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Keywords:

  • New anticonvulsant drug;
  • Status epilepticus;
  • Pilocarpine-induced SE rat model;
  • Soman-induced SE rat model;
  • Pharmacokinetic-pharmacodynamic correlation

Summary

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Summary
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Purpose: sec-Butyl-propylacetamide (SPD) is a one-carbon homolog of valnoctamide (VCD), a central nervous system (CNS)–active amide derivative of valproic acid (VPA) currently in phase II clinical trials. The study reported herein evaluated the anticonvulsant activity of SPD in a battery of rodent seizure and epilepsy models and assessed its efficacy in rat and guinea pig models of status epilepticus (SE) and neuroprotection in an organotypic hippocampal slice model of excitotoxic cell death.

Methods:  The anticonvulsant activity of SPD was evaluated in several rodent seizure and epilepsy models, including maximal electroshock (MES), 6-Hz psychomotor; subcutaneous (s.c.) metrazol-, s.c. picrotoxin, s.c. bicuculline, and audiogenic, corneal, and hippocampal kindled seizures following intraperitoneal administration. Results obtained with SPD are discussed in relationship to those obtained with VPA and VCD. SPD was also evaluated for its ability to block benzodiazepine-resistant SE induced by pilocarpine (rats) and soman (rats and guinea pigs) following intraperitoneal administration. SPD was tested for its ability to block excitotoxic cell death induced by the glutamate agonists N-methyl-d-aspartate (NMDA) and kainic acid (KA) using organotypic hippocampal slices and SE-induced hippocampal cell death using FluoroJade B staining. The cognitive function of SPD-treated rats that were protected against pilocarpine-induced convulsive SE was examined 10–14 days post-SE using the Morris water maze (MWM). The relationship between the pharmacokinetic profile of SPD and its efficacy against soman-induced SE was evaluated in two parallel studies following SPD (60 mg/kg, i.p.) administration in the soman SE rat model.

Key Findings:  SPD was highly effective and displayed a wide protective index (PI = median neurotoxic dose/median effective dose [TD50/ED50]) in the standardized seizure and epilepsy models employed. The wide PI values of SPD demonstrate that it is effective at doses well below those that produce behavioral impairment. Unlike VCD, SPD also displayed anticonvulsant activity in the rat pilocarpine model of SE. Thirty minutes after the induction of SE, the calculated rat ED50 for SPD against convulsive SE in this model was 84 mg/kg. SPD was not neuroprotective in the organotypic hippocampal slice preparation; however, it did display hippocampal neuroprotection in both SE models and cognitive sparing in the MWM, which was associated with its antiseizure effect against pilocarpine-induced SE. When administered 20 and 40 min after SE onset, SPD (100–174 mg/kg) produced long-lasting efficacy (e.g., 4–8 h) against soman-induced convulsive and electrographic SE in both rats and guinea pigs. SPD ED50 values in guinea pigs were 67 and 92 mg/kg when administered at SE onset or 40 min after SE onset, respectively. Assuming linear pharmacokinetics (PK), the PK–PD (pharmacodynamic) results (rats) suggests that effective SPD plasma levels ranged between 8 and 40 mg/L (20 min after the onset of soman-induced seizures) and 12–50 mg/L (40 min after the onset of soman-induced seizures). The time to peak (tmax) pharmacodynamic effect (PD-tmax) occurred after the PK-tmax, suggesting that SPD undergoes slow distribution to extraplasmatic sites, which is likely responsible for antiseizure activity of SPD.

Significance:  The results demonstrate that SPD is a broad-spectrum antiseizure compound that blocks SE induced by pilocarpine and soman and affords in vivo neuroprotection that is associated with cognitive sparing. Its activity against SE is superior to that of diazepam in terms of rapid onset, potency, and its effect on animal mortality and functional improvement.

Since 1993 more than a dozen new antiepileptic drugs (AEDs) have been brought to the market for the treatment of epilepsy. Each new therapy has provided substantial benefit to patients in the way of improved seizure control, fewer adverse events, and/or more favorable pharmacokinetics (PK). Despite the availability of several new AEDs, there still remains a considerable need for developing new medications for patients with highly refractory epilepsy. In fact, it is estimated that 30% of the patients with epilepsy still have uncontrolled seizures. All new AEDs for the symptomatic treatment of epilepsy have evolved from early evaluation in highly predictive animal seizure and epilepsy models (Bialer et al., 2004; Rogawski, 2006; Bialer & White, 2010). The investigational AED sec-butyl-propylacetamide (SPD; Fig. 1), a one-carbon homolog of valnoctamide (VCD; Fig. 1), was submitted to the National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (NINDS) Anticonvulsant Screening Program (ASP) in September 2008. Over the following 3 years, SPD was evaluated in a battery of rodent electrical and chemoconvulsant seizure models. The results from this investigation, reported herein, suggest that SPD possesses a unique and broad-spectrum antiseizure profile that is comparable to that of valproic acid (VPA).

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Figure 1.   Chemical structures of valproic acid (VPA), valpromide (VPD), valnoctamide (VCD), and sec-butyl-propyl carboxamide (SPD). Asterisks denote chiral centers.

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Status epilepticus (SE) is not a disease, but is a manifestation of an underlying central nervous system (CNS) insult or systemic pathology that affects CNS function. SE results when there is a failure of those inherent factors that would normally function to stop seizures. SE can result when there is a decrease in inhibition or an increase in excitation, or a combination of both. Based on two large population-based studies in Richmond, VA (DeLorenzo et al., 1996) and Rochester, MN (Hesdorffer et al., 1998), it is estimated that there are between 60,000 and 150,000 episodes of SE and 55,000 SE-related deaths each year. Between 4% and 16% of patients with epilepsy will have at least one episode of SE, and one third of the cases of SE occur as the presenting symptom in patients with a first unprovoked seizure (Hauser et al., 1990).

