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Strong anticonvulsant effect of thalidomide on amygdaloid kindling
Epilepsy Research (2011) 95, 263—269
journal homepage: www.elsevier.com/locate/epilepsyres
Strong anticonvulsant effect of thalidomide on amygdaloid kindling Guadalupe Palencia a, Carmen Rubio b, Veronica Custodio-Ramirez b, Carlos Paz b, Julio Sotelo a,∗ a
Neuroimmunology Unit, National Institute of Neurology and Neurosurgery of Mexico, Insurgentes Sur 3877, Mexico City 14269, Mexico b Neurophysiology Laboratory, National Institute of Neurology and Neurosurgery of Mexico, Insurgentes Sur 3877, Mexico City 14269, Mexico Received 4 February 2011; received in revised form 7 April 2011; accepted 17 April 2011 Available online 17 May 2011
KEYWORDS Epilepsy therapy; Complex seizures; Refractory epilepsy; Thalidomide; Kindling
Summary Thalidomide was synthesized more than 50 years ago as hypnotic sedative with unique pharmacologic properties. Recently, we have described a notorious anticonvulsant effect of thalidomide on pentylenetetrazole-induced seizures. Here, we report the results of thalidomide administration on amygdaloid kindling. A total of 100 male Wistar rats were implanted with brain electrodes in the basolateral amygdaloid nucleus and the sensory motor cortex. After surgery the animals received a daily electric stimulus through the amygdaline electrode (500 A intensity, 60 Hz frequency, 1ms duration) until seizures appeared. The following treatment groups were made: (a) controls; (b) rats treated daily with thalidomide (10 mg/kg) or with topiramate (80 mg/kg); (c) rats treated with different doses of thalidomide. Significant reduction in the after-discharge and retard of behavioral stages were observed in rats treated with thalidomide or with topiramate as compared with controls (p < 0.01): Also, a similar anticonvulsant outcome of thalidomide therapy was obtained with doses of either 2.5, 5, 10 or 50 mg/kg; at 100 mg/kg all epileptic activity was suppressed. Anticonvulsant efficacy of thalidomide was superior in most parameters than that obtained with topiramate. In amygdaloid kindling, which simulates human epilepsy characterized by focal seizures secondarily generalized, low doses of thalidomide display strong anticonvulsant properties. © 2011 Elsevier B.V. All rights reserved.
Introduction
∗ Corresponding author. Tel.: +52 55 5553 7306; fax: +52 55 5553 7106. E-mail address: [email protected] (J. Sotelo).
Thalidomide was synthesized more than 50 years ago as hypnotic, sedative and antiemetic (Bergstroem et al., 1964; Frederickson et al., 1977). However, the tragic discovery in the early 50’s of severe teratogenic effects of thalidomide in humans, which were widely covered by the lay press, led to the abandonment of research, simultaneous
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264 to the public repudiation of the drug (Kanno, 1987; Parman et al., 1999). Nevertheless, during the last decades various peculiar therapeutic properties of thalidomide in cellular substrates other than in the brain have been described, mainly as immunomodulator and antineoplasic (Palencia et al., 2002; Richardson et al., 2002; Cuadrado et al., 2005; Chim et al., 2010; Clark et al., 2010). Currently, there are several medical recommendations for thalidomide therapy in human diseases of infectious, neoplastic or autoimmune origin (Calabrese & Fleischer, 2000; Richardson et al., 2002; Lehman et al., 2004); however, it has not been used for treatment of neurological or psychiatric diseases, which is ironic for a drug originally developed for disturbances of the central nervous system (Somers, 1960; Frederickson et al., 1977; Palencia et al., 2007). Interestingly, few studies have been conducted on the neurologic effects of thalidomide (Keller et al., 1956; Somers, 1960; Frederickson et al., 1977; Teo et al., 1999). There are two isolated cases of a favorable anticonvulsant result in patients with Rasmussen encephalitis treated with thalidomide (Ravenscroft et al., 1998; Marjanovic et al., 2003), although in both instances the authors ascribed the clinical response to the actions of thalidomide on the immune system (Medhi et al., 2009; Rao et al., 2010), rather than on the neural substratum. Experimentally, Friderichs (1982) found inhibition of electroshock-induced seizures by thalidomide. On experimental studies thalidomide has also been proposed as a potential neuroprotector for cerebral ischemia (Zhang et al., 2010). We have described an intense anticonvulsant effect of thalidomide in rats with seizures chemically-induced by pentylenetetrazole (PTZ) (Arrieta et al., 2005; Palencia et al., 2007). Additionally, we have recently reported strong anticonvulsant properties of thalidomide in humans: In a preliminary, open labeled study, thalidomide was added to the standard antiepileptic treatment of six male patients with severe refractory epilepsy; the mean number of seizures diminished from 26 ± 4 per month before thalidomide to 7 ± 1 per month during one year of thalidomide therapy, supporting the idea that thalidomide is a promising treatment for selected cases of epilepsy (Palencia et al., 2010). The hypnotic and sedative properties of thalidomide, as well as its rather peculiar affinity for selective neurological substrates on the basal forebrain and brainstem suggest potential anti-epileptic attributes (Palencia et al., 2007). The efficacy of anticonvulsant drugs must be experimentally tested on animal models that simulate human epilepsy; amygdaloid kindling is a widely accepted animal model of generalized seizures (Bertram, 2007), which replicates in the laboratory a common form of human epilepsy characterized by complex partial seizures secondarily generalized (Goddard, 1967; Borowicz et al., 2003; Chen et al., 2009). Thus, the testing of any substance in the model of amygdaloid kindling provides reliable information on its potential anticonvulsant properties (Goddard et al., 1969; Löscher et al., 1993). The aim of the present study was to compare the effects of thalidomide administration with those obtained with topiramate, a widely used antiepileptic (Nakamura et al., 1994; Privitera et al., 2003) with well known effectiveness in amygdaloid kindling (Engel et al., 1978; Engel, 1991; Wauquier & Zhou, 1996; Borowicz et al., 2003).
