Review

10.1517/13543780902762850 © 2009 Informa UK Ltd ISSN 1354-3784 399All rights reserved: reproduction in whole or in part not permitted

IKur/Kv1.5channelblockersforthetreatmentofatrialfibrillationJuan Tamargo†, Ricardo Caballero, Ricardo Gómez & Eva Delpón†Universidad Complutense, School of Medicine, Department of Pharmacology, Madrid, Spain

Atrial fibrillation (AF) is the most common sustained arrhythmia. Anti-arrhythmic drugs remain the mainstay of therapy, but the available class I and III anti-arrhythmic drugs are only moderately effective in long-term restoring/maintaining sinus rhythm (SR) and can produce potentially fatal ventricular pro-arrhythmia. In an attempt to identify safer and more effective anti-arrhythmic drugs, drug discovery efforts have focused on ‘atrial selective drugs’ that target cardiac ion channel(s) that are exclusively or predominantly expressed in the atria. The ultra-rapid activating delayed rectifier K+ current (IKur), carried by Kv1.5 channels, is a major repolarizing current in human atria, but seems to play no role in the ventricle. This finding offers the possibility of developing selective IKur blockers to restore and maintain SR without a risk of ventricular pro-arrhythmia. Several IKur blockers are now being developed but clinical data are still limited, so the precise role of these agents in the treatment of AF remains to be defined. In this review we analyze the possible advantages and disadvantages of the developmental IKur blockers as they represent the first step for the development of potential atrial selective drugs for a more effective and safer treatment and prevention of AF.

Keywords: atrial fibrillation, cardiac action potential, cardiac ion channels, IKur, Kv1.5 channels, pharmacology

Expert Opin. Investig. Drugs (2009) 18(4):399-416

1. Introduction

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice, with an estimated prevalence of 0.4 – 1% in the general population, increasing with age to 8% in patients older than 80 years [1]. AF impairs cardiac output, exercise tolerance and quality of life, increases stroke risk (five-fold), exacerbates heart failure, and is independently associated with a two-fold increase in mortality [1]. Overall, hospitalizations for AF have increased two- to three-fold in recent years, accounting for approximately one third of hospitalizations for cardiac rhythm disturbances [1,2]. Thus, AF represents a considerable financial burden on the healthcare system. Although AF can occur in the absence of underlying disease (idiopathic or lone AF), it is most commonly seen in patients with structural heart disease, including heart failure, arterial hypertension, coronary artery disease, cardiothoracic surgery, mitral valvular disease or thyrotoxicosis. Moreover, familial AF occurrence is more common than previously recognized, an observation supporting a genetic susceptibility for developing this dysrhythmia [1].

1.1 Atrialfibrillationisoftenaself-perpetuatingdiseaseIn many patients, initial paroxysms of AF (self-terminating) become more frequent and of longer duration to the point where AF becomes persistent (i.e., AF requires cardioversion to restore sinus rhythm; SR) and then permanent over time (AF cannot be converted to SR). This progression involves changes in the electrophysiological, structural and contractile properties of the atria (atrial remodeling)

1. Introduction

2. Atrial action potential

3. Treatment of atrial fibrillation

4. Structure of Kv1.5 channels

encoding IKur

5. The ultrarapid activating

delayed rectifier K+ current, IKur

6. Electrophysiological effects of

IKur blockers

7. The future of new IKur blockers

(the two sides of the coin)

8. Expert opinion

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in a way that promotes the maintenance and recurrence of the arrhythmia. AF produces a rapid (within hours) and inhomogeneous shortening of the atrial action potential duration (APD) and effective refractory period (AERP) and a lack of adaptation of APD to fast heart rates [1,3-5]. Long-term AF also slows intra-atrial conduction, which together with the structural changes in the atrial wall facilitates AF perpetuation after changes in AERP are stabilized. Thus, atrial remodeling explains why paroxysmal AF tends to become persistent and/or permanent, chronic AF becomes resistant to anti-arrhythmic drugs, the higher success rate of cardioversion when AF is of short duration and the early recurrences of AF after cardioversion. The development of more effective anti-arrhythmic drugs should be based on a better understanding of the electrophysiological mechanisms underlying AF. AF is caused by a variety of mechanisms, including local ectopic activity and single- and multiple-circuit re-entry, which are not mutually exclusive and may coexist [1,3]. Over the last 50 years, the multiple wavelet re-entry hypothesis has been the most widely accepted conceptual model to explain AF [1,3]. Accordingly, AF is maintained by multiple continuously propagating waves of atrial re-entry (wavelets) separated by lines of conduction block, fixed (anatomical obstacles, branching, scars or areas of fibrosis) or functional (non-uniform recovery repolarization, regional stretch, gap-junction uncoupling), resulting in a short excitable gap between the fibrillation waves [3-5]. The number of wavelets that can coexist is determined by the atrial size and the wavelength (WL) (product of conduction velocity and AERP) of the atrial impulse. A large atrial mass with a short AERP and slow impulse conduction increases the number of wavelets, favor-ing the onset and maintenance of AF. The electrophysiologi-cal atrial remodeling shortens the atrial APD and refractoriness and slows conduction velocity in a heteroge-nous manner, providing a substrate for multiple-circuit re-entry. Thus, according to the multiple wavelet re-entry hypothesis, an effective way to prevent or terminate AF is to prolong the AERP by lengthening the atrial APD. This would increase the wavelength, thereby reducing the number of wavelets that can coexist in the atria to the point that AF fails to be sustained.

2. Atrialactionpotential

The human atrial action potential reflects a sequential activation and inactivation of ion channels that conduct depolarizing, inward (Na+ and Ca2+), and repolarizing, outward (K+), currents [6,7]. Figure 1 shows the ionic currents involved in the genesis of the five phases of the human atrial and ventricular action potential. The initial upstroke of the atrial cells (Phase 0) is due to the opening of Na+ channels. Most cardiac Na+ channels open transiently within milliseconds upon membrane depolarization, resulting in the peak transient current (INa), which determines excitation and conduction.

However, a small percentage of Na+ channels may continue to open and close spontaneously, carrying the so-called per-sistent or late Na+ current (INaL), which plays a major con-tribution to the shape and duration of the action potential plateau. The initial rapid repolarization (Phase 1) is a conse-quence of the rapid voltage-dependent closure (inactivation) of Na+ channels, and the activation of the transient outward current (Ito) and the ultrarapid component of the delayed rectifier K+ current (IKur). During Phase 2, inward depolar-izing currents through Na+ (slowly inactivated, INaL) and L-type Ca2+ channels (ICaL) are balanced by the different components of the delayed rectifier K+ current (IKur, rapid-IKr and slow-IKs). The net outward current during Phase 3 of repolarization is provided by the delayed rectifier K+ cur-rents, along with the inactivation of the ICaL. The strong inward rectifier K+ current (IK1) contributes to the terminal Phase 3 of repolarization and sets the resting potential (around − 80 mV) close to the K+ equilibrium potential (Phase 4). In atrial cells, acetylcholine released on vagal stimulation binds to muscarinic (M2) receptors and activates another inward rectifier K+ current (IKAch). Activation of IKAch hyper-polarizes the resting membrane potential and produces a non-uniform shortening of atrial APD and AERP, which decreases the WL of re-entrant circuits and promotes the stability of AF. The differences in action potential wave-forms between atrial and ventricular cells reflect differences in ion channel expression levels (Figure 1) [4,6,7]. The greater density of outward repolarizing currents (Ito and IKur) com-pared with inward currents can explain why the human atrial action potential is shorter and the plateau occurs at lower potentials than the ventricular action potential. Moreover, IKur and Ito are the main repolarizing currents in the human atria, while IKr and IKs are the main repolarizing currents in ventricular myocytes and Purkinje fibers. Most important, IKur is a major repolarizing current in human atria but is virtually absent in human ventricle [8-10]. In addition, atrial cells display a more gradual Phase III repo-larization, present a more depolarized resting membrane potential, possibly due to a lower density of IK1 [11] and a more negative diastolic threshold of excitation than that of ventricular cells.

