Effects of sildenafil on pentylenetetrazol-induced convulsions in mice and amygdala-kindled seizures in rats

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1 Pharmacological Reports 2010, 62, ISSN Copyright 2010 by Institute of Pharmacology Polish Academy of Sciences Short communication Effects of sildenafil on pentylenetetrazol-induced convulsions in mice and amygdala-kindled seizures in rats Dorota Nieoczym 1, Katarzyna Soca³a 1, Chris Rundfeldt 2, Piotr WlaŸ 1 Department of Animal Physiology, Institute of Biology, Maria Curie-Sk³odowska University, Akademicka 19, PL Lublin, Poland Drug-Consult Net, Toepfferspark 2a, D Magdeburg, Germany Correspondence: Piotr WlaŸ, piotr.wlaz@umcs.lublin.pl Abstract: Sildenafil is the first marketed phosphodiesterase 5 inhibitor for the treatment of erectile dysfunction and recently, for pulmonary hypertension. While the treatment was found to be highly effective, several adverse effects are associated with this compound. Among numerous central nervous system-related untoward effects, proconvulsant activity was reported. The purpose of this study was to assess the effect of sildenafil on seizure threshold in rodents. Two seizure models/tests were used: the timed intravenous (iv) pentylenetetrazol (PTZ) infusion test in mice and the amygdala-kindling model in rats. Sildenafil was administered intraperitoneally 30 min before induction of seizures. In the iv PTZ paradigm, the first myoclonic twitch, generalized clonus with loss of the righting reflex, and forelimb tonus were recorded. In the amygdala-kindling model in rats, the following parameters were analyzed: threshold for induction of epileptiform discharges in the stimulated amygdala (afterdischarge threshold, ADT), seizure severity, seizure duration, and afterdischarge duration. Sildenafil (dosage range of 5 40 mg/kg) did not significantly affect the threshold for myoclonic twitches in the timed iv PTZ infusion test in mice but significantly decreased the threshold for clonic seizures at a dose of 20 mg/kg. Sildenafil at all doses tested neither significantly influenced the focal seizure threshold in the amygdala-kindling model of epilepsy in rats nor influenced seizure severity. Sildenafil significantly shortened afterdischarge duration and seizure duration recorded at the ADT current, indicative of a weak anticonvulsant activity. Our results show that sildenafil may have both pro- and anticonvulsant activity, which depends on the experimental model of epilepsy, on animal species and the dose of sildenafil. Based on these data and in view of the clinical observations, sildenafil should be used in patients suffering from epilepsy with caution and only based on a careful individual risk/benefit evaluation. Key words: sildenafil, epilepsy, animal models of epilepsy, pentylenetetrazol, amygdala-kindling, mice, rats Introduction Sildenafil, a selective phosphodiesterase 5 (PDE5) inhibitor, is widely used to alleviate male erectile dysfunction and recently, for treatment of pulmonary hypertension. Its pharmacological activity is associated with the nitric oxide (NO)/cyclic guanosine monophosphate (cgmp) pathway, which plays a key role in the regulation of the contractile state of smooth muscle tissues such as that in corpus cavernosum in the penis. PDE5 hydrolyzes cgmp to guanosine mono- Pharmacological Reports, 2010, 62,

2 phosphate (GMP), and targeting this enzyme with sildenafil causes an increase in the cgmp level [43]. PDE5 is also found in the lungs, platelets, other smooth muscles, heart, placenta, skeletal muscles, pancreas, liver, several gastrointestinal tissues, and in some brain areas [4]. The ability of sildenafil to cross the blood-brain barrier and the presence of PDE5 and other components of the NO/cGMP pathway in the brain [22] predispose this drug to exert some central nervous system (CNS) effects. It was reported that sildenafil enhances neurogenesis [49, 50], which may be beneficial for the treatment of stroke. Its influence on learning and memory consolidation processes was extensively studied in animal models and the results suggest that this PDE5 inhibitor may be useful as a new therapy for memory impairments [8, 28, 32]. In addition, antinociceptive activity of sildenafil in experimental models was also reported [3, 23]. However, some animal studies have also documented a potential of sildenafil