G. Palencia et al.
Methods Development of amygdaloid kindling: One hundred male Wistar rats weighing 280—310 g were kept under controlled environmental conditions (20—23 ◦ C and 12/12-h light/dark cycle) for at least 2 weeks before surgery. At the time of surgery the rats were anesthetized (Ketamine 100 mg/kg, i.p.) and placed on a stereotaxic apparatus to implant bipolar electrodes for stimulation and recording in the left basolateral amygdaloid nucleus (coordinates: anterior 6.2 mm, lateral 5.0 mm, height 1.5 mm using the interaural line as reference point), and in the right sensory—motor cortex (coordinates: anterior 6.7 mm, lateral 2.5 mm, height 9.0 mm), in order to record the electrographic propagation of activity. Electrodes were led to their loci using the stereotaxic atlas of Paxinos and Watson (1986). Each electrode consisted of two twisted insulated wires (0.005 in. diameter) made of stainless steel and coated with Teflon, except for the tips. A screw implanted in the skull served as indifferent source of reference. Electrodes were arranged and soldered to a mini connector and secured to the skull with dental acrylic. Skin cuts were sutured while exposing the mini-connector. After 10 days of post-operative recovery, the rats were placed in a soundproof chamber while their mini-connectors were connected to an amplifier (BioScience Vector PSG32) by flexible cables which allowed free movements. Electrographic activity records were stored and digitized in a computer provided with software Stellate Systems, the stimulation parameters for amygdaloid responses were 1.0 ms rectangular pulses at 60 Hz for 1 s with intensity of 500 A delivered by an electronic circuit breaker device which permitted us to record and stimulate through the same electrode. The stimuli were applied once daily until 10 stage-5 seizures were obtained (Wada et al., 1974; Rubio et al., 2004). Once the electrographic recordings were completed the rats were anesthetized and sacrificed by intra-cardiac perfusion with formalin at 5% in saline solution. The brains were extracted, embedded in paraffin and sliced (15 m). The sections were stained with hematoxylin—eosin in order to visualize the correct position of the electrodes. Anticonvulsant treatment: Effects of drug treatment on the amygdaloid kindling were studied according to the following experimental design, to achieve 4 main goals:
(I) To compare the potential antiepileptic activity of thalidomide with that of topiramate; 3 sub-groups of rats (n = 5 each) were made; (Ia) controls, only kindling development; (Ib) oral administration of thalidomide at a daily dose of 10 mg/kg, 30 min before the electrical stimulation; (Ic) oral administration of topiramate 80 mg/kg daily 30 min before electrical stimulation. (II) To compare the antiepileptic activity of thalidomide with that of topiramate once the kindling phenomenon had been achieved 3 additional sub-groups of rats (n = 5 each) were made: (IIa) controls, only kindling development; (IIb) identical daily doses of thalidomide as those from sub-group Ib, but the treatment began when the rats had already presented 3 stage5 generalized seizures; (IIc) identical doses of topiramate as to those from sub-group Ic, but the treatment began when the rats had already presented 3 stage-5 generalized seizures. (III) To define the antiepileptic dose-response to thalidomide therapy administered from the beginning of kindling the following sub-groups were made: (IIIa) controls (n = 5), only kindling development; (IIIb) thalidomide in doses of either 2.5, 5, 10, 50 or 100 mg/kg (n = 5 for each dose); (IIIc) (n = 5) topiramate 80 mg/kg. Treatments started the first day of electrical stimulation. (IV) To define the antiepileptic dose-response to thalidomide therapy administered once the generalized kindled seizures had developed the following sub-groups were made: (IVa) controls (n = 5); (IVb) thalidomide in doses of either 2.5, 5, 10, 50 or
Strong anticonvulsant effect of thalidomide on amygdaloid kindling
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Fig. 1 (A) Comparisons between number of trials and duration of the amygdaloid afterdischarge in rats treated daily, beginning at the time of the first electrical stimulation, with 10 mg/kg of thalidomide (TM) or with 80 mg/kg of topiramate (TP) and controls (C). Animals treated either with TM or with TP showed a minor duration of the afterdischarge as compared with controls (*p < 0.01). (B) Comparisons between number of trials and behavioral stages reached; animals treated either with TM or with TP presented significant inhibition to the progress of behavioral stages as compared with controls (*p < 0.01).
100 mg/kg (n = 5 for each dose); (IVc) topiramate 80 mg/kg. Treatments started on day 14 of electrical stimulation. The doses of thalidomide used in experiments I and II were identical to those used by us in the experimental model of pentylenetetrazole-induced seizures in rats as previously reported (Palencia et al., 2007); topiramate doses employed in this study were identical as those reported by Wauquier & Zhou (1996). The following two parameters were individually measured: duration of the electrographic activity from the basolateral amygdaloid nucleus characterized by epileptic spikes called afterdischarge (AD) and the number of trials required to reach each behavioral stage following Racine’s criteria (1975). Briefly: Stage-1 mouth and facial movements, Stage-2 head nodding, Stage-3 forelimb clonus, Stage4 rearing, Stage-5 rearing and falling. The number of trials to reach each of these behavioral stages, and the duration of amygdaloid AD along kindling development and during 10 episodes of stage-5 seizures were compared between groups. Differences of the AD and behavior stages in control and experimental groups were analyzed by a two-way repeated measures (ANOVA) considering different doses of treatment. Subsequent comparisons within conditions were made using a Turkey’s test. Differences of the AD duration were determined by one-way repeated measures of variance (ANOVA) The cut- off for the nominal level of significance was p < 0.05. Values were expressed as means ± SE.
