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BJP British Journal of Pharmacology DOI:10.1111/bph.12943 www.brjpharmacol.org REVIEW Correspondence David J Grieve, Centre for Experimental Medicine, Queens University Belfast, Institute of Clinical Science Block A, Grosvenor Road, Belfast BT12 6BA, UK E-mail: d.grieve@qub.ac.uk Selective targeting of glucagon-like peptide-1 signalling as a novel therapeutic approach for cardiovascular disease in diabetes Commissioning Editor: Barbara McDermott Received 13 June 2014 Revised 21 August 2014 Accepted 14 September 2014 Mitchel Tate1, Aaron Chong1, Emma Robinson1, Brian D Green2 and David J Grieve1 Centre for Experimental Medicine, Queens University Belfast, Belfast, UK, and 2Institute for Global Food Security, Queens University Belfast, Belfast, UK Glucagon-like peptide-1 (GLP-1) is an incretin hormone whose glucose-dependent insulinotropic actions have been harnessed as a novel therapy for glycaemic control in type diabetes Although it has been known for some time that the GLP-1 receptor is expressed in the CVS where it mediates important physiological actions, it is only recently that specific cardiovascular effects of GLP-1 in the setting of diabetes have been described GLP-1 confers indirect benefits in cardiovascular disease (CVD) under both normal and hyperglycaemic conditions via reducing established risk factors, such as hypertension, dyslipidaemia and obesity, which are markedly increased in diabetes Emerging evidence indicates that GLP-1 also exerts direct effects on specific aspects of diabetic CVD, such as endothelial dysfunction, inflammation, angiogenesis and adverse cardiac remodelling However, the majority of studies have employed experimental models of diabetic CVD and information on the effects of GLP-1 in the clinical setting is limited, although several large-scale trials are ongoing It is clearly important to gain a detailed knowledge of the cardiovascular actions of GLP-1 in diabetes given the large number of patients currently receiving GLP-1-based therapies This review will therefore discuss current understanding of the effects of GLP-1 on both cardiovascular risk factors in diabetes and direct actions on the heart and vasculature in this setting and the evidence implicating specific targeting of GLP-1 as a novel therapy for CVD in diabetes Abbreviations ANP, atrial natriuretic peptide; CAD, coronary artery disease; CVD, cardiovascular disease; DPP-4, dipeptidyl peptidase-4; eNOS, endothelial NOS; GLP-1, glucagon-like peptide-1; hs-CRP, high sensitivity C-reactive protein; ICAM-1, intercellular adhesion molecule-1; MI, myocardial infarction; PAI-1, plasminogen activator inhibitor-1; STZ, streptozotocin; T1DM, type diabetes mellitus; T2DM, type diabetes mellitus; TLR, toll-like receptor; UKPDS, United Kingdom Prospective Diabetes Study; VCAM-1, vascular cell adhesion molecule-1 â 2014 The British Pharmacological Society British Journal of Pharmacology (2015) 172 721736 721 BJP M Tate et al Tables of Links TARGETS LIGANDS GPCRsa Enzymese ACh 1-adrenoceptor Akt (PKB) Adiponectin Leptin 2-adrenoceptor DPP-4 Alogliptin Linagliptin GLP-1 receptor eNOS ANP Liraglutide Ion channelsb ERK1/2 Byetta Lixisenatide KATP channel MMP-2 cAMP Metformin Catalytic receptorsc MMP-9 Exenatide Nitric oxide (NO) Toll-like receptor-2 p38-MAPK Exendin-4 Rosiglitazone Toll-like receptor-4 p42-MAPK Glimepiride Saxagliptin Other protein target p44-MAPK Glipizide Sitagliptin TNF- PI3K GLP-1 Stromal cell-derived factor-1 Transportersd PKA GLP-1(9-36) VCAM-1 Ca2+ ATPase Src Glyburide VEGF-A ICAM-1 Victoza IL-6 Vildagliptin Insulin These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http:// www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b,c,d,eAlexander et al., 2013a,b,c,d,e) Introduction The prevalence of type diabetes mellitus (T2DM) is increasing alarmingly with the 2013 figure of 382 million estimated to rise to 592 million by 2035 (International Diabetes Federation, 2014) A change in lifestyle coupled with an increase in obesity has led to a global epidemic, with diabetics typically carrying a fivefold greater mortality risk as a result of cardiovascular disease (CVD) compared with non-diabetics (Stamler et al., 1993), and coronary artery disease (CAD) being the leading underlying cause (Bertoni et al., 2004) It is well established that hyperglycaemia plays a central role in development and progression of CVD associated with diabetes (Nathan, 1996) Indeed, two long-term clinical trials, the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications study and the United Kingdom Prospective Diabetes Study (UKPDS), have demonstrated that intensive glucose-lowering strategies are effective in markedly reducing the incidence of microvascular (e.g retinopathy, nephropathy) and macrovascular (e.g CAD, stroke) complications in both type diabetes mellitus (T1DM) and T2DM (Holman et al., 2008), although several similar large-scale trials have reported limited benefits (Action to Control Cardiovascular Risk in Diabetes Study Group, 2008; Duckworth et al., 2009; Ginsberg, 2011) Nonetheless, there remains a significant incidence of CVD even in optimally treated diabetic patients, so it is clear that more effective strategies are required In this regard, the incretin peptide hormone, glucagon-like peptide-1 (GLP-1), has received considerable recent attention The incretin effect is responsible for augmenting insulin secretion following nutrient ingestion and GLP-1 together 722 British Journal of Pharmacology (2015) 172 721736 with its sister hormone, gastrointestinal peptide, account for up to 60% of post-prandial insulin secretion, leading to rapid blood glucose reduction (Nauck et al., 1986; Drucker et al., 1987) Furthermore, they possess an inherent ability to reduce glucagon secretion (Kreymann et al., 1987), delay gastric emptying (Nọslund et al., 1998a) and promote satiety (Flint et al., 1998) The metabolic actions of GLP-1 are mediated by GLP-1 receptor activation and stimulation of cAMP and several downstream kinases, including ERK1/2, PI3K and PKA Under physiological conditions, GLP-1 has a short halflife (2 min) as it is rapidly degraded by its endogenous inhibitor, dipeptidyl peptidase-4 (DPP-4) (Deacon et al., 1995), resulting in cleavage of two amino acids from native GLP-1(7-36) to produce GLP-1(9-36), which acts as a weak GLP-1 receptor antagonist lacking insulinotropic activity (Green et al., 2004) However, emerging evidence suggests that metabolically inactive GLP-1(9-36) may itself be an important signalling molecule (Ban et al., 2010; Gardiner et al., 2010) More detailed information on GLP-1 biology and signalling is provided by recent review articles (Grieve et al., 2009; Donnelly, 2012; Pabreja et al., 2013) The unique ability of GLP-1 to promote insulin secretion in a glucose-dependent manner has been harnessed for treatment of T2DM, with GLP-1 receptor agonists resistant to DPP-4 (exenatide, Byettađ; liraglutide, Victozađ), and DPP-4 inhibitors (e.g sitagliptin, vildagliptin) now widely used for effective glycaemic control Interestingly, it is well recognized that GLP-1 exerts wide-ranging extra-pancreatic actions occurring independently of its established metabolic effects Indeed, GLP-1 signalling is reported to play several important roles in the CVS in both health and disease (Grieve et al., 2009), although it appears that the GLP-1 receptor may not GLP-1 and cardiovascular disease in diabetes be as widely expressed as previously thought For example, recent work suggests that cardiac GLP-1 receptor expression may be localized to atrial tissue, sinoatrial node and vasculature, with some species variation (Kim et al., 2013; Pyke et al., 2014; Richards et al., 2014), and that earlier reports of more ubiquitous expression may be questionable because of poor antibody selectivity and sensitivity (Panjwani et al., 2012) Nonetheless, it is clear that GLP-1 exerts important cardiovascular actions, although it is only recently that its effects in the setting of diabetes, a condition synonymous with micro/ macrovascular complications, have been explored This is clearly important because of the large number of patients receiving GLP-1-based therapies, in which its cardiovascular actions are largely unknown This review will therefore discuss the current understanding of the effects of GLP-1 on both cardiovascular risk factors in diabetes and direct actions on the heart/vasculature in this setting and the evidence implicating specific targeting of GLP-1 as a novel therapy for CVD in diabetes, with a primary focus on the role of GLP-1 receptor agonists More detailed discussion of the pleiotropic actions of DPP-4 inhibitors in this setting is provided by recent review articles specifically focused on this important aspect of cardiovascular GLP-1 signalling (Scheen, 2013; Aroor et al., 2014) Influence of GLP-1 on cardiovascular risk factors in diabetes BP and hypertension Increased BP is an established risk factor for CVD in both normoglycaemia and T2DM (Turner et al., 1998; Vasan et al., 2001) Notably, therapeutic reductions in BP and circulating glucose have an additive effect in decreasing cardiovascular complications in T2DM patients, as highlighted by the UKPDS (Stratton et al., 2006) Indeed, in an experimental setting, chronic GLP-1 infusion inhibits development of hypertension in Dahl salt-sensitive rats, as well as reducing cardiac fibrosis and hypertrophy, effects which appear to occur via a natriuretic/diuretic mechanism independently of blood glucose (Yu et al., 2003), suggesting that GLP-1 may confer additional benefits which could be harnessed for the treatment of hypertension associated with T2DM Consistent with an indirect BP-lowering effect, it was recently reported that liraglutide-stimulated reduction of angiotensin IIinduced hypertension in mice was blocked by the natriuretic peptide receptor antagonist, anantin, in a GLP-1 receptordependent manner, but unaltered by the NOS inhibitor, NGmonomethyl-L-arginine, and that liraglutide induced rapid increases in atrial natriuretic peptide (ANP) secretion both in vivo and in isolated perfused hearts, suggesting that observed BP reduction occurred at least partly via direct activation of cardiac ANP (Kim et al., 2013) Importantly, in the context of diabetes, the GLP-1 mimetic, exendin-4, inhibited development of both spontaneous and high salt-induced hypertension in obese db/db mice via beneficial actions on renal sodium handling (Hirata et al., 2009) Furthermore, it was recently reported that treatment of insulin-resistant Zucker rats with the DPP-4 inhibitor, linagliptin, for weeks reduced BP and improved diastolic function (Aroor et al., 2013) BJP Interestingly, although chronic administration of GLP-1 may prevent development of hypertension, it is widely reported that acute GLP-1 exposure is associated with increased BP and heart rate, which predisposes to CVD For example, acute infusion of GLP-1(7-36) increased systolic/ diastolic BP and heart rate in both normal and insulindeficient streptozotocin (STZ)-induced T1DM rats (Barragỏn et al., 1994), with the same group reporting that exendin-4induced increases in BP and heart rate were reversed by the GLP-1 receptor antagonist, exendin(9-39) (Barragỏn et al., 1996), suggesting that these effects occurred via an insulinindependent mechanism but involving GLP-1 receptor activation Although similar increases in BP and heart rate after short-term GLP-1 administration have been reported in various experimental models (Grieve et al., 2009), the data from clinical studies are less clear For example, GLP-1 infusion in a small number of T2DM patients with or without CAD for 105 and 48 h, respectively, had no effect on heart rate or systolic/diastolic BP (Toft-Nielsen et al., 1999; Nystrửm et al., 2004), whereas 48 h GLP-1 infusion in patients with ischaemic heart failure (Halbirk et al., 2010) and treatment of T2DM patients with an exendin-transferrin fusion protein or exenatide for or 10 days, respectively (Kothare et al., 2008; Gustavson et al., 2011), resulted in elevated heart rate and diastolic BP However, the data from longer-term GLP-1 clinical trials are more consistent, with the majority reporting decreased BP and minimal effects on heart rate For example, the Liraglutide Effect and Action in Diabetes (LEAD)-4 study, investigating 26 week liraglutide treatment in combination with metformin in T2DM patients, reported a modest reduction in systolic BP of mmHg compared with placebo (1.1 mmHg) (Zinman et al., 2009) Similarly, the LEAD-2 study, assessing liraglutide combination therapy, reported a 23 mmHg decrease in systolic BP versus a small increase (0.4 mmHg) in the glimepiride control group (Nauck et al., 2009) Furthermore, a 30 week trial comparing once-weekly versus twice-daily exenatide injection in drugnaùve T2DM patients observed a significant decrease in systolic/diastolic BP compared with baseline (Drucker et al., 2008), while another 20 week trial in obese patients reported reduced systolic/diastolic BP in response