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`QT interval prolongation by non-
`cardiovascular drugs: issues and
`solutions for novel drug development
`William Crumb and Icilio Cavero
`
`In 1997, the Committee for Proprietary Medicinal Products (CPMP)
`
`issued a document concerning the potential of non-cardiovascular
`
`drugs to cause prolongation of the QT interval of the electrocardio-
`
`gram. This article reviews several aspects of this complex problem,
`
`including a preclinical strategy (in vitro electrophysiology in human
`
`cardiac cells and in vivo pharmacologically validated conscious dogs)
`
`to satisfy the expectations of the CPMP. In particular, the discussion
`
`stresses the danger of drugs prolonging the QT interval in patients
`
`with concurrent cardiac risk factors and the need for rigorous clinical
`
`testing to determine the risk of fatal cardiac events for drugs with
`
`the propensity to prolong QT.
`
`William Crumb
`Zenas Technologies L.L.C.
`5896 Fleur de Lis Drive
`New Orleans
`LA 70124
`USA
`tel: 11 504 584 2416
`fax: 11 504 837 5546
`
`Icilio Cavero
`Department of Lead Discovery
`Rhône-Poulenc Rorer
`Centre de Recherche de Vitry-
`Alfortville
`13 Quai J. Guesde
`B.P. 14
`F-94400 Vitry sur Seine
`France
`
`In December 1997, the European Agency
`for Evaluation of Medicinal Products of the
`Committee for Proprietary Medicinal Products
`(CPMP) issued a statement (Note CPMP/986/96)
`entitled ‘Points to Consider:The Assessment of the Potential for
`QT Interval Prolongation by Non-cardiovascular Medicinal
`Products’1.As explained in the following section, the
`QT interval of the electrocardiogram (ECG) is a
`widely used measure of the ventricular repolariz-
`ation process and its prolongation may be asso-
`ciated with a risk of sudden death.
`The foundations of the CPMP document are
`based on a substantial number of serious cardiac
`events produced by a wide range of non-cardio-
`vascular therapeutic agents that are not expected
`on the basis of their mechanism of action to pro-
`long QT. Such agents belong to different phar-
`macological classes, such as psychotropic drugs
`(tricyclic-amitriptiline and tetracyclic antide-
`pressants, phenothiazine derivatives, haloperi-
`dol, pimozide, risperidone and sertindole), pro-
`
`kinetic (cisapride), antimalarial medicines (halo-
`fantrine, quinine and chloroquine), antibiotics
`belonging
`to
`several
`chemical
`classes
`(azithromycin, erythromycin, clarithromycin,
`spiramycin, pentamidine,
`trimethoprim-sul-
`famethoxazole and sparfloxacin), antifungal
`agents (ketoconazole, fuconazole and itracona-
`zole), an agent for treating urinary incontinence
`(terodiline), and certain histamine H1-receptor
`antagonists (astemizole, terfenadine and dephen-
`hydramine). These drugs, in certain very rare in-
`stances, can trigger life-threatening polymorphic
`ventricular tachycardias, such as torsade de pointes,
`often in the presence of additional factors favour-
`ing, directly or indirectly, proarrhythmic events.
`The relevant factors include congenital or ac-
`quired long-QT syndrome, ischemic heart dis-
`ease, congestive heart failure, severe hepatic or
`renal dysfunction, bradycardia, electrolyte im-
`balance (hypokalemia due to diuretic treatment,
`hypomagnesemia, hypocalcemia, acidosis and
`intracellular Ca11 loading), intentional or acci-
`dental overdose, and concomitant treatment with
`ion channel blocking drugs or agents that inhibit
`the drug detoxification processes2–4.
`The CPMP guideline should be considered to
`be a strong signal sent by public Health
`Authorities to drug developers that the problem
`of QT prolongation by non-cardiovascular drugs
`is now very significant and, thus, it requires care-
`ful scrutiny and research efforts for any com-
`pound undergoing future development.
