Intranasal pitavastatin attenuates seizures in different experimental models of epilepsy in mice
a b s t r a c t
This study was carried out to evaluate the effect of intranasal pitavastatin (PVS) on pentylenetetrazole (PTZ)- induced seizures, increasing current electroshock (ICES) seizures, and status epilepticus in mice. Intranasal PVS, 0.5 and 1.0 mg/kg, showed significant increase in latency to PTZ-induced seizures and ICES seizure threshold compared to control; however, the effects were dose-dependent and were more significant at higher dose. Further, intranasal PVS (1.0 mg/kg) but not intravenous PVS (50.0 mg/kg) showed effective protection against PTZ-induced status epilepticus. No impairment in cognitive functions was observed following intranasal PVS (1.0 mg/kg), thus making it a prospective therapeutic approach for acute seizures and status epilepticus.
1.Introduction
Epilepsy is a serious CNS disorder affecting more than 70 million people worldwide, making it one of the most common neurological dis- eases globally [1]. Epilepsy is characterized by recurrent, unprovoked seizures as a result of electrical instabilities in the brain that can range from brief lapses of attention or muscle jerks to severe and prolonged convulsions [1]. The 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) are potent cholesterol-lowering drugs which also possess beneficial antiinflammatory, antioxidant, immuno- modulatory, and antiexcitotoxic effects [2–4]. Statins have proven neuroprotective effects in several neurological diseases such as Alzheimer’s and Parkinson’s disease, cerebral ischemia, multiple sclero- sis, and traumatic brain injury. Several in vitro and in vivo studies have demonstrated the potential antiseizure properties of statins, par- ticularly atorvastatin [5], simvastatin [6], fluvastatin [7], lovastatin [8], and pitavastatin [9] in epilepsy induced by pentylenetetrazole (PTZ), pilocarpine, or increasing current electroshock (ICES). Most of the pub- lished reports demonstrated the antiseizure activity of statins following oral or intravenous doses at high concentration. Probably, statin has to cross highly lipophilic blood–brain barrier, following oral or intravenous dosing, in order to produce the effect resulting in use of their high doses [4]. Further, dose-dependent side effects of statins, in particular myopa- thy, rhabdomyolysis, and cognitive impairment are of concern [10,11]. Recent development in drug delivery highlights the use of intranasal administration as a novel route for rapid administration of drugs to brain [12–15]. Intranasal drug administration is painless, does not re- quire skilled personnel, and is immediately available for all patients. The nasal cavity permits topically administered drugs to rapidly achieve effective blood levels avoiding gastrointestinal destruction and hepatic first-pass metabolism, improving bioavailability compared with oral ad- ministration [13]. Additionally, the intranasal delivery exploits nose– brain pathway to bypass the blood–brain barrier and deliver the drug directly to the brain [14,15]. The present study explores the neuropro- tective effect of intranasal pitavastatin (PVS), a new statin that exhibits potent antihyperlipidemic activity, against seizures induced by PTZ [16], ICES [17], and in emergency condition—status epilepticus [18]. Further, the effect of intranasal PVS on cognitive impairment was assessed to
evaluate the safety of the treatment.
2.Materials and methods
Pitavastatin was received as gift sample from Mylan Laboratories Limited (Hyderabad, India). Pentylenetetrazole (PTZ) and phenytoin
were purchased from Sigma Aldrich, Inc. (St. Louis, MO, USA). Pitavastatin solution was prepared by dissolving its weighed quantity in aqueous solution of PEG 400. Pentylenetetrazole and phenytoin solu- tions were prepared in normal saline.Healthy Swiss albino male mice weighing between 20 g and 35 g were used for the studies. The animals were housed in a temperature-controlled room (18–24 °C) and provided with free access to food and water. All care and handling of animals were in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), with the approval (Approval No. 114) of Institutional Animal Ethics Committee, Jamia Hamdard, New Delhi.To assess the effects of intranasal PVS on seizures, status epilepticus, and cognition, the animals were divided into different groups with each group containing 6 mice (n = 6). Five groups were used for PTZ- and ICES-induced seizure response study. Two groups received different doses of intranasal PVS, i.e., 0.5 and 1.0 mg/kg. Untreated animals and vehicle-treated (intranasal PEG aqueous solution) animals were used as controls, whereas group receiving intravenous PVS (50.0 mg/kg) was used for comparison. To study the acute effects of intranasal PVS on seizures, the experiments were performed on the seventh day of dosing, 30 min after the administration of PVS [5]. For status epilepticus studies, three groups (n = 6) were used. One group received intranasal PVS (1.0 mg/kg), untreated animals served as the control, and intrave- nous PVS (50.0 mg/kg) was used for comparison. To study the effects of intranasal PVS in status epilepticus, where immediate action is required, a single-dose protocol was followed, and the experiment was performed 30 min after a single dose of PVS [5]. For cognitive assessment, two groups (n = 6) were used, and intranasal PVS (1.0 mg/kg) was evaluated against untreated animals as control. The ex- periments were performed on the seventh day of dosing, 30 min after the administration of PVS.