to liraglutide, which persisted for the year follow-up period (Astrup et al., 2012) Interestingly, meta-analysis of six clinical trials comprising 2171 T2DM patients found that exenatide treatment for months produced maximal systolic BP reduction in individuals with abnormally high baseline levels, whereas no effects were observed in normotensive subjects (Okerson et al., 2010) It should be noted that BP reduction is positively correlated with weight loss (Neter et al., 2003), so it is possible that the observed changes after chronic GLP-1 treatment may occur secondary to its metabolic effects However, although beneficial effects of GLP-1 on body weight are associated with improved hypertension, it is clear that this cannot solely account for its vascular effects as several studies have reported a BP reduction prior to weight loss For example, a combined meta-analysis of three 26 week liraglutide trials reported decreased BP after only weeks, while maximal weight loss did not occur until weeks (Gallwitz et al., 2010) Indeed, heart rate, which is not linked to body weight, was increased by chronic administration of both liraglutide and exenatide in T2DM patients in parallel with reduced systolic BP (Garber British Journal of Pharmacology (2015) 172 721736 723 BJP M Tate et al et al., 2009; Gill et al., 2010) Interestingly, it was recently reported that GLP-1 secretory function increases with age and is negatively correlated with systolic BP, suggesting that this may represent an adaptive response (Yoshihara et al., 2013) Dyslipidaemia The pathophysiology of diabetes is commonly considered largely in terms of associated hyperglycaemia However, it is increasingly apparent that dyslipidaemia is equally important and represents a significant risk factor for CVD in diabetic patients (Reiner et al., 2011) It is likely that impaired insulin sensitivity contributes to dyslipidaemia in T2DM, which is associated with reduced GLP-1 secretion Indeed, in addition to their established glycaemic actions, GLP-1 receptor activation and DPP-4 inhibition are reported to improve lipid profiles in both experimental and clinical diabetes For example, short-term infusion of GLP-1 in normoglycaemic Syrian golden hamsters decreased lipid absorption and triglyceride levels, an effect potentiated by oral glucose (Hein et al., 2013), suggesting that its incretin action may inhibit intestinal production of chylomicrons, which are strongly linked to atherosclerosis (Nakano et al., 2008) Similarly, circulating triglycerides and fat pad mass in rats with diet-induced obesity were reduced after week treatment with liraglutide (Madsen et al., 2010), while 40 day administration of exendin-4 ameliorated systemic and cardiac insulin resistance and dyslipidaemia in both genetic KKAy and dietinduced T2DM mouse models (Monji et al., 2013) Furthermore, chronic GLP-1 receptor activation with both the GLP-1 analogue, CNTO3649, and exendin-4 in apolipoprotein E3-Leiden transgenic mice, which develop severe hypercholesterolaemia after high-fat feeding, resulted in reduced very-low-density lipoprotein (VLDL)-triglyceride and apolipoprotein B synthesis in parallel with decreased hepatic triglyceride, cholesterol and phospholipids and lipogenesis gene expression (Parlevliet et al., 2012) The longacting DPP-4 inhibitor, teneligliptin, is also reported to decrease circulating triglyceride and free fatty acid levels in insulin-resistant Zucker fatty rats after week treatment (Fukuda-Tsuru et al., 2012) Importantly, the majority of clinical studies have also demonstrated beneficial outcomes of GLP-1 administration on lipid metabolism in T2DM, such as reduced circulating triglycerides and low-density lipoprotein (LDL) cholesterol (Flock et al., 2007; Drucker et al., 2008; Tremblay et al., 2011), although one study in which patients were treated with exenatide for 24 weeks reported a similar plasma lipid profile versus controls (Moretto et al., 2008) For example, decreased circulating levels of atherogenic triglyceride-rich lipoproteins were observed following week vildagliptin monotherapy in drug-naùve T2DM patients, characterized by specific reductions in total plasma and chylomicron triglycerides, together with apolipoprotein B-48 and cholesterol in the chylomicron subfraction (Matikainen et al., 2006) Furthermore, the LEAD-4 study found that 26 week liraglutide treatment in combination with metformin and rosiglitazone decreased circulating LDL cholesterol, triglycerides and free fatty acids in T2DM patients compared with placebo controls, although it is interesting to note that these changes were greater in response to low-dose treatment (Zinman et al., 2009) Indeed, similar results are reported for DPP-4 inhibitors, which 724 British Journal of Pharmacology (2015) 172 721736 produce much lower circulating levels of GLP-1 For example, twice-daily sitagliptin led to a significant reduction in circulating triglycerides and free fatty acids in a large number of T2DM patients compared with placebo and glipizide control groups, despite similar decreases in fasting plasma glucose and HbA1c levels (Scott et al., 2007) Interestingly, a single injection of exenatide was shown to attenuate postprandial increases in triglycerides, apolipoprotein B-48 and CIII/ remnant lipoprotein cholesterol for up to h in patients with impaired glucose tolerance and recent-onset T2DM (Schwartz et al., 2010), suggesting that such lipid profile benefits may not be explained solely by chronic changes in body weight, glucose levels and insulin resistance Indeed, it was recently reported that exendin-4 completely reverses hepatic steatosis in mice fed a high-fat diet via a GLP-1 receptor-dependent mechanism resulting in reduced numbers/size of circulating VLDL-triglyceride and VLDL-apolipoprotein B particles (Parlevliet et al., 2012), suggesting that GLP-1 may exert direct effects on dyslipidaemia in diabetes Obesity Although it is well known that obesity significantly increases the risk of T2DM (Willett et al., 1999), and both are independent risk factors for CVD (Hubert et al., 1983), many established diabetes therapies, including sulfonylureas and thiazolidinediones, may increase body weight However, GLP-1 reduces body weight because of beneficial effects on glucagon secretion, gastric emptying and satiety (Kreymann et al., 1987; Flint et al., 1998; Nọslund et al., 1998a), so it seems likely that impaired GLP-1 secretion observed in nondiabetic obese individuals (Holst et al., 1982; Nọslund et al., 1998b) may at least partly account for their increased body weight Indeed, weight loss improves the postprandial GLP-1 response in severely obese patients (Verdich et al., 2001), suggesting that the two are interlinked Furthermore, a 20 week treatment with liraglutide was reported to cause significant weight loss in obese individuals and to reduce the incidence of prediabetes (Astrup et al., 2009), confirming an apparent role for GLP-1 in weight control which may be harnessed for therapeutic benefit This assertion is supported by the LEAD trials which have consistently reported a reduction in body weight in T2DM patients following liraglutide treatment (Moretto et al., 2008; Buse et al., 2009; Garber et al., 2009; Nauck et al., 2009; 2013; Zinman et al., 2009) For example, in the LEAD-2 trial, 26 week combination therapy of liraglutide with metformin in T2DM patients resulted in increased weight loss compared with metformin alone (Nauck et al., 2013) Importantly, weight loss associated with both liraglutide and exenatide treatment is reported to be linked to improved cardiovascular risk factors, such as HbA1c and BP, and to persist for at least years (Klonoff et al., 2007; Astrup et al., 2009; 2012), highlighting important benefits of GLP-1 which may not be related to its insulinotropic actions The long-term effects of GLP-1 on weight loss may be particularly important as conventional weight loss is typically poorly maintained in T2DM patients Despite GLP-1 receptor agonists promoting weight loss in both diabetic and non-diabetic obese subjects, DPP-4 inhibitors appear to be weight-neutral (Amori et al., 2007), suggesting that GLP-1 receptor agonists may exert direct gastrointestinal effects in addition to improving insulin resistance (Rask et al., 2001), although this GLP-1 and cardiovascular disease in diabetes could simply be due to differences in circulating GLP-1 levels However, postprandial GLP-1 levels are reported to be increased immediately after gastric bypass surgery, despite patients remaining obese, indicating that GLP-1 may regulate appetite and food intake directly (Morinigo et al., 2006) Indeed, in T2DM patients, GLP-1 promotes satiety, thereby reducing energy consumption (Gutzwiller et al., 1999), while in healthy individuals i.v administration of GLP-1(7-36) slows gastric emptying in a dose-dependent manner (Nauck et al., 1997) GLP-1 and vascular disease Vascular function Impaired endothelial and vascular function are established as key initiating factors underlying the development of microvascular and macrovascular complications associated with diabetes Indeed, it has been known for some time that native GLP-1(7-36) induces ex vivo dose-dependent vasodilatation in a number of isolated rodent vessels, including aorta (Golpon et al., 2001; Green et al., 2008), pulmonary artery (Richter et al., 1993; Golpon et al., 2001), femoral artery (Nystrửm et al., 2005) and mesenteric artery (Ban et al., 2008), although several different mechanisms have been proposed For example, some studies indicate that the vasorelaxant actions of GLP-1 are dependent upon endothelium-derived NO (Golpon et al., 2001; Ban et al., 2008; Gaspari et al., 2011), whereas others have proposed endothelium-independent mechanisms involving mediators such as KATP channels, cAMP and 2-adrenoceptor activation (Nystrửm et al., 2005; Gardiner et al., 2008; Green et al., 2008) Interestingly, although several studies suggest that the vascular actions of GLP-1 are dependent upon the GLP-1 receptor (Gaspari et al., 2011; Chai et al., 2012), it appears that they may also be mediated, at least partly, by its truncated metabolite, GLP1(9-36), which induces dose-dependent relaxation in both isolated mouse mesenteric artery (Ban et al., 2008) and rat aorta (Green et al., 2008) It should be noted that although the synthetic GLP-1 mimetic, exendin-4, exerts similar actions in rat aorta (Golpon et al., 2001; Green et al., 2008), they are of reduced magnitude compared with GLP-1(7-36) and are absent in mouse mesenteric artery (Ban et al., 2008) Importantly, the vasorelaxant actions of GLP-1 are also reported in vivo For example, systemic administration of GLP-1(7-36) by both bolus dose and short-term infusion in rats induced hindquarters vasodilatation (Gardiner et al., 2010) Interestingly, however, GLP-1 promoted vasoconstriction in both mesenteric and renal arteries, while exendin-4 exerted similar vasoconstriction in mesenteric artery but induced vasodilatation in both hindquarters and renal artery (Gardiner et al., 2008), suggesting differential vascular effects Indeed, a similar study demonstrated that GLP-1(7-36) infusion acutely increased muscle microvascular blood volume in the absence of changes in microvascular blood flow velocity or femoral blood flow, in association with increased plasma NO, muscle insulin clearance/uptake, hindlimb glucose extraction and muscle interstitial oxygen saturation (Chai et al., 2012) It should be noted that in contrast to the ex vivo studies, GLP-1(9-36) failed to modulate vascular function in BJP rats in vivo when given as either a bolus dose or via short-term infusion, which together with the fact that DPP-4 inhibitors prolonged the vascular actions of native GLP-1(7-36) in this setting (Gardiner et al., 2010), suggest that the actions of this inactive metabolite may not be significant in vivo Importantly, it appears that the vascular effects of GLP-1 are also evident in the setting of diabetes, where they are reported to promote beneficial actions Chronic treatment of STZ/nicotinamide T1DM rats with either GLP-1(7-36) or exendin-4 was shown to prevent endothelial dysfunction in parallel with reduction of blood glucose (ệzyazgan et al., 2005), effects which may be mediated via activation of endothelial NOS (eNOS) (Goyal et al., 2010) Although similar studies have not been performed in the setting of overt T2DM, it was recently reported that chronic treatment of insulin-resistant Zucker rats with the DPP-4 inhibitor, linagliptin, resulted in reduced hypertension in parallel with increased expression of total/phosphorylated eNOS (Aroor et al., 2013) Furthermore, high-fat fed apolipoprotein E-deficient mice treated with a different DPP-4 inhibitor, desfluoro-sitagliptin, demonstrated attenuation of endothelial dysfunction in parallel with eNOS activation and improved glucose tolerance (Matsubara et al., 2012) Interestingly, however, endothelial dysfunction in rat femoral artery induced by short-term triglyceride exposure was not affected by exendin-4 (Nathanson et al., 