`In an attempt to offer an overview of the mul-
`tiple aspects of this complex and now political
`problem, this article will briefly review some as-
`pects of cardiac electrophysiology, species het-
`erogeneity in ion channels intervening in cardiac
`repolarization, and congenital cardiac-channel
`
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`abnormalities responsible for fatal arrhythmic events. The re-
`view will also describe a preclinical strategy (in vitro electro-
`physiology in human cardiac cells and in vivo pharmacologi-
`cally validated conscious dogs) that should answer the basic
`expectations of the CPMP guideline. The discussion section
`stresses several problems associated with QT prolongation and,
`in particular, the danger of drugs prolonging QT in patients
`with concurrent cardiac risk factors.
`
`Cardiac electrophysiology
`The cardiac action potential
`The cardiac action potential is the pattern of electrical activity
`associated with excitable heart cells. It is the result of numerous,
`distinct, successively activated currents generated by the passage
`of biologically important ions (Na1, Ca11 and K1) through spe-
`cialized membrane structures such as ionic pumps and exchang-
`ers and, most importantly, voltage-gated ion channels. These
`currents are considered to be depolarizing when they carry ex-
`tracellular positive charges into the cell and to be repolarizing
`when they carry positive charges to the cell exterior5.
`The cardiac action-potential recorded from either an atrial or
`a ventricular human myocyte can be dissected into five distinct
`phases (Fig. 1).The phase 0, or action potential upstroke, is gen-
`erated by the rapid, transient influx of Na1 into myocytes via
`Na1 channels (inward current: INa). Phase 2, or plateau of the
`action potential, is essentially because of the entry of extracellu-
`lar Ca11 into the cells through L-type Ca11 channels (inward
`current: ICa). Phases 1 and 3 describe, respectively, the early and
`late repolarization process and are mediated by the efflux of K1
`from the cell through the opening of several distinct K1 chan-
`nels. The transient outward current (Ito) contributes to the ter-
`mination of the upstroke of the action potential by causing an
`early phase of rapid repolarization (Phase 1), whereas several
`
`Figure 1. Example of an action potential recorded from a myocyte of
`human atrium and Na1 (INa), Ca11 (ICa), K1 (IKr, Isus, IKs and IK1) ion
`channel currents, which underlie each of its phases (0, 1, 2, 3 and 4).
`
`Figure 2. Examples of action potentials recorded from canine, guinea
`pig, rat and human atrial myocytes. Note the dramatic differences in
`morphology and duration. Action potentials were recorded at 37 6
`18C using the whole-cell patch clamp technique. Action potentials
`were elicited by a 4 ms current pulse of 1.5–2 times the threshold
`level. The solution bathing the cell consisted of (in mmol L21): 137
`NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 11 Glucose, 10 HEPES; adjusted to a
`pH of 7.4 with NaOH. Glass pipettes were filled with an ‘internal’
`solution that consisted of (in mmol L21): 120 K-aspartate, 20 KCl,
`4 Na-ATP, 5 EGTA, 5 HEPES; adjusted to a pH of 7.2 with KOH.
`
`distinct K1 channels contribute to Phase 3 repolarization [IKr or
`Human Ether-a-go-go Related Gene (HERG) current, Isus and
`IKs] by opposing Ca11 influx during the plateau phase.
`Finally, the inward rectifier (IK1), though mainly responsible
`for maintaining resting potential (Phase 4) also plays a promi-
`nent role in the final repolarization process in many species,
`although to a lesser extent in the human heart, and clamps the
`cardiac myocyte at its resting potential (Fig. 1).
`All of the currents described in human ventricle have also
`been shown to be present in human atrium, although their dis-
`tribution and amplitudes are a tissue-specific feature.These re-
`sults imply that human atrial myocytes, as proposed in a later
`section, could be used for determining the cardiac electrophysi-
`ological safety of novel drug candidates because human atrial
`tissue can be obtained from virtually normal atria.
