Download Teratogenicity as a Consequence of Drug-Induced

Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Pharmacy 235
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Teratogenicity as a Consequence
of Drug-Induced Embryonic
Cardiac Arrhythmia
Common Mechanism for Almokalant, Sotalol, Cisapride,
and Phenytoin via Inhibition of IKr
BY
ANNA-CARIN SKÖLD
ACTA UNIVERSITATIS UPSALIENSIS
UPPSALA 2000
Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) in Toxicology
presented at Uppsala University in 2000
ABSTRACT
Sköld, A.-C. 2000. Teratogenicity as a consequence of drug-induced embryonic cardiac
arrhythmia. Common mechanism for almokalant, sotalol, cisapride, and phenytoin via
inhibition of IKr. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala
Dissertations from the Faculty of Pharmacy 235. 51 pp. Uppsala. ISBN 91-554-4828-3.
Drugs prolonging the repolarisation phase of the myocardial action potential, via inhibition
of the rapid component of the delayed-rectifying potassium channel (IKr), may cause death
due to arrhythmia. Selective IKr-blockers are also embryotoxic in rats. The aim of this thesis
was to determine if drugs with the ability to block IKr cause embryotoxicity across species by
a common mechanism: pharmacologically induced episodes of embryonic arrhythmia leading
to hypoxia-related damage in the embryo.
The selective IKr -blocker almokalant (ALM) induced, in both mice and rats, concentrationdependent embryonic dysrhythmia in vitro, and embryonic death, growth retardation and
malformations in vivo. These results agree with data indicating that IKr is expressed and
functional during a restricted period in rodent embryonic life (gestational days 10-14 in rats),
but not thereafter. Single doses during this period caused stage-specific external, visceral and
skeletal defects in rats. The later the treatment was during this period, the more caudal the site
of the skeletal defect. Overall, the pattern and stage-specificity of defects were very similar to
that reported after transient hypoxia. Pre-treatment with the spin-trapping agent PBN reduced
ALM teratogenicity, suggesting involvement of reactive species generated during the
reoxygenation phase.
In rabbits, which depend on IKr in adult life, the non-selective IKr -blocker sotalol induced
embryonic death at the same developmental stage as ALM treatment in rats. Embryos seem to
be more susceptible to arrhythmia-induced death than the adult. Cisapride, a gastrointestinal
stimulating agent with IKr-blockage as a side effect, induced embryonic arrhythmia and
identical stage-specific digital defects as found with ALM in rats. Phenytoin, a non-selective
IKr-blocking anticonvulsant, induced embryonic arrhythmia and malformations in rats. The
concentrations required to induce embryonic arrhythmia correlated well with maternal plasma
concentrations causing malformation (orofacial cleft).
In conclusion, the results support the hypothesis of a common hypoxia-related mechanism
resulting in developmental toxicity across species for both selective and non-selective
IKr-blockers. The use of an in vitro embryo culture model may be a tool for detection of drugs
with arrhythmogenic potential.
Key words: IKr, delayed rectifying K+ channel, embryonic cardiac arrhythmia, teratogenicity.
Anna-Carin Sköld, Department of Pharmaceutical Biosciences, Division of Toxicology,
Uppsala Universtiy, Box 594, SE-751 24 Uppsala
© Anna-Carin Sköld 2000
ISSN 0282-7484
ISBN 91-554-4828-3
Printed in Sweden by Lindbergs Grafiska HB, Uppsala 2000
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Till minne av
farmor och morfar
3
The thesis is based on the following papers, which will be referred to in the text by
Roman numerals:
I.
Anna-Carin Sköld, Katrin Wellfelt, and Bengt Danielsson. Stage specific
skeletal and visceral defects by the IKr blocker almokalant: Further evidence
for teratogenicity via a hypoxia-related mechanism. Manuscript submitted.
II.
Katrin Wellfelt, Anna-Carin Sköld, Alf Wallin, and Bengt Danielsson (1999)
Teratogenicity of the class III-antiarrhythmic drug almokalant - role of
hypoxia and reactive oxygen species. Reproductive Toxicology 13, 93-101.
III.
Anna-Carin Sköld and Bengt Danielsson (2000). Developmental toxicity of
the class III antiarrhythmic agent almokalant in mice: adverse effects mediated
via induction of embryonic heart rhythm abnormalities. Arzneimittel
Forschung/ Drug Research 50, 520-525.
IV.
Anna-Carin Sköld and Bengt Danielsson. The IKr blocking drug sotalol causes
developmental toxicity in the pregnant rabbit. Manuscript accepted for
publication in Pharmacology and Toxicology.
V.
Anna-Carin Sköld and Bengt Danielsson. Teratogenicity of cisapride:
Evidence of relation to its ability to block IKr channels and cause embryonic
cardiac arrhythmia. Manuscript submitted.
VI.
Bengt Danielsson, Anna-Carin Sköld, Faranak Azarbayjani, Inger Öhman, and
William Webster (2000). Pharmacokinetic data support pharmacologically
induced embryonic dysrhythmia as explanation to fetal hydantoin syndrome in
rats. Toxicology and Applied Pharmacology 163, 164-75.
Reprints were made by kind permission from the publishers: Elsevier Science
(Paper II), Editio Cantor Verlag (Paper III), Pharmacology & Toxicology (Paper IV),
and Academic Press (Paper VI).
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CONTENTS
INTRODUCTION...................................................................................................................................7
THE HEART ............................................................................................................................................7
CARDIAC ARRHYTHMIA .........................................................................................................................8
Definitions........................................................................................................................................8
Causes ..............................................................................................................................................9
Treatment .........................................................................................................................................9
PHARMACOLOGICALLY INDUCED ARRHYTHMIA ..................................................................................10
Arrhythmia in humans....................................................................................................................10
Arrhythmia in animals....................................................................................................................11
EMBRYONIC ARRHYTHMIA AND ITS RELATION TO HYPOXIA AND DEVELOPMENTAL TOXICITY ...........12
COMPOUNDS STUDIED .........................................................................................................................14
Almokalant .....................................................................................................................................14
Sotalol ............................................................................................................................................15
Cisapride and Mosapride...............................................................................................................15
Phenytoin .......................................................................................................................................16
MATERIALS AND METHODS .........................................................................................................17
DEVELOPMENTAL TOXICITY STUDIES ..................................................................................................17
EFFECTS ON THE EMBRYONIC HEART...................................................................................................18
DETERMINATION OF TEST COMPOUND IN MATERNAL PLASMA.............................................................19
RESULTS AND DISCUSSION ...........................................................................................................20
EMBRYONIC ARRHYTHMIA AND HYPOXIA IN DEVELOPMENTAL TOXICITY (PAPERS I AND II) ..............20
Almokalant as model compound for IKr-related developmental toxicity ........................................20
Similarities in teratogenicity between hypoxia and IKr-blockers....................................................22
Hypoxia versus reoxygenation damage..........................................................................................26
IKR-BLOCKERS, EMBRYONIC ARRHYTHMIA AND DEVELOPMENTAL TOXICITY IN OTHER SPECIES
THAN THE RAT (PAPERS III AND IV)....................................................................................................28
Almokalant .....................................................................................................................................28
Developmental toxicity in the mouse.........................................................................................................28
Effects on the mouse embryonic heart .......................................................................................................29
Correlation between effects on the embryonic heart and developmental toxicity ......................................29
Sotalol ............................................................................................................................................30
Developmental toxicity in the rabbit..........................................................................................................30
Correlation between the effects on the embryonic heart and developmental toxicity ................................30
Comments on developmental toxicity........................................................................................................31
DRUGS THAT BLOCK IKR AS A SIDE EFFECT (PAPERS V AND VI) ..........................................................31
Cisapride and mosapride ...............................................................................................................32
Effects on the embryonic heart ..................................................................................................................32
Developmental toxicity..............................................................................................................................32
Correlation between the effects on the embryonic heart and developmental toxicity ................................33
Comments on developmental toxicity........................................................................................................34
Phenytoin .......................................................................................................................................34
Developmental toxicity related to maternal plasma concentrations ...........................................................35
Effects on the embryonic heart ..................................................................................................................35
Correlation between effects on the embryonic heart and developmental toxicity ......................................36
Comments on developmental toxicity........................................................................................................36
WEC: AN INDICATOR FOR DEVELOPMENTAL TOXICITY AND FOR PROARRHYTHMIC POTENTIAL
IN ADULTS? ........................................................................................................................................36
HUMAN RELEVANCE AND FUTURE STUDIES .........................................................................................39
CONCLUDING REMARKS ...............................................................................................................40
ACKNOWLEDGEMENTS..................................................................................................................41
REFERENCES......................................................................................................................................43
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ABBREVIATIONS
ALM
AV node
bpm
ECG
FDA
GD
HERG
IK
IKr
ip
iv
OTC
PBN
po
ROS
WEC
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almokalant
atrioventricular node
beats per minute
electrocardiogram
Food and Drug Administration (US regulatory authority)
gestation day
human ether-a-gogo related gene
the delayed rectifying potassium current
rapid component of IK
intraperitoneal
intravenous
(-)-2-oxo-4-thiazolidine carboxylic acid
α-phenyl-N-t-butylnitrone
per os
reactive oxygen species
whole embryo culture
INTRODUCTION
Cardiac arrhythmia in connection with cardiovascular disease has long been a wellknown cause of death. During the last five-six years, increased interest has been
focussed on pharmacologically induced death due to arrhythmia. This thesis will deal
with arrhythmias induced pharmacologically by a special type of drug, which
prolongs the repolarisation phase of the myocardial action potential, due to inhibition
of the rapid component of the delayed-rectifying potassium channel (IKr). In addition
to possessing arrhythmogenic potential, selective IKr-blockers also have embryotoxic
and teratogenic properties in animals. A hypothesis has been presented proposing that
these developmental toxic effects are a result of pharmacologically induced episodes
of embryonic cardiac arrhythmias leading to hypoxia-related damage in the embryo
(Danielsson, 1993).
The aim of this thesis is to investigate the proposed mechanism, and to study if also
drugs with primarily another pharmacological action, for which the IKr-blocking
property is a side effect, cause similar developmental toxicity. An introduction to the
electrophysiology of the heart follows as a brief background together with a brief
introduction of the classification, origin, and treatment of arrhythmias in general.
More detailed information is then given on pharmacologically induced arrhythmias,
together with the data available at the start of this work on embryonic arrhythmia in
relation to hypoxia and developmental toxicity.
The heart
The heart rate is regulated by electrical impulses. Pacemaker cells in the sinus node
generate action potentials that propagate to the atrium, to the atrioventricular node
(AV node) and to the rest of the ventricular muscle via Purkinje fibres. The changes in
action potential are mediated via the in- and outflow of different ions through ion
channels. In figure 1 a schematic illustration of an action potential and an ECG are
shown (Abrahamsson, 1996; Crumb and Cavero, 2000).
