Lack of effect of sildenafil on cocaine-induced convulsions in mice

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1 Pharmacological Reports 2009, 61, ISSN Copyright 2009 by Institute of Pharmacology Polish Academy of Sciences Short communication Lack of effect of sildenafil on cocaine-induced convulsions in mice Dorota Nieoczym, Katarzyna Soca³a, Piotr WlaŸ Department of Animal Physiology, Institute of Biology, Maria Curie-Sk³odowska University, Akademicka 19, PL Lublin, Poland Correspondence: Piotr WlaŸ, Abstract: The convulsant action of cocaine and the proconvulsant effects of sildenafil, a drug which is widely used in the treatment of erectile dysfunction, have been documented both in humans and mice. Since it was reported that sildenafil alone, and in conjunction with cocaine, is used recreationally, the present study was performed to examine the influence of sildenafil on cocaine-induced seizures in mice. We showed that administration of sildenafil (5 20 mg/kg, ip) did not affect latency to clonic seizures induced by ip administration of cocaine at a dose of 85 mg/kg, nor did it influence seizure incidence and mortality. We conclude that sildenafil does not significantly increase the risk of seizures when co-administered with cocaine. Key words: sildenafil, cocaine, convulsions, clonus, mice Introduction Cocaine (benzoylmethyl ecgonine) is a psychostimulatory and highly addictive alkaloid obtained from the leaves of Erythroxylon coca. Cocaine administration in humans causes euphoria, decreased fatigue and hunger, and increased energy and motor activity [6, 9]. However, cocaine overdose can exert deleterious effects on the cardiovascular and central nervous systems [8, 19]. Central hyperexcitation usually leads to a single tonic-clonic seizure episode both in naive and heavy cocaine users, but multiple seizures and status epilepticus were also noted [6]. Both single administration of a high dose of cocaine and repetitive administration of sub-convulsive doses (pharmacological kindling) elicit convulsions in animals [8, 12, 13, 20, 23]. Sildenafil is predominantly used in the treatment of erectile dysfunction of various etiologies. The pharmacological action of sildenafil is closely associated with the nitric oxide (NO)/cyclic guanosine monophosphate (cgmp) pathway. Sildenafil competitively inhibits phosphodiesterase 5 (PDE5), which catalyzes the breakdown of cgmp to guanosine monophosphate (GMP). The increase in cgmp levels enhances the effect of NO and leads to relaxation of the smooth muscle in the corpus cavernosum, thus improving blood flow into the penis. The ability of sildenafil to cross the blood-brain barrier and the presence of PDE5 in different brain areas implicates that this drug may affect various central nervous system functions [21, 34]. Sildenafil has been shown to improve memory and learning processes, exhibits prodepressant and anxiogenic activity, and has proaggressive potential [10, 15, 18, 21, 24, 27, 36]. Antinociceptive activity of silde- 930 Pharmacological Reports, 2009, 61,

2 Sildenafil and cocaine-induced seizures in mice Dorota Nieoczym et al. nafil in animals has also been reported [4, 5], and the proconvulsant activity of sildenafil has been reported in humans [14]. Furthermore, sildenafil decreased the threshold for clonic seizures induced by intravenous (iv) administration of pentylenetetrazole (PTZ) and bicuculine and abolished the adenosine-induced increase in threshold for myoclonic jerks and tonic extension in the iv PTZ seizure threshold test in mice [2, 29]. It was reported that the NO/cGMP pathway affects both glutamatergic and GABAergic neurotransmission so that it may influence seizure susceptibility [26]. Interviews with customers of night clubs in North West England documented that sildenafil was used as recreational drug by 3% of responders. Most of them combined sildenafil with illegal or illicit drugs and alcohol. Some of them took sildenafil concomitantly with cocaine [3]. Considering the proconvulsant effects of both cocaine and sildenafil, one could suspect that sildenafil might enhance the risk of seizures after the use of cocaine. Therefore, the aim of the present study was to examine the effect of sildenafil on seizure activity induced by acute cocaine administration in mice. Materials and Methods The experiments were carried out on male Albino Swiss mice weighing g. The animals were housed in Makrolon cages under the strictly controlled laboratory conditions (ambient temperature of C, relative humidity of 45 55%, 12/12 h light /dark cycle, light on at 6:00 a.m., chow pellets and tap water continuously available). After acclimatization, mice were randomly divided into four experimental groups, each of which consisted of 9 10 mice. The experimental protocol was approved by the Local Ethics Committee at the Medical University of Lublin (license no. 16/2007), and all procedures were in strict compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Sildenafil (1-{[3-(4,7-dihydro-1-methyl-7-oxo-3-propyl- 1H-pyrazolo(4,3-d)pyrimidin-5-yl)-4-ethoxyphenyl]- sulfonyl-}4-methylpiperazine citrate, kindly provided by Polpharma S.A., Starogard Gdañski, Poland) and cocaine (as hydrochloride salt, Merck, Darmstadt, Germany) were dissolved in saline. The animals were given sildenafil at doses of 5, 10, and 20 mg/kg. Control groups received vehicle (saline) only. Sildenafil was administered 30 min before cocaine treatment. All drug solutions were administered intraperitoneally (ip) at a volume of 10 ml/kg. The pretreatment time was the same as that used in previous studies [18, 29] and our pilot experiments. The mice were injected with cocaine at a dose of 85 mg/kg. Immediately afterwards, they were individually placed in transparent boxes and were continuously observed for a period of 12 min, and the latency to the first clonic seizure was measured according to Shimosato et al. [31]. Clonic seizures were defined as episodes of clonic movements of the whole body with loss of righting reflex lasting at least three seconds. Animals which did not convulse during the whole observation period were, thus, given a maximum latency of 720 seconds. In addition, seizure incidence and mortality over the whole 12 min observation period were noted. Sildenafil-induced motor deficits were detected and quantified by using the chimney test, as described by Boissier et al. [7]. In this test, the inability of an animal to climb backwards up through a Plexiglas tube [3.2 cm (inner diameter) 30 cm (length)] within 60 seconds is an indication of motor impairment. To evaluate the influence of sildenafil on the latency to cocaine-induced clonic seizures, the data were analyzed with non-parametric Kruskal-Wallis analysis of variance (ANOVA) on ranks. Statistical analysis of seizure incidence and cocaine-induced mortality was done with the chi-square test. A p value of less than or equal to 0.05 was accepted as a significant difference. Results Observation of the animals in test cages showed no grossly observable behavioral changes in the groups that received sildenafil. The chimney test did not indicate any neurotoxic effects of sildenafil at the doses tested (not illustrated). As shown in Figure 1, cocaine administered at a dose of 85 mg/kg produced seizures in mice, both in control animals and in animals which were pretreated with sildenafil (dosage range 5 20 mg/kg). The treatment with sildenafil slightly prolonged the latency to clonic seizures induced by cocaine. However, Krus- Pharmacological Reports, 2009, 61,

3 Tab. 1. Effects of sildenafil on seizure incidence and mortality following administration of cocaine in mice Sildenafil (mg/kg) Seizure incidence Mortality 0 (control) 7/10 (70%) 4/10 (40%) 5 2/9 (22.2%) 2/9 (22.2%) 10 4/10 (40%) 2/10 (20%) 20 6/10 (60%) 5/10 (50%) The mice were treated with sildenafil (5, 10, or 20 mg/kg; ip) or saline (control group) 30 min prior to cocaine administration (85 mg/kg; ip). Chi-square: seizure incidence? = 5.156, DF = 3, p = 0.161; mortality? = 2.750, DF = 3, p = Fig. 1. The effect of sildenafil on the latency of clonic seizure activity induced by cocaine in mice. The animals were treated with sildenafil (5, 10, or 20 mg/kg, ip; hatched bars) or saline (control group; open bar) 30 min before cocaine (85 mg/kg; ip) injection. The bars represent the mean values (± SEM) of the latency to the onset of clonic seizures. Each group consisted of 9 10 animals. Kruskal- Wallis ANOVA on ranks: H = 4.514, DF = 3, p = kal-wallis ANOVA on ranks did not reveal statistically significant differences between the control and experimental groups. Likewise, no statistically significant differences were found with respect to the influence of different doses of sildenafil on seizure incidence and mortality produced by administration of cocaine at a dose of 85 mg/kg (Tab. 1). Discussion Proconvulsant activity of cocaine and sildenafil has been previously reported both in humans and in mice [2, 14, 23, 29]. Cocaine-induced seizures are caused mainly by the increase in concentration of monoamines (serotonin, dopamine and noradrenaline) in the synaptic cleft, although participation of other neurotransmitter systems has also been reported [8, 23, 35]. The mechanism of proconvulsant activity of sildenafil has not been precisely delineated, but it seems to be connected with an enhancement of the NO effect upon -aminobutyric acid (GABA)- and glutamate-mediated neurotransmission [29]. NO is a neuronal messenger synthesized by NO synthase (NOS). At high levels, it activates guanylyl cyclase (GC) to produce the intracellular second messenger, cgmp [34]. Interactions between the NO/cGMP pathway and neurotransmitter systems, especially glutamatergic and GABAergic systems, have been previously reported [1, 26]. Furthermore, previous reports have shown that NOS inhibitors may exhibit both proconvulsant and anticonvulsant activity or no effect on seizure activity depending on the type of seizures, animal species, and experimental seizure model [25, 28, 30, 32]. The involvement of the NO/cGMP pathway in cocaineinduced seizures has been previously suggested [16, 17, 23]. However, our present results did not uncover any notable impact of sildenafil on the latency of clonic seizures evoked by cocaine administration. Cocaine-induced behavioral effects are believed to be mediated mainly by interaction with monoaminergic neurotransmission. The influence of sildenafil on this neurotransmission was investigated both in vitro and in vivo [11, 36]. Zhu et al. [36] reported that sildenafil enhanced activity of serotonin transporters (SERT) in a time- and dose-dependent fashion in RBL-2H3 cells; no effects on noradrenaline and dopamine transporters were noted. It may be supposed that sildenafil abolished or at least decreased the influence of cocaine on SERT and, thus, might counteract its proconvulsant activity in the present experimental seizure model. On the other hand, studies carried out on rats revealed that sildenafil did not affect the content of monoamines in nigrostriatal tract in the central nervous system. Sildenafil also did not prevent the fall in monoamine levels in 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP)-treated rats. Nonetheless, the data provided by Zhu et al. [36] and Uthayathas et al. [33] do not undoubtedly delineate the influence of sildenafil on monoamine pathways in the central nervous system. 932 Pharmacological Reports, 2009, 61,

4 Sildenafil and cocaine-induced seizures in mice Dorota Nieoczym et al. Park et al. [22] reported that the NO/cGMP pathway, especially NOS which is responsible for NO synthesis and accumulation, participates in cocaineinduced kindling. Inducible NOS (inos) inhibitors (e.g., L-N 6 -(1-iminoethyl)-lysine and ( )-epigallocatechin gallate) prevented development of kindling and protected animals against death. According to that report, enhancement of the NO effect by sildenafil may have a proconvulsant effect, although that result was not confirmed by our study. This study allows us to hypothesize that the mechanism of the proconvulsant action of cocaine in pharmacological kindling substantially differs from that of acute treatment. In opposition to the previous reports that demonstrated a proconvulsant effect of sildenafil both in mice and humans, our results did not show any influence of the PDE5 inhibitor on the latency of cocaineinduced clonic seizures [2, 14, 29]. Proconvulsant activity of sildenafil was previously reported only in models of seizures induced by GABA antagonists, i.e., PTZ and bicuculline, while cocaine-induced seizures are mediated mainly by the enhancement of monoaminergic neurotransmissions. The influence of sildenafil on these systems in the central nervous system has not been precisely elucidated and it seems that only serotonergic neurotransmission is affected by sildenafil [36]. In conclusion, our study suggests that sildenafil does not significantly increase the risk of seizures when co-administered with cocaine. Acknowledgments: The authors wish to thank Prof. Dr. W³adys³aw Lasoñ (Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland) and Polpharma S.A. (Starogard Gdañski, Poland) for a generous gift of cocaine hydrochloride and sildenafil, respectively. References: 1. Ahern GP, Klyachko VA, Jackson MB: cgmp and S- nitrosylation: two routes for modulation of neuronal excitability by NO. 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