`The shape and duration of the cardiac action potential are fea-
`tures specific to each animal species (Fig. 2). They reflect subtle
`differences in type, structure, cellular distribution and relative
`contribution to the generation of the cardiac-action potential of
`the transmembrane-current through the various channels.
`
`Species heterogeneity in cardiac ion channels
`K1 channels represent the class of channels with the greatest
`species-dependent heterogeneity. Differences in the makeup,
`identity and pharmacology of cardiac ion channels suggest that
`the results obtained from tissue derived from experimental ani-
`mals may not adequately predict drug effects in the human myo-
`cardium. Extrapolation of such data to human tissue requires
`great caution and may not always be valid (Table 1).
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`Table 1. Electrophysiological effects of selected drugs on the action potential duration (APD) and certain K1 currents in
`guinea pig, dog, rat and man and their adverse clinical effects
`
`Preclinical effect in
`
`Drug
`
`Indication
`
`Guinea pig
`
`Dog
`
`Terodiline
`
`Urinary incontinence No change APD (Ref. 29)
`
`Terfenadine Antihistamine
`
`No change (Ref. 30) or
`increased APD (Ref. 32)
`
`Increased APD
`(Ref. 30)
`
`Rat
`
`?
`
`Man
`
`?
`
`Adverse
`clinical effect
`
`Arrhythmias (Ref. 31)
`
`Decreased K1
`currents
`(Ref. 33)
`No change K1
`currents
`(Refs 33, 35, 36)
`No change
`APD (Ref. 39)
`
`Arrhythmias (Ref. 35)
`
`Decreased K1
`currents
`(Ref. 34)
`Decreased HERG Arrhythmias (Ref. 2)
`current (Ref. 34)
`
`Decreased HERG Arrhythmias (Ref. 40)
`current (Ref. 15)
`
`both congenital and acquired forms of long QT syndrome
`(LQTS). Electrophysiological studies performed on HERG, the
`protein product of the gene believed to be responsible for IKr, in
`human heart indicate dramatic interspecies differences in this
`channel. For instance, the Class III antiarrhythmic dofetilide is
`100-times more potent in blocking HERG than in blocking
`Bovine Ether-A-go-Go (BEAG), the channel believed to be re-
`sponsible for IKr in bovine15.This remarkable difference is the re-
`sult of a single-point mutation occurring in the a-subunit of the
`channel.Thus, very subtle changes in the protein sequence con-
`stituting a channel can dramatically affect ionic-channel pharma-
`cology. Mutations in both of the proteins (KVLQT1 and minK)
`that are believed to co-assemble and form the slow component of
`the delayed rectifier, IKs, have also been reported and may play a
`role in congenital and acquired forms of LQTS (Ref. 16).
`However, a recent study questions whether IKs play an important
`role in the repolarization of the human cardiac-action potential17.
`
`Possible cardiac adverse-effects of drugs modulating
`cardiac ion channels
`Drugs that modify the normal flux of ions through channels
`may modify certain aspects of the action potential and, thus,
`affect cardiac function.Therefore, blockers of Na1 channels re-
`duce the rate of rise of the action potential (Vmax) and can
`produce disturbances in cardiac conduction, which, if severe,
`may be life-threatening. Drugs that decrease the rate of Na-cur-
`rent inactivation and increase residual Na-current can prolong
`the duration of the action potential (ADP), prolong QT inter-
`val and thus may trigger torsades de pointes arrhythmias. Blockers
`of Ca11 channels decrease ADP, reduce the rate of A-V conduc-
`tion and produce cardiac depression, whereas Ca11 channel-
`activators prolong ADP and may cause arrhythmias. Finally, K1
`channel-blockers prolong ADP and QT (Fig. 3) and can provoke
`
`Loratadine
`
`Antihistamine
`
`No change K1 currents
`(Ref. 33)
`
`?