Results Rats that received the treatment with thalidomide (10 mg/kg) or with topiramate (80 mg/kg) at the beginning of electrical stimulation showed a significant reduction on the duration of the AD as compared with controls F(2,14) = 8.167, p < 0.004 (Fig. 1A). Similarly, when studied the development of behavioral stages, the administration of thalidomide (10 mg/kg) or topiramate (80 mg/kg) since the beginning of electrical stimulation prevented the development of behavioral stages beyond stage-1 F(2,14) = 15.334, p < 0.01 (Fig. 1B). When the treatment was started on day 14th of electrical stimulation a significant decrease in the AD duration was seen in rats administered either with thalidomide or with topiramate as compared with controls (p ≤ 0.01); when compared thalidomide with topiramate, thalidomide was more effective (p ≤ 0.01) F(2,14) = 15.304, p < 0.01 (Fig. 2A). Similarly, when the treatment started on day 14 of electrical stimulation thalidomide inhibited kindled behavioral stages more efficiently than topiramate; no significant differences were found when compared topiramate with controls (2,14) = 5.403, p < 0.01 (Fig. 2B).
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Figure 2 (A) Comparisons between number of trials and duration of the amygdaloid afterdischarge in rats treated daily beginning at day 14 (arrow), at the time of the first generalized seizure, with 10 mg/kg of thalidomide (TM) or with 80 mg/kg of topiramate (TP) and controls (C). Animals treated either with TM or with TP showed a minor duration of the afterdischarge as compared with controls (*p < 0.01). (B) Comparisons between number of trials and behavioral stages reached; animals treated either with TM or with TP presented significant inhibition to the progress of behavioral stages as compared with controls (*p < 0.01).
Once the anticonvulsant effect of thalidomide at doses of 10 mg/kg was demonstrated different doses were compared under identical experimental conditions. When thalidomide or topiramate were administered since the beginning of electrical stimulation thalidomide in doses either of 2.5, 5, 10 or 50 mg/kg were similar on their anticonvulsant effectiveness; nevertheless, when a high dose of thalidomide was used (100 mg/kg) all epileptic activity was suppressed (Fig. 3A). When compared thalidomide with topiramate, no significant differences were obtained. When the same doses of thalidomide were administered once the generalized seizures of amygdaloid kindling had developed (on day 14 of kindling stimulation) a paradoxical anticonvulsant effect was observed, minor doses of thalidomide (2.5 mg/kg) were more effective than high doses of 50 and 100 mg/kg (Fig. 3B), in this experiments low doses of thalidomide (2.5, 5 and 10 mg/kg) were also more effective than topiramate. In these experiments, in contrast with AD results, different doses of thalidomide did not induce significant changes of behavioral stages (results not shown). Although no toxicity tests were made, no evident signs of acute toxicity (such as drowsiness or motor disturbances) were observed along the experiments, even in animals treated with high doses of thalidomide.
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Figure 3 Mean duration of the afterdischarge along the kindling development in animals treated with various doses (2.5, 5, 10, 50 or 100 mg/kg) of thalidomide (TM) or with topiramate (TP) 80 mg/kg and controls (C). (A) When the treatments began the day that kindling stimulation started antiepileptic effect of TM was similar with doses of 2.5, 5, 10 and 50 mg/kg (p < 0.01); at a very high dose of 100 mg/kg all epileptic activity abated. (B) When the treatments began on day 14, once the generalized seizures had developed. A paradoxical response was obtained, low doses of TM were more effective than high doses or than topiramate. The data represent the mean values of all recordings after the beginning of treatment (*p < 0.05).
Discussion Our results showed that thalidomide had a strong anticonvulsant effect; in some parameters it was more effective than topiramate. In previous reports we have shown that thalidomide is effective against PTZ-induced in rats (Palencia et al., 2007), which is an experimental model similar to tonic clonic seizures in humans (Fisher, 1989). Antiepileptic drugs that are effective against kindled seizures are usually also effective against partial complex seizures in humans originated in the amygdala or in neighbor limbic areas (McNamara et al., 1980; Fujiwara et al., 2010). The fact that in our studies thalidomide had anticonvulsant activity comparable, and in some parameters superior to that of topiramate, particularly in the duration of the AD, opens new possibilities for research on thalidomide therapy in epilepsy. Although thalidomide is a rather old substance it constitutes a novel antiepileptic whose pharmacological mechanisms are singular, no other sedative with anticonvul-
Strong anticonvulsant effect of thalidomide on amygdaloid kindling sant effect shares the same neural substrates and pathways where thalidomide shows molecular affinity. Nonetheless, the severe teratogenicity of thalidomide restricts its potential clinical use mostly for male epilepsy patients or for females in whom the possibility of pregnancy during therapy is totally excluded. One of the many paradoxes of thalidomide therapy in humans, besides its severe teratogenicity (Parman et al., 1999), is its minor toxicity at low doses is notorious, only long-term peripheral neuropathy is a concern on lasting therapeutic regimes (Tosi et al., 2005). It is notorious the absence of acute toxicity of thalidomide in multiple animal experiments (Somers, 1960); even large doses are devoid of acute side-effects; in the initial toxicological studies the median lethal doses could not be found due to the remarkable tolerance of experimental animals to very high doses of thalidomide (Somers, 1960). Neurological research has shown that in contrast with other sedatives and hypnotics (like phenobarbitone), thalidomide does not produce hypermotility due to excitation or hypomotility due to narcosis. Thalidomide was devoid of narcosis even after maximal doses, in rats it does not affect temperature, heart-rate, blood pressure, respiration or analgesia (Somers, 1960). High doses of thalidomide had a minor effect on motor-coordination in the electroshock model in mice (Friderichs, 1982) but does not impair spontaneous locomotor activity (Somers, 1960; Teo et al., 1999). However, similarly with other hypnotics and sedatives, thalidomide potentiates the narcotic and sedative action of barbiturates, alcohol, chlorpromazine and reserpine. It also counteracts the effects of methylamphetamine and methylphenidate. Nonetheless, in contrast with other sedatives, no addiction to thalidomide has been reported in humans or acute drug toxicity as documented in more than twenty cases of accidental or intentional over dosage (Somers, 1960). In our previous studies using thalidomide in PTZ-induced seizures the antiepileptic effect of thalidomide was more evident in the brainstem component of seizures responsible for the tonic—clonic seizure, than on the forebrain component, responsible for the myoclonic jerks (Palencia et al., 2010; Eells et al., 2004); this dichotomy shows a peculiar mechanism of action of thalidomide as compared