2009), suggesting that the reported in vivo protective actions may occur via indirect mechanisms In this regard, it is important to note that the vascular actions of GLP-1 in diabetes are likely to occur, at least partly, secondary to stimulation of insulin, which induces vascular relaxation via Ca2+-dependent activation of eNOS (Han et al., 1995; Kahn et al., 1998) In addition to the data supporting important vascular actions of GLP-1 in experimental diabetes, several studies have reported beneficial functional effects in the clinical setting For example, in T2DM patients with stable CAD, acute GLP-1 administration improved brachial artery flowmediated vasodilatation, an effect not observed in healthy individuals (Nystrửm et al., 2004) Comparable effects were observed in insulin-resistant patients with obesity-related metabolic syndrome, where acute treatment with GLP-1(736) enhanced insulin-mediated forearm blood flow responses to both ACh and sodium nitroprusside in the absence of changes in forearm glucose extraction/uptake, while GLP1(9-36) did not affect vascular function (Tesauro et al., 2013) Similarly, in patients with T1DM, brachial artery endothelial dysfunction induced by acute blood glucose modulation was counteracted by simultaneous infusion of GLP-1 (Ceriello et al., 2013) Interestingly, GLP-1-induced enhancement of endothelium-dependent peripheral vasodilatation observed in non-diabetic individuals is differentially modulated by sulphonylureas, with glyburide abolishing GLP-1-induced ACh-mediated responses which are unaltered by glimepiride (Basu et al., 2007) Furthermore exenatide, which is commonly used for hyperglycaemic control in T2DM, also increased postprandial endothelial function assessed by peripheral arterial tonometry in patients with recent-onset disease when given as a single dose, largely secondary to a reduction in circulating triglycerides (Koska et al., 2010), although chronic treatment for months in obese prediabetic patients had no additional effect when compared with British Journal of Pharmacology (2015) 172 721736 725 BJP M Tate et al those receiving metformin (Kelly et al., 2012) While it appears that clinical GLP-1 administration exerts acute vascular effects in T2DM, data on its chronic actions in this setting are variable T2DM patients who received exenatide for a period of months as an adjunct to standard metformin therapy demonstrated improved brachial artery flowmediated dilatation, indicated by elevated peak dilatation and shear rate which are reflective of improved macrovascular and microvascular function respectively (Irace et al., 2013) Notably, enhanced vasodilatation in exenatide-treated patients in this study was significantly greater than that observed in those receiving glimepiride as an add-on to metformin therapy Furthermore, liraglutide treatment for 12 weeks in T2DM patients well controlled on metformin monotherapy resulted in an improvement in both circulating markers of vascular function [asymmetric dimethylarginine, plasminogen activator inhibitor-1 (PAI-1), E-selectin] and retinal microvascular endothelial function (Forst et al., 2012) However, a similar study in a small number of severely obese T2DM patients chronically treated with GLP-1 receptor agonists for months reported that neither exenatide or liraglutide had any effect on brachial artery endothelial-dependent flow-mediated dilation (Hopkins et al., 2013), indicating potential for other confounding factors in this setting which may need to be considered Furthermore, week treatment with sitagliptin or alogliptin significantly reduced flowmediated dilatation in male T2DM patients (Ayaori et al., 2013), suggesting that chronically increased physiological levels of GLP-1 may exert unfavourable vascular actions, although it is possible that this could be a class-specific effect of DPP-4 inhibitors warranting further investigation Inflammation and atherosclerosis It is well known that the incidence and progression of endothelial dysfunction is exacerbated in T2DM, secondary to established risk factors, such as insulin resistance, dyslipidaemia and hyperglycaemia Endothelial dysfunction in this setting is characterized by an elevation in circulating adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and an increased propensity to develop atherosclerosis which is typified by inflammatory cell infiltration and plaque formation (Van Gaal et al., 2006) Interestingly, several recent clinical and experimental studies appear to indicate that GLP-1 exerts both anti-inflammatory and anti-atherogenic actions For example, GLP-1 treatment in T2DM patients is associated with beneficial effects on a number of established CVD biomarkers, including high sensitivity C-reactive protein (hs-CRP) and PAI-1, which are important in atherosclerosis development (Haffner, 2006) Similarly, 14 week treatment of T2DM patients with liraglutide resulted in significantly reduced PAI-1 levels, and a dose-dependent decrease in plasma hs-CRP levels (Vilsbứll et al., 2007; Courrốges et al., 2008), an effect that was also observed after 26 week treatment with exenatide (Bergenstal et al., 2010) and was over and above that seen in patients treated with insulin glargine (Diamant et al., 2010) Importantly, the beneficial effects of exenatide on circulating hs-CRP appear to persist at year treatment in T2DM patients receiving standard metformin therapy, resulting in reduced levels of both hs-CRP and leptin (Bunck et al., 2010) DPP-4 inhibitors seem 726 British Journal of Pharmacology (2015) 172 721736 to exert similar effects as T2DM patients receiving sitagliptin for months also demonstrated significant reductions in plasma hs-CRP, together with VCAM-1 and associated albuminuria, which may attenuate glucose excursion and inhibit vascular inflammation (Horvỏth et al., 2009; Hattori, 2010) Interestingly, a recent study demonstrated that cessation of exenatide treatment resulted in the reversal of benefits on circulating hs-CRP within months (Varanasi et al., 2011) It should be noted that although these studies support the suggestion that GLP-1 may protect against inflammation and atherosclerosis in the clinical setting, it is not possible to draw clear conclusions because of the absence of longer-term studies specifically assessing effects on disease development Interestingly, a recent study has reported a positive correlation between circulating GLP-1 levels and CAD in both diabetic and non-diabetic patients undergoing angiography because of typical or atypical chest pain, highlighting the possibility that GLP-1 may exert detrimental effects in this setting (Piotrowski et al., 2013) Nonetheless, the majority of clinical data are broadly supportive of the anti-inflammatory actions of GLP-1, which persist for up to 12 weeks in obese T2DM patients after a single exenatide injection (Chaudhuri et al., 2011) Indeed, in this study, GLP-1 was associated with a specific reduction of several inflammatory mediators, including TNF-, toll-like receptor-2 (TLR-2) and TLR-4, in parallel with suppression of NF-B signalling and MMP-9 activity, which are key initiating factors of atherosclerosis Furthermore, in obese T2DM patients, week liraglutide treatment is reported to decrease levels of the inflammatory macrophage activation molecule, sCD163, and pro-inflammatory cytokines, TNF- and IL-6, while increasing levels of the anti-inflammatory adipokine, adiponectin (Hogan et al., 2014) Importantly, these clinical observations are supported by a number of experimental studies which have specifically assessed the effects of GLP-1 on development and progression of atherosclerosis For example, continuous infusion of exendin-4 in both wild-type and apolipoprotein E-deficient normoglycaemic mice was reported to decrease monocyte adhesion and development of atherosclerotic lesions in thoracic aorta, effects proposed to occur via cAMP/PKA-dependent suppression of inflammation (Arakawa et al., 2010) These findings were confirmed by a different group who demonstrated reduced aortic macrophage recruitment, foam cell formation and atherosclerotic lesion development in apolipoprotein E-deficient mice after GLP-1 infusion (Nagashima et al., 2011) Furthermore, chemokine-induced migration of CD4+ lymphocytes is inhibited by both GLP-1(7-36) and exendin-4 in a GLP-1 receptordependent manner (Marx et al., 2010), while liraglutide suppresses NF-B signalling in HUVECs and THP-1 monocyte adhesion in human aortic endothelial cells via downstream activation of several proinflammatory and cell adhesion molecules, including TNF-, VCAM-1 and E-selectin (Shiraki et al., 2012; Krasner et al., 2014) Indeed, liraglutide inhibits TNF- in human vascular endothelial cells and reduces hyperglycaemia-mediated PAI-1, ICAM-1 and VCAM-1 activation, which is associated with endothelial dysfunction and accelerated atherogenesis in T2DM (Liu et al., 2009) Furthermore, the DPP-4 inhibitor, des-fluoro-sitagliptin, is reported to exert cAMP-dependent anti-inflammatory actions in cultured human macrophages by increasing GLP-1 levels and to GLP-1 and cardiovascular disease in diabetes reduce atherosclerotic lesion formation in apolipoprotein E-deficient mice (Matsubara et al., 2012), while alogliptin inhibits vascular monocyte/macrophage recruitment and reduces atherosclerotic burden in high-fat diet-fed, LDL receptor-deficient mice in association with improvement of metabolic indices (Shah et al., 2011) Taken together, these experimental data clearly support an important role for GLP-1 in protecting against vascular inflammation and atherogenesis, effects which are borne out by the reported clinical benefits of GLP-1 treatment on circulating inflammatory mediators and CVD biomarkers However, it is evident that long-term studies specifically investigating effects on atherosclerotic disease development and progression are required to ascertain whether the apparent protective effects of GLP-1 under both normoglycaemic and diabetic conditions may translate to the clinical setting Angiogenesis Abnormal angiogenesis is a hallmark of CVD which is exacerbated by diabetes, with impaired neovascularization contributing significantly to progression of ischaemic disease associated with peripheral and coronary arteries Interestingly, it is becoming apparent that GLP-1 may modulate angiogenesis suggesting that such actions may underlie some of its reported beneficial cardiovascular effects For example, exendin-4 stimulates proliferation of human coronary artery endothelial cells in a GLP-1 receptor-dependent manner via downstream activation of eNOS, PKA and PI3K/Akt signalling (Erdogdu et al., 2010) and promotes in vitro HUVEC migration, ex vivo aortic sprouting angiogenesis and in vivo blood vessel formation in Matrigel plugs (Kang et al., 2013), while native GLP-1(7-36) stimulates in vitro angiogenesis in HUVECs via Akt, Src and PKC-dependent pathways (Aronis et al., 2013), suggesting that GLP-1 may directly modulate neovascularization Importantly, these effects appear to translate to the pathological situation, with several recent studies reporting that GLP-1 can promote the pro-angiogenic actions of mesenchymal stem cells in different disease settings Intracoronary artery delivery of GLP-1 eluting encapsulated human mesenchymal stem cells in a porcine model of experimental myocardial infarction (MI) resulted in improved left ventricular function and remodelling which was associated with increased infarct zone angiogenesis (Wright et al., 2012), while peri-adventitial treatment of porcine vein grafts with these cells inhibited neointima formation in parallel with accelerated adventitial angiogenesis (Huang et al., 2013) Furthermore, the addition of GLP-1 to encapsulated human mesenchymal cells significantly improved blood flow recovery and foot salvage in a mouse model of hindlimb ischaemia via increased capillary and arteriole formation secondary to paracrine activation of VEGF-A (Katare et al., 2013) Although the majority of work investigating the pro-angiogenic actions of GLP-1 has been performed in normoglycaemic models, a recent study reported similar beneficial effects in the setting of diabetes Impaired myocardial angiogenesis in STZ-treated T1DM rats and associated fibrosis and diastolic dysfunction were reversed by genetic deletion of DPP-4 or pharmacological inhibition with vildagliptin (Shigeta et al., 2012) Interestingly, this study identified DPP-4 as being membrane-bound and localized to the cardiac capillary endothelium with increased expression in diabetes, which together with a BJP report of increased binding affinity of GLP-1 to the coronary endothelium but not cardiomyocytes in isolated perfused T1DM rat hearts (Barakat et al., 2011), supports a key endothelial-specific role of GLP-1 in this setting Although these data provide supportive evidence for pro-angiogenic actions of GLP-1 in diabetes, it is clear that additional mechanistic studies are required using different CVD models in order to define its precise role Furthermore, it is important to assess the