The prognosis of the patient with SE depends on the etiology, duration of SE, and age at time of presentation (Goodkin & Riviello, 2011). The mortality rate associated with SE ranges between 4% and 37% and is highly dependent on the age of the person, the presence of an acute precipitant, prior history of epilepsy, and a number of other factors, (see Goodkin & Riviello, 2011 for review and references). Although many people will survive an episode of SE with no, or only limited, untoward effects, SE is life-threatening and is associated with long-term neurologic sequela that include an elevated risk for developing epilepsy and substantial cognitive decline. Although controversial, nonconvulsive SE is also associated with high morbidity and mortality (Krumholz, 1999).

Treatment of SE is aimed at controlling convulsive seizures as quickly as possible before compensatory mechanisms fail and the patient enters into a “refractory” state. The benzodiazepines (lorazepam and diazepam), phenytoin or its parenteral prodrug phosphenytoin, and phenobarbital are generally considered the first-line antiseizure drugs for the early treatment of SE. Second-line antiseizure drugs include intravenous valproic acid, levetiracetam, and lacosamide. SE can quickly become pharmacologically refractory when initial attempts to control the seizures fail despite adequate treatment. The patient with refractory SE is often placed into a drug-induced coma with an intravenous seizure suppressive agent to control both clinical and electrographic seizures. In addition to diazepam and lorazepam, other agents that might be considered for the patient in refractory SE include pentobarbital, midazolam, thiopental, propofol, and ketamine. There is a clear need for more effective treatments for refractory SE that display rapid onset and effective seizure control without producing dose-limiting sedation and respiratory depression. Furthermore, the development of an effective therapy that attenuates refractory SE, offers some neuroprotective potential, and prevents the cognitive decline associated with SE would represent an important advance in the treatment of SE.

In addition to the recognized need for better treatments of status arising from traditional causes, there are efforts underway to treat insults resulting in status from potential human exposure to chemical nerve agents. In 2006, the NIH/NINDS (ASP) initiated a multidisciplinary medical countermeasure screening effort designed to identify drugs that are effective in the treatment of medically refractory SE precipitated by exposure to nerve agents. As a proof of principle, several pilocarpine status models were employed to assess if compounds found effective in these models could also be used to treat status induced by actual chemical nerve agents (e.g., soman).

Beyond the traditional testing offered by the ASP, SPD was evaluated for its ability to block excitotoxin-induced neurotoxicity in an organotypic cultured (rats) hippocampal slice preparation using the glutamate receptor ligands N-methyl-d-aspartate (NMDA) and kainic acid (KA). SPD was simultaneously examined in vivo in Sprague-Dawley rats for its ability to acutely interrupt diazepam-sensitive and diazepam-resistant convulsive SE induced by lithium-pilocarpine. Based on the promising results obtained from this evaluation, SPD was submitted to more extensive evaluation to assess its ability to block the cognitive decline associated with pilocarpine-induced SE. SPD was then evaluated for its ability to prevent soman-induced SE at the U.S. Army Medical Research Institute of Chemical Defense (MRICD). The MRICD is well known for its ability to study the arrest of nerve agent (soman)–induced convulsive and nonconvulsive SE and neuronal degeneration. In addition, the pharmacokinetic profile of SPD was evaluated following intraperitoneal administration (60 mg/kg) to rats and a pharmacokinetic-pharmacodynamic (PK–PD; protection against soman-induced seizures) correlation was performed. The results obtained with SPD provide proof-of-concept that a blinded collaborative multifaceted approach can identify drugs that are effective against diazepam-resistant and soman-induced SE and preserve cognitive function following SE. The results with SPD using this approach demonstrate that SPD possesses a unique and more favorable profile compared to that obtained with the established antiseizure drugs diazepam and valproic acid and they provide the basis for this report.

Results

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Summary
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Anticonvulsant efficacy of SPD in seizure and epilepsy models

As summarized in Table 1, SPD displayed a broad spectrum of anticonvulsant activity in a variety of seizure and epilepsy models including audiogenic seizure (AGS)-susceptible Frings mice, the maximal electroshock (MES), 6 Hz, subcutaneous metrazol (scMet), subcutaneous bicuculline (scBic), and subcutaneous picrotoxin (scPic) seizure tests. SPD also displayed dose-dependent efficacy in two kindling models of partial seizures.

Table 1.   SPD anticonvulsant activity (in comparison to valnoctamide-VCD) in various mouse (i.p.) and rat (p.o.) models for epilepsy
Anticonvulsant testSPD-ED50 (mg/kg)95% confidence interval (mg/kg)VCD-ED50 (mg/kg)
  1. –, not tested. [Correction added after online publication 9-Dec-2011: Columns in Table 1 have been reordered.]