One of the topics in this thesis is disturbance of the heart rhythm - arrhythmia or
dysrhythmia. Semantics dictate that the term “arrhythmia” strictly means an absence
of rhythm, while “dysrhythmia” refers to a distortion of cardiac rhythm. In this thesis
the two terms are used interchangeably, with arrhythmia being used in its colloquial
and popular sense.
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(mV)
R
1
0
P
2
T
3
0
Q
S
- 80
4
5
A
4
(time)
B
QT interval
Figure 1: A) a schematic action potential of a human cardiac conduction cell correlated with
electrolyte shifts. Phase 0: depolarisation, inward sodium current (INa). Phase 1: rapid
repolarisation, outward potassium current (Ito). Phase 2: plateau of the action potential,
L-type Ca2+-channels, inward calcium current (ICa). Phase 3: repolarisation, outward
potassium currents (IKr, Isus, and possibly IKs) and decay of L-type Ca2+ current. Phase 4:
maintaining resting potential, inward rectifier current (IK1). 5: refractory period (during
which a new action potential may not be elicited). B) a schematic picture of a surface
ECG.
Cardiac arrhythmia
Definitions
Disturbances in the heart rhythm are very common. If an ECG is recorded for
24 hours, most people show single extra beats. This is normal and usually
asymptomatic and has no negative prognostic implications. Symptoms due to changes
in the heart rhythm are a frequent reason for patients to contact doctors. The
symptoms are usually palpitations, fatigue, and vertigo. Although many arrhythmias
are benign, others can lead to more serious symptoms needing treatment, especially
due to negative effects on the circulation. Cardiac arrhythmias are the result of
disturbed electrical activity of the heart and are classified according to their site of
origin, supraventricular (the atrium or AV node) and ventricular, and according to
whether the heart rate is increased (tachycardia) or decreased (bradycardia). It is often
unclear which of the various mechanisms mentioned below are responsible for the
arrhythmia. The most common mechanism causing arrhythmias is re-entry, which is
an abnormal conduction circuit. This can occur in both micro- and macro-circuits in
different parts of the heart. The micro-circuits involve only a very small part of the
cardiac muscle, whereas macro-circuits involve larger structures of the heart. There
are three other basic mechanisms underlying arrhythmias: delayed
after-depolarisation, abnormal pacemaker activity in parts of the heart other than the
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nodal and conducting tissues, and heart block. The heart block can be complete, in
which the atria and ventricles beat independently at rates determined by their own
pacemakers, or partial, in which a proportion of the sinus beats from the atria are
transmitted to the ventricle (Noguchi and Gill, 1992; Rang et al., 1999).
Causes
The cause of arrhythmias may be pathological changes, “non-cardiac”, or
pharmacological. This thesis will discuss pharmacologically induced arrhythmias of a
special type, induced by class III antiarrhythmic drugs. The most common cause of
arrhythmia is pathological damage to cardiac tissue due to infarction. The cardiac
muscle or the conduction system becomes scarred and fibrous. The macro-circuits
mentioned above can also originate from congenital extra impulse propagation
pathways, also known as accessory pathways. The cause of the arrhythmia may also
be “non-cardiac”, such as intoxication, metabolic disorder, and thyroid toxicosis, etc.
Treatment
When the cause of the arrhythmia is “non-cardiac”, then the therapy should of course
be aimed at eliminating the underlying cause. The treatment of “cardiac” arrhythmias
can be either pharmacological or non-pharmacological or a combination of both. The
non-pharmacological treatments are electric or surgical methods such as defibrillation,
use of a pacemaker, and radio frequency ablation. Vaughan-Williams (Rang et al.,
1999) developed in 1970 a classification system for antiarrhythmic drugs based on
pharmacological actions and gross electrophysiological properties. It consists of four
major groups.
Class I: drugs that block sodium channels, thus reducing the excitability of the nonnodal regions of the heart where the inward sodium current is important for
propagation of the action potential. This class is further subdivided into C (slow
dissociation), B (fast dissociation), and A (speed between B and C). Class II:
β-adrenoceptor antagonists. Class III: drugs that prolong the refractory period of the
myocardium, thus tending to suppress re-entrant rhythms. Class IV: calcium
antagonists, which block calcium channels and thus impair impulse propagation in
nodal areas and in damaged areas of the myocardium. Some drugs, including digitalis
and adenosin, do not fit neatly into any of the above categories, but are widely used.
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Pharmacologically induced arrhythmia
Arrhythmia in humans
Treatment of cardiac arrhythmias with antiarrhythmic drugs may lead to the induction
of new arrhythmias. This may sound like a paradox, but considering the complexity of
cardiac electrophysiology, it is not surprising that interfering with any particular
mechanism may lead to unpredictable consequences (Abrahamsson, 1996).
In the early part of the 1980s, there was a great deal of interest in developing class IC
sodium channel blocking drugs because of their profound effect on myocardial
conduction and potent suppression of ventricular ectopic beats. These drugs have a
lesser effect on myocardial refractoriness. Their use- and rate-dependent properties
made them attractive agents for the management of both supraventricular and
ventricular tachyarrhythmias. The popularity of these drugs was profoundly lessened,
however, by the findings of the Cardiac Arrhythmia Suppression Trial (CAST)
(Anonymous, 1989; Anonymous, 1992). The study showed, quite unexpectedly, that
the long-term mortality was increased in patients who were treated with class I
antiarrhythmics. It was thought that the increased mortality was due to proarrhythmic
properties of these drugs. The interest turned instead to agents with class III
properties. The target for most of these agents was found to be the rapid component of
the delayed rectifying potassium channel (Colatsky and Argentieri, 1994).
By definition, class III antiarrhythmic drugs prolong the duration of the action
potential. The result is a lengthening of the effective refractory period with associated
antiarrhythmic activity. The antiarrhythmic efficacy was verified in clinical trials of
these new compounds (Bashir et al., 1995; Darpö and Edvardsson, 1995; Echt et al.,
1995; Sager et al., 1993). The effects of these drugs on myocardial cells manifests
itself as a prolongation of the QT interval (corrected QT interval) on the surface ECG.
The consequences of this QT prolongation can be therapeutic or toxic. Therefore it
was disappointing to find that class III antiarrhythmic drugs are also
proarrhythmogenic. Treatment with these class III antiarrhythmics was associated
with the risk for a special kind of arrhythmia called Torsade de Pointes (Roden,
1994). This is a characteristic type of polymorphous ventricular tachycardia in which
the QRS axis of each successive beat differs slightly from the preceding one and
seems to rotate around the isoelectric line: Torsade de Pointes - twisting of the points
(Viskin, 1999).
In the development of Torsade de Pointes, there is a complex interaction of prolonged
repolarisation, the development of early afterdepolarisations with the capacity to
propagate and capture the whole heart, and bradycardia (Doig, 1997). The rapid
component of the delayed rectifying potassium channel, IKr, is the primary ion
channel involved in repolarisation. The gene HERG (human ether-a-gogo related
gene) seems to represent the molecular basis of IKr because the K+ currents encoded
by HERG display biophysical and pharmacological characteristics similar to those of
IKr. In addition, HERG mutations are responsible for one form of the
long QT syndrome. Thus HERG is a primary target for both congenital
10
(mutation-induced) and acquired (drug-induced) action potential prolongation,
delayed repolarisation, and cardiac arrhythmias (Taglialatela et al., 1998).
There has been a great deal of interest regarding the safety assessment of
proarrhythmic potential in antiarrhythmic agents, both from the authorities and from
the pharmaceutical companies. During research in this area it has also been found that
several classes of non-cardiovascular drugs, such as psychotropic, prokinetic,
antimalarial, antifungal and antihistamine drugs, can also induce prolongation of the
QT interval and Torsade de Pointes. This side effect may be even more important
since the indications for many of these non-cardiovascular drugs are rather mild
(Crumb and Cavero, 2000). The European Agency for Evaluation of Medicinal
Products (CPMP) wanted to establish a European consensus concerning how drugs
with effects on the QT-interval should be assessed (preclinically and clinically). Their
work resulted in a “Points to consider”-document: “The assessment of the potential
for QT-interval prolongation by non-cardiovascular medicinal products” (CPMP
986/96/1997). The document proposes practical approaches for preclinical in vitro and
in vivo testing of drug candidates to detect the potential for QT prolongation and
arrhythmias, and clinical trial design to determine this potential. In the US, the FDA
has focussed on this issue and withdrawn approval for some drugs with relatively mild
indications. Even though the concentrations at which the proarrythmia occurs are
usually higher than normal therapeutic concentrations, and the number of patients
affected has been small, the withdrawel is motivated because of interaction effects,
and the increased susceptibility of some patients to develop arrhythmias due to
differences in genetic background.
Arrhythmia in animals
The proarrhythmogenicity of class III antiarrhythmics has also been shown in
animals. The rabbit heart seems to be especially vulnerable towards the induction of
Torsade de Pointes, and the rabbit has been used as a good animal model for testing
this property of new drugs (Abrahamsson, 1996; Carlsson et al., 1993). The delayed
rectifier K+ current (IK) represents the sum of two currents: A slow (IKs) and a
relatively rapid activating delayed rectifier (IKr) (Carmeliet, 1992). Class III agents
specifically block the latter current. Not all mammalian hearts have both kinetically
distinct components. For example, both IKr and IKs appear to exist in guinea pig
ventricular and atrial myocytes, sheep and calf Purkinje fibres, embryonic chick atrial
cells, and human atrial cells, whereas only IKr appears to exist in rabbit heart and cat
ventricle (Wiesfeld et al., 1996). In contrast, the pure IKr-blockers almokalant and
dofetilide have no effect on the action potential duration in rats (Abrahamsson et al.,
1994; Wang et al., 1996), which is why the rat has been considered as an unsuitable
species for testing proarrhythmic potential. The dominant repolarising outward
current in the rat is Ito and it appears that the action potential is too short to allow for
activation of the IK to any considerable extent (Abrahamsson et al., 1994).
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Embryonic arrhythmia and its relation to hypoxia and
developmental toxicity
The observed species differences regarding IKr in the adult seem to be non-existing in
the embryo. In contrast to adult rodents, experimental studies indicate that the IKr
channel is expressed and functional in the early mouse and rat embryo as soon as the
heart starts beating (Abrahamsson et al., 1994; Ban et al., 1994; Davies et al., 1996;
Wang et al., 1996; Webster et al., 1996). In the mouse, IKr is expressed in the atrium
at an early stage of embryonic development, when it is the dominating repolarisation
channel (Davies et al., 1996). In view of this finding, it has been proposed that IKr
plays an important role in the early development of the mammalian cardiac electrical
system (Abrahamsson et al., 1994). The presence of IKr in the early embryo in species
where this ion channel is suppressed in favour of other ion channels later in
development, suggests a universal functional role for the IKr channel across all
mammalian species in early embryonic development (Davies et al., 1996; Wang et al.,
1996).