effects of GLP-1 therapy in diabetic patients in order to investigate whether the apparent pro-angiogenic effects of GLP-1 translate to the clinical setting and are of functional relevance GLP-1 and the diabetic myocardium The heart is one of the major organ targets of GLP-1 and an increasing number of studies have investigated the actions of native GLP-1(7-36), GLP-1 receptor agonists and DPP-4 inhibitors in the context of cardioprotection The majority of experimental studies have focused on the effects of GLP-1 in cardiac ischaemia and its apparent ability to protect against acute myocardial damage Indeed, it is well established that GLP-1 pretreatment and chronic DPP-4 inhibition reduce infarct size after experimental ischaemia in both small and large animal models, which is associated with increased survival and improved cardiac function (Bose et al., 2005; Ban et al., 2008; 2010; Timmers et al., 2009) Interestingly, a recent study employing a rabbit model of ischaemia/ reperfusion injury reported protective actions of transferrinstabilized GLP-1, when given both 12 h prior to ischaemia and immediately upon reperfusion, suggesting that GLP-1 may limit infarct size and contractile dysfunction directly, rather than by preconditioning the heart against ischaemia, as suggested by previous reports (Matsubara et al., 2011) In addition to its established beneficial actions against acute ischaemic myocardial damage, GLP-1 also confers protection against contractile dysfunction associated with experimental chronic post-MI remodelling, dilated cardiomyopathy and hypertensive heart failure (Nikolaidis et al., 2004a; Poornima et al., 2008; Liu et al., 2010), with similar results reported in the clinical setting in response to short-term GLP-1 treatment (Nikolaidis et al., 2004b; Sokos et al., 2006) Until recently, only limited data were available on the cardiac actions of GLP-1 in diabetes, but it is becoming increasingly apparent that GLP-1 also plays a key cardioprotective role in this setting This is important as it is well known that hyperglycaemia is associated with increased susceptibility to cardiac disease and poor outcomes in both humans and experimental models (Shiomi et al., 2003; Liu et al., 2005; Greer et al., 2006; Vergốs et al., 2007) Chronic DPP-4 inhibition with linagliptin improves obesity-related diastolic dysfunction in insulin-resistant Zucker rats, but has no effect on cardiomyocyte hypertrophy and fibrosis (Aroor et al., 2013) Indeed, exendin-4 has been reported to directly protect isolated rat cardiomyocytes from high glucoseinduced apoptosis via inhibition of endoplasmic reticulum stress and activation of sarcoplasmic reticulum Ca2+ ATPase 2a (Younce et al., 2013) GLP-1 also appears to protect against diabetic cardiomyopathy, which is defined as cardiac dysfunction in the absence of, or disproportionate to, associated British Journal of Pharmacology (2015) 172 721736 727 BJP M Tate et al hypertension and CAD and is characterized by marked collagen accumulation and impaired diastolic function (Bugger and Abel, 2014) Both GLP-1 receptor activation and DPP-4 inhibition attenuate development of cardiac dysfunction, extracellular matrix remodelling, cardiomyocyte hypertrophy and apoptosis in experimental models of T1DM and T2DM, with various mechanisms proposed including reduction of lipid accumulation, oxidative stress and myocardial inflammation, and modulation of the MMP-2/tissue inhibitor of MMP-2 axis, endoplasmic reticulum stress and microvascular barrier function (Shigeta et al., 2012; Liu et al., 2013; Monji et al., 2013; Picatoste et al., 2013; Wang et al., 2013) Furthermore, it appears that GLP-1 also confers infarctreducing actions in diabetes, which is associated with increased susceptibility to myocardial ischaemia For example, mice made diabetic by a combination of STZ Figure Summary of the cardiovascular actions of GLP-1 in diabetes GLP-1 exerts indirect cardiovascular benefits in diabetes secondary to its established metabolic actions and subsequent reduction of cardiovascular risk factors In addition, GLP-1 promotes direct cardiovascular benefits which confer protection against CVD and heart failure, the latter of which may occur via direct myocardial actions or secondary to reduced hypertension and coronary atherosclerosis 728 British Journal of Pharmacology (2015) 172 721736 GLP-1 and cardiovascular disease in diabetes injection and high-fat feeding and treated with the GLP-1 receptor agonist, liraglutide, prior to coronary artery ligation, demonstrated reduced infarct development and improved survival compared with those treated with the glucoselowering drug, metformin, suggesting that the observed effects occurred via direct actions on the heart and not secondary to reduced blood glucose (Noyan-Ashraf et al., 2009) Similar cardioprotective effects have been reported with DPP-4 inhibition in experimental diet-induced obesity (Huisamen et al., 2013), while the infarct-limiting effects of exendin-4 in mice with T2DM were shown to be mediated by cAMP-induced PKA activation (Ye et al., 2013) Interestingly, it has recently been suggested that the infarct-reducing actions of DPP-4 inhibitors may be glucose-dependent, as both sitagliptin and vildagliptin were found to only decrease infarct size in isolated rat hearts subjected to ischaemiareperfusion injury when they were perfused with elevated glucose concentrations mmol L1, with similar results observed in vivo in diabetic, but not normoglycaemic rats BJP (Hausenloy et al., 2013) This raises the intriguing possibility that glucose-lowering may counteract the cardioprotective actions of GLP-1 and explain why several large-scale clinical trials focused on intensive glucose control in T2DM have failed to demonstrate significant cardiovascular benefits (Giorgino et al., 2013) Furthermore, it appears that at least part of the observed beneficial actions of DPP-4 inhibitors against ischaemia-reperfusion injury may be mediated by the chemokine, stromal cell-derived factor in a GLP-1independent manner (Bromage et al., 2014) In addition to the experimental data highlighting a protective role for GLP-1 in the diabetic heart, importantly, a small number of studies have assessed its cardiac actions in patients with diabetes It has been known for some time that short-term GLP-1 treatment exerts beneficial effects in clinical heart failure in both normoglycaemic and diabetic patients For example, in a small number of heart failure patients (New York Heart Association class III/IV), week infusion with GLP-1 plus standard therapy improved left Figure Proposed mechanisms underlying the reported cardiovascular actions of GLP-1 Although it is well established that GLP-1 exerts several beneficial effects on the CVS relevant to diabetes, such as reduction of metabolic CVD risk factors, BP modulation, improved vascular function, decreased atherosclerosis, promotion of angiogenesis and attenuation of adverse cardiac remodelling, the precise mechanisms are yet to be established, although several pathways have been proposed which are the focus of further investigation EC, endothelial cell; ECM, extracellular matrix; SDF, stromal cell-derived factor 1; SR, sarcoplasmic reticulum British Journal of Pharmacology (2015) 172 721736 729 730 British Journal of Pharmacology (2015) 172 721736 NCT01243424 September 2018 CV, cardiovascular; MI, myocardial infarction; NCT, National Clinical Trial Time to CV death, non-fatal MI, non-fatal stroke and hospitalization for unstable angina pectoris 000 Linagliptin CARMELINA p.o., mg, once daily NCT00790205 NCT01897532 January 2018 December 2014 Time to first CV event Time to CV death, non-fatal MI, non-fatal stroke and hospitalization for unstable angina pectoris 300 14 000 Linagliptin CAROLINA Not specified Sitagliptin TECOS p.o., 50 or 100 mg, once daily NCT01394952 August 2019 Time to CV death, non-fatal MI or non-fatal stroke 9622 Dulaglutide REWIND s.c injection, 1.5 mg, once weekly NCT01179048 NCT01147250 January 2015 Lixisenatide 000 NCT01144338 ELIXA s.c injection, 20 g, once daily Time to first primary CV event October 2015 December 2017 Rate of CV death, non-fatal MI or non-fatal stroke Time to CV death, non-fatal MI or non-fatal stroke 340 14 000 Liraglutide LEADER s.c injection, 1.8 mg, once daily Exenatide EXSCEL s.c injection, mg, once weekly Expected end date Primary outcome(s) Drug Study name Administration Estimated patient enrollment Current long-term clinical trials of GLP-1-based therapies on cardiovascular outcomes ventricular ejection fraction and myocardial oxygen consumption compared with those receiving standard therapy alone, effects that were seen in both diabetic and nondiabetic patients (Sokos et al., 2006) Furthermore, a small non-randomized trial of 72 h GLP-1 infusion following primary angioplasty after acute MI led to improved cardiac function in both non-diabetic and diabetic patients which was still evident upon 120 day follow-up (Nikolaidis et al., 2004b) More recently, a larger randomized trial in patients presenting with ST-segment elevation MI reported that exenatide infusion for 15 prior to primary angioplasty continued until h post-reperfusion resulted in improved myocardial salvage at months although no functional benefits were observed (Lứnborg et al., 2012) Indeed, two current clinical trials are assessing the potential of using exenatide as a post-conditioning agent to reduce reperfusion injury following percutaneous coronary intervention (Effect of Additional Treatment With EXenatide in Patients With an Acute Myocardial Infarction, the EXAMI trial, NCT01254123; Pharmacological Postconditioning to Reduce Infarct Size Following Primary PCI, POSTCON II, NCT00835848) Interestingly, in patients with left ventricular diastolic dysfunction, DPP-4 activity in the coronary sinus and peripheral circulation is reported to be negatively correlated with diastolic function and increased by co-morbid diabetes (Shigeta et al., 2012), suggesting that reduced GLP-1 levels in diabetes may underlie the associated cardiac dysfunction Exenatide has also been found to modulate myocardial glucose transport and uptake in T2DM patients dependent upon the degree of insulin resistance (Gejl et al., 2012), although a similar study reported that GLP-1-induced increases in resting myocardial glucose uptake in lean individuals were absent in obese T2DM patients, with parallel studies in pigs suggesting that this was due to impaired p38-MAPK signalling (Moberly et al., 2013) Interestingly, a recent experimental study found that exendin-4 reduced contractile function and was unable to stimulate glucose utilization in normal rat hearts in the presence of fatty acids (Nguyen et al., 2013), despite previous reports of increased myocardial glucose uptake in response to GLP-1 in experimental myocardial ischaemia and dilated cardiomyopathy (Nikolaidis et al., 2005; Zhao et al., 2006; Bhashyam et al., 2010) Such findings highlight the need for detailed investigation of the effects of GLP-1 on altered myocardial metabolism in diabetic patients both with and without cardiac complications, in which the effects of GLP-1 may diverge Although these clinical and experimental data are clearly supportive of an important role for GLP-1 signalling in the diabetic heart, they are largely descriptive with limited focus on underlying mechanisms Previous studies in experimental models of heart failure have highlighted several pathways which may mediate the cardioprotective effects of GLP-1, including cAMP/PKA, PI3K/Akt, p44/p42MAPK, ERK1/2 (Bose et al., 2005; Timmers et al., 2009; Ravassa et al., 2011), together with suggestions of GLP-1 receptor-independent signalling (Nikolaidis et al., 2005; Sonne et al., 2008) However, the precise mechanisms underlying the apparent protective actions of GLP-1 in the diabetic heart, in which GLP-1 signalling is likely to be different, remain unknown and clearly need to be defined in order to fully assess the therapeutic potential of GLP-1 in this setting NCT identifier M Tate et al Table BJP Molecular insights into Kv11.1 (hERG) cardiotoxicity BJP Table Continued Therapeutic class TdPa Redfern categoryb Status in the United Statesa Cisapride Gastroprokinetic ++ Withdrawn Ranolazine Antianginal + Restricted Sotalol Antiarrhythmic ++ Restricted Thioridazine Antipsychotic ++ Restricted Name Chemical structure +, drugs with possible TdP risk and ++, drugs with known TdP risk Information on these compounds was retrieved from CredibleMedsđ available at: http://crediblemeds.org/ (accessed 10 April 2014) b Redfern categories for most compounds were derived from the literature (Redfern et al., 2003); they are 1: Class Ia and III antiarrythmics; 2: Withdrawn from market due to TdP; 3: Measurable incidence/numerous reports of TdP in humans; 4: Isolated reports of TdP in humans; 5: No reports of TdP in humans Redfern categories for these compounds were deduced according to the definition for different categories a Radioligand saturation assay Membrane aliquots containing 20 g of protein were