Frings audiogenic seizures2018–22
Maximal electroshock seizure (mice-MES)7155–9058
Maximal electroshock seizure (rats-MES)ip: 20 po:2915–27 1–53po: 29
Metrazol-induced seizure (mice-scMet)6247–7132
Metrazol-induced seizure (rats-scMet)1813–2554
Picrotoxin-induced seizure (mice-Pic)179–28
Bicuculine-induced seizure (mice-Bic)9487–103
Corneal kindled mouse3931–45
Hippocampal kindled rats1913–28∼40
6 Hz-32 mA (mice)2724–3037
Mice-neurotoxicity (TD50)8881–9577
Rat-neurotoxicity (TD50)ip: 49 po: 13143–55 94–175po:58

In the two kindling models evaluated (i.e., the corneal-kindled mouse and the hippocampal-kindled rat), SPD produced a dose-dependent reduction in the secondarily generalized seizure (median effective dose [ED50] 39 and 19 mg/kg, respectively) and seizure severity as estimated by the Racine seizure score (Racine, 1972). In the hippocampal kindled rat model, SPD produced a decrease in the seizure score from a predrug level of 4.6 to 1.3 at a dose of 32 mg/kg. This effect on seizure severity was also associated with a decrease in the afterdischarge duration from 39 ± 3.0 to 25.0 ± 4.0 s (mean ± standard error of the mean [SE]). In the corneal-kindled mouse, 60 mg/kg SPD decreased the seizure score from 5 to <1. Lastly, SPD was effective against refractory 6-Hz limbic seizures (ED50 27 mg/kg). Collectively, these results suggest that SPD has the ability to decrease both the focal and the secondarily generalized seizure at doses that are devoid of behavioral toxicity.

In a separate study, SPD was found to produce a marked and significant increase in intravenous Metrazol seizure threshold at the two dose levels tested. At a dose equivalent to the MES ED50 (70 mg/kg, i.p.), the threshold for first twitch and clonus was increased >200 and >600-fold, respectively. A slightly greater increase in seizure threshold was noted at a dose equivalent to the rotorod median neurotoxic dose (TD50) (Table S1).

It is notable that SPD was found to be active following two routes of administration (i.p. and oral) in two different rodent species (mouse and rat) at doses that did not impair motor function. The ED50 values ranged between 17 and 29 mg/kg. Furthermore, SPD was found to possess a wide safety margin or protective indexes (PI = TD50/ED50) ranging between 4.4 and 7.7.

SPD blocks convulsive seizures induced by the cholinergic agonist, pilocarpine

Administration of lithium-pilocarpine induces SE characterized by convulsive and nonconvulsive seizures that can last for several hours. SE is then followed by a latent phase, characterized by synaptic remodeling and neuronal plasticity, extensive neuronal loss, and subsequent cognitive deficits and the precipitation of spontaneous recurrent seizures, the hallmark of epilepsy. From a behavioral perspective, the number and severity of the observed convulsive seizures following pilocarpine administration were similar in the two treatment groups (pilocarpine alone and pilocarpine + SPD). The first convulsive stage 3 or greater seizure was observed 12 min after pilocarpine administration. Within the succeeding 30 min, rats were observed to have 4.9 ± 0.2 seizures with an interseizure interval of 3–5 min. On average, each convulsive seizure lasted for approximately 60 s. SPD, administered 30 min after the first observed stage 3 motor seizure (which was to mark the onset of SE), prevented the expression of further convulsive seizures in the pilocarpine + SPD group (ED50 = 84 mg/kg; Table 2) in a dose-dependent manner. In those animals in which SPD was observed to halt the convulsive seizure activity, onset was recorded as immediate and complete over the next 90 min of observation. The only other AED found to exert an effect when administered under the same experimental conditions was carbamazepine (ED50 = 50 mg/kg). The other comparator prototypical AEDs tested in this model—clonazepam, diazepam, valproic acid, and phenobarbital—were all ineffective at the highest dose tested: that is, 40, 10, 300, and 40 mg/kg, respectively. VCD was equipotent to SPD when given at SE onset, but in contrast to SPD VCD lost its activity when administered 30 min after the SE onset (Table 2).

Table 2.   Comparative efficacy of SPD, VCD, and several prototypical AEDs against benzodiazepine-resistant convulsive seizures in the lithium-pilocarpine model of SE
Compound testedED50– mg/kg (95% CI)
0 min post SE onset30 min post SE onset
  1. aData on file with the Anticonvulsant Screening Program, NINDS, NIH. The 0 min post SE onset data are given only as comparators.

SPD8/8 protected at 65 mg/kg84 (62–103)
Valnoctamide (VCD)8/8 protected at 65 mg/kg0/8 at 80 mg/kg
Clonazepama1.3 (0.6–2.5)>40 mg/kg
Carbamazepinea45 (38–52)50 (44–56)
Diazepama3.0 (1.6–4.4)0/8 at 10 mg/kg
Valproic acida366 (23–575)0/8 at 300 mg/kg
Phenobarbitala31 (18–46)0/8 at 40 mg/kg

SPD prevents cognitive impairment associated with pilocarpine-induced SE

In the Morris water maze (MWM) spatial memory and learning task, animals in all three treatment groups (non-SE naive, pilocarpine-SE, and pilocarpine + SPD) displayed obvious learning as evidenced by a decrease in their escape latencies over the course of each training session (Fig. 2). However, when compared to naive non-SE and SPD-treated rats, rats in the nontreated pilocarpine-SE group took a longer time to find the platform (Fig. 2), experienced a significantly higher number of missed platform encounters, and traveled greater distance before finding the platform in both the hidden and visible trials (Fig. 3). Together, these results confirm that SE leads to substantial cognitive decline as measured by impaired performance in the MWM. Regardless of the outcome measure employed—time to reach the platform (Fig. 2) or total distance travelled (Fig. 3)—rats that received SPD 30 min after the onset of SE performed better than those that received vehicle only. Indeed, the SPD-treated rats performed as well as naive control rats; that is, there was no statistically significant difference between the two groups [p > 0.05, one way analysis of variance (ANOVA)]. The results from this study suggest that SPD treatment at a time when SE is normally refractory to the benzodiazepine diazepam (i.e., 30 min after the onset of SE) prevented the cognitive decline associated with pilocarpine-induced SE.

