Optimization of an Imidazo[1,2-a]pyridine Series to Afford Highly Selective Type I1/2 Dual Mer/Axl Kinase Inhibitors with In Vivo Efficacy.

Inhibition of Mer and Axl kinases has been implicated as a potential way to improve the efficacy of current immuno-oncology therapeutics by restoring the innate immune response in the tumor microenvironment. Highly selective dual Mer/Axl kinase inhibitors are required to validate this hypothesis. Starting from hits from a DNA-encoded library screen, we optimized an imidazo[1,2-a]pyridine series using structure-based compound design to improve potency and reduce lipophilicity, resulting in a highly selective in vivo probe compound 32. We demonstrated dose-dependent in vivo efficacy and target engagement in Mer- and Axl-dependent efficacy models using two structurally differentiated and selective dual Mer/Axl inhibitors. Additionally, in vivo efficacy was observed in a preclinical MC38 immuno-oncology model in combination with anti-PD1 antibodies and ionizing radiation.

Cardiovascular Toxicity of Targeted Therapies for Cancer: An Overview of Systematic Reviews.

BACKGROUND: Several targeted therapies for cancer have been associated with cardiovascular toxicity. The evidence for this association has not been synthesized systematically nor has the quality of evidence been considered. We synthesized systematic review evidence of cardiovascular toxicity of individual targeted agents. METHODS: We searched MEDLINE, Embase, and the Cochrane Database of Systematic Reviews for systematic reviews with meta-analyses of cardiovascular outcomes for individual agents published to May 2020. We selected reviews according to prespecified eligibility criteria (International Prospective Register of Systematic Reviews CRD42017080014). We classified evidence of cardiovascular toxicity as sufficient, probable, possible, or indeterminate for specific cardiovascular outcomes based on statistical significance, study quality, and size. RESULTS: From 113 systematic reviews, we found at least probable systematic review evidence of cardiovascular toxicity for 18 agents, including high- and all-grade hypertension for bevacizumab, ramucirumab, axitinib, cediranib, pazopanib, sorafenib, sunitinib, vandetanib, aflibercept, abiraterone, and enzalutamide, and all-grade hypertension for nintedanib; high- and all-grade arterial thromboembolism (includes cardiac and/or cerebral events) for bevacizumab and abiraterone, high-grade arterial thromboembolism for trastuzumab, and all-grade arterial thromboembolism for sorafenib and tamoxifen; high- and all-grade venous thromboembolism (VTE) for lenalidomide and thalidomide, high-grade VTE for cetuximab and panitumumab, and all-grade VTE for bevacizumab; high- and all-grade left ventricular ejection fraction decline or congestive heart failure for bevacizumab and trastuzumab, and all-grade left ventricular ejection fraction decline/congestive heart failure for pazopanib and sunitinib; and all-grade corrected QT interval prolongation for vandetanib. CONCLUSIONS: Our review provides an accessible summary of the cardiovascular toxicity of targeted therapy to assist clinicians and patients when managing cardiovascular health.

A flexible high content imaging assay for profiling macrophage efferocytosis.

Macrophages are a diverse population of cells originating from the myeloid lineage, which form an important component of the innate immune system, helping to regulate immune response through secretion of pro/anti-inflammatory cytokines. However they also have an important homeostatic role - helping to remove cellular debris and apoptotic cells from the body (a phagocytic process known as efferocytosis). Here we describe a robust 384 well microplate based imaging assay, using apoptotic target cells for the specific quantification of efferocytosis in human primary monocyte derived macrophages. The methodology described allows for the assay to run in either fixed end-point or live-cell format (the former offering multiple morphological and intensity-based readouts, whilst the latter opens the possibility for future expansion of the methodology to encompass kinetic profiling). Within the methodology described we couple high content image acquisition (on the Cell Voyager 7000S) with multi-parametric image analysis - using Perkin Elmer Columbus combined with GeneData Screener.

Cardiovascular toxicity of targeted therapies for cancer: a protocol for an overview of systematic reviews.