It is an interesting but not unexpected finding, in view of the presence and
functionality of IKr in the early embryo, that the embryonic rat heart reacts with
dysrhythmia when exposed to selective IKr-blockers. Thus, embryonic bradycardia has
been described for the selective IKr-blockers almokalant, dofetilide, and L-691,121
(Abrahamsson et al., 1994; Ban et al., 1994; Spence et al., 1994; Webster et al., 1996)
in rat embryos cultured in vitro during the period when the IKr-channel seems to play
an important role. The severity of the bradycardia depends on concentration,
arrhythmia and cardiac arrest occur at sufficiently high concentrations. The embryonic
heart during this stage of development is clearly visible and the heart beats
prominently, making it possible to examine the adverse embryonic cardiac effects by
visual inspection using a stereomicroscope with low magnification.
The effects of almokalant on the rodent embryonic heart in our in vitro model agree
with the results obtained with more traditional electrophysiological methods. Figure 2
shows transmembrane recordings of action potentials from the atrium of the
spontaneously beating rat embryo hearts treated with different doses of almokalant
during the same stage as almokalant was tested in whole embryo culture (WEC)
(Abrahamsson et al., 1994 versus Webster et al., 1996). As seen in figure 2,
almokalant induced concentration-dependent bradycardia and arrhythmia in the rat
embryo. This may be the result of increased duration of the action potential, leading to
decreased heart rate. After extensive prolongation of the action potential, almokalant
induced spontaneous deflection of the membrane potential consistent with early
afterdepolarisations, which in our in vitro model is manifested as arrhythmia. In this
context, it should be mentioned that the transmembrane recordings seen in figure 2,
are very similar to those obtained in a sensitive adult rabbit model that mimics the
course of events in sensitive patients who developed almokalant induced arrhythmia
(Abrahamsson et al., 1994). This may also indicate that our in vitro model may be
used as a sensitive indicator for the detection of arrhythmogenic potential of IKrblocking compounds.
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a)
b)
c)
Figure 2: Reprinted from Cardiovascular Research, 28, Abrahamsson et al. Induction of
rhythm abnormalities in the fetal rat heart. A tentative mechanism for the embryotoxic effect
of the class III antiarrhythmic agent almokalant, Copyright (1994), with permission from
Elsevier Science.
a): Action potential duration at the 75% repolarisation level (APD75, triangles) and intrinsic
heart rate (HR, circles) over time in foetal rat myocardium on gestational day 13. Ordinate:
APD75 expressed in ms and HR as beats min-1. Values are means, bars=SD, n=4.
b): The effect of increasing concentrations of almokalant on the spontaneously beating foetal
rat heart on gestational day 13. Circles = frequency in beats per minute (beats min-1).
Tringles=action potential duration at the 75% repolarisation level (APD75, ms). Values are
means, bars=SD. The number of experiments decreases due to the occurrence of early
afterdepolarisations. *p<0.05, †p<0.01, ‡p<0.005 v the control value.
c): Transmembrane recordings of action potentials from the atrium of the spontaneously
beating foetal rat heart at gestational day 13. (A) In the control situation; (B) After 0.1 µM
almokalant; (C) After 1 µM almokalant; (D) After 10 µM almokalant.
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Several investigators have proposed that the severe bradycardia and arrhythmia that
are induced explain the observed high incidence of embryonic/foetal death due to
hypoxia following administration of selective IKr-blockers. The mean foetal weights
of the few surviving foetuses were decreased and there was increased incidence of
malformations, showing a teratogenic potential of almokalant and of L-691,121
(Abrahamsson et al., 1994; Ban et al., 1994; Danielsson, 1993; Spence et al., 1994).
It has been suggested that the malformations reported following administration of
selective IKr-blockers are related to episodes of hypoxia as a consequence of
embryonic arrhythmia (Danielsson, 1993). This theory was based on the observation
that similar malformations as those produced by selective IKr-blockers, as well as
increased mortality and decreased foetal weights, are caused by embryonic hypoxia,
induced by temporary clamping of the uterine vessels in rats (Brent and Franklin,
1960; Franklin and Brent, 1964; Leist and Grauwiler, 1974; Webster et al., 1987).
In the studies with whole embryo culture and L-691,121 and dofetilide (Ban et al.,
1994; Spence et al., 1994), wash-out experiments were performed. In these
experiments, the heart rates were examined during exposure to the drug and after
rinsing and transfer to drug-free medium. The heart rates returned almost to baseline
levels, which indicates a reversible pharmacological effect. The in vivo situation for
drugs with a short half-life resembles the conditions with the wash-out experiments.
During peak concentrations of the compound there might be a short period of
arrhythmia, then the heart rhythm might return to normal when the concentrations
decline. That would mean that a short period of severe hypoxia occurred due to
embryonic cardiac arrhythmia, followed by reoxygenation when the heart starts to
beat normally again. This may be what occurs when uterine vessels are clamped for a
short period.
Compounds studied
Almokalant
Almokalant was developed by Astra Hässle in the 1980s as an antiarrhythmic drug. It
is pharmacologically classified as a class III antiarrhythmic agent and it is a selective
inhibitor of the rapid component of the delayed rectifying potassium channel (IKr)
(Abrahamsson, 1996). Almokalant also has proarrhythmic potential and the
development of almokalant was discontinued due to induction of Torsade de Pointes,
14
which occurred in some susceptible patients during the clinical trials (data supplied by
the manufacturer).
Almokalant is a known animal teratogen, but there is no information regarding human
relevance (since no pregnant women have been treated with almokalant).
Sotalol
Sotalol is a non-selective β-adrenoceptor antagonist and a class III antiarrhythmic
agent. It has been available as a β-blocker since the early 1960s and in the early 1970s
it was also characterised as a class III agent via inhibition of IKr. The indications for
its use include hypertension, angina, and cardiac arrhythmias (Kowey et al., 1997).
The β-blocking properties are primarily due to the l-isomer (Beyer et al., 1993), while
the class III activity is present in both the l- and d-isomers (Sanguinetti and
Jurkiewicz, 1990). In clinical practice the racemate of sotalol is used.
The incidence of Torsade de Pointes has been reported to be 2-5% for patients treated
with sotalol (MacNeil, 1997). Despite the wide clinical use of sotalol, there are no
experimental embryo-foetal developmental toxicity studies available in the literature.
There is also very limited information on human pregnancy outcome data.
Cisapride and Mosapride
Mosapride
Cisapride
Cisapride and mosapride are two structurally related benzamide derivates which
facilitate or restore motility in the gastrointestinal tract, most likely via action on
seretonin-4 receptors (5-HT4 receptor agonist) (Wiseman and Faulds, 1994; Yoshida
et al., 1993). Cisapride has been on the market for several years, whereas mosapride is
15
presently undergoing extensive preclinical and clinical evaluation as a novel
prokinetic agent. Recently, it was reported that treatment with cisapride may
occasionally give rise to arrhyhtmia (1996a; 1996b; Bran et al., 1995; Lewin et al.,
1996; Wysowski and Bacsanyi, 1996). It has been shown that cisapride, but not
mosapride, inhibits IKr at clinically relevant concentrations (Carlsson et al., 1997). In
view of the discovered proarrhythmic potential, the use of cisapride has been
restricted in many countries and the drug was removed from the US market on July
14, 2000 (Vincent 2000).
There are no published experimental teratology studies on cisapride or mosapride in
the literature, and only limited human cisapride data on pregnancy outcome.
Phenytoin
Phenytoin (PHT) has been used for treating convulsive disorders for over six decades
(Merritt and Putnam, 1938) for a review, see Rogawski and Porter (1990). It has also
been used as an antiarrhythmic drug in a more limited manner, primarily for treating
digitalis intoxication (Bigger and Hoffman, 1980). Phenytoin is pharmacologically
active by stabilising the membranes of excitable cells, such as neurones and cardiac
cells, via interference with voltage-dependent sodium, calcium, and potassium ion
channels, although the exact mechanism is not known. (Kuo and Bean, 1994;
Macdonald, 1989; Ragsdale et al., 1991; Yaari et al., 1987). It has recently been
shown that phenytoin inhibits the delayed rectified potassium channel IK, which
makes the substance particularly interesting for my thesis (Nobile and Lagostena,
1998; Nobile and Vercellino, 1997). We also have new data (Danielsson et al.
unpublished) that shows that it is the rapid component of IK that is inhibited by
phenytoin. Previous work has suggested that PHT has proarrhythmic potential, and
eight cases of PHT-induced arrhythmia evolving to asystole and death have been
recorded, as has marked sinus bradycardia leading to syncope in four of 15 young
volunteers given PHT by intravenous injection, for a review see Tomson and
Kennebäck (1997).
Phenytoin is an established human teratogen and foetal adverse effects have been
reported in mice, rats, rabbits and cats. The pattern of foetal adverse effects, described
as “Foetal Hydantoin Syndrome” in humans (Hanson and Smith, 1975), is very
similar across species, for a review see Finnell and Dansky (1991). It consists of
embryonic death, intra-uterine growth retardation, mild CNS dysfunction, craniofacial
abnormalities (including orofacial clefts) and limb reduction defects.
16
AIMS OF THE STUDY
•
To elucidate the role of hypoxia and hypoxia/ reoxygenation damage in
developmental toxicity of selective IKr-blockers mediated via embryonic
arrhythmia, using almokalant as a model compound.
•
To investigate if two established IKr-blockers, almokalant and sotalol, induce
developmental toxicity/ embryonic arrhythmia in other species than the rat.
•
To investigate if cisapride and phenytoin (gastrointestinal motility stimulating-,
and antiepileptic drug, respectively), which have recently been shown to have
IKr-blocking properties as a side effect, induce embryonic arrhythmia, embryonic
death and hypoxia-related malformations.
MATERIALS AND METHODS
All animal work was approved by the ethical committees in Uppsala and Stockholm.
In all studies performed (both in vivo and in vitro) day 0 of pregnancy was determined
by observing the day of occurrence of vaginal plug and/or sperms in the vaginal smear
in the rat and mouse or observance of mating in the rabbit. Details of how the studies
were performed are given in each paper. Below is a summary of the main procedures
used.
