons by increasing oxidative stress, suppressing glial neuroprotective functions and enhancing the aggregation of the neurotoxic protein, α-synuclein. Collectively, these results reinforce the negative effects of chronic stress on the viability of dopaminergic cells within the SN.