4-dimensional functional profiling in the convulsant-treated larval zebrafish brain

This is the final version of the article. Available from Springer Nature via the DOI in this record.Functional neuroimaging, using genetically-encoded Ca(2+) sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.This work was funded by the Biological and Biotechnology Research Council (CASE studentship BB/L502510/1, with AstraZeneca Safety Health and Environment), and by the University of Exeter and AstraZeneca

Requirements for a lead compound to become a clinical candidate

A drug candidate suitable for clinical testing is expected to bind selectively to the receptor site on the target, to elicit the desired functional response of the target molecule, and to have adequate bioavailability and biodistribution to elicit the desired responses in animals and humans; it must also pass formal toxicity evaluation in animals. The path from lead to clinical drug candidate represents the most idiosyncratic segment of drug discovery and development. Each program is unique and setbacks are common, making it difficult to predict accurately the duration or costs of this segment. Because of incidents of unpredicted human toxicity seen in recent years, the regulatory agencies and public demands for safety of new drug candidates have become very strict, and safety issues are dominant when identifying a clinical drug candidate

Functional brain imaging in larval zebrafish for characterising the effects of seizurogenic compounds acting via a range of pharmacological mechanisms

This is the final version. Available on open access from Wiley via the DOI in this recordData availability statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.Background and Purpose Functional brain imaging using genetically encoded Ca2+ sensors in larval zebrafish is being developed for studying seizures and epilepsy as a more ethical alternative to rodent models. Despite this, few data have been generated on pharmacological mechanisms of action other than GABAA antagonism. Assessing larval responsiveness across multiple mechanisms is vital to test the translational power of this approach, as well as assessing its validity for detecting unwanted drug‐induced seizures and testing antiepileptic drug efficacy. Experimental Approach Using light‐sheet imaging, we systematically analysed the responsiveness of 4 days post fertilisation (dpf; which are not considered protected under European animal experiment legislation) transgenic larval zebrafish to treatment with 57 compounds spanning more than 12 drug classes with a link to seizure generation in mammals, alongside eight compounds with no such link. Key Results We show 4dpf zebrafish are responsive to a wide range of mechanisms implicated in seizure generation, with cerebellar circuitry activated regardless of the initiating pharmacology. Analysis of functional connectivity revealed compounds targeting cholinergic and monoaminergic reuptake, in particular, showed phenotypic consistency broadly mapping onto what is known about neurotransmitter‐specific circuitry in the larval zebrafish brain. Many seizure‐associated compounds also exhibited altered whole brain functional connectivity compared with controls. Conclusions and Implications This work represents a significant step forward in understanding the translational power of 4dpf larval zebrafish for use in neuropharmacological studies and for studying the events driving transition from small‐scale pharmacological activation of local circuits, to the large network‐wide abnormal synchronous activity associated with seizures.Biotechnology and Biological Sciences Research Council (BBSRC)National Centre for the Replacement Refinement and Reduction of Animals in ResearchUniversity of ExeterMedical Research Council (MRC)AstraZenecaEuropean Unio

Molecular targeting of retinoic acid metabolism in neuroblastoma: the role of the CYP26 inhibitor R116010 in vitro and in vivo

Isomerisation to all-trans-retinoic acid (ATRA) is widely accepted as the key mechanism underlying the favourable clinical properties of 13-cis-retinoic acid (13cisRA). As intracellular metabolism of ATRA by CYP26 may result in clinical resistance to 13cisRA, an increase in efficacy may be achieved through modulation of this metabolic pathway. We have evaluated the effect of the CYP26 inhibitor R116010 on retinoid metabolism in neuroblastoma cell lines and a xenograft model. In neuroblastoma cells, which showed a high level of CYP26 induction in response to ATRA, R116010 selectively inhibited ATRA metabolism. In addition, siRNA-mediated knockdown of CYP26 selectively increased ATRA levels and the expression of retinoid-responsive marker genes was potentiated by R116010. Treatment of mice bearing SH-SY5Y xenografts with 13cisRA (100 mg kg−1) revealed substantial levels (16%) of intratumoral ATRA after 6 h, despite plasma ATRA levels representing only 1% total retinoids under these conditions. Co-administration of R116010 with 13cisRA in this mouse model resulted in significant increases in plasma ATRA and 13cisRA concentrations. Furthermore, R116010 induced significant decreases in levels of 4-oxo metabolites in hepatic tissue after co-administration with either ATRA or 13cisRA. These data suggest considerable potential for CYP26 inhibitors in the future treatment of neuroblastoma with 13cisRA

Large-Scale Assessment of the Zebrafish Embryo as a Possible Predictive Model in Toxicity Testing

Background: In the drug discovery pipeline, safety pharmacology is a major issue. The zebrafish has been proposed as a model that can bridge the gap in this field between cell assays (which are cost-effective, but low in data content) and rodent assays (which are high in data content, but less cost-efficient). However, zebrafish assays are only likely to be useful if they can be shown to have high predictive power. We examined this issue by assaying 60 water-soluble compounds representing a range of chemical classes and toxicological mechanisms. Methodology/Principal Findings: Over 20,000 wild-type zebrafish embryos (including controls) were cultured individually in defined buffer in 96-well plates. Embryos were exposed for a 96 hour period starting at 24 hours post fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for LC 50 determination. Zebrafish embryo LC50 (log mmol/L), and published data on rodent LD50 (log mmol/kg), were found to be strongly correlated (using Kendall’s rank correlation tau and Pearson’s product-moment correlation). The slope of the regression line for the full set of compounds was 0.73403. However, we found that the slope was strongly influenced by compound class. Thus, while most compounds had a similar toxicity level in both species, some compounds were markedly more toxic in zebrafish than in rodents, or vice versa. Conclusions: For the substances examined here, in aggregate, the zebrafish embryo model has good predictivity for toxicit

Age and gender dependent heart rate circadian model development and performance verification on the proarrhythmic drug case study

BACKGROUND: There are two main reasons for drug withdrawals at the various levels of the development path – hepatic and cardiac toxicity. The latter one is mainly connected with the proarrhythmic potency and according to the present practice is supposed to be recognized at the pre-clinical (in vitro and animal in vivo) or clinical level (human in vivo studies). There are, although, some limitations to all the above mentioned methods which have led to novel in vitro – in vivo extrapolation methods being introduced. With the use of in silico implemented mathematical and statistical modelling it is possible to translate the in vitro findings into the human in vivo situation at the population level. Human physiology is influenced by many parameters and one of them which needs to be properly accounted for is a heart rate which follows the circadian rhythm. We described such phenomenon statistically which enabled the improved assessment of the drug proarrhythmic potency. METHODS: A publicly available data set describing the circadian changes of the heart rate of 18 healthy subjects, 5 males (average age 36, range 26–45) and 13 females (average age 34, range 20–50) was used for the heart rate model development. External validation was done with the use of a clinical research database containing heart rate measurements derived from 67 healthy subjects, 34 males and 33 females (average age 33, range 17–72). The developed heart rate model was then incorporated into the ToxComp platform to simulate the impact of circadian variation in the heart rate on QTc interval. The usability of the combined models was assessed with moxifloxacin (MOXI) as a model drug. RESULTS: The developed heart rate model fitted well, both to the training data set (RMSE = 128 ms and MAPE = 12.3%) and the validation data set (RMSE = 165 ms and MAPE = 17.1%). Simulations performed at the population level proved that the combination of the IVIVE platform and the population variability description allows for the precise prediction of the circadian variation of drugs proarrhythmic effect. CONCLUSIONS: It can be concluded that a flexible and practically useful model describing the heart rate circadian variation has been developed and its performance was verified

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research