INTRODUCTION: The introduction of targeted therapies for cancer has contributed to dramatic improvements in patient survival. Nevertheless, several targeted therapies have been associated with 'off-target' adverse effects, based on varying levels of evidence. To date, this evidence has not been systematically synthesised. We will synthesise published systematic review evidence of cardiovascular toxicity associated with targeted cancer therapies. METHODS AND ANALYSIS: We will include systematic reviews of randomised controlled trials and observational studies that report on cardiovascular outcomes for individual agents. We will identify systematic reviews by applying predeveloped, standardised search strategies within Embase, Medline and Cochrane Central. Two independent reviewers will identify reviews published up to 31 December 2016 using predefined eligibility criteria. They will resolve ambiguous cases through consensus, arbitrated by a third reviewer if required. The reviewers will extract and report data according to methodological guidelines for overviews provided by the Cochrane Collaboration, Joanna Briggs Institute and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols. They will assess the quality of included reviews by applying the Assessment of Multiple Systematic Reviews tool. They will judge the quality of evidence in included reviews based on their assessment of bias and incorporation into the interpretation of findings. In synthesising the evidence, we will classify agents based on systematic review evidence of toxicity (sufficient, probable, possible or indeterminate) for specific cardiovascular outcomes (congestive heart failure, myocardial infarction, ischaemic heart disease, left ventricular ejection fraction decline, cerebrovascular disease, pulmonary embolism, thrombosis and hypertension). This will provide clinicians and patients with an accessible synthesis based on robust methodology. ETHICS AND DISSEMINATION: Ethics approval is not required for overviews. We will conduct the study in collaboration with consumer representatives. We will submit results for peer-review publication, and disseminate them through established clinical and consumer networks. PROSPERO REGISTRATION NUMBER: CRD42017080014.

Reliability of thermodilution derived cardiac output with different operator characteristics.

Cardiac output (CO) is commonly measured using the thermodilution technique at the time of right heart catheterisation (RHC). However inter-operator variability, and the operator characteristics which may influence that, has not been quantified. Therefore, this study aimed to assess inter-operator variability with the thermodilution technique using a mock circulation loop (MCL) with calibrated flow sensors. Participants were blinded and asked to determine 4 levels of CO using the thermodilution technique, which was compared with the MCL calibrated flow sensors. The MCL was used to randomly generate CO between 3.0 and 7.0 L/min through changes in heart rate, contractility and vascular resistance with a RHC inserted through the MCL pulmonary artery. Participant characteristics including gender, specialty, age, height, weight, body-mass index, grip strength and RHC experience were recorded and compared to determine their relationship with CO measurement accuracy. In total, there were 15 participants, made up of consultant cardiologists (6), advanced trainees in cardiology (5) and intensive care consultants (4). The majority (9) had performed 26-100 previous RHCs, while 4 had performed more than 100 RHCs. Compared to the MCL-measured CO, participants overestimated CO using the thermodilution technique with a mean difference of +0.75 ± 0.71 L/min. The overall r2 value for actual vs measured CO was 0.85. The difference between MCL and thermodilution derived CO declined significantly with increasing RHC experience (P < 0.001), increasing body mass index (P < 0.001) and decreasing grip strength (P = 0.033). This study demonstrated that the thermodilution technique is a reasonable method to determine CO, and that operator experience was the only participant characteristic related to CO measurement accuracy. Our results suggest that adequate exposure to, and training in, the thermodilution technique is required for clinicians who perform RHC.

From the Cover: Development and Application of a Dual Rat and Human AHR Activation Assay.

Significant prolonged aryl hydrocarbon receptor (AHR) activation, classically exhibited following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, can cause a variety of undesirable toxicological effects. Novel pharmaceutical chemistries also have the potential to cause activation of AHR and consequent toxicities in pre-clinical species and man. Previous methods either employed relatively expensive and low-throughput primary hepatocyte dosing with PCR endpoint, or low resolution overexpressing reporter gene assays. We have developed, validated and applied an in vitro microtitre plate imaging-based medium throughput screening assay for the assessment of endogenous species-specific AHR activation potential via detection of induction of the surrogate transcriptional target Cytochrome P450 CYP1A1. Routine testing of pharmaceutical drug development candidate chemistries using this assay can influence the chemical design process and highlight AHR liabilities. This assay should be introduced such that human AHR activation liability is flagged early for confirmatory testing.

Phenotypic Screen for Cardiac Regeneration Identifies Molecules with Differential Activity in Human Epicardium-Derived Cells versus Cardiac Fibroblasts.