with most other anticonvulsants tested on PTZ-induced seizures (Palencia et al., 2007). In amigdaloid kindling independent mechanisms are elicited in the partial and in the generalized seizures (Sikes et al., 1977); C cerebral structures involved in the partial seizures are primarily limbic, at the hippocampus and amygdala, whereas in the generalized seizure participate the substance nigra, globus pallidus, neocortex, thalamus and hippocampus. It seems interesting that in the kindling model the anticonvulsant effect of thalidomide was also manifest in the brainstem as well as in the cortical components. In our study the effects of Thalidomide administration were more evident on the duration of the AD than on changes of behavioral scores; also, the amigdaloid AD did not differ from the cortical AD. It is important to stress the fact that in the model of amygdaloid kindling the treatments that affect the duration of AD do not necessarily modify the behavioral stages (Sikes et al., 1977). In contrast with our results, early experiments on anticonvulsant activity had shown no effects of thalidomide on leptazol or strychnine-induced convulsions (Somers, 1960). The seda-
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tive effects of thalidomide are mediated by neural pathways different from those followed by other sedative substances (Frederickson et al., 1977) which indicate unique actions of thalidomide on the brain (Ionov, 2009), exemplified by the absence in humans of drug dependence and the lack of narcosis induced by thalidomide even after the ingestion of very large doses (Somers, 1960). Thalidomide administration inhibits the expression of tumor necrosis factor (De Sanctis et al., 2010); it seems possible that this effect on cytokines might influence seizures (Li et al., 2011). In contrast with the neural effects of most other sedatives, thalidomide acts selectively as analog of glutamic acid. In comparative studies of hypnotic drugs in cats, thalidomide had hypnotic activity comparable with that of pentobarbital but, in contrast with the latter, thalidomide did not induce ataxia but enhanced the sleep induced by electrical stimulation of the basal forebrain, suggesting a peculiar mechanism of action based on forebrain activation (Frederickson et al., 1977). It is important the fact that the strong antiepileptic properties of thalidomide in amygdaloid kindling were obtained with low doses of the drug (2.5 mg/kg), and it was not substantially increased with much higher doses (50 mg/kg); this unexpected finding which was more evident when thalidomide was started after the seizures had already developed (Fig. 3B) opens the possibility of long-term antiepileptic treatment using low doses of thalidomide in selected patients. If low doses of thalidomide are indeed effective in human epilepsy, secondary effects other than teratogenicity, such as peripheral neuropathy could be prevented. Recently, we have conducted a clinical open labeled pilot study of thalidomide therapy in male patients with chronic refractory epilepsy, intense anticonvulsant results were documented, the mean number of monthly seizures reduced from 26 ± 4 to 7 ± 1 along thalidomide administration (Palencia et al., 2010). Clinical research on the potential therapeutic use of thalidomide in epilepsy seems warranted. If proven successful as antiepileptic, chemical manipulations on the basic molecule of thalidomide, aiming to eliminate its iatrogenic potential (Sumrall et al., 2010) but retaining its anticonvulsant properties would be a valuable avenue for further pharmacological investigation.
Conflict of interests None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The drugs used in these experiments were purchased by the authors. No pharmaceutical company participated in this study in any form.
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G. Palencia et al. Keller, H., Kunz, W., Muckter, H., 1956. N-phthalyl-glutamic acid imide; experimental studies on a new synthetic product with sedative properties. Arzneimittelforschung 6, 426— 430. Lehman, T.J., Schechter, S.J., Sundel, R.P., Oliveira, S.K., Huttenlocher, A., Onel, K.B., 2004. Thalidomide for severe systemic onset juvenile rheumatoid arthritis: a multicenter study. J. Pediatr. 145, 856—857. Li, G., Bauer, S., Nowak, M., Norwood, B., Tackenberg, B., Rosenow, F., Knake, S., Oertel, W.H., Hamer, H.M., 2011. Cytokines and epilepsy. Seizure Apr. 20 (3), 249—256. Löscher, W., Rundfeldt, C., Hönack, D., 1993. Pharmacological characterization of phenytoin-resistant amygdala-kindled rats, a new model of drug-resistant partial epilepsy. Epilepsy Res. 15, 207—219. McNamara, J.O., Byrne, M.C., Dasheiff, R.M., Fitz, J.G., 1980. The kindling model of epilepsy: a review. Prog. Neurobiol. 15, 139—159. Marjanovic, B.D., Stojanov, L.M., Zdravkovic, D.S., Kravljanac, R.M., Djordjevic, M.S., 2003. Rasmussen syndrome and longterm response to thalidomide. Pediatr. Neurol. 29, 151—156. Medhi, B., Prakash, A., Avti, P.K., Chakrabarti, A., Khanduja, K.L., 2009. Intestinal inflammation and seizure susceptibility: understanding the role of tumour necrosis factor-alpha in a rat model. J. Pharm. Pharmacol. 61, 1359—1364. Nakamura, J., Tamura, S., Kanda, T., Ishii, A., Ishihara, K., Serikawa, T., Yamada, J., Sasa, M., 1994. Inhibition by topiramate of seizures in spontaneously epileptic rats and DBA/2 mice. Eur. J. Pharmacol. 254, 83—89. Palencia, G., Arrieta, O., Ríos, C., Altagracia, M., Kravzov, J., Sotelo, J., 2002. Effect of thalidomide in different tumors in rodents. J. Exp. Ther. Oncol. 2, 158—162. Palencia, G., Calderon, A., Sotelo, J., 2007. Thalidomide inhibits pentylenetetrazole-induced seizures. J. Neurol. Sci. 258, 128—131. Palencia, G., Martinez-Juarez, I.E., Calderon, A., Artigas, C., Sotelo, J., 2010. Thalidomide for treatment of refractory epilepsy. Epilepsy Res. 92, 253—257. Parman, T., Wiley, M.J., Wells, P.G., 1999. Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity. Nat. Med. 5, 582—585. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney. Privitera, M.D., Brodie, M.J., Mattson, R.H., Chadwick, D.W., Neto, W., Wang, S., 2003. EPMN 105 Study Group Topiramate, carbamazepine and valproate monotherapy: double-blind comparison in newly diagnosed epilepsy. Acta Neurol. Scand. 107, 165—175. Racine, R.J., 1975. Modification of seizure activity by electrical stimulation: cortical areas. Electroencephalogr. Clin. Neurophysiol. 38, 1—12. Rao, R.S., Medhi, B., Khanduja, K.L., Pandhi, P., 2010. Correlation of seizures and biochemical parameters of oxidative stress in experimentally induced inflammatory rat models. Fundam. Clin. Pharmacol. 24, 325—331. Ravenscroft, A., Schoeman, J.F., Pretorious, M.L., Rickman, R.C., 1998. Rasmussen’s encephalitis: case presentation and discussion on the role of specific immune modulation. Brain Dev. 20, 398. Richardson, P., Hideshima, T., Anderson, K., 2002. Thalidomide: emerging role in cancer medicine. Annu. Rev. Med. 53, 629—657. Rubio, C., Custodio, V., Juárez, F., Paz, C., 2004. Stimulation of the superior cerebellar peduncle during the development of amygdaloid kindling in rats. Brain Res. 1010, 151—155. Sikes, R.W., Chronister, R.B., White Jr., L.E., 1977. Origin of the direct hippocampus — anterior thalamic bundle in the rat: a combined horseradish peroxidase — golgi analysis. Exp. Neurol. 57, 379—395.
