Childs Nerv Syst (2016) 32:1715–1719
`DOI 10.1007/s00381-016-3181-4
`
`ORIGINAL PAPER
`
`Effects of lacosamide Ba novel antiepileptic drug^ in the early
`stages of chicken embryo development
`
`Mesut Mete 1 & Beyhan Gurcu 2 & Fatih Collu 2 & Ulkun Unlu Unsal 1 &
`Yusuf Kurtulus Duransoy 1 & Mehmet Ibrahim Tuglu 3 & Mehmet Selcuki 1
`
`Received: 24 June 2016 /Accepted: 5 July 2016 /Published online: 29 July 2016
`# Springer-Verlag Berlin Heidelberg 2016
`
`Abstract
`Introduction Antiepileptic drugs (AEDs) are teratogens
`and confer a risk of congenital malformation. The estimat-
`ed prevalence of major congenital malformations such as
`cardiac defects, facial clefts, hypospadias, and neural tube
`defects in epileptic women is 4–10 %, which represents a
`two- to fourfold increase in pregnant women compared to
`the general population. However, there are no clear data
`for newer drugs. Lacosamide (LCM), a novel AED, is the
`first of the third-generation AEDs to be approved as ad-
`junctive therapy for the treatment of partial-onset seizures.
`There are no data on the pharmacokinetics of LCM during
`pregnancy, and only some published data have reported
`on its effects during pregnancy.
`Methods In this study, three different doses of LCM (0.12,
`0.5, and 1.60 mg in 0.18 mL) were applied under the embry-
`onic disks of specific pathogen-free Leghorn chicken embryos
`after a 30-h incubation. Incubation was continued for 80 h, at
`which time all embryos were evaluated macroscopically and
`microscopically.
`Results There was growth retardation in all of the LCM-treated
`groups. Major malformations increased in a dose-dependent
`manner and were mostly observed in the supratherapeutic group.
`
`* Mesut Mete
`dr.mmete@hotmail.com
`
`1 Department Neurosurgery, Celal Bayar University School of
`Medicine, Yelken Evleri Sitesi B Blok k:5 d:16 Güzelyurt,
`Manisa, Turkey
`2 Department of Biology, Faculty of Science and Letters Zoology
`Section, Celal Bayar University, Manisa, Turkey
`3 Department of Histology-Embryology, Celal Bayar University
`School of Medicine, Manisa, Turkey
`
`Conclusion Based on our data, LCM may cause growth retar-
`dation or major congenital malformations. Nevertheless, more
`extensive investigations of its reliability are needed.
`
`Keywords Anti-epileptic . Chick embryo . Lacosamide .
`Malformation
`
`Introduction
`
`In the USA, over 1 million women of childbearing age have
`epilepsy and the continued use of antiepileptic drugs (AEDs)
`is recommended to reduce the maternal and fetal trauma asso-
`ciated with seizures [1]. However, prenatal exposure to AEDs
`can cause major congenital malformations, growth retarda-
`tion, and intelligence deficits in the developing fetus [1–3].
`The estimated prevalence of major congenital malformations,
`such as cardiac defects, facial clefts, hypospadias, and neural
`tube defects, in the children of epileptic women is 4–10 %,
`which represents a two- to fourfold increase compared to the
`general population [3]. In this group of patients, the treatment
`target with mono- or polytherapy should be optimal seizure
`control with minimum fetal exposure to AEDs.
`The risks of the AEDs valproic acid, phenytoin, phenobarbi-
`tal, levetiracetam, oxcarbazepine, and topiramate have been re-
`ported [1–4]. The North American Antiepileptic Drug
`Pregnancy Registry assessed the risk of major congenital mal-
`formation with AED exposure as 9.3 % for valproate, 5.5 % for
`phenobarbital, 4.2 % for topiramate, 3.0 % for carbamazepine,
`2.9 % for phenytoin, 2.4 % for levetiracetam, and 2.0 % for
`lamotrigine [4]. However, there are limited data for newer drugs.
`Lacosamide (LCM), a novel third-generation AED, was
`first approved as adjunctive therapy for the treatment of
`partial-onset seizures in 2008 and for monotherapy in 2014
`[5]. It has been suggested that LCM is a safe, effective, well-
`
`ARGENTUM Exhibit 1149
` Argentum Pharmaceuticals LLC v. Research Corporation Technologies, Inc.