incubated in a total volume of 100 L of incubation buffer (10 mM HEPES, 130 mM NaCl, 60 mM KCl, 0.8 mM MgCl2, mM EGTA, 10 mM glucose, 0.1% BSA, pH 7.4) at 25C for 120 to ensure that the equilibrium was reached at all concentrations of radioligand Total binding was determined at a range of concentrations (0.222 nM) of [3H]-dofetilide, whereas non-specific binding was determined at three different concentrations of radioligand in the presence of 10 M astemizole and analysed by linear regression Incubations were terminated by dilution with ice-cold wash buffer (25 mM Tris-HCl, 130 mM NaCl, 60 mM KCl, 0.8 mM MgCl2, 0.05 mM CaCl2, 0.05% BSA, pH 7.4) Separation of bound from free radioligand was performed by rapid filtration through Whatman GF/B filters (GE Healthcare, Buckinghamshire, UK) using a Brandel harvester (Brandel, MD, USA) Filters were subsequently washed six times with mL ice-cold wash buffer Filter-bound radioactivity was determined by scintillation spectrometry using a liquid Scintillation Analyzer (Tri-Carb 2900TR, PerkinElmer, Groningen, The Netherlands) after addition of 3.5 mL of Packard Emulsifier-Safe (PerkinElmer) and h extraction Radioligand association and dissociation assay Kinetic association experiments were performed by incubating membrane aliquots containing 20 g of protein in a total volume of 100 L incubation buffer at 25C for 120 with 16 different concentrations (0.716 nM) of [3H]-dofetilide The amount of radioligand bound to the receptor was measured at various time intervals during the incubation Incubations were terminated and samples were obtained and analysed as described in radioligand saturation assay Further traditional association and dissociation assays were performed as described previously (Yu et al., 2014) Radioligand displacement assay The [3H]-dofetilide binding assay for the Kv11.1 channel was performed as described previously (Yu et al., 2014) In short, membrane aliquots containing 20 g protein were incubated in a total volume of 100 L incubation buffer at 25C for 60 Radioligand displacement experiments were conducted using 11 concentrations of the competing ligand in the presence of nM [3H]-dofetilide At this concentration, total radioligand binding did not exceed 10% of the radioligand added to prevent ligand depletion Non-specific binding was determined in the presence of 10 M astemizole and represented approximately 15% of the total binding [3H]dofetilide did not bind to membranes prepared from empty HEK293 cells lacking the Kv11.1 channel (data not shown) Total binding was determined in the presence of incubation buffer and was set at 100% in all experiments, whereas nonspecific binding was set at 0% Incubations were terminated by dilution with ice-cold wash buffer Separation of bound from free radioligand was performed by rapid filtration British Journal of Pharmacology (2015) 172 940955 943 BJP Z Yu et al through a 96-well GF/B filter plate using a PerkinElmer Filtermate-harvester (PerkinElmer) Filters were subsequently washed 12 times with ice-cold wash buffer The filter-bound radioactivity was determined by scintillation spectrometry using the P-E 1450 Microbeta Wallac Trilux scintillation counter (PerkinElmer) after addition of 25 L Microscint (PerkinElmer) and h extraction Radioligand competition association assay The binding kinetics of unlabelled reference compounds were determined at 25C using the competition association assay according to a previously published method (Motulsky and Mahan, 1984) In a standard assay, three different concentrations (0.3-, one- and threefold of their Ki values) of unlabelled dofetilide, astemizole and E-4031 were tested We assessed the binding kinetics of all other unlabelled reference compounds in a simplified one-concentration competition association assay based on Guo et al (2012) The experiments were initiated by incubating membrane aliquots containing 20 g of protein in a total volume of 100 L of incubation buffer in the absence (control) or presence of a certain concentration of unlabelled ligands at 25C for 120 with nM [3H]dofetilide The amounts of radioligand bound to the receptor were measured at various time intervals during the incubation Incubations were terminated and samples were obtained and analysed as described in radioligand displacement assay Determination of logKW-C8 and logKW-IAM parameters by HPLC LogKW-C8 values were measured on a Supelcosil LC-ABZ, cm ì 4.6 mm, m column (Supelco, Bellefonte, PA, USA) according to a methodology described previously (Lombardo et al., 2001; Heitman et al., 2009) In short, retention times of the compounds were determined at three different methanol percentages These retention times were converted to k values by using the formula k = (tR t0) / tR in which tR is the retention time and t0 is the retention time of a non-delayed compound (pure methanol) The calculated logk values were plotted against the methanol concentrations and extrapolated to a 0% methanol situation yielding the logKW-C8 values for 15 reference compounds (intercept of Y axis) An isocratic method was applied to measure the logKW-IAM values of all tested compounds on a 10 cm ì 4.6 mm, 10 m Regis IAM PC DD2 column (Regis, Morton Grove, IL, USA) (Valko et al., 2000) Retention times of the compounds were determined at three different concentrations of acetonitrile The kIAM values were calculated by the equation kIAM = (tR t0) / tR in which tR represents retention times of tested compounds, whereas t0 is determined by injecting a sodium nitrate solution in the HPLC system The logkIAM values for a compound were plotted against the applied acetonitrile concentrations The intercept with the Y axis of the straight line through these data points yielded the extrapolated logKW-IAM values for the 15 reference compounds Data analysis All data of radioligand binding assays were analysed using the non-linear regression curve fitting program Prism v 5.1 (GraphPad, San Diego, CA, USA) KD and Bmax values of [3H]dofetilide at HEK293Kv11.1 membranes were obtained by 944 British Journal of Pharmacology (2015) 172 940955 computational analysis of saturation curves Apparent inhibitory binding constants (Ki values) were derived from the IC50 values according to the Cheng and Prusoff equation Ki = IC50 / (1 + [L*] / KD), where [L*] was the concentration of radioligand and KD was its dissociation constant from the saturation assay (Cheng and Prusoff, 1973) In the kinetic association experiments, the on- and off-rates were derived from the linear regression analysis using the equation kobs = kon[L*] + koff, where the kobs value was obtained by computer analysis of the exponential association of [3H]-dofetilide bound to the receptor with [L*] being the concentration of radioligand The association and dissociation rates were used to calculate the kinetic KD value using the following equation KD = koff / kon The association and dissociation rates for unlabelled compounds were calculated by fitting the data into the competition association model using kinetics of competitive binding (Motulsky and Mahan, 1984): K A = k1 [ L ] + k2 K B = k3 [ I ] + k4 S = ( K A K B )2 + 4k1k3 LI 10 18 K F = 0.5 ( K A + K B + S ) KS = 0.5 ( K A + K B S ) Q= Y = Q Bmax k1 L10 K F KS k4 ( K F KS ) k4 K F ( KF X ) k4 KS ( KS X ) + e e KS K F KS KF Where X is the time (min), Y the specific binding of [3H]dofetilide, k1 and k2 are the kon (M1ãmin1) and koff (min1) of [3H]-dofetilide obtained from the traditional association and dissociation assay, L the concentration of [3H]-dofetilide (nM), Bmax the maximum specific binding (dpm) and I the concentration of the unlabelled compound (nM) Fixing these parameters allowed the following parameters to be calculated: k3, which is the kon value (M1ãmin1) of the unlabelled compound and k4, which is the koff value (min1) of the unlabelled compound LogKW-C8 and logKW-IAM values were derived from linear regression analysis as mentioned earlier The MW, logP and pKa values were calculated using a structure-based calculation plug-in provided by ChemAxon (Budapest, Hungary) All values obtained from radioligand binding assays in this study are means of at least three independent experiments performed in duplicate Statistical analysis was performed with Students two-tailed unpaired t-test Materials Astemizole, sertindole, terfenadine, moxifloxacin, amiodarone, chlorpromazine, ibutilide, clofilium, pimozide, cisapride, ranolazine, sotalol, thioridazine and all the solutes for HPLC determinations were purchased from Sigma Aldrich (Zwijndrecht, The Netherlands) Dofetilide and E-4031 were synthesized in our own laboratory (Shagufta et al., 2009; Vilums et al., 2012) Tritium-labelled dofetilide (specific activity 6587 Ciãmmol1) was purchased from PerkinElmer (Groningen, The Netherlands) BSA (fraction V) was purchased from Sigma (St Louis, MO, USA) G418 was obtained from Molecular insights into Kv11.1 (hERG) cardiotoxicity BJP [3H]-dofetilide association and dissociation assay Stratagene (Cedar Creek, TX, USA) The chemicals for HPLC were of HPLC grade; all the other chemicals were of analytical grade and achieved from standard commercial sources HEK293Kv11.1 cells were kindly provided by Dr Eckhard Ficker (University of Cleveland, Cleveland, OH, USA) The molecular target nomenclature (Kv11.1) conforms to The Concise Guide to PHARMACOLOGY 2013/14: Ion Channels (Alexander et al., 2013b) Initial experiments were performed to fully characterize the association and dissociation rates of [3H]-dofetilide to and from HEK293Kv11.1 membranes respectively As the association rate of a ligand is dependent upon the concentration used, kinetic association experiments with a range of [3H]dofetilide concentrations were conducted In Figure 2A, curves are shown for four of such concentrations (0.76, 2.7, 3.8 and 7.1 nM) A plot of the kobs values against more, including higher, concentrations of [3H]-dofetilide (Figure 2B) was consistent with a linear correlation (r2 = 0.86, P < 0.0001), indicating that the binding of [3H]-dofetilide to the Kv11.1 channel followed the law of mass action for a simple bimolecular interaction and that the equation kon = (kobs koff) / [L*] was applicable in this study The kon and koff values obtained from this plot were 0.017 nM1ãmin1 and 0.12 min1 respectively (Table 2) When koff was divided by kon, a kinetically derived KD value of 7.1 nM was obtained These values were in agreement with values for apparent onand off-rates and kinetic KD of [3H]-dofetilide assessed at one (5 nM) concentration (kon = 0.032 0.003 nM1ãmin1, koff = Results [3H]-dofetilide saturation assay The binding of [3H]-dofetilide to HEK293Kv11.1 cell membranes was saturable and best described by a one-site binding model A representative saturation curve and the averaged data of three independent experiments performed in duplicate are shown in Figure and Table respectively The KD and Bmax values obtained from this assay were 2.4 0.1 nM and 1.6 0.1 pmolãmg1 protein respectively The KD value for [3H]-dofetilide from this assay was used to calculate Ki rather than IC50 values from the displacement assay for 15 reference compounds Figure Saturation of [3H]-dofetilide binding to HEK293Kv11.1 membranes Total binding was determined at increasing concentrations of [3H]-dofetilide Non-specific binding was determined at three concentrations of [3H]-dofetilide and non-specific binding at other concentrations of radioligand was extrapolated by linear regression Specific binding was calculated as the difference between the total and non-specific binding The KD value was 2.4 0.1 nM and the Bmax value was 1.6 0.1 pmolãmg1 protein Data shown are representative results from a single experiment performed in duplicate Table Binding parameters of dofetilide from different equilibrium and kinetic binding assays Binding parameters Saturation assay Kinetic association assaya Traditional association and dissociation assayb Displacement assay Competition association assay kon (nM1ãmin1) 0.017 koff (min1) 0.12 0.032 0.003 0.048 0.011 0.20 0.03 KD (nM) 2.4 0.1 7.1c 0.13 0.02 6.4 1.3c 2.7 0.3c Ki (nM) 5.4 0.8 Values are means (SEM) of three independent assays performed in duplicate a Data were derived from linear regression of one independent association assay of [3H]-dofetilide at different concentrations b Data from our previous study (Yu et al., 2014) c Kinetic KD = koff / kon British Journal of Pharmacology (2015) 172 940955 945 BJP Z Yu et al A B Figure Characterization of the association and dissociation rates of [3H]-dofetilide to HEK293Kv11.1 membranes in the kinetic association assay (A) Representative association curves of [3H]-dofetilide at four different concentrations (B) A plot of kobs values versus the concentration of [3H]-dofetilide Data shown are representative results from a single experiment performed in duplicate 0.20 0.03 min1 and KD = 6.4 1.3 nM) derived from the traditional association and dissociation assays published previously (Yu et al., 2014) [3H]-dofetilide displacement assay Competition binding assays were performed to generate Ki