3. Treatmentofatrialfibrillation

Although several non-pharmacological therapeutic approaches (i.e., surgery, catheter ablation, internal atrial defibrillators and pacing) have been developed, anti-arrhythmic drug therapy remains the mainstay of managing AF. The anti-arrhythmic treatment of AF involves two main strategies: to restore and maintain normal SR (rhythm control) with class I and III anti-arrhythmic drugs and/or to allow patients to remain in AF, but control the ventricular response (rate control) using atrio-ventricular nodal blocking drugs (beta-blockers, amiodarone, digitalis glycosides, or nondihydropyridine calcium channel antagonists) [1].

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3.1 RateversusrhythmcontrolWhile restoring and maintaining SR has several theoretical advantages over rate control, until recently there has been little evidence supporting the comparative advantages of either strategy. Five recent randomized clinical trials (PIAF, AFFIRM, RACE, STAF, HOT-CAFE) [12-16] compare rate with rhythm control in patients with recurrent AF or high risk of recurrences [1] (see [17] for a review). No significant differences were observed between both strategies in all-cause mortality, symptoms, quality of life, stroke or in any other point examined, although a non-significant trend for excess mortality in the rhythm control strategy was observed [1,17]. Moreover, rate control was associated with fewer hospitalizations and fewer adverse drug effects, being more cost-effective [17]. Thus, in appropriate patients with persistent AF or high risk of recurrence, a strategy of ventricular rate control, in combination with oral anti-coagulation, can be at least as effective as rhythm control in preventing clinical outcomes and may, in fact, prove to be superior. There are several explanations for these results [17]. One possible explanation is that the beneficial antiarrhythmic effect (e.g., maintenance of SR) on survival may be offset by the low efficacy of rhythm control to maintain SR (the percentage of patients in SR at the end of the study ranged between 38% and 63.5%) and the high frequency of serious adverse effects (pro-arrhythmia, bradycardia, organ toxicity) associated with anti-arrhythmic drug therapy, which, additionally, led to a higher rate of early drug discontinuation [12,14,18]. Additionally, premature discontinuation of oral anticoagulation in some patients who were believed

to be in SR could have contributed to an increased incidence of thromboembolic events. AF is common in patients with heart failure and is associated with an adverse outcome [1]. The AFFIRM trial suggests that patients without congestive heart failure fared significantly better with rate control [19]. Very recently, the AF-CHF (Atrial Fibrillation and Congestive Heart Failure) trial enrolled 1376 patients with systolic heart failure (left ventricular ejection fraction ≤ 35%, and New York Heart Association (NYHA) functional class II – III) and AF (two thirds had persistent AF) receiving optimal heart failure therapy and anticoagulation [20]. Over a follow-up period of 37 months, the study found no differences between rhythm- (amiodarone in 82% of patients) and rate-control strategies (β-blockers and digoxin) in reducing cardiovascular deaths or other secondary outcomes (death from any cause, worsening of heart failure, or stroke). However, the rate-control strategy eliminated the need for repeated cardioversion and reduced the rate of hospitalization. These findings suggest that rate control should be considered a primary approach for patients with AF and congestive heart failure. Nevertheless, there are several concerns with the study, including a high crossover rate (21%) between study groups and the suboptimal control of SR in the rhythm-control group (58% of patients had at least 1 recurrent episode of AF); at 12 months, significantly fewer patients received β-blockers in the rhythm-control group which could have influenced mortality; and, finally, during the follow-up, the percentage of patients in the rhythm-control group receiving amiodarone dropped to 73% at 36 months, which might have negated the potential benefits of this strategy.

A. B.

IK1, IKAch, IKATPINa

Ito1

IKur

ICaL

IKr, IKs

INaLPhase 0

Phase 1

Phase 2

Phase 3

Phase 4

Ito1

IKr

IKs

IK1, IKATP

Phase 0

Phase 1

Phase 2

Phase 3

Phase 4

ICaL

INa

INaL

Figure 1. Ionic currents involved in shaping of human atrial (A) and ventricular (B) action potentials. The initial upstroke (Phase 0) of the action potential is due to the activation of the inward Na+ current (INa). Repolarization is mainly due to outward efflux of K+ (blue arrows) that counterbalances the depolarizing effects of inward Ca2+ (ICaL) and Na+ (peak INa and late-INaL) influx (red arrows). The main voltage-dependent human K+ currents (Ito, IKur, IKr and IKs) are represented together with the inward rectifier currents (IK1, IKACh and IKATP). Some currents are exclusive to the atria (IKur) or are more important (Ito, IKACh) in the atria than in the ventricles and vice versa (IKr). See text for a proper explanation.

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402 ExpertOpin.Investig.Drugs(2009) 18(4)

3.2 Rhythm-controlinAFClass IA and IC (Na+ channel blockers) and class III anti-arrhythmic drugs (which prolong atrial APD and AERP without slowing impulse conduction) have been widely used to restore and maintain normal SR for cardioversion of AF to SR [1]. Class IA and IC drugs prolong AERP by slowing the recovery of availability of Na+ channels, but they also exhibit other ancillary properties that contribute to their anti-arrhythmic action, including the blockade of several K+ currents [21]. Class III agents prolong atrial APD and AERP primarily by blocking one or several outward repolarizing K+ currents [22]. However, available drugs exhibit modest efficacy for maintaining SR (< 50% of patients remain in SR 1 year after cardioversion), produce potentially serious adverse effects and most of them are contraindicated in patients with several cardiovascular conditions commonly associated with AF, including heart failure, coronary artery disease and hypertension [1]. Moreover, these drugs prolong atrial APD via blockade of the IKr and/or IKs, which are expressed in both human atria and ventricle [1,3-5]; and, thus, they prolong ventricular repolarization and the corrected QT interval for heart rate (QTc) on the electrocardiogram (ECG) and increase the risk for ventricular tachyarrhythmias, such as torsades de pointes (TdP), which limits their utility in the treatment of AF.

In an attempt to identify safer and more effective drugs to terminate/prevent AF without the risk of ventricular pro-arrhythmia, drug discovery efforts have focused on ‘atrial selective drugs’, i.e., drugs that target cardiac ion channel(s) that are exclusively or predominantly expressed in the atria. Because IKur carried by Kv1.5 channels is present only in the atria, but not in the ventricles [6-10], IKur blockers would be expected to prolong human atrial APD and AERP, convert AF to SR and/or prevent recurrences of AF without undesired effects on impulse propagation, ventricular repolarization (QTc interval) or cardiac output and with a minimum risk of ventricular pro-arrhythmia.

In this review, we address the molecular basis and biophysical properties of IKur and the pharmacological properties, advantages and disadvantages of the new IKur blockers, as an example of atrial-selective drugs that might convert AF to SR and prevent recurrences of AF without the pro-arrhythmic risk of many currently available class I and III agents.