`IPR2016-00204
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`Childs Nerv Syst (2016) 32:1715–1719
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`Table 1 Numbers and percentages of normal and abnormal embryos after incubation with physiological saline and varying amounts of LCM
`
`Groups
`
`Embryos n (%)
`
`Lethal n (%)
`
`Observed n (%)
`
`Growth retardation n (%)
`
`Malformation n (%)
`
`Sham
`Subtherapeutic
`Therapeutic
`Supratherapeutic
`
`7 (100 %)
`12 (100 %)
`11 (100 %)
`12 (100 %)
`
`0 (0)
`4 (33 %)
`3 (27 %)
`2 (16 %)
`
`7 (100 %)
`8 (66 %)
`8 (72 %)
`10 (83 %)
`
`7 (100 %)
`7 (58 %)
`5 (45 %)
`5 (41 %)
`
`0 (0)
`1 (8 %)
`3 (27 %)
`5 (41 %)
`
`tolerated adjunctive treatment for reducing seizure frequency
`in patients with highly refractory partial seizures [6]. For sei-
`zures, its recommended daily dose is 200–600 mg [7].
`However, there are no data on the pharmacokinetics of LCM
`during pregnancy and only two reports on its effects during
`pregnancy [8, 9]. Therefore, we examined the effects of 0.12
`(subtherapeutic), 0.5 (therapeutic), and 1.6 (supratherapeutic)
`mg doses of LCM on chick embryos in ovo.
`
`Material and methods
`
`This study was conducted with the cooperation of the Histology
`Department Research Laboratory of Celal Bayar University
`Medical School. Fertilized, specific-pathogen-free Leghorn
`chicken eggs were supplied by the Republic of Turkey
`Ministry of Agriculture and Rural Affairs, Bornova Veterinary
`Control and Research Institute. All experiments were conduct-
`ed in accordance with the animal research protocol of Celal
`Bayar University Ethics Committee (no. 77.637.4335–27).
`
`Incubation and injection
`
`Forty-two eggs weighing 65 ± 5 g (mean ± SD) were incubat-
`ed at 37.5 ± 0.2 °C and 60–80 % relative humidity. Each egg
`was repositioned on its axis every 2 h. After 30 h of incuba-
`tion, each egg was opened under ×4 optical magnification
`[10–12], when at Hamburger–Hamilton stage 9 [11]. They
`were rinsed with 70 % ethanol, a piece of plastic tape was
`placed close to the egg air cavity, and a small hole was opened
`for injections. In each group, the embryonic disks were iden-
`tified and the same volume of liquid (total 180 μL) was
`injected under each disk with a 30-gauge syringe.
`
`Drug preparation
`
`Intravenous LCM solution is available in a 10-mg/mL prepa-
`ration. LCM solutions of three concentrations were prepared.
`The doses given were calculated according to the weight of the
`eggs with reference to the daily dose range used in humans. In
`the LCM-treated groups, 12, 50, or 160 μL LCM solution was
`diluted with physiological saline to a total volume of 180 μL.
`
`Groups
`
`The eggs were divided into sham (group 1, n = 7) and LCM-
`treated (n = 35) groups. The LCM-treated group was
`subdivided into three groups according to the drug dose:
`0.12 mg (subtherapeutic group, n = 12), 0.5 mg (therapeutic
`group, n = 11), and 1.6 mg (supratherapeutic group, n = 12). In
`all of the groups, the eggs were closed with sterile tape after
`injection and incubation was continued for 80 h, at which time
`the eggs were reopened and the embryos were dissected from
`the embryonic membranes with adherence to microsurgical
`rules, using the water-floating technique. Then all embryos
`were put into a 10 % formalin solution for 24 h. The embryos
`were viewed under an Olympus (SZX7) stereomicroscope.
`
`Results
`
`Embryos from the sham and treatment groups were ob-
`served and photographed macroscopica lly and
`
`Fig. 1 Normal development of chick embryo after physiological saline
`solution injection. Telencephalon (black square), diencephalon (black
`star), mesencephalon (asterisk), metencephalon (black diamond suit),
`heart (triangle-headed rightwards arrow), eye (black rightwards
`arrowhead), ALB anterior limb bud, PLB posterior limb bud
`
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`Childs Nerv Syst (2016) 32:1715–1719
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`1717
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`Fig. 2 a and b demonstrated the subtherapeutic group of embryos with
`growth retardation and major malformations after LCM injection,
`respectively. Growth retardation in brain vesicles (small telencephalon
`(black square), mesencephalon (asterisk) and metencephalon (black
`diamond suit)),shrinkage in eyes/microphthalmia (black rightwards
`
`arrowhead), anomaly in heart development (rightwards dashed arrow),
`excessive growth and expansion in blood vessels (triangle-headed right-
`wards arrow), super-twisted body (shrinkage) (black curved downwards
`and rightwards arrow), short tail (
`)
`
`microscopically. Table 1 summarizes the characteristics
`of the normal and abnormal embryos after incubation
`with varying amounts of LCM and physiological saline.