Developmental toxicity studies
It is generally accepted in developmental toxicology that there is a higher probability
that the observed adverse effects occur in humans if they have been recreated in more
than one animal species. This is the reason why new drugs must be tested in one
rodent (usually rat) and one non-rodent species (usually rabbit) according to
international guidelines. The animals are kept under specified conditions regarding
temperature, humidity and light/dark cycle, usually having free access to feed and
water. All animals are inspected daily for clinical signs. The treatment period should
cover organogenesis, which means that it should cover the period from implantation
to closure of the hard palate. In the mouse, the dosing starts on gestation day (GD) 6
and ends on GD 15, in the rat GD 6-16 and in the rabbit GD 6-18. Generally three
dose levels are used: a low dose near the expected therapeutic concentration/exposure,
a high dose where minimal toxicity is observed, usually as a decrease in maternal
body weight gain, and an intermediate dose. The control group is often given the
vehicle or water. Near time of delivery (mouse GD 18, rat GD 21, and rabbit GD 29)
a caesarean section is performed. The uterine contents are examined with respect to
17
number of corpora lutea, number of implantation sites, sex and number of viable
foetuses, number of dead foetuses, early and late intrauterine deaths, foetal weights,
and external abnormalities. Approximately half of the foetuses in each litter are
preserved in Bouin’s solution for subsequent freehand sectioning in order to detect
visceral anomalies (Barrow and Taylor, 1969). The remaining foetuses are preserved
in 95% alcohol for clearing and staining of the skeleton with alizarin red in order to
detect skeletal anomalies. Abnormalities can be classified into the following three
categories: 1) major; rare and/or probably lethal, 2) minor; minor differences from
'normal' that are detected relatively frequently and 3) variants; alternative structures
that also occur regularly in the control population.
In this thesis the conventional design has been complemented with more specific
investigations, including examination of foetal adverse effects after administration on
single days of pregnancy.
Effects on the embryonic heart
Whole embryo culture of rats and mice has been used in this thesis as a tool for
studying effects on the embryonic heart rhythm. As described in the section
“Embryonic arrhythmia and its relation to hypoxia and developmental toxicity” the
observed effects, bradycardia and arrhythmia, agree with results from more traditional
electrophysiological methods. Explanted embryos were in all experimental designs
cultured using modified techniques of New (1978). The examination of effects on the
heart in NMRI mouse embryos were performed on GD 10 and in Sprague Dawley rat
embryos on GD 11. The dams were sacrificed by cervical dislocation (the rats first
being lightly anaesthetised by enflurane or CO2), the abdomen was then opened, the
uterus excised and the decidual swellings removed under aseptic conditions. The
decidua and Reichert’s membrane were removed from each swelling and the embryos,
with the yolk sac and ectoplacental cone intact, were placed in sterile culture bottles
with one embryo per culture flask. The culture medium consisted of either 20% or
~75% immediately centrifuged heat-inactivated rat serum and either 80% or ~25%
Tyrode’s salt solution. The culture bottles were gassed with 55% N2, 40% O2 and
5% CO2 (vol./vol./vol.) for 5 minutes, and capped tightly and cultured in a rotating
culture system at 37°C and 40 rounds per minute. The embryos were allowed to
recover for 60 minutes until an initial heart rate reading was taken under a stereo
microscope. Embryos were removed from the culture medium for approximately
25 seconds per examination. The embryos were kept on a heated stage to maintain the
bottles at 37°C. Embryos were selected for each experiment based on the following
criteria:
1) Morphologically normal appearance
2) Good yolk sac circulation
3) A heart rate of at least 120 beats per minute (bpm) for mice and 140 bpm for rats.
18
Embryos not fulfilling all three criteria were discarded. After the selection of
embryos, the test substance was added to each culture. Controls were treated with
either water or vehicle in corresponding volumes. The embryos were then kept in
culture up to two hours, during which the embryos were examined for heart rate,
incidence of arrhythmias and cardiac arrest, one to three times, each 15 seconds. The
embryos were video-taped during all observation periods. Their heart rhythms were
evaluated from these videotapes, which increases the accuracy in detecting arrhythmia
and bradycardia.
Determination of test compound in maternal plasma
Blood samples were taken for analysis of the test compounds in maternal plasma.
Almokalant and cisapride were analysed at Astra Hässle by reversed phase liquid
chromatography and fluorescence detection.
Sotalol was analysed at the Department of Medicine and Care, Division of
Pharmacology at the University of Linköping. The plasma was analysed with a liquid
chromatographic method using fluorescence detection (Gustavsson et al., 1997).
Phenytoin was analysed at the Department of Clinical Pharmacology, Karolinska
Institute at Karolinska Hospital. Concentrations of total and free phenytoin were
determined with FPIA (fluorescence polarization immunoassay) technology (Axsym
Phenytoin package insert 1997 and TDx/TDxFLx Free phenytoin package insert 1997,
Abbott Laboratories) (Jolley, 1981; Thomas et al., 1991). Ultrafiltration was used to
isolate the free fraction (Gurley et al., 1995).
19
RESULTS AND DISCUSSION
Embryonic arrhythmia and hypoxia in developmental toxicity (Papers
I and II)
Papers I and II elucidate the role of hypoxia in the teratogenicity of IKr-blocking
agents. We investigated whether IKr-blockers induced the same type of stage-specific
defects as episodic hypoxia induces. Almokalant was used as a model substance.
Another aim was to study if pre-treatment with PBN, a radical spin-trapping agent, or
OTC, an antioxidant, could reduce the incidence of almokalant-induced defects. Such
pre-treatment may indicate if the defects induced are mainly caused by “pure
hypoxia” or by reoxygenation damage after an episode of severe embryonic
dysrhythmia.
Almokalant as model compound for IKr-related developmental toxicity
Almokalant was administered as single doses during the period when the rat
embryonic heart was extremely sensitive to selective IKr-blockers (Ban et al., 1994;
Spence et al., 1994; Webster et al., 1996). Almokalant was given orally on GDs 10,
11, 12, 13 or 14 and the doses were different on different days, adjusted in order to
allow the majority of the foetuses to survive until term. No adverse clinical signs were
observed in the dams that could be related to the treatment with almokalant.
Almokalant increased the incidence of embryonic/foetal death, decreased the mean
foetal weights and induced external malformations in similarity to what has been
shown with other IKr blockers in conventionally designed teratology studies (Ban et
al., 1994; Marks and Terry, 1996; Spence et al., 1994). The main results are
summarised in table 1.
We also showed that these external defects were phase-specific, with orofacial clefts
after administration on GD 11 (figure 3) and digital reduction defects on the forepaw
after administration on GD 13 (figure 4) and on the hindpaw after administration
GD 14. Our results regarding almokalant-induced external defects confirm results of
Webster et al. (1996). The similarities in developmental toxicity between the different
IKr-blockers strongly support the idea that the teratogenicity is a class effect for
IKr-blockers that is mediated by a common mechanism.
20
Figure 3: A rat foetus on GD 21
with cleft lip and palate after
administration of 125 mg/kg
ALM on GD 11.
Figure 4: A rat foetus with
brachydactyly of the left forepaw
after administration of 25 mg/kg
ALM on GD 13.
Almokalant, dofetilide, and L-691,121 all induced bradycardia and cardiac arrest in
whole embryo culture. It is generally accepted that the observed dose-dependent high
incidences of foetal death for these substances are a consequence of observed adverse
effects on the embryonic heart (Abrahamsson et al., 1994; Ban et al., 1994; Spence et
al., 1994; Webster et al., 1996). The similarity in the pattern of malformations may
also indicate a common hypoxia-related mechanism as explanation to their
teratogenicity. The induced embryonic bradycardia and arrhythmia/cardiac arrest
could lead to hypoxia and the generation of free radicals at reoxygenation in the
embryo, which in turn could cause the malformations. This mechanism will be
discussed further in the following sections.
21
Table 1: A summary of the main effects induced by almokalant in Papers I and II,
together with reported effects for other selective IKr-blockers.
Abnormality
Almokalant
Dofetilide
Ibutilide
L-691,121
Rodent whole embryo culture
Bradycardia
Yes
Yes
Yes
Arrhythmia
Yes
Yes
Yes
Cardiac arrest
Yes
Yes
Yes
Embryonic/ foetal death
Yes
Yes
Yes
Yes
Decreased foetal weights
Yes
Yes
Yes
Yes
Cleft lip/ palate
GD 11
GD 11
GD 6-15
GD 6-17
Short/ kinked/ threadlike/ absent tail
GD 11-12
GD 11
GD 6-15
GD 6-17
Anal atresia
GD 11
Digital defects: forepaw
GD 13
GD 13
GD 6-15
Digital defects: hindpaw
GD (13) 14
GD (13) 14
Oedema
GD 15
Reproductive data
Defects observed externally
GD 6-15
a
GD 6-17
a
GD 6-15
a
GD 6-17
GD 6-15
b
a
GD 10, 12
a
No distinction was made between the fore- and the hindpaw.
In whole embryo culture
b
Similarities in teratogenicity between hypoxia and IKr-blockers
Hypoxia was one of the first teratogenic agents to be identified and studied, for review
see Grabowski (1970). Embryonic hypoxia can be obtained by for example, reducing
the oxygen tension in the air or by mechanically clamping the uterine blood vessels
leading to impaired uteroplacental blood flow. It is also secondary to pharmacological
effects by various drugs. The early postimplantion rat embryo, during the period
GD 6- GD 11, resists the induced hypoxia relatively well, and many embryos survive
90-120 min of uterine vessel clamping (Franklin and Brent, 1964; Leist and
Grauwiler, 1973). A low incidence of congenital malformations is seen in the
surviving foetuses including CNS defects such as dilated ventricles (hydrocephalus),
orofacial clefts, and heart defects. By GD 12 the embryos become more sensitive, and
during GDs 13 - 16 most embryos are unable to survive 60 min of hypoxia. Shorter
periods of hypoxia on these days, 30-45 min, induce a range of malformations, the
most common of which are distal digital defects. Other less frequently seen defects
include tail, face, and urogenital abnormalities (Leist and Grauwiler, 1974; Webster et
al., 1987). Pathological examination (2-24 h after the clamping procedure) showed
that malformations such as cleft palate and digital defects are preceded by oedema,
dilated blood vessels, haemorrhage, blisters and the subsequent development of
22
necrosis (Leist and Grauwiler, 1974; Webster et al., 1987). After GD 16, the foetus
again becomes less sensitive to hypoxia induced by uterine vessel clamping (Leist and
Grauwiler, 1973). Hypoxia could also induce a variety of skeletal defects. The type
and localisation depended on the timing of the hypoxia episode relative to
organogenesis: a clear-cut head to tail shift of a variety of skeletal defects was
observed as the developmental stage of the induced oxygen deficiency advanced
(Degenhardt, 1958; Ingalls and Curley, 1957; Leist and Grauwiler, 1973; Morawa and
Han, 1968; Murakami and Kameyama, 1963).
A summary of phase-specific external, visceral, and skeletal malformations induced by
almokalant in our studies (in the rat) is presented in table 2, together with defects
reported to be induced by hypoxia. Almokalant induced a relatively high incidence of
cardiac interventricular septum defects (~10%) and vascular malformations (~12%)
consisting of the absence (for example lack of inominate), abnormal vessels (for
example enlarged aorta with reduced pulmonary trunk) or transposition of vessels
(such as the azygos vein, and retroesophageal right subclavian) after dosing on GD 10
and 11 and to a lesser degree on GD 12 and 13. In view of this, it is interesting that
episodic hypoxia also causes phase-specific interventricular septal defects and
abnormalities of the aortic arch (Leist and Grauwiler, 1973).
