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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5329_Библиотеки_им_академика_М_И_Перельмана
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high intracellular Kþconcentrations. These differences in intracellular and extracellular ion concentrations and charges across the plasma membrane are called the
electrochemical gradients and generate the driving force of the diffusion-controlled
flow of ions through their selective ion channels.
At rest, inwardly rectifying potassium channels are open and keep the membrane
potential approximately at –80 mV.
During the excitation of the cardiomyocyte, the membrane potential depolarizes
from the rest potential to þ40 mV. The membrane is kept at positive potentials
throughout phases 0–2 of the action potential due to the subsequent opening of the
voltage-gated sodium channel (Na
v
1.5, gene symbol SCN5A)andL-type calcium
channels (protein symbol for the pore forming a-subunit is Ca
v
1.2, gene symbol
CACNA1C). The opening of these channels leads to a rise in [Na
þ
]iand the release of
Ca
2 þ
from intracellular stores, which subsequently increases [Ca
2 þ
]i. The cardiac
sodium–calcium exchangerand the ATP-drivenpumps sodium–potassium ATPase and
calciumATPase account for the [Na
þ
]iand [Ca
2 þ
]irestorationfound at rest potentials.
Figure 6.1 Schematic diagram to illustrate the correlation between an ECG and the action
potential of a human ventricular myocyte. P: The P wave displays the excitation of the
pacemaker and atrium. Q, R, S: The Q, R, and S spikes built a complex, which mirrors the
excitation and the propagation of the excitation in the ventricles. T: The T wave reflects the
recovery of the ventricles from excitation. The QT interval is the duration from the beginning of
the excitation in the ventricles (Q spike) to the end of their recovery (end of Twave). The action
potential of a ventricular myocyte starts with depolarization in phase 0 and ends after the
membrane potential returned to rest potential in phase 4.
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Finally, voltage-gated pota ssium channels accomplish the repolarization to rest
potentials. The primary potassium channel involved in human ventricular repolarization is the human ether a-go-go related gene product (hERG, protein symbol
K
v
11.1, gene symbol KCNH2). The current related to this channel is the rapidly
activating delayed rectifier current I
Kr
.
6.2.2 Delayed Repolarization: Mechanisms and Models
6.2.2.1 hERG/I
Kr
Block Since repolarization is essentially achieved by the
hERG/I
Kr
current, it becomes plausible that a block of this current presumably
causes a delay of ventricular repolarization and consequently will increase a
torsadogenic risk. A block of hERG/I
Kr
current is not a sufficient condition to predict
neither the potentials of a compound to delay ventricular repolarization nor a
torsadogenic risk (see Sections 6.2.2.2–6.2.2.4 and 6.2.3) [4]. Virtually all drugs
associated with TdP, however, are hERG/I
Kr
blockers and prolong QT/QTc intervals
of the heart and action potential durations of isolated cardiomyocytes [9]. Consequently, in vitro models were developed to address hERG liabilities to assess cardiac
risk in an early stage of the drug development process.
hERG blockers comprise compounds with diverse structures and from different
therapeutic classes, including antiarrhythmics, antipsychotics, antimicrobials, and
antihistamines. In early drug development, 70% of all compounds have hERG
blocker potentials. It is suggested that the presence of multiple aromatic side-chains in
the large central cavity of the channel may contribute to the promiscuity of hERG
blockers [10–12]. As a safety margin for potential drugs, a 30-fold difference between
the maximum therapeutic-free plasma concentration and the IC
50
for hERG activity is
proposed [13].
Impairment of clinically relevant metabolic pathways is able to increase cardiac
risk. A key enzyme in the clearance of pharmaceutical drugs is the cytochrome P450
(CYP) 3A4. This enzyme can be affected by a number of compounds including the
antifungal drug ketoconazole and grape fruit juice. It was shown that coadministration
of these bloc kers with drugs such as terfenadine, astemizole, cisapride, or pimozide
with known hERG blocker properties reveal a higher risk for TdP arrhythmias in
patients (Figure 6.2) [14].
A cost-effective high-throughput assay system is the hERG-binding assay. Membrane fractions of cells stably expressing the hERG channel are extracted and
incubated with a high affinity hERG channel ligand labeled with radioactive isotope,
for example, [
3
H]astemizole. The nonradioactive test compound is added to the [3H]
astemizole-labeled membrane fraction. The decrease of radioactivity with rising
concentrations of the test compound is analyzed to determine the IC
50
values for the
inhibition of [
3
H]astemizole approximation of the dissociation constant Kdand
the binding site density B
max
of the test compound to the hERG channel
(Figure 6.3a) [15].