Strong anticonvulsant effect of thalidomide on amygdaloid kindling Somers, G.F., 1960. Pharmacological properties of thalidomide (alpha-phthalimido glutarimide), a new sedative hypnotic drug. Br. J. Pharmacol. Chemother. 15, 111—116. Sumrall, A., Fredericks, R., Berthold, A., Shumaker, G., 2010. Lenalidomide stops progression of multifocal epithelioid hemangioendothelioma including intracranial disease. J. Neurooncol. 97, 275—277. Teo, S.K., Trigg, N.J., Shaw, M.E., Morgan, J.M., Thomas, S.D., 1999. Subchronic toxicity of thalidomide in rodents after 13 weeks of oral administration. Int. J. Toxicol. 18, 337—352. Tosi, P., Zamagni, E., Cellini, C., Plasmati, R., Cangini, D., Tacchetti, P., Perrone, G., Pastorelli, F., Tura, S., Baccarani, M.,
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journal homepage: www.elsevier.com/locate/epilepsyres
Strong anticonvulsant effect of thalidomide on amygdaloid kindling Guadalupe Palencia a, Carmen Rubio b, Veronica Custodio-Ramirez b, Carlos Paz b, Julio Sotelo a,∗ a
Neuroimmunology Unit, National Institute of Neurology and Neurosurgery of Mexico, Insurgentes Sur 3877, Mexico City 14269, Mexico b Neurophysiology Laboratory, National Institute of Neurology and Neurosurgery of Mexico, Insurgentes Sur 3877, Mexico City 14269, Mexico Received 4 February 2011; received in revised form 7 April 2011; accepted 17 April 2011 Available online 17 May 2011
KEYWORDS Epilepsy therapy; Complex seizures; Refractory epilepsy; Thalidomide; Kindling
Summary Thalidomide was synthesized more than 50 years ago as hypnotic sedative with unique pharmacologic properties. Recently, we have described a notorious anticonvulsant effect of thalidomide on pentylenetetrazole-induced seizures. Here, we report the results of thalidomide administration on amygdaloid kindling. A total of 100 male Wistar rats were implanted with brain electrodes in the basolateral amygdaloid nucleus and the sensory motor cortex. After surgery the animals received a daily electric stimulus through the amygdaline electrode (500 A intensity, 60 Hz frequency, 1ms duration) until seizures appeared. The following treatment groups were made: (a) controls; (b) rats treated daily with thalidomide (10 mg/kg) or with topiramate (80 mg/kg); (c) rats treated with different doses of thalidomide. Significant reduction in the after-discharge and retard of behavioral stages were observed in rats treated with thalidomide or with topiramate as compared with controls (p < 0.01): Also, a similar anticonvulsant outcome of thalidomide therapy was obtained with doses of either 2.5, 5, 10 or 50 mg/kg; at 100 mg/kg all epileptic activity was suppressed. Anticonvulsant efficacy of thalidomide was superior in most parameters than that obtained with topiramate. In amygdaloid kindling, which simulates human epilepsy characterized by focal seizures secondarily generalized, low doses of thalidomide display strong anticonvulsant properties. © 2011 Elsevier B.V. All rights reserved.
Introduction
∗ Corresponding author. Tel.: +52 55 5553 7306; fax: +52 55 5553 7106. E-mail address: [email protected] (J. Sotelo).