values for 15 reference compounds Compounds were selected based on structural diversity and having a wide range of Kv11.1 binding affinities, and included both antiarrhythmic drugs and drugs for other therapeutic areas (e.g astemizole and terfenadine for the treatment of allergic conditions) All compounds produced a concentrationdependent inhibition of specific [3H]-dofetilide binding and their displacement curves were best described by a one-site competition model (Figure 3) All Ki values are listed in Table Among the 15 compounds, clofilium had the highest affinity to the Kv11.1 channel, displacing [3H]-dofetilide with a Ki value of 0.55 0.09 nM, whereas moxifloxacin exhibited the lowest affinity of 252 121 M Ranolazine and sotalol showed similar and relatively weak inhibition of the channel with Ki values of 21 and 25 M respectively Addition946 British Journal of Pharmacology (2015) 172 940955 ally, amiodarone, thioridazine and chlorpromazine displayed modest Kv11.1 blockade with Ki values from 0.3 to M All other compounds demonstrated relatively high affinity to the Kv11.1 channel, between 2.5 0.2 nM (astemizole) and 63 nM (terfenadine) [3H]-dofetilide competition association assay With the kon (k1) and koff (k2) values of [3H]-dofetilide obtained from the traditional association and dissociation assays, it was possible to determine the kon (k3) and koff (k4) values of unlabelled compounds by performing the so-called competition association experiments Firstly, we validated the competition association assay at the Kv11.1 channel using three concentrations of cold dofetilide equivalent to 0.3-, oneand threefold of its Ki value A representative experiment is shown in Figure The kon (k3) and koff (k4) values for dofetilide determined in this assay were 0.048 0.011 nM1ãmin1 and 0.13 0.02 min1, respectively, and in good agreement with the kinetic parameters determined in the traditional association and dissociation assays, as shown in Table Furthermore, the kinetic KD value (2.7 0.3 nM) derived from Molecular insights into Kv11.1 (hERG) cardiotoxicity BJP A B Figure Displacement curves of [3H]-dofetilide from HEK293Kv11.1 membranes by different known Kv11.1 channel blockers (A) Dofetilide, astemizole, E-4031, sertindole, terfenadine, moxifloxacin, amiodarone and chlorpromazine; (B) ibutilide, clofilium, pimozide, cisapride, ranolazine, sotalol and thioridazine Data shown are representative results from a single experiment performed in duplicate this assay was similar to the KD value (2.4 0.1 nM) obtained from the [3H]-dofetilide saturation assay In addition, these KD values were in the same range as dofetilides affinity constant that stemmed from the displacement assay (Ki = 5.4 0.8 nM) and the kinetically derived KD values from kinetic association assay and traditional kinetic experiments (7.1 nM or 6.4 1.3 nM respectively) Overall, the results presented here demonstrated that the competition association assay could be applied to determine the association and dissociation rates of other unlabelled ligands at the Kv11.1 channel It is noteworthy that a good experimental window was achieved using a concentration of dofetilide at onefold of its Ki value in the competition association assay, as displayed in Figure Reassuringly, when astemizole and E-4031 were tested in this standard three-concentration assay, similar findings were observed as well (data not shown) Thus, the other unlabelled ligands were only tested at onefold of their Ki values rather than three different concentrations in the further competition association experiments in order to improve the throughput of this method Subsequently, kinetic parameters of the 12 other known Kv11.1 blockers were evaluated and representative normalized curves for several compounds are depicted in Figure The on- and off-rates of all compounds determined by these experiments are shown in Table The association rates for all the compounds were quite distinct with kon values ranging from (4.7 1.0) ì 106 nM1ãmin1 (moxifloxacin) to 0.23 0.07 nM1ãmin1 (clofilium), i.e an almost 50 000-fold difference between the fastest and slowest associating compounds On the other hand, the dissociation rates of these 15 compounds were more similar, with the highest value of 0.86 0.17 min1 for sertindole and lowest koff of 0.083 0.003 min1 for astemizole, i.e only a 10-fold difference Considering the kinetically derived KD values shown in Table 3, clofilium was the most potent inhibitor to the Kv11.1 channel with a kinetic KD value of 0.54 0.17 nM, while moxifloxacin had the lowest affinity (KD = 65 10 M) to the channel These results were in the same rank order as the Ki values derived from the equilibrium displacement assay Correlations of equilibrium Ki with kinetic KD, kon and koff values A plot of the logarithms of kinetic KD values (i.e koff/kon) derived from the [3H]-dofetilide competition association assays and the logarithms of equilibrium Ki values obtained from the displacement experiments was made and a significant correlation (Figure 6A) was observed This showed an excellent consistency of the results from two different methods and indicated a high reliability of the [3H]-dofetilide competition association assay More interestingly, a signifiBritish Journal of Pharmacology (2015) 172 940955 947 BJP Z Yu et al Table The affinity constants and kinetic parameters of 15 compounds at the Kv11.1 channel obtained from the [3H]-dofetilide displacement and competition association assay Compound Ki (nM)a kon (nM1ãmin1)b koff (min1)b KD (nM)c Dofetilide 5.4 0.8 0.048 0.011 0.13 0.02 2.7 0.3 Astemizole 2.5 0.2 0.17 0.03 0.083 0.003 0.53 0.07 E-4031 13 0.7 0.026 0.003 0.27 0.02 10 Sertindole 34 0.048 0.007 0.86 0.17 18 Terfenadine 63 0.0071 0.0025 0.25 0.03 39 Moxifloxacin 252 347 120 995 (4.7 1.0) ì 106 0.28 0.06 64 531 10 276 0.23 0.02 387 37 (1.3 0.2) ì 104 0.36 0.05 714 154 Amiodarone Chlorpromazine 308 33 2518 301 (6.0 0.7) ì 10 Ibutilide 5.1 0.4 0.046 0.006 0.20 0.02 4.6 1.1 Clofilium 0.55 0.09 0.23 0.07 0.10 0.01 0.54 0.17 3.2 0.4 Pimozide 28 0.071 0.020 0.22 0.07 Cisapride 54 10 0.031 0.009 0.59 0.14 20 Ranolazine 21 379 776 (1.5 0.4) ì 105 0.23 0.04 16 672 820 Sotalol 24 663 379 (1.7 0.3) ì 105 0.32 0.03 19 740 591 0.24 0.05 Thioridazine 065 41 (2.3 0.4) ì 10 050 48 Values are means (SEM) of three independent assays performed in duplicate a Ki values were derived from the [3H]dofetilide displacement assay b kon (k3) and koff (k4) values of unlabelled compounds were determined in the [3H]-dofetilide competition association assay c Kinetic KD = koff / kon Figure Competition association assay of [3H]-dofetilide in the absence (control) or presence of 0.3-, one- and threefold of unlabelled dofetilides Ki value Data shown are representative results from a single experiment performed in duplicate cant inverse relationship was also found between pkon and pKi values for unlabelled compounds in Figure 6B In contrast, there was no significant linear relationship between pkoff and pKi values (r2 = 0.15, P = 0.15, data not shown) Together, this suggested that the [3H]-dofetilide competition association assay was successfully validated for assessing the kinetics of other unlabelled competitive compounds and that the affinity of these compounds at the Kv11.1 channel was mainly controlled by their on-rates rather than off-rates 948 British Journal of Pharmacology (2015) 172 940955 Lipophilicity (logKW-C8) and membrane partition coefficient (logKW-IAM) of Kv11.1 blockers The isocratical logKW-C8 values (lipophilicity) were evaluated at pH 7.4 and are detailed in Table The lipophilicity of the 15 reference compounds covered a wide numerical range, varying from 0.56 (sotalol) to 5.52 (amiodarone) We also calculated logP values as a measure for lipophilicity and Molecular insights into Kv11.1 (hERG) cardiotoxicity BJP Figure Representative competition association curves for [3H]-dofetilide in the absence (control) or presence of unlabelled sertindole, clofilium and cisapride at a concentration of onefold their Ki values Data shown are representative results from a single experiment performed in duplicate Table The lipophilicity, membrane partition coefficients and other physicochemical properties of 15 Kv11.1 blockers Compound LogKW-C8a Dofetilide 0.84 Astemizole 3.52 E-4031 LogKW-IAMb MWc LogPc pKac 2.08 441.57 0.59 3.40 458.57 5.39 8.75 1.29 1.98 401.52 1.73 8.01 Sertindole 3.97 3.38 440.94 3.77 8.59 Terfenadine 4.05 4.01 471.67 6.48 9.02 Moxifloxacin 1.12 1.57 401.43 1.97 9.42 Amiodarone 5.52 3.30 645.31 7.64 8.47 Chlorpromazine 3.39 3.36 318.86 4.54 9.20 Ibutilide 0.90 2.53 384.58 3.25 10.40 Clofilium 2.00 0.35 338.98 2.91 nad Pimozide 4.69 3.80 461.55 5.83 8.38 Cisapride 3.12 2.66 465.95 2.49 8.24 Ranolazine 2.17 2.39 427.54 2.83 7.17 Sotalol 0.56 0.66 272.36 0.05 9.43 Thioridazine 3.53 3.80 370.58 5.47 8.93 8.99 LogKW-C8 values were derived from HPLC experiments on a Supelcosil LC-ABZ, cm ì 4.6 mm, m column LogKW-IAM values were derived from HPLC experiments on a 10 cm ì 4.6 mm, 10 m Regis IAM PC DD2 column c Values were derived from the structure-based calculation plug-in by ChemAxon d na, not applicable; this compound is permanently charged a b plotted these against the logKW-C8 data (Table 4) A significant correlation was found between them (r2 = 0.81, P < 0.0001), which implied that for this series of compounds, calculated logP values can be used interchangeably with the experimentally determined values To mimic the interactions of our ligands with membrane phospholipids, an IAM HPLC column that is a reflection of the lipid environment of a fluid cell membrane on a solid matrix was used to determine membrane partition coefficients (logKW-IAM) for all reference compounds Their logKW-IAM values were measured at pH 7.4 and are summarized in Table Terfenadine had the highest logKW-IAM value of 4.01, indicating that this compound possessed the highest affinity for membrane phospholipids On the contrary, the logKW-IAM British Journal of Pharmacology (2015) 172 940955 949 BJP Z Yu et al A Figure Correlation between logKW-C8 and logKW-IAM values at pH 7.4 for 15 Kv11.1 inhibitors (r2 = 0.52, P < 0.0024) LogKW-C8 and logKW-IAM values are listed in Table B binding kinetics of these ligands at the Kv11.1 channel Similarly, there were no correlations between logKW-C8 and pKi, pkon or pkoff values (data not shown) Role of other calculated physicochemical properties in ligand-receptor binding kinetics Figure Correlations between the affinity constant (Ki) and (A) the kinetically derived equilibrium dissociation constants, KD (r2 = 0.98, P < 0.0001) and (B) the association rates, kon, for unlabelled ligands at the Kv11.1 channel (r2 = 0.95, P < 0.0001) Ki, KD and kon values are listed in Table for clofilium was only 0.35, most likely due to its quaternary ammonium moiety, which demonstrated that this compound hardly interacted with phospholipid membranes Subsequently, the possible correlation between logKW-C8 and logKW-IAM was studied and the result is shown in Figure A significant linear relationship (r2 = 0.52, P < 0.0024) was observed for these two parameters even when including the outlier clofilium Obviously, a similar significant correlation was also found between calculated logP and logKW-IAM values (r2 = 0.52, P = 0.0022, data not shown) Next, the relationship between affinity constants or kinetic rate constants of the 15 Kv11.1 inhibitors and their membrane interactions were investigated, as shown in Figure 8AC Apparently, no relationship was found for any of them (P > 0.05), demonstrating that membrane interactions did not affect affinity and 950 British Journal of Pharmacology (2015) 172 940955 Lastly, two other physicochemical properties of the unlabelled compounds, MW and acid/base constant (pKa) (Table 4), were compared with their on-/off-rates and dissociation constants As depicted in Figure 9, no significant correlations were observed between the logarithms of on-rates of the compounds in this study and their molecular properties Moreover, there were no obvious relationships between the logarithms of off-rates or equilibrium Ki values and these physicochemical properties either (P > 0.05, data not shown) These results implied that the binding kinetics of compounds at the Kv11.1 channel was not governed by their overall, macroscopic, physicochemical properties Discussion and conclusions Since the introduction of the competition association assay (Motulsky and Mahan, 1984), more and more researchers have utilized this method to study the binding kinetics of unlabelled ligands at their targets, such as muscarinic receptors and the adenosine A2A receptor (Schreiber et al., 1985; Dowling and Charlton, 2006; Guo et al., 2012) In addition, a few examples in the literature have been reported to apply this technique for the determination of ligand binding kinetics at ligand-gated ion channels (Hawkinson and Casida, 1992) To the best of our knowledge, the current study is the first to assess binding kinetics of ligands interacting with voltage-gated ion channels, in particular the