Teratogenicity of the class III antiarrhythmic drug almokalant. Role of hypoxia and reactive oxygen species

Reproductive Toxicology, Vol. 13, No. 2, pp. 93–101, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0890-6238/99/$–see front matter

PII S0890-6238(98)00066-5

TERATOGENICITY OF THE CLASS III ANTIARRHYTHMIC DRUG ALMOKALANT. ROLE OF HYPOXIA AND REACTIVE OXYGEN SPECIES KATRIN WELLFELT*†, ANNA-CARIN SKO¨ LD*†, ALF WALLIN† and BENGT R. DANIELSSON*† *Department of Pharmaceutical Biosciences, Division of Toxicology, Uppsala University, Uppsala, Sweden and †Astra AB, Safety Assessment, So¨derta¨lje, Sweden Abstract — The class III antiarrhythmic drug almokalant (ALM) was given to pregnant rats on Gestation Day 11 (125 mmol/kg) or 13 (25 mmol/kg). Other groups were pretreated with a-phenyl-N-t-butylnitrone, (PBN; 850 mmol/kg intraperitoneally) 1 h before administration of ALM or given (-)-2-oxo-4-thiazolidine carboxylic acid (OTC; 250 mmol/kg subcutaneously) 4 h before administration of ALM. PBN is a spin-trapping agent that can capture reactive oxygen species (ROS), and OTC is an antioxidant. Controls received tap water only. All groups (eight in total) consisted of 7 to 10 pregnant rats. ALM induced cardiovascular defects, orofacial clefts, and tail defects after administration on Day 11, and reduced the size of digits on Day 13. Pretreatment with PBN prevented induction of all the above-mentioned malformations by ALM. The results also indicated that OTC may have some protective effect against ALM-induced teratogenicity but not to the same extent as PBN. The results support the hypothesis that almokalant induces malformations via induction of episodes of embryonic arrhythmia/cardiac arrest, which result in hypoxia followed by reoxygenation and generation of ROS. Key Words: Almokalant; class III antiarrhythmics; OTC; PBN; protection by antioxidants; reactive oxygen species; ROS; teratogenicity.

cleft lip and/or palate, and short or missing tail (7). In mechanistically orientated studies, several investigators have proposed that the observed embryolethality in rats (3–5,8) and mice (9) is caused by pharmacologically induced embryonic bradycardia/permanent cardiac arrest. The observed foetal adverse effects at term (malformations and decreased foetal weights) may be a consequence of embryonic hypoxia secondary to the embryonic bradycardia. However, in addition to bradycardia, class III antiarrhythmics have been reported to cause embryonic arrhythmia and temporary cardiac arrest (8), resulting in episodes of severe embryonic hypoxia followed by reoxygenation. Recently, evidence has been presented indicating that embryotoxic reactive oxygen species (ROS) are generated within the embryo during such conditions (10). In adult tissues, generation of ROS 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 (11,12). Malformations caused by class III antiarrhythmics have been preceded by early pathologic changes within 48 h, such as haemorrhage and haematomas in the same areas that are deformed (orofacial area and distal parts of the limb bud) in the late foetus (5,7). Identical early changes in the embryo and the same type of digital defects (fusion, reduced size, and absence of digits) induced by almokalant could be produced by inducing

INTRODUCTION Almokalant is pharmacologically characterised as a class III antiarrhythmic agent. Class III antiarrhythmic drugs prolong the repolarisation phase of the myocardial action potential and thus lengthen the refractory period of the myocardium, which prevents arrhythmias in the adult heart. This pharmacologic effect is most likely mediated by inhibition of one component of the potassium K1 channel, the delayed-rectifying potassium current (Ik) (1). Several class III antiarrhythmic drugs like ibutilide, dofetilide, d-sotalol, and L-691,121 are used therapeutically or are under clinical development. In teratology studies, all the above-mentioned class III antiarrhythmic drugs have induced dose-dependent embryolethality at doses causing no signs of adverse effects in the pregnant dams (2–6). Lower doses than those causing embryolethality have induced phase-specific teratogenic effects as well as retarded embryonic/ foetal growth (2,5). The external malformation pattern has been very similar for all class III antiarrhythmics studied and consists of missing or reduced size of digits, This work was supported by Swedish Medical Research Council (MFR grant K97-17x). Address correspondence to Bengt Danielsson, MD, PhD, Astra AB Safety Assessment, S-151 85, So¨derta¨lje, Sweden. Phone: 1 46 8 553 265 29; Fax: 1 46 8 553 288 23; E-mail: bengt.danielsson@ astra.se.astra.com Received 29 June 1998; Revision received 6 November 1998; Accepted 7 November 1998. 93

