Characterization of a high throughput human stem cell cardiomyocyte assay to predict drug-induced changes in clinical electrocardiogram parameters.

Human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CM's) play an increasingly important role in the safety profiling of candidate drugs. For such models to have utility a clear understanding of clinical translation is required. In the present study we examined the ability of our hIPSC-CM model to predict the clinically observed effects of a diverse set of compounds on several electrocardiogram endpoints, including changes in QT and QRS intervals. To achieve this, compounds were profiled in a novel high throughput voltage-sensitive dye platform. Measurements were taken acutely (30 min) and chronically (24 h) to ensure that responses from compounds with slow onset kinetics or that affected surface ion channel expression would be captured. In addition, to avoid issues associated with changes in free drug levels due to protein binding, assays were run in serum free conditions. Changes in hIPSC-CM threshold APD90 values correlated with compound plasma exposures that produced a +10 ms change in clinical QTc (Pearson r2 = 0.80). In addition, randomForest modeling showed high predictivity in defining TdP risk (AUROC value = 0.938). Risk associated with QRS prolongation correlated with an increase in action potential rise-time (AUROC value = 0.982). The in-depth understanding of the clinical translatability of our hIPSC-CM model positions this assay to play a key role in defining cardiac risk early in drug development. Moreover, the ability to perform longer term studies enables the detection of compounds that may not be highlighted by more acute assay formats, such as inhibitors of hERG trafficking.

Translation of in vitro cannabinoid 1 receptor agonist activity to in vivo pharmacodynamic endpoints.

INTRODUCTION: Building an understanding of in vivo efficacy based on the evaluation of in vitro affinity or potency is critical in expediting early decision making in drug discovery and can significantly reduce the need for animal studies. The aim of the present study was to understand the translation of in vitro to in vivo endpoints for the cannabinoid receptor 1 (CB1). METHODS: Using a selection of CB1 agonists we describe an evaluation of in vitro to in vivo translation comparing in vitro receptor affinity or functional potency, using both cAMP and ß-arrestin endpoints, to various in vivo CB1 agonist-associated endpoints. RESULTS: We demonstrate that in vitro CB1 agonism significantly correlates with the CB1-induced cue in the drug discrimination model in vivo, but not with other purported CB1 agonist-mediated in vivo endpoints, including hypothermia and sedation. Thus, these data challenge common perceptions regarding CB1 agonist-induced tetrad effects in rodents. DISCUSSION: This work exemplifies how in vitro profiling of receptor affinity or potency can predict in vivo pharmacodynamic effects, using the CB1 as an example system. The translatability of in vitro activity to in vivo efficacy allows for the ability to rapidly contextualize off-target CB1 in vitro findings, allowing clear and rapid definition of the risk posed by such activity without the need for extensive animal studies. This has significant implications in terms of early decision making in drug discovery and reducing the use of animals in research, while also outlining a template for expanding the approach for additional targets.

Late-Stage Lead Diversification Coupled with Quantitative Nuclear Magnetic Resonance Spectroscopy to Identify New Structure-Activity Relationship Vectors at Nanomole-Scale Synthesis: Application to Loratadine, a Human Histamine H1 Receptor Inverse Agonist.

An experimental approach is described for late-stage lead diversification of frontrunner drug candidates using nanomole-scale amounts of lead compounds for structure-activity relationship development. The process utilizes C-H bond activation methods to explore chemical space by transforming candidates into newly functionalized leads. A key to success is the utilization of microcryoprobe nuclear magnetic resonance (NMR) spectroscopy, which permits the use of low amounts of lead compounds (1-5 µmol). The approach delivers multiple analogues from a single lead at nanomole-scale amounts as DMSO-d6 stock solutions with a known structure and concentration for in vitro pharmacology and absorption, distribution, metabolism, and excretion testing. To demonstrate the feasibility of this approach, we have used the antihistamine agent loratadine (1). Twenty-six analogues of loratadine were isolated and fully characterized by NMR. Informative SAR analogues were identified, which display potent affinity for the human histamine H1 receptor and improved metabolic stability.