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Figure 2.   SPD prevents lithium-pilocarpine SE–induced cognitive decline. Summarized results representing the average time (mean ± SEM) required for rats to find the escape platform (latency) of rats trained in the Morris water maze. Trial days one through five consisted of four training sessions with a hidden platform and days 6 and 7 consisted of four trials per day to find the visible platform using the acquired spatial map. There was a progressive decrease in escape latencies over the training days in all three groups. Animals in the naive and drug-treated group (Pilo + SPD) learned to navigate quickly using the visual cues, and there was no statistically significant difference between the two groups in their ability to find the escape platform. Pilocarpine-treated SE animals had a significantly higher escape latency and performed poorly in acquisition and retention of spatial memory, in comparison to the naive control group and the SPD group *p < 0.05, one-way ANOVA with Newman-Keuls multiple comparison test.

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Figure 3.   SPD treatment 30 min after the first convulsive seizure induced by lithium-pilocarpine decreased the total distance travelled in the Morris water maze. Results are expressed as the mean ± SEM. Similar to the escape latency, there was a progressive decrease in the distance travelled by the rats in all three groups over the course of training. Animals in the naive and Pilo + SPD group learned to directly swim toward the platform and spent most of their time in the quadrant where the platform was located. In contrast, pilocarpine- treated animals took a significantly longer time and traveled greater distance to find the escape platform (p < 0.05, one-way ANOVA with Newman-Keuls multiple comparison test; *as compared to naive and to the Pilo + SPD).

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SPD protects against hippocampal cell death associated with pilocarpine-induced SE

Pilocarpine-induced SE results in marked cell loss in the hippocampus (Fig. 4), as evidenced by increased FluroJade B staining in the dentate gyrus (DG), CA1, and CA3 cell layers. Administration of SPD within 30 min of SE prevented the hippocampal cell death in a majority of animals (7 of 15). It is interesting to note that SPD showed variability in its neuroprotective effects: 47% of animals showed complete neuroprotection in all areas of hippocampus. Forty percent of animals show neuroprotection in CA3 and DG, while showing some damage in CA1 area, whereas 93% of animals showed complete neuroprotection of dentate hilar neurons. Only 13% of animals showed significant damage in CA1, CA3, or DG (Fig. 4C,D). However, irrespective of the neuronal damage observed, all of the SPD-treated rats showed significantly higher cognitive performance than rats in the pilocarpine SE group. In those animals where SPD was observed to halt the convulsive seizure activity, onset was recorded as immediate and complete over the next 90 min of observation.

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Figure 4.   SPD-attenuated hippocampal pathology associated with lithium-pilocarpine SE. Images of representative hippocampal sections (n = 24 in each group) stained for FluoroJade B to determine the extent of cell death in naive and treated group of rats. (A, B) Pilocarpine-induced convulsive SE resulted in substantial cell loss in the dentate gyrus (DG), CA1, and CA3 hippocampal neurons, as evidenced by increased fluorescence. (C, D) SPD, when administered within 30 min of the first convulsive seizure (stage 3 or higher) conferred significant neuroprotection in the pyramidal cell layers. (C) Representative slices from rats showing partial neuroprotection, with significant cell death in CA1 neurons, whereas DG and CA3 neurons were protected. (D) Representative slices from animals showing full neuroprotection.

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SPD did not prevent excitotoxic cell death induced by NMDA or kainic acid (KA) in organotypic hippocampal slice cultures

In hippocampal slice cultures, exposure to NMDA (10 μm) or KA (20 μm) induced substantial cell death as determined by significant PI uptake after 24 h. SPD was added to the slice cultures at 10 and 100 μm to assess the neuroprotective effect against each glutamate receptor agonist. No significant difference in the extent of cell death was observed with this compound with either concentration against either NMDA or KA (n = 8).

SPD blocks electrographic and convulsive seizures induced by soman in rats and guinea pigs

As discussed in the preceding text, SPD was found to attenuate conclusive SE in the lithium-pilocarpine-SE rat model, which closely resembles nerve agent–induced SE. In the rat nerve agent seizure model, SPD was administered at various doses along with the standard medical countermeasures at treatment delays of 20 or 40 min after the onset of soman-induced seizures to determine the effective dose for termination of soman-induced seizures. SPD was capable of stopping soman-induced seizures at both treatment delay times. The ED50 for seizure control at the 20-min treatment delay was calculated by probit analysis to be 149 mg/kg (p = 0.06) (Fig. 5); an ED50 dose for the 40-min treatment delay could not be calculated. Following administration of SPD at the 20-min treatment, the average latency for seizure termination at the 20-min treatment delay time was 11.4 ± 1.4 min (standard error of the mean, SEM), whereas the seizure termination latencies for the 40-min treatment delay time group averaged 31.3 ± 2.7 min (SEM). The seizure termination latencies of the 20-min treatment delay group were significantly shorter than those of the 40-min treatment delay group (Fig. 6). In these initial tests of SPD, the drug was suspended in 0.5% methyl cellulose. Following some pilot formulation studies, additional testing of SPD in rats was conducted using the multisol vehicle (a pure solution rather than a suspension). In limited tests at the 20-min treatment delay, the anticonvulsant ED50 was 71 mg/kg (p < 0.05) and seizure termination latency was 389 s (6.8 min). When compared to the study conducted using methylcellulose as the vehicle, results obtained using the multisol vehicle resulted in significant reductions in both measures of approximately 50%.