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Table 1. Dose dependent developmental toxicity of almokalant given orally on GD 11 Almokalant dose (mmol/kg) Treatment GD 11 No. of pregnancies No. of litters No. of live foetuses Mean foetal weight, g, (SD) Embryonic/foetal death, % (No. of affected litters) Orofacial clefts (%) Tail defects (%) Digital defects (%)

75

100

125

150

175

6 6 68 4.7 (0.6) 13.9 (4)

6 6 71 4.6 (0.4) 9.0 (4)

6 6 49 4.3 (0.5) 38.0 (6)

6 3 13 4.4 (0.3) 84.2 (6)

5 1 1 3.9 (2) 98.5 (5)

6 (8.5) 20 (28) 0

6 (12) 21 (43) 0

1 (7.7) 12 (92) 0

1 (1.5) 0 0

temporary hypoxia by clamping of uterine vessels on Gestation Day (GD) 14 (13). Hypoxia and haemorrhage have also been proposed to be involved in the pathogenic mechanism for development of orofacial clefts during the same susceptible period in which these defects could be produced by almokalant (14–16). The main purpose of this study was to investigate the role of ROS in reported external malformations (orofacial clefts and digital and tail reductions) at doses of almokalant allowing the majority of almokalantexposed embryos to survive until term. This was done by investigating the effects of pretreatment with the spintrapping agent PBN (a-phenyl-N-t-butylnitrone), with the capacity to capture ROS, and the antioxidant OTC ((2)-2-oxo-4-thiazolidine carboxylic acid), a promoter of glutathione synthesis. Another purpose was to investigate whether almokalant had potential to cause cardiovascular defects, as has been reported for other class III antiarrhythmic drugs (3,6). MATERIALS AND METHODS Animal maintenance and mating procedure Sprague-Dawley rats (Mo¨llegaard Breeding Centre Ltd, Denmark) were kept under specified conditions at a constant 12-h light/dark cycle at Safety Assessment, Astra AB, So¨derta¨lje. They had free access to food and water. Matings were allowed overnight, and the day of occurrence of a vaginal plug and/or sperm in the vaginal smear was considered Day 0 of pregnancy. Substances Almokalant (100 mmol/mL) in citrate buffer solution, pH 5 (Astra Ha¨ssle AB), was diluted with sterile water to give the appropriate concentrations (5 and 25 mmol/mL). OTC, (-)-2-oxo-4-thiazolidine carboxylic acid (Sigma Chemical Company) was dissolved in 50 mM phosphate-buffered saline (PBS) to a final concentration of 68 mmol/mL. PBN, a-phenyl-N-t-butylnitrone (Sigma Chemical Company) was dissolved in 50 mM PBS to a final concentration of 113 mmol/mL.