`
`Dofetilide
`
`Antiarrythmic
`
`Decrease IKr (Ref. 37),
`increase APD (Ref. 38)
`
`Transient outward current
`This current (Ito) responsible for Phase 1 of the action potential
`is present in cardiac myocytes of several species, including rat,
`dog, cat and man. However, this current is not present in guinea
`pig myocytes. In addition to species differences in the expres-
`sion of Ito, there are also differences in the molecular identity of
`Ito in those species that do possess it. For example, rabbit heart
`Ito is most likely the protein product of the Kv1.4 gene, whereas
`that of the rat heart appears to be encoded by the Kv4.2 gene
`and possibly Kv4.3 gene6–9. In contrast, human heart Ito is be-
`lieved to predominantly be the product of the Kv4.3 gene8.
`The importance of Ito in the normal electrical activity of the
`heart is illustrated by the fact that the blockade of this channel
`by tedisamil, an Ito blocker, can result in changes in cardiac-
`action potential duration10. Furthermore, in a canine model of
`ventricular arrhythmias, a reduction in Ito amplitude is believed
`to be an underlying arrythmogenic factor11.
`
`Sustained current
`This current is referred to as the sustained (ISUS) or pedestal
`current. In the rat heart, ISUS is entirely due to IKv1.5, a current
`highly sensitive to blockade by 4-aminopyridine12. However,
`this 4-aminopyridine-sensitive current has not yet been de-
`scribed in guinea pig or dog heart and, therefore, it cannot ac-
`count for the ISUS observed in these species. IKv1.5 partly medi-
`ates the human atrium ISUS, with the remaining portion being
`due to a novel, specific, non-selective cation channel13. This
`channel also appears to be responsible for ISUS in the human
`ventricle, where no Kv1.5-like current can be recorded14.
`
`Delayed rectifier
`The rapid component of the delayed rectifier K1 current (IKr) has
`been the topic of much research, because of its involvement in
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`K+
`
`K+
`B
`
`K+
`
`B
`
`B
`
`Extracellular
`
`Intracellular
`
`EAD
`
`B
`
`QT interval
`
`K+ channel(cid:0)
`blockade
`
`ADP prolongation(cid:0)
`and early after(cid:0)
`depolarization(cid:0)
`(EAD)
`
`R
`
`P
`
`T
`
`Q
`
`S
`
`QT interval
`
`QT(cid:0)
`prolongation
`
`Torsades de(cid:0)
`pointes
`
`Figure 3. Simplified diagram showing the effect produced by a drug
`blocking cardiac K1 channel (B) on the duration of the action poten-
`tial duration and on electrocardiogram (ECG). This drug can produce
`prolongation of the unicellular action potential followed by early
`post-depolarization, QT interval prolongation and torsades de pointes.
`
`arrhythmias, whereas K1 channel-activators shorten ADP and
`can also trigger arrhythmia. It should be noted that Na1, K1
`and Ca11 channel-blockers can also be useful antiarrhythmics
`in patients with existing arrhythmias.
`
`QT-interval of the electrocardiogram
`The electrical activity of the whole heart is reflected in the ECG.
`The wave sequence comprising the ECG-trace during a normal
`cardiac cycle results from the sum of the elementary electrical
`activities of each excitable cell in the heart chambers (Fig. 3).
`QT-interval duration represents the sum of both ventricular
`depolarization (QRS interval) and ventricular repolarization
`(QT minus QRS). However, QT prolongations very rarely result
`from widening of the QRS complex. The QT segment of the
`ECG itself or its heart rate-corrected form (QTc), according to
`the formulae of Bazzett (QTcB 5 QT/˛RR) or Fridericia (QTcF
`5 QT/3˛RR), are clinically used indices of the cardiac repolar-
`ization process. Its value is influenced by several factors, such as
`heart rate (the correction formula by Bazzett is relatively inac-
`curate because it under- or over-estimates the true duration of
`repolarization at low and high rates, respectively), extent of the
`sympathetic and parasympathetic drive to the heart, the ECG-
`lead selected to measure this parameter and even the person
`performing the manual measurement of the QT. For this rea-
`son, the section of the CPMP QT guideline dealing with
`
`methodologies requires that the electrocardiographic effects of
`drugs should be evaluated by cardiologists experienced in such
`evaluations. In particular, measurements of QT interval and QT
`dispersion should be assessed as the mean value derived from
`3–5 cardiac cycles and excluding the U-wave from the QT in-
`terval measurement, unless the T- and U-waves merge together1.
