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Cardiac Drug Development Guide
METHODS IN PHARMACOLOGY AND TOXICOLOGY
Y. James Kang, MD, SERIES EDITOR

In Vitro Neurotoxicology: Principles and Challenges, edited by

Evelyn Tiffany-Castiglioni, 2004
Cardiac Drug Development Guide, edited by Michael K. Pugsley, 2003
Methods in Biological Oxidative Stress, edited by Kenneth Hensley and
Robert A. Floyd, 2003
Apoptosis Methods in Pharmacology and Toxicology: Approaches to Measurement
and Quantification, edited by Myrtle A. Davis, 2002
Ion Channel Localization: Methods and Protocols, edited by Anatoli N. Lopatin
and Colin G. Nichols, 2001
METHODS IN PHARMACOLOGY AND TOXICOLOGY

Cardiac Drug
Development Guide
Edited by

Michael K. Pugsley
Department of Pharmacology, Forest Research Institute,
Jersey City, NJ
© 2003 Humana Press Inc.
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Totowa, NJ 07512

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Library of Congress Cataloging-in-Publication Data

Cardiac drug development guide / edited by Michael K. Pugsley.

p. ; cm. -- (Methods in pharmacology and toxicology)
Includes bibliographical references and index.
ISBN 1-58829-097-2 (alk. paper) eISBN: 1-59259-404-2
1. Cardiovascular agents. 2. Drug development.
[DNLM: 1. Cardiovascular Agents--pharmacology. 2. Drug Design. 3.
Drug Evaluation, Preclinical--methods. QV 150 C2633 2003] I. Pugsley,
Michael K. II. Series.
RM345.C34 2003
615’.71--dc21
2003012876
 Preface

Cardiac Drug Development Guide outlines, in detail, the therapeutics of cardiac

medicine currently at the cutting edge of scientific research and development around
the world. This volume integrates basic and clinical cardiac pharmacology by com-
bining, for the first time, both classical and molecular aspects of therapeutic drug
development. The chapters comprise a broad spectrum of therapeutic areas and hence
involve a comprehensive discussion of molecular, biochemical, and electrophysi-
ological concepts based on years of in vitro as well as in vivo pharmacological stud-
ies. In addition, the latter part of the book includes comprehensive clinical cardiac
chapters that describe important topics in molecular medicine. These chapters also
discuss current clinical therapeutic trends in medicine and provide an evaluation of
the efficacy of novel drugs in these areas.
Cardiac Drug Development Guide has many distinctive and outstanding features
that set it apart from other cardiac pharmacology books. This book introduces topics
in an easily understandable format for researchers in many varying disciplines by
integrating and thereby simplifying concepts not usually discussed across a broad
range of cardiac disciplines and in a highly technical field. Each chapter not only
introduces and describes the physiology, pharmacology, and pathophysiology of the
disease, but also overviews the clinical implications of drug development, what stages
these areas are currently in, and also reviews some of the methodologies involved in
drug discovery and development. As a result, this book provides a comprehensive
overview of the most advanced procedures in cardiac pharmacology today. It is hoped
that Cardiac Drug Development Guide provides useful information to graduate stu-
dents, academic scientists, clinicians, and those researchers in the pharmaceutical
and biotechnology sectors of the drug industry.
Cardiac Drug Development Guide was fashioned from my many years of experience
both learning pharmacology as a graduate student in Canada and as an MRC postdoctoral
research fellow and applying that knowledge in industry as a senior pharmacological
scientist. I wish to thank all my colleagues around the world for contributing to this
book and to my family, especially my loving wife, Suzanne, for all their support.
Michael K. Pugsley

v
 Contents

Preface ................................................................................................................... v
Contributors ....................................................................................................... ix
I INTRODUCTION
1 Cardiac Drug Development: From Animal Models to Clinical Trials
Michael K. Pugsley ............................................................................................. 3

II NOVEL MOLECULAR TARGETS FOR CARDIAC DRUG DEVELOPMENT

2 The Role of Cardiac Pacemaker Currents in Antiarrhythmic
Drug Discovery
David A. Saint ................................................................................................... 27
3 Oxygen Free Radicals in Heart Disease: Novel Therapies
Elizabeth Roth and Laszlo Hejjel .................................................................. 47
4 Mitogen-Activated Protein Kinases-Mediated Signaling
in Cardiac Pathology: A Perspective of Novel Therapeutic Targets?
Tanya Ravingerova, Miroslav Barancik, and Monika Strniskova ........ 67
5 Development of Anti-Inflammatory Drugs for Cardiovascular Disease
Melanie B. Smith and David J. Lefer ............................................................. 87
6 Development and Use of Platelet Glycoprotein Antagonists
in Heart Disease
Joel S. Bennett .................................................................................................. 107
7 The Sodium–Hydrogen Exchange System in the Heart:
A Target for the Protection of the Ischemic and Reperfused Heart
and Inhibition of Heart Failure
Morris Karmazyn ............................................................................................ 123
8 Nitric Oxide and Cardiac Ischemia
Mariarosaria Bucci, Inmaculada Posadas Mayo,
and Giuseppe Cirino ................................................................................... 141

III FUNCTIONAL ENDPOINTS EVALUATING CARDIAC DRUG ACTIVITY

9 Ionic Mechanisms of Atrial Fibrillation
J. Christian Hesketh and David Fedida ...................................................... 163
10 Targeting Ischemic Ventricular Arrhythmias
Michael J. A. Walker and Leon J. Guppy .................................................... 175
11 Biochemical Mediators of Ventricular Arrhythmias
in Ischemic Heart Disease
Hugh Clements-Jewery and Michael J. Curtis ........................................... 203

vii
viii Contents

12 Heterologous Expression Systems and Screening Technologies

in Ion Channel Drug Discovery
Maurizio Taglialatela .................................................................................... 227
13 Molecular Diversity of Ion Channels in the Mouse Heart:
A Suitable Model for Cardiac Drug Development?
Jeanne M. Nerbonne ........................................................................................ 245
14 Delayed Protection of the Myocardium:
A Novel Therapeutic Window for Cardiac Drug Development
László Szekeres and James Parratt ............................................................. 271

IV CLINICAL ASPECTS IN CARDIAC DRUG DEVELOPMENT

15 Cardiac Troponin Testing for Detection of Myocardial Infarction:
Clinical Utility and Analytical Issues
Fred S. Apple .................................................................................................... 297
16 A Genetic Basis for Cardiac Arrhythmias:
Current Status and the Future
Jeffrey A. Towbin, Matteo Vatta, Hua Li, and Neil E. Bowles ............. 317
17 Myocardial Adenoviral Vector Delivery for Cardiovascular
Gene Therapy
Hendrik T. Tevaearai, Andrea D. Eckhart, and Walter J. Koch ............. 351
18 The Role of Pharmacometrics in Cardiovascular
Drug Development
Paul J. Williams, Amit Desai, and Ene Ette .............................................. 365
19 Gender Differences in Heart Failure:
Concerns for Drug Development
Mark A. Sussman ............................................................................................ 389
20 Angiogenesis Therapies for Coronary Artery Disease:
Trials and Tribulations
Michael Simons ............................................................................................... 407
Index ................................................................................................................... 421
 Contributors

FRED S. APPLE • Clinical Laboratories, Hennepin County Medical Center

and Department of Laboratory Medicine and Pathology, University of Minnesota,
School of Medicine, Minneapolis, MN
MIROSLAV BARANCIK • Department of Cardiovascular Physiology, Institute
for Heart Research, Slovak Academy of Sciences, Slovak Republic
JOEL S. B ENNETT • Hematology-Oncology Division, Department of Medicine,
University of Pennsylvania School of Medicine, Philadelphia, PA
NEIL E. BOWLES • Department of Pediatrics, Baylor College of Medicine, Houston, TX
MARIAROSARIA BUCCI • Department of Experimental Pharmacology,
Faculty of Pharmacy, University of Naples, Naples, Italy
GIUSEPPE CIRINO • Department of Experimental Pharmacology,
Faculty of Pharmacy, University of Naples, Naples, Italy
HUGH CLEMENTS-JEWERY • Centre for Cardiovascular Biology and Medicine,
King’s College London, London, UK
MICHAEL J. CURTIS • Centre for Cardiovascular Biology and Medicine,
King’s College London, London, UK
AMIT DESAI • Department of Pharmacy Practice, School of Pharmacy,
University of the Pacific, Stockton, CA
ANDREA D. ECKHART • Department of Surgery, Duke University Medical Center,
Durham, NC
ENE ETTE • Vertex Pharmaceutical Corporation, Cambridge, MA
DAVID FEDIDA • Department of Physiology, University of British Columbia,
Vancouver, Canada
LEON J. GUPPY • Department of Pharmacology and Therapeutics,
University of British Columbia, Vancouver, Canada
LASZLO HEJJEL • Division of Cardiac Surgery, Heart Institute, Medical Faculty,
University of Pecs, Pecs, Hungary
J. CHRISTIAN HESKETH • Department of Experimental Medicine, Cardiome Pharma
Corporation, Vancouver, Canada
MORRIS KARMAZYN • Department of Physiology and Pharmacology,
University of Western Ontario, Ontario, Canada
WALTER J. KOCH • Department of Surgery, Duke University Medical Center,
Durham, NC
DAVID J. LEFER • Department of Molecular and Cellular Physiology,
Louisiana State University Health Sciences Center, Shreveport, LA
HUA LI • Department of Pediatrics, Baylor College of Medicine, Houston, TX

ix
x Contributors

JEANNE M. NERBONNE • Department of Molecular Biology and Pharmacology,

Washington University Medical School, St. Louis, MO
JAMES PARRATT • Department of Physiology and Pharmacology,
University of Strathclyde, Strathclyde Institute for Biomedical Sciences,
Glasgow, Scotland
INMACULADA POSADAS MAYO • Department of Experimental Pharmacology,
Faculty of Pharmacy, University of Naples, Naples, Italy
M ICHAEL K. P UGSLEY • Department of Pharmacology, Forest Research Institute,
Jersey City, NJ
TANYA RAVINGEROVA • Department of Cardiovascular Physiology,
Institute for Heart Research, Slovak Academy of Sciences, Slovak Republic
ELIZABETH ROTH • Department of Experimental Surgery, Medical Faculty,
University of Pecs, Pecs, Hungary
DAVID A. SAINT • Cellular Biophysics Laboratory, Department of Physiology,
University of Adelaide, Adelaide, Australia
MICHAEL SIMONS • Section of Cardiology, Dartmouth Medical School,
Dartmouth Hitchcock Medical Center, Hanover, NH
MELANIE B. SMITH • Department of Molecular and Cellular Physiology,
Louisiana State University Health Sciences Center, Shreveport, LA
MONIKA STRNISKOVA • Department of Cardiovascular Physiology,
Institute for Heart Research, Slovak Academy of Sciences, Slovak Republic
MARK A. SUSSMAN • Division of Molecular Cardiovascular Biology,
The Children’s Hospital and Research Foundation, Cincinnati, OH
LÁSZLÓ SZEKERES • Department of Pharmacology and Pharmacotherapy,
University of Szeged, Szeged, Hungary
MAURIZIO TAGLIALATELA • Section of Pharmacology, Department of Neuroscience,
School of Medicine, University of Naples Federico II, Naples, Italy
HENDRICK T. TEVAEARAI • Department of Surgery, Duke University Medical
Center, Durham, NC
JEFFREY A. TOWBIN • Department of Pediatrics, Cardiovascular Sciences
and Molecular Human Genetics, Baylor College of Medicine, Houston, TX
MATTEO VATTA • Department of Pediatrics, Baylor College of Medicine, Houston, TX
MICHAEL J. A. WALKER • Department of Pharmacology and Therapeutics,
University of British Columbia, Vancouver, Canada
PAUL J. WILLIAMS • Department of Pharmacy Practice, School of Pharmacy,
University of the Pacific, Stockton, CA
Cardiac Drug Development 1

I
INTRODUCTION
Cardiac Drug Development 3

1
Cardiac Drug Development
From Animal Models to Clinical Trials

Michael K. Pugsley

CONTENTS
CARDIAC DRUGS: THE MARKET POTENTIAL FOR ANTIARRHYTHMIC DRUG DEVELOPMENT
GENESIS OF THE CARDIAC ACTION POTENTIAL (CAP)
ISCHEMIA-INDUCED CHANGES IN MYOCYTES PRODUCE CHANGES IN THE EKG
THE ROLE OF CARDIAC ION CHANNELS IN HEART DISEASE
SAFETY PHARMACOLOGY
METHODS USED TO INVESTIGATE THE ROLE OF ION CHANNELS
IN CARDIAC DRUG DEVELOPMENT
HIGH THROUGHPUT SCREENING (HTS) METHODS IN CARDIAC DRUG DEVELOPMENT
CLINICAL DRUG TRIALS
REFERENCES

1. CARDIAC DRUGS: THE MARKET POTENTIAL FOR

ANTIARRHYTHMIC DRUG DEVELOPMENT
Statistics from the American Heart Association show that the total number of
arrhythmia-related mortalities is approx 500,000 of an estimated 2,000,000 US deaths
per year, or nearly one-quarter of all cardiovascular-related deaths (1). The majority of
such deaths, which have remained at a nearly constant ratio since the 1970s when car-
diac drug development programs were formally being established within the pharma-
ceutical industry, are caused by ventricular fibrillation (VF; Fig. 1).
The development of novel, effective cardiac (antiarrhythmic) drugs was fueled by
the need for agents that would possess the so-called ideal pharmacological properties,
including high oral bioavailability, marked efficacy and selectively for the abolition of
ectopic ventricular arrhythmias, and a reduced adverse events profile (e.g., reduced
hypotensive actions and lack of proarrhythmic tendencies). The majority of drugs
developed at that time were analogs of lidocaine; however, structurally distinct classes
of drugs were developed, including the trifluoroethoxybenzamides (flecainide). These
compounds, according to the Vaughan Williams classification scheme (2), were class I
antiarrhythmic agents, that is, those that reduce the influx of sodium (Na+) ions during
phase 0 of the action potential (AP). In the mid-1980s, the development of this abun-
dant group of diverse new chemical entities with activity in animal models of
From: Cardiac Drug Development Guide
Edited by: M. K. Pugsley © Humana Press Inc., Totowa, NJ

3
4 Pugsley

Fig. 1. The ischemic heart produces a broad spectrum of arrhythmias that precipitate sudden
cardiac death. Antiarrhythmic drug therapy (ion channel-blocking drugs) can suppress fatal
arrhythmias and produce a normal EKG rhythm. Unfortunately, although these drugs are benefi-
cial, many possess side effects, including myocardial depression and proarrhythmic tendencies.

arrhythmias and efficacy in the clinic (phase 1 and 2 studies) resulted in their evalua-
tion in larger phase 3 clinical trials. The Cardiac Arrhythmia Suppression Trial (CAST-
I) study was undertaken to examine whether the incidence of cardiac death in patients
with asymptomatic or mild ventricular arrhythmias, post-myocardial infarction (MI),
could be reduced with class I antiarrhythmic drugs (3). The CAST-I clinical trial with
flecainide, encainide, and later moricizine in CAST-II (4) and mexiletine in the Inter-
national Mexiletine and Placebo Antiarrhythmic Coronary Trial (IMPACT) trial
showed an abnormally high incidence of death in drug-treated groups when compared
with placebo controls (5). Thus, these clinical trials, albeit not showing marked effi-
cacy of these drugs, provided a valuable lesson as to the complex interrelationship that
exists among the antiarrhythmic drug used, the arrhythmogenic substrate, and resulting
drug efficacy.
Thus, despite the large number of experiments and clinical trials that have been
conducted, remarkably few Na+ channel-blocking drugs are used clinically to suppress
arrhythmias. This low number of drugs accentuates the findings from the CAST-I and
CAST-II trial as well as others (3,4). Many studies with a variety of Na+ channel-
blocking antiarrhythmic agents suggest that these drugs can effectively suppress
arrhythmias; however, this does not necessarily result in an improved survival rate
after a myocardial infarction in patients (6). Quinidine, for example, was shown clini-
cally to increase the incidence of mortality as compared with mexiletine (7), and
Cardiac Drug Development 5

lidocaine, although abolishing fatal VF, does not significantly improve survival in post-
MI patients (8).
The class II (beta-blocking) antiarrhythmic agents, typified by propranolol, atenolol,
and esmolol, are the only drugs that have been consistently shown to produce an
increased time to onset of ventricular tachycardia (VT; ref. 9), a reduction in arrhyth-
mia incidence (10), and to improve survival post-MI (11). A true appreciation for these
actions seems to have been overlooked despite the overwhelming abundance of data
describing the efficacy of this class of antiarrhythmic drug (12). Both the class III K+
channel-blocking and class IV Ca2+ channel-blocking agents have, in contrast, exhib-
ited poor results in post-MI clinical trials (13).
An examination of the developmental patterns for antiarrhythmic drugs suggests
that because of the poor performance of antiarrhythmics in the past, there must be
continued research in this area. Therefore, novel molecular targets must be determined
and drugs for these targets developed. Some novel cardiac targets include a pathologi-
cally targeted approach whereby drugs have been developed that show marked selec-
tivity for myocardial ischemia and have a greater therapeutic index when compared
with prevous antiarrhythmic drugs (14). Sodium-hydrogen exchange inhibitor drugs,
such as cariporide, which target the cardiac-specific isoform of this membrane pump,
may also have activity against arrhythmias and efficacy in congestive heart failure
(15). Thus, because of the large number of mortalities each year from cardiac disease,
there is a very large unmet medical need for more efficacious antiarrhythmic drugs.
Current medical expenditures in the United States for arrhythmias and arrhythmia-
related conditions are over $1 billion, and the unmet medical market is estimated to be
well over $3 billion.

2. GENESIS OF THE CARDIAC ACTION POTENTIAL (CAP)

To develop effective cardiac drugs, it is necessary to understand the basic tenets of
heart function. The coordinated electrical activity of cardiac muscle results from the
establishment of transmembrane potentials that culminate from an integration of many
different ion channels. The corresponding changes in ionic permeability results in the
flow of current producing contraction of heart muscle.APs in excitable cells result
from the presence of voltage-gated ion channels (16) that open and close depending
on the voltage across the membrane. Depolarization of the resting membrane poten-
tial causes the opening of voltage-gated Na+ channels. This increase in Na+ perme-
ability results in the development of an inward current that enhances depolarization.
Membrane potential is re-established by the rapid inactivation of voltage-gated Na+
channels and opening of voltage-gated K+ channels. In the heart membrane, potential
is complicated by the presence of voltage-gated Ca2+ channels. These channels medi-
ate the slow inward current (Isi) that is responsible for the plateau phase of the AP. In
some tissues, such as the sinoatrial and atrioventricular nodes, voltage-gated Ca2+
channels predominate and produce the AP.
The CAP (Fig. 2A) for a ventricular myoctye consists of 4 phases. A rapid upstroke
(phase 0) is followed by a brief peak (phase 1) followed by a sustained plateau (phase 2).
A rapid repolarization (phase 3) begins after several hundred milliseconds, and this is
followed by phase 4 that persists until the next rapid upstroke event. Thus, the shape of
the AP is governed by ionic current flux via gated channels in the membrane for Na+,
6 Pugsley

Fig. 2. The temporal association between some cardiac ion channels involved in the genesis
of the AP and the EKG. (A), a ventricular AP is depicted with ion currents involved in its
genesis. Although ion channels vary in species the fast upward spike generated by Na+ (INa)
represents the QRS complex of the EKG (B). Ventricular repolarization results from the com-
bined actions of many K+ currents and is delineated in the EKG by the upward deflection of the
T-wave (B).

Ca2+ and K+ (Fig. 2A). Membrane pumps and exchangers are involved as well. The
properties of the AP change moderately among tissue types. In pacemaker cells of the
nodal tissues phase 4 is characterized by a slow, steady depolarization from the resting
membrane potential (Vm) that leads to a threshold potential. When this potential is met,
a rapid upstroke (phase 0) results and a nodal AP develops that is composed of similar
phases as in ventricular or other cardiac cells. Although there may be some differences
regarding phase 4 development in various cardiac tissue, the fundamentals of AP gen-
eration are unchanged. The pacemaker current shapes the periodicity of oscillations in
the heart because this current is activated by the hyperpolarized cell membrane at the
conclusion of the AP.
Cardiac Drug Development 7

The inward voltage-gated Na+ channel is responsible for producing phase 0 and the
rapid upstroke of the cardiac AP. Although Na+ channels rapidly inactivate as Vm
approaches the equilibrium potential (0 mV), a second voltage-gated ion channel is
activated that is carried by Ca2+ ions. Calcium channels carry Isi that is responsible for
the plateau phase of the AP (Fig. 2A). Although many Ca2+ channel subtypes occur,
there are at least two isoforms found in the heart: the L and T types.
Within a short period of time (125 ms), cardiac Ca2+ channels inactivate and K+
channels activate. Repolarization is rapid when the total outward K+ current becomes
appreciably greater than inward Ca 2+ current. A large number of voltage- and
nonvoltage-gated K+ currents are involved in repolarization of the cardiac AP. The
voltage-gated K+ currents include the transient outward K+ current (Ito), one of the
earliest channels to open; the outward or delayed rectifier current (IK), which opens at
the end of phase 2 and is the main K+ current responsible for ventricular repolarization.
The last to open is the inward rectifier K+ current (IK1) which, unlike other K+ currents,
closes during depolarization and is responsible for maintenance of the resting mem-
brane potential (Fig. 2A).

2.1. The Cardiac Na+ Channel

Depolarization of the cell membrane opens Na+ channels. Molecular studies have
revealed characteristics of the Na+ channel. All voltage-gated Na+ channels are com-
prised of ~2000 amino acids and contain four homologous internal repeats (DI–DIV),
each of which has six putative transmembrane (SI–S6) segments (17). The subunit is
the protein that forms the ion channel pore (17). Recently, Sato et al. (18) determined
the crystal structure of the Na+ channel and suggests that the Na+ channel α-subunit is
a bell-shaped membrane protein (18).
Most Na+ channels are heterotrimeric complexes in the membrane. The α-subunit
(~260 kDa) interacts with at least two small auxiliary β subunit proteins. The β1 sub-
unit (~36 kDa) regulates current amplitude and refines channel kinetics for neuronal
isoforms but not the cardiac isoform of the channel (19). The β2 subunit (~33 kDa)
modulates Na+ channel localization in tissue (20). Many subtypes of cardiac voltage-
gated Na+ channels have been described.
Despite many isoforms, ionic conductance of the Na+ channel is transient. Activa-
tion occurs rapidly, and prolonged depolarization produces Na+ channel inactivation,
preventing the continued influx of Na+ into the myocyte. Molecular studies reveal that
the α-helical intracellular linker between domains III and IV (DIII–DIV) of the Na+
channel is responsible for inactivation and channel closure (21).
Local anesthetics and antiarrhythmic drugs interact with the inactivation gate (22).
The inactivation produced by a change in membrane potential and drug block of the
channel are interacting processes. These occur as a result of drug binding to a site on or
near the inactivation gate in a voltage-, time-, and channel state-dependent manner
according to the modulated receptor hypothesis. This model suggests that as Na+ chan-
nels change states antiarrhythmic drugs can associate or dissociate from these states
(23,24). Evidence exists for a specific binding site on the Na+ channel for drugs.
Ragsdale et al. (25) identified a putative antiarrhythmic drug binding site on the S6
transmembrane spanning region of domain IV (DIVS6) that lines the pore of the Na+
channel. Thus there exists a greater promise for cardiac drug development as a result of
the molecular localization of drug action in the heart. A return of the membrane poten-
8 Pugsley

tial to its predepolarizing (resting) level begins with activation of Ca2+ current and
repolarizing K+ currents.
2.2. Cardiac Ca2+ Channels
Voltage-gated Ca2+ channels are important regulators of electrical signaling and
mechanical function in the heart. In the myocyte, Ca2+ is highly regulated by voltage-
gated Ca2+ channels, Ca2+ pumps and by the Na+/Ca2+ exchanger.
Calcium channels are responsible for the genesis of APs in cardiac pacemaker cells,
the propagation of APs in sinoatrial and atrioventricular node cells and in the control of
depolarization-induced Ca2+ entry responsible for the plateau (phase 2) of the CAP
(Fig. 2A).
Voltage-gated Ca2+ channels are hetero-oligomeric protein complexes that are com-
prised of a α1 (~240 kDa) subunit, a β subunit (~60 kDa), and an accessory α2-δ (~175
kDa) subunit (26). Currently, six classes of voltage-gated Ca2+ channel have been char-
acterized. In the heart, there is a single L- and T-type Ca2+ channel and each mediate an
important action in the genesis of the AP.
The α1 subunit of the Ca2+ channel (~1800 amino acid residues) is the major protein
constituent that contains the ionic pore, selectivity filter, and gating machinery. In car-
diac ventricular muscle the α1C subunit encoding the L-type Ca2+ channel is found at
appreciably high levels (>80%) whereas α1D subunit expression dominates atrial
muscle (26). Of the three isoforms of the α1 subunit that encode for the T-type channel
only α1G and α1H are found in cardiac tissue (26,27).
The α1 subunit also contains the binding domain for Ca2+ channel-blocking drugs.
The L-type channel is blocked by three groups of drugs. The phenylalkylamine (e.g.,
verapamil) and benzothiazepine (e.g., diltiazem) blockers are effective clinically used
antiarrhythmics whereas the 1,4-dihydropyridines (e.g., nifedipine) are useful anti-
hypertensive agents. Chemically, Ca2+ channels show a marked structural homology
to each other and to voltage-gated Na+ channels. This subunit is composed of four
homologous domains (DI–DIV) each of which is composed of six transmembrane
spanning α-helical proteins that form a pore in the membrane.
Calcium channels also require auxiliary subunits for functional expression. Cur-
rently, four mammalian isoforms of the β subunit exist but the cardiac L- and T-type
Ca2+ channels are only co-expressed with the β2-subunit isoform (28). Of the three α2-
δsubunit isoforms that have been detected in various tissues, only the α2-δ1 and α2-δ2
types are expressed in the heart (28,29). The cardiac L-type Ca2+ channels possess the
high affinity, stereoselective-binding sites for channel block. Inhibition produces anti-
arrhythmic activity against supraventricular arrhythmias. The S6 regions of DIII and
DIV may contain the actual high affinity binding sites for channel blocking drugs (26).
2.3. Cardiac K+ Channels
Interest in the development of drugs that prolong refractoriness, that is, possess class
III antiarrhythmic action, increased markedly after the negative results of the CAST
trials. Repolarization and the configuration of phase 3 of the AP in cardiac tissue
seemed the next likely target for antiarrhythmic drug development. This was especially
tantalizing because repolarization resulted from the complex interaction of multiple K+
channels providing numerous targets for drug development (30).
However, whereas individual K+ currents overlap in contribution to the total mem-
brane current during the AP, the relative importance of each may vary under differ-
Cardiac Drug Development 9

ent physiological conditions. During ischaemia, changes in cell properties may alter
the degree to which different channels contribute to the CAP. Despite these short-
comings, intensive development has continued for selective K+ channel-blocking
antiarrhyhmic drugs.
Mammalian K+ channels have been categorized into three main families: the volt-
age-gated K+ channels (Kv), the inward rectifying K+ channels (K1), and the two
pore domain channels (K2P). All K+ currents have a similar primary amino acid
sequence with highly conserved structural regions. The molecular structures of K+
channels are described as having one or two pore-forming domains and two, four, or
six transmembrane-spanning domains. The molecular diversity of K+ channels is
largely to the result of variability in the heteromeric association of pore-forming α
subunits and accessory or β-subunits (31). Voltage-dependent K+ channel α subunits
have six transmembrane spanning sequences and a pore-forming region. The
inwardly rectifying K+ channel α subunit is composed of two transmembrane span-
ning sequences and one pore-forming region.
The voltage-dependent activation of K+ currents plays a considerable role in the
repolarization of the cardiac cell membrane. Voltage-dependent inactivation may pro-
ceed either rapidly or slowly by N- and C-type inactivation, respectively (32). The
differential distribution of currents carried by K+ channels is extremely important in
the regulation of myocardial cell resting potential and repolarization and thus to the
configuration of the cardiac AP within different cells of the heart and electrocardio-
gram (EKG) morphology between species.
Thus, the heterogeneity of K+ channels provides a large potential for the develop-
ment of K+ channel-blocking drugs (33). However, class III agents are bradycardic and
prolong the action potential duration (APD) at low heart rates more effectively than
high heart rates, reducing their efficacy.
This reverse-use dependence limits their beneficial actions in the arrhythmic condi-
tion (34). The resulting bradycardia associated with new potent K+ channel blockers,
such as dofetilide and sematilide, is associated with torsade des pointes arrhythmias
(35). Note, however, that some drugs, such as amiodarone, lack this effect (34).
Thus, although many K+ channels exist in cardiac muscle, a complete overview of
only those K+ conductances, which carry most of the outward repolarizing current, is
beyond the scope of this article. Note, however, that the K+ currents that do contribute
include the transient outward K+ current, the delayed rectifier K+ (IK) current (and its
components, IKr and IKs), and the inward rectifier (IKIR) current.

3. ISCHEMIA-INDUCED CHANGES IN MYOCYTES

PRODUCE CHANGES IN THE EKG
Electrical activity in excitable cells results from the opening and closing of ion chan-
nels in a voltage- and time-dependent manner. The depolarization of a single cardiac
cell results in the electrotonic spread of electrical activity to adjacent cells and the
production of current that flows in the direction of depolarization. A second, repolariz-
ing current, is established to restore electrical excitability to cells. If these currents are
recorded in individual cells, an AP is observed (Fig. 2A); if they are recorded on the
surface of the body, an EKG is observed (Fig. 2B).
Thus, the EKG is defined as the global summation of all the electrical activity that is
generated by cells within the heart. It is the rate of change of voltage across the cell
10 Pugsley

membrane as a function of time (ΔdV/dt). The intervals that are defined by the EKG
present the clinician and basic researcher with a fundamental tool to diagnose disease
and investigate drug activity.
Whereas electrical activity generated by atrial depolarization is recorded by the EKG
as the P-wave (Fig. 2B), ventricular depolarization produces a QRS complex. Repolar-
ization of the ventricles is recorded by the EKG as the T-wave. The QT interval repre-
sents the ventricular refractory period and includes depolarization and repolarization
of ventricular muscle. In contrast to the atria, the AP in ventricular tissue is long (~300
ms) and similar to the duration of the QT interval. Thus, the QT interval is an approxi-
mate measure of ventricular repolarization and thus K+ channel function.
The ST segment is an important measure of the EKG because it represents the early
phase of ventricular repolarization. Clinically, depression in this segment can be used
to diagnose conditions, such as angina. Elevation in this segment occurs in a damaged
area of the ventricular wall that may be associated with myocardial ischemia or infarc-
tion. However, any abnormality in these measures may be indicative of some underly-
ing pathophysiological process that alters the AP in cardiac cells that are a consequence
of changes in voltage-gated ion channel(s) in tissue (36).
Because the EKG is a composite of voltage-gated ion channels, it is not consistent
between various animal species. The variability is quantitative, that is, measureable
differences in current densities can be recorded and qualitative, that is, EKG shape
varies because of expression of ion channel(s) distinct from that found in the human
heart (Fig. 3). Although little disparity exists regarding the role of both Na+ and Ca2+
channels in the hearts of various species, important species and regional differences
exist in the contribution K+ channels make to repolarization of the cardiac AP. This
disparity is of particular importance when determining which species to use in estab-
lishing relevant in vivo and in vitro animal models to assess the activity of novel car-
diac drugs.

4. THE ROLE OF CARDIAC ION CHANNELS IN HEART DISEASE

Implicit in the development of novel cardiac drugs is an understanding of the
molecular mechanism(s) responsible for arrhythmogenesis and the proarrhythmic pro-
pensity observed for many antiarrhythmic drugs. The blockade of Na+ channels in the
heart by antiarrhythmic drugs reflects limited selectivity to ischemic tissue. Therefore,
drug blockade produces an increase in the rate of delayed conduction in ischemic-
damaged muscle without either the abolition of excitability or the production of com-
plete conduction block (37). This disparity increases the probability for the
development of re-entrant arrhythmias. Therefore, slowed conduction in injured heart
tissue along with a reduction in electrical conduction in normal tissue surrounding the
damaged area may be an important factor in arrhythmogenesis.
Mutations in the cardiac Na+ channel has directly implicated it in inherited cardiac
disease. In the heart, mutations in NaV1.5 produce dramatic changes in the Na+ chan-
nel. Usually inherited as autosomal-dominant mutations, these changes prolong the QT
interval. Mutations result from an amino acid deficit in the DIII–IV linker responsible
for normal channel inactivation (38). The predominant intragenic mutation, a deletion
of the amino acids K1505P1506Q1507 (ΔKPQ) and two missense mutations (R1644H and
N1325S) produce channels, but with altered inactivation properties (38,39).
Cardiac Drug Development 11

Fig. 3. A comparison of some EKG recordings from various species. (A) depicts a human
EKG, (B) from a rat, and (C) from a rabbit. Note that the EKG tracings are not to scale.

As with many diseases, there is a prominent genetic component to the etiology of

cardiac arrhythmias that relates to alterations in ion channel function. Because many
ion channels mediate a basis for electrical activity in myocytes, it is of no surprise that,
in a manner similar to that for Na+ channels, altered Ca2+ and K+ ion channel properties
produce disease. The search is now on to determine the functional consequences asso-
ciated with aberrant ion channel proteins for which novel antiarrhythmic drugs may be
developed. Mechanistically, these findings will provide crucial information as to
whether these disorders result from faulty voltage-gated ion channels themselves or
from mutations in the regulatory protein components of the electrical-excitation cou-
pling system in the heart.
The best characterized of the cardiac disorders involving a K+ channel is the long-
QT syndrome (LQTS). It is a specific cardiac disorder related to acquired or inherited
alterations in IK channel function and displays a torsade des pointes phenotype. The
majority of genetic loci (11p15 in humans) that have been identified contain the genes
responsible for LQTS and encode those subunits for the IK channel (39,40). In mam-
mals a gene from the KCNQ (KvLQT) subfamily, KCNQ1, when co-expressed with
the auxiliary subunit, minK (or KCNE1), produces functional K+ channels (LQTS1)
similar to the slowly activating component of the IK current (IKs; ref. 40).
12 Pugsley

However, the association of human ether-a-go-go (HERG; or KCNH2) with KCNE1

(minK) or KCNE2 (MiRP, a minK homolog) produces the molecular equivalent of the
rapidly activating Ik (IKr) (41). Many mutations within the HERG α subunit suggest it is
a mediator of LQTS2 (42).
Before the delineation of the molecular correlates for LQTS, the development of
potent and selective K+ channel blockers resulted in QT prolongation and induction of
torsade arrhythmias (42). Studies indicated that methanesulfonamide drugs were potent
blockers of native IKr channels in cardiac tissue and the molecular correlates of IKr, the
HERG K+ channels. Thus these drugs produce heterogeneity of refractoriness and pre-
dispose one to arrhythmia incidence. This is now a concern for the pharmaceutical and
biotechnology industries, as well as government agencies (e.g., Food and Drug Admin-
istration [FDA]) because many cardiac (and noncardiac) drugs exhibit QT interval pro-
longation and may also block HERG K+ channels. Thus, the potential for these adverse
actions of a drug, unrelated to their primary pharmacological actions, has prompted the
establishment of regulatory requirements for safety pharmacology studies that outline,
using good laboratory practice methods to investigate novel cardiovascular (and
noncardiovascular) drugs.

5. SAFETY PHARMACOLOGY
In the simplest sense, pharmacological studies can be separated essentially into three
main categories that characterize (1) the primary pharmacodynamic, (2) the secondary
pharmacodynamic actions of a new drug, which determines the mechanism of drug
action, and (3) the safety pharmacology of the new drug.
Safety pharmacology is defined as those studies that investigate any potential
undesirable pharmacological effects of a drug on normal physiological function (43).
These studies are not toxicological in nature but rather are an attempt to characterize
whether or not a dose–response relationship exists between a drug and the observed
response.These studies are conducted before drug administration to humans (phase 1
clinical trials) to reduce the likelihood of adverse events from occurring in the clini-
cal setting. The information excogitated from such studies provides supplemental
knowledge relating to the mechanism of action responsible for the observed benefi-
cial, as well as any potential adverse, pharmacological effects. Safety pharmacology
studies primarily examine the effects of a drug on three main vital organ systems
using a core battery of in vitro and in vivo test systems. These vital organ systems are
the central nervous system, respiratory system, and the cardiovascular system. All
studies are conducted, optimized, and validated according to International Confer-
ence on Harmonization (ICH) ethics and scientific quality standards that reflect proper
study design, conduct of experiments, data-recording methods, and data-
reporting practices.
Studies are conducted according to good laboratory practice regulations set forth
by global regulatory agencies (43,44). Thus, safety pharmacology is a unique new
discipline that bridges the gap that currently exists between pharmacology and toxi-
cology. Thus, it is an important consequential means by which to ensure the safety
of drugs, especially those being developed for cardiac disease, before their use in
human studies. Thus, all new drugs must undergo rigorous safety pharmacology
screening methods.
Cardiac Drug Development 13

6. METHODS USED TO INVESTIGATE THE ROLE

OF ION CHANNELS IN CARDIAC DRUG DEVELOPMENT
6.1. In Vivo Methods
The following describes some of the methods that can used to characterize the
pharmacological actions of novel drugs on the heart. These methods are not defined
according to specific use in either general or safety pharmacology studies, but are
simply divided into in vivo and in vitro methods. In vivo methods require the use of
whole animals (mice to primates) where the heart is functionally connected to the
circulation. Selection of animal species may vary with the proposed candidate mol-
ecule, thus, a thorough understanding of the physiology and functional ion channel
status of the species selected should be known. These methods are concerned with
measuring the effect of the drug on electrical and mechanical heart performance. The
effect on the mechanical performance of the heart in vivo can be evaluated in terms
of cardiac output and ventricular pressure whereas actions on electrical properties
are characterized using the EKG.
Many direct and indirect methods exist to determine drug activity on cardiac output.
Direct measurement of cardiac output is best accomplished using flow probes. Flow
probes can be surgically implanted around blood vessels and the volume of blood flow-
ing through the vessel measured. Although many different types of flow probes are
available for use, the most common are ultrasonic and electromagnetic (45).
Electromagnetic flow probes provide a greater accuracy for absolute measures of blood
flow in a vessel than do ultrasonic flow probes. Ultrasonic flow probes, however, are useful
for measuring patterns of changes in flow because they use the Doppler principle of signals
reflected from moving blood in the vessel rather than the distortion created by moving red
blood cells in the magnetic field of the electromagnetic flow probe. This should be consid-
ered when investigating the hemodynamic actions of a drug.
Rather than the direct measurement of cardiac output by flow probes, it is possible to
use indirect dye dilution techniques that use tracer materials, such as indocyanine green
or Evan’s blue dye (46). The introduction of thermodilution methods has allowed for
the precise measurement of cardiac output. This clinically used technique has been
adapted for use in animal experiments; however, validation and optimization of the
method is critical for accurate determination of the effects of novel cardiac drugs.
Cardiac output measurement is the preeminent method for evaluation of the effects
of a drug on heart function. However, there are other physical indices of cardiac func-
tion that can complement cardiac output studies. Cardiac contractility (performance)
depends upon filling pressure in the ventricle and the initial or diastolic fiber length.
Therefore, drug effects on such parameters should be assessed. End diastolic pressures
are easily measured in the right ventricle in vivo by the direct positioning of a catheter
into the right ventricle. Left ventricular pressure recording is best accomplished by
measuring pulmonary wedge pressures.
The methods described above provide the best indices of the functional state of the
heart. Effective cardiac contraction, in the absence and presence of the candidate drug
molecule, can only occur in vivo if adequate delivery of oxygen is supplied to the
myocardium and metabolic waste products removed. Thus, coronary blood flow should
be measured globally and regionally in these studies. Coronary blood flow can be mea-
sured using flow probes placed around a coronary artery or by using microspheres (47).
14 Pugsley

Electrical activity in the heart can be examined at many levels providing the
researcher with the ability to quantitate the effects of novel cardiac drugs at all levels.
Global electrical activity is most easily quantified using the EKG. The electrical activ-
ity can be examined in different chambers and in various anatomical locations within
the heart by insertion of recording electrodes into those areas (or chambers) of the
heart (48). With the placement of stimulating electrodes into any of these areas, it is
possible to electrically challenge the heart. The electrical stimulation method allows
for an assessment of the ion channel status (Na+ and K+) within cardiac tissue in
the absence and presence of cardiac drug (48). This method also permits for an evalu-
ation of the vulnerability of the ventricle to arrhythmia (premature ventricular contrac-
tion [PVC] or VF) induction, thus, is an excellent method with which to rapidly screen
novel drugs for cardiac activity.
Extracellular monophasic action potentials (MAPs) can be recorded from the
endocardium or epicardium of atria, ventricles, or the whole heart (49). The MAP is an
extracellularly recorded potential that approximates the time course of the intracellular
AP. The MAP is generated by the application of pressure to the contact electrode against
the cardiac muscle wall. The MAP can be used to record myocardial activation time,
repolarization (phases 1–3) of the CAP, dispersion of APD, postrepolarization refrac-
toriness, early after-depolarization development, ischemia-induced changes in the
APD, and the actions of cardiac drugs (49).
Arrhythmias can seriously impair mechanical function of the heart. Thus, although
VT limits or impairs cardiac output, VF terminates cardiac output. Arrhythmias can be
detected experimentally by changes in both blood pressure and the EKG. The experimen-
tal methods used to induce arrhythmias are both numerous and complex. Arrhythmias
may be produced electrically (as discussed previously), chemically or by ischemia (50).
The induction of arrhythmias by chemical methods is usually performed in small
animal species. Standard chemical methods for the induction of arrhythmias include
the use of cardiac glycosides in guinea pigs, chloroform in mice, aconitine in rats and
catecholamines to dogs (48,50). Note that the types of arrhythmias that result are spe-
cific to the chemical agent used and are species dependent. Although the arrhythmias
that result are reproducible and exhibit characteristic sensitivity to historical or control
antiarrhythmic drugs, the mechanism(s) responsible for the development of the arrhyth-
mia are not well defined. However, the use of these proarrhythmic models provide a
rapid in vivo screening system with which to investigate the potential efficacy of novel
cardiac drugs that can be compared with current, clinically used drugs.
Appropriate electrical stimulation, applied though the implantation of electrodes into
any part of the heart, can result in the induction of arrhythmia (48). A large variety of
defined stimulation protocols can be used and can be chosen for either the induction of
arrhythmias or for indirectly probing the functional status of either Na+ or K+ channels.
For example, electrical currents and pulse widths required to induce single extra beats
in the myocardium reflect Na+ channel availability and excitability (or i-t curves)
whereas refractory periods or effective refractory period (ERP) reflect Na+ channel
status but are highly dependent on K+ channels which control repolarization (46).
The types of arrhythmias induced by electrical stimulation include single extra
beats, VT, or VF. These studies can be conducted in normal intact hearts or in previ-
ously ischemic or infarcted hearts. Thus, the flexibility that results from the avail-
ability of the numerous stimulation protocols to probe arrhythmia propensity and ion
Cardiac Drug Development 15

channel activity also makes this a very useful method with which to investigate novel
cardiac drugs.
However, the most clinically relevant method for arrhythmia induction results from
the occlusion of a coronary artery. This deprives blood flow to the cardiac region
subserved by the artery, results in ischemia, and produces changes representative of a
heart attack. The continued occlusion of the coronary artery results in MI. Both the
duration of coronary artery occlusion and the size of the developed ischemic area
determine the extent of infarction. These factors then determine the severity of
arrhythmias that develop (51). Reperfusion, which results from the release of the
coronary artery from occlusion, produces arrhythmias that appear similar in mor-
phology to those that result from ischemia alone but vary markedly in their develop-
ment characteristic (51).
VT and VF in these animal models occur at critical times after coronary artery
occlusion or reperfusion subsequent to a preceding ischemic period. These methods
can be used in all species but care should be taken when selecting the species with
which to conduct arrhythmia studies because coronary collateralization in the heart is
critical to the size of the developing ischemic area or infarct (52).
Collateralization varies to a large degree among the hearts of different animal spe-
cies. The hearts of guinea pigs have an abundance of coronary collateral vessels, which
prevents its use in the study of regional myocardial ischemia (46). Arrhythmic
responses are also not uniform in dogs because of the extent of pre-existing coronary
collateralization (50). Coronary collaterals are not present to any significant degree in
the hearts of species such as rats, pigs, rabbits, and primates; this makes them better
candidates as models with which to investigate responses to ischemia and reperfusion
in the heart (50).
To accurately assess the efficacy of a novel cardiac drug, it is necessary to quantitate
the area of ischemia or infarction that is produced by the most physiologically relevant
animal model. Many diverse experimental methods can be used to quantitate the size of
the infarction produced in the heart. The most accurate methods are those that directly
quantitate the anatomical size of the infarct. Such gross anatomical methods include
cross-sectioning the ventricle into slices and estimating the area of infarction found in
each slice. Demarcation between infarcted, ischemic, and normal myocardial tissue
can be resolved using stains that chemically interact with normal tissue and not necrotic
tissue (46). These provide an accurate estimate of infarct size that is easily determined
by visual examination providing reliable results in drug assessment.