0 0 0

Study design Pregnant rats were given a single oral dose of almokalant on GD 11 (125 mmol/kg) or on GD 13 (25 mmol/kg). On both days, one group was given almokalant only, a second group was given a subcutaneous dose of OTC, 250 mmol/kg, 4 h before administration of almokalant, and a third group was given PBN, 850 mmol/kg, intraperitoneally 1 h before administration of almokalant. On both GD 11 and 13, an additional group was given tap water only and served as a control group. No groups dosed with PBN or OTC only were included in the study, since published studies do not indicate any teratogenic potential of PBN or OTC (17,18). Selection of doses The selection of doses of almokalant was based on data from our previous studies in rats with almokalant (2,5) and a pilot study on GD 11 (Table 1). The doses of almokalant in this study were selected to let the majority of the exposed embryos survive until term. Higher doses of almokalant, e.g. 150 mmol/kg on GD 11 (Table 1) and 100 mmol/kg on GD 13 (5), result in higher incidences of foetuses with malformations. However, at such dose levels, a great proportion of the embryos die in utero, manifested as a high percentage (;50 to 70%) of embryonic/foetal death at term (Table 1). Administration of even-higher doses (300 and 1000 mmol/kg) result in total embryonic loss (2,8). Administration of doses resulting in a high incidence of embryonic death may mask the teratogenic potential of class III-antiarrhythmic drugs. (See also the 175 mmol/kg group in Table 1.) The doses selected (25 and 125 mmol/kg on GD 11 and 13, respectively), allow the majority of exposed embryos to survive until term, but still induce a biologically and statistically significant increase in malformations. The doses of OTC and PBN were based on findings in a study in mice (18). In that study, the doses of OTC (250 mmol/kg) and PBN (1700 mmol/kg) did not severely interfere with maternal well-being. However, the PBN dose had to be reduced by 50% to 850 mmol/kg in the present

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study in rats, compared with the study in mice (18) because of maternal toxicity in rats at the higher dose level. Endpoints The maternal body weights were recorded regularly during the study (between 0800 h and noon on GD 0, 3, 6, 9, 11, 15, 18, and 21 1 GD 12 and 13 in the groups dosed on GD 11 and 13, respectively), and all animals were inspected daily for clinical signs. This was done to evaluate how the treatments interfered with maternal well-being, which could influence the outcome. On Day 21 of pregnancy, the animals were sacrificed and the uterine contents were examined. The litter data recorded were embryonic/foetal death and foetal weights. The foetuses were examined for external malformations, and subsequently, every second foetus in each litter was fixed in Bouin’s solution for internal examination of possible cardiovascular defects (19). For further details regarding number of treated dams and litter data, see Tables 2 and 3. Evaluation and statistics When evaluating the outcome data, it is not only of interest to evaluate whether the incidence of the effects in offspring from treated dams is merely statistically different from controls; the biologic significance also has to be taken into account. Therefore, we have included historical control data from our laboratory regarding the foetal defects. These data consist of 12 studies preceding the present study (eight dose-finding studies—only external examination and four main teratology studies—both external and internal examination). The rats were of the same strain and from the same breeder as in the present study. The statistical analysis for dam body weight and mean foetal weight was performed using a linear model (ANCOVA), with treatment group as a factor and the number of foetuses as a covariate for both variables, and additionally the body weight at GD 9 as a covariate for the dam body weight. The variables embryonic/foetal death and external and cardiovascular defects were analysed with Kruskal-Wallis and Wilcoxon-MannWhitney tests. The tested variable for defects was the number of defects in a litter divided by the number of foetuses in that litter, hence based on the litter as experimental unit. In all cases, a value of P , 0.05 was considered statistically significant. RESULTS Maternal data Clinical observations No signs of dysfunction were observed in the dams that could be related to treatment with almokalant or in dams pretreated with OTC before dosing with al-

Fig. 1. Effects of almokalant and combined treatment with ALM1OTC and ALM1PBN on maternal body weight: a) dosing on GD 11 (ALM 125 mmol/kg) and b) dosing on GD 13 (ALM 25 mmol/kg).

mokalant. However, the dams pretreated with PBN showed toxicity, manifested as piloerection, squinting, and decreased motor activity, and the dams did not eat or drink. Despite these severe symptoms, which lasted for

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Table 2a. Effects of OTC and PBN on almokalant pregnancy outcome data when treated on GD 11 Treatment GD 11 (ALM: 125mmol/kg) No. of pregnancies No. of litters No. of live foetuses Mean foetal weight, g, (SD) Embryonic/foetal death, % (No. of affected litters)