The disadvantage of this assay system is that it only assesses the physical
interaction of a compound with the channel and does not provide information
regarding the effect on its physiological properties.
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The “gold standard” assay system to display interactions of compounds with the
electrical properties of the hERG channel are conventional voltage clamp recordings
from recombinant cell lines such as CHO (Chinese hamster ovary) or HEK (human
embryonic kidney) cells stably expressing the hERG channel. The typical hERG tail
current can be recorded by applying a two-step repolarizing voltage protocol after the
initial depolarization. The block of the tail current can be observed by subsequently
repeated application of the voltage protocol in the presence of the test compound
(Figure 6.3b) [15].
N
H
N
N
F
F
O
Pimozide
ON
N
H
O
Cl
H
2
N
OMe
OMe
F
Cisapride
N
OH
OH
Terfenadine
NN
NH
N
MeO
F
Astemizole
ONN
O
O
O
N
N
Cl
Cl
H
Ketoconazole
Figure 6.2 Compounds that impair clinically relevant metabolic pathways and increase
cardiac risk. Ketoconazole, terfenadine, astemizole, cisapride, and pimozide.
Figure 6.3 (a) Inhibition of [3H]astemizole binding in HERG transfected membranes by
typical hERG blocking compounds. (b) A representative hERG/I
Kr
current tracing before and
after astemizole in HERG transfected HEK293 cell
(with permission from Dr. Peter
Chiu [14]).
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Today, the low throughput of conventional patch clamp can be overcome by
automated planar patch clamp systems that are able to analyze up to 384 cells in
parallel. The throughput of the assay is dependent on the success rate to obtain high
quality resistance called “seals” (in the range of giga ohms) of the cell membrane to
the patch substrate and whole cells recordings with appropriate current amplitudes.
These issues can be overcome by the population patch approach where a single
amplifier is able to average the whole-cell currents from multiple cells at once
leading to very high success rates (>95%). However, comparisons between
conventional and high-throughput patch clamp reveal some significant differences [16, 17].
The hERG assay can be modified to reach high-throughput screening (HTS)
capacities by using membrane potential-sensitive fluorescent dyes or the assessment
of rubidium (Rb
þ
) efflux by either spectrophotometric analysis in an atomic
absorption spectrometer or using the radioactive nuclide
86Rbþ
. Nonetheless, all
these assays provide a decreased sensitivity for compound-induced hERG block and
lack an ideal correlation with conventional voltage clamp experiments [16, 18].
The hERG channel blocker effects described above are due to acute (seconds to
minutes) compound interactions on the channel itself. Another mechanism, which
leadstoablockoftheI
Kr
current, is a chronic action (hours to days) on the trafficking
of the hERG channel during its synthesis. Th is provides retent ion of the protein in
the endoplasm atic reticulum, which subsequently decreases the channel density on
the cell surface and I
Kr
currents [19–21]. Drugs with solely chronic blocking
capabilities with no acute effect on the electrical properties of the hERG channel
were identified [20]. Although these drugs would not be registered by conventional
(acute) hERG assays and would be not identified in preclinical cardiac safety assays,
no experimental protocols to address this phenomenon are currently issued in the
regulatory guidelines.
6.2.2.2 KvLQT1/MinK or IKs Block The slowly activating delayed rectifier
potassium current I
Ks
is involved in ventricular repolarization. This current requires
the coupling of the pore forming a-subunit K
v
LQT1 (protein symbol Kv7.1, gene
symbol KCNQ1) with its b-subunit MinK (gene symbol KCNE1) to generate
functional I
Ks
. Mutations in the encoding genes KCNQ1 and KCNE1 have been
linked to the LQT1 or the LQT5 syndromes, respectively. I
Ks
is a reserve repolarizing
current that is activated with increased heart rate and by beta-adrenergic agonists as
well as serum- and glucocorticoid-inducible kinase (SGK) 1 [22]. The modulations
are dependent on the presence of the functional b-subunit MinK.