4. StructureofKv1.5channelsencodingIKur

Kv1.5 channels belong to the superfamily of voltage-gated K+ channels (Kv), formed by coassembly of four identical pore-forming α-subunits encoded by the KCNA5 gene (human chromosome 12p13.3) and four accessory β-subunits (Kvβ1.2, Kvβ1.3, and Kvβ2.1) that bind to the N-terminus of the α-subunit to form α4β4 complexes. Each α-subunit (613 amino acids) contains six transmembrane-spanning segments (S1 – S6) with cytoplasmic N-and C-terminal domains (Figure 2) [6,7,23-25]. The S1 – S4 module represents

the voltage sensor, which reacts to potential changes across the membrane and regulates the gating of the channel. The S4 segment contains positively charged residues (Arg or Lys) at every third position and serves as the principal voltage sensor. The highly conserved selectivity filter signature (Gly-Tyr-Gly) is located between S5 and S6 and is connected to S5 via the pore helix and a short loop located outside the membrane. The pore of the channel, through which K+ ions flow across the plasma membrane, is formed by S5, S6, the selectivity filter and the pore helix. The intracellular part of the pore is arranged in what has been dubbed an ‘inverted teepee’ cavity, with the selectivity filter located at its wide end [23]. The large water-filled pore cavity is lined with highly hydrophobic amino acids and has been identified as the binding site for IKur blockers. Kv1.5 α-subunits coassemble with Kvβ1.2 subunits to form the IKur in human atrium [6,7,26]. Immunohistochemical studies show that, in human atria, Kv1.5 channels are concentrated at the level of the intercalated disks (close to N-cadherin) and T-tubules, with some staining at the cell surface [27,28]. Although Kv1.5 channels are expressed both at the mRNA and protein levels in human ventricular tissues, functional IKur only exists in atrial cells [7,26,29].

5. TheultrarapidactivatingdelayedrectifierK+current,IKur

5.1 CurrentfeaturesandmolecularbasisIKur has been recorded in human [8,9,29], rat [30] and canine [31] atrial myocytes and in adult mouse ventricular myocytes (IK,slow1) [32]. The biophysical and pharmacological characteristics of the currents generated through Kv1.5 channels in heterologous expression systems are similar to the IKur recorded in human atria [7], and antisense-targeting Kv1.5 specifically suppresses IKur in human and rat atrial myocytes [29], which confirms that Kv1.5-based channels (10 – 14 pS) are the molecular basis for IKur. Kinetic differences of activation, inactivation and recovery from inactivation discriminate between various K+ currents. At 37°C, human atrial IKur presents a rapid activation (τ < 2 ms at 0 mV, midpoint (Vh) − 7 mV and slope factor (k) 6 – 12 mV) and outward rectification and a slow and incomplete inactivation during the time course of the action potential [6,8,9,33]. Recovery from inactivation proceeds slowly (time constants of 0.42 and 7.9 s at − 80 mV), which confers substantial rate-dependent properties on IKur over a physiologically relevant range of frequencies [33]. Coexpression of Kvβ1.2 subunits with Kv1.5 channels causes a hyperpolarizing shift in the voltage dependence of activation and inactivation gating and slows the rate of Kv1.5 channel deactivation [34]. It is widely accepted that IKur inactivates by a ‘type C’ mechanism as a result of a concerted constriction of the external mouth of the pore involving all four α-subunits [6,7,35]. C-type inactivation is thought to be voltage independent and occurs predominantly from the open state, is slowed by external tetraethylammonium (TEA) or elevation of extracellular K+

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concentration ([K+]o) or when the residue Arg485, located in the outer pore mouth, is substituted (Arg485Val) [36]. Binding of external K+ ions within the conducting pathway inhibits inac-tivation via a ‘foot-in-the-door’ mechanism that prevents con-striction of the outer pore mouth [36]. However, IKur does not present N-type inactivation because Kv1.5 channels do not have a long N-terminal domain [37].

5.2 PharmacologyofIKur

IKur is relatively insensitive to TEA, Ba2+ and selective IKr blockers (dofetilide, E-4031 and MK-499), but is highly sensitive to low concentrations of 4-aminopyridine (4-AP, ∼ 10 μM) [6,8,37]. Several anti-arrhythmic drugs (ambasilide, amiodarone, clofilium, dronedarone, flecainide, quinidine, propafenone, 5-hydroxy-propafenone, tedisamil) [6,37-40], local anesthetics (bupivacaine [41,42] and benzocaine [43,44]), angiotensin II type 1 receptor antagonists (losartan, candesartan, irbesartan) [45-47], spironolactone [47,48], the pacemaker-current inhibitors (zatebradine [49], ivabradine [50]), atorvastatin [51] and some calcium antagonists (verapamil, diltiazem, nifedipine) [39,52,53] inhibit IKur, in addition to their effects on other cardiac ionic channels. However, in the last decade, several pharmaceutical companies have developed selective IKur blockers in an attempt to prevent and treat AF (see [54-56] for a review). However, most of these agents can also inhibit cardiac Na+,

Ca2+ and a variety of K+ channels. The ion channel blocking profile and the chemical structures of some investigational IKur blockers is summarized in Table 1. In general, IKur blockers appear to act via a preferential interaction with Kv1.5 channels in their open state [6,57]. This assessment is based on the following findings.

They produce a rapid current decay upon depolariza-1. tion and upon repolarization slow current deactivation producing a crossover of tail current traces recorded in the absence and in the presence of the drug. These results indicate that the drugs enter into the channel cavity only after channel opening and inhibit IKur by binding to a receptor site located at this level, preventing normal closure of the inactivation gate (foot-in-the-door); thus, the drug must dissociate from their receptor site before the channel can close.They display a reduced affinity for channels in the 2. resting-closed (i.e., at a holding potential of – 80 mV) or inactivated state (i.e., at positive potentials).Kv1.5 channel block increases with the stimulation 3. frequency, which represents an advantageous property because channels would be most affected at fast action potential firing rates characteristics for AF and less affected in SR.

NH2 COOH(613)

Kv1.5 × 4

-

-

--

-

-

In

Out

Voltage sensorPore

+

+

++

Po

A.

α

α

α

α

β

ββ

SF

S5

S6

S5

S6

Po Po

V505I508V512V516

T479, T480

B.

Kvβ

S1 S2 S3 S4 S5 S6

β

Figure2.MolecularstructureoftheKv1.5channel(A)andviewoftheS5-S6domainoftwoα-subunitsoftheKv1.5channelshowingtheresiduesfacingtheporecavityinvolvedintheinhibitioninducedbyvernakalant,S9947andAVE0118(B).(A) Upper part: schematic representation of the structure of one Kv1.5 α-subunit with 6 membrane-spanning domains and the intracytoplasmatic accesory

β-subunits (Kvβ and KChiP). Lower part: α and accessory β-subunits coassemble as tetramers to form the functional channel. (B) Po: Pore helix;SF: Selectivity filter.

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Tab

le1

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of

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r/K

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Spec

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[ref

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n].

CH

O: C

hine

se h

amst

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vary

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ls; G

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aci

d (2

-pyr

idin

-3-y

l-eth

yl)-

amid

e.

AZD

7009

: ter

t-Bu

tyl-2

-(7-

[(2S)

-3-(

4-cy

anop

heno

xy)-

2-hy

drox

ypro

pyl]-

9-ox

a-3,

7-di

azab

icyc

lo[3

.3.1

]non

-3-y

l)eth

ylca

rbam

ate.