6.2. In Vitro Methods

Many types of in vitro cardiac preparations are used to study cardiac function and
the electrophysiological effects of drugs on myocardial cells. Methods include single
myocardial cells, isolated cardiac tissue preparations, the Langendorff isolated rat heart,
and heterologous expression systems.
Although single adult myocardial cells are isolated from many different animal spe-
cies, including humans, the rat and guinea pig remain the principle species used.
Enzymes are perfused into isolated hearts and the dissociated cells harvested for use in
electrophysiological and biochemical study. The main advantage of the use of single
isolated myocytes is that the unknown electrophysiological actions of a newly devel-
oped cardiac drug can be investigated independent of other cells in the heart.
16 Pugsley

The patch clamp technique revolutionized cardiac drug discovery because it was
possible to record current flow through ion channels in the many cells that comprise
the CAP. The electrophysiological actions of a drug are investigated using many con-
figurations of the patch clamp. Whole-cell recording, in which the total ionic current
that flows across the cell membrane is recorded, and patch clamp recording, in which
the current that only flows across a small membrane patch is recorded, are the most
commonly used (53). However, for drug study, the whole-cell configuration is pre-
ferred. This method allows the investigator to determine the effects of the drug on
many different ion channels using electrophysiological protocols that probe the func-
tional properties of the channel. Many properties of the channel can be examined in the
absence and presence of various concentrations of the investigational drug. The goal of
these studies is to characterize the molecular actions of the drug in an attempt to eluci-
date both its site of action and putative mechanism.
Isolated tissue preparations have become a cornerstone of the physiological and
pharmacological evaluation of natural and synthetic drugs. Note, however, that the
extrapolation of drug effects on isolated tissue preparations to the whole animal is
limited by the complexity of mechanisms present in intact animals. Nevertheless, the
assessment of the pharmacological action of antiarrhythmic drugs in isolated tissues
is an essential step in clarifying the actions of a novel drug and in determining further
studies in intact animals.
The cardiovascular system is a rich source of tissues for in vitro studies. Therefore,
to investigate drug action some of the most widely used cardiac preparations include
isolated atrial or ventricular tissue, papillary muscles and the isolated, coronary-per-
fused right ventricular wall (54). The use of these tissue preparations in drug investiga-
tions is numerous. The small size of most cardiac tissues allows for the rapid and
continuous diffusion of oxygen and nutrients to subcellular layers, resulting in viable,
stable preparations. These preparations from different animal species are extensively
used to represent the heart; therefore, the type of isolated tissue preparation used when
attempting to study problems concerning drug actions or cardiac function must be care-
fully considered.
The isolated perfused whole heart is the most physiologically relevant isolated car-
diac tissue. This preparation has many advantages compared with either isolated
myocytes or isolated tissues because it lends itself to the study of the actions of cardiac
drugs on mechanical, electrical, and biochemical properties of the heart. The isolated
Langendorff heart is a simple preparation with which to screen for the cardiac actions
of drugs because it uses physiological buffers to maintain normal heart function (55).
The greatest function of the isolated perfused heart is its usefulness in assessing
drug actions on the rate of generation of ventricular pressure. It is also a sensitive
indicator of the chronotropic and inotropic actions of drugs on the heart. Coronary flow
and a surface EKG can also be recorded as supplementary indices of drug action on
coronary vasculature resistance and ion channels. Drug activity can also be assessed in
diseased hearts.
Equally, ischemia and reperfusion arrhythmias can be investigated in isolated per-
fused hearts. Occlusion of a coronary artery in an isolated perfused heart produces
arrhythmias. The antiarrhythmic efficacy of the drug under investigation can readily be
compared with that observed in vivo in the absence of blood. Isolated hearts can also
be rendered globally or regionally ischemic and the effect of the drug can be investi-
Cardiac Drug Development 17

gated on measures, such as oxygen consumption and other markers of cardiac metabo-
lism associated with these pathological states. Thus, the extensive variety of convinc-
ing applications for use of this method in normal and diseases hearts will ensure its
continued use in the preclinical assessment of novel cardiac and antiarrhythmic drugs.
The use of heterologous expression systems allows for the electrophysiological mea-
surement of the properties of cardiac drugs on isolated ion channels from the heart
using voltage and patch clamp techniques. These systems are used to express high
levels of the desired functional protein that is not endogenous to the system. Some
available expression systems include Xenopus oocytes, transfected mammalian cells,
such as human embryonic kidney (and Chinese hamster ovary cells, and baculovirus-
infected insect cell lines, such as Sf9 and Hi5. The expression system selected should
be chosen (as with isolated tissues described above) appropriately based on the ques-
tions addressed because each system has certain advantages and disadvantages. For
example, one system may be appropriate for the study of drugs on ion channel function
(e.g., Xenopus oocytes) whereas another may be more useful to synthesize large
amounts of protein for biochemical analysis (e.g., baculovirus-insect cell lines).
Xenopus laevis oocytes are one of the most commonly used expression systems with
which to examine cDNA (or mRNA) encoding cardiac ion channels. Their usefulness
resides in the fact that oocytes do not express significant levels of endogenous Na+, K+,
or Ca2+ currents. RNA that is injected into oocytes is efficiently transcribed so that the
effects of cardiac and antiarrhythmic drugs on exogenous channels can be studied. One
disadvantage of the oocyte system is that proteins are modified by the oocyte. In mam-
malian cells, protein synthesis involves many sequential processing steps that result in
a unique structural or functional protein. Studies of translational mRNA processes in
oocytes suggest that although similar to mammalian cells, some post-translational pro-
cessing differences exist. Glycosylation of the protein in oocytes may be different from
mammalian glycosylation possibly resulting in altered functional properties. However,
despite this caveat, oocytes have become a gold standard for functional expression of
ion channels.Today, many distinct mammalian cell lines can be used to express cloned
human cardiac ion channels. The most relevant to the examination of cardiac or antiar-
rhythmic drugs are those cell lines that stably express the HERG K+ ion channel that is
implicated in torsade des pointes arrhythmias or the LQTS.

7. HIGH THROUGHPUT SCREENING (HTS) METHODS

IN CARDIAC DRUG DEVELOPMENT
HTS defines a series of usually in vitro methodologies that can be used to rapidly
screen many (thousands) potential cardiac drug candidates for activity against ion chan-
nels that eventually may exhibit efficacy in humans. Recently, the defined involvement
of ion channels in multiple disease conditions (i.e., channelopathies) have re-invigo-
rated drug development for arrhythmias. The implication of ion channels, such as IKr in
torsade des pointes and the LQTS, are such an example. Thus, although the voltage-
gated ion channel so-called flavor of the day may vary, ion channels in the heart remain
viable targets for drug development. However, as is inherent to pharmacology, there
remains a continued lag in available HTS processes that accurately quantitate the car-
diac efficacy of a potentially novel molecule. Thus, the pharmacologist cannot hope to
compete with chemists that develop structure–activity relationships for novel drugs. It
18 Pugsley

is understood that using in vivo screens, isolated tissues, dissociated myocytes, iso-
lated hearts, or heterologous expression systems to evaluate new drug candidates is a
gravely slower, and more tedious process, than drug synthesis.
However, attempts to improve upon early screening procedures using in vitro or
biochemical methods may begin to close the Grand Canyon-size gap that currently
exists between chemical synthesis and determining drug efficacy and safety. Because
ion channels really do not contain functionally defined binding sites and are not a
receptor this further complicates the development of HTS methods.
Sodium and other ion channels characteristically contain binding sites for neuro-
toxins and other drugs that activate, block, or modulate channel response to drugs
through known mechanisms. Thus, attempts have been made to use radioligand-
binding methods to develop HTS assays for ion channels. However, complications
arise because the activity of many neurotoxins involves a state-dependent binding to
the Na+ channel (56). The utility of such a preliminary assay as a screen is attenuated
because drugs that do not interact with the state of the channel produced by the neu-
rotoxin would not be detected.
Fluorescence-based cellular methods may more appropriately reflect the selectivity
and sensitivity that is required for HTS assays (56). Briefly, the fluorescence methods
use a heterocycle fluorophore or fluorescence dye molecule that localizes in a region of
the cell (such as the cytoplasm) and responds to a stimulus. Fluorophores for Ca2+ ions
include dyes, such as fura-2 and fluo-4 (Molecular Probes, Eugene, OR), whereas oth-
ers are available for Na+ and K+ ions.
Probes such as these are measured using technologies, such as the Fluorometric
Imaging Plate Recorder or FLIPR384 (Molecular Devices, Sunnyvale, CA). Because
these systems contain enhanced automation features, including potential robot inte-
gration, assays that quantitate intracellular constituents, such as Ca2+, pH and Na+ and
Vm, they may be modified to conduct HTS assay development. Cell responses can be
monitored in real time, kinetic data for drugs can be derived, relative drug potencies
can be determined, and drug-ion channel interaction kinetics can be determined.
Radiotracers can also be used in HTS assays. Denyer et al. (56) describe a novel
cell-based Na+ channel assay using Cytostar-T scintillating microplates (Amersham
Life Science, Piscataway, NJ). These plates have a transparent scintillant base that
emits light as it is excited by the decay of the radioisotope in close proximity. Cells are
grown to confluence on the microplates and various neurotoxins are used to maintain
open Na+ channels. Permeant 14C-labeled guanidinium is added to the cells. Once suf-
ficient radiotracer incorporation into the cells occurs, tetrodotoxin is used to block the
channels. Blockade or inhibition of channel opening by the test drug alters the signal
emitted from the scintillation base of the microplate and provides an index of activity
on the Na+ channel. Complex assays such as this may provide the necessary steps in the
development of HTS screening methods for ion channels.
Coronary artery occlusion and MI development results in progressive changes in
the cellular architecture of both infarcted and noninfarcted ventricular muscle. These
changes are caused by remodeling within the heart. Remodeling involves changes in
cellular events that modulate adaptation and include hypertrophy of myocytes, deposi-
tion of extracellular matrix components, and proliferation of fibroblasts (57). These
processes ultimately are responsible for ensuing dysfunction that transpires post-MI.
Diminution in cardiac contractility derives from muscle dystrophy and fibrosis, events
Cardiac Drug Development 19

subsequent to scar formation within the damaged ventricular muscle. For this to occur
within the heart, there must be an altered response and downregulation of many
homeostatic mechanisms responsible for normal contractility. Ultimately such changes
derive from altered gene expression, and this can be studied at the subcellular level
using microarray technology.
DNA microarrays are a powerful new method by which to determine the pattern of
gene expression in both normal and diseased tissues. Microarrays currently provide an
initial central point for use in drug target and validation. These methods allow for the
identification of those genes, whether up- or downregulated in expression that may
be involved in disease. It is now recognized that many cardiac disease processes
involve multiple modifications in genes. These concurrent changes may then culmi-
nate in the disease process. Thus, therapy can now ideally be directed at the cause of
the disease and not simply at the symptoms.
Microarray technology may have an application in cardiac disease because it pro-
vides researchers with the ability to characterize the gene expression levels of thou-
sands of genes in parallel (58). The development of DNA microarrays for application
to disease is a multistep process. Genes to be arrayed are identified and obtained from
private or public genebanks. Both normal and diseased tissues are used and usually
~15,000 genes are identified that may characterize the cardiac disease under investiga-
tion. Selected genes are usually maintained in bacterial plasmids and subject to poly-
merase chain reaction amplification procedures before hybridization on microarray
plates. The polymerase chain reaction amplification product is arrayed at a high den-
sity to either glass slides radiolabeled with a fluorescent dye (such as C4S) or nylon
membranes radiolabeled with phosphorus (33P). The signal intensity of the hybridized
genetic sample determines the expression level of the corresponding gene and the pro-
file is then processed using bioinformatic methods.
Although the potential impact of microarray use in many cardiac diseases is sub-
stantial, its application remains at an elementary stage. Several studies have been con-
ducted in models of heart failure, MI, and ventricular hypertrophy (57,59). In these
studies, no fewer than 55 known (and unknown) genes were detected that could be
involved in these cardiac diseases. Functional relevance remains inconclusive and many
caveats exist that relate to the use of this methodology.Microarrays, although high
throughput in nature, have a low sensitivity, and genes that exhibit ~70% homology are
indistinguishable (60). Quality control issues, computational resources, and a require-
ment that a large number of diseased and nondiseased tissues must be profiled to derive
conclusions concerning the reproducibility of gene expression patterns are issues that
require resolution. Owing to the ingenuity of researchers in this field, resolution is only
time dependent.