3.Experimental design
PTZ seizures were induced by intraperitoneal injection of PTZ solu- tion at a dose of 60 mg/kg. This dose of PTZ produced myoclonic jerks and generalized seizures in all the animals without mortality. The laten- cy to myoclonic jerks and generalized seizures was observed immedi- ately after PTZ injection for a period of 30 min. In the absence of seizures, 30 min (1800 s) was taken as the latency time [16].The ICES test was used to evaluate the anticonvulsant effect of PVS. Electroshock, starting with a current of 2 mA, was applied via ear electrode (forceps style) using electro-convulsometer (Microteknik, India). The electroshock was given as a single train of pulse for 0.2 s with linearly increasing intensity of 2 mA/2 s. The current at which tonic hind limb extension (HLE) occurred was recorded as the seizure threshold current. If no tonic HLE was observed by a current of 30 mA, electroshock was terminated, and this cutoff current was used in the analysis [17].Status epilepticus was induced in the mice by subcutaneously injecting PTZ at a dose of 80 mg/kg in the loose skin behind the neck [18]. The volume of the injection was 0.1 mL/10 g body weight. Two hours prior to PTZ administration, phenytoin sodium (40 mg/kg) dissolved in alkalinized saline was administered intraperitoneally (volume: 0.1 mL/10 g body weight) to prevent the terminal tonic HLE produced by PTZ. The time required for the development of unequivocal sustained clonic seizure activity involving the limbs (isolated myoclonic jerks were not counted) was carefully noted. Seizure-free state for a period of 30 min was taken as protection. Observations were made 30 min after administration of PVS.Cook’s pole climbing apparatus consists of a grid floor composed of stainless steel rods capable of delivering electric shock.
A pole, 2.5 cm in diameter, hangs inside the chamber through a hole in the upper cen- ter of the chamber. The animal under investigation was gently placed on the wooden platform set in the center of the grid floor. This platform served as a shock-free zone. When the mouse stepped down and placed all its paws on the grid floor, electric shock (20 V, 50 Hz, 1 mA, 1 s) was delivered for 15 s and the step-down latency (SDL) was recorded. SDL was defined as the time taken by the mouse to step down from wooden platform to grid floor with its entire paw on the grid floor. Mice showing SDL in the range (2–15 s) during the first test were used for the second session and the retention test. The second session was carried out 90 min after the first test. When mice stepped down before 60 s, electric shock was delivered for 15 s. During the second test, animals were re- moved from shock-free zone if they did not step down for a period of 60 s. Memory retention was tested after 24 h in a similar manner, except that electric shocks were not applied to the grid floor. Each subject was again placed on the platform, and the SDL was recorded, with an upper cutoff time of 600 s.Mice was dropped one at a time in a plexiglass cylinder (height 25 cm and diameter 10 cm) containing water up to a height of 9 cm at room temperature and left for 15 min. After a brief spell of vigorous activity, they showed a posture of immobility which is characterized by floating motionless in the water, making only those movements nec- essary to keep the head above water. This immobility reflects a state of depression. After allowing 1 min for acclimatization, each animal was observed for 15 min for immobility.
Thus, immobility time (i.e., total du- ration of immobility in a period of 5 min) was recorded for each animal.The elevated plus maze served as the exteroceptive behavioral model (wherein the stimulus existed outside the body) to evaluate learning and memory in mice. The apparatus consisted of two open arms (50 cm × 10 cm) and two covered arms (50 cm × 40 cm × 10 cm). The arms extended from a central platform (10 cm × 10 cm) and the maze was elevated to a height of 50 cm from the floor. The an- imals were placed individually at the either end of the open arms and allowed to enter the closed arms. If the animal fails to enter the closed arm within 180 s during first screening, it was not included in the exper- iment. During training, if the animal did not enter the closed arm within 180 s, it was gently pushed in the closed arm. To become acquainted with the maze, the animals were allowed to explore the maze for 20 s after reaching the closed arm and then returned to their home cage. The learning was tested 30 min later on the same day, and the animals were re-tested 24 h after the first day training to test the retention of memory. The time taken by the animal to move from the open arm to the closed arm is noted as transfer latency (TL). A time of 180 s was taken as cutoff, and animals not entering the closed arm in this period were assigned the TL of 180 s. A long latency period to reach enclosed arm indicates poor retention compared to significantly shorter latencies. All results are expressed as mean ± standard error of the mean (SEM) for 6 animals (n = 6). The data obtained were analyzed using one-way analysis of variance (ANOVA) followed by Student’s t-test. A value of p b 0.05 was considered significant for comparison.