23
Table 2: Stage specific abnormalities induced by almokalant (Papers I and II) or hypoxia.
Abnormality
Almokalant
Hypoxia
Embryonic/ foetal death
Yes
Yes
Decreased foetal weights
Yes
Yes
Cleft lip/palate
GD 11
GD ~7-15
Digital defects of the forepaw
GD 13
GD 13 (14)
Digital defects of the hindpaw
GD 14 (13)
GD 14 (13)
Short/ kinked/ absent tail
GD 11-12
GD ~7-12
CNS/ Brain defects
GD 11
GD ~ 8-12
Cardiac ventricular septum
GD 10-11 (12)
Cardiovascular abnormalities
e.g. absence or transposition of
vessels
GD 10-11 (12-13)
Absence/ malposition of
postcaval lung lobe
GD 10 (12)
Attenuated diaphragm
GD 10-11 (12)
Dilated urethras
GD 10-11 (12)
Abnormalities of the kidney
GD 10-11 (12)
GD ~ 7-12
Undescended testis
GD 11-12
GD ~ 12
Reproductive data
External/visceral defects
GD 8-10
Skeletal defects
Skull bones
GD 10-14
Thoracic
GD 10-11 (lower
12-14)
GD ~ 10
Lumbar
GD 10-11
GD ~ 10
Sacral
GD 11
GD ~ 11
Caudal
GD 11-12
GD ~ 11
Sternebrae
GD 10-14
GD 14-15
th
27 presacral
GD 10-11
Ribs/extra rib
GD 10
GD ~ 10
Almokalant induces lung abnormalities and imperforation of the anus, resembling the
effects of ibutilide. The latter drug leads to portions of lung being absent and anal
atresia after dosing during GD 6 - 16 (Marks and Terry, 1996). For almokalant the
lung defects were most accentuated after dosing on GD 10, and ~20 % of the foetuses
and litters had malposition/absence of the post caval lobe of the lung, while anal
atresia was induced most effectively by dosing on GD 11. Some CNS defects, such as
dilated ventricles, and zigzag-shaped pia mater were also induced after dosing on
GD 11. New malformations, not previously described for IKr-channel blocking drugs,
were found in this study. These consisted of urogenital defects such as increased renal
24
pelvic cavitation, or absence of the renal papilla and undescended testes. These
defects were mainly observed after treatment on GD 10 and 11.
Figure 5: A rat foetus
skeleton with an example of
almokalant-induced defects
on the thoracic level after
administration on GD 10.
In this respect it is worth noticing that undescended testis (Franklin and Brent, 1964;
Ingalls et al., 1952) and renal agenesis / hypoplasia (Brent and Franklin, 1960;
Franklin and Brent, 1964) and hydronephrosis (Leist and Grauwiler, 1973) have been
induced after episodic hypoxia. The same is true for attenuated diaphragm /
abdominal wall defects and other occasionally observed malformations in this study
(abnormal structure of endocrine glands) and in the study with ibutilide
(aplebharia/agnathia) (Marks and Terry, 1996). In all cases similar defects have been
found after embryonic hypoxia due to clamping of uterine vessels during GDs 7-12 in
the rat (Brent and Franklin, 1960; Leist and Grauwiler, 1973; Leist and Grauwiler,
1974).
Almokalant caused skeletal defects in the present study (in the rat) on all
administration days between GD 10 and GD 14. Skeletal examination showed a high
incidence of vertebral abnormalities on the thoracic level on GD 10 (an example is
shown in figure 6) and on lower thoracic- to caudal level on GD 11, compared to
controls. Both vertebral centra and arches were affected, as were the ribs, with defects
that involved misalignment, incomplete ossification of varied type and extent, and
fusion. On GD 10 a very high incidence of an extra (27th) presacral vertebrae was
noticed compared to controls. Increased incidences of sternebral defects, compared to
controls were obtained on all days, while almost no vertebral defects were observed
on GD 13 or 14. On GD 13 and 14 digital defects such as brachydactyly, syndactyly,
and oligodactyly were induced on the forepaw and hindpaw, respectively. Skeletal
defects showed a clear trend; the later the treatment the more caudal was the site of
the defect. The phase-specificity for induction of skeletal defects by almokalant is
25
thus almost completely in accordance with that which has been reported for skeletal
malformations caused by hypoxia in rodents (Ingalls and Curley, 1957; Leist and
Grauwiler, 1973; Morawa and Han, 1968) and rabbits (Degenhardt, 1958).
Hypoxia versus reoxygenation damage
The exact mechanism that gives rise to hypoxia-related malformations is not fully
understood. It has long been known that a reduced oxidation rate will primarily affect
organs that are at a rapid proliferation phase at the time, causing discontinuation in
development. The hypoxia may result either in organs that consist of sensitive cells
dying, becoming retarded or having their differentiation capacity affected in some
manner (Naujoks, 1953). This could result in an overall embryonic growth retardation
that would be more pronounced in the areas with most cell proliferation, such as facial
prominences, leading for example to failure of closing the palate (Bronsky et al.,
1986). A new paper has studied how hypoxia plays a global role in developmental
physiology and morphogenesis in rat embryos on GD 9 – 11 (Chen et al., 1999). The
hypoxia may act as a physiological signal co-ordinating some group of genes
important in development
However, several studies indicate that damage after hypoxic episodes is related to free
radicals that are generated during the reoxygenation phase. In adult tissues, generation
of free radicals is a well established mechanism for tissue damage during
reoxygenation of the ischemic heart after myocardial infarction, and the central
nervous system (CNS) after stroke (Gutteridge, 1993; McCord, 1993). The major
cause of damage is readmission of oxygen and not the re-establishment of flow
(Kalyanaraman et al., 1995). Reactive oxygen species (ROS) have also been
identified in embryonic tissues after episodes of hypoxia followed by normoxia. In a
study by Fantel et al. ROS (superoxide anion radicals generated in mitochondria)
were detected in the embryonic tissues corresponding to the distal parts of the digits
after two transient 30-minute episodes of hypoxia separated by normoxia in GD 14 rat
embryos cultured in vitro (Fantel et al., 1995).
To investigate the role of free radicals in almokalant-induced developmental toxicity,
the effects of pre-treatment with the spin-trapping agent PBN, (α-phenyl-N-tbutylnitrone) with capacity to capture free radicals, and the antioxidant OTC (2-oxo4-thiazolidine carboxylic acid) -a promotor of gluthathione synthesis, were studied.
Pre-treatment with PBN resulted in a decrease in the incidence of almokalant-induced
defects both on GD 11 and 13. No foetuses with orofacial clefts, ventricular septum
defects, tail defects or distal digital defects were observed in almokalant-treated dams
after pre-treatment with PBN. The only observed visceral malformations in the PBN
pre-treated dams was a vessel defect (malposition of the aortic arch) in one foetus, and
one foetus with dilated urethras treated on GD 11. Pre-treatment with OTC on GD 11
reduced the incidence of septal defects. On GD 13 there was a reduction in digital
defects, compared to dams given almokalant only. However, pre-treatment with OTC
did not cause a reduction in almokalant-induced orofacial clefts or tail defects. The
26
statistical evaluation showed that OTC did not significantly alter the incidence of
almokalant-induced internal or external malformations on GD 11 or 13.
These results suggest that the almokalant-induced defects are mainly caused by
reoxygenation damage after an episode of severe embryonic dysrhythmia, rather than
“pure hypoxia”. The mechanism for such embryonic damage is unknown, but may
involve generation of free radicals, such as reactive oxygen and nitric oxide species
during the reoxygenation phase (Fantel, 1996; Fantel et al., 1999).
The fact that PBN prevented almokalant-induced malformations to a much higher
extent than OTC, may have several explanations. PBN is a lipophilic radical spintrapping agent, which rapidly penetrates cell membranes and is most likely distributed
both inside and outside of cells in embryonic tissues. PBN thus seems to have the
capacity to directly capture (trap) generated reactive oxygen species both within the
cell and in the circulation. OTC in contrast, acts indirectly by promoting glutathione
synthesis by functioning as an intracellular delivery system (Williamson et al., 1982).
Glutathione is the major intracellular reductant present in developing embryonic
tissues (Allen and Balin, 1989). Recent studies suggest that glutathione is released
into the circulation, presumably as a defence against ROS during
hypoxia/reoxygenation (Liu et al., 1994). However, the synthesis of glutathione (via
OTC) and the release of extracellular glutathione as a response to generated free
radicals are probably slower processes, with a lower capacity for detoxification than
immediate trapping of free radicals by PBN, available in high concentrations both
intra - and extracellularily. An increased protective effect by PBN, compared to OTC,
was also seen in a study by Zimmerman et al. (1994). PBN, but not OTC, completely
prevented cocaine-induced embryonic vascular disruption thought to be mediated by
ROS.
The only cardiovascular malformation detected in the group pre-treated with PBN was
a defect of the great vessels (malposition of the aortic arch) on GD 11. Even if this
finding does not exclude hypoxia - reoxygenation damage, it suggests that
abnormalities of the great vessels arise through another mechanism. In view of this, it
is of great interest to note that abnormalities of the great vessels have been induced
experimentally by modifying the normal blood flow pattern (Jaffee, 1967; Sweeney et
al., 1979) and by production of episodes of embryonic bradycardia, ventricular
arrhythmia and temporary cardiac arrest (Rajala et al., 1984). A wide variation of
vessel defects, including the malposition of the great vessels, can be induced by
selectively increasing, decreasing, or misdirecting blood flow in the developing
cardiovascular system by mechanical methods or by cardioactive substances (Gilbert
et al., 1980; Kirby, 1987; Gilbert et al., 1980). Lack of sufficient blood flow and
pressure changes have been related to the induction of abnormally absent vessels.
Thus, the results suggest that the observed cardiovascular defects are result of both the
induced embryonic arrhythmia per se, resulting in pressure changes and misdirection
of embryonic blood flow, and of the hypoxia-related damage.
27
As mentioned previously, it is generally accepted that the increased incidence of
embryonic/foetal death by selective IKr-blockers is a consequence of severe
bradycardia or of cardiac arrest. The results in this study support this hypothesis, since
pre-treatment with PBN or OTC did not significantly decrease the incidence of
embryonic death. Further, the decreased foetal weights in this and in other studies
with class III antiarrhythmics may be directly related to hypoxia, (due to bradycardia)
in embryos surviving until term.
IKr-blockers, embryonic arrhythmia and developmental toxicity in other
species than the rat (Papers III and IV).