Thalidomide was synthesized more than 50 years ago as hypnotic, sedative and antiemetic (Bergstroem et al., 1964; Frederickson et al., 1977). However, the tragic discovery in the early 50’s of severe teratogenic effects of thalidomide in humans, which were widely covered by the lay press, led to the abandonment of research, simultaneous
0920-1211/$ — see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2011.04.008
264 to the public repudiation of the drug (Kanno, 1987; Parman et al., 1999). Nevertheless, during the last decades various peculiar therapeutic properties of thalidomide in cellular substrates other than in the brain have been described, mainly as immunomodulator and antineoplasic (Palencia et al., 2002; Richardson et al., 2002; Cuadrado et al., 2005; Chim et al., 2010; Clark et al., 2010). Currently, there are several medical recommendations for thalidomide therapy in human diseases of infectious, neoplastic or autoimmune origin (Calabrese & Fleischer, 2000; Richardson et al., 2002; Lehman et al., 2004); however, it has not been used for treatment of neurological or psychiatric diseases, which is ironic for a drug originally developed for disturbances of the central nervous system (Somers, 1960; Frederickson et al., 1977; Palencia et al., 2007). Interestingly, few studies have been conducted on the neurologic effects of thalidomide (Keller et al., 1956; Somers, 1960; Frederickson et al., 1977; Teo et al., 1999). There are two isolated cases of a favorable anticonvulsant result in patients with Rasmussen encephalitis treated with thalidomide (Ravenscroft et al., 1998; Marjanovic et al., 2003), although in both instances the authors ascribed the clinical response to the actions of thalidomide on the immune system (Medhi et al., 2009; Rao et al., 2010), rather than on the neural substratum. Experimentally, Friderichs (1982) found inhibition of electroshock-induced seizures by thalidomide. On experimental studies thalidomide has also been proposed as a potential neuroprotector for cerebral ischemia (Zhang et al., 2010). We have described an intense anticonvulsant effect of thalidomide in rats with seizures chemically-induced by pentylenetetrazole (PTZ) (Arrieta et al., 2005; Palencia et al., 2007). Additionally, we have recently reported strong anticonvulsant properties of thalidomide in humans: In a preliminary, open labeled study, thalidomide was added to the standard antiepileptic treatment of six male patients with severe refractory epilepsy; the mean number of seizures diminished from 26 ± 4 per month before thalidomide to 7 ± 1 per month during one year of thalidomide therapy, supporting the idea that thalidomide is a promising treatment for selected cases of epilepsy (Palencia et al., 2010). The hypnotic and sedative properties of thalidomide, as well as its rather peculiar affinity for selective neurological substrates on the basal forebrain and brainstem suggest potential anti-epileptic attributes (Palencia et al., 2007). The efficacy of anticonvulsant drugs must be experimentally tested on animal models that simulate human epilepsy; amygdaloid kindling is a widely accepted animal model of generalized seizures (Bertram, 2007), which replicates in the laboratory a common form of human epilepsy characterized by complex partial seizures secondarily generalized (Goddard, 1967; Borowicz et al., 2003; Chen et al., 2009). Thus, the testing of any substance in the model of amygdaloid kindling provides reliable information on its potential anticonvulsant properties (Goddard et al., 1969; Löscher et al., 1993). The aim of the present study was to compare the effects of thalidomide administration with those obtained with topiramate, a widely used antiepileptic (Nakamura et al., 1994; Privitera et al., 2003) with well known effectiveness in amygdaloid kindling (Engel et al., 1978; Engel, 1991; Wauquier & Zhou, 1996; Borowicz et al., 2003).
G. Palencia et al.
Methods Development of amygdaloid kindling: One hundred male Wistar rats weighing 280—310 g were kept under controlled environmental conditions (20—23 ◦ C and 12/12-h light/dark cycle) for at least 2 weeks before surgery. At the time of surgery the rats were anesthetized (Ketamine 100 mg/kg, i.p.) and placed on a stereotaxic apparatus to implant bipolar electrodes for stimulation and recording in the left basolateral amygdaloid nucleus (coordinates: anterior 6.2 mm, lateral 5.0 mm, height 1.5 mm using the interaural line as reference point), and in the right sensory—motor cortex (coordinates: anterior 6.7 mm, lateral 2.5 mm, height 9.0 mm), in order to record the electrographic propagation of activity. Electrodes were led to their loci using the stereotaxic atlas of Paxinos and Watson (1986). Each electrode consisted of two twisted insulated wires (0.005 in. diameter) made of stainless steel and coated with Teflon, except for the tips. A screw implanted in the skull served as indifferent source of reference. Electrodes were arranged and soldered to a mini connector and secured to the skull with dental acrylic. Skin cuts were sutured while exposing the mini-connector. After 10 days of post-operative recovery, the rats were placed in a soundproof chamber while their mini-connectors were connected to an amplifier (BioScience Vector PSG32) by flexible cables which allowed free movements. Electrographic activity records were stored and digitized in a computer provided with software Stellate Systems, the stimulation parameters for amygdaloid responses were 1.0 ms rectangular pulses at 60 Hz for 1 s with intensity of 500 A delivered by an electronic circuit breaker device which permitted us to record and stimulate through the same electrode. The stimuli were applied once daily until 10 stage-5 seizures were obtained (Wada et al., 1974; Rubio et al., 2004). Once the electrographic recordings were completed the rats were anesthetized and sacrificed by intra-cardiac perfusion with formalin at 5% in saline solution. The brains were extracted, embedded in paraffin and sliced (15 m). The sections were stained with hematoxylin—eosin in order to visualize the correct position of the electrodes. Anticonvulsant treatment: Effects of drug treatment on the amygdaloid kindling were studied according to the following experimental design, to achieve 4 main goals:
(I) To compare the potential antiepileptic activity of thalidomide with that of topiramate; 3 sub-groups of rats (n = 5 each) were made; (Ia) controls, only kindling development; (Ib) oral administration of thalidomide at a daily dose of 10 mg/kg, 30 min before the electrical stimulation; (Ic) oral administration of topiramate 80 mg/kg daily 30 min before electrical stimulation. (II) To compare the antiepileptic activity of thalidomide with that of topiramate once the kindling phenomenon had been achieved 3 additional sub-groups of rats (n = 5 each) were made: (IIa) controls, only kindling development; (IIb) identical daily doses of thalidomide as those from sub-group Ib, but the treatment began when the rats had already presented 3 stage5 generalized seizures; (IIc) identical doses of topiramate as to those from sub-group Ic, but the treatment began when the rats had already presented 3 stage-5 generalized seizures. (III) To define the antiepileptic dose-response to thalidomide therapy administered from the beginning of kindling the following sub-groups were made: (IIIa) controls (n = 5), only kindling development; (IIIb) thalidomide in doses of either 2.5, 5, 10, 50 or 100 mg/kg (n = 5 for each dose); (IIIc) (n = 5) topiramate 80 mg/kg. Treatments started the first day of electrical stimulation. (IV) To define the antiepileptic dose-response to thalidomide therapy administered once the generalized kindled seizures had developed the following sub-groups were made: (IVa) controls (n = 5); (IVb) thalidomide in doses of either 2.5, 5, 10, 50 or
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Fig. 1 (A) Comparisons between number of trials and duration of the amygdaloid afterdischarge in rats treated daily, beginning at the time of the first electrical stimulation, with 10 mg/kg of thalidomide (TM) or with 80 mg/kg of topiramate (TP) and controls (C). Animals treated either with TM or with TP showed a minor duration of the afterdischarge as compared with controls (*p < 0.01). (B) Comparisons between number of trials and behavioral stages reached; animals treated either with TM or with TP presented significant inhibition to the progress of behavioral stages as compared with controls (*p < 0.01).