to induce aggression [22], to increase anxiety levels [19, 44], and to have abuse potential [41]. Men who use sildenafil have also complained of some common side effects like dizziness, headache, lightheadedness, visual changes, nervousness, insomnia and abnormal dreams [24]. Anxiogenic and proaggressive activity, cognitive enhancement and insomnia induction may have a common underlying mechanism, i.e., excitatory activity in certain CNS structures. Such activation may also result in proconvulsant activity. Central stimulants with cognitive enhancing potential such as ginkgo biloba or caffeine have been shown to precipitate seizures in epilepsy patients [6, 13]. Among the numerous central nervous system untoward effects, the potential proconvulsant activity of sildenafil was indeed reported in clinical use. Gilad et al. [12] were first to describe generalized tonic-clonic seizures in two otherwise healthy men soon after the usage of sildenafil. Furthermore, seizures were also noted during the clinical trials of the drug [12]. Sildenafil decreases the threshold for clonic pentylenetetrazol (PTZ)- and bicuculline-induced seizures in mice [35] and abolishes anticonvulsant activity of adenosine, which increases the threshold for myoclonic and tonic seizures in the iv PTZ seizure threshold test in mice [2]. Proconvulsant activity was also reported for another PDE5 inhibitor, vardenafil [18, 40]. Taken together, it may be suggested that the NO/cGMP pathway may have an influence on seizure activity. The components of this pathway, such as NO and cgmp, affect both excitatory and inhibitory neurotransmission [1, 31], and the imbalance between neural excitation and inhibition is believed to be the main causative factor of epileptic disorders. Men with epilepsy often complain of some sexual disturbances, which include reduction of sexual desire and arousal as well as erectile dysfunctions. These dysfunctions are caused by epilepsy itself but they may result from antiepileptic medication as well. Sildenafil is widely accepted as a safe and effective medication to treat erectile dysfunction in men with epilepsy [39]. Unfortunately, recent results suggest that this drug may bear a certain risk for men suffering from epilepsy. To date it cannot be ruled out that sildenafil may increase the frequency and/or severity of seizures. This prompted us to investigate the activity of sildenafil in two experimental seizure models in rodents, i.e., the timed iv PTZ infusion test in mice and the amygdala-kindling model of epilepsy in rats. Both models are sensitive to detect both proconvulsant and anticonvulsant effects, including effects on seizure threshold. Materials and Methods Animals The experiments were conducted on male Albino Swiss mice weighing g and male Wistar rats weighing g. They were used in this study after at least one week of acclimatization. The animals were housed in polycarbonate cages under the strictly controlled laboratory conditions (ambient temperature of C, relative humidity of 45 55%, a 12/12 light/dark cycle with the light on at 6:00 h; chow pellets and tap water continuously available). The rats were kept individually in cages while mice were housed in groups of ten. The experimental protocol was approved by the Ethical Committee of the Medical University, Lublin, and all the procedures were in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). 384 Pharmacological Reports, 2010, 62,

3 Sildenafil and seizures in animal models Dorota Nieoczym et al. Drugs Sildenafil, (5-[2-ethoxy-5-(4-methylpiperazin-1-yl)sulfonylphenyl]-1-methyl-3-propyl-4H-pyrazolo[4,3-d]pyrimidin-7-one citrate, kindly provided by Fako Ýlaçlarý, Istanbul, Turkey) was dissolved in sterile saline (experiments on mice) or suspended in a 1% aqueous solution of Tween 80 (POCH, Gliwice, Poland) (experiments on rats). The solutions/suspensions were prepared freshly and administered intraperitoneally (ip) at a volume of 10 ml/kg (mice) or 3 ml/kg (rats). The control animals received respective vehicles at appropriate volumes. The pretreatment times were taken from studies evaluating CNS effects of sildenafil [19, 35]. PTZ (Sigma) was freshly dissolved in sterile saline and administered iv as described below. The timed iv PTZ infusion test in mice Thirty minutes following ip administration of sildenafil or its vehicle, the mice were placed in the