The purpose of this part of the thesis was to investigate if established IKr-blockers
induce embryonic dysrhythmia and developmental toxicity in other species than the
rat. Almokalant was studied in NMRI-mice in a conventional developmental toxicity
study and in whole embryo culture. Further, sotalol was studied in New Zealand
White rabbits in a limited embryo-foetal developmental toxicity study, which was
divided into three parts, with different dosing regimes.
Almokalant
Developmental toxicity in the mouse
Almokalant was administered in the diet during GDs 6-15 in doses of 50, 125, and
300 µmol/kg. No signs of dysfunction were observed in the pregnant dams. A dosedependent embryonic lethality was observed resulting in a 100% embryo mortality at
the highest tested concentration in agrement with previous studies (Abrahamsson et
al., 1994; Danielsson, 1993) . The mouse embryos are thought to have died during
GDs 9-12 as indicated by the decreased maternal body weight gain from GDs 9-12
and onwards in the group with total litter loss. The plasma concentration of
almokalant was measured in satellite animals that had been dosed with 300 µmol/kg,
which resulted in a mean maternal plasma concentration of 1.3 µmol/l on GD 15.
The surviving foetuses in the control, 50 and 125 µmol/kg groups were examined for
external, visceral and skeletal defects. There were no significant differences between
the treated groups concerning external- and visceral malformations. The mean foetal
weight was statistically significantly reduced in the group treated with 125 mg/kg. In
the 50 and 125 µmol/kg groups, the incidence of some minor skeletal defects was
statistically significantly increased compared with the control group. These minor
skeletal defects were manifested in the intermediate dose group by a higher incidence
of unossified centers of cervical vertebrae (~12% affected foetuses versus ~7% and
~6% in the low dose and control groups, respectively), and a decreased degree of
calcification of the forepaw and hindpaw (~17% affected foetuses versus ~8% and
~9% in the low dose and control groups, respectively). The decreased calcification
was mainly localised to the distal phalanges. These results may reflect slight digital
reduction defects of the same type as those observed in the rat (Webster et al., 1996;
28
Papers I and II). In the low dose group, the increase was due to a higher incidence of
cervical ribs and an extra sternebral ossification center compared with the control
group. The percentage of foetuses showing skeletal variants (extra ribs) was
significantly increased in the low and intermediate dose groups compared with
controls. Overall, the results show that almokalant is a developmental toxicant in
mice, causing dose-dependent increase in embryonic death and skeletal defects, as
well as decreased mean foetal weights. The absence of external, visceral, and some
skeletal defects (compared to the rat), may be explained by the wide dose interval
between the intermediate and high dose groups. Further, single dose exposure on
specific days of gestation might increase the chance of observing / inducing these
effects. An example of such a dose regime with cisapride is presented in the next
section (pages 31-34).
Effects on the mouse embryonic heart
Almokalant was tested in GD 10 mouse embryos cultured in vitro. This day
corresponds in developmental stage to GD 11 in the rat, when almokalant was tested.
Almokalant caused bradycardia/arrhythmia at concentrations higher than 325 nmol/l,
in a concentration-dependent manner. A statistically significant decrease in heart rate
(bradycardia) was obtained compared to control embryos as follows; 650 nmol: 13%,
1.3 µmol: 20%, 2.6 µmol: 27% and 5.2 µmol: 42%. At the concentration 2.6 µmol,
arrhythmia (irregular heart rhythm and/or episodes of cardiac arrest) was observed in
one out of 17 examined embryos, and at 5.2 µmol, arrhythmia was observed in 7 out
of 15 examined embryos. The results obtained when mice embryos were cultured in
vitro with almokalant were similar to those obtained when rats were cultured with
almokalant and other pure IKr-blockers (Ban et al., 1994; Spence et al., 1994; Webster
et al., 1996).
Correlation between effects on the embryonic heart and developmental toxicity
The concentration (650 nmol/l) where almokalant reduced the embryonic heart rate in
vitro correlates well with the observation that a concentration of 1.3 µmol/l in vivo,
caused embryonic death in the present study. This strengthens the theory that the
pharmacologically induced embryonic bradycardia/arrhythmia that might lead to
impaired embryonic circulation and hypoxia as a secondarily effect mediates the
developmental toxicity of IKr-blockers not only in rats, but also in mice. The main
adverse manifestations in this study (increased incidence of embryo lethality in the
300 and 125 µmol/kg groups and decreased foetal weight in the 125 µmol/kg group)
are well known adverse manifestations produced by hypoxia due to different causes
(Grabowski, 1970). In view of the similarities in adverse effects of the embryonic
heart in the mouse and in the rat, we conclude that almokalant has teratogenic
potential also in the mouse.
29
Sotalol
Developmental toxicity in the rabbit
This study aimed to investigate if the IKr-blocker sotalol could induce developmental
toxicity in rabbits during the corresponding part of embryonic development as IKrblockers had caused developmental toxicity in the rat. Initially, repeated dosing was
performed on GDs 13-16. Administration of 150-300 mg/kg sotalol during this period
resulted in 100% embryonic death. Thereafter, single doses were given on GD 8, 9,
10, 12, 13, 14, 15, 16, or 17. Single doses of 100 mg/kg of sotalol on GD 8, 9, or 10,
resulted in a slight increase in the incidence of embryonic death, compared to
historical controls. Marked increased embryonic death (55-90%) was noticed after a
single dose of 100-150 mg/kg of sotalol on GD 12, 13, 14, 15 or 16. No external or
visceral abnormalities were observed in the surviving foetuses. In a subsequent part of
the study, different doses of sotalol (50, 60, 75, 85, or 100 mg/kg) were administered
on GD 14, one of the most sensitive days for induction of embryonic/foetal death.
There was total litter loss (100% embryonic death) at 85 and 100 mg/kg.
Administration of 60 and 75 mg/kg resulted in increased embryonic death, decreased
litter size and a corresponding increase in mean foetal weight. The dose of 50 mg/kg
gave a statistically significant elevated level of embryonic/foetal death (28.8%), but
did not severely affect litter size or mean foetal weight. No external or visceral
abnormalities were observed in the surviving foetuses.
In contrast to the adult rat, the adult rabbit shows a high susceptibility of developing
arrhythmias (Torsade de Pointes type) when exposed to IKr-blocking drugs such as
almokalant and dofetilide (Carlsson et al., 1993). In this study, two animals died
without any adverse clinical signs preceding death. It is reasonable to assume that the
two cases of maternal death (one at 225 mg/kg after three days of dosing and one after
a single dose of 100 mg/kg) were secondary to maternal cardiac arrhythmia. The
observation that dose levels in the range of 100 – 225 mg/kg induced death in only
two out of 22 adult animals (9%), compared to the high incidence of embryonic death
(approximately 70%), indicates that the early embryonic heart is more susceptible to
react with arrhythmias than the adult heart when exposed to IKr-blockers.
Correlation between the effects on the embryonic heart and developmental toxicity
It is reasonable to assume that the sotalol-exposed rabbit embryos die as a
consequence of embryonic bradyarrhythmia/ cardiac arrest, in the same way as has
been proposed happens after embryonic exposure to IKr-blockers in the rat. This
assumption is strengthened by results from studies in rat WEC where sotalol caused
dose-dependent bradyarrhythmia in the embryonic heart on GDs 11 and 13. Sotalol
caused a statistically significant embryonic cardiac effect at 50 µM (Webster et al.,
1996). The magnitude of these cardiac adverse effects has been related to embryonic
death in vivo in this species. In this study in rabbits, a single dose of sotalol
(50 mg/kg) on GD 14 resulted in embryonic death (28% of the embryos) at a Cmax of
~46 µM. These results indicates that the rabbit embryonic heart is at least as likely to
react with lethal bradyarrhythmia as the rat embryonic heart.
30
Comments on developmental toxicity
The results clearly showed that the IKr-blocker sotalol causes phase- and
concentration-dependent embryonic death in the rabbit in the same way and during
the corresponding period as IKr-blockers cause embryo lethality in the rat. It could be
argued that it is the β-blocking property of sotalol that is responsible for the
embryonic bradycardia and subsequent developmental toxicity, and not the IKrblockage. However, results from rodents indicate that this is not the case. In the rat,
the heart begins to beat around GD 10, but does not receive the extrinsic autonomic
innervation until about GD 16-18 (Adolph, 1965; Gomez, 1958; Hogg, 1957). The
sinus node is innervated on about GD 16. In early embryonic life, there is a slow fixed
heart rate, which increases progressively with age. β-adrenergic receptors are present
in the non-innervated embryonic rodent heart (Martin et al., 1973; Robkin et al.,
1974). For example, in the foetal heart β1-receptors are already in place in early
development (GD 12), but receptor coupling to adenylate cyclase (indicating some
aspect of physiological function) is low at this stage. In contrast, coupling shows a
spike at GD 18 and furthermore, on this day the concentration of β-receptors in foetal
tissues is five times higher than on GD 12 (Slotkin et al., 1994). These data suggest an
association between β-receptor expression and cell differentiation in late foetal stages
and may contribute to explaining the relative absence of effects of β-antagonists
(despite the occurrence of beta-receptors) on the embryonic/foetal heart. Altogether,
these results suggest that the ability of β-blockers to cause embryonic/foetal
bradycardia by direct pharmacological action is low during the susceptible stages for
induction of hypoxia-related malformations. This is also supported by the fact that
β-blockers as a group have not shown any developmental toxic potential in teratology
studies (Danielsson and Webster, 1997).
Drugs that block IKr as a side effect (Papers V and VI)
In this part of the thesis the purpose was to investigate if two compounds, cisapride
and phenytoin, whose primary action is not IKr-blockage, also have the potential to
cause embryonic arrhythmia, embryonic death and hypoxia-related malformations in
the same way as selective IKr-blockers. Both compounds have recently been shown to
have IKr-blocking properties as a side effect. Cisapride – (Carlsson et al., 1997),
Phenytoin – IK (Nobile and Vercellino, 1997), phenytoin – IKr (Danielsson et al.
unpublished data). Mosapride was used as a reference compound and included in the
whole embryo culture. Mosapride is structurally related to cisapride and has the same
pharmacological activity (gastrointestinal motility stimulation) but it does not block
IKr at therapeutic concentrations (Carlsson et al., 1997). Phenytoin is an antiepileptic
drug (Rogawski and Porter, 1990).
The background data available for cisapride and phenytoin was completely different.
The proarrhythmic potential of cisapride in adults and its IKr-blocking properties were
known, while the information regarding developmental toxicity was very scarce.