100 mg/kg (n = 5 for each dose); (IVc) topiramate 80 mg/kg. Treatments started on day 14 of electrical stimulation. The doses of thalidomide used in experiments I and II were identical to those used by us in the experimental model of pentylenetetrazole-induced seizures in rats as previously reported (Palencia et al., 2007); topiramate doses employed in this study were identical as those reported by Wauquier & Zhou (1996). The following two parameters were individually measured: duration of the electrographic activity from the basolateral amygdaloid nucleus characterized by epileptic spikes called afterdischarge (AD) and the number of trials required to reach each behavioral stage following Racine’s criteria (1975). Briefly: Stage-1 mouth and facial movements, Stage-2 head nodding, Stage-3 forelimb clonus, Stage4 rearing, Stage-5 rearing and falling. The number of trials to reach each of these behavioral stages, and the duration of amygdaloid AD along kindling development and during 10 episodes of stage-5 seizures were compared between groups. Differences of the AD and behavior stages in control and experimental groups were analyzed by a two-way repeated measures (ANOVA) considering different doses of treatment. Subsequent comparisons within conditions were made using a Turkey’s test. Differences of the AD duration were determined by one-way repeated measures of variance (ANOVA) The cut- off for the nominal level of significance was p < 0.05. Values were expressed as means ± SE.
Results Rats that received the treatment with thalidomide (10 mg/kg) or with topiramate (80 mg/kg) at the beginning of electrical stimulation showed a significant reduction on the duration of the AD as compared with controls F(2,14) = 8.167, p < 0.004 (Fig. 1A). Similarly, when studied the development of behavioral stages, the administration of thalidomide (10 mg/kg) or topiramate (80 mg/kg) since the beginning of electrical stimulation prevented the development of behavioral stages beyond stage-1 F(2,14) = 15.334, p < 0.01 (Fig. 1B). When the treatment was started on day 14th of electrical stimulation a significant decrease in the AD duration was seen in rats administered either with thalidomide or with topiramate as compared with controls (p ≤ 0.01); when compared thalidomide with topiramate, thalidomide was more effective (p ≤ 0.01) F(2,14) = 15.304, p < 0.01 (Fig. 2A). Similarly, when the treatment started on day 14 of electrical stimulation thalidomide inhibited kindled behavioral stages more efficiently than topiramate; no significant differences were found when compared topiramate with controls (2,14) = 5.403, p < 0.01 (Fig. 2B).
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Figure 2 (A) Comparisons between number of trials and duration of the amygdaloid afterdischarge in rats treated daily beginning at day 14 (arrow), at the time of the first generalized seizure, with 10 mg/kg of thalidomide (TM) or with 80 mg/kg of topiramate (TP) and controls (C). Animals treated either with TM or with TP showed a minor duration of the afterdischarge as compared with controls (*p < 0.01). (B) Comparisons between number of trials and behavioral stages reached; animals treated either with TM or with TP presented significant inhibition to the progress of behavioral stages as compared with controls (*p < 0.01).
Once the anticonvulsant effect of thalidomide at doses of 10 mg/kg was demonstrated different doses were compared under identical experimental conditions. When thalidomide or topiramate were administered since the beginning of electrical stimulation thalidomide in doses either of 2.5, 5, 10 or 50 mg/kg were similar on their anticonvulsant effectiveness; nevertheless, when a high dose of thalidomide was used (100 mg/kg) all epileptic activity was suppressed (Fig. 3A). When compared thalidomide with topiramate, no significant differences were obtained. When the same doses of thalidomide were administered once the generalized seizures of amygdaloid kindling had developed (on day 14 of kindling stimulation) a paradoxical anticonvulsant effect was observed, minor doses of thalidomide (2.5 mg/kg) were more effective than high doses of 50 and 100 mg/kg (Fig. 3B), in this experiments low doses of thalidomide (2.5, 5 and 10 mg/kg) were also more effective than topiramate. In these experiments, in contrast with AD results, different doses of thalidomide did not induce significant changes of behavioral stages (results not shown). Although no toxicity tests were made, no evident signs of acute toxicity (such as drowsiness or motor disturbances) were observed along the experiments, even in animals treated with high doses of thalidomide.
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Figure 3 Mean duration of the afterdischarge along the kindling development in animals treated with various doses (2.5, 5, 10, 50 or 100 mg/kg) of thalidomide (TM) or with topiramate (TP) 80 mg/kg and controls (C). (A) When the treatments began the day that kindling stimulation started antiepileptic effect of TM was similar with doses of 2.5, 5, 10 and 50 mg/kg (p < 0.01); at a very high dose of 100 mg/kg all epileptic activity abated. (B) When the treatments began on day 14, once the generalized seizures had developed. A paradoxical response was obtained, low doses of TM were more effective than high doses or than topiramate. The data represent the mean values of all recordings after the beginning of treatment (*p < 0.05).