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Figure 5.   Anticonvulsant dose–response curve of SPD administered 20 min after the onset of soman-induced seizures in rats.

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Figure 6.   Latency for seizure control—the time from when SPD was administered to rats until the last epileptiform event could be detected on the EEG record. Asterisks indicate a significantly (p < 0.001) shorter latency at the 20-min treatment time than the 40-min treatment time.

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When SPD was tested in the guinea pig soman status model, SPD administered at seizure onset was found to be effective with an ED50 of 67 mg/kg (p = 0.06); mean time seizure termination was 11.9 min from injection to spike termination. SPD administered at 40 min after seizure onset produced an anticonvulsant effect with an ED50 of 92 mg/kg (p = 0.01); mean time to seizure termination was equivalent to 5.8 min from injection to spike termination. These ED50 curves and seizure termination latencies are displayed in Figs 7 and 8. Figure 9 depicts the rapid anticonvulsant effect of SPD in the guinea pig model when given 40 min after SE onset.

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Figure 7.   Probit-derived dose–effect curves for the anticonvulsant action of SPD at the two different test times in the guinea pig model.

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Figure 8.   Latency (mean and SEM) for seizure control—the time from when SPD was administered to the guinea pig until the last epileptiform event could be detected in the EEG record. There were no statistical differences between the two treatment times.

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Figure 9.   An example of the rapid anticonvulsant effect of SPD in the guinea pig model. Shown is a compressed electroencephalography (EEG) file of one animal, with time of day on the x-axis. This animal was administered soman at 9:02:54 a.m. (left arrow); seizures began at 9:08:19 and were allowed to continue for 40 min. SPD, 100 mg/kg (i.p.), was administered at 9:49:04 (right arrow) and seizure activity promptly terminated at 9:51:30, with no further indication of seizure activity throughout the rest of the recording session.

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Effect of SPD on soman-induced histopathology in rats

Following treatment with SPD, animals were categorized as either having seizure turned off (regardless of SPD dose) or not having seizures turned off. As depicted in Table 3, brains from these animals showed that seizure control prevented neuropathology, whereas animals that had seizures continued to display extensive neuropathology.

Table 3.   Number of animals and degree of neuropathology (mean neuropathology score) as a function of soman-induced seizure control by SPD in rats
 No neuropathologyNeuropathology
  1. χ2 = 55.67, d.f. = 3, p < 0.0001.

Seizure controlled38 (X = 0)2 (X = 11.5)
Seizure not controlled2 (X = 0)29 (X = 17.5)

SPD pharmacokinetics in rats

The pharmacokinetics (PK) of SPD were studied following intraperitoneal administration (60 mg/kg) to naive rats (not treated with soman). The water solubility of SPD is 1.5 mg/ml; therefore, SPD was administered to rats in a solution of propylene glycol, alcohol and water for injection 5:1:4, where its solubility was increased to 27 mg/ml. The plasma concentration–time plots of SPD are presented in Fig. S1. SPD PK parameters, calculated by noncompartmental analysis, are summarized in Table 4. SPD total clearance (CL) of 0.48 L/h was mainly metabolic, with only 0.1% of the dose or CL being excreted unchanged in the urine. In rats, SPD displayed a sevenfold higher CL than VCD, and due to its higher lipophilicity SPD volume of distribution (V) was three-times more than that of VCD (Blotnik et al., 1996). As a consequence of these opposite trends in CL and V, SPD half-life (t1/2) was similar to that of VCD. The dose was chosen as the intermediate dose among SPD various ED50 values, assuming linear pharmacokinetics. Rat liver blood flow is 60–70 ml/min/kg (Altman & Dittmer, 1974). Assuming that SPD metabolism occurs primarily in the liver and that the SPD blood-to-plasma ratio is about 1, the SPD liver extraction ratio (E) is E = CL/Q = CLm/Q = 32/65 = 0.48. [Correction added after online publication 9-Dec-2011: 32/65 in previous equation updated from 20/65.] If these rat data can be extrapolated in humans it may indicate that SPD might be slightly susceptible to hepatic first-pass effect following oral dosing.

Table 4.   PK parameters of SPD obtained following intraperitoneal administration (60 mg/kg) to rats
ParameterSPD (i.p.)
  1. t1/2, half-life; CL, total clearance; CL/F, extravascular (oral) clearance calculated after extravascular (i.p.) dosing; AUC, area under plasma drug concentration–time curve; AUMC, area under the first moment curve; MRT, mean residence time; Vz, volume of distribution based on linear terminal slope; Vz/F, apparent volume of distribution calculated after extravascular dosing; Cmax, peak plasma drug concentration; tmax, time to reach Cmax; fm, fraction metabolized of the systemically available drug; fe, fraction of the systemically available drug excreted unchanged in the urine.

t1/2 (h)0.84
CL/F (L/h/kg)1.9
AUC (mg/L × h)32.1
AUMC (mg/L × h2)61
MRT (h)1.9
Vz/F (L/kg)2.3
Cmax (mg/L)14.1
tmax (h)0.75
fe (%)0.1

SPD pharmacokinetic-pharmacodynamic (PK-PD) correlation (rats)