Control

ALM

ALM1OTC

10 10 121 5.3 (0.4) 5.5 (4)

9 9 85 4.3c (0.6) 22.7c (7)

7 7 73 4.7c (0.4) 26.3 (5)

ALM1PBN 8 8 96 5.0a (0.4) 16.5c (7)

a

Significantly different from ALM only. Significantly different from the control.

c

more than 8 h, the dams showed no adverse signs 24 h after PBN administration. Maternal body weights The signs of maternal toxicity were accompanied by an absolute loss in body weight during the 1 to 2 days after treatment with PBN (Figure 1), reaching statistical significance in relation to controls and almokalanttreated animals on GD 13. The PBN-treated dams regained most of this body weight loss, and the body weight gain was essentially the same in all groups from 2 days after dosing until caesarean section (Figure 1). Litter data Foetal weights (Table 2) Mean foetal weights were statistically significantly decreased in the groups receiving almokalant and almokalant 1 OTC on GD 11, compared with controls. In the almokalant 1 PBN-treated group, the mean foetal weights were statistically significantly increased compared with the almokalant-treated group, approaching the values in the controls. There were no obvious differences in foetal weight in the groups dosed with almokalant on GD 13 compared with controls, except for the almokalant 1 PBN-treated group that was statistically decreased from the controls. Embryonic/foetal death On GD 11, the incidence of embryonic/foetal death was increased in all almokalant-treated groups compared

with controls, reaching statistical significance in the almokalant and almokalant 1 PBN-treated groups (Table 2a). The incidence of embryonic/foetal death in almokalant-treated groups was also increased after treatment on GD 13 (Table 2b), but the increase was not as high as in the animals treated on GD 11. The increase was statistically significant when the almokalant group was compared with the controls. Foetal examination Almokalant-only-treated groups Almokalant administered on GD 11 of pregnancy caused malformations visible at external examination (orofacial clefts and short tail) in the pilot study in a dose-dependent manner (Table 1) and an increased incidence of these external defects compared with controls and historical control data in the main study (Table 3a). Both cleft lip and cleft palate were observed in all foetuses with orofacial clefts (Table 1 and 3a, Figure 2b), which is of some interest since previously only cleft lip has been reported (5). Visceral examination revealed both a biologically and a statistically significant increased incidence of cardiovascular defects (ventricular septum defects and great vessel abnormalities, Figure 2a, Table 3a). Digital defects were the only external visible malformations after dosing on GD 13 (Table 3b). The incidence of digital defects was statistically significantly increased in the almokalant and almokalant 1 OTC groups in the main study compared with control.

Table 2b. Effects of OTC and PBN on almokalant pregnancy outcome data when treated on GD 13 Treatment GD 13 (ALM: 25mmol/kg) No. of pregnancies No. of litters No. of live foetuses Mean foetal weight, g, (SD) Embryonic/foetal death, % (No. of affected litters) c

Statistically different from the control.

Control 9 9 116 5.2 (0.4) 1.7 (2)

ALM 10 10 107 5.1 (0.4) 8.5c (7)

ALM1OTC

ALM1PBN

9 9 90 5.2 (0.5) 16.7 (5)

9 9 103 4.9c (0.5) 5.5 (5)

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Table 3a. Effect of OTC and PBN on almokalant teratogenicity when treated on GD 11 Treatment GD 11 (ALM: 125 mmol/kg)

Control

No. of litters No. of foetuses examined externally No. of foetuses examined internally External defects Orofacial clefts Short tail Kinked tail Syndactyly Total number of external defects No. of affected foetuses (%) No. of affected litters (%) Statistical evaluation Cardiovascular defects Septum defects Vessel defects Total number of cardiovascular defects No. of affected foetuses (%) No. of affected litters (%) Statistical evaluation

10 121 59

Pooled ALM

ALM 9 85 42

0 0 0 0 0 0 0 –

ALM1OTC

15 134 –

1 9 1 0 11 9 (11) 3 (33) ns

ALM1PBN

7 73 36

7 25 6 0 38 29 (22) 9 (60) c

3 6 0 1 10 10 (14) 5 (71) c

Historical controls

8 96 48

127 1609 466

0 0 0 0 0 0 0

1 0 0 0

apool

0 0 0

7 2 9

2 1 3

0 1 1

0 0 –

7 (17) 5 (56) c

3 (8.3) 3 (43) c

1 (2.1) 1 (13) a

0 0

The statistical evaluation was based on the litter as experimental unit (no. of defects in a litter/no. of foetuses in that litter). a Significantly different from ALM only, apool significantly different from animals treated with ALM only, pooled from the pilot and main studies. c Significantly different from controls. ns 5 Not significant.

Effects of pretreatment with PBN Both on Days 11 and 13, pretreatment with PBN resulted in a decrease in the incidence of almokalantinduced external and cardiovascular malformations (Table 3a and 3b). No foetuses with orofacial clefts, ventricular septum defects, tail defects, or distal digital defects were observed in almokalant-treated dams after pretreatment with PBN. The only observed malformation in the PBN-pretreated dams was a vessel defect (malpo-

sition of the aortic arch) in one foetus, treated on GD 11 (Table 3). PBN pretreatment significantly decreased almokalant-induced internal and external (if results from the pilot and the main studies were combined) defects on GD 11 and external defects on GD 13. Effects of pretreatment with OTC Pretreatment with OTC on GD 11 reduced the incidence of septal defects. On GD 13, there was a

Table 3b. Effects of OTC and PBN teratogenicity when treated on GD 13 Treatment GD 13 (ALM: 25 mmol/kg) No. of litters No. of foetuses examined externally No. of foetuses examined internally External defects Brachydactyly forepaw Oligodactyly forepaw Syndactyly forepaw Total number of external defects No. of affected foetuses (%) No. of affected litters (%) Statistical evaluation Cardiovascular defects Vessel defects Total number of cardiovascular defects No. of affected foetuses (%) No. of affected litters (%) Statistical evaluation

Control 9 116 58 0 0 0 0 0 0 – 0 0 0 0 –

ALM 10 107 55 16 1 1 18 14 (13) 5 (50) c 2 2 1 (1.8) 1 (10) ns

ALM1PBN

Historical controls

9 103 52

127 1609 466

8 0 1 9 5 (5.6) 4 (44) c

0 0 0 0 0 0 a

0 0 0

1 1

0 0

0

ALM1OTC 9 90 45

1 (2.2) 1 (11) ns

0 0 ns

The statistical evaluation was based on the litter as experimental unit (no. of defects in a litter/no. of foetuses in that litter). a Significantly different from ALM only. c Significantly different from controls. ns 5 Not significant.

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Fig. 2. Almokalant-induced intra-ventricular septum defect (a) and cleft lip and palate (b).

reduction in digital defects, compared with dams given almokalant only. However, pretreatment with OTC did not reduce almokalant-induced orofacial clefts or tail defects. The statistical evaluation showed that OTC did not significantly alter the incidence of almokalant-induced internal or external malformations on GD 11 or 13. DISCUSSION In the present study, the class III antiarrhythmic drug almokalant caused phase-specific malformations in the same manner as in a previous study (5). Orofacial clefts and tail defects were observed after dosing on GD 11 while administration on GD 13 resulted in digital defects. Also, as in previous investigations, an increased incidence of embryonic death was observed in all groups treated with almokalant. Published studies with class III antiarrhythmic drugs (2–6,8) suggest that the embryonic death caused by almokalant and drugs like dofetilide, ibutilide, and L-691,121 is a consequence of embryonic circulatory collapse due to severe bradycardia/permanent

cardiac arrest. The results of this study support this assumption, since pretreatment with PBN or OTC did not significantly decrease the incidence of embryonic death. Also, the decreased foetal weights in this and in previously mentioned studies with class III antiarrhythmics may be directly related to hypoxia (due to bradycardia) in embryos surviving until term. The result that pretreatment with PBN completely prevented induction of externally visible malformations (orofacial clefts and digital and tail defects) by almokalant indicates that these malformations are a consequence of reoxygenation damage rather than hypoxia. The surviving foetuses at term most likely had been exposed to transient episodes of hypoxia followed by reoxygenation and generation of ROS as a result of almokalant-induced episodes of arrhythmias/temporary cardiac arrest. During the susceptible period for almokalant teratogenicity, GD 10 to 14 in the rat, the embryonic heart dose-dependently reacts with arrhythmia and temporary cardiac arrest in response to exposure to almokalant (5,8). Reactive oxygen species (ROS) also

ROS-induced teratogenicity ● K. JONSSON

have been identified in embryonic tissues after episodes of hypoxia followed by normoxia. In a study by Fantel et al., superoxide anion radicals generated in mitochondria were detected in the embryonic tissues corresponding to the distal parts of the digits after two transient 30-min episodes of hypoxia separated by normoxia in GD 14 rat embryos cultured in vitro (20). Induction of a transient episode of hypoxia by clamping of uterine vessels for 30 to 45 min on GD 14 in the pregnant rat in vivo results in reduced size of digits in the foetus at term (10). In similarity to almokalant, episodes of transient hypoxia, both in vivo (10) and in vitro (20), have also been shown to induce identical early changes in the embryo. These changes were located in the same areas (orofacial and distal part of phalanges) as those deformed in the late foetus. This is the first study reporting cardiovascular defects (ventricular septum defects and malformations of great vessels) with almokalant, but such defects have previously been reported for other class III antiarrhythmics like L-691,121 (3) and ibutilide (6) as well as cardiovascular-active calcium antagonists exerting effects on the L-type of the calcium channel (21–23). Such calcium antagonists have also been reported to cause bradycardia in embryos during the sensitive period for induction of cardiovascular malformations (21,22). Both the Ikr channel and L-type of the Ca11receptor are expressed in the embryo during the susceptible period for induction of malformations and have been proposed to be of major importance in the early stages of development of the mammalian cardiac electrical system (24). Pretreatment with PBN prevented almokalant-induced septum defects. This observation indicates that the septum defects may be associated with generation of ROS in the same way as the orofacial, tail, and digital defects. The only malformation detected in the group pretreated with PBN was a great vessel defect (malposition of the aortic arch) on GD 11. Even if this finding does not exclude hypoxia–reoxygenation damage, it may suggest another mechanism as an explanation of abnormalities of great vessels. In view of this, it is of great interest to note that abnormalities of great vessels have been induced experimentally by modifying normal blood flow patterns (25,26) and by production of episodes of embryonic bradycardia, ventricular arrhythmia, and temporary cardiac arrest (27). A wide variation of vessel defects, including malposition of great vessels, can be induced by selectively increasing, decreasing, or misdirecting blood flow in the developing cardiovascular system by mechanical methods or cardioactive substances (28,29). Lack of sufficient blood flow and pressure changes have been related to induction of abnormally absent vessels (19). As discussed in a previous work, the Ikr channel