Activation and proliferation of resident cardiac progenitor cells has therapeutic potential to repair the heart after injury. However, research has been impeded by a lack of well-defined and characterized cell sources and difficulties in translation to screening platforms. Here, we describe the development, validation, and use of a 384-well phenotypic assay in primary human epicardium-derived cells (EPDCs) to identify compounds that induce proliferation while maintaining the progenitor phenotype. Using this assay, we screened 7400 structurally diverse compounds where greater than 90% are biologically annotated and known to modulate a broad range of biological targets. From the primary screen, we identified and validated hits and expanded upon the lead molecules of interest. A counterscreen was developed in human cardiac fibroblasts to filter out compounds with a general proliferative effect, after which the activity of selected molecules was confirmed across multiple EPDC donors. To further examine the mechanism of action of compounds with annotated targets, we performed knockdown experiments to understand whether a single known target was responsible for the proliferative effect, confirming results with protein expression and activity assays. Here, we were able to show that the annotated targets of compounds of interest were not responsible for the proliferative effect, which highlights potential differences in cell types and signaling pathways and possible polypharmacology. These studies demonstrate the feasibility of using relevant human primary cells in a phenotypic screen to identify compounds as novel biological tools and starting points for drug discovery projects, and we disclose the first small molecules to proliferate human primary EPDCs.

Assessment of extracellular field potential and Ca2+ transient signals for early QT/pro-arrhythmia detection using human induced pluripotent stem cell-derived cardiomyocytes.

Cardiovascular toxicity is a prominent reason for failures in drug development, resulting in the demand for assays that can predict this liability in early drug discovery. We investigated whether iCell® cardiomyocytes have utility as an early QT/TdP screen. Thirty clinical drugs with known QT/TdP outcomes were evaluated blind using label-free microelectrode array (parameters measured were beating period (BP), field potential duration (FPD), fast Na+ amplitude and slope) and live cell, fast kinetic fluorescent Ca2+ transient FLIPR® Tetra (parameters measured were peak count, width, amplitude) systems. Many FPD-altering drugs also altered BP. Correction for BP, using a Log-Log (LL) model, was required to appropriately interpret direct drug effects on FPD. In comparison with human QT effects and when drug activity was to be predicted at top test concentration (TTC), LL-corrected FPD and peak count had poor assay sensitivity and specificity values: 13%/64% and 65%/11%, respectively. If effective free therapeutic plasma concentration (EFTPC) was used instead of TTC, the values were 0%/100% and 6%/100%, respectively. When compared to LL-corrected FPD and peak count, predictive values of uncorrected FPD, BP, width and amplitude were not much different. If pro-arrhythmic risk was to be predicted using Ca2+ transient data, the values were 67%/100% and 78%/53% at EFTPC and TTC, respectively. Thus, iCell® cardiomyocytes have limited value as an integrated QT/TdP assay, highlighting the urgent need for improved experimental alternatives that may offer an accurate integrated cardiomyocyte safety model for supporting the development of new drugs without QT/TdP effects.

Fractal dimension: a novel clot microstructure biomarker use in ST elevation myocardial infarction patients.

OBJECTIVES: Changes in clot microstructure are increasingly implicated in the pathology of atherosclerosis although most data are from techniques in the remote laboratory using altered blood. We validate the novel biomarker Gel Point in STEMI patients and assess therapeutic interventions. Gel Point marks the transition of blood from a visco-elastic liquid to visco-elastic solid and is rapidly measured using unadulterated blood. The Gel Point provides measurements of three parameters to reflect clot microstructure (fractal dimension (df)), real-time clot formation time (TGP) and blood clot strength (elasticity at the Gel Point (G'GP)). METHODS: We prospectively recruited 38 consecutive patients with STEMI undergoing primary percutaneous coronary intervention (pPCI). Venous blood samples were collected on admission, after pPCI and 24 h after admission for assessment of the new biomarkers, standard coagulation tests and scanning electron microscopy (SEM). RESULTS: df after pPCI was lower than df on admission (mean 1.631 [SD 0.063] vs 1.751 [0.052], p < 0.000001) whereas df at 24 h was similar to that on admission. G'GP also showed similar trend to df (p < 0.001). TGP was prolonged at after-PCI measurement compared with admission (median 854 s [IQR 581-1801] vs 217 [179-305], p < 0.00001). Changes in the values of df and G'GP were consistent with changes in the SEM images of the mature clot. CONCLUSIONS: We characterise Gel Point derived markers of clot microstructure in patients admitted with emergency arterial thrombosis. This point of care test can potentially be used to assess the efficacy of therapeutic interventions by measuring changes in clot microstructure.