A pharmacokinetic-pharmacodynamic (PK-PD) correlation between SPD plasma profile and SPD activity in terminating soman-induced seizures at delay time of 20 and 40 min is presented in Figs 10 and 11, respectively. When SPD (100–132 mg/kg) was administered at 20 min after (soman-induced) seizure onset, the maximal effect was reached at 30 min and lasted for 4–6 h (Fig. 10). When SPD (145–158 mg/kg) was administered at 40 min after (soman-induced) seizure onset, the maximal effect was reached at 1 h and lasted for 3–5 h. Assuming linear PK the PK-PD correlation depicted in Figs 10 and 11 shows that the SPD effective plasma levels ranged between 8 and 40 mg/L (20 min after seizure onset) and 12–50 mg/L (40 min after seizure onset). The time to peak effect (PD-tmax) occurred after the PK-tmax and may indicate slow distribution of SPD to the extraplasmatic active site responsible for SPD activity. This slower distribution to active site may contribute to the fact that SPD effect (responders rate) declined significantly slower than SPD plasma levels and in a few rats at the 20 min post seizure onset lasted for 24 h.

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Figure 10.   PK–PD) correlation. Solid line indicates plasma concentrations following intraperitoneal administration of SPD (60 mg/kg) to rats. Dashed lines indicate percent responders to SPD administered 20 min after seizure onset, measured as the number of “protected” rats (rats without soman-induced seizures) divided by total number of rats tested at each time point.

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Figure 11.   PK–PD correlation. Solid line indicates plasma concentrations following intraperitoneal administration of SPD (60 mg/kg) to rats. Dashed lines indicate percent responders to SPD administered 40 min after seizure onset, measured as the number of “protected” rats (rats without soman-induced seizures) divided by total number of rats tested at each time point.

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Discussion

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Summary
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

SPD displays broad-spectrum antiseizure activity in rodent seizure and epilepsy models

In the present investigation, we describe the acute antiseizure activity of a VPA amide analog (or a VCD one-carbon homolog); that is, SPD in a battery of traditional seizure and epilepsy models often employed in the search for novel AEDs. The results obtained from these investigations demonstrate that SPD, like VPA, possesses a broad-spectrum anticonvulsant profile in models of focal and generalized seizures. Moreover, SPD was found to be effective in the 6 Hz seizure model of pharmacoresistant epilepsy. Although little can be said about the molecular mechanism through which SPD exerts its acute antiseizure effects, the results from the anticonvulsant testing conducted thus far would support the conclusion that it exerts its effects through an ability to prevent seizure spread and elevate seizure threshold. This conclusion is based on the marked effect exerted by SPD in the mouse and rat MES test (seizure spread) and its ability to elevate seizure threshold in the intravenous Metrazol seizure threshold test. Further evidence supporting this conclusion is provided by the results obtained in the two kindling models of partial seizures, that is, the hippocampal kindled rat and the corneal kindled mouse. In both of these animal models, SPD prevented the expression of secondarily generalized seizures (i.e., decreased the Racine seizure score from 5 to 1 or less). Consistent with this observation, SPD was found to decrease the afterdischarge duration in the hippocampal kindled rat, an indirect measure of seizure spread. The results at the whole animal level are not necessarily surprising given the close structural similarity between SPD and VPA. Ongoing studies at the cellular level will hopefully provide some insight into the specific molecular mechanisms underlying the pronounced antiseizure activity of SPD.

Anticonvulsant effects of SPD in animal models of SE

SE is initially treated with a benzodiazepine such as diazepam or lorazepam. Both are extremely effective when given early in SE; however, the benzodiazepines lose their efficacy when given after 30 min of spontaneous self-sustaining seizures; for example, animals that experience prolonged SE quickly develop pharmacoresistant SE if treatment is not initiated within a short period of time (Jones et al., 2002). There are few data concerning the treatment of refractory SE.

SPD was found to be a highly effective antiseizure drug in two distinctly different models of SE: the lithium-pilocarpine convulsive SE model and the soman-induced SE model. The results obtained in these two models demonstrate that SPD has the ability to arrest ongoing seizure activity when administered after 30 min in the lithium-pilocarpine-SE model and after 20 or 40 min in the soman-induced SE model.

Comparative effect of SPD in lithium-pilocarpine SE model

As summarized in Table 1, SPD clearly differentiated itself in the lithium-pilocarpine-SE convulsive model from other AEDs that have been evaluated under identical conditions: that is, diazepam, clonazepam, valproic acid, phenobarbital, and VCD. The only other traditional AED that has displayed a similar antiseizure effect in the lithium-pilocarpine model is carbamazepine. Carbamazepine halted the progression of convulsive SE when administered 30 min after the first observable seizure with an ED50 of 50 mg/kg. However, the antiseizure effect of carbamazepine is not correlated with a similar degree of neuroprotection and/or cognitive sparing (A. B. Alex and H. S. White, unpublished data) to that observed with SPD (discussed below).

SPD activity in soman-induced SE in rats and guinea pigs

The time required for seizure control in the soman-rat model following SPD treatment at the 20-min treatment delay time was 11.4 min. In general, SPD provided faster seizure control against soman-induced seizures than the anticholinergics or benzodiazepines under comparable conditions (McDonough & Shih, 1993; Shih et al., 1999). In addition, the increase in the latencies for seizure control with a longer treatment delay (e.g., 40 vs. 20 min) is commonly observed with other drugs in this model. However, it seems clear that the vehicle in which SPD was prepared (0.5% methyl cellulose vs. multisol) had a significant effect on the ED50 and latency for seizure control in the rat model; for example the multisol vehicle significantly enhanced the pharmacodynamic effect of the drug. Based on our previous studies (McDonough & Shih, 1993; Shih et al., 1999; McDonough et al., 2000), few drugs show the rapid anticonvulsant effectiveness against soman-induced seizures in rats or guinea pigs at long (40-min) treatment delays as was seen with SPD. Similar to the neuroprotective effects seen in the lithium-pilocarpine SE model, control of soman-induced seizures with SPD successfully protected animals against the severe neuropathology that is typically observed in the brains of animals exposed to nerve agents (Carpentier et al., 1990; Lallement et al., 1994; McDonough et al., 1995).

