3.1 Anticonvulsant Efficacy of SPD in Seizure and Epilepsy Models
As summarized in SPD displayed a broad spectrum of anticonvulsant activity in a variety of seizure and epilepsy models including AGS-susceptible Frings mice, the MES, 6 Hz, scMet, scBic, and scPic seizure tests. SPD also displayed dose-dependent efficacy in two kindling models of partial seizures.
SPD anticonvulsant activity (in comparison to valnoctamide-VCD) in various mouse (ip) & rat (po) models for epilepsy
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 (ED50=39 and 19 mg/kg, respectively) and seizure severity as estimated by the Racine seizure score. In the hippocampal kindled rat model, SPD produced a decrease in the seizure score from a pre-drug 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 sec to 25.0 ± 4.0 sec. In the corneal kindled mouse, 60 mg/kg SPD decreased the seizure score from 5 to less than 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 i.v. 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 rotarod TD50
Importantly, 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 17-29 mg/kg. Furthermore, SPD was found to possess a wide safety margin or protective indexes (PI= TD50/ED50) ranging between 4.4-7.7.
3.2 SPD blocks convulsive seizures induced by cholinergic agonist, pilocarpine
Administration of lithium-pilocarpine induces SE characterized by convulsive and non-convulsive 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 hall mark 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 minutes, rats were observed to have 4.8 4.9±0.2 seizures with an inter seizure interval of 3-5 minutes. On average, each convulsive seizure lasted for approximately 60 sec. SPD, administered 30 minafter the first observed stage 3 motor seizure (which was to mark the onset of SE), dose-dependently prevented the expression of further convulsive seizures in the pilocarpine + SPD group (ED50=84 mg/kg; ). In those animals where SPD was observed to halt the convulsive seizure activity, onset was recorded as immediate and complete over the next 90 minutes 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; i.e., 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 ().
Comparative efficacy of SPD, VCD and several prototypical AEDs against benzodiazepine-resistant convulsive seizures in the lithium-pilocarpine model of SE
3.3 SPD prevents cognitive impairment associated with pilocarpine-induced SE
In the Morris water maze spatial memory and learning task, animals in all three treatment groups (non-SE naïve, pilocarpine-SE, and pilocarpine + SPD) groups displayed obvious learning as evidenced by a decrease in their escape latencies over the course of each training session (). However, when compared to naïve non-SE and SPD-treated rats, rats in the non-treated pilocarpine-SE group took a longer time to find the platform (), experienced a significantly higher number of missed platform encounters and traveled greater distance before finding the platform in both the hidden and visible trials (). Together, these results confirm that SE leads to substantial cognitive decline as measured by impaired performance in the Morris water maze. Regardless of the outcome measure employed; time to reach the platform () or total distance travelled (), 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 naïve control rats; e.g., there was no statistically significant difference between the two groups (p > 0.05, one way 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.
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 (more ...)
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 (more ...)
3.4 SPD protects against hippocampal cell death associated with pilocarpine-induced SE
Pilocarpine-induced SE results in marked cell loss in the hippocampus (), as evidenced by increased FluroJade B staining in the dentate gyrus (DG), CA1, and CA3 cell layers. Administration of SPