Fetal safety of erythromycin. An update of Swedish data.

PURPOSE: In previous studies from the Swedish Medical Birth Register, a possible association between erythromycin therapy and an increased risk for cardiovascular defects was found. Other studies using different methodology have not verified this observation. The finding resulted in a warning for the use of erythromycin in early pregnancy, followed by a marked decline in such use. The present study was conducted to follow up on the previous observations and to find methodological explanations for the variation in results in these different published studies. METHODS: Data on Swedish women who gave birth during the period 1996-2011 were studied using the drug information in the Medical Birth Registry, obtained from midwife interviews conducted toward the end of the first trimester. Data on the presence of congenital malformations were ascertained from national health registers. Odds ratios were then determined using the Mantel-Haenszel methodology. RESULTS: A persistent association was seen between erythromycin use and the occurrence of cardiovascular defects, with a risk estimate of 1.70 (95 % confidence interval (CI): 1.26-2.39), of similar strength during the first and last 8 years of observation. We discussed the contradictory findings of the other published studies and pointed out possible methodological problems that may explain the absence of an effect in studies based on prescription registers. CONCLUSIONS: Our study verified an association between early pregnancy erythromycin use and infant cardiovascular defects; most defects were mild. The cause of this association is unclear.

Maternal toxicity.

Although demonstration of some degree of maternal toxicity is required in regulatory developmental toxicology studies, marked maternal toxicity may be a confounding factor in data interpretation. Reduction in maternal body weight gain is the far most frequently used endpoint of toxicity, but alternative endpoints, like organ toxicity or exaggerated pharmacological response, can also be taken into consideration. The following conclusions are based on literature data and discussions at maternal toxicity workshops attended by representatives from regulatory agencies, academia, and industry: (1) Available results do not support that maternal toxicity (defined as clinical signs, decreased body weight gain or absolute body weight loss of up to 15% in rats or 7% in rabbits) can be used to explain the occurrence of major malformations. (2) There is clear evidence that substantial reductions in maternal weight gain (or absolute weight loss) are linked with other manifestations of developmental toxicity. Among these can be mentioned decreased fetal weight, and skeletal anomalies (e.g., wavy ribs) in rats and decreased fetal weights, post implantation loss, abortions, and some skeletal anomalies in rabbits. (3) There are several examples of misinterpretation among companies, where it was incorrectly expected that regulatory authorities would not label chemicals/drugs as "teratogens/developmental toxicants" because embryo fetal adverse effects were only observed at doses also causing signs of maternal toxicity. (4) Similarly, even if mechanistic studies indicate that a substance causes developmental toxicity via exaggerated pharmacological effects in the mother, such a mechanism does not automatically negate the observed fetal adverse effects.From a regulatory perspective, an observed developmental toxic finding is considered to be of potential human relevance (even if it is mediated via maternal pharmacological effects or occur at doses causing signs of maternal toxicity) unless the company can provide appropriate mechanistic and/or other convincing evidence to the contrary.

ILSI/HESI maternal toxicity workshop summary: maternal toxicity and its impact on study design and data interpretation.

Workshops on maternal toxicity were held at the annual Society of Toxicology, Teratology Society, and European Teratology Society meetings in 2009. Speakers presented background information prior to a general discussion on this topic. The following recommendations/options are based on the outcome of the discussions at the workshops: 1. A comprehensive evaluation of all available data from general toxicity studies, range-finding Developmental and Reproductive Toxicology (DART) studies, class effects, structure-activity relationships, exposure studies, etc. is essential for appropriate dose selection for definitive DART studies. The intent is to avoid marked maternal toxicity leading to mortality or decreased body weight gains of greater than 20% for prolonged periods. (a) Evaluate alternative endpoints for dose selection and data interpretation (e.g., target tissue effects and pharmacology) for biotherapeutics. (B) Evaluate additional maternal parameters based on effects and/or target organs observed in short-term (e.g., 2- or 4-week) general toxicity studies. 2. Evaluate all available data to determine a cause-effect relationship for developmental toxicity. (a) Conduct a pair-feeding/pair-watering study as a follow-up. (b) Evaluate individual data demonstrating maternal toxicity in the mother with adverse embryo-fetal outcomes in the litter associated with the affected mother. (c) Conduct single-dose studies at increasing doses as a complement to conventional embryo-fetal toxicity studies for certain classes of compounds that affect the hERG channel. 3. Support statements that embryo-fetal effects are caused by maternal toxicity and/or exaggerated pharmacology, especially for malformations. (a) Provide mechanistic or other supporting data. (b) Establish the relevance of the DART findings in animals for human exposures. Birth Defects Res (Part B) 92:36-51, 2010. © 2011 Wiley-Liss, Inc.

New proposals for testing drugs with IKr-blocking activity to determine their teratogenic potential.

Drugs blocking the potassium current IKr, either as an intended pharmacologic effect (eg antiarrhythmics dofetilide and almokalant) or as an unwanted side-effect (eg antihistamine astemizole, propulsive drug cisapride, antidepressive drugs and macrolide antibiotics) are potential human teratogens. It is the contention of this paper that the existing repeat dose regimen used in teratology studies to fulfil regulatory requirements, does not properly identify the teratogenic risk of these drugs. Results from conventional studies for dofetilide and almokalant showed high rates of postimplantation embryonic death with few malformed fetuses. For astemizole and cisapride only embryonic death was seen. These latter results were not considered important because they occurred either in the presence of maternal toxicity and/or at high doses. Subsequent studies have shown that IKr-blockers are highly teratogenic when administered on single gestational days (GD) during a sensitive period of rat pregnancy (GD 10-14) when they induce a high incidence of stage-specific malformations. This teratogenic activity of astemizole and cisapride was missed in the original teratology studies. Mechanistically IKr-blockers cause bradycardia and arrhythmia of the embryonic heart and while an embryo may be able to survive a single day exposure to a teratogenic dose, repeat dosing often leads to death of the embryo. With this review we suggest that new drugs identified at the preclinical stage of development as having IKr-blocking properties, should undergo more comprehensive teratology testing including single GD dosing and studies using embryo culture. This would further help identify and characterise their teratogenic potential.

Polytherapy with hERG-blocking antiepileptic drugs: increased risk for embryonic cardiac arrhythmia and teratogenicity.

BACKGROUND: The antiepileptic drugs (AEDs) phenytoin, phenobarbital, dimethadione, and carbamazepine cause a similar pattern of malformations in humans, with an increased risk after polytherapy. The teratogenicity has been linked to cardiac rhythm disturbances and hypoxic damage as a consequence of their common potential to inhibit a specific potassium ion current (IKr). The IKr is of major importance for embryonic cardiac repolarization and rhythm regulation. This study investigated whether these AEDs cause irregular rhythm and if various combinations of AEDs result in higher arrhythmia risk than exposure to a single AED. METHODS: The effects on heart rhythm of a single AED (monotherapy), and of various combinations (polytherapy) of AEDs, in gestational day 10 C57BL mouse embryos in culture were analyzed and graphically illustrated during a 25 s recording with a digitalization technique. RESULTS: All of the studied AEDs caused increased intervals between heartbeats (resulting in bradycardia) and large variations in the interval between heartbeats (resulting in irregular rhythm) in a concentration-dependent manner in cultured mouse embryos. Dimethadione caused irregular rhythm at concentrations within and phenytoin slightly above the therapeutic ranges. Polytherapy resulted in more substantial prolongation of the mean interval between heartbeats (>60 ms) than monotherapy at clinically relevant concentrations. CONCLUSIONS: The results suggest that polytherapy more than monotherapy causes substantial prolongation of the cardiac repolarization, a marker associated with high risk of developing irregular rhythm during longer exposure periods (days to months). This supports the idea that the increased risk for malformations following polytherapy is linked to an increased risk for cardiac rhythm disturbances.

Embryonic cardiac arrhythmia and generation of reactive oxygen species: common teratogenic mechanism for IKr blocking drugs.

In the adult organism, it is well established that hypoxia followed by reperfusion may be fatal and result in generation of reactive oxygen species (ROS) and subsequent tissue damage. There is also considerable evidence that temporary decrease or interruption in oxygen supply to the embryo and ROS generation during reperfusion result in tissue damage in embryonic tissues. A wide spectrum of different malformations by transient embryonic hypoxia could be produced, depending on the duration, extent, and timing of the hypoxic event. It is the contention of this paper that drugs that block the potassium channel IKr, either as an intended pharmacologic effect or as an unwanted side-effect, are potentially teratogenic by a common ROS related mechanism. Drugs blocking the IKr channel, such as almokalant, dofetilide, phenytoin, cisapride and astemizole, do all produce a similar pattern of hypoxia-related malformations. Mechanistic studies show that the malformations are preceded by embryonic cardiac arrhythmia and periods of hypoxia/reoxygenation in embryonic tissues. Pretreatment or simultaneous treatment with radical scavengers with capacity to capture ROS, markedly decrease the teratogenicity of different IKr blocking drugs. A second aim of this review is to demonstrate that the conventional design of teratology studies is not optimal to detect malformations caused by IKr blocking drugs. Repeated high doses result in high incidences of embryonic death due embryonic cardiac arrhythmia, thus masking their teratogenic potential. Instead, single dosing on specific days is proposed to be a better way to characterize the teratogenic potential of Ikr blocking drugs.

Increased susceptibility to phenytoin teratogenicity: excessive generation of reactive oxygen species or impaired antioxidant defense?

Phenytoin is a human and animal teratogen. Accumulating evidence suggests that the teratogenicity is associated with a potential of phenytoin to cause embryonic cardiac arrhythmia and resultant generation of toxic reactive oxygen species via hypoxia-reoxygenation mechanisms. The A/J mouse is more susceptible to phenytoin teratogenicity than other mouse strains. The aim of this study was to investigate whether A/J mice have other antioxidant enzyme activities than C57BL/6J and CD-1 mice. Also, strain differences in phenytoin effects on embryonic heart rate and rhythm were determined. Another objective was to determine whether a spin trapping agent with capacity to capture reactive oxygen species alter the developmental toxicity of phenytoin. Treatment with this agent resulted in a marked decrease in phenytoin teratogenicity, which supports the idea that reactive oxygen species are important mediators for the teratogenic action of phenytoin. The A/J mice embryos were most susceptible to the adverse cardiac effects of phenytoin and had the highest activity of superoxide dismutase and glutathione peroxidase, while the activity of catalase was the same in embryos of the three different strains. The high activities of antioxidant enzymes in the A/J stain indicate that the sensitivity to develop malformations is caused by excessive arrhythmia-related generation of reactive oxygen species rather than impaired antioxidant defense.

Is erythromycin therapy teratogenic in humans?

The possible teratogenic effect of erythromycin therapy, noted previously, was studied. Women who had taken erythromycin or penicillin V in early pregnancy and their infants were studied, using the Swedish Medical Birth Register where information on drug use during pregnancy was recorded based on interviews in early pregnancy. The risk for any congenital malformation after erythromycin therapy (but not after penicillin V therapy) was increased (odds ratio 1.24, 95% confidence interval: 1.01-1.51) and this was due to an effect on cardiovascular malformations (odds ratio 1.92, 95% CI: 1.37-2.68). There was also an indicated increased risk for pyloric stenosis (risk ratio 3.0, 95% CI: 1.1-8.5 after exposure in early pregnancy). Various explanations to the finding are discussed, one of them linked to the fact that erythromycin inhibits a specific cardiac potassium channel (IKr) which seems to play a major role in cardiac rhythm regulation in the early embryo. Potent blocking drugs cause as a class effect cardiac defects in animal experiments.

Phenytoin teratogenicity: hypoxia marker and effects on embryonic heart rhythm suggest an hERG-related mechanism.

BACKGROUND: The antiepileptic drug phenytoin (PHT) is a human and animal teratogen. The teratogenicity has been linked to PHT-induced embryonic cardiac arrhythmia and hypoxic damage during a period when regulation of embryonic heart rhythm is highly dependent on a specific K(+) ion current (I(Kr)). PHT has been shown to inhibit I(Kr). The aims of this study were to investigate whether teratogenic doses cause embryonic hypoxia during and after the I(Kr) susceptible period and to further characterize PHT effects on embryonic heart rhythm. METHODS: Pregnant C57BL mice were administered the hypoxia marker pimonidazole followed by PHT or saline (controls) on GD 10 or GD 15. The embryos were fixed and sectioned, and the immunostained sections were analyzed with a computer assisted image analysis. Effects of PHT (0-250 microM) on heart rhythm in GD 10 embryos cultured in vitro were videotaped and then analyzed by using a digitalization technique. RESULTS: PHT dose-dependently increased the hypoxia staining (6- and 11-fold after maternal dosing of 100 and 150 mg/kg, respectively) during the period I(Kr) is expressed and functional (GD 10). In contrast, there were no differences between the PHT doses in hypoxia staining, and much less pronounced hypoxia after this period (GD 15). With increasing PHT concentrations, increased length of the interval (bradycardia) and large variations in length between individual heartbeats (arrhythmia) were recorded. CONCLUSIONS: PHT induced bradycardia/arrhythmia and severe embryonic hypoxia during the I(Kr) susceptible period, supporting the idea of an I(Kr)-arrhythmia-hypoxia-related teratogenic mechanism.

Effects of the antiepileptic drugs lamotrigine, topiramate and gabapentin on hERG potassium currents.

Drugs that inhibit the cardiac rapid delayed rectifier potassium ion current (I(Kr)) can be proarrhythmic and their clinical use has been associated with sudden unexpected death (SUD) due to cardiac arrhythmia. SUD is 20-40 times more common among people with epilepsy than in the general population and case-control studies have identified polytherapy with antiepileptic drugs (AEDs) as a risk factor. In a previous study, it was described that the old AEDs phenytoin and phenobarbital had the potential to inhibit the I(Kr) channel and it was suggested that this could contribute to the increased risk for SUD in patients with epilepsy. In this study, we have investigated the I(Kr) blocking potential of some more recently introduced AEDs, lamotrigine (LTG), topiramate (TPM) and gapapentin (GBP). The whole cell patch-clamp recording technique was used to study the effects on I(Kr) channels expressed by the human ether-a-go-go related gene (hERG) stably expressed in human embryo kidney (HEK) 293 cells. Tail currents, which are purely related to hERG currents, were blocked with IC50 and IC20 (the concentrations when 50% and 20% inhibition was obtained compared to control values) of 229 and 21 microM, respectively, for LTG. A 40% inhibition of tail currents was obtained at GBP concentrations of 100 mM and a 20% inhibition at 54 mM. A 35% inhibition of tail currents was obtained at TPM concentrations of 1000 microM and a 20% inhibition at 87 microM, respectively. Collective data show that drugs with the same margins (ratio hERG IC50/unbound therapeutic concentration) as LTG, may have arrhythmogenic potential. The risk for arrhythmia may be clinically significant in the presence of predisposing factors such as seizure-induced acidosis and in the case of concurrent treatment with other I(Kr) blocking drugs, or in case of pharmacokinetic drug-drug interactions resulting in excessively high concentrations of LTG.

Teratogenicity by the hERG potassium channel blocking drug almokalant: use of hypoxia marker gives evidence for a hypoxia-related mechanism mediated via embryonic arrhythmia.

The rapid component of the delayed rectifying potassium ion current (IKr), plays an important role in cardiac repolarization. In rats, potent IKr channel blocking drugs cause similar stage-specific malformations (such as orofacial clefts and digital reductions) on gestational days (GDs) 10-14 as after periods of embryonic oxygen deprivation (hypoxia). The idea of a hypoxia-related teratogenic mechanism is supported by studies using rat embryos cultured in vitro. These studies show that the embryonic heart reacts with concentration-dependent bradycardia, arrhythmia, and cardiac arrest when exposed to IKr blockers on GDs 10-14. The main purpose of this study was to investigate whether previously shown teratogenic doses on GD 11 and 13 of the selective IKr blocker almokalant (ALM) induce hypoxia in rat embryos in vivo by using the hypoxia marker pimonidazole hydrochloride (PIM). Rats were orally dosed with almokalant or tap water on GD 11 (150 micromol/kg), 13 (50 micromol/kg), or 16 (800 micromol/kg), followed by PIM intravenously 30 min later. Two hours after the PIM dose, the embryonic heart activity was videotaped and analysed, and the embryos were fixed, sectioned, and immunostained. Computer-assisted image analysis showed a two- and threefold increase in hypoxia staining in embryos exposed to teratogenic doses of ALM on GDs 11 and 13. Embryonic arrhythmia was observed in almokalant groups on these GDs, but not in controls. In contrast, dosing on GD 16, with a much higher dose (800 micromol/kg), caused neither hypoxia nor any effects on heart rhythm. The results support the IKr-related arrhythmia-hypoxia hypothesis, by showing that the potent IKr-blocking drug, almokalant, (1) causes severe embryonic hypoxia and arrhythmia at stages (GDs 11 and 13) when developmental toxicity could be induced and IKr is functional and (2) does not cause hypoxia or affect heart rhythm at a developmental stage when IKr is suppressed (GD 16) and potent IKr blockers do not induce developmental toxicity.

Phenytoin and phenobarbital inhibit human HERG potassium channels.

Drugs that inhibit the cardiac rapid delayed rectifier potassium ion current (IKr) channel can be proarrhythmic and their clinical use has been associated with sudden unexpected death (SUD). Since SUD is about 20 times more common among people with epilepsy than in the general population, and some data indicate that drug treatment may contribute, we tested the hypothesis that the classic antiepileptic drugs phenytoin (PHT), carbamazepine (CBZ), and phenobarbital (PB) have a potential to block IKr. The whole cell patch-clamp recording technique was used to study the effects on IKr channels expressed by the human ether-a-go-go related gene (HERG) stably expressed in Human Embryo Kidney (HEK) 293 cells. Tail currents, which are purely related to HERG, were blocked with an IC50 (the concentration when 50% inhibition was obtained compared to control values) of 240 microM for PHT and 3 mM for PB. A 20% inhibition of tail currents was obtained at CBZ concentrations of 250 and 500 microM. Collective data show that drugs with the same margins (ratio HERG IC50/unbound therapeutic concentration), as PHT and PB, may have arrhythmogenic potential, especially when used in predisposed patients and in the case of drug-drug interactions. SUD in epilepsy is generally a seizure-related phenomenon. However, our data suggest that PHT and PB may play a contributing role, perhaps by making some patients more vulnerable to the cardiovascular depression induced by seizures.

Teratogenicity of the I(Kr)-blocker cisapride: relation to embryonic cardiac arrhythmia.

Cisapride and mosapride are structurally and pharmacologically related prokinetic agents. In contrast to mosapride, cisapride causes embryonic lethality in teratology studies, and has been related to fatal cardiac arrhythmia in the adult. The arrhythmogenic potential of cisapride is linked to its potential to inhibit a specific ion channel (I(Kr)) as a side effect. Mosapride lacks I(Kr)-blocking properties. The aims of this study were (1) to compare the effects of cisapride and mosapride on embryonic heart rhythm in vitro and (2) to investigate if cisapride in vivo, has potential to induce hypoxia-related teratogenic effects as has been shown for selective I(Kr)-blockers. Cisapride induced severe embryonic bradycardia (approximately 60% decrease), and arrhythmia in 94% of the cultured rat embryos at 1000 ng/ml. Mosapride did not induce any bradycardia or arrhythmia up to 2000 ng/ml. In vivo, single dose administration of cisapride to rats on gestational day (GD) 13 caused digital reductions (8/108 fetuses, 4/9 litters) at 75 mg/kg and high incidence of embryonic death (55-82%) at 100-200 mg/kg. Identical developmental toxic effects have been described after temporary interruption of oxygen supply, and after single dose administration of selective I(Kr)-blockers, on the same GD. The results support the idea that all potent I(Kr)-blocking agents have the potential to cause embryolethality and teratogenicity, and that the adverse effects are mediated via hypoxic episodes due to embryonic arrhythmia.

Embryonic arrhythmia by inhibition of HERG channels: a common hypoxia-related teratogenic mechanism for antiepileptic drugs?

PURPOSE: There is evidence that drug-induced embryonic arrhythmia initiates phenytoin (PHT) teratogenicity. The arrhythmia, which links to the potential of PHT to inhibit a specific potassium channel (Ikr), may result in episodes of embryonic ischemia and generation of reactive oxygen species (ROS) at reperfusion. This study sought to determine whether the proposed mechanism might be relevant for the teratogenic antiepileptic drug trimethadione (TMO). METHODS: Effects on embryonic heart rhythm during various stages of organogenesis were examined in CD-1 mice after maternal administration (125-1,000 mg/kg) of dimethadione (DMO), the pharmacologically active metabolite of TMO. Palatal development was examined after administration of a teratogenic dose of DMO and after simultaneous treatment with DMO and a ROS-capturing agent (alpha-phenyl-N-tert-butyl-nitrone; PBN). The Ikr blocking potentials of TMO and DMO were investigated in HERG-transfected cells by using voltage patch-clamping tests. RESULTS: DMO caused stage-specific (gestation days 9-13 only) and dose-dependent embryonic bradycardia and arrhythmia at clinically relevant maternal plasma concentrations (3-11 mM). Hemorrhage in the nasopharyngeal part of the embryonic palate (within 24 h) preceded cleft palate in fetuses at term. Simultaneous treatment with PBN significantly reduced the incidence of DMO-induced cleft palate, from 40 to 13%. Voltage patch-clamping studies showed that particularly DMO (70% inhibition), but also TMO, had Ikr blocking potential at clinically relevant concentrations. CONCLUSIONS: TMO teratogenicity, in the same way as previously shown for PHT, was associated with Ikr-mediated episodes of embryonic cardiac arrhythmia and hypoxia/reoxygenation damage.