Discussion Our results showed that thalidomide had a strong anticonvulsant effect; in some parameters it was more effective than topiramate. In previous reports we have shown that thalidomide is effective against PTZ-induced in rats (Palencia et al., 2007), which is an experimental model similar to tonic clonic seizures in humans (Fisher, 1989). Antiepileptic drugs that are effective against kindled seizures are usually also effective against partial complex seizures in humans originated in the amygdala or in neighbor limbic areas (McNamara et al., 1980; Fujiwara et al., 2010). The fact that in our studies thalidomide had anticonvulsant activity comparable, and in some parameters superior to that of topiramate, particularly in the duration of the AD, opens new possibilities for research on thalidomide therapy in epilepsy. Although thalidomide is a rather old substance it constitutes a novel antiepileptic whose pharmacological mechanisms are singular, no other sedative with anticonvul-
Strong anticonvulsant effect of thalidomide on amygdaloid kindling sant effect shares the same neural substrates and pathways where thalidomide shows molecular affinity. Nonetheless, the severe teratogenicity of thalidomide restricts its potential clinical use mostly for male epilepsy patients or for females in whom the possibility of pregnancy during therapy is totally excluded. One of the many paradoxes of thalidomide therapy in humans, besides its severe teratogenicity (Parman et al., 1999), is its minor toxicity at low doses is notorious, only long-term peripheral neuropathy is a concern on lasting therapeutic regimes (Tosi et al., 2005). It is notorious the absence of acute toxicity of thalidomide in multiple animal experiments (Somers, 1960); even large doses are devoid of acute side-effects; in the initial toxicological studies the median lethal doses could not be found due to the remarkable tolerance of experimental animals to very high doses of thalidomide (Somers, 1960). Neurological research has shown that in contrast with other sedatives and hypnotics (like phenobarbitone), thalidomide does not produce hypermotility due to excitation or hypomotility due to narcosis. Thalidomide was devoid of narcosis even after maximal doses, in rats it does not affect temperature, heart-rate, blood pressure, respiration or analgesia (Somers, 1960). High doses of thalidomide had a minor effect on motor-coordination in the electroshock model in mice (Friderichs, 1982) but does not impair spontaneous locomotor activity (Somers, 1960; Teo et al., 1999). However, similarly with other hypnotics and sedatives, thalidomide potentiates the narcotic and sedative action of barbiturates, alcohol, chlorpromazine and reserpine. It also counteracts the effects of methylamphetamine and methylphenidate. Nonetheless, in contrast with other sedatives, no addiction to thalidomide has been reported in humans or acute drug toxicity as documented in more than twenty cases of accidental or intentional over dosage (Somers, 1960). In our previous studies using thalidomide in PTZ-induced seizures the antiepileptic effect of thalidomide was more evident in the brainstem component of seizures responsible for the tonic—clonic seizure, than on the forebrain component, responsible for the myoclonic jerks (Palencia et al., 2010; Eells et al., 2004); this dichotomy shows a peculiar mechanism of action of thalidomide as compared with most other anticonvulsants tested on PTZ-induced seizures (Palencia et al., 2007). In amigdaloid kindling independent mechanisms are elicited in the partial and in the generalized seizures (Sikes et al., 1977); C cerebral structures involved in the partial seizures are primarily limbic, at the hippocampus and amygdala, whereas in the generalized seizure participate the substance nigra, globus pallidus, neocortex, thalamus and hippocampus. It seems interesting that in the kindling model the anticonvulsant effect of thalidomide was also manifest in the brainstem as well as in the cortical components. In our study the effects of Thalidomide administration were more evident on the duration of the AD than on changes of behavioral scores; also, the amigdaloid AD did not differ from the cortical AD. It is important to stress the fact that in the model of amygdaloid kindling the treatments that affect the duration of AD do not necessarily modify the behavioral stages (Sikes et al., 1977). In contrast with our results, early experiments on anticonvulsant activity had shown no effects of thalidomide on leptazol or strychnine-induced convulsions (Somers, 1960). The seda-
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tive effects of thalidomide are mediated by neural pathways different from those followed by other sedative substances (Frederickson et al., 1977) which indicate unique actions of thalidomide on the brain (Ionov, 2009), exemplified by the absence in humans of drug dependence and the lack of narcosis induced by thalidomide even after the ingestion of very large doses (Somers, 1960). Thalidomide administration inhibits the expression of tumor necrosis factor (De Sanctis et al., 2010); it seems possible that this effect on cytokines might influence seizures (Li et al., 2011). In contrast with the neural effects of most other sedatives, thalidomide acts selectively as analog of glutamic acid. In comparative studies of hypnotic drugs in cats, thalidomide had hypnotic activity comparable with that of pentobarbital but, in contrast with the latter, thalidomide did not induce ataxia but enhanced the sleep induced by electrical stimulation of the basal forebrain, suggesting a peculiar mechanism of action based on forebrain activation (Frederickson et al., 1977). It is important the fact that the strong antiepileptic properties of thalidomide in amygdaloid kindling were obtained with low doses of the drug (2.5 mg/kg), and it was not substantially increased with much higher doses (50 mg/kg); this unexpected finding which was more evident when thalidomide was started after the seizures had already developed (Fig. 3B) opens the possibility of long-term antiepileptic treatment using low doses of thalidomide in selected patients. If low doses of thalidomide are indeed effective in human epilepsy, secondary effects other than teratogenicity, such as peripheral neuropathy could be prevented. Recently, we have conducted a clinical open labeled pilot study of thalidomide therapy in male patients with chronic refractory epilepsy, intense anticonvulsant results were documented, the mean number of monthly seizures reduced from 26 ± 4 to 7 ± 1 along thalidomide administration (Palencia et al., 2010). Clinical research on the potential therapeutic use of thalidomide in epilepsy seems warranted. If proven successful as antiepileptic, chemical manipulations on the basic molecule of thalidomide, aiming to eliminate its iatrogenic potential (Sumrall et al., 2010) but retaining its anticonvulsant properties would be a valuable avenue for further pharmacological investigation.
Conflict of interests None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The drugs used in these experiments were purchased by the authors. No pharmaceutical company participated in this study in any form.
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