DPO

-1(M

erck

and

Co.

): 2-

isop

ropy

l-5-m

ethy

lcyc

lohe

xyl)

diph

enyl

phos

phin

e ox

ide.

ICA

GEN

-4: N

-[3-

(4-e

thyl

-ben

cene

-sul

fony

lam

ino)

-2-h

ydro

xy-in

dan-

5-yl

]-3-

met

hoxy

-ben

zam

ide.

ISQ

-1 (M

erck

& C

o.):

3-[(d

imet

hyla

min

o)m

ethy

l]-6-

met

hoxy

-2-m

ethy

l-4-p

heny

lisoq

uino

lin-1

(2H

)-on

e.

NIP

-142

(Nis

san

Che

mic

al In

dust

ries)

: (3R

*, 4

S*)-

4-cy

clop

ropy

lam

ino-

3,4-

dihy

dro-

2,2-

dim

ethy

l-6-(

4-m

etho

xyph

enyl

acet

hyla

min

o)-7

-nitr

o-2H

-1-b

enzo

pyra

n-2-

ol h

ydro

chlo

ride.

S994

7 (s

anofi

-ave

ntis

): 2′

-(be

nzyl

oxyc

arbo

nyla

min

omet

hyl)-

biph

enyl

-2-c

arbo

xylic

aci

d 2,

4-di

fluor

o-be

nzyl

amid

e.

TAEA

(Mer

ck a

nd C

o.):

2-ph

enyl

-1,1

-dip

yrid

in-3

-yl-2

-pyr

rolid

in-1

-yl-e

than

ol.

Ver

naka

lant

: 3-P

yrro

lidin

ol, 1

-[(1

R,2R

)-2-

[2-(

3,4-

dim

etho

xyph

enyl

) eth

oxy]

cycl

ohex

yl]-

,hyd

roch

lorid

e, (3

R).

AV

E123

1 (s

anofi

-ave

ntis

), C

9356

(Car

diom

e/A

stel

las

Phar

ma)

and

XEN

-D01

01 (X

entio

n): s

truc

ture

indi

sclo

sed.

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However, in human atrial myocytes IKur amplitude is markedly reduced at rapid activation rates [58,59], so that it is possible that, during AF, IKur may be already so reduced that there is little current to block [60,61]. On the contrary, in a rat model, very rapid atrial pacing produces an immediate, transient and rate-dependent increase in Kv1.5 mRNA (peak effect at 2 h, but returns to baseline after 8 h) and a gradual decrease of Kv4.2 and Kv4.3 mRNA, while mRNA levels of Kv1.2, Kv1.4, Kv2.1, rERG, KvLQT1 and minK remain unchanged [62]. Thus, it is possible that the increased expression of the Kv1.5 channel protein might contribute, at least in part, to the rapid shortening of the AERP at the onset of AF. If so, it is possible that selective IKur blockers might antagonize the shortening of the atrial APD and AERP and prevent new episodes of AF, being more effective in preventing AF (because of the large amplitude of IKur at slow heart rates), than in terminating the arrhythmia [58].

5.3 MoleculardeterminantsofKv1.5channelblockThe Kv1.5 channel pore cavity has been identified as the binding site for open channel blockers. In fact, the potency of anti-arrhythmic drugs (quinidine) and local anesthetics (bupivacaine, benzocaine) to block Kv1.5 channels is affected by introducing specific mutations in residues located at the base of the pore helix (Thr479 and Thr-480) near the selectivity filter and in the S6 (Thr-507, Leu-510 and Val-514) [42,44,57]. Therefore, it was predicted that these latter residues are located facing towards the central cavity of the channel pore and identified as potential binding sites for these drugs. However, residues Thr-507, Leu-510 and Val-514 that determine the Kv1.5 channel blockade induced by irbesartan [46] and S0100176 [63] are not facing the channel pore. More recently, Ala-scanning mutagenesis of the pore domain, homology modeling of the Kv1.5 pore and docking simulation of drug-channel interaction confirm that two residues located in the base of the pore helix (Thr-479 and Thr-480) and several residues located in S6 facing the pore cavity are the potential sites for IKur block induced by AVE0118 (Val-505, Ile-508, Val-512 and Val-516), S0100176 (Val-505, Ile-508 and Val-512) and vernakalant (Val-505 and Ile-508) [63-65]. The positions of these residues are highly conserved among different cardiac voltage-dependent K+ channels (i.e., hERG or Kv11.1, KCNQ1 or Kv7.1, and Kv1.3), which explains why the development of selective IKur blockers is a difficult task and most of these compounds might also affect other K+ currents with affinities similar to that of Kv1.5 (Table 1) [63]. β-subunits can modulate drug-induced Kv channel blockade. Even when most of the IKur blockers seem to interact with a receptor site located in the channel cavity accessible from the cytoplasmic mouth of the pore, other alternative access routes for drugs to their binding site of Kv1.5 channels have been proposed, such as through a crevice between S5 and S6, in a similar way to that described for local anesthetics in voltage gated Na+ channels [51,66]. Furthermore, some evidence suggests that the external mouth of

the channel pore, formed by the P loop and adjacent S5 – S6 segments, is the binding site for some long-chain poly- unsaturated fatty acids (arachidonic and docosahexaenoic acids) [67] and for nifedipine [53]. The finding that R(+)-N-methyl-bupivacaine, a membrane-impermeant permanently charged drug, produces different effects on IKur when applied from the intra- or the extracellular side of the membrane, confirms the existence of an external binding site for bupivacaine-like local anesthetics [68].

6. ElectrophysiologicaleffectsofIKurblockers

Selective IKur blockers would be expected to prolong atrial APD and AERP, to decrease atrial vulnerability and reverse AF to SR without affecting ventricular repolarization (QTc interval) or increasing the risk of ventricular pro- arrhythmia. At low micromolar concentrations (10 – 50 μmol/L) 4-AP fully inhibits IKur without affecting other cur-rents [9,69,70], and thus it can be considered a selective IKur blocker. However, the reported effects of 4-AP on atrial APD90 are contradictory. Thus, 4-AP shortens atrial APD90 and ERP in isolated human atria [71] and in canine coro-nary-perfused right atrial preparations [60,72] and in human myocytes obtained from patients in SR (nonremodeled ‘healthy’ atria), these effects are accompanied by an increase in the height and a prolongation of the duration of the pla-teau phase (APD20) [73]. On the contrary, it has been reported that 4-AP prolongs APD90 in atrial myocytes iso-lated from canine atria [69,74], patients undergoing coronary bypass or cardiac surgery [9,75] and in anesthetized dogs, where 4-AP, at plasma concentrations between 25 and 50 μmol/L, significantly lengthens AERP without affecting ven-tricular refractoriness [70]. These contradictory results can be attributed to the different driving rates and experimental protocols (isolated atrial myocytes, isolated atria, in vivo vs. in vitro) and to the effects of 4-AP on autonomic tone [70,76]. Furthermore, in isolated myocytes from patients with chronic AF (‘remodeled atria’), 4-AP only slightly prolongs the APD90 [72,73]. AVE0118, at a concentration (6 μmol/L) at which Ito, IKAch and some IKr are also blocked (Table 1), reproduces the effects of 4-AP, so that it elevates the plateau potential and shortens the APD90 in atrial trabeculae in SR, but prolongs APD90 in AF [73]. It has been reported that both IKur density and Kv1.5 protein expression can be reduced by 50% in atrial cardiomyocytes from patients with valvular disease and persistent AF [77], a result that has not been confirmed in subsequent studies [75,78], probably reflecting differences in patient populations, AF duration or concomitant medications [4]. However, this does not imply that IKur blockers would not be effective in prolonging AERP in patients with AF. Using a mathematical model of the human atrial AP (Figure 3), it was predicted that inhibition of IKur by 90% leads to an increased activation of the ICaL, which shifts the plateau level to positive potentials at which IKr is rapidly activated, so that mid and late repolarization are