8. CLINICAL DRUG TRIALS

Clinical drug trials test the activity of the drug on the disease or symptom. The
extensive general and safety pharmacology studies that are conducted prior to the ini-
tiation of the testing of the drug in humans require years. However, it is critical that
these studies define the pharmacological effects and the potential for serious drug tox-
icity before use in humans and are the sum of in vitro and in vivo studies conducted in
both animal and human preparations (as described above). If these preclinical studies
20 Pugsley

Fig. 4. The preclinical and clinical relationships for new drug development and the approval
process.

are successful, then the pharmaceutical or biotechnology company provides the data in
the form of an Investivational New Drug application to governmental agencies such as
the FDA in the United States or to the European Agency for the Evaluation of Medici-
nal Products (EMEA) in Europe.
Clinical drug trials of novel antiarrhythmic or cardiovascular experimental drugs (as
for all drugs) are conducted in four phases (Fig. 4). The early phases of drug testing in
humans are usually broad in perspective and provide information regarding dose, drug
kinetics, and tolerance. Thus these early phases concern the testing of the safety of the
drug (phase 1) and the efficacy of the drug (phase 2). The attrition rate for new drugs
reaches approx 60% by the end of this phase of clinical trial testing. In later trials, large
Cardiac Drug Development 21

controlled studies that delineate the range of the drug’s effectiveness; incidence of
adverse events and the benefit to potential patient population (phase 3) are conducted.
The attrition rate for drugs is reduced to approx 10–20%. Successful completion allows
the drug company to file a New Drug Application with the FDA or EMEA. If granted,
the drug is then conditionally approved for marketing.
This conditional approval is given before phase 4 studies. However, these phase 4
(or late phase 3) studies are intended to provide for postmarketing surveillance and
allow for the monitored release of the drug, whereby patients may be given the drug
under specified supervision by physicians at selected medical centers.
These studies are an indispensable part of medical research and offer the only means
by which to provide new therapies for cardiovascular diseases that afflict humans.
However, as many clinical cardiovascular trials have taught (and cost!) the drug indus-
try, this is a very arduous process and many cardiovascular clinical drug trials have
failed. However, despite these failures, a re-evaluation of clinical trial data provides
for suggestions as to how to improve clinical trials involving cardiac drugs. These
suggestions include the regular requirement of phase 4 trials with surrogate endpoints,
a clear definition of phase 3 study endpoints, and also by possibly reassessment of
thestructure of the clinical trial hierarchy itself (61,62). Recently, Lipicky (61) sug-
gested that the traditional distinctions between study phases for clinical trials not be
used. It is reasoned that conforming to the rigid phases currently used to conduct clini-
cal trials with cardiac (particulary antiarrhythmic) drugs may be detrimental to drug
development (61). Rather, it is suggested that combining phase 2 with phase 3 trials
may reduce unnecessary delays associated with independent, consecutive trials and be
of a clear benefit to drug development. Additionally, it is suggested that multiple drug
doses should be studied, the type of arrhythmia be defined and the doses be related to
some measure of cardiac activity (61). Consideration of suggestions such as these may
reduce the attrition rate associated with drug development for cardiac disease, improve
the safety profile for these drugs in patients, and ultimately provide novel drugs that
improve symptoms associated with the disease or prevent the disease itself.

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Role of Cardiac Pacemaker Currents 25

II
Novel Molecular Targets
for Cardiac Drug Development
Role of Cardiac Pacemaker Currents 27

2
The Role of Cardiac Pacemaker Currents
in Antiarrhythmic Drug Discovery

David A. Saint

CONTENTS
THE PACEMAKER CURRENT AS A TARGET FOR THERAPY
THE CHARACTERISTICS OF I(F)
VARIABILITY OF CHARACTERISTICS OF I(F) IN DIFFERENT TISSUES
MOLECULAR BIOLOGY OF I(F) AND I(H)
DISTRIBUTION OF HCN CHANNELS
RELEVANCE OF I(F) TO CLINICAL CONDITIONS
TYPES OF DRUGS UNDER CURRENT DEVELOPMENT
CONCLUSIONS
REFERENCES

1. THE PACEMAKER CURRENT AS A TARGET FOR THERAPY

From even the most basic physiological observations, it is obvious that parts of the
heart can generate their own intrinsic contractile rhythm. With the development of
techniques to record electrical activity from tissues, it became clear that contraction of
the heart is triggered by an action potential and that most areas of the heart can, under
some circumstances, generate rhythmic action potentials, although it is cells in the
sinoatrial node (SAN) that have the highest intrinsic frequency, and hence it is these
cells that normally provide the drive for the rest of the heart. Even a single, isolated
SAN cell will generate rhythmic action potentials separated by a period of slow dias-
tolic depolarization. It is the diastolic depolarization that repeatedly drives the mem-
brane potential towards threshold for action potential firing. The mechanism underlying
this diastolic depolarization has been a subject of much puzzlement. By the 1970s, it
was clear that to provide a slow depolarization, there must be a slow increase in net
inward current, although it was not clear whether this came about as a consequence of
the slow inactivation of an outward current (presumed to be a carried by potassium) or
the slow activation of an inward current (1). Using the intracellular recording and volt-
age clamp techniques available at the time, it was difficult to resolve the different com-
ponents of the current flowing during the diastolic depolarization (2–4). With the advent
of patch-clamp techniques, coupled with techniques for preparation of isolated cells
From: Cardiac Drug Development Guide
Edited by: M. K. Pugsley © Humana Press Inc., Totowa, NJ

27
28 Saint

from various parts of the heart, resolution of individual currents became possible. The
mechanism of the diastolic depolarization was greatly clarified by the discovery in
pacemaker cells of an inward current that was activated by hyperpolarization (5–8).
The current was shown to activate relatively slowly on hyperpolarization and to have
low selectivity among cations. These properties are just what one would expect for a
pacemaking current
Around the same time as it was discovered in cardiac myocytes, a similar current
was identified in photoreceptors (9). Subsequently, similar currents were discovered
in other tissues, particularly in parts of the central nervous system (CNS). The cur-
rent in cardiac tissue was christened I(f) (f for funny, because of its funny properties)
and in most other tissues it was called I(h) (h, because of the activation on hyperpolar-
ization). The terms are often used interchangeably.
I(f) or I(h) has been implicated in cardiac (10) and neuronal (11–13) rhythmogenesis,
sensory adaptation (14,15), shaping of synaptic potentials (16), and control of synaptic
transmitter release (17,18).

2. THE CHARACTERISTICS OF I(F)

As noted above, the critical and unusual feature of I(f) is its voltage dependence. I(f)
is activated by hyperpolarizations with a threshold of approx –40 to –50 mV in the
SAN. Figure 1 shows a typical activation curve, which depicts the relative fraction of
channels open at steady state as a function of membrane voltage. This relation indi-
cates that the current is activated at voltages near the range of the diastolic depolariza-
tion in SAN cells. The fully activated current/voltage (I–V) relation reverses near +10
to +20 mV in physiological solutions as a consequence of the channel having a mixed
permeability to Na+ and K+. The activation by hyperpolarization and permeability to
Na+ and K+ are critical properties with respect to the role of I(f) in the generation of
diastolic depolarization and hence of spontaneous activity. Hence, membrane poten-
tials around the maximum diastolic potential in SAN cells activate the current, which,
because it is a nonspecific cation current, produces an inward, depolarizing current at
these potentials. To a large extent, it is this slow activation of I(f) that tends to drive the
slow diastolic depolarization until the membrane potential reaches threshold for the
triggering of a new action potential.
However, when it was first described, there was initially some skepticism regarding
the role of I(f) in pacemaking and, indeed, there is still some degree of controversy as
to just how important I(f) is to pacemaking (19,20). Although I(f) seems to be a good
candidate for the role of a pacemaker current, the real situation is somewhat more
complex. An important caveat is that, rather than being driven by one current, the pace-
making activity of SAN cells is a product of the interplay of many currents (21). This
assertion is reinforced by the observation that pacemaking continues (albeit at a slowed
rate) when I(f) is blocked (22) and from the observation that the smo mutant of zebra
fish (which has a mutation in pacemaking channels carrying the fast kinetic component
of the current which renders them nonfunctional) still has an operational pacemaker,
albeit once again at a greatly slowed rate (23). Hence, it seems that the autorhythmicity
of SAN cells is driven and stabilized by an interplay of several currents, none of which
is crucial, but all of which influence rhythm. This situation provides redundancy and
pleiotropism in the regulation of rhythm (21). Nevertheless, it is certainly true that one
Role of Cardiac Pacemaker Currents 29

Fig. 1. The biophysical properties of I(f). (A) Spontaneous activity of a single SAN cell.
Note that the maximum diastolic potential is about –65 mV. (B) I(f) current induced in a
single SAN cell by a hyperpolarising pulse to the voltages indicated from a holding potential
of –35 mV. (C) Voltage dependence of activation of I(f), (Y∞) and fully activated I/V relation-
ship. Reprinted from DiFrancesco, D. (1995), The onset and autonomic regulation of cardiac
pacemaker activity: relevance of the f current. Cardiovasc. Res. 29, 4493–4556, with permis-
sion from Elsevier Science.

of the major influences on auto rhythmicity is I(f) and, hence, it is sensible to use I(f) as
a target in attempts to modulate rhythmicity.
If I(f) is indeed responsible for driving the diastolic depolarization, one would imag-
ine that it should be modulated by sympathetic and parasympathetic stimulation in a
way consistent with their chronotropic effects. I(f) is indeed modulated by adrenergic
agonists in a way consistent with an increase in heart rate (24).
In 1986, DiFrancesco (25) showed that I(f) was carried by channels having a single
channel conductance of about 1 pS, and that modulation of I(f) by adrenaline at the
single channel level consists of an increase in the open probability without a change in
single channel conductance. This effect of adrenaline is mediated via the G-protein/
adenyl cyclase/cAMP pathway. Using patch clamp techniques, it has been shown that
I(f) channels are modulated by a direct effect of intracellular cAMP at the intracellular
side of the channel, independent of any phosphorylation effects (26). The effect of
cAMP consists of a facilitation of the opening of single I(f) channels in response to
hyperpolarization. Measurement of the voltage dependence of the channel open prob-
ability shows that cAMP shifts the probability curve to more positive voltages without
modifying its shape (27).
Interestingly, the modulation itself seems to be subject to other influences, notably
the action of phosphorylation. Hence, the phosphatase inhibitor calyculin A increases
I(f) and potentates the beta-adrenergic mediated response (28), suggesting that there is
a so-called tonic level of phosphorylation of the channels that regulates their activity
(29). The channels carrying I(f) have also been reported to be modulated by various
hormones and growth factors. Epidermal growth factor increases maximal I(f) conduc-
tance, apparently acting through tyrosine kinase (30). Vasoactive intestinal peptide
produces a slight shift in the voltage dependence of activation of I(f) (28), an effect that
may be related to the role of vasoactive intestinal peptide as a cotransmitter with ace-
30 Saint

tylcholine released from vagal nerve endings. The thyroid hormone T3 increases the
current density of I(f) in rabbit SAN cells without changing the voltage dependence, an
effect that may underlie the sinus tachycardia commonly seen in hyperthyroidism (31).
Similar results have been reported with parathyroid hormones (32). In addition, I(f) is
modulated by muscarinic agonists in a way consistent with the decrease in heart rate
seen with these agents (33).

3. VARIABILITY OF CHARACTERISTICS
OF I(F) IN DIFFERENT TISSUES
Reflecting this wide range of tissues in which it is found and the differing physi-
ological functions with which it is therefore involved, the properties of I(f) (or I[h])
differ significantly in their voltage dependence, activation kinetics, and sensitivity to
cAMP in different tissues (12,34). This heterogeneity of properties of I(f) is found not
only between tissues but also within organs or tissue types themselves. Hence, bio-
physical studies have demonstrated that I(f) has a dramatically different voltage depen-
dence of activation in different cardiac regions, with the threshold for activation being
–50 mV in SA node, –85 mV in Purkinje myocytes, and –120 mV in adult ventricular
myocytes (35). This difference in the voltage-dependent properties of I(f) is highly
correlated with the intrinsic pacemaker activity of these tissues; the I(f) current having
the most positive activation is found in the tissue with the highest pacing rate (in SA
node), whereas the current having the most negative activation is found in tissue that
normally exhibits no diastolic depolarization at all (ventricular myocytes). Although
some of these differences in current properties may be the result of post-translational
modification of the channels or functional modulation by phosphorylation, the most likely
explanation is that I(f) currents are carried by different isoforms of the channel in differ-
ent tissues. Current density is also an important factor, presumably controlled by the
level of gene expression; as well as different voltage dependence, I(f) in ventricular cells
is present at a very low current density (Fig. 2). With the recent cloning of the channels
responsible for I(f), the factors controlling the level of gene expression, and which isoform
of the channel is expressed, have become amenable to investigation.