It was reported that only a small number of compounds described to be specific I
Ks
blockers which show benign effects in vivo (e.g., chromanol 293B or HMR 1556) can
potentiate torsadogenic risk after preceding I
Kr
block in rabbits [23]. Moreover, more
potent I
Ks
blockers were shown to prolong QT/QTc interval and induce severe TdP
arrhythmias in conscious dogs [24].
A higher throughput analysis to record I
Ks
and address safety pharmacological
aspects is established and validated by the application of a recombinant cell line stably
expressing both genes, KCNQ1 and KCNE1, in a planar automated patch clamp
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system [25]. In addition, functional modulation of the current in such recombinant cell
lines by adrenergic agonists has been reported [26].
6.2.2.3 Na
v
1.5 Activator The excitation of ventricular cardiomyocytes is initiated
by the fast activation of voltage-gated sodium channel resulting in inward sodium
current (I
Na
) and the fast upstroke in phase 0 of the cardiac action potential (Figure 6.1).
The voltage-gated sodium channel in the heart primarily consists of the pore forming
a-subunit Na
v
1.5, which largely accounts for the properties of the cardiac sodium
current and complex of gene products from all four known b-subunits (gene symbols
SCN1B, SCN2B, SCN3B, and SCN4B) [27]. The influence of the b-subunits on the
propertiesof the cardiac sodium current is still not defined but the report that a mutation
in SCN4B causes a Brugada-like LQTS indicates their significance [28].
The fast activation of I
Na
is responsible for the propagation of the excitation in the
heart. Approximately 20% of all patients with Brugada syndrome carry mutations in
the gene SCN5A encoding the Na
v
1.5 channel (LQT3 syndrome), which account for a
defective inactivation of the channel and a sustained inward I
Na
in the late phase of the
action potential. The persisting I
Na
induces delayed ventricular repolarization ac-
companied by an increased torsadogenic risk [3].
A few compounds including the neurotoxic peptides ATX-II [29], BmK 1 [30],
Anthopleurin-C [31], the alkaloid veratridine, and the S-enantiomers of the compounds DPI 201-106 and Carsatrin [32–34] are described to disturb accurate
inactivation of cardiac Na
v
1.5 and activate persistent late INa(Figure 6.4). In addition,
the a
1
-adrenergic agonist Alfuzosin (Figure 6.4), which is approved by the FDA/for
the treatment of benign prostatic hyperplasia, revealed QT/QTc interval prolongation
properties by activating late I
Na
[35]. Heterologous expression of cardiac sodium
channels Na
v
1.5 have been reported to study drug-induced modulations of the gating
of channel [31].
6.2.2.4
L-Type Calcium Channel Activator The most important calcium channel
in the adult human heart is the voltage-gated
L-type (long-lasting activation) calcium
channel. The pore forming a-subunit Ca
v
1.2 requires coupling to the d - and b -sub-
units (a2d and b2a) to generate functional
L-type calcium currents (I
Ca,L
) [36].
Mutations in the exons 8 and 8a in the encoding gene for the a-subunit CACNA1C can
lead to Timothy’s (LQT8) syndrome. These mutations are attributed to cause
improper voltage-dependent inactivation of the channel encompassing a prolonged
calcium-inward current, a delay in ventricular repolarization with a high risk for
sudden cardiac death, and severe complications in other tissues [37]. To date, two
compounds have been identified that activate I
Ca,L
directly,namely FPL 64176 and the
(S)-( )-BayK 8644 (Figure 6.5) [38–41]. Both compounds are able to induce TdPlike arrhythmias in perfused cardiac wedges or Langendorff-perfused hearts [42, 43].
Furthermore, b-adrenergic receptor agonists are able to increase I
Ca,L
currents [44].
Recombinant expression systems have been established and allow the recording of
functional
L-type calcium channels in planar automated patch clamp systems.
Protocols are proposed to overcome typical current run-down effects and (S)( )-BayK 8644 induced elevation of I
Ca,L
was revealed [36, 45]. It is still not
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reported if these recombinant cell models are capable of indicating correct GPCR
modulation of I
Ca,L
.
6.2.3 Shortened Ventricular Repolarization
The causes for acquired QT shortening have been reported to be hypercalcaemia,
hyperkalemia, elevated plasma acetylcholine or catecholamine concentrations, hyperthermia, myocardial ischemia, and VT [46, 47]. Rare incidents for inherited short
N
NN
H
NM
eO
MeO
NH
2
O
O
O
N
OH
N
N
H
CN
N
N
S
N
OH
N
F
F
N
H
N
O
OH
H
OH
H
OH
N
O
H
H
O
H
H
HO
O
O
MeO
MeO
DPI 201-106Veratridine
Carsatrin
Alfuzosin
Figure 6.4 Compounds that disturb accurate inactivation of cardiac Nav1.5 and/or activate
persistent late I
Na
. Veratridine, DPI 201-106, Carsatrin, and Alfuzosin.