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accelerated and thus, the APD remains unchanged during SR [79]. In accordance with the model predictions, in the presence of an IKur blocker (E-4031), 4-AP no longer shortens but prolongs atrial APD90 in SR. However, AF-induced electrical remodeling reduces ICaL and Ito and markedly shortens the atrial APD, so that inhibition of IKur increases the plateau potential over a range (below 0 mV) where the activation of other K+ repolarizing currents (IKr and IKs) is less pronounced, and so a prolongation of the APD90 is observed. These findings suggest that the shortening of atrial APD associated with AF increases the contribution of IKur to atrial repolarization even when the current can be downregulated [73,79]. Thus, selective IKur blockers may be more, rather than less, effective in prolonging APD in patients with AF than in controls.

6.1 EffectsofinvestigationalIKurblockersinanimalmodelsThe effects of some novel IKur blockers on cardiac ion currents recorded in native cells as well as in heterologous expression systems are summarized in Table 1. In anesthetized and/or conscious dogs [80-82,88-90,92,96-98], goats [82,89,99,100], pigs [81,82,92] and African green monkeys [88,89], novel IKur blockers produce a dose-dependent prolongation of atrial APD and ERP, an effect that is stronger on left atria (LA) (which has shorter refractoriness) than on right atria (RA), and decrease LA vulnerability to AF, without affecting intra-atrial conduction velocity, ventricular ERP or QTc intervals [55,90,92,97]. In fact, they are highly effective in terminating/preventing AF (induced by rapid atrial pacing, vagal stimulation, aconitine or heart failure) and/or atrial flutter (AFL; induced by intracaval and atrial free wall lesions, canine sterile pericarditis) without affecting ventricu-lar ERP or the QT interval [89-92,95,96,101-103] (see [55] for a review). By contrast, selective IKr blockers (sotalol, dofetilide, MK-499) and class III anti-arrhythmic drugs (azimilide, ibutilide) prolong RA ERP stronger than LA ERP and pro-long ventricular ERP and the QTc interval [88,89,92,97]. DPO-1 shows selectivity for hKv1.5/IKur over Ito (8-fold), IK1, IKr and IKs (20-fold) [86]. In human atrial myocytes, DPO-1 produces larger prolongation of the APD at the plateau level (APD20) than at 90% of repolarization (120% vs 25% at 0.2 μM), but this effect is not evident in human ventricular myocytes [87]. In addition, in African green monkeys, DPO-1 produces a significant prolongation of AERP without changes in atrial excitation threshold, ventricular refractory period, ECG intervals, heart rate, or blood pressure [104], and in a canine model of atrial flutter, DPO-1 terminates atrial arrhythmia at doses that increase AERP but have no effects on ventricular ERP or PR, QRS or QT intervals of the ECG [102]. Furthermore, in goats and pigs after 48 – 72 h of AF-induced electrical remodeling, IKur blockers (AVE0118, AVE1231) prolong AERP and AF wavelength in a dose-dependent manner, terminate AF and reduce AF inducibility; by contrast, dofetilide prolongs the

AERP in sinus rhythm, but not in remodeled atria [82,90,97,99,100]. These findings suggest that the effects of IKur blockers persist in electrically remodeled atria and support the selective inhibition of IKur as an approach to terminate AF. In isolated human atrial trabeculae from SR patients, 4-AP and AVE0118 shift the plateau to more positive values and increase contractile force [73,105], but this effect is not observed in ventricular trabeculae. Similarly, in goats with chronic AF, AVE0118 fully restores atrial contractility after cardioversion of AF to SR and this effect is accompanied by a significant prolongation of the atrial APD20 [106]. This inotropic effect is abolished by KBR7943, a blocker of the reverse mode Na+/Ca2+-exchange, but is independent of changes in peak ICaL [105]. Thus, the positive inotropic effect is atrial-specific and due to an increase in Ca2+ entry via reverse mode Na+/Ca2+ exchange. It can be hypothetized that this increase in atrial contractility may reduce the risk of thromboembolic events associated with atrial stunning following cardioversion [90,105,106].

6.2 MethodologicalconsiderationsAnimal models represent important tools to analyze the efficacy of new investigational IKur blockers, but because of the differences in the K+ current profiles responsible for atrial repolarization among different species, at this early stage of development caution should be exerted before extrapolating results observed in these models to patients with AF. Kv1.5 is the molecular correlate of IKur in human [8,9,29], dog [31], pig [107] and rat atria [30,108], but the molecular counterpart of IKur and its functional role in atrial repolarization in the rabbit [109], goat [110], guinea pig [55] and African green monkey [88,89] remains to be clarified. Atrial action potential is shorter and the degree of electrical remodeling is more pronounced in goats than in humans, so, in these two species, the relative contribution of IKur and Ito to repolarization and the effect of blockade of these currents on the shape of the action potential may differ [82,100]. Furthermore, atrial cellular electrophysiology in pigs and goats is incompletely explored. IKur is present in rat atrial and ventricular myocytes [7,30] and thus, IKur blockers prolong APD and ERP in both tissues [87,88,92]. Therefore, the rat model provides a measure of in vivo efficacy but no direct measurement of selectivity. Additionally, in rodents, Ito plays an important role in the repolarization [7], so they cannot differentiate Kv4.2/3 from Kv1.5 channel blockers [55]. Dog models are the most widely used to analyze the electrophysiological effects of the new IKur blockers against AF/AFL. Kv1.5 mRNA and protein are the molecular counterparts of IKur in canine atria and ventricle [31,74], so IKur blockers can prolong ventricular APD90 and ERP as as the QTc interval. In fact, DPO-1 prolongs canine atrial and ventricular APD [74,104], whereas it has no effect on the APD in human ventricular myocytes [86]. Another important difference is that, in

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contrast to chronic human AF, in a canine model of chronic AF, IKur remains unchanged [111]. Finally, a potential effect of IKur blockade on in vivo canine ventricular repolarization might be obscured by pentobarbital, which blocks several K+ repolarizing currents, and prolongs canine ventricular APD50 and APD90 at concentrations achieved during general anesthesia [112]. This may explain, at least in part, the lack of effect of DPO-1 on canine ventricular repolarization in dogs anesthetized with pentobarbital [102].

6.3 ClinicaltrialsOnly a few new IKur blockers have been studied in clinical trials. Two drugs from the Icagen/Bristol-Myers Squibb program progressed to Phase I trials, but were discontinued [55,113]. AVE0118 was studied in a Phase II trial, but the results have not been published. Because it undergoes rapid first-pass hepatic metabolism and presents a short half-life in dogs and pigs (0.2 – 0.4 h) [55,97,98], AVE0118 has been replaced by the orally active drug

NAP

NAP

to 9 pA/pF

to 9 pA/pF

Ica, L

Ito

Ikur

Ik

AFAP

AFAPAFAP + Ikur inhibition

AFAP + Ikur inhibition

0

5

-5

5

0

0 200 400 600-5

0

-90

V (

mV

)I (

pA

/pF

)I (

pA

/pF

)

∆APD-60 = 0 ms∆APD-60 = + 25 ms

NAP + Ikur inhibition

NAP + Ikur inhibition

t (ms)

0 200 400 600

t (ms)

Figure3.EffectofIKurinhibition(90%reductioninmaximalconductance)onthenormal(NAP)andatrialfibrillation(AF)-modifiedhumanatrialactionpotentials(AFAP).The top panels show the control action potential morphologies and the action potentials after IKur inhibition. The net change in action potential duration is given.