`In conditions of stable sinus rhythm, QTc values greater than
`430 msec (adult males) or 450 msec (adult females) and QT
`dispersion values ,40–60 msec or drug-induced changes
`,100% from the baseline value are generally considered to re-
`flect some abnormality in the repolarization process1.
`
`Long QT syndrome, a genetic disease
`Long QT syndrome is a clinically heterogeneous group of disor-
`ders of cardiac repolarization, which may result from the use of
`a drug or from a pathological condition with a genetic or non-
`genetic basis. The essential electophysiological mechanism un-
`derlying this condition is a reduction in the intensity of the net
`outward-current responsible for the repolarization process.This
`can result from either delayed inactivation of the inward Na1
`current or a decrease in the current carried by one or more K1
`channels (gain and loss of function mutations, respectively, for
`congenital LQTS patients). These pathological changes in ion
`channel function lead to a delay in the repolarization process,
`which can trigger the development of early- and late post-depo-
`larizations (particularly at the level of the Purkinje conducting
`system) followed by episodes of torsades de pointes (Fig. 3).
`Studies utilizing genetic analysis and molecular biological
`techniques have identified mutations in genes encoding proteins
`that form ion channels in individuals afflicted with congenital
`LQTS. Recently, three ion channel encoding genes (KvLQT1,
`HERG and SCN5A) have been found to be sites of one or more
`mutations, which produce unfavourable changes in the structure
`of the encoded channel protein16,18. Hence, the Na1 channel
`with such a mutated a-subunit (SCN5A) exhibits a markedly
`enhanced time-dependent residual current compared with the
`equivalent current carried by the wild-type channel. This is
`responsible for the prolongation in action potential duration.
`Several mutations in the HERG gene that encodes the a-sub-
`unit of the K1 channel carrying the rapid component of the
`delayed rectifier (IKr) have also been described. Mutations have
`also been identified in the KvLQT1 a-subunit that coassembles
`with the minK b-subunit to form the K1 channel carrying the
`slow component of the delayed rectifier (IKs). Recently, mu-
`tations have also been reported in the minK b-subunit. These
`mutations are associated either with a reduction in the ampli-
`tude of a repolarizing K1 current or with abnormally operat-
`ing or non-functional channels. The phenotypic manifestation
`of such alterations is generally a prolongation in action potential
`duration accompanied by a particular susceptibility to triggers
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`[sympathetic (arousal) and parasympathetic (resting) acti-
`vation, hypokalemia, drugs prolonging repolarization] of
`arrhythmic events of the torsade de pointes type19.
`
`Strategies to determine the potential
`of a drug to prolong QT
`Preclinical tests recommended by CPMP
`The CPMP QT document proposes preclinical and clinical stud-
`ies that are likely to unveil the QT prolongation potential of
`non-cardiovascular medicines and to provide reassurance con-
`cerning their safe clinical usage1. Preclinical, in vivo cardiovas-
`cular safety pharmacology studies are required to be rigorous,
`and to use a range of escalating doses. Furthermore, they
`should include measurements (typically in the dog) of heart
`rate, blood pressure and ECG analysis. In addition, before first-
`use in humans it is also recommended that an in vitro electro-
`physiological study be performed using a suitable cardiac
`preparation and physiologically relevant conditions. In fact, in
`vitro Purkinje fibers or papillary muscles taken from the
`myocardium of an established laboratory animal species (such
`as rabbit, guinea pig, dog or pig) are considered suitable, be-
`cause it is believed the major ionic currents underlying their
`action potentials resemble those contributing to the repolar-
`ization process of the human heart.
`In addition, these studies should be extended to inspection
`for a reverse rate-dependency phenomenon if the compound
`under study is found to prolong the action potential duration.
`The concentrations of the drug to be tested are expected to
`cover and well exceed (in our opinion, 10–30-fold) the antici-
`pated maximal therapeutic plasma concentrations of the drug
`candidate. Furthermore, these studies should take into consid-
`eration certain aspects of the drug’s pharmacokinetics, such as
`the existence of major active metabolites.The effect of the drug
`on the action potential prolongation at 90% of repolarization
`(ADP90), on possible early post-depolarization events and sub-
`sequent triggered activities are considered of primary rel-
`evance in the context of a proarrhythmic potential accompany-
`ing the prolongation of QT interval. Additional parameters to
`be measured are ADP30, ADP60, membrane resting potential,
`action potential amplitude and upstroke velocity (Vmax), be-
`cause they can provide additional information on the cardiac
`electrophyisological safety of the compound.
`If the results of all these studies indicate that the novel agent
`does not prolong the QT interval in an unacceptable manner,
`then the drug candidate can be cleared for safety assessment
`studies in healthy volunteers provided all other normal safety
`requirements are met1.
`The preclinical in vitro electrophysiologic approach pro-
`posed by the CPMP guideline and as outlined above is a classi-
`cal one. However, it may not be the best one available, because
`
`274
`
`it makes the basic assumption that the effects of compounds
`on cardiac ion channels present in the heart of the animal
`species selected can be directly translated to the human heart.
`As explained above and illustrated in Table 1, there are many
`examples of species-differences in cardiac ion channel expres-
`sion and pharmacology. In order to overcome this problem,
`we propose that in vitro cardiac safety electrophysiology stud-
`ies should, whenever possible, be performed using human
`heart ion channels.
`
`Use of human cardiac myocytes to determine the
`electrophysiological effects of a drug candidate
`The in vitro electrophysiologic profile of candidate drugs should
`be performed whenever possible on ion channels involved in
`the electrical activity of the human heart myocytes [INa, ICa, Ito,
`Isus, IK1 and Ikr (HERG)]. If recording a particular current in na-
`tive human heart cells (IKr) is difficult, then the cloned human
`channel (HERG) expressed stably in a human cell line (such as
`HEK cells) is a suitable alternative. In addition, the experimen-
`tal conditions (temperature, holding potential and ionic solu-
`tions) for these studies should be as close as possible to those
`existing on a physiological level. The concentrations studied
`should cover a 2–3 log unit range, with the highest concen-
`tration studied being at least 10- to 30-fold higher than the
`anticipated plasma or tissue concentration necessary for the
`therapeutic activity. Results obtained for the compound under
`study using this assay should then be compared to reference
`compounds known to block a particular ion channel and
`which are clinically associated with arrhythmias. Furthermore,
`if a concentration of the drug under study has an effect on an
`ion channel, it is essential to determine the possible existence
`of a rate-dependency relationship for this effect (Fig. 4, Box 1).
`The proposed departure from the classical study of the action
`potential profile on ventricular preparations from the hearts of
`experimental animals is supported by the fact that ion channels
`are ultimately responsible for any drug-induced change in the
`action potential pattern. Although ion pumps and exchangers
`do contribute to the morphology of the action potential, virtu-
`ally every drug that has been associated with arrhythmic events
`in man has been found to affect one or more human cardiac
`ion channels. Finally, the extent of the effects produced by a
`compound on the whole action potential profile may depend
`on the morphology of the action potential at the time of the
`study, making interpretation of such experiments difficult20.
`The main objection against any novel approach to determine
`safety is whether the proposed tests are sufficiently powerful
`to reveal, at least as well as established methods, a possible ad-
`verse effect of a compound. Figure 5 illustrates an example of
`the ion channel-blocking effects of three clinically used drugs,
`terfenadine, haloperidol, and cisapride, which have been
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`In vitro tests in human cardiac ion channels(cid:0)(cid:0)
`Study of the effects of the drug candidate on native human ion currents using
`appropriate experimental conditions (Box 1)
`
`Figure 4. Proposed flow-chart of studies
`designed to reveal the potential of non-
`cardiovascular drugs to prolong QT.
`
`Evidence of ion-channel block?
`
`Abandon or proceed to
`further electrophysiological
`studies
`
`Yes
`
`No
`
`In vivo tests in awake, trained, experimentally validated dogs(cid:0)
`implanted with telemetry systems(cid:0)(cid:0)
`Measurement of aortic blood pressure, heart rate and ECG (PR, QRS, QT interval,
`T- and U-wave morphology, arrhythmic events) for the duration of action of the
`compound administered at escalating doses in successive days. The highest dose
`should give plasma levels that are at least 10-fold higher than the plasma
`concentration expected to afford the desired therapeutic effect in man. The
`interpretation of the results is subject to caution if the compound increases the
`baseline heart rate
`
`Evidence of QT prolongation?
`
`Abandon
`
`Yes
`
`No
`
`Check for possible active metabolites and study
`them in in vitro electrophysiological tests. In
`vivo electrophysiology in anaesthetized dogs to
`measure cardiac conductance and refractory
`periods
`
`Phase I clinical studies
`
`Abandon non-innovative(cid:0)
`drugs increasing QT >10 ms
`
`reported to produce malignant arryhthmias2,21,22.These drugs,
`in the nanomolar range, block the HERG K1 channel carrying
`IKr. The blockade of this human cardiac channel is presently
`considered the most likely mechanism to underlie the pro-
`arrhythmic risks associated with the use of these drugs under
`certain clinical conditions.
`To avoid the disastrous consequences of finding, during the
`final preclinical development process, that the drug candidate
`chosen has properties conferring arrhythmogenic potential,
`the electrophysiological studies proposed should be performed
`as early as is feasible and preferably during the early preclinical
`selection phases. An ideal scenario would be one in which sev-
`eral possible drug candidates possessing the desired pharma-
`cological profile – as far as their efficacy, preliminary pharma-
`cokinetics and drug safety properties are concerned – are
`rapidly screened against cardiac ion channels and ranked ac-
`cording to their adverse activity (generally blockade of K1,
`Ca11 and Na1 channels). The safest compounds should be se-
`lected for further development studies. By moving this evalu-
`ation process to earlier in the selection process of an acceptable
`drug candidate, the latter apprehension surrounding com-
`
`pound cardiac-safety evaluation and discovery of possible side-
`effects can be substantially reduced.
`Furthermore, it would help the decision making process to
`extend, whenever considered necessary, the in vitro cardiac
`safety screening evaluation of a drug candidate, by performing
`studies using conditions that mimic pathological processes
`(acidosis, hypokalemia and low resting potentials) known to
`favour the proarrhythmic activity of compounds blocking car-
`diac repolarization channels. Such an evaluation, as outlined in
`Box 1, would provide the kind of database necessary to grade
`potential preclinical risks during the preparation of the devel-
`opment plans for a compound.
`In addition to the use of native human cardiac tissue to de-
`fine the ion channel-blocking profile of a given drug, investi-
`gators have used various expression systems, both stable and
`transient, which express a desired channel. These expression
`systems range from frog eggs or Xenopus oocytes, perhaps the
`most commonly used expression system, to insect cell lines to
`a variety of mammalian cell lines, including non-cardiac
`human cell lines. Although convenient to use, these systems
`have many potential limitations. For instance, ion channels in
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`CFAD V. UPENN
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`reviews
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`research focus
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`PSTT Vol. 2, No. 7 July 1999
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`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`HERG block (%)
`
`10-4
`
`10-2
`
`100
`Drug (nM)
`
`102
`
`104
`
`Figure 5. Effects of the cisapride, haloperidol and terfenadine on IKr
`current, recorded from stably expressed human ether-a-go-go related
`gene (HERG) channels in HEK-293 cells. Experiments were performed
`at 378C using the whole cell patch clamp technique. Symbols are
`means 6 SE (n 5 5–7). Fits of the mean concentration-response
`curves yielded IC50 values of 208 nM, 12 nM, and 28.1 nM for
`terfenadine, cisapride and haloperidol, respectively. HERG current was
`elicited by a 400 ms voltage pulse to 110 mV from a holding potential
`of 275 mV. Current was measured upon return to 240 mV (400 ms).
`The solution bathing the cell consisted of (in mmol L21): 137 NaCl,
`4 KCl, 1 MgCl2, 1.8 CaCl2, 11 Glucose, 10 HEPES; adjusted to a pH of
`7.4 with NaOH. Glass pipettes were filled with an ‘internal’ solution
`that consisted of (in mmol L21): 120 K-aspartate, 20 KCl, 4 Na-ATP,
`5 EGTA, 5 HEPES; adjusted to a pH of 7.2 with KOH.
`
`would be routinely performed with any drug undergoing
`preclinical development for first-use-in-man, provided that they
`are performed with the use of recent technological advances. In
`particular, heart rate, blood pressure and ECG parameters (PR,
`QRS and QT intervals,T- and U-wave morphology and arrhyth-
`mic episodes) should be determined in unrestrained dogs with
`implanted telemetry systems, allowing these parameters to be
`monitored through the full duration of the biological effects
`from the administered dose. Increasing doses of the drug can-
`didate (given by the same route as that to be used clinically)
`can be studied in successive days, with the highest one being
`selected to yield plasma (or more correctly, myocardial) levels
`of the active species [or the major metabolite(s)] that are at least
`10-times higher than that expected to produce the desired ef-
`fect in man (Box 1). If relevant plasma concentrations can only
`be obtained with repeated drug dosing, this pharmacodynamic
`study should be performed by using a repeated-dose regimen,
`with the cardiovascular parameters measured during the first
`
`Box 1. In vitro electrophysiology studies in native
`cardiac channels present in human atrial myocytes
`or in cloned channels expressed stably in
`mammalian cells for determining the potential of
`non-cardiovascular drugs to prolong QT interval
`
`Basic in vitro electrophysiology studies in human cardiac
`channels under physiologically relevant conditions
`Determination of the effects of INa, ICa, Ito, IKl, Isus and IKr human
`ether-a-go-go related gene (HERG) on:
`• five concentrations (covering 2–3 log units) of the drug
`candidate in six experimentally viable preparations using
`physiological ionic solutions, 1 Hz and 378C temperature
`(except for INa, which can be correctly recorded only at a
`lower temperature). These tests should be validated by
`studying aprropriate reference compounds simultaneously.
`• frequency (0.1, 1, and 2 Hz) dependence studies for any
`relevant adverse effect produced by the test compound.
`
`Advanced in vitro electrophysiology studies in human
`cardiac channels using pathologically relevant conditions
`Determination of the effects on INa, ICa, Ito, IKl, Isus and IKr (HERG) on:
`• the drug using the same parameters listed in the previous
`paragraph, but by using certain experimental conditions
`that simulate some of those known to predispose to
`arrhythmia (acidosis, hypokalemia, low resting potentials).
`
`the native cardiac cell have subunits or accessory proteins that
`are important for its normal functioning. Typically, in expres-
`sion systems only the a-subunit is generally expressed without
`any of these potential modulating subunits. In addition, there
`are some prominent potassium channels that play an impor-
`tant role in the repolarization of the heart that have no cloned
`correlate, such as the transient outward channel in human
`heart. Furthermore, the expression system used can have dra-
`matic effects on the pharmacology of an io