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Defining an appropriate and efficient assessment of drug‐induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc‐prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14‐based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B‐based “double‐negative” nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high‐dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double‐negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

Our goal is to accurately predict all types of cardiovascular events in patients utilising nonclinical cardiovascular safety data. In the past two decades, cardiovascular safety science has primarily focused on events associated with the electrocardiogram. Broadening out to other cardiovascular parameters, we share real-life case studies that highlight our progress towards improved and better-informed project progression based upon use of disease models, mechanism-based translation and structure-function relationships. To fulfil this goal, further advances in patient-relevant humanised models will be required to enable cardiovascular safety science to keep pace with the ever-changing landscape of novel therapeutic paradigms.

Off-target pharmacology may contribute to both adverse and beneficial effects of a new drug. In vitro pharmacological profiling is often applied early in drug discovery; there are fewer reports addressing the relevance of broad profiles to clinical adverse effects. Here, we have characterized the pharmacological profile of the active metabolite of fostamatinib, R406, linking an understanding of drug selectivity to the increase in blood pressure observed in clinical studies. R406 was profiled in a broad range of in vitro assays to generate a comprehensive pharmacological profile and key targets were further investigated using functional and cellular assay systems. A combination of traditional literature searches and text-mining approaches established potential mechanistic links between the profile of R406 and clinical side effects. R406 was selective outside the kinase domain, with only antagonist activity at the adenosine A3 receptor in the range relevant to clinical effects. R406 was less selective in the kinase domain, having activity at many protein kinases at therapeutically relevant concentrations when tested in multiple in vitro systems. Systematic literature analyses identified KDR as the probable target underlying the blood pressure increase observed in patients. While the in vitro pharmacological profile of R406 suggests a lack of selectivity among kinases, a combination of classical searching and text-mining approaches rationalized the complex profile establishing linkage between off-target pharmacology and clinically observed effects. These results demonstrate the utility of in vitro pharmacological profiling for a compound in late-stage clinical development.

SummaryG-protein-coupled P2Y1 and P2Y12 receptors play key roles in platelet activation, however the importance of ionotropic P2X1 receptors remains unclear. Platelet P2X1 responses are highly labile in vitro, but were greatly enhanced by increasing [Ca2+]o in the range 1–10 mM. The P2X1 agonist α,β-MeATP stimulated a shape change which saturated at peak [Ca2+]i of ≥ 400 nM, without evidence for aggregation. The maximal P2X1-evoked transmission decrease was 82% of that obtained via P2Y1 receptors. α., β-MeATP caused a disc to sphere transformation in virtually all platelets, but lacked the long processes produced by ADP. Following block of P2Y1 receptors with A3P5PS, co-stimulation with α., β-MeATP and ADP failed to induce aggregation despite the generation of peak [Ca2+]i responses similar to those stimulated via P2Y1 receptors. Therefore early, transient Ca2+ influx via P2X1 receptors can contribute to platelet activation by stimulating a significant morphological change, but does not readily synergise with P2Y12 receptors to support aggregation.

We have examined the role of ATP-dependent P2X1 receptors in megakaryocytes (MKs) and platelets using receptor-deficient mice and selective agonists. α,β-meATP- and ATP- evoked ionotropic inward currents were absent in whole-cell recordings from MKs of P2X1−/− mice, demonstrating that the P2X receptor phenotype in MKs, and by inference, platelets, is due to expression of homomeric P2X1 receptors. P2X1 receptor deficiency had no effect on MK (CD 41) numbers or size distribution, showing that it is not essential for normal MK development. P2Y receptor-stimulated [Ca2+]i responses were unaffected in MKs from P2X1−/− mice, however the inward cation current associated with Ca2+ release was reduced by ∼50%, suggesting an interaction between the membrane conductances activated by P2X1 and P2Y receptors. Interaction between P2X1 and P2Y receptors in human platelets was also examined using [Ca2+]i recordings from cell suspensions. α,β-meATP (10 μM) evoked a rapid transient P2X1 receptor-mediated increase in [Ca2+]i, whereas ADP-(10 μM) evoked P2Y receptor responses were slower, peaked at a higher level and remained elevated for longer periods. Co-application of α,β-meATP and ADP resulted in marked acceleration and amplification of the peak [Ca2+]i response. We conclude that ionotropic P2X1 receptors may play a priming role in the subsequent activation of metabotropic P2Y receptors during platelet stimulation. British Journal of Pharmacology (2002) 135, 363–372; doi:10.1038/sj.bjp.0704486