restrainer and a needle (27G, ¾ in., Sterican, B. Braun Melsungen AG, Melsungen, Germany) was inserted into the lateral tail vein [46]. The needle was attached, using polyethylene tubing (PE20RW, Plastics One Inc., Roanoke, VA, USA), to a plastic syringe that was mounted in a syringe pump (model 351, Sage Instruments, White Plains, NY, USA). The syringe contained a 1% PTZ solution in saline, which was administered into the vein of unrestrained animals at a constant rate of 0.2 ml/min. The time intervals from the start of infusion of PTZ solution to the appearance of three separate endpoints, i.e., first myoclonic twitch, generalized clonus with loss of righting reflex, and forelimb tonus were recorded. The threshold, in mg of PTZ per kg of the body weight, for each endpoint was calculated according to the following formula: infusion duration (s) infusion rate (ml/s) PTZ concentration (mg/ml) PTZ (mg/kg) = weight (kg) The amygdala-kindling model in rats The rats were anesthetized with chloral hydrate (360 mg/kg, ip) and received stereotaxic implantation of a bipolar electrode in the right basolateral amygdala. The electrode consisted of two twisted Teflon - coated stainless steel wires (250 µm in diameter, Science Products GmbH, Hofheim, Germany) separated at the tip by 0.5 mm. The following stereotaxic coordinates were used according to the brain atlas of Paxinos and Watson [30]: AP 2.2 mm, L 4.8 mm, V 8.5 mm. All coordinates were measured from the bregma. One stainless steel screw, placed over the left parietal cortex, served as the indifferent reference electrode, two others were screwed in the skull to anchor the electrode assembly. Both bipolar and reference electrodes were connected to miniature female plugs, and the assembly was fixed on the skull with dental acrylic cement. After electrode implantation, the animals were treated with antibiotics for one week to prevent infection. After a post-operative period of two weeks, constant current stimulations (500 µa, 1 ms, monophasic square-wave pulses at 50 Hz for 1 s), generated by a stimulator (S48, Grass Technologies, West Warwick, RI, USA) with an isolated constant-current unit (PSIU6, Grass Technologies) were delivered to the amygdala once daily (five times per week) until at least 10 fully kindled stage 5 seizures were elicited. Seizure severity was ranked according to a modified [47, 48] scoring system of Racine [33]: 1, immobility, eye closure, ear twitching, twitching of vibrissae, sniffing, facial clonus; 2, head nodding associated with more severe facial clonus; 3, clonus of one forelimb; 3.5, bilateral forelimb clonus without rearing; 4, bilateral forelimb clonus accompanied by rearing; 4.5, falling on their side (without rearing), loss of righting reflex accompanied by generalized clonic seizures; 5, rearing and falling on the back accompanied by generalized clonic seizures. Seizure duration was the duration of limbic seizures (stage 1 2) and/or motor seizures (stage 3 5); limbic seizure activity sometimes occurring after termination of motor seizures was not included in seizure duration. Afterdischarges were defined as spikes with a frequency of at least 1 Hz and with amplitude of at least twice the pre-stimulation baseline in the EEG recorded from the site of stimulation. Secondary afterdischarges that sometimes followed primary afterdischarges generated by the epileptic focus [9] were not included in afterdischarge duration. Pharmacological Reports, 2010, 62,

4 Afterdischarge threshold (ADT), i.e., the threshold for induction of epileptiform discharges in the stimulated amygdala, was evaluated in eight fully-kindled rats. ADT was determined after administration of sildenafil or its vehicle by administering a series of stimulations at intervals of 1 min with increasing steps of about 20% of the previously applied current until an afterdischarge of at least a 3 s duration was evoked. Electrical stimulations for each individual animal began about three 20%-steps below its control ADT value taken 2 3 days before drug experiment. At the ADT current, seizure severity, seizure duration and afterdischarge duration were recorded and analyzed. Statistics All data are presented as the means ± SEM. Statistical analysis of data from the iv PTZ seizure threshold test in mice was performed by the one-way analysis of variance (ANOVA) followed by the Tukey s