N
F
3
C
O
2
NC
O
2
M
e
N
MeO
2
C
O
S( )-(–)-BayK8644
FPL 64176
Figure 6.5 Compounds that activate L-type calcium channels (I
Ca,L
). FPL 64176 and
(S)-( )-BayK 8644.
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QT syndromes suggest potential targets for drug-induced QT shortening with
increased proarrhythmic risk including gain of function mutation s in KCNH2,
KCNQ1,orKCNJ2 and loss of function mutation in the genes SCN5A, CACNA1C,
and CACNB2B [5, 48].
It was recently shown that drugs can induce shortening of repolarization, and this
effect is also associated with an increased proarrhythmic risk. In the same study,
evidence was provided that the in vitro hERG assay alone delivers insufficient
information to predict potentials to delayed repolarization. From a group of 92
compounds identified as hERG blockers, 28.3% and 16.3% revealed no effect and
induced shortening in repolarization assays (either intracellular recordings from
isolated rabbit Purkinje fibers or Langendorff-perfused rabbit hearts), respectively.
Due to these observations, the shortening of repolarization has to be accounted for
cardiac safety studies [4, 5]. There are at least two mechanisms for drug-induced
shortening of ventricular repolarization linked to a proarrhythmic risk described in the
literature and are discussed in Sections 6.2.3.1 and 6.2.3.2 [5].
6.2.3.1 hERG/I
Kr
Activation In contrast to the numbers of well-known hERG/I
Kr
blockers, only a few activators are reported including RPR260243, the two urea
derivatives N1643 and N3623, PD-118057, and mallotoxin (Figure 6.6) [49–53]. The
compounds increase I
Kr
current by different mechanism. RPR260243 slows
the deactivation and attenuates inactivation of the hERG channel (type-1 agonist).
The type-2 agonists, such as PD-118057, attenuate inactivation but do not slow
deactivation [49, 54].
N
CO
2
H
F
F
F
O
N
OMe
RPR260243
Figure 6.6 Compounds that activate hERG/IKr. RPR260243 and mallotoxin.
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Although the hERG activation compounds have been proposed to be potentially
antiarrhythmic drugs [52], evidence for the proarrhyhmic potentials have been clearly
provided [4].
6.2.3.2 Activation of ATP-Sensitive Inward Rectifier Potassium Currents The
a-subunits of inward rectifier potassium channels expressed in the heart belong to the
Kir2, Kir3, and Kir6 family. The term “inward rectifier” is related to the electrophysiological properties of the channels. These channels have a high conductance at
negative membrane potentials and the conductance does not increase during depolarization when the membrane potential turns to more positive values [55].
I
KATP
is the only inward rectifier current described to be implicated in druginduced short QT syndromes. Levcromakalim, pinacidil, and the approved antianginal drug Nicorandil are potent activators of the inward rectifier potassium channel
Kir6.2 (Figure 6.7). This channel is the pore-forming a-subunit underlying the
cardiac ATP-sensitive potassium current (I
KATP
) [55]. These compounds induce
shortening of repolarization, increased in dispersion of repolarization, and arrhythmia
in Langendorff-perfused hearts [4, 56, 57].
In vitro analysis of the current was established in heterologous expression of
KCNJ11 in Xenopus oocytes and radioactive
86Rbþ
flux assay with liposomes
enriched with purified human Kir6.2 protein, which was expressed in Saccharomyces
cerevisiae [58, 59].
6.2.4 Alterations in Intracellular Ca
2 þ
Handling
L-type calcium channels are activated during the cardiac action potential
(Section 6.2.2.4). The influx of calcium ions triggers the calcium release from
intracellular stores [calcium-induced calcium release (CICR)] and the contraction
of the cardiac myocytes. Mutations in the ryanodine receptor 2 (RyR2) are responsible
for catecholaminergic polymorphic ventricular tachyarrhythmias. These are associated with calcium overload, delayed afterdepolarizations, and a higher risk for sudden
cardiac death [60].