The middle and lower panels show the evolution of several membrane currents in the absence or presence of IKur. Ito: Transient outward K+ current; IK: Delayed

rectifier K+ current; IKur: Ultrarapid delayed rectifier current; ICaL: L-type calcium current. Taken from Courtemanche etal. [79] (with permission).

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AVE1231, which is currently in Phase I [55]. A Phase I trial with XEN-D0101 was announced in 2005, but the results of this study have not been reported. AZD7009 was studied in a Phase II study in 122 patients with persistent AF or AFL of 48 h-90 days that were randomized to placebo or intravenous (IV) AZD7009 (3-h infusion). AZD7009 was given in doses to obtain target pseudo-steady-state plasma levels between 0.25 and 2.5 μmol/L after 30 min of infusion [114]. At the three highest doses (1.5, 2 and 2.5 μmol/L), AZD7009 converted AF to SR in 45, 64 and 70% of patients after a mean time of 62, 55 and 26 min, respectively, whereas no placebo-treated patients converted. Conversion to SR occurred in 78% of patients with AF ≤ 1 week and in 52% of those with a duration > 1 week; SR was maintained for 24 h in 21 of 22 patients. At plasma concentrations ≥ 0.75 μmol/L, AZD7009 increased the QTc interval (40 ms to 80 ms) and a number of outliers with QTc values > 550 ms were seen, particularly after conversion to SR. Clinical adverse effects (dizziness, bradycardia, hypotension and nausea) were significantly more common in the AZD7009 group than in the placebo group, and one patient exhibited a non-sustained, polymorphic ventricular tachycardia of eight beats with TdP-like features after AZD7009 infusion. The potential of AZD7009 to prolong the QTc interval and possibly cause TdP clearly limits its clinical development.

Vernakalant (RSD1235) is a mixed Na+ (INa and INaL) and atria-preferred K+ channel (IKur, Ito and IKACh) blocker under development for the acute cardioversion of AF to SR [56,65,93-96]. Vernakalant produces a voltage- and frequency-dependent open channel INa block, increasing in depolarized tissues (from an IC50 of 107 μM at -120 mV to 31 μM at -60 mV) and at high heart rates (from an IC50 of 2 μM at 1 Hz, to an IC50 of 0.5 μM at 10 Hz), so under these con-ditions vernakalant cannot be considered as a selective IKur blocker [93-95]. In animal models of AF, the overall rate of conversion with vernakalant is close to 52% within 90 min of infusion (placebo conversion rate 3.8%), but the drug has no effect on PR or QTc intervals [56,115]. The efficacy and safety of oral and IV vernakalant have been analyzed in seven clinical trials. The characteristics of these trials are summarized in Table 2. In the Phase II dose-step Conversion of Rapid Atrial Fibrillation (CRAFT) trial, patients were randomized to vernakalant or placebo [116]. Vernakalant (2 mg/kg followed by 3 mg/kg) shows significant differences over placebo in termination of AF (61 vs 5%), proportion of patients in SR at 30 min (56 vs 5%) and 1 h post-infu-sion (53 vs 5%) and median time to conversion to SR (14 vs 162 min), while no changes in QTc and QRS intervals were observed. In the Atrial Arrhythmia Conversion Trials (ACT) I and III, patients were randomized to vernakalant or placebo. Vernakalant produces a higher conversion rate to SR within 90 min than placebo in patients with AF lasting 3 h to 7 days (52% vs 3.8%) or 3 h to 45 days (37% vs 3%), than in patients in AF for

8 – 45 days (8% vs 0%), the mean time of conversion being 11 min and 8 min in the ACT I and ACT III trials, respectively [115,117-119]. However, in these two trials, less than 8% of the patients with AFL were converted to SR by vernakalant. The ACT II trial compared the efficacy and safety of the IV vernakalant and placebo in patients who developed AF or AFL following coronary artery bypass graft and/or valve replacement surgery [120]. A higher percentage of patients treated with vernakalant converted to SR within 90 min compared with placebo-treated patients; the mean time to conversion was 12 min and 75% converted on the first dose. The open-label ACT IV trial evaluated the safety of vernakalant in patients with symptomatic AF sustained for > 3 h but ≤ 45 days duration [121]. The overall conversion rates to SR for patients with recent-onset AF (< 7 days) was 51% within a median time of 14 min, while conversion rate for those with AF of 8 days or longer was < 10%. These findings confirm that vernakalant is effective and safe to convert recent AF to SR. Intravenous vernakalant has a very short half-life (2.9 – 3.4 h), is metabolized primarily by the cytochrome P450 CYP2D6 and presents a mean steady-state volume of distribution of 88.5 L for extensive metabolizers (111.5 L for poor metabolizers) [115,122]. The most common side effects were dysgeusia, paresthesia, nausea, cough, pruritus, sneezing, bradycardia and hypotension, but no death or cases of TdP or ventricular fibrillation were reported [115,119,122]. In three randomized studies, 39 (5.3%) of 737 patients treated with vernakalant developed any ventricular arrhythmia within 2 h after infusion and further 69 (9.4%) had an event between 2 and 24 h after infusion compared with 20 (6.3%) and 41 (13%) patients in the placebo arm [122]. Discontinuation rate was reported to be 3.8%. However, the safety of vernakalant in patients with heart failure (they were excluded from trials), bradicardia, coronary artery disease or QTc prolongation (> 440 ms) has yet to be determined. An oral formulation of vernakalant is under development for the long-term maintenance of SR after cardioversion. A Phase Ib study confirmed that the drug is orally bioavailable (78%), reaching peak plasma concentration after 1 – 2 h [122]. In a Phase IIa trial in patients with AF lasting 3 – 180 days, vernakalant (300 mg b.i.d.) added to conventional therapy was superior to placebo in maintaining SR postcardioversion (57% vs 39%) [123]. In this study, the most common adverse events were bradycardia and first-degree AV block, but no TdP was observed. An ongoing dose-ranging Phase IIb study analyzes the safety and tolerability, pharmacokinetics and efficacy of vernakalant (150, 300 or 500 mg b.i.d.) in 735 patients at risk of recurrent AF. Patients in AF were electrically cardioverted and then, randomized to vernakalant or placebo for the next 90 days. Very recently, Cardiome announced the results of the interim analysis of 446 patients, indicating that the median time to recurrence of AF was greater than 90 days in patients treated with vernakalant, 500 mg b.i.d., compared with 39 days in those of the placebo group [124].

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Tab

le2

.Clin

ical

dev

elo

pm

ent

of

vern

akal

ant.

Stu

dy

[ref

]N

Phas

ed

esig

nPa

tien

tch

arac

teri

stic

sD

ose

(m

g/k

g)

Prim

ary

end

po

int

Ou

tco

me

vs

pla

ceb

oC

om

men

ts

CRA

FT [9

5]56

II, R

, DB,

PC

AF

3-72

h0.