4. MOLECULAR BIOLOGY OF I(F) AND I(H)

Although the properties of I(f) and the single channels underlying the current have
been investigated for nearly three decades, particularly because of its importance to
cardiac pacemaking (10), cloning of the gene coding for I(f) was only achieved nearly
three decades after the original description of I(f), and even then the clone was discov-
ered serendipitously. While searching for proteins interacting with the SH3 binding
domain of neural Src, Santoro et al. (36) identified a putative member of a new family
of channels cloned from mouse brain. Using BLAST searches of expressed sequence
tag databases, along with reverse-transcription polymerase chain reaction and screen-
ing of cDNA libraries, four isoforms of the channel were subsequently cloned in mam-
mals (37–40). Functional expression of these channels resulted in currents with the
hallmarks of the cardiac I(f) or its neuronal equivalent I(h). Although the properties of
different isoforms differ quantitatively, all isoforms except one yield currents that are
activated by hyperpolarization, are permeable to both K+ and Na+, are blocked by
Role of Cardiac Pacemaker Currents 31

Fig. 2. The relative magnitudes of I(f) in rabbit ventricle and SAN cells. I(f) was evoked
by hyperpolarizing voltage steps to the voltages indicated from a holding potential of –35
mV in either a single rabbit SAN cell (A) or ventricle cell (B). Note the different current
scales. Reprinted from Shi, W., Wymore, R., Yu, H., Wu, J., Wymore, R. T., Pan, Z.,
Robinsin, R., Dixon, J. E., McKinnon, D., and Cohen, I. S. (1999), Distribution and preva-
lence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tis-
sues. Circ. Res. 85, e1–e6, with permission from Lippincott, Williams & Wilkins Publishers.

cesium (Cs+) in a voltage-dependent way, and are modulated by a direct action of cAMP
on the cytoplasmic side of the channel (38,39,41).
Because of the serendipitous nature of their discovery, the early nomenclature of the
gene and its isoforms for pacemaker channels is somewhat inconsistent. Some
investigators referred to the isoforms as HAC1 or HAC2, denoting hyperpolarizing acti-
vated channel. However, because of the direct action of cAMP and gating by
hyperpolarization, the nomenclature suggested by Clapham (42) and Biel et al. (43) has
been generally adopted; that is, of referring to the clones as HCN (hyperpolarization-
activated, cyclic nucleotide-gated) channels. Compared with the previous nomenclature,
HCN1 corresponds to HAC2 (mBCNG-1); HCN2 corresponds to HAC1 (mBCNG-2);
and HCN3 corresponds to HAC3 (mBCHG-4).
Cloning of the gene for HCN channels and the consequent ability to perform site-
directed mutagenesis and functional studies of channels expressed in heterologous sys-
tems has enabled rapid progress in defining the structure and function of the channel
protein. The gene transcripts code for a protein of between 800 and 1200 amino acids,
with the different protein isoforms having an overall sequence identity of 60%. They
are members of the voltage-gated cation-channel superfamily, members of which are
characterized by six membrane-spanning segments (S1–S6), including a voltage-sens-
ing S4 segment, and an ion-conducting pore between S5 and S6. It seems safe to
assume, by analogy with the voltage-gated family of potassium channels, that func-
tional HCN channels are formed as tetramers of subunits, with each of the four sub-
units contributing to the pore region. In a structure analogous to the S4 voltage-sensing
32 Saint

region of voltage-gated K+ channels, the S4 region of HCN channels has two regions
each containing five positively charged amino acids (10 positive charges compared
with 5–7 in potassium channels). This raises some intriguing questions because the two
families of channels are gated in opposite ways, the Kv family of potassium channels
being opened by depolarization and HCN channels by hyperpolarization. This conun-
drum of the biophysics of gating of HCN channels has yet to be resolved (44).
The pore region of HCN channels is also related to that of K+ channels, having a
signature GYG triplet (45). However, the aspartate that follows the GYG signature
sequence in most K+ channels is replaced in HCN channels by arginine, alanine, or
glutamine. This presumably underlies the high relative permeability to Na+ of HCN
channels compared to classic K+ channels (46); values of PNa/PK for cloned HCN chan-
nels are between 0.25 to 0.41 (37,38,41).
In the carboxy terminus, HCN channels contain a sequence with a high degree of
homology to the cyclic-nucleotide-binding proteins, such as cyclic nucleotide gated
(CNG) channels of photoreceptors and olfactory neurons (47), cAMP-dependent pro-
tein kinases, and the catabolic-activator protein of Escherichia coli. The mechanism of
modulation of the channel by cAMP is thought to consist of a COOH-terminus medi-
ated inhibition of opening, which is partially relieved by cAMP binding, as revealed by
site directed mutagenesis of the COOH linker in HCN1 and HCN2 isoforms (48).
When expressed in heterologous systems, three of the four HCN genes have been
shown to generate hyperpolarization-activated currents with distinct biophysical prop-
erties. HCN1 channels activate most quickly and require the least amount of hyperpo-
larization to open, with a half-maximal voltage, V1/2, of about –73 mV (40). HCN2
channels activate more slowly, require stronger hyperpolarizations (V1/2 of –92 mV),
but are strongly modulated by cAMP (38,49,50). HCN4 may activate at even more
negative potentials and with the slowest kinetics (37,49,51), although V1/2 values
reported in the literature are variable. To date, HCN3 channels have not been found to
form functional homomultimers. It is possible that this is caused by the lack of a sub-
unit necessary for full functionality because HCN channels have been shown to be
modulated by co-expression of a MinK-related peptide (52).
The existence of different isoforms of the HCN channel provides a convincing
molecular basis for the heterogeneity in I(f) among different cells, with SAN cells
expressing one isoform and ventricular cells another, consistent with the macroscopic
properties of I(f), and hence meeting the physiological requirements of the cell. How-
ever, more complicated scenarios are possible. A given cell, for example, could express
a mixture of HCN channel isoforms with different properties, a supposition reinforced
by the finding that the I(f) current displays fast and slow components in heart (24,53)
and in neurons (54). Consistent with idea of several different components of I(f) being
present simultaneously, a recent zebra fish mutant that displays a slow heart rate was
found to be deficient in the fast kinetic component of I(f), whereas the slow component
was unchanged (23). As a further intriguing possibility because functional HCN channels
are likely tetrameric assemblies of subunits, hybrid channels, or heterotetrameric assem-
blies of subunits, may also be synthesized in some cells. Although most investigations
have been performed to date on homomeric channels, there is evidence that channels can
be formed as heteromeric assemblies, which display properties intermediate between the
two isoforms (55). This additional complexity may be important in the fine control of
pacemaking and may underlie the heterogeneity of cardiac tissue (56).
Role of Cardiac Pacemaker Currents 33

5. DISTRIBUTION OF HCN CHANNELS

As a logical development of earlier studies demonstrating the presence and proper-
ties of I(f) in a variety of tissues, efforts have now been directed to studies defining the
relative expression levels of the different isoforms of the HCN gene.
The relative expression levels of the different isoforms varies considerably through-
out the heart, consistent with the different properties of I(f) and the different intrinsic
pacemaker activity of the tissues. Hence, in SAN, the dominant isoform of the channel
appears to be HCN4, with lesser amounts of HCN1, whereas in Purkinje fibers, there
are equal amounts of HCN1 and HCN4 with a smaller amount of HCN2. In ventricle,
the dominant isoform appears to be primarily HCN2 (57). It should be noted that het-
erogeneity of cellular properties in the heart is found not only at the “gross” level (i.e.,
between SAN, atria, and ventricles), but that this heterogeneity can be traced down
almost to a single cell level. For instance, the SAN shows a gradation of properties
throughout its structure, in terms of the electrophysiology of the tissue and the ion
channels expressed (58,59). It may be that this phenotypic heterogeneity is a reflection
of the different mix of HCN4 and HCN1 expressed in these cells, giving central pace-
maker cells a higher intrinsic rate than peripheral cells.
As well as a high level of expression in heart, HCN isoforms are also expressed in
various parts of the CNS, where HCN1-4 show distinct but overlapping patterns of
mRNA expression (14,39,50,60,61). HCN1 is expressed selectively in specific brain
regions, including the hippocampus, layer 5 cells of the neocortex, and Purkinje cells
of the cerebellum. HCN2 is widely expressed throughout brain, including neocortex,
hippocampus, and thalamus. Finally, HCN4 is expressed in a restricted manner in sub-
cortical and lower brain regions.
HCN expression can also be found in the periphery, notably in smooth muscle
(62). Indeed, it seems likely that HCN channels are expressed in any tissue that dis-
plays intrinsic rhythmic activity. Note also that HCN may be involved in some unex-
pected functions, for example, sour taste perception (63). Importantly, in the context
of side effects of specific bradycardic agents, HCN1 channels are highly expressed in
the retina (60).

6. RELEVANCE OF I(F) TO CLINICAL CONDITIONS

An increased heart rate increases myocardial oxygen demand and decreases time for
myocardial relaxation and diastolic ventricular filling. Because of the increased trans-
mural pressure on the coronary perfusion vessels during systole, perfusion is greatly
limited, or prevented entirely, during much of the cardiac cycle and perfusion occurs
mostly during diastole. Hence, for example, in the presence of a compromised coro-
nary flow because of coronary artery stenosis, a decrease in diastolic perfusion time
secondary to an increase in heart rate may further reduce overall coronary perfusion, to
the point where the myocardium can become ischemic. Generally, the subendocardium
is most vulnerable to ischemia, and tachycardia has indeed been shown to exacerbate
this vulnerability (64,65). Under these circumstances, drugs that block sinus tachycar-
dia, reduce heart rate at rest, or both could be expected to increase the diastolic coro-
nary perfusion time and hence improve overall perfusion and function of the ischemic
subendocardium (66,67). This, at least partly, is the rationale behind the use of beta-
blocking agents and the rate-lowering Ca2+ channel-blocking agents, such as diltiazem
34 Saint

and verapamil. However, interpretation of the action of these agents, particularly the
calcium channel blockers, is complicated by their tendency to reduce blood pressure
and myocardial contractility, both of which reduce myocardial oxygen demand and,
indirectly, affect coronary perfusion. One would nevertheless predict that a pure
bradycardic agent without these attendant pressor effects should be beneficial in
improving coronary perfusion and resistance of the myocardium to ischemia. This pre-
diction is born out in practice: slowing heart rate with ZD 7288 in dogs reduces the
severity of myocardial ischemia produced by left anterior descending coronary artery
(LAD) occlusion (68), with similar results having been shown in pigs (69). Because
I(f) is the primary determinant of heart rate, I(f) blockers therefore have a promising
role as anti-ischemic agents.
In addition, blockers of I(f) may be useful in contexts other than when the myocar-
dium is vulnerable to ischemia. I(f) appears to be upregulated in the ventricle during
cardiac failure, leading to enhanced autorhythmicity (70); however, note the results
from a previous study (71). It seems possible that this enhanced autorhythmicity may
be responsible for the greater propensity to arrhythmia in cardiac failure, and hence
blockers of I(f) may also be useful as antiarrhythmic agents in this situation. This would
constitute an action separate from their bradycardic effects, although the bradycardic
effect would still be present, and perhaps provide an additional benefit.

7. TYPES OF DRUGS UNDER CURRENT DEVELOPMENT

One of the earliest blockers of I(f) described was Cs+ (72), which is moderately
specific for I(f), although it does block other ionic currents in addition, especially at
higher doses (73). Difficulties with using Cs+ in physiological solutions and its lack of
specificity sparked the search for organic blockers of I(f) in the hope of generating a
more specific and more potent compounds. The following two broad lineages of com-
pounds have emerged from these efforts:
1. A series of compounds related to phentolamine and clonidine, the prototype being alinidine
(N-allyl-clonidine), with the latest compound being ZD-7288 (74).
2. A series of compounds based on modifications of verapamil, the prototype being falipamil (or
AQ-A 39), from which zatebradine (75) and ivabradine (S-16257-2) have been developed.