SummarySimultaneous measurements of [Ca2+]i and light transmission were used to examine the relationship between P2X1 receptor activation and functional platelet responses. The P2X1 agonist α,β-MeATP evoked a transient [Ca2+]i increase and a reversible decrease in light transmission; both responses required external Ca2+ and the nucleotidase apyrase. The transmission response was due to shape change only, verified by scanning electron microscopy and insensitivity to Reopro, a GPIIbIIIa antagonist. α,β-MeATP stimulated smaller shape changes than ADP, however P2X1 responses had a lifespan of 50 nM was required for detectable shape change. Overlap of concentration-response relationships for α,β-MeATP-evoked [Ca2+]i and shape change suggests that other second messengers are not involved. Therefore, the physiological P2X1 agonist ATP can contribute to platelet activation, in contrast to its previously described inhibitory action at metabotropic platelet purinoceptors.

ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X1, P2Y1 and P2TAC. Given the low potency of ADP at P2X1 receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re-examined the activation of P2X1 receptors by ADP following HPLC and enzymatic purification. Native P2X1 receptor currents in megakaryocytes were activated by α,β-meATP (10 μM) and commercial samples of ADP (10 μM), but not by purified ADP (10–100 μM). Purified ADP (up to 1 mM) was also inactive at recombinant human P2X1 receptors expressed in Xenopus oocytes. Purification did not modify the ability of ADP to activate P2Y receptors coupled to Ca2+ mobilization in rat megakaryocytes. In human platelets, P2X1 and P2Y receptor-mediated [Ca2+]i responses were distinguished by their different kinetics at 13°C. In 1 mM Ca2+ saline, α,β-meATP (10 μM) and commercial ADP (40 μM) activated a rapid [Ca2+]i increase (lag time 0.5 s) through the activation of P2X1 receptors. Hexokinase treatment of ADP shifted the lag time by ∼2 s, indicating loss of the P2X1 receptor-mediated response. A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X1 receptors, whereas ADP is a selective agonist at metabotropic (P2Y1 and P2TAC) receptors. British Journal of Pharmacology (2000) 131, 108–114; doi:10.1038/sj.bjp.0703517

Pharmacological investigations during the drug discovery and development process are broadly divided into three distinct categories: primary pharmacodynamic, secondary pharmacodynamic, and safety pharmacological studies. Secondary pharmacodynamic studies form an important aspect of pharmacological investigations during the drug discovery process. This article discusses drivers for secondary pharmacodynamic studies, different strategic approaches to such studies, and the impact of pharmacological profiling on the drug discovery process. Keywords: secondary pharmacodynamics; drug discovery; drug development; pharmacological profiling; medicine–research

Purine nucleotides acting through P2 receptors play key roles in platelet signaling. The P2X1 receptor is an adenosine triphosphate (ATP)-gated ion channel that mediates a rapid calcium influx signal, but can also synergize with subsequent adenosine diphosphate (ADP)-evoked P2Y1 receptor-mediated responses and thus may contribute to platelet activation during hemostasis. Recent studies have shown that P2X1 receptors contribute to the formation of platelet thrombi, particularly under conditions of high shear stress. Based on intracellular Ca2+ measurements a previous report has suggested that a splice variant of the P2X1 receptor, P2X1del, is expressed in platelets and, in contrast to the full-length P2X1WT receptor, is activated by ADP. In the present study we show that the P2X1del receptor fails to form functional ion channels and is below the limit of detection in human platelets. Furthermore, ADP does not contribute to the rapid ionotropic P2X receptor-mediated response in platelets. These results support the notion that ATP is the principal physiologic agonist at P2X1 receptors and that it plays a role in the activation of platelets. (Blood. 2003;102:3646-3651)