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seems to be expressed in the embryonic heart of the rat from GD 9 to GD 14 (5). Before GD 9 and at GD 15 and onward, the embryos are insensitive to the teratogenic and embryotoxic effects of almokalant as well as other class III antiarrhythmics. These results suggest a “downregulation” in favour of other repolarising currents with increasing age. The dose of almokalant needed to cause embryotoxicity/teratogenicity tended to decrease from Day 10 to Day 13. Available data indicate that this may reflect the progressive increase in embryonic susceptibility to hypoxia (as shown in clamping experiments during this period; 30) rather than any increase in the sensitivity of the Ikr-channel. In fact, the gestational Day 13 heart seems to be less susceptible to drug-induced bradycardia than the Day 11 heart. This has been shown for both almokalant and dofetilide (4,5). The fact that PBN prevented almokalant-induced malformations to a much higher extent than OTC may have several explanations. PBN is a lipophilic radical spin-trapping agent that rapidly penetrates cell membranes and is most likely distributed both intra- and extracellularly in embryonic tissues. PBN thus seems to have the capacity to directly capture generated reactive oxygen species both within the cell and in the circulation. In contrast, OTC acts indirectly by promoting glutathione synthesis by functioning as an intracellular delivery system (31). Glutathione is the major intracellular reductant present in developing embryonic tissues (32). Recent studies suggest that glutathione may also be released into the circulation, presumably as a defence against ROS during hypoxia/reoxygenation (33). However, the synthesis of glutathione (via OTC) and the release of extracellular glutathione as a response to generated ROS is probably a slower process with a lower capacity for detoxification when compared with immediate trapping of ROS by PBN, which is available in high concentrations both intra- and extracellularly. An increased protective effect by PBN, compared with OTC, was also seen in a study by Zimmerman (18). In that study, PBN, but not OTC, completely prevented cocaine-induced embryonic vascular disruption, which was thought to be mediated by ROS. As mentioned in the introduction, there is substantial evidence that drugs with class III antiarrhythmic activity, especially those that inhibit a specific component (Ikr) of the time-dependent delayed rectifier K1 current (Ik), have teratogenic and embryolethal properties that are common for all drugs in this class. In view of this, it is very interesting that the human teratogen phenytoin has been shown in a recent study to inhibit Ikr at therapeutic concentrations, and that this pharmacologic property has been reported as the basis for phenytoin anticonvulsant action (34). Phenytoin, as well as some other anticonvulsants (e.g. trimethadione), has

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been associated with similar foetal adverse effects as class III antiarrhythmics, consisting mainly of a) orofacial clefts, b) cardiovascular defects, c) distal digital hypoplasia, and d) embryonic/foetal growth retardation in numerous clinical reports and animal studies (35–37). All these manifestations have also been recreated in animal models using nonepileptic animals. Malformations caused by phenytoin have been shown to be preceded by the same early pathologic changes (vascular disruption and haemorrhage), both in the orofacial (38) and the limb regions (39,40), as seen with class III antiarrhythmics and temporary clamping of uterine vessels. Phenytoin, trimethadione, and carbamazepine, which all are known to, at least partly, exert their pharmacologic effects on K1 and/or Ca11 channels (41), have recently also been shown to have the capacity to cause embryonic bradycardia and arrhythmias in the same way as class III antiarrhythmics (42,43). These adverse effects on the embryonic heart by phenytoin and the active metabolite of trimethadione could be induced at concentrations comparable with those in the upper range of the human therapeutic interval. PBN has also been reported to decrease phenytoin teratogenicity (17). In view of the great similarities in teratogenic action between the established human teratogen phenytoin and class III antiarrhythmic drugs, it is suggested that one consider all class III antiarrhythmics as potential human teratogens. In conclusion, the results of this study support the idea that teratogenicity and embryotoxicity of almokalant (and probably all class III antiarrhythmics) are initiated via pharmacologically induced embryonic bradycardia and arrhythmia/cardiac arrest leading to: 1. circulatory collapse and embryonic death due to severe hypoxia 2. hypoxia, resulting in growth retardation 3. episodes of severe transient hypoxia followed by reoxygenation and generation of ROS causing orofacial clefts, ventricular septum defects, and distal digital defects 4. alterations in embryonic blood flow and pressure leading to great vessel defects. Furthermore, in view of the similarities between class III antiarrhythmics (almokalant and dofetilide) and the human teratogen phenytoin (in pharmacologic effects, phase specificity, pattern of malformations, and early changes preceding the defects), we propose that class III antiarrhythmics are considered as potential human teratogens. REFERENCES 1. Carmeliet E. Use-dependent block and use-dependent unblock of the delayed rectifier K1 current by almokalant in rabbit ventricular myocytes. Circulation Research. 1993;73:857– 68.

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