`In the sham group, no growth retardation or major congen-
`ital malformation was detected in any of the seven embryos
`(Table 1, Fig. 1). In the subtherapeutic group, 4 of 12 embryos
`died during the procedure. Of the remaining 8, 7 showed
`growth retardation and 1 had a major malformation (Table 1,
`Fig. 2). In the therapeutic group, 3 of 11 embryos died during
`the procedure. Of the remaining 8, 5 showed growth retarda-
`tion and 3 had major malformations (Table 1, Fig. 3). In the
`supratherapeutic group, 2 of 12 embryos died during the
`
`procedure. Of the remaining 10, 5 had growth retardation
`and 5 had major malformations (Table 1, Fig. 4).
`
`Discussion
`
`The pharmacological treatment of epilepsy during pregnancy
`is problematic. It is necessary to balance the potential terato-
`genic effects of AEDs on the fetus with the irreversible dam-
`age of uncontrolled epilepsy done to the mother and fetus in
`the management of epilepsy during pregnancy [10 ].
`Complications related to seizures include fetal death, poorer
`
`Fig. 3 a and b demonstrated the embryos with growth retardation and
`major malformations in therapeutic group after LCM injection
`respectively. Anencephaly (rightwards white arrow), shrinkage in
`eyes/microphthalmia (black rightwards arrowhead), abnormal heart
`
`looping (rightwards dashed arrow), excessive growth and expansion of
`blood vessels (triangle-headed rightwards arrow), reduced size of limbs
`)
`(triangle-headed rightwards arrow), short tail (
`
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`Childs Nerv Syst (2016) 32:1715–1719
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`Fig. 4 a and b demonstrated the embryos with growth retardation and
`major malformations in supratherapeutic group after LCM injection,
`respectively. Anencephaly (rightwards white arrow), shrinkage in eyes/
`microphthalmia (black rightwards arrowhead), anomaly in heart
`
`development (rightwards dashed arrow), shortening and thickening of
`the body (⤴), reduced size of limbs (triangle-headed rightwards arrow),
`reduction in vascularization (black circle), super-twisted body
`(shrinkage) (black curved downwards and rightwards arrow)
`
`cognitive development, preterm labor, and maternal injury.
`Congenital malformations (such as cardiac defects, facial
`clefts, extremity abnormality, neural tube defects) and growth
`retardation could be seen related to the teratogenic effects of
`AEDs [2, 13, 14]. Therefore, treatment requires extra care and
`the goal should be to control generalized tonic-clonic seizures
`with minimal in utero fetal AED exposure [1, 13].
`Many AEDs have been reported to have teratogenic ef-
`fects; these include carbamazepine, gabapentin, lamotrigine,
`levetiracetam, phenobarbital, phenytoin, topiramate, and
`valproate. Lamotrigine and levetiracetam appear to confer
`low risks for both anatomical and behavioral teratogenesis
`[15]. However, less is known about the teratogenic effects of
`newer AEDs, which necessitates studies in animal models due
`to the limitations inherent in human epidemiological and clin-
`ical AED studies. In humans, pregnancies exposed to AEDs in
`the first trimester are at increased risk of major congenital
`malformations. The early chick embryo model is an ideal
`model that corresponds to the first month of embryonic devel-
`opment in mammals. It is also suitable for investigating the
`effects of chemical agents on embryo development [10].
`Therefore, we observed chick embryos at 80 h of development
`after injecting LCM and physiological saline solution.
`LCM is a newer AED with a dual mode of action. It selec-
`tively enhances the slow inactivation of voltage-gated sodium
`channels without affecting fast inactivation, and modulates col-
`lapsing response mediator protein 2 (CRMP-2) [6, 16]. CRMP-
`2 is a part of the signal transduction cascade of neurotrophic
`factors and has neuroprotective effects. The ability of LCM to
`modulate CRMP-2 contributes to the decreased neuronal loss
`observed in status epilepticus [6]. It is also efficacious for
`treating neuropathic pain and neuroprotection [17, 18].
`The Food and Drug Administration has classified LCM as
`a human pregnancy class BC^ compound [9], which means
`that animal reproduction studies have shown an adverse effect
`
`on the fetus and there are no adequate, well-controlled studies
`in humans, but the potential benefits may warrant use of the
`drug in pregnant women despite the potential risks. A litera-
`ture review of PubMed found only one case study and one
`experimental study that have reported its effects during preg-
`nancy [8, 9]. Ylikotila et al. treated a 7-week-pregnant woman
`who had cerebral venous thrombosis and status epilepticus
`with a combination of LCM and levetiracetam and reported
`that the infant was born without malformations, but was small
`for gestational age [8]. In their case study, the antiepileptic
`treatment was started in the late organogenesis period [8]. In
`an experimental study, Lee et al. [9] investigated the terato-
`genic potential of LCM using a zebrafish model and reported
`that LCM induced head and tail malformation, scoliosis, and
`growth retardation, and was teratogenic; in addition, there
`were significant differences among dose levels. Our study
`examined chicken embryos at the Hamburger and Hamilton
`[11] stage coinciding with 80 h of embryogenesis. In the
`LCM-treated groups, although most of the embryos appeared
`normal, growth retardation was obvious. The growth retarda-
`tion was the least in the subtherapeutic group, while major
`malformations increased with the dose and were mostly ob-
`served in the supratherapeutic group (Table 1).