A new biomarker quantifies differences in clot microstructure in patients with venous thromboembolism.

This study compared patients with venous thromboembolism (VTE) to non-VTE patients using a biomarker of clot microstructure (df ) and clot formation time (TGP ). df was the only marker that identified a significant difference (P < 0·001) between the VTE (n = 60) and non-VTE cohorts (n = 69). The 'abnormal' clot microstructures in the VTE patients suggests either inadequate response to anticoagulant therapy or the presence of a procoagulant state not detected by other markers of coagulation (i.e., International Normalized Ratio). Furthermore, elevated values of df in first time VTE patients who later develop a secondary event indicates that df may identify those at risk of recurrence.

A new structural biomarker that quantifies and predicts changes in clot strength and quality in a model of progressive haemodilution.

INTRODUCTION: We investigated the effect of progressive haemodilution on the dynamics of fibrin clot formation and clot microstructure using a novel rheological method. The technique measures clotting time (TGP), clot strength (G`GP), and quantifies clot microstructure (df) at the incipient stages of fibrin formation. We use computational modelling to examine the relationship between structure and mass, as well as helium ion microscopy (HIM) to compare morphological changes in the fully formed clot to that of the incipient clot. METHODS: This is an in vitro study; 90 healthy volunteers were recruited with informed consent and a 20ml sample of whole blood obtained from each volunteer. Five clinically relevant dilutions were investigated using 0.9w.v isotonic saline (0, 10, 20, 40 and 60%, n=18 for each dilution). The rheological method of assessing structural clot changes was compared against conventional coagulation screen and fibrinogen estimation. RESULTS: Fractal dimension (df) and final clot microstructure both decreased with progressive dilution (significant at a dilution of 20%) with similar relationships observed for final clot characteristics in HIM images. Significant correlations were observed between df and G`GP (clot strength) (0.345, p=0.02), as well as clotting time (PT: -0.690, p>0.001; APTT: -0.672, p>0.001; TGP: -0.385, p=0.006). CONCLUSIONS: This study provides new insight into the effects of haemodilution by isotonic saline on clotting time (TGP), clot strength (G'GP) and clot microstructure (df). Previous studies have attempted to link clot microstructure to clot quality/strength, however this study provides a significant step in quantifying these relationships.

Right ventricular thrombus detection and multimodality imaging using contrast echocardiography and cardiac magnetic resonance imaging.

We present the case of right ventricular thrombus formation associated with a right ventricular infarct secondary to a proximal right coronary artery thrombus, which was not evident on transthoracic echocardiography but detected on both delayed gadolinium enhanced magnetic resonance imaging and microsphere contrast echocardiography. The diagnosis of right ventricular thrombosis altered the decision to place an implantable cardiac defibrillator in this patient. Anticoagulation with warfarin resulted in resolution of the thrombus. This case highlights the utility of multimodality imaging in the detection and follow-up of right ventricular thrombus in the setting of right ventricular myocardial infarction, and the effectiveness of anticoagulation therapy.

Accuracy of 3-dimensional transoesophageal echocardiography in assessment of prosthetic mitral valve dehiscence with comparison to anatomical specimens.

The evolution of echocardiography from 2-Dimensional Transthoracic Echo through to real time 3-Dimensional Transoesophageal Echo has enabled more accurate visualisation and quantification of valvular disorders especially prosthetic mitral valve paravalvular regurgitation. However, validation of accuracy is rarely confirmed by surgical or post-mortem specimens. We present a case directly comparing different echocardiographic modality images to post mortem specimens in a patient with prosthetic mitral valve paravalvular regurgitation.

Analgesic patches and defibrillators: a cautionary tale.

Implantable cardioverter defibrillator (ICD) insertions have increased significantly over the last decade. Transdermal patches are increasingly used for drug delivery. Skin burns associated with metal containing transdermal patches have been reported with magnetic resonance imaging and external cardiac defibrillation. However, there are no reports of dermal injury secondary to an ICD shock and a transdermal drug delivery patch. We report the first known case of a patient who suffered a dermal burn following a defibrillation due to a transdermal patch being positioned over the ICD.