5 +

1 IV

2 +

3 IV

CSR

dur

ing

infu

sion

or

with

in 3

0 m

inC

SR: 6

1 vs

5%

(P <

0.0

1)M

TC: 1

4 vs

162

min

AC

T I [

98]

336

60A

FLIII

P, R

, MC

, PC

AF

3 h-

45 d

3 h

– 7

d, n

=220

8 –

45 d

, n=1

16

3 m

g/kg

for

10

min

plu

s 2

mg/

kg a

fter

15

min

IV

CSR

with

in 9

0 m

in

CSR

: 52

vs 4

%(P

< 0

.01)

Hig

hest

con

vers

ion

A

F ≤

72 h

(78%

)*M

TC: 1

1 m

in

AC

T II

[99]

150

AF

3 -7

2 h

afte

r

card

iac

surg

ery

CSR

: 47

vs 1

4%M

TC: 1

2 m

in

AC

T III

[98]

262

23A

FLA

F 3

h-45

d3

h –

7 d,

n=1

708–

45

d, n

=116

CSR

: 51.

2 vs

3.6

%(P

< 0

.01)

Hig

hest

con

vers

ion

A

F ≤

72 h

(71%

)*M

TC: 8

min

AC

T IV

[100

]16

7III

Ope

nA

F 3

h-45

d3

h –

7 d,

n=1

708

– 45

d, n

=69

CSR

: 51%

MTC

: 14

min

Prev

entio

n [1

01]

221

IIa, P

CPe

rsis

tent

AF

post

card

iove

rsio

nO

ral 3

00 o

r 60

0 b.

i.d.

SR a

t 1

mon

th61

vs

43%

Diff

eren

ce a

t 30

0 m

g b.

i.d.*

Prev

entio

n73

5IIb

, R, D

B, P

CO

ral 1

50, 3

00 o

r 50

0 b.

i.d.

SR a

t 3

mon

ths

52 v

s 39

%(P

< 0

.01)

MTR

on

500

mg

90 v

s 39

day

s

*p <

0.0

5

AFL

: Atr

ial fl

utte

r; C

SR: C

onve

rsio

n to

sin

us r

hyth

m; d

: Day

s; D

B: D

oubl

e-bl

ind;

h: H

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7. ThefutureofnewIKurblockers(thetwosidesofthecoin)

Data from experimental models and clinical evidence in AF patients confirm that some new IKur blockers prolong atrial APD and AERP and increase atrial contractility at concentrations at which these drugs have almost no effect on intracardiac conduction and ventricular repolarization (QTc interval). Thus, IKur blockers have the potential to become a new therapeutic approach for cardioversion of AF to SR and prevent recurrences. However, most of the new drugs developed as selective IKur blockers display mixed ion channel activity, blocking other cardiac K+ (Ito, IKr and IKAch) Ca2+ and Na+ currents with affinities comparable with that of IKur, i.e., they produce a non-selective IKur blockade (Table 1). Only S9947, ISQ-1, DPO-1 and C9356 might be regarded as selective IKur blockers because their blocking activity on this current clearly dominates the activity on the currents (IKr or IKs) that determine ventricular repolarization [54,55]. The lack of atrial selectivity may explain why the clinical development of some IKur blockers has been withheld due to effects on ventricular repolarization. However, it should be mentioned that the effects of these four drugs on several important ionic currents are unknown (see Table 1), the effects of C9356 have been studied only in HEK-cells and the electrophysiological effects of ISQ-1, DPO-1 and S9947 have been studied in dogs and pigs anesthetized with pentobarbital [88,89,92,104]. Therefore, it is uncertain whether these drugs can be considered as selective IKur blockers. The question is whether the limited selectivity of IKur blockers is beneficial or detrimental in the treatment of AF. Since several different ionic currents contribute to the human atrial AP, agents that affect more than one of these currents would probably be much more effective than a blocker of just one current [54]. Chronic AF leads to an increase of the agonist-independent, constitutively active IKACh channels that may contribute to the shortening of atrial APD [125]. Thus, probably the most attractive combination for the development of atrio-selective drugs might be a mixed blockade of IKur and constitutive IKACh, because both currents are present in atrial cells with little or no activity in ventricular myocytes [4-6]. In some patients AF can be triggered by ectopic beats originating from rapidly firing atrial foci mainly located within or near the pulmonary veins [1]. Furthermore, in certain cases, the maintenance of AF may depend upon the periodic activity of a small number of sustained, high-frequency, functional re-entrant sources (rotors) localized mainly in the LA [126]. A recent simulation study showed that specific blockade of IKur and/or Ito (but not of IKr or IKs) can terminate rotor activity [127]. The underlying mechanism is related to a maximal prolongation of atrial APD at the plateau phase, rather than at the terminal phase of repolarization. This leads to random tip meander and wavebreak, resulting in rotor termination. But despite these suggestive findings, the therapeutic role of IKur blockers

on atrial ectopic and/rotor activity is unknown. Furthermore, important electrical heterogeneities have been found in canine isolated arterially perfused RA, characterized by regional (epicardial vs endocardial) and transmural distribution of repolarization [72], and Ito/IKur block with low concentrations of 4-AP inhomogeneously abbreviates APD leading to a canine perfused RA leading to a reduction in dispersion of repolarization. Interestingly, several IKur blockers (AVE0118, C9356, NIP-142) prolong human APD at the plateau level, while the APD90 values remain unaltered [31,73,86,105] and inhibit Ito (Table 1), the Ito/IKur blocking ratio ranging between 1.6 and 8. Thus, inhibition of Ito may contribute to the atrio-selective effects of IKur blockers and the termination of AF under certain circumstances. Recent evidence suggests the existence of atrial-selective INa blockers that specifically or predominantly depress Na+ channels in atria versus ventricles [128] and it has been proposed that atrial-selective Na+ channel blockers may represent a new strategy in the treatment of AF. There are major differences in action potential characteristics (Figure 1) and biophysical properties of Na+ channels of atrial and ventricular myocytes, so that the voltage-dependence of inactivation is shifted to more negative potentials, the resting membrane potential is less negative and recovery of INa from inactivation is slower in atria than in ventricles [128,129]. Therefore, a larger fraction of atrial versus ventricular Na+ channels would be inactivated at a given take-off potential. At fast stimulation rates (e.g., during AF), the take-off potential is progressively depolarized and decreases the diastolic time for drug unblock from the channels. Both effects increase the fraction of inactivated Na+ channels in the atria, which results in a greater inhibition of INa, and the development of use-dependent postrepolarization refractoriness (PRR) [21,128,130]. Therefore, an IKur blocker with a high affinity for the inactivated state of the Na+ channel may be more effective in blocking atrial than ventricular Na+ channels, leading to an atrial-selective prolongation of AERP at rapid activation rates.