4.Results
The results of the PTZ-induced seizure studies are shown in Table 1. In the untreated group (control), the latency to myoclonic jerk and generalized seizures was found to be 68.9 ± 2.15 and 91.7 ± 2.89 s, re- spectively. The vehicle-treated group showed an insignificant increase (p N 0.5) in latency to myoclonic jerk (71.0 ± 2.18 s) and generalized seizures (94.2 ± 4.20 s) compared with the control group. Intranasal PVS, 0.5 and 1.0 mg/kg, increased the latency to myoclonic jerk (106.0± 2.90 and 138.0 ± 4.15 s, respectively) and generalized seizures (148.2 ± 3.47 and 210.1 ± 6.80 s, respectively) dose-dependently, and the increase in latency was significant (p b 0.001 and p b 0.0001, re- spectively) compared with the control group.Following intravenous PVS treatment (50.0 mg/kg), the increase in latency to myoclonic jerk (102.8 ± 1.97 s) and generalized seizures (146.0 ± 5.55 s) was significant compared with the control group; how- ever, the increase was insignificantly different (p N 0.5) from lower dose (0.5 mg/kg) intranasal PVS.The results of the ICES studies showed that intranasal PVS, at both the doses, i.e., 0.5 and 1.0 mg/kg, but not intravenous PVS (50.0 mg/kg), increased the seizure threshold current to hind limb ex- tension and showed significant (p b 0.001 and p b 0.0001, respectively) protection against ICES-seizures compared to vehicle-treated and con- trol group. The increase in seizure threshold current by intranasal PVS was found to be dose-dependent. In PTZ-induced status epilepticus, no significant difference was ob- served in latency to generalized seizure and duration of clonic seizures be- tween intravenous PVS (3.50 ± 0.45 and 31.15 ± 1.99 min, respectively) and control group (3.34 ± 0.29 and 30.21 ± 2.96 min, respectively). However, intranasal PVS (1.0 mg/kg) significantly (p b 0.0001) in- creased the latency time (25.01 ± 1.14 min), decreased the duration of clonic seizures (16.88 ± 1.17 min), and showed effective protection against status epilepticus.
In addition, the result showed that treatment with intranasal PVS decreased the mortality rate to 33.3% (2/6) com- pared to 100.0% (6/6) in control group.Pole climbing test, forced swim test and elevated plus maze test were performed to evaluate the effect of intranasal PVS treatment on cognitive impairment (memory loss and confusion), one of the most se- rious side-effect of statin class of drugs. The results of these studies are shown in Table 2. The results showed that intranasal PVS did not signif- icantly alter the acquired and transfer latency time in pole climbing test (p N 0.5), immobility time in forced swim test (p N 0.5) and the acquired and transfer latency time in elevated plus maze test (p N 0.5) compared with control group. The results suggested that the intranasal PVS, over the treatment period used in the present study, did not cause cognitive impairment in the animals.
5.Discussion
This study evaluates the protective role of intranasal PVS against PTZ-induced seizures, ICES-induced seizures and PTZ-induced status epilepticus. The results of the present study showed that PVS exert pro- tective effect against PTZ seizures. Our results are in agreement with the recent studies which also reported the similar effects of PVS [9]. Inter- estingly, high dose intranasal PVS showed better protection against PTZ seizures compared to intravenous PVS. Also, intranasal PVS at both doses, but not intravenous PVS, increased the seizure threshold current and showed protection against ICES seizures. This could possibly be due to intranasal route, which is known to circumvent high- ly restrictive blood–brain barrier. Intranasal delivery exploits the olfac- tory and trigeminal neuronal pathways, bypass the blood–brain barrier, and deliver the drug directly to the brain [14,15]. Improved brain avail- ability for treatment of epilepsy via intranasal administration has been reported for variety of drugs [19].
In addition, PVS is highly bound to plasma protein which further reduces its activity in brain following intravenous administration. The results presented here evidently suggest that intranasal PVS has beneficial neuroprotective effects against both myoclonic and generalized seizures. The molecular mechanism of the anticonvulsant properties of PVS has not been established in the present study; however, it could be postulated that PVS, like other statins, might be effective against seizures via combination of more than one cholesterol-independent pleiotropic effects including (1) reduction of protein isoprenylation, (2) lipid raft disruption, (3) suppression of in- flammatory response, and (4) excitoprotection mediated through the inhibition of calcium-dependent calpain activation, ROCK inhibition, activation of the PI3K pathway, and increased APP cleavage [4].Since, intranasal route is considered as a rapid means of delivering drugs to the brain, we further evaluated the effect of intranasal PVS in emergency conditions like status epilepticus induced in animals by PTZ. We found that intranasal PVS (1.0 mg/kg) significantly increases latency to status epilepticus, decreases duration of seizure, and decreases mortality compared to intravenous PVS and control group. The noninvasive nature and ease of self-administration shows that in- tranasal PVS has the potential for emergency conditions such as status epilepticus.Various studies have shown the potential risk of cognitive impair- ment following statin treatment [10,11], and thus, we evaluated the effect of intranasal PVS on cognitive impairment using forced swim test, pole climbing test and elevated plus maze test. In the present study, no decrements in cognitive functions were observed following intranasal PVS treatment. The study highlights the safety of intranasal PVS treatment.
6.Conclusion
Intranasal PVS showed beneficial neuroprotection against acute seizures as well as against emergency condition—status epilepticus without producing any cognitive impairment. Our current findings will encourage new investigations disclosing the molecular mecha- nisms to establish the efficacy and safety of intranasal Pitavastatin PVS.