`
`Conclusion
`
`Based on our data, LCM is not safe for developing embryos
`and may cause growth retardation or major congenital
`malformations. Nevertheless, more extensive investigations
`of its reliability are needed.
`
`Acknowledgments The authors would like to thank to Dr. Nayif
`YILMAZ for his assistance in obtaining the drugs.
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`Childs Nerv Syst (2016) 32:1715–1719
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`1719
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`Compliance with ethical standards All experiments were conducted
`in accordance with the animal research protocol of Celal Bayar University
`Ethics Committee (no. 77.637.4335–27).
`
`Conflict of interest Authors declare that there is no conflict of interest.
`
`References
`
`1. Patel SI, Pennell PB (2016 Mar) Management of epilepsy during
`pregnancy: an update. Ther Adv Neurol Disord 9(2):118–129
`2. Gerard EE, Meador KJ (2016 Feb) Managing epilepsy in women.
`Continuum (Minneap Minn) 22(1 Epilepsy):204–226
`3. Bhakta J, Bainbridge J, Borgelt L (2015) Teratogenic medications
`and concurrent contraceptive use in women of childbearing ability
`with epilepsy. Epilepsy Behav 52(Pt A):212–217
`4. Hernández-Díaz S, Smith CR, Shen A, Mittendorf R, Hauser WA,
`Yerby M, Holmes LB, North American AED Pregnancy Registry;
`North American AED Pregnancy Registry (2012) Comparative
`safety of antiepileptic drugs during pregnancy. Neurology
`22;78(21):1692–1699
`5. Foldvary-Schaefer N, Fong JS, Morrison S, Wang L, Bena J
`(2016) Lacosamide tolerability in adult patients with partial-
`onset seizures: impact of planned reduction and mechanism
`of action of concomitant antiepileptic drugs. Epilepsy Behav
`57(Pt A):155–160
`6. Patyar S, Medhi B (2010) Lacosamide, a newer antiepileptic.
`Neurosciences (Riyadh) 15(1):3–6
`. Doty P, Rudd GD, Stoehr T, Thomas D. Lacosamide
`Neurotherapeutics 2007;4(1):145–148
`
`7.
`
`8. Ylikotila P, Ketola RA, Timonen S, Malm H, Ruuskanen JO (2015)
`Early pregnancy cerebral venous thrombosis and status epilepticus
`treated with levetiracetam and lacosamide throughout pregnancy.
`Reprod Toxicol 57:204–206
`9. Lee SH, Kang JW, Lin T, Lee JE, Jin DI (2013) Teratogenic poten-
`tial of antiepileptic drugs in the zebrafish model. Biomed Res Int
`2013:726478
`10. Guney O (2003) The effects of folic acid in the prevention of neural
`tube development defects caused by phenytoin in early chick em-
`bryos. Spine 28:442–445
`11. Hamburger V, Hamilton HL (1951) A series of normal stages in the
`development of the chick embryo. J Morphol 88(1):49–92
`12. Lindhout D, Omtzigt JGC, Cornel MC (1992) Spectrum of neural
`tube defects in 34 infants prenatally exposed to antiepileptic drugs.
`Neurology 42:111–118
`13. Tomson T, Landmark CJ, Battino D (2013) Antiepileptic drug treat-
`ment in pregnancy: changes in drug disposition and their clinical
`implications. Epilepsia 54(3):405–414
`14. Sveberg L, Svalheim S, Taubøll E (2015) The impact of seizures on
`pregnancy and delivery. Seizure 28:35–38
`15. Meador KJ, Loring DW (2016) Developmental effects of antiepi-
`leptic drugs and the need for improved regulations. Neurology
`19;86(3):297–306
`16. Beyreuther BK, Freitag J, Heers C, Krebsfänger N, Scharfenecker
`U, Stöhr T (2007) Lacosamide: a review of preclinical properties.
`CNS Drug Rev 13(1):21–42
`17. Alcantara-Montero A, Sanchez-Carnerero CI (2016) Lacosamide
`and neuropathic pain, a review. Rev Neurol 1;62(5):223–229
`18. Pitkänen A, Immonen R, Ndode-Ekane X, Gröhn O, Stöhr T,
`Nissinen J (2014) Effect of lacosamide on structural damage and
`functional recovery after traumatic brain injury in rats. Epilepsy Res
`108(4):653–665
`
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