The selectivity of IKur blockers to prolong atrial versus ventricular ERP can be explained by the presence of IKur in atria but not in ventricles. However, in healthy non-remodeled atria, IKur blockers shorten atrial APD90 and prolong AERP; for example, they prolong the ERP/APD90 ratio and PRR. This prolongation of the ERP/APD90 ratio is a typical prop-erty of Na+ channel blockers, but not of K+ channel block-ers, which suggests that the prolongation of AERP can be related to the blockade of INa. Some IKur blockers (AZD7009 and vernakalant) inhibit the INa (Table 1) and slow conduction velocity and increase diastolic threshold of excitation in atria but not in ventricles [56,94,101,131]. In isolated canine atria, AZD7009 concentration-dependently reduces the Vmax (an indirect index of INa) and increases the ERP through APD90 lengthening and PRR [131]. By contrast, the increase in ventricular ERP is small and ventricular conduction time is unaffected by AZD7009 [101,131]. Thus, effects on both repolarization and INa may contribute to the atrial-selective

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prolongation of AERP and the anti-arrhythmic effects of AZD7009, particularly at rapid activation rates. A similar mechanism has been proposed to explain the atrial-selective effects of some Na+ channel blockers [128,130] and AVE0118 [61]. From the clinical point of view, only a few investigational IKur blockers have been studied in clinical trials, and there are no comparative studies with class I and III anti-arrhythmic drugs. At present, AVE1231 and XEN-D0101 are in Phase I and only the multichannel blocker vernakalant has progressed to Phase III. Additionally, the pharmacodynamic and pharmacokinetic properties and the long-term safety profile of most of the new IKur blockers are presently unknown. Because Kv1.5 channels are also present in other tissues (i.e., brain, macrophages and vascular, intestinal, and airway smooth muscle), the potential effects of these drugs on non-cardiac tissues should also be carefully addressed. Therefore, at present the clinical efficacy and safety (pro-arrhythmia) and the precise role of IKur blockers to restore and/or maintain SR in patients with AF remains to be defined. However, if selective IKur blockers are found effective and safe in long-term prospective clinical trials, they will represent the first step in the development of atrial-specific anti-arrhythmic drugs that may circumvent the risk of ventricular pro-arrhythmia and the negative inotropism observed with class I and III anti-arrhythmic drugs.

Therefore, the final question is whether a selective IKur blocker can suppress AF. It has been hypothesized [130] that a selective IKur blocker would not be able of effectively suppress AF, because IKur is markedly reduced at fast stimulation rates (e.g., during AF), so that in patients with AF IKur may already be greatly reduced without much left to block [58,59]. However, and even when in remodeled atria, a selective IKur blocker would slightly prolong atrial APD90 and AERP, when IKur block is combined with the inhibition of INa and Ito (and perhaps with IKACh) it can suppress AF/AFL both in experimental models [99,101,130] and in clinical studies [114,116,119]. Unfortunately, we do not have any ‘pure’ IKur blocker, so the hypothesis that a selective IKur blocker can (or cannot) suppress AF cannot be tested.

7.1 CritiquetoIkurblockadeasatherapeuticapproachTwo findings two findings raise the possibility that blocking of IKur can provide the substrate for development of AF. First, loss-of-function mutations of KCNA5 gene located at the N-terminus (A115V), S1 (A251T), S1-S2 linker (P307S, P310L), and C-terminus (P532L and R578K) have been reported in three ethnic groups with idiopathic-lone AF [132,133]. Kv1.5 currents generated by the KCN5A variants are indistinguishable from those generated by wild-type (WT) channels, but C-terminus variants are strikingly less sensitive to blocking by quinidine and propafenone (IC50 = 8.4 μM for WT vs 133 μM for the variants). Thus, mutations may also contribute to variability in drug-induced blockade. Another mutation (E375X) found in a patient with lone AF truncates the Kv1.5 channel protein, eliminating

S4 – S6, the pore region and the C-terminus [134]. Heterologously expressed E375X hKv1.5 channels fail to generate IKur and exert a dominant-negative effect on WT current. Exposure of the proband to isoproterenol induces irregular atrial discharges degenerating into AF. In human atrial myocytes, 4-AP (50 μM) prolongs atrial APD50 and APD90 values, and this effect is associated with early after-depolarizations and triggered activity upon isoproterenol challenge [134]. These findings indicate that defective Kv1.5 channels may predispose the atria to electrical instability and arrhythmogenesis. Second, it is well known that a shortening of AERP can provide the substrate for the development of AF and selective IKur blockers shorten APD90 in healthy human trabeculae [73,79]. Moreover, in isolated canine coronary-perfused right atrial preparations in which AF could not be induced with programmed stimulation, low concentrations of 4-AP (10 – 25 μM, that selectively inhibit IKur) shorten atrial APD90 and AERP, and now non-sustained episodes of AF could be induced in a third of the preparations [60]. However, in acutely remodeled atria (following the administration of acetylcholine or ischemia/reperfusion plus isoproterenol), 4-AP produces almost no prolongation of the APD90, and only at concen-trations ≥ 50 μM it prolongs atrial APD90 and AERP and exerts a mild anti-arrhythmic effect. From this data, it was proposed that blocking of IKur can provide the substrate for the development of AF in healthy canine atria [60]. Even when these results should be interpreted with caution because they were obtained in atrial preparations lacking a number of principal electrical and structural changes observed in chronically remodeled atria, they raise the question of whether IKur blockade may be pro-arrhythmic in healthy atria through the shortening of atrial APD and ERP.

8. Expertopinion

Atrial fibrillation, the most common cardiac arrhythmia, is associated with significant morbidity and mortality and impairment of quality of life. Despite significant advances in the non-pharmacologic treatment of AF, anti-arrhythmic drug therapy remains the mainstay of managing AF for most patients. However, class I and III anti-arrhythmic drugs commonly used to restore and maintain SR exhibit suboptimal efficacy (less than 50% of patients remain in SR 1 year after a successful cardioversion), produce serious adverse effects (including pro-arrhythmia, negative inotropy and extracardiac organ toxicity) that may offset the benefits of SR, and are contraindicated in patients with specific cardiovascular conditions commonly associated with AF, including heart failure, coronary artery disease and structural heart diseases. In an attempt to develop new anti-arrhythmic agents, safer and more effective than those presently used, in the last decade drug discovery efforts have focused on ‘atrial-selective drugs’ that target cardiac ion channel(s) that are exclusively or predominantly expressed in the atria. IKur

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is a major repolarizing current in human atria, but not in the ventricles, so that blocking of IKur is thought to be a promising target for atrial-specific therapy of AF. Hence, selective inhibition of IKur would be expected to prolong human atrial APD and refractoriness, and to restore and maintain SR with a minimum risk of ventricular pro- arrhythmia. In the last decade, several IKur blockers have been developed and are now being tested in experimental models. However, only a few drugs can be regarded as selective IKur blockers, while the majority of the new compounds block a variety of cardiac K+ channels that determine ven-tricular repolarization as well as Na+ channels with affinities comparable with that of IKur. Moreover, only a few investiga-tional IKur blockers have been studied in clinical trial, and there are no comparative studies with class I and III anti-arrhythmic drugs. At present, AVE1231 and XEN-D0101 are in Phase I

and only the multichannel blocker vernakalant has progressed to Phase III. Therefore, at present, adequately powered, pro-spective, randomized, controlled trials are needed to assess the long-term clinical efficacy and safety of these newly developed agents to restore and/or maintain SR in patients with AF. Nevertheless, these drugs represent the first step in the development of atrial-specific, anti-arrhythmic drugs that may circumvent the risk of ventricular pro-arrhythmia and the negative inotropism observed with the class I and III anti-arrhythmic drugs available at present.

Declarationofinterest

This work was supported by CICYT (SAF2005 – 04609 and SAF2008-04903), FIS-FEDER (PI080665), Red HERACLES (RD06/0009) and Lilly Foundation Grants.

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AffiliationJuan Tamargo†, Ricardo Caballero, Ricardo Gómez & Eva Delpón †Author for correspondenceUniversidad Complutense, School of Medicine, Department of Pharmacology, 28040 Madrid, Spain Tel: +34 91 3941472; Fax: +34 91 3941470; E-mail: jtamargo@med.ucm.es

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