However, if the theory about IKr blockage being the cause of developmental toxicity
via induction of embryonic cardiac arrhythmia and hypoxia/reoyxgenation damage is
31
correct, then cisapride ought to induce similar effects as pure IKr-blockers such as
almokalant. With phenytoin it was the other way round: the potential to cause
developmental toxicity both in animals and humans was well established, but not the
mechanism. Different theories regarding the mechanism have been proposed, for
example the formation of reactive metabolites (Dansky and Finnell, 1991; Finnell and
Dansky, 1991). In view of the similarities reported in the malformation patterns and
early changes preceding the defects, including haemorrhage and necrosis of distal
parts of digits between PHT (Danielsson et al., 1992) and IKr-blockers (Webster et al.,
1996) in animals studies, we decided to investigate if PHT would affect the
embryonic heart at concentrations inducing developmental toxicity.
Cisapride and mosapride
Effects on the embryonic heart
In rat whole embryo culture, the control group heart rate decreased slightly (~6%) at
the end of the study period (at 2 hours), compared to the original heart rate. Mosapride
did not statistically significantly affect the embryonic heart rhythm at any of the tested
concentrations; 100, 1,000 and 2,000 ng/ml. The heart rate was approximately 90% of
the heart rate recorded before administration of mosapride at the end of the study
period. Cisapride (100 ng/ml) caused slight bradycardia (~10 %) but no irregular heart
rhythm, similar to what had been obtained with mosapride. In contrast, exposure to
1,000 ng/ml of cisapride resulted in severe bradycardia manifested as a mean decrease
in heart rate of 34 % and an irregular heart rhythm observed in almost all embryos (15
out of 16). The arrhythmia was of the same kind as been described for the selective
IKr-blocker almokalant in previous studies (rat – Webster et al., 1996, mouse –
Paper III). Cisapride caused decreased heart rate and arrhythmia in rat embryos during
the same period as that in which selective IKr-blockers induce identical embryonic
cardiac adverse effects (Abrahamsson et al., 1994; Ban et al., 1994; Spence et al.,
1994; Webster et al., 1996).
Developmental toxicity
Since mosapride did not induce embryonic cardiac arrhythmia in vitro, only cisapride
was investigated for hypoxia-related malformations in vivo in the rat. Data supplied
by the manufacturer (unpublished) showed no evidence of any embryotoxic or
teratogenic effects of mosapride in the rat and in the rabbit in conventional teratology
studies even at very high doses (300 mg/kg - rat, 125 mg/kg - rabbit). This data agree
with the observed absence of arrhythmogenic potential in cultured embryos.
32
Figure 6: Defects of the left forepaw after administration
of cisapride to pregnant rats on GD 13.
A single dose of cisapride on GD 13 caused deteriorated condition such as pilo
erection and decreased motor activity in this study. The signs of maternal toxicity
were dose-dependent, with the most severe signs of the longest duration (up to two
days) after 200 mg/kg. Dosing with 75 mg/kg and 50 mg/kg did not seem to affect the
dams. Cisapride was shown to have both embryotoxic and teratogenic potential. As
has been observed in studies with different selective IKr-blockers (Danielsson,
1993),(Ban et al., 1994; Spence et al., 1994; Webster et al., 1996) high incidences
(55-82%) of embryonic death and a decrease in mean foetal weight in surviving
foetuses were seen after administration of high doses (100, 160 and 200 mg/kg) of
cisapride. When the dose was lowered to 75 mg/kg, the incidence of embryonic death
decreased considerably (15%) and at 50 mg/kg there was no statistically significant
difference in comparison to controls. In both these dose groups (50 and 75 mg/kg) the
majority of foetuses could be examined for external malformations. Identical digital
reduction defects (brachydactyly) and fusion of digits (syndactyly) as those seen after
single dose administration of the selective IKr-blockers almokalant and dofetilide as
well as hypoxia (Papers I and II; Leist and Grauwiler, 1973; Webster et al., 1996), of
the same stage of development (GD 13) were obtained with cisapride (figure 6).
Correlation between the effects on the embryonic heart and developmental toxicity
Blood samples taken one hour after oral administration showed that a maternal plasma
concentration of 1,100 ng/ml resulted in adverse effects on the embryonic/foetal
development (embryonic death and malformations). Corresponding concentrations of
cisapride, 1,000 ng/ml in the whole embryo culture system, caused severe bradycardia
and arrhythmia.
33
Comments on developmental toxicity
These results indicate that drugs with IKr-blocking properties as a side effect also
cause embryonic death and phase-specific defects due to induced embryonic
arrhythmia, in the same way as selective IKr-blockers do. The only known major
difference between cisapride and mosapride is the ability of cisapride to block the IKr.
The IKr-blocking potential of cisapride is thus most likely of major importance to
explain why almost all rat embryos exposed to cisapride had arrhythmia and severe
bradycardia at 1,000 ng/m in this study, and while no embryos exposed to 2,000 ng/ml
of mosapride showed such effects. These results further strengthen the idea that
cisapride’s potential to cause embryonic arrhythmia and malformations are a
consequence of its ability to exert pharmacological action via inhibition of IKr. In vitro
studies show that the potency of cisapride to block IKr is similar to the selective
IKr-blocker dofetilide (Carlsson et al., 1997). The differences in developmental
toxicity between cisapride and mosapride could not be attributed to differences in
pharmacokinetics, since both drugs are readily absorbed, with a similar half-life in
rats (1-2 hours) and transfer to the foetus in this species (cispride- (Michiels et al.,
1987), mosapride- unpublished data). Neither can the differences be attributed to
differences in their chemical structures or primary pharmacological effect (Carlsson et
al., 1997).
There are no published experimental teratology studies on cisapride in the open
literature. The FDA pregnancy labelling, which summarises non-clinical studies
conducted by the manufacturer, mentions embryotoxicity/foetotoxicity at
160 mg/kg/day and reduced birth weight of pups and adversely affected pup survival
at 40 and 160 mg/kg/day in rats. In rabbits, cisapride was embryotoxic and foetotoxic
at 20 mg/kg/day or higher. The pregnancy text mentions that there was no evidence of
a teratogenic potential of cisapride. In this study, we have observed a sharp dose
response curve, with no adverse effects at 50 mg/kg, pronounced embryonic death and
teratogenicity at 75 mg/kg, and almost total litter loss at 160 mg. A likely reason for
missing the teratogenic potential in the original rat teratology study is thus the wide
dose interval between the two doses used (40mg/kg/day and 160 mg/kg/day).
Phenytoin
Phenytoin has been associated with a variety of developmental toxicity manifestations
including embryonic death, intrauterine growth retardation/decreased foetal weights,
and a variety of malformations including orofacial clefts, limb defects, skeletal
ossification defects, heart and urogenital abnormalities, as well as CNS defects such
as dilated ventricles (hydrocephalus) in both animal and human studies (Dansky and
Finnell, 1991; Finnell and Dansky, 1991). Both almokalant and hypoxia have been
associated with a very similar pattern of developmental toxic effects (see table 2). The
purpose of this study was to investigate if PHT would affect the embryonic heart at
concentrations that induce developmental toxicity. In this study in the rat, embryonic
death, decreased foetal weights and orofacial clefts were chosen to represent
34
developmental toxicity after a single dose of PHT on GD 11, since they are easily
recognised. Further orofacial cleft was induced with almokalant on GD 11. The
maternal plasma concentrations, both total and free, of PHT were followed during
24 hours after dosing with PHT.
Developmental toxicity related to maternal plasma concentrations
A non-toxic dosing regime of 150 mg/kg po (Rowland et al., 1990) did not induce any
developmental toxicity. The plasma concentration of PHT peaked 4 hours after dosing
(Cmax total 63 µM and Cmax free 7 µM). It declined slowly over the next 20 hours to
41 µM (total) and 4.4 µM (free) at the end of the sampling period.
A dosing regime, 100 mg/kg ip, which has been reported to be slightly
developmentally toxic (Harbison and Becker, 1972), induced a small decrease in
mean foetal weight. This dose resulted in peak concentrations at around 8 hours
(Cmax total 150 µM and Cmax free 16 µM) which declined to 47 µM (total) and 6.3 µM
(free) by 24 hours. During the 8 hours after dosing the free plasma levels after
100 mg/kg ip were 2-7 times higher than those seen after administration of 150 mg/kg
po. The AUC for this dose was more than double that of the 150 mg/kg oral dose. An
established developmentally toxic dosing regime, 150 mg/kg ip (Harbison and
Becker, 1972), induced a relatively high incidence of early embryonic death (24%)
with almost all litters (9/11) affected. Foetal weight was statistically significantly
reduced compared to control. As predicted, this single dose of PHT on GD 11 induced
teratogenicity, specifically orofacial clefts, which occurred in 12 out of 105 foetuses.
There was persistently high plasma concentrations (Cmax total ∼240 µM and Cmax free
33 µM) for the entire 24 hours following dosing giving an AUC (total or free) double
that of the 100 mg/kg ip dose.
Effects on the embryonic heart
Phenytoin caused a dose-dependent decrease in heart rate in rat WEC. When rat
embryos were cultured in 75% rat serum, the embryonic heart rates were statistically
significantly decreased after the addition of 200 µM (-32%) or 100 µM (-16%) of
phenytoin, when compared to the heart rates before addition of phenytoin. This agrees
with earlier studies by our group, where the heart rates were compared to controls
(Danielsson et al., 1997).
A “bridging” experiment between in vivo and in vitro conditions was also performed.
The dams were administered phenytoin in vivo, and the embryos were removed from
the uterus after four hours, placed in culture medium in vitro, and the heart rates were
examined after 30 minutes in culture. The doses and routes of administration were
chosen to represent both developmentally toxic and non-toxic levels of phenytoin as
observed in the in vivo part of this study. The embryos from the dams treated with the
developmentally toxic dose of 150 mg/kg phenytoin ip had a statistically significantly
decreased heart rate, 25% lower than those from the control dams. After treatment
with the developmentally non-toxic dose of 150 mg/kg phenytoin po, there was a
slight, but not statistically significant decrease of 7%. In general, the embryos with
35
slower heart rates were paler and the contraction force of the heartbeats seemed lower
compared to the embryos with normal heart rates.
Correlation between effects on the embryonic heart and developmental toxicity
A dosing regime of phenytoin that does not cause any foetal adverse effects
(150 mg/kg po) resulted in a total plasma concentration that was lower than that
which causes embryonic bradyarrhythmia.
In contrast, dosing regimes that resulted in total and free plasma concentrations that
caused slight (100 mg/kg ip) and marked (150 mg/kg ip) bradyarrhythmia caused in
this experiment slight and marked developmental toxicity, respectively.
Comments on developmental toxicity
The close correlation that we have shown between embryonic arrhythmia and
developmental toxicity for phenytoin strengthens the idea that non-selective IKrblocking drugs should be considered as potential developmental toxicants, due to their
ability to cause embryonic arrhythmia. In addition to acting on K+ channels,
phenytoin acts on both Na+ and Ca2+ channels. In view of this it cannot be excluded
that the actions of Na+ and Ca2+ ions contribute to the cardiac dysrhythmia in the
embryo. However, in view of the considerable similarities in developmental toxic
effects between selective IKr-blockers, and our very recent data showing that PHT
inhibits IKr (Danielsson et al. unpublished), it is reasonable to assume that action on
IKr is the major reason for the observed embryonic dysrhythmia.