post-hoc test for multiple comparisons. Differences in the ADT and seizure severity were calculated by the Wilcoxon signed-rank test for paired replicates, while afterdischarge duration and seizure duration were analyzed by Student s t-test for paired replicates. A p value less than or equal to 0.05 was considered a statistically significant difference. These statistical calculations were performed with SigmaStat 2.03 (SPSS, Erkrath, Germany). Results Effects of sildenafil in the timed iv PTZ infusion test in mice Sildenafil (dosage range of 5 40 mg/kg) significantly decreased the threshold for clonic seizures in the timed iv PTZ infusion test in mice (one-way ANOVA: F(4, 44) = 3.194; p = 0.022). Post-hoc analysis revealed that only at a dose of 20 mg/kg did the drug reduce the threshold significantly (60.00 ± 2.47 mg/kg in control group to ± 2.52 mg/kg, p = 0.009) (Fig. 1B). The compound did not significantly affect threshold for the myoclonic twitches (one-way ANOVA: F(4, 43) = 2.328; p = 0.071). It should, however, be noted that there was a tendency towards Fig. 1. Effect of sildenafil on the threshold for the first myoclonic twitch (panel A), generalized clonic seizures (panel B) and forelimb tonic extension (panel C) intheiv PTZ seizure threshold test in mice. Data are presented as the means + SEM (n = 7 11). Sildenafil or its vehicle was administered ip 30 min before the test. Data were analyzed by the one-way analysis of variance (ANOVA) followed by Tukey s post-hoc test. *p < 0.05 vs. control group threshold-decreasing activity (Fig. 1A). The susceptibility of the mice to the PTZ-induced forelimb tonic seizures was not changed significantly by the dosage regimen used (Fig. 1C, one-way ANOVA: F(4, 35) = 0.689; p = 0.605). 386 Pharmacological Reports, 2010, 62,

5 Sildenafil and seizures in animal models Dorota Nieoczym et al. Fig. 2. Effect of sildenafil on ADT (panel A), seizure severity (panel B), seizure duration (panel C), and afterdischarge duration (panel D) in the amygdala-kindling model in rats. Data are presented as the means + SEM (n = 7 8). Grey bars indicate data obtained following sildenafil administration; open bars indicate control groups, which were treated with 0.5% Tween 80 in saline. Sildenafil (5, 10, 20 and 40 mg/kg) or its vehicle was administered ip 30 min before the test. Control readings from the same group of rats were obtained 2 3 days before the respective drug experiments. Data were analyzed by Wilcoxon signed-rank test for paired replicates (ADT and seizure severity) and Student s t-test for paired replicates (seizure duration and afterdischarge duration). *p < 0.05 vs. respective control experiment Effect of sildenafil on the focal seizure threshold in amygdala-kindled rats The pre-stimulus EEG recorded after administration of sildenafil was not different from that of the vehicle sessions, as far as could be concluded from visual inspection. Sildenafil at the doses tested (range of 5 40 mg/kg) did not significantly affect the focal seizure threshold (Fig. 2A) nor did it influence seizure severity (Fig. 2B). Instead, some tendency towards threshold-increasing activity was visible. Sildenafil significantly reduced afterdischarge duration and seizure duration recorded at the ADT current. Specifically, sildenafil at doses of 5 mg/kg and 40 mg/kg re- Pharmacological Reports, 2010, 62,

6 duced seizure duration by 14% (p = 0.046) and 24% (p = 0.018), respectively (Fig. 2C). A reduction of 25% at the dose level of 20 mg/kg did not reach level of significance. As depicted in Figure 2D, a statistically significant reduction of afterdischarge duration was produced by sildenafil at doses of 10 and 40 mg/kg (p = and p = 0.042, respectively) while a similar reduction seen at 20 mg/kg did not reach level of significance. Discussion In the present study, we demonstrated that sildenafil, a selective PDE5 inhibitor, decreased the threshold for clonic seizures produced by an iv administration of PTZ in mice, while in amygdala-kindled rats, no indications of a proconvulsant reduction of the seizure threshold was found. Both models are very sensitive methods for assessing seizure threshold [21, 46]. Instead, in the amygdala-kindling model some anticonvulsant activity became apparent since duration of afterdischarges and seizure duration induced at the afterdischarge threshold was reduced. However, both