SPD is a water-insoluble compound; therefore, in the rat study it was suspended in 0.5% methyl cellulose (MC). The intraperitoneal injection of a MC SPD suspension into an animal using anything less than an18-gauge needle proved problematic. Although other solvents were not tested in the rats, it can be presumed that administering SPD in a pure solution (multisol, like in the guinea pig study) could only help absorption and improve the SPD anticonvulsant (pharmacodynamic) response. In summary, SPD was effective against generalized SE seizures induced by the nerve agent soman in the rat and guinea pigs model when treatment was delayed 20 and 40 min after seizure onset. SPD showed a unique ability to immediately stop soman-induced devastating seizures even at the 40-min treatment delay. In contrast to other AEDs that have been studied using this model, SPD was successful (even after a 40-min delay) in terminating soman-induced seizures.

It was noted that many of the animals treated with SPD had an initial anticonvulsant effect (i.e., the seizures stopped), but that later in the day, or more typically by the recording session the next morning, the seizures had returned. This occurred almost exclusively when SPD was formulated with the 0.5% methylcellulose. The rats in which the seizures were terminated by SPD displayed no neuropathology, whereas animals in which the treatment was not successful in stopping the seizures displayed significant levels of neuropathology in those brain areas susceptible to nerve agent-induced damage.

SPD attenuates hippocampal sclerosis

Prolonged SE and chronic epilepsy are associated with marked hippocampal sclerosis, spontaneous seizures, and progressive cognitive decline (Mazarati et al., 2004; Löscher & Brandt, 2010). Furthermore, the frequency, duration, and severity of seizures are variably associated with greater risk of cognitive impairment (Meador, 2002). Using an acute animal model of SE, the NIH-NINDS-ASP was able to identify a novel drug candidate; that is, SPD, which possesses substantial antiseizure activity against benzodiazepine-resistant SE that was subsequently shown to prevent SE-induced hippocampal sclerosis in both the lithium-pilocarpine and soman models and cognitive impairment in the lithium-pilocarpine model of SE.

Diazepam has been found to be effective in preventing neuronal damage in SE (Ben-Ari et al., 1980; Pitkänen et al., 2005). However, our studies have shown that diazepam (20 mg/kg, i.p.) is most effective when given early after the onset of pilocarpine-induced SE and the neuroprotective effect wanes after 30 min of uninterrupted SE (A. B. Alex and H. S. White, unpublished data). It is also important to note that diazepam’s apparent neuroprotective properties are almost exclusively associated with its ability to interrupt SE, that is, insult modification. In the current study, SPD (130 mg/kg, i.p.) when administered after 30 min of pilocarpine-induced SE, significantly reduced the hippocampal neuronal loss in a majority of animals when sacrificed 3–4 weeks after the initial insult. Bolanos et al. (1998) have shown that VPA (600 mg/kg, twice a day, for 1 month) reduced cell death in CA1 damage, but did not protect CA3 or dentate hilar neurons. Considering all the available data so far, a single dose of SPD exhibits superior neuroprotection over VPA and other standard AEDs when administered shortly after SE induction (Bolanos et al., 1998; Klitgaard, 2001; Brandt et al., 2006; François et al., 2006; Zheng et al., 2010). As discussed in the preceding paragraph, the ability of SPD to prevent hippocampal neuronal cell loss was most likely due to its acute anticonvulsant effect (insult modification), and as discussed below, not a result of any inherent neuroprotective properties.

In contrast to MK-801, which is neuroprotective in vitro in organotypic hippocampal slice cultures and in vivo when administered 30 min after SE onset (Lee et al., 1997; Kristensen et al., 2001), SPD was only neuroprotective in vivo. In contrast to SPD, MK-801 did not display substantial antiseizure effects when administered 30 min after SE onset. These results suggest that the neuroprotective action of MK-801 is related to its activity as an NMDA-receptor antagonist, whereas, the neuroprotective effects observed in SPD-treated rats are more likely the result of its marked antiseizure activity in vivo. These results demonstrate the benefit associated with early intervention of SE with a highly effective antiseizure agent.

Early treatment with SPD mitigates lithium pilocarpine SE-induced cognitive impairment

Previous investigations have demonstrated that pilocarpine-induced SE induces neuronal loss, particularly in the hippocampal subfields CA1, CA3, and dentate hilus, chronic spontaneous seizures, and long-term deficits in learning and memory (Mello et al., 1993; Cunha et al., 2009). Place navigation in the Morris water maze requires place representations or cognitive maps, and the hippocampus is thought to be critical for computing place representations. The present study evaluated the ability of SPD to prevent the short- and long-term consequences associated with experimentally induced SE, for example, hippocampal sclerosis and SE-induced learning deficits.