7.1. Alinidine and Congeners

Alinidine (or ST567) was shown to be bradycardic as long as 20 yr ago in dogs (76)
and in humans (77). Although it blocks I(f), alinidine is not very specific in its action,
also blocking the slow inward current and outward currents in rabbit SAN cells (78).
There also have been suggestions that alinidine can block anion channels (79), an action
that may underlie its sometimes reported negative inotropic effect (80). In addition,
alinidine displays a variety of pharmacological properties, for example, it antagonizes
cromakalim and hence inhibits KATP channel opening (81). It has been shown that
alinidine exhibits rate-independent cardioprotective effects, perhaps as a consequence
of adenosine antagonist properties (82,83). Alinidine has also been shown to be
antimuscarinic—in paced left rat atria, alinidine acted as competitive antagonist against
oxotremorine with a pA2 of 5.82, and in guinea pig papillary muscle it antagonized
carbachol with a pA2 value of 5.58 (84). Early reports that alinidine slows conduction
through the atrioventricular (AV) node (76) have not been confirmed in later studies
Role of Cardiac Pacemaker Currents 35

(85). Although alinidine has been shown to prolong the effective refractory period in
the AV node of dogs subject to coronary artery occlusion (86,87), an effect on refrac-
tory period was not noted in humans at a dose of 40 mg (88).
Despite the potential difficulties with nonspecific actions, alinidine has been
shown to have very few acute side effects in humans (89,90). Note, however, that the
latter study (albeit with a limited numbers of subjects) reported that alinidine did not
appear to enhance myocardial salvage or preservation of left ventricular function or
to reduce the incidence of major arrhythmias in the early phase of myocardial infarc-
tion. Nevertheless, interest in alinidine congeners continues with a number of com-
pounds still under investigation (91). Many of these exhibit most of the nonspecific
effects of alinidine and so are unlikely to be any more useful than alinidine as
bradycardic agents. However, one compound, ZD7288, has been developed that
shows some promise as a therapeutic agent.

7.1.1. ZD 7288
ZD 7288 (Fig.3A) has been shown to be bradycardic in rabbits and guinea pig (92)
and to block I(f) in dissociated guinea pig SAN cells (93). The block is not use depen-
dent (in contrast to that produced by zatebradine) and has been suggested to be caused
by an action of the very hydrophobic ZD 7288 molecule at an intracellular site on the
HCN channel (94). In sheep Purkinje fibers, the blocking of I(f) by ZD 7288 appears to
show so-called reverse use-dependence, and it has been suggested that this may limit
its efficacy under physiological conditions (95). ZD 7288 is more selective than
alinidine or UL-FS 49, producing less prolongation of the action potential in SAN cells
at bradycardic concentrations (96).
As noted previously, HCN channels are widely expressed in CNS and some periph-
eral tissues, and one could expect that blockers of HCN channels will have effects in
these tissues as well as in the heart. In this context, most interest in ZD 7288 at present
seems to be centered on its actions in neurons (97,98). ZD 7288 inhibits I(h) in rod
photoreceptors (99), a finding that has relevance to one of the side effects of HCN
channel blockers, which is that they produce visual disturbances.
As well as side effects produced by blockade of HCN channels in extracardiac tis-
sues, which will be a common effect to all specific bradycardic agents, a difficulty with
the clinical use of ZD 7288 may arise as a consequence of its kinetics; in in vitro
preparations, the action appears to be essentially irreversible (95).

7.2. Falipamil (AQ-A 39) and Congeners

An alternative development track for blockers of I(f) has been made through modi-
fications to the benzenacetonitrile Ca2+ channel antagonist verapamil. Structural modi-
fication by replacement of the lipophilic alpha-isopropylacetonitrile moiety by various
heterocyclic ring systems has led to a new range of molecules with specific bradycardic
activity, the prototype being falipamil (AQ-A 39). Falipamil has been shown to be
bradycardic in many isolated tissue preparations, including guinea pig (100); rabbit
(101); dog (102); anesthetized cats, dogs (103), and pigs (104); and in humans (105).
Despite the production of similar effects on heart rate, AQ-A 39 differs from
alinidine and mixidine in that it does not depress cardiac contractility in anesthetized
dogs with spontaneous heart rates (106). A similar observation has been made in anes-
thetized pigs, at least for arterial plasma concentrations lower than 1500 ng/mL (104).
36 Saint

Fig. 3. Depicts the chemical structures of several novel I(f) blockers in development. (A) shows
ZD 7288 (4-[N-ethyl-N-phenylamino]-1,2-dimethyl-6-[methylamino] pyridinium chloride),
(B) shows Zatebradine (UL-FS 49 or 1,3,4,5-tetrahydro-7,8-dimethoxy-3-[3-][2-(3,4-dimeth-
oxyphenyl)-ethyl) methylimino]propyl]-2H-3-benzazepin-2-on-hydrochloride), and (C) shows
(±) S 15544 (the racemic parent of Ivabradine; (±) S 15544 or 7,8-dimethoxy3-[3-][(4,5-
dimethoxybenzocyclobutan-1-yl) methyl] methyl-amino] propyl}1,3,4,5-tetrahydro-2H-3-
benzazepin-2-one). Note that ivabradine has a chiral carbon center.

Consistent with these bradycardic actions, falipamil shows protective effects against
ischemia in anesthetized dogs subjected to 15 min of coronary artery occlusion, fol-
lowed by 3 h of reperfusion. In this study, both AQ-AH 208 and AQ-A 39 (falipamil)
produced similar decreases in heart rate (24%) and increases in the endocardial/epicar-
dial distribution of collateral blood flow. During occlusion and throughout reperfusion,
both compounds also produced a significant improvement in the percentage of shorten-
ing of the ventricle in the ischemic-reperfused region (107). A similar result has been
reported in humans; in a randomized, controlled study 10 male patients with
Role of Cardiac Pacemaker Currents 37

angiographically confirmed ischemic heart disease received AQ-A 39 (falipamil) in a

single intravenous dose (2 mg/kg). After submaximal exercise, heart rate during pla-
cebo was 129 ± 3, and during AQ-A 39, it was 113 ± 3 beats min–1. AQ-A 39 did not
affect systolic arterial pressure and improved exercise tolerance (108).
Some problems in the clinical use of falipamil may arise because of its pharmacoki-
netic properties, such as its terminal half-life (t1/2). In human plasma, the t1/2 was deter-
mined to be 1.8 ± 0.6 h (109). In addition, falipamil has been reported to have
anticholinergic effects in isolated SAN preparations in addition to its rate-dependent
block of I(f) (110). In intact animals, this anticholinergic effect can be manifest in
vagolytic effects, and these can result in a paradoxical increase in heart rate in some
circumstances (111).
As further development has proceeded, falipamil has been submitted to further opti-
mization mainly by manipulation of the phthalmidine moiety, resulting in a second
generation of specific bradycardic agents with increased potency and selectively and a
prolonged duration of action, represented by the benzazepinone-derivative UL-FS 49
(zatebradine; ref. 112).
7.2.1. Zatebradine (UL-FS 49)
Zatebradine (Fig. 3B) blocks sinus tachycardia; it has been shown to markedly
attenuate exercise-induced heart rate both in animal models (113–116) and in humans
(117–120) at concentrations that do not affect the inotropic or lusitropic state or vascu-
lar tone. Zatebradine exhibits a less anticholinergic effect than falipamil (121). In rab-
bit SAN cells (75) and sheep Purkinje fibers (122), the bradycardic effect of
zatebradine has been attributed to a use-dependent inhibition of I(f), caused by inter-
action with the open state of the channel that reduces open probability (123). This is in
contrast to the mode of action of ZD 7288 (and other alinidine-related compounds)
that interact with the HCN channel in a way that reduces the single-channel conduc-
tance and that do not show the same use dependence. At concentrations that block I(f),
zatebradine has minimal effects on the L-type Ca2+ current (ICa) or the delayed recti-
fier K+ current (IKr) in rabbit sinoatrial cells (75), although in spontaneously beating
rabbit SAN cells zatebradine prolonged the duration of the action potential, suggest-
ing a K+ channel block (75,124). More recent electrophysiological studies demon-
strate that zatebradine also prolongs action potential duration in guinea pig papillary
muscles and rabbit Purkinje fibers (125,126), an effect that was more prominent in
Purkinje fibers than in papillary muscles. Consistent with its bradycardic effects,
zatebradine has been shown to confer protection against exercise-induced regional
contractile dysfunction in dogs (66,67,127) and in anesthetized pigs (69).
In 1999, zatebradine was in Phase III clinical trials. However, the Zatebradine Study
Group, from a randomized double-blind, placebo-controlled, multicenter study in pa-
tients with chronic stable angina pectoris taking extended-release nifedipine, concluded
that zatebradine seemed to provide no additional antianginal benefit. This group raised
questions regarding the benefit of heart rate reduction alone as an antianginal approach
to patients with chronic stable angina (128). This conclusion was later reinforced by
Glasser et al. (129), who noted that “despite significant reductions in resting and exer-
cise heart rate, there were no clinically significant effects on myocardial ischemia,
suggesting that the anti-ischemic effect of heart rate reduction should be re-evaluated.”
These studies raise some questions about the likely usefulness of specific
bradycardic agents in cardiac disease, although it may be that their conclusions can
38 Saint

only be interpreted as being relevant to zatebradine. In addition to these doubts about

efficacy, the class III actions of zatebradine (130) have spurred efforts to improve the
specificity of its I(f)-blocking actions. This has resulted in development of the com-
pound ivabradine.
7.2.2. Ivabradine (S-16257-2)
Ivabradine is the (+)-enantiomer of the racemic compound S15544 (note that
zatebradine is nonchiral). It is similarly bradycardic as zatebradine in isolated tissues
(125), by the same mechanism, that is, a block of I(f) (131). The major improvement
of ivabradine over zatebradine is that it produces much less prolongation of the action
potential in the bradycardic concentration range (125). Because the prototype com-
pound for ivabradine, S15544, is a racemate, it is possible to compare the isomers
and to “dissect out” the action potential prolonging action (see Fig. 3B for chemical
structure). Most of the action potential prolonging effect resides in the (–)-enanti-
omer (S16260), with the (+)-enantiomer (S16257, ivabradine) having a much smaller
effect (132).
Ivabradine is bradycardic and alters neither myocardial contractility nor coronary
vasomotion at rest and during exercise in normal dogs (133). It has been shown to be
effective against exercise-induced myocardial ischemia in dogs (134). In humans, iv
ivabradine in the first Phase I study was shown to produce a decrease of maximal heart
rate during exercise (135). In later Phase I trials, pharmacokinetic studies have been
conducted in humans (136). Ivabradine is currently in later stage clinical trials. It
remains to be seen whether it will prove to be a more effective anti-ischemic agent than
zatebradine, thus proving the concept that pure bradycardic agents can be useful as
therapeutic agents for angina and other conditions producing myocardial ischemia.

8. CONCLUSIONS
There are a range of side effects typical for specific bradycardic agents, such as
effects on the lungs. Animal studies show that relatively high iv doses of zatebradine
contract guinea pig airways by a histamine-like mechanism, and zatebradine can reduce
FEV1 in humans (137). Besides these generalized extraneous effects of specific
bradycardic agents and the usual problems of pharmacokinetics and metabolism that
must be overcome, a range of side effects is possible that are related to the fundamental
pharmacodynamics of these agents. Because HCN channels are found in a variety of
extracardiac tissues, notably the CNS, it may be unavoidable that these agents will
produce CNS actions and these may limit their usefulness as antianginal agents. Indeed,
one of the common side effects already documented for these agents is a disturbance of
vision, a consequence of the presence of HCN channels in the photoreceptors. How-
ever, this problem of a diffuse target for therapeutic intervention has not inhibited the
development of other drugs, such as class I or class III antiarrhythmic agents. The
target for these agents, sodium channels, or potassium channels, respectively, are wide-
spread in extracardiac tissues, but this does not necessarily impede the clinical use of
these agents. There is no reason to suppose that the presence of HCN channels in
extracardiac tissues will be more of an impediment to the usefulness of specific
bradycardic agents, although side effects may inevitably occur. In fact, the situation
with specific bradycardic agents may offer more scope for drug development than has
currently been exploited. Because HCN channels exist in at least four isoforms, one
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