Kv11.1 channel It might be argued that our study has some limitations The stably transfected HEK293Kv11.1 cells lack the (co)expression of two ancillary -subunits, minK and MiRP1 (Vandenberg et al., 2001) Discrepancies could also exist Molecular insights into Kv11.1 (hERG) cardiotoxicity A A B B BJP C Figure Lack of correlation between the association rates (pkon values) of selected compounds and their physicochemical properties (A) No significant correlation was observed with MW (r2 = 0.049, P = 0.43); (B) no significant correlation was observed with pKa (r2 = 0.000076, P = 0.98) MW and pKa values are shown in Table Figure Correlation of logKW-IAM values and binding parameters of 15 Kv11.1 inhibitors No significant relationship between logKW-IAM and values of (A) pKi (r2 = 0.00011, P = 0.97), (B) pkon (r2 = 0.0087, P = 0.74) or (C) pkoff was observed (r2 = 0.051, P = 0.42) All data used in these plots are listed in Tables and between membrane binding assays and whole-cell experiments, which are more relevant to the channels natural orientation For instance, the kinetics of the drugKv11.1 interaction have been reported to be use- and frequencydependent in whole-cell patch-clamp assays due to special gating kinetics of the channel (Stork et al., 2007; Windisch et al., 2011) In our membrane binding assays, we speculate that the channels maintain the same configurations and thus the kinetic parameters in this investigation should resemble the actual binding process between Kv11.1 blockers and the channel To perform straightforward and accurate kinetic determinations, experiments were carried out at 25C with membrane preparations of stably transfected HEK293Kv11.1 cells We reason that all parameters obtained at 25C in this investigation lead to a similar compound rank order as those at physiological temperature In fact, this has been shown for another target based on vant Hoff and Eyring equations (Mondal et al., 2013) British Journal of Pharmacology (2015) 172 940955 951 BJP Z Yu et al The linear plot of kobs values obtained at increasing concentrations of [3H]-dofetilide (Figure 2B) strongly supported a pseudo-first-order kinetic behaviour of its binding to the Kv11.1 channel, which provided the theoretical foundation for all kinetic analyses in our present study Other mechanisms of action such as induced-fit or conformational selection would have resulted in deviations from linearity; in such cases, the Motulsky and Mahan model and equations would not have been applicable (Tummino and Copeland, 2008; Vogt and Di Cera, 2012) All 15 reference compounds had a pseudo-Hill coefficient close to unity in the [3H]-dofetilide displacement assay (data not shown), which indicated a competitive mode of inhibition with regard to the radioligand This is another indication of the simple bimolecular interaction model and corroborated a pseudo-first-order kinetic behaviour of the binding to the Kv11.1 channel This finding was in accordance with previous studies describing the presence of a single affinity state for Kv11.1 inhibitors at the channel (Finlayson et al., 2001a,b) Subsequently, a newly developed [3H]-dofetilide competition association assay was applied to study the binding kinetics of 15 unlabelled reference compounds with diverse chemical structures at the Kv11.1 channel (Table 1) The kon, koff and KD values of dofetilide from this assay were comparable with the values derived from saturation and kinetic association experiments and traditional association and dissociation assays (Yu et al., 2014), indicative of the accuracy and reliability of this new assay Moreover, the excellent linear correlation between these kinetically derived KD values of 15 reference compounds and their Ki values from the equilibrium displacement assay further supported the latter (Figure 6A) Taken together, this led us to conclude that a novel [3H]-dofetilide competition association assay was successfully developed and validated Correlations between the affinity data (Ki) and the kinetic parameters (on- and off-rates) were also investigated in the present study Surprisingly, there was a significant inverse correlation between pKi and pkon values for reference compounds (Figure 6B), whereas no relationship was found between pKi and pkoff values (data not shown) Although this correlation between affinity and association rates has been shown to be the case for 2-adrenoceptor agonists (Sykes and Charlton, 2012) and OX2-receptor antagonists (Mould et al., 2014), this phenomenon is supposed to be unusual and counterintuitive Dogma has it that the collision theory sets the maximum value for kon as the diffusion limit for a ligand and a target, which is about 108109 M1ãs1 (Smith, 2009) In this view, there can only be small variations in the on-rate constants and thus the equilibrium affinity changes are mainly dictated by the off-rates of ligands (Copeland et al., 2006; Smith, 2009; Lu and Tonge, 2010) In contrast to these classical assumptions, association rates of our selected structurally diverse compounds varied around 50 000-fold, whereas their dissociation rates differed only 10-fold This clearly indicated that apart from off-rates, on-rates also play a pivotal role in regulating affinity of ligands to their targeted receptors, at least for the Kv11.1 channel Recently, it has been reported that drug-receptor association rates and corresponding affinity were enhanced due to concentrating effects and lateral diffusion of membrane associated 2-adrenoceptor ligands and kon values were positively 952 British Journal of Pharmacology (2015) 172 940955 correlated to their lipophilicity and membrane interactions (Hanson et al., 2012; Sykes et al., 2014) Furthermore, Sykes et al (2014) observed that dissociation rates were much more correlated to the corrected true affinity, which considers drug concentration gradients in the local environment due to membrane affinity than the apparent affinity Additionally, it was recommended that any prediction of pharmacodynamic properties should take membrane interactions as well as lipophilicity into account (Taillardat-Bertschinger et al., 2003) Hence, the potential influence of membrane affinity on ligand binding to the Kv11.1 channel was investigated by the determination of both logKW-C8 and logKW-IAM values However, when pKi and pkon were compared with logKW-C8 and logKW-IAM, no significant relationships were observed in our case (Figure 8) This indicated that membrane interactions of our ligands did not influence their association rates and binding affinity to the Kv11.1 channel and that the apparent affinity of these reference compounds is their true affinity Interestingly, when logKW-C8 and logKW-IAM were compared with each other, clofilium was observed to deviate from the significant linear correlation (Figure 7) Herein, we hypothesized that the lipophilic alkyl chains of clofilium compensated for its hydrophilic quaternary ammonium group and thus dominated its lipophilicity in the octanol-water system, while the positive charge at the nitrogen played a more critical role in the IAM system and weakened its membrane interactions With regard to dissociation rates, no correlations were found to either logKW-C8 or logKWIAM values as well (data not shown) This was in accordance with previous findings (Mason et al., 1991; Sykes et al., 2014) and demonstrated that the dissociation rate of a drug was not affected by its local concentrations and thus independent of membrane affinity Of note, typical Kv11.1 inhibitors are known to bind to the inner cavity in the intracellular part of the channel (Vandenberg et al., 2001) However, all experiments in the present study were performed with HEK293Kv11.1 membranes instead of intact cells and were thus independent of transmembrane transport of ligands From Figure 6B, it follows that a significant relationship between affinity and association rates for 15 Kv11.1 inhibitors was found, which showed that ligand-Kv11.1 rather than ligand-membrane association controlled their affinity Apparently, an aqueous entry pathway predominates the binding of Kv11.1 blockers to the channel and an additional compartment induced by the lipid membranes does not play a significant role We questioned whether it would be possible to correlate other general physicochemical properties of these molecules to their biological profile (Figure 9) This was not the case, as typical features such as MW and the basicity of the Kv11.1 blockers were not correlated to any of the equilibrium or kinetic binding parameters tested However, it has been stated previously that physicochemical properties of drugs are relevant for their kinetic parameters (Shaikh et al., 2007; Smith, 2009) For instance, Miller et al (2012) reported that MW was one of the most important factors to affect the dissociation kinetics of ligands from their biological targets including enzymes (kinases) and GPCRs From the present study, it follows that the effects of general molecular properties on the binding kinetics of a ligand to Kv11.1 channel are negligible compared with other targets Molecular insights into Kv11.1 (hERG) cardiotoxicity Previous studies have also suggested that koff values, or drug-target residence times (RTs, the reciprocal of koff), play an important role in the duration of pharmacological effects (Copeland et al., 2006; Lu and Tonge, 2010; Guo et al., 2012) In other words, an increased drug-target RT could elicit an improved or prolonged drug effect (Dahl and kerud, 2013) Nevertheless, when adverse effects result directly from drug occupancy at the pharmacological target, such as chlorpromazine binding to D2 receptor or clopidogrel to P2Y12 receptor, a long RT would result in so-called on-target drug toxicity, and thus a short RT is favoured (Copeland, 2010) In our hands, however, the dissociation rates and hence RTs of selected 15 Kv11.1 inhibitors were very similar (Table 3) In contrast, association rates of our reference compounds varied widely and are therefore potentially more important to predict the safety of Kv11.1 inhibitors Previously, Redfern et al (2003) have assigned drugs into five categories of torsadogenic propensity and proposed a 30-fold safety margin between Cmax and Kv11.1 IC50 values as a marker to predict TdP However, it is known that TdP are not only induced by inhibition of the Kv11.1 channel but also regulated by other potassium, sodium and calcium channels (Redfern et al., 2003; Sager et al., 2014) Therefore, a comprehensive in vitro pro-arrhythmia assay has recently been introduced to assess drug-induced pro-arrhythmic risk more efficiently and accurately (Cavero, 2014; Sager et al., 2014) The kinetics of drug block and unblock were suggested to be incorporated together with drug potency at ion channels in arrhythmia evaluation during the interpretation of this paradigm (Sager et al., 2014) Although the present study focuses on the Kv11.1 channel only, it provides a new medium throughput method to determine the association and dissociation rates of Kv11.1 blockers, which can be used subsequently for other ion channels as well, if a radioligand is available Interestingly, Veroli et al (2014) derived from mathematical models that fast binding Kv11.1 blockers in the untrapped configuration and trapped blockers induced greater action potential and QT prolongation This strengthens the application of our method in the future and suggests that using slow association and/or fast dissociation characteristics as a novel marker might be beneficial for reducing Kv11.1 cardiotoxicity of drug candidates In conclusion, a novel [3H]-dofetilide competition association assay has been successfully developed and validated to characterize the kinetic binding parameters of unlabelled compounds at the Kv11.1 channel Importantly, association rates of Kv11.1 blockers were divergent, i.e not diffusion limited, and excellently correlated to their affinity values In addition, membrane interactions and other molecular properties not influence the affinity and kinetic binding parameters of ligands at the Kv11.1 channel Altogether, this is quite unlike the mechanisms of interaction proposed for other drug target classes, such as kinases and GPCRs Hence, we postulate that association rates can be used to assess a compounds Kv11.1 liability, apart from its affinity However, further studies involving compounds with a wide range of koff values are required to assess the effect of RT on drug-induced Kv11.1 cardiotoxicity Overall, we believe that this research provides novel insights into the kinetic study of ion channels, which can hopefully help to avoid Kv11.1-induced cardiotoxicity of drug candidates in the future BJP Acknowledgements Z Y is supported by a scholarship from the Chinese Scholarship Council We thank Dr Steven Charlton (Novartis, UK) for helpful discussions, particularly about the IAM column Author contributions Z Y designed and conducted the experiments, analysed the data and wrote the manuscript A P I designed the experiments, assessed the results and wrote the manuscript L H H designed