Hippocampal neuronal preservation has a direct relation to the functional outcome in patients with epilepsy (Meador, 2002). Although transient disruption of cognitive encoding may occur with paroxysmal focal or generalized epileptic discharges, epileptogenesis-related neuronal plasticity, reorganization, and sprouting may contribute to the progressive cognitive decline following brain injury induced by SE and other brain insults (Pitkänen et al., 2002; Hamed, 2009). There are only limited studies that compare the long-term functional outcome of SE and behavioral responses. Data from experimental models of SE indicate that AEDs preventing hippocampal damage usually afford protection against SE-induced behavioral responses (Bolanos et al., 1998; Cha et al., 2002). Reduction of cell loss in VPA-treated rats has been correlated with an increased performance in visuospatial learning and memory tasks in KA-induced SE rats, whereas phenobarbital did not prevent the hippocampal damage or the cognitive impairment associated with SE (Bolanos et al., 1998). However, in another study, VPA did not improve the performance of rats in Morris water maze (Brandt et al., 2006) and it has been suggested that the VPA-induced cognitive deficits may be due to its negative effect on hippocampal neurogenesis (Umka et al., 2010). Interestingly, clinical studies have not demonstrated any positive effect with VPA on epileptogenesis or on cognition in patients with traumatic brain injury (Temkin et al., 1999). Topiramate a broad-spectrum AED, is neuroprotective, but only partially prevented pilocarpine-induced cognitive impairment (Cha et al., 2002; Frisch et al., 2007). Compared to the available data obtained with a number of currently available AEDs, SPD prevented visuospatial learning and memory deficits induced by pilocarpine-SE without causing any motor or behavioral toxicity. Again, the present results support the value of early treatment of SE with an effective antiseizure drug such as SPD in preventing neuronal death and ultimately preserving cognitive function. Regardless of the mechanism responsible for this outcome, the goal is to provide the patient with therapies that improve their overall quality of life.

Summary

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Summary
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

SPD was found to be a highly effective anticonvulsant in a battery of well-defined acute and chronic seizure models and may represent a novel alternative for the symptomatic treatment of epilepsy. Corroborating results obtained in two different SE models (i.e., lithium-pilocarpine and soman) using two different species (rats and guinea pigs) demonstrate that SPD is extremely effective in preventing pilocarpine or soman-induced benzodiazepine-resistant seizures while offering neuroprotection and cognitive sparing. These effects suggest that SPD may offer long-term functional benefit following SE.

In the interest of developing a truly antiepileptogenic therapy, it must be recognized that effective suppression of all early seizures, even nonclinical seizures, may interfere with the process of epileptogenesis. Therefore, control of symptomatic seizures following SE or other brain injuries with an effective drug therapy may also contribute to disease modification or antiepileptogenesis (Dichter, 2009). SPD, a novel VPA amide derivative, was effective in blocking behavioral seizures induced by pilocarpine or soman. The fact that SPD is a very close homolog of VCD that has >40 years of clinical experience in Europe contributes to SPD’s promising clinical potential as an antinerve gas agent and new antiepileptic and CNS drug that is effective following oral and parenteral administration.

Lastly, the early identification of the marked antiseizure effect of SPD in both the lithium-pilocarpine and soman models of SE that was the result of a unique collaborative effort among different government agencies (the NIH and the U.S. Army’s MRICD) and the actual owners of the candidate compounds represented by either academia or various pharma groups, or in this case The Hebrew University of Jerusalem, is an example of a new type of collaboration. Such partnerships are a testament to how future drug development will likely be performed. Crafting “win-win” relationships with previously unlikely partners is becoming more common and even essential for the successful translation of new therapeutics.

Acknowledgments

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Summary
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Studies conducted at the University of Utah were supported by NINDS, NIH Contract No. NO1-NS-4-2359 (HSW, ABA, KSW, ALP). The studies conducted at MRICD were supported by an Inter-Agency Agreement between NIH/NINDS (Y1-O6-9613-01) and USAMRICD (A120-B.P2009-2) and the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division. The views expressed in this paper are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.

Disclosures

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Summary
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Dr. Meir Bialer has received in the last 3 years speakers or consultancy fees from Bial, CTS Chemicals, Desitin, Janssen-Cilag, Johnson & Johnson, Medgenics, Rekah, Sepracor, Teva, UCB Pharma, and Upsher-Smith. In the last 5 years, the author received research grants from Jazz Pharmaceuticals, Johnson & Johnson, and The Epilepsy Therapy Development Project and has been involved in the design and development of new antiepileptics and CNS drugs as well as new formulations of existing drugs. Dr. H. Steve White is the Director of the University of Utah Anticonvulsant Drug Development Program and Principal Investigator of the NINDS, NIH Contract that funded the University of Utah Research reported herein.

Dr. White also wishes to disclose that in the last 3 years he has served as a paid consultant to Johnson & Johnson Pharmaceutical Research and Development, GlaxoSmithKline, Valeant Pharmaceuticals, Eli Lilly & Co., and Upsher-Smith Laboratories, Inc., is a member of the UCB Pharma Speakers Bureau, the NeuroTherapeutics Pharma Scientific Advisory Board, and has received research funding from NeuroAdjuvants, Inc. Lastly, Dr. White is one of two scientific cofounders of NeuroAdjuvants, Inc., Salt Lake City, UT. None of the other 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.

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  5. Discussion
  6. Summary
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Summary
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Data S1. Experimental section: Materials and methods.

Figure S1. Plasma concentration-time plot obtained following intraperitoneal administration of SPD (60 mg/kg) to rats.

Table S1. Effect of SPD on the threshold for minimal seizures induced by the timed intravenous infusion of Metrazol in mice following intraperitoneal administration.

FilenameFormatSizeDescription
EPI_3338_sm_SI.doc113KSupporting info item
EPI_3338_sm_FigS1.tif108KSupporting info item
EPI_3338_sm_Support-Infrev.doc104KSupporting info item

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