effects were not strictly dose-dependent. Proconvulsant activity of sildenafil was previously reported both in humans [12] and mice [35]. The mechanism of proconvulsant effect of sildenafil is not clearly determined, but the experimental evidence points to the involvement of the NO/cGMP pathway because interactions between this pathway and both the excitatory and inhibitory neurotransmissions were previously found [1, 5, 31]. Targeting PDE5 with sildenafil leads to increases in the cgmp levels in cells. High levels of cgmp may activate protein kinase G (PKG), which phosphorylates the 2 and 3 subunits of the GABA receptor complex, thereby reducing the GABA-mediated inhibition. The decrease in GABAergic transmission intensifies glutamate release which may precipitate seizures [1]. The interplay between the NO/cGMP pathway and neurotransmitter systems is not fully understood. Stimulation of N-methyl-D-aspartate (NMDA) receptors leads to an increase in intracellular Ca 2+ concentration and activates NO and cgmp production. The components of the NO/cGMP pathway may regulate release of neurotransmitters by negative feedback mechanisms. NO and its donors may both enhance and inhibit the glutamate neurotransmission [5, 31]. High levels of NO in cells may also inhibit the reuptake of glutamate [5]. The glutamate-mediated neurotransmission is also directly controlled by cgmp. Increases in cgmp concentration, treatment with its analogs or phosphodiesterase inhibition limit the release of glutamate from neurons in the hippocampus and cerebral cortex [31]. In addition, the influence of cgmp on the glutamatergic system seems to be biphasic because very high doses of cgmp increase the release of glutamate [31]. The previous reports suggest that high levels of cgmp, as caused by inhibiting PDE5 with sildenafil, may affect neuronal excitability and eventually predispose to convulsions. So far, the influence of cgmp on seizure activity has not been extensively investigated. An increase in the concentration of cyclic nucleotides, including cgmp, was noted in different brain structures and in the cerebrospinal fluid in laboratory animals made to convulse [11, 16, 26, 27, 38]. Previous reports do not allow for the conclusion as to whether the proconvulsant activity of sildenafil is directly related to the accumulation of cgmp in the cells or only to the enhancement of the NO effects. NO synthase inhibitors may exhibit proconvulsant [34, 37, 42], anticonvulsant [29, 37, 42] or no effect on seizure activity [25, 37, 42]; the effect was dependent on the animal species, type of seizures, and experimental seizure models. Moreover, it was shown that low concentrations of NO exert neuroprotective activity while its high concentrations may actually be neurotoxic [7]. Both our results and results presented by Riazi et al. [35] demonstrate that sildenafil decreases the threshold for clonic seizures but it does not influence generalized tonic seizures. Moreover, our study revealed its lack of effect on myoclonic twitches in the iv PTZ seizure threshold test in mice. The previous report also revealed that inhibition of guanylyl cyclase by methylene blue and NOS by N G -nitro-l-arginine- -methyl-ester reversed the effect of sildenafil on the clonic seizures, while L-arginine (NOS substrate) and sodium nitroprusside (NO donor) enhance its proconvulsant activity. It was thus postulated that the differences in the effect of sildenafil on clonic and tonic seizures are dependent on the brain structures, which control these two seizure types [35]. Clonic seizures are governed by forebrain, while tonic seizures are generated by neurons in the brain stem [10]. The participation of the NO/cGMP pathway in myoclonic twitches and tonic seizures cannot be unequivocally 388 Pharmacological Reports, 2010, 62,