Ligand biological activity predicted by cleaning positive and negative chemical correlations.

Predicting ligand biological activity is a key challenge in drug discovery. Ligand-based statistical approaches are often hampered by noise due to undersampling: The number of molecules known to be active or inactive is vastly less than the number of possible chemical features that might determine binding. We derive a statistical framework inspired by random matrix theory and combine the framework with high-quality negative data to discover important chemical differences between active and inactive molecules by disentangling undersampling noise. Our model outperforms standard benchmarks when tested against a set of challenging retrospective tests. We prospectively apply our model to the human muscarinic acetylcholine receptor M1, finding four experimentally confirmed agonists that are chemically dissimilar to all known ligands. The hit rate of our model is significantly higher than the state of the art. Our model can be interpreted and visualized to offer chemical insights about the molecular motifs that are synergistic or antagonistic to M1 agonism, which we have prospectively experimentally verified.

Cardiac sodium channel antagonism - Translation of preclinical in vitro assays to clinical QRS prolongation.

INTRODUCTION: Cardiac sodium channel antagonists have historically been used to treat cardiac arrhythmias by preventing the reentry of the electrical impulse that could occur following myocardial damage. However, clinical studies have highlighted a significant increase in mortality associated with such treatment. Cardiac sodium channel antagonist activity is now seen as an off-target pharmacology that should be mitigated during the drug development process. The aim of this study was to examine the correlation between in vitro/ex vivo assays that are routinely used to measure Nav1.5 activity and determine the translatability of the individual assays to QRS prolongation in the clinic. METHODS: A set of clinical compounds with known Nav1.5 activity was profiled in several in vitro/ex vivo assays (binding, membrane potential, patch clamp and the Langendorff isolated heart). Clinical data comprising compound exposure levels and changes in QRS interval were obtained from the literature. Sensitivity/specificity analysis was performed with respect to the clinical outcome. RESULTS: The in vitro assays showed utility in predicting QRS prolongation in the clinic. Optimal thresholds were defined for each assay (binding: IC20; membrane potential: IC10; patch clamp: IC20) and sensitivity (69-88%) and specificity (53-84%) values were shown to be similar between assay formats. DISCUSSION: The data provide clear statistical insight into the translatability of Nav1.5 antagonism data generated in vitro to potential clinical outcomes. These results improve our ability to understand the liability posed by such activity in novel development compounds at an early stage.

L-type calcium channel antagonism - Translation from in vitro to in vivo.

INTRODUCTION: Although therapeutically beneficial in the treatment of certain diseases, L-type calcium channel antagonism can result in unwanted off-target pharmacology leading to adverse drug reactions and to the termination of the development of otherwise promising compounds. In the present study three marketed calcium channel inhibitors, nifedipine, verapamil and diltiazem were profiled in a series of in vitro and ex-vivo assays in an effort to determine the ability of these assays to discriminate, between dihydropyridine versus non-dihydropyridine-like compounds, and how well they can predict the cardiovascular effects observed in a conscious telemetered rat model. METHODS: Standard calcium channel antagonists were profiled in radioligand binding, patch clamp and calcium flux assays. In addition, cardiovascular endpoints related to calcium channel activity were also examined in ex vivo tissue bath preparations, including relaxation of pre-constricted rat aorta and the guinea pig Langendorff isolated heart model. The data generated were correlated with in vivo blood pressure and heart rate data from conscious telemetered rats. RESULTS: Our results show that the binding, FLIPR and aorta assays allow differentiation of the compounds in two distinct classes of L-type calcium channel antagonists, and are good predictors of in vivo outcomes. DISCUSSION: These results suggest that in vitro and ex vivo profiling remains a valuable tool in predicting potential in vivo cardiovascular safety issues, and can aid in the selection of novel development compounds that show inherent inhibitory activity against L-type calcium channels.