WEC: an indicator for developmental toxicity and for proarrhythmic
potential in adults?
Interest in reliable and easy tests for proarrhythmic potential of especially noncardiovascular drugs has increased dramatically. Various tests have been proposed by
the regulatory agencies, the medicinal industry themselves, and by Contract Research
Organisations (CROs), including studies with Purkinje fibres, heart organ baths from
species expressing functional IKr, and cell-lines expressing HERG. One of the major
drawbacks has been that the relatively simple mouse and rat are not suitable due to the
fact that their hearts lack IKr-dependence. This is true for the adult rodent heart, but
not for the rodent embryonic heart as has been shown in this thesis. We propose that
observation/recording of heart rhythm in rodent embryos cultured in vitro during a
restricted sensitive period of development is a good test for studying the potential of
substances to induce developmental toxicity, and arrhythmia in sensitive adults.
Table 3 shows a compilation of compounds tested in WEC. The drugs have been
tested up to 20 times the clinical concentration. Some of the tested compounds are
anti-epileptic drugs such as trimethadione, carbamazepine and phenobarbital that have
been associated with a similar spectrum of foetal adverse effects as phenytoin
(Dansky and Finnell, 1991; Finnell and Dansky, 1991). Their common capacity to
36
cause embryonic dysrhythmia during the sensitive period via interference with ion
channels seems to be a likely unifying mechanism for their foetal adverse effects.
Another interesting pair of compounds are the antihistamines terfenadine and
fexofenadine. Terfenadine has induced Torsade de Pointes in some susceptible
patients and it blocks IKr (Taglialatela et al., 1998). Fexofenadine is a metabolite of
terfenadine and is today often used instead of terfenadine since it seems to lack
proarrhythmic potential.
Although not all data for all compounds are available the trend is clear. Induction of
severe embryonic bradycardia (= statistically significant and usually at least ~20%
decrease in heart rate) and arrhythmia seems to be a good indicator of both
developmental toxicity and proarrhythmic potential in adults. Using rodent whole
embryo culture may have some of these advantages over other proposed test systems:
•
•
•
•
•
it is a relatively simple method to carry out
it is based on the rat or the mouse, which are common laboratory animals used in
toxicology testing
the effects are tested on a whole organ, not only a specific cell type, which could
increase the precision of the test
since it is an intact, although immature, heart it may react with arrhythmia also for
compounds acting on other channels than IKr, such as the L-type Ca2+ and Na+
channels, which are expressed at least from GD 11 in the mouse embryo, and
other types of arrhythmia than those induced by IKr may also be of importance.
it is probably possible to further develop the method by including ECG recordings
of the embryonic heart (as in the study by (Abrahamsson et al., 1994)).
Further studies testing more compounds, however, are needed before the whole
embryo culture can be established as a good test system for developmental toxic and
proarrhythmic potential.
37
Yes
Yes
Phenytoin
No
Yes
No
Vigabatrin
Cisapride
38
Fexofenadine
Terfenadine
Mosapride
Yes, prel data
No, prel data
No
Valproic acid
Trimethadione/
dimethadione
Yes
Yes
Carbamazepine
Phenobarbital
Sotalol
L 691,121
Dofetilide
Yes
Yes
Yes
Yes
Almokalant
Substance
WEC
Severe
bradycardia
No, prel data
No, prel data
Yes
No
No
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
WEC
Arrhythmia,
Cardiac arrest
No information
No information
No information
Yes
No information
No information
Yes
No
Yes
No information
No information
No information
No information
No information
Yes
No information
Yes
Yes
Yes
Yes
Yes
Clinically
Arrhythmia,
Cardiac arrest,
Torsade de Pointes
No information
No information
hypoxia rel.)
No information
No (yes, but not
hypoxia rel.)
Yes
Yes
Yes
Yes
No information
No information
No information
No information
Teratogenicity
Clinically
No (yes, but not
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Animal models
Embryotoxicity/
hypoxia-related
teratogenicity
Yes
Yes
No
? Under
investigation
? Under
investigation
? Under
investigation
Yes, prel data
? Under
investigation
Yes
Yes
Yes
Yes
Yes
IKr-blockage
Table 3: Summary of compounds tested in whole embryo culture.
Human relevance and future studies
The human relevance of the embryonic arrhythmia related mechanism by IKr-blockers
teratogenicity is unknown. However, indirect evidence indicates that the proposed
mechanism may be relevant to humans. Experimental data indicates that the early
embryo expresses IKr in mice, rats, and rabbits, and that it reacts with bradycardia and
arrhythmia when exposed to IKr-blockers. These data suggest a universal functional
role across all mammalian species (including humans) for IKr channels in the
development of the mammalian cardiac electrical system.
Phenytoin is an established human teratogen and has recently been shown to inhibit
IKr (Danielsson et al. unpublished). Phenytoin causes very similar patterns of
developmental toxic effects in a variety of species (including humans, rabbits, rats,
and mice), which strengthens the idea of a common mechanism across species. The
foetal hydantoin syndrome in humans includes both major malformations such as
orofacial clefts and minor malformations such as distal digital defects, and growth
retardation. It has been possible to recreate these manifestations in animal models at
therapeutic concentrations. Both phenytoin and selective IKr-blockers induce orofacial
clefts and distal digital defects in animals preceded by identical damage with respect
to vascular disruption and haemorrhage. In clinical practice, it is well known that
episodes of severe hypoxia during labour may result in foetal intracranial
haemorrhage and permanent tissue damage. Little information is available, however,
regarding the consequences of hypoxia in the human embryo in early pregnancy. It is
not unreasonable to assume that the human embryo may respond to hypoxic episodes
with stage-specific damage, in the same way as other mammalian species.
It would be interesting to investigate if human embryos express functional
IKr-channels, and if so, during which stage of development. Another interesting study
would be to determine whether children born with PHT-induced malformations suffer
from long QT syndrome, being more susceptible towards drug-induced prolongation
of the QT interval. Regarding the promising method of monitoring rodent embryonic
heart rhythms with WEC, further studies will tell if it can be used as one reliable test
for proarrhtymic potential in adults as well as teratogenicity of non-cardiovascular
drugs – a “cardiac Ames test”.
39
CONCLUDING REMARKS
During the work with this thesis, general interest in compounds acting on IKr, also for
drugs not being developed as class III antiarrhythmics, increased dramatically, due to
their proarrhythmic potential. The interest has been focussed on cardiac arrhythmia in
adults and not on their potential for causing developmental toxicity. But, as has been
shown in this thesis, these two characteristics are strongly linked.
Referring to the aims with this study:
•
The different studies (Papers I-III) with the selective IKr-blocker almokalant
strongly support a hypoxia-related mechanism due to embryonic arrhythmia
across species (rat, mouse).
•
The decrease in almokalant-induced malformations by the spin-trapping agent
PBN (Papers I-II) suggests that the defects are mainly caused by reoxygenation
damage after episodes of severe embryonic dysrhythmia, rather than “pure
hypoxia”.
•
Sotalol induced embryonic death in the rabbit (Paper IV). The adult rabbit heart
expresses functional IKr and is a sensitive species for IKr-induced arrhythmia. The
embryonic heart seems to be more sensitive than the adult heart, reacting with
arrhythmia at lower concentrations than the adult heart, as indicated in this study.
•
The observed developmental toxicity has been linked to the IKr-inhibition not only
for the selective IKr-blocker almokalant, but also for non-selective IKr-blockers
such as sotalol, cisapride, and phenytoin. This leads to the hypothesis that if a
compound exerts a blocking effect on IKr as the therapeutic mechanism or as a side
effect, it has the potential to cause embryonic/foetal toxicity and teratogenicity.
This might occur irrespective of the original indications that the compound was
developed for.
•
Since the heart in rodent embryos, in contrast to adult hearts in rodents, expresses
a functional IKr-channel, WEC (whole embryo culture) with observation of the
heart rhythm may become a new, relatively simple screening method. Thus, the
embryonic heart may during a restricted period serve as an indicator for
proarrhythmic potential of different compounds, and of their potential to induce
developmental toxicity.
40
ACKNOWLEDGEMENTS
This work was performed at AstraZeneca R&D, Södertälje, Safety Assessment, and at
the Department of Pharmaceutical Biosciences, Division of Toxicology, Uppsala
University. I wish to express my sincere gratitude to everyone who made this work
possible, and especially:
To Bengt Danielsson, my supervisor, who offered me the opportunity to become a
Ph.D. student (and a study director at the same time). Thank you for sharing your
knowledge in reproductive toxicology and for always being enthusiastic, even when
the results did not turn out as expected.
To Peter Moldéus, Head of Safety Assessment, for his generosity in giving me the
time and opportunity to do this work.
To Lennart Dencker, Head of the Division of Toxicology, for taking me in as an
internal/external Ph.D. student and for your kind support and interest in my work.
To my co-authors Alf Wallin, Faranak Azarbayjani, Inger Öhman, and William
Webster for a pleasant, fruitful collaboration and valuable scientific discussions.
To Katrin Wellfelt collaborator and co-author, for “skilful technical contribution”,
friendship, laughs and talk about any- or everything, and especially for the work
during your maternity leave.
To Arvid Wellfelt for accompanying your mother to Safety Assessment (no choice
I suppose), even though I wasn’t too happy about your throwing up on me.
To all present and past friends and colleagues at the Department of Pharmaceutical
Biosciences, Division of Toxicology.
To all present and past friends and colleagues at the Department of Reproductive
Toxicology, Safety Assessment, especially those involved in my studies, and that
means more or less everyone.
To my liaison officer Anita Annas for friendship and helping me to organise things in
Uppsala after I left for Gärtuna.
To Raili Engdahl and Lena Norgren for all help and pleasant company in the lab in
Uppsala, especially for helping me to manage to watchmaker forceps under the
microscope in the beginning of this work.
To Terri Mitchard and Elaine Gordon for taking urgent measures in revisions of the
English language now and then.
41
To Karin Cederbrant my “friend-PhD-student” at Safety Assessment for
encouragement in every aspect of work and life, and nice company during late nights
of the writing process. Fuskigt att du blev doktor före mig, hm.
To my parents Gerd and Erling, who have always believed in me, encouraged me and
done everything in their power to support me, remember all the crumpled up
papers.....
To my husband Johan for endless love, support, encouragement, patience, and
delicious meals. I’m really looking forward to spending more time with you.
To all my other (BMC-) friends, who I’ve spent many joyful hours with, and who
haven’t tired on repeated phone-calls when I needed encouragement or just a break.
To MFR, CFN, and AstraZeneca for financial support.
42
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Abrahamsson, C. Proarrhythmias related to delayed repolarisation. Importance of
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