7 Sildenafil and seizures in animal models Dorota Nieoczym et al. excluded because sildenafil reversed anticonvulsant activity of adenosine in relation to these two kinds of seizures in the iv PTZ seizure threshold test in mice [2]. Inhibition of NO synthesis by NOS inhibitors exhibited anticonvulsant activity against tonic [15, 17] and clonic seizures [35] induced by PTZ. It seems that sildenafil shows proconvulsant activity in seizures generated by antagonists of the GABA A receptor. While in the PTZ model seizures are induced due to a direct pharmacological interaction with the most important inhibitory neurotransmitter system, the increased seizure susceptibility in the amygdalakindling model is related to a complex neuronal adaptation involving neuronal plasticity. While the PTZ model is considered to be a model of seizures, the amygdala-kindling model is considered to be a model of epilepsy in rats. In this model, sildenafil did not lower the threshold for induction of neuronal synchronization, but instead tended to show anticonvulsant activity; it limited duration of behavioral seizures and duration of afterdischarges, both recorded at the ADT current. Previous results showed that downregulation of the NO/cGMP pathway may induce changes in neuronal excitability, which may lead to convulsant events. N G -nitro-l-arginine, an inhibitor of NOS, increases kindling rate in the amygdala-kindling model of epilepsy in rats, particularly in its initial stage [36]. Other NOS inhibitors, like methyl-l-arginine and nitro-l-arginine, facilitate the initiation of interictal-like spontaneous bursts in the rats hippocampal slices in in vitro model of kindling [14]. The activation of the cgmp/no pathway by sildenafil may thus exhibit the opposite effects, leading to a possible explanation of the weak anticonvulsant activity observed in this study. Increases in cgmp concentrations have been noted in kindled rats directly after seizures [38, 45]. These increased levels may be an effect of the enhancement of NMDA receptors activity, which is followed by activation of the NO/cGMP pathway. Furthermore, this change may have a protective effect. This hypothesis is confirmed by the present study because sildenafil limited seizure activity in the kindling model. It is known that a high level of cgmp may diminish the presynaptic release of glutamate, which in turn reduces excitability and decreases seizure susceptibility [31]. Inhibition of PDE5 and increases in cgmp concentrations did not influence seizure threshold in amygdala-kindled rats. The effect of sildenafil on seizure duration and afterdischarge duration suggests that PDE5 inhibition limits spread of the convulsive activity from the focus [20]. In conclusion, our results show that sildenafil may have both pro- and anticonvulsant activity, which depends on the experimental model of epilepsy, animal species, and the dose of sildenafil. Our data indicate that the NO/cGMP pathway contributes to the formation and spreading of seizure activity in the CNS. Our findings and similar findings made by others [2, 35] also substantiate the case observations made during the clinical development and clinical use of sildenafil. The data indicate that not all patients with epilepsy may be at risk to precipitate seizures upon intake of sildenafil. Only patients in whom impairment of GA- BAergic inhibition is the underlying mechanism of epilepsy may be particularly at risk for precipitation of seizures. However, the underlying cause of epilepsy is often not known, making it difficult to identify the sildenafil related risk beforehand. This medication should thus be used with caution in patients suffering from epilepsy and only based on a careful individual risk/benefit evaluation. However, further studies are needed to estimate the risk/benefit ratio. Acknowledgment: The authors wish to thank Fako Ýlaçlarý (Istanbul, Turkey) for the generous gift of sildenafil. References: 1. Ahern GP, Klyachko VA, Jackson MB: cgmp and S-nitrosylation: two routes for modulation of neuronal excitability by NO. Trends Neurosci, 2002, 25, Akula KK, Dhir A, Kulkarni SK: Nitric oxide signaling pathway in the anti-convulsant effect of adenosine against pentylenetetrazol-induced seizure threshold in mice. Eur J Pharmacol, 2008, 587, Ambriz-Tututi M, Velázquez-Zamora DA, Urquiza- Marín H, Granados-Soto V: Analysis of the mechanism underlying the peripheral antinociceptive action of sildenafil in the formalin test. Eur J Pharmacol, 2005, 512, Bender AT, Beavo JA: Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev, 2006, 58, Bogdanov MB, Wurtman RJ: Possible involvement of nitric oxide in NMDA-induced glutamate release in the rat striatum: an in vivo microdialysis study. Neurosci Lett, 1997, 221, Bonilha L, Li LM: Heavy coffee drinking and epilepsy. Seizure, 2004, 13, Pharmacological Reports, 2010, 62,

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