Delayed after depolarizations (DADs) are caused by [Ca
2 þ
]ioverload and/or
abnormal spontaneous openings of ryanodine receptors in the diastole. Known drugs,
which interfere with the calcium homeostasis are the cardiac glycosides such as
O
N
O
OHNC
N
N
H
N
N
H
CN
Levcromakalim Pinacidil
N
N
H
N
+
OOO
O
Nicorandil
Figure 6.7 Potent activators of the inward rectifier potassium channel Kir6.2. Levcromakalim,
pinacidil, and Nicorandil.
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digitoxin (Figure 6.8). A block of the Naþ/Kþ-ATPases and subsequently increasing
[Na]
i
and decreasing the Naþ/Ca
2 þ
exchanger in the SR are the supposed proposed
mechanisms for the mode of action for this class of compounds. Interestingly, theses
drugs cause ventricular arrhythmia without prolongation of the QT/QTc interval and
might not be detected in repolarizing assays. For this reason, it is proposed to include
assays that monitor the modulation of the calcium handling into cardiac safety
studies [61].
6.2.5 Preclinical Models for Assessment of Ion Channel-Related
Cardiotoxicity
As outlined above, there are several cardiac ion channels with implication in
cardiotoxicity. Recombinant or heterologous expression systems are established for
almost all cardiac voltage-gated ion channels to study compound-induced modulation
of the currents in different assay systems such as manual or paralleled automated
patch clamp, voltage-sensitive fluorescent, or radioactive or nonradioactive rubidium
flux assays. These models are necessary to study drug effects on the separated currents
in relatively inexpensive assay systems with preferably high throughput and assess
potential cardiotoxicity at an early stage in the drug development process.
The results of these in vitro assays are now incorporated into in silico prediction of
drug interaction with ion channels such as the quantitative structure–activity relationship (QSAR) for hERG channel [62–64].
The comprehensive study published in 2008 by Lu and colleagues obviated that
assessment of hERG/I
Kr
current modulation alone was not predictive for druginduced delay of ventricular repolarization [4]. Moreover, evidence was provided
that detection of delayed repolarization was not sufficient to determine proarrhythmic
potentials. Lastly, preclinical safety pharmacology studies were suggested to include
assessment of the potentials to shorten ventricular repolarization [4–6].
Until recently, there was a gap between the demands to assess the potentials for the
modulation of cardiac repolarization in high-content models such as primary cardiac
myocytes and tissues or explanted heart and the ability to apply them in highthroughput assay systems. Primary cardiomyocytes derived from either animal or
human hearts are difficult to generate in a standardized fashion and their preparation is
Figure 6.8 Cardiac glycoside, digitoxin, which interferes with calcium homeostasis.
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time consuming and costly. Furthermore, the use of primary adult cardiomyocytes in
high-throughput assay systems is hampered by their tendency to dedifferentiate in
culture within a few days and the nonhomogeneous nature of the culture.
A promising solution to bridge this gap is provided by the pluripotent stem cell
science. Genetically selected mouse embryonic stem cell-derived cardiomyocytes
(smESCM) have been shown to be physiologically relevant due to their ability to
integrate the cryo-infracted hearts and to recover the contractile function of the
injured heart [65]. Moreover, these cells are electrophysiologically characterized and
can be applied to automated planar patch clamp systems [66, 67]. Figure 6.9a–c
shows typical voltage clamp recording of I
Na
, I
Ca,L
, and IKrfrom smESCM.
Moreover, in Figure 6.9D a series of current clamp recording of action potentials
in the absence and presence of the hERG blocker dofetilide (Figure 6.10) are
provided, which reveal the potential of these cells to monitor I
Kr
blocker. All patch
clamp experiments including the current recordings were conducted with the
automated planar patch clamp systems Port-a-Patch and Patchliner from Nanion
(Munich, Germany) allowing the assessment of the modulation of cardiac repolarization in a higher throughput.
Figure 6.9 Automated patch clamp recordings from selected mouse embryonic stem cellderived cardiomyocytes (smESCM) in the Patchliner from Nanion (Munich, Germany). a–c: (a)
Voltage clamp recordings of I
Na
, (b) I
Ca,L
, and (c) IKr. Series of current clamp recording
from smESCM in the Port-a-Patch (Nanion) reveals reversible action potential
prolongation of the hERG blocker dofetilide.
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