the experiments, assessed the results and wrote the manuscript Conflict of interest None References Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M et al (2013a) The Concise Guide to PHARMACOLOGY 2013/14: G protein-coupled receptors Br J Pharmacol 170: 14591581 Alexander SP, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Catterall WA et al (2013b) The concise guide to PHARMACOLOGY 2013/14: ion channels Br J Pharmacol 170: 16071651 Cavero I (2014) 13th Annual Meeting of the Safety Pharmacology Society: focus on novel technologies and safety pharmacology frontiers Expert Opin Drug Saf 13: 12711281 Cheng Y-C, Prusoff WH (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction Biochem Pharmacol 22: 30993108 Chiu PJ, Marcoe KF, Bounds SE, Lin C-H, Feng J-J, Lin A et al (2004) Validation of a [3H] astemizole binding assay in HEK293 cells expressing HERG K+ channels J Pharmacol Sci 95: 311319 Copeland RA (2010) The dynamics of drug-target interactions: drug-target residence time and its impact on efficacy and safety Expert Opin Drug Discov 5: 305310 Copeland RA, Pompliano DL, Meek TD (2006) Drug-target residence time and its implications for lead optimization Nat Rev Drug Discov 5: 730739 Dahl G, kerud T (2013) Pharmacokinetics and the drug-target residence time concept Drug Discov Today 18: 697707 Diaz GJ, Daniell K, Leitza ST, Martin RL, Su Z, McDermott JS et al (2004) The [3H]dofetilide binding assay is a predictive screening tool for hERG blockade and proarrhythmia: comparison of intact cell and membrane preparations and effects of altering [K+]o J Pharmacol Toxicol Methods 50: 187199 Dowling MR, Charlton SJ (2006) Quantifying the association and dissociation rates of unlabelled antagonists at the muscarinic M3 receptor Br J Pharmacol 148: 927937 British Journal of Pharmacology (2015) 172 940955 953 BJP Z Yu et al Doyle DA, Cabral JM, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL et al (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity Science 280: 6977 Nerbonne JM (2000) Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium J Physiol 525: 285298 Finlayson K, Pennington AJ, Kelly JS (2001a) [3H]dofetilide binding in SHSY5Y and HEK293 cells expressing a HERG-like K+ channel? Eur J Pharmacol 412: 203212 Noble D (2008) Computational models of the heart and their use in assessing the actions of drugs J Pharmacol Sci 107: 107117 Finlayson K, Turnbull L, January CT, Sharkey J, Kelly JS (2001b) [3H]dofetilide binding to HERG transfected membranes: a potential high throughput preclinical screen Eur J Pharmacol 430: 147148 Fitzgerald PT, Ackerman MJ (2005) Drug-induced torsades de pointes: the evolving role of pharmacogenetics Heart Rhythm 2: S30S37 Guo D, Mulder-Krieger T, IJzerman AP, Heitman LH (2012) Functional efficacy of adenosine A2A receptor agonists is positively correlated to their receptor residence time Br J Pharmacol 166: 18461859 Hancox JC, McPate MJ, El Harchi A, Zhang YH (2008) The hERG potassium channel and hERG screening for drug-induced torsades de pointes Pharmacol Ther 119: 118132 Hanson MA, Roth CB, Jo E, Griffith MT, Scott FL, Reinhart G et al (2012) Crystal structure of a lipid G protein-coupled receptor Science 335: 851855 Hawkinson J, Casida JE (1992) Binding kinetics of gamma-aminobutyric acidA receptor noncompetitive antagonists: trioxabicyclooctane, dithiane, and cyclodiene insecticide-induced slow transition to blocked chloride channel conformation Mol Pharmacol 42: 10691076 Heijman J, Voigt N, Carlsson LG, Dobrev D (2014) Cardiac safety assays Curr Opin Pharmacol 15: 1621 Heitman LH, Narlawar R, de Vries H, Willemsen MN, Wolfram D, Brussee J et al (2009) Substituted terphenyl compounds as the first class of low molecular weight allosteric inhibitors of the luteinizing hormone receptor J Med Chem 52: 20362042 Pan AC, Borhani DW, Dror RO, Shaw DE (2013) Molecular determinants of drugreceptor binding kinetics Drug Discov Today 18: 667673 Pawson AJ, Sharman JL, Benson HE, Faccenda E, Alexander SP, Buneman OP et al.; NC-IUPHAR (2014) The IUPHAR/BPS Guide to PHARMACOLOGY: an expert-driven knowledgebase of drug targets and their ligands Nucl Acids Res 42 (Database Issue): D1098106 Rampe D, Brown AM (2013) A history of the role of the hERG channel in cardiac risk assessment J Pharmacol Toxicol Methods 68: 1322 Redfern W, Carlsson L, Davis A, Lynch W, MacKenzie I, Palethorpe S et al (2003) Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development Cardiovasc Res 58: 3245 Sager PT, Gintant G, Turner JR, Pettit S, Stockbridge N (2014) Rechanneling the cardiac proarrhythmia safety paradigm: a meeting report from the Cardiac Safety Research Consortium Am Heart J 167: 292300 Sanguinetti MC, Tristani-Firouzi M (2006) hERG potassium channels and cardiac arrhythmia Nature 440: 463469 Schreiber G, Henis YI, Sokolovsky M (1985) Analysis of ligand binding to receptors by competition kinetics Application to muscarinic antagonists in rat brain cortex J Biol Chem 260: 87898794 Krohn KA (2001) The physical chemistry of ligand-receptor binding identifies some limitations to the analysis of receptor images Nucl Med Biol 28: 477483 Shagufta, Guo D, Klaasse E, de Vries H, Brussee J, Nalos L et al (2009) Exploring chemical substructures essential for hERG K+ channel blockade by synthesis and biological evaluation of dofetilide analogues ChemMedChem 4: 17221732 Lombardo F, Shalaeva MY, Tupper KA, Gao F (2001) ElogDoct: a tool for lipophilicity determination in drug discovery Basic and neutral compounds J Med Chem 44: 24902497 Shaikh SA, Jain T, Sandhu G, Latha N, Jayaram B (2007) From drug target to leads-sketching a physicochemical pathway for lead molecule design in silico Curr Pharm Des 13: 34543470 Lu H, Tonge PJ (2010) Drugtarget residence time: critical information for lead optimization Curr Opin Chem Biol 14: 467474 Smith GF (2009) Medicinal chemistry by the numbers: the physicochemistry, thermodynamics and kinetics of modern drug design Prog Med Chem 48: 129 Mason RP, Rhodes DG, Herbette LG (1991) Reevaluating equilibrium and kinetic binding parameters for lipophilic drugs based on a structural model for drug interactions with biological membranes J Med Chem 34: 869877 Stork D, Timin E, Berjukow S, Huber C, Hohaus A, Auer M et al (2007) State dependent dissociation of HERG channel inhibitors Br J Pharmacol 151: 13681376 Miller DC, Lunn G, Jones P, Sabnis Y, Davies NL, Driscoll P (2012) Investigation of the effect of molecular properties on the binding kinetics of a ligand to its biological target Medchemcomm 3: 449452 Sykes D, Parry C, Reilly J, Wright P, Fairhurst R, Charlton SJ (2014) Observed drug-receptor association rates are governed by membrane affinity: the importance of establishing micro PK/PD relationships at the 2-adrenoceptor Mol Pharmacol 85: 608617 Mondal K, Regnstrom K, Morishige W, Barbour R, Probst G, Xu Y-Z et al (2013) Thermodynamic and kinetic characterization of hydroxyethylamine -secretase-1 inhibitors Biochem Biophys Res Commun 441: 291296 Sykes DA, Charlton SJ (2012) Slow receptor dissociation is not a key factor in the duration of action of inhaled long-acting 2-adrenoceptor agonists Br J Pharmacol 165: 26722683 Motulsky HJ, Mahan L (1984) The kinetics of competitive radioligand binding predicted by the law of mass action Mol Pharmacol 25: 19 Taillardat-Bertschinger A, Carrupt P-A, Barbato F, Testa B (2003) Immobilized artificial membrane HPLC in drug research J Med Chem 46: 655665 Mould R, Brown J, Marshall FH, Langmead C (2014) Binding kinetics differentiates functional antagonism of orexin-2 receptor ligands Br J Pharmacol 171: 351363 Tummino PJ, Copeland RA (2008) Residence time of receptor-ligand complexes and its effect on biological function Biochemistry 47: 54815492 954 British Journal of Pharmacology (2015) 172 940955 Molecular insights into Kv11.1 (hERG) cardiotoxicity BJP Valko K, Du CM, Bevan CD, Reynolds DP, Abraham MH (2000) Rapid-gradient HPLC method for measuring drug interactions with immobilized artificial membrane: comparison with other lipophilicity measures J Pharm Sci 89: 10851096 Vilums M, Overman J, Klaasse E, Scheel O, Brussee J, IJzerman AP (2012) Understanding of molecular substructures that contribute to hERG K+ channel blockade: synthesis and biological evaluation of E-4031 analogues ChemMedChem 7: 107113 Vandenberg JI, Walker BD, Campbell TJ (2001) HERG K+ channels: friend and foe Trends Pharmacol Sci 22: 240246 Vogt AD, Di Cera E (2012) Conformational selection or induced fit? A critical appraisal of the kinetic mechanism Biochemistry 51: 58945902 Vandenberg JI, Perry MD, Perrin MJ, Mann SA, Ke Y, Hill AP (2012) hERG K+ channels: structure, function, and clinical significance Physiol Rev 92: 13931478 Veroli GYD, Davies MR, Zhang H, Abi-Gerges N, Boyett MR (2014) hERG inhibitors with similar potency but different binding kinetics not pose the same proarrhythmic risk: implications for drug safety assessment J Cardiovasc Electrophysiol 25: 197207 Windisch A, Timin E, Schwarz T, Stork-Riedler D, Erker T, Ecker G et al (2011) Trapping and dissociation of propafenone derivatives in HERG channels Br J Pharmacol 162: 15421552 Yu Z, Klaasse E, Heitman LH, Ijzerman AP (2014) Allosteric modulators of the hERG K+ channel: radioligand binding assays reveal allosteric characteristics of dofetilide analogs Toxicol Appl Pharmacol 274: 7886 British Journal of Pharmacology (2015) 172 940955 955 ISSN 0007-1188 (print) ISSN 1476-5381 (online) www.brjpharmacol.org BJP British Journal of Pharmacology www.bps.ac.uk Editor-in-Chief J.C (Ian) McGrath Glasgow, UK & Sydney, Australia Senior Editors Amrita Ahluwalia London, UK Richard Bond Houston, USA Michael J Curtis London, UK David MacEwan Liverpool, UK Susan Wonnacott Bath, UK Mark Giembycz Calgary, Canada Daniel Hoyer Melbourne, Australia Paul Insel La Jolla, USA Reviews Editors Senior Online Editor Stephen Alexander Nottingham, UK Andrew Lawrence Melbourne, Australia Annette Gilchrist Downers Grove, USA Press Editors Y.S Bakhle Caroline Wedmore Editorial Board Ruth Andrew Edinburgh, UK Alexis Bailey Guildford, UK Chris Bailey Bath, UK Phillip Beart Melbourne, Australia Heather Bradshaw Bloomington, USA Keith Brain Birmingham, UK John Challiss Leicester, UK Victoria Chapman Nottingham, UK Steven Charlton Horsham, UK Diana Chow Houston, USA Macdonald Christie Sydney, Australia Sandy Clanachan Edmonton, Canada Lucie Clapp London, UK David Cowan London, UK John Cryan Cork, Ireland Nicola Curtin Newcastle upon Tyne, UK Gerhard Cvirn Graz, Austria Anthony Davenport Cambridge, UK Peter Doris Houston, USA Pedro DOrlộans-Juste Sherbrooke, Canada Claire Edwards Oxford, UK Michael Emerson London, UK Peter Ferdinandy Szeged, Hungary Anthony Ford San Mateo, USA Chris George Cardiff, UK Heather Giles London, UK Derek Gilroy London, UK Gary Gintant Illinois, USA Michelle Glass Auckland, New Zealand Fiona Gribble Cambridge, UK John Hartley London, UK Robin Hiley Cambridge, UK Andrea Hohmann Georgia, USA Miles Houslay London, UK Jackie Hunter Weston, UK Ryuji Inoue Fukuoka, Japan Paul Insel San Diego, USA Angelo A Izzo Naples, Italy Yong Ji Nanjing, China Eamonn Kelly Bristol, UK Melanie Kelly Halifax, Canada Terry Kenakin Durham, USA Dave Kendall Nottingham, UK Charles Kennedy Glasgow, UK Chris Langmead Welwyn Garden City, UK Rebecca Lever London, UK Eliot Lilley Redhill, UK Jon Lundberg Stockholm, Sweden Mhairi Macrae Glasgow, UK Ziad Mallat Cambridge, UK Karen McCloskey Belfast, UK Barbara McDermott Belfast, UK Alister McNeish Reading, UK Olivier Micheau Dijon, France Paula Moreira Coimbra, Portugal Maria Moro Madrid, Spain Fiona Murray San Diego, USA Anne Negre-Salvayre Toulouse, France Janet Nicholson Biberach an der Riss, Germany Eliot Ohlstein Pennsylvania, USA Saoirse OSullivan Nottingham, UK Hiroshi Ozaki Tokyo, Japan Reynold Panettieri Jr Philadelphia, USA Sandy Pang Toronto, Canada The British Journal of Pharmacology is a broad-based journal giving leading international coverage of all aspects of experimental pharmacology.The Editorial Board represents a wide range of expertise and ensures that well-presented work is published as promptly as possible, consistent with maintaining the overall quality of the journal Disclaimer The Publisher, British Pharmacological Society and Editors cannot be held responsible for errors or any consequences arising from the use of information contained in this journal; 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SR, sarcoplasmic reticulum British Journal of Pharmacology (2015) 172 721 736 729 730 British Journal of Pharmacology (2015) 172 721 736 NCT012 434 24 September 2018 CV, cardiovascular;... larger group of studies (such as PMSF usage) were absent in studies of CBD and THCV British Journal of Pharmacology (2015) 172 737 7 53 7 43 BJP J M McPartland et al The low affinity of CBD at CB1... 9-tetrahydrocannabivarin â 2014 The British Pharmacological Society British Journal of Pharmacology (2015) 172 737 7 53 737 BJP J M McPartland et al Tables of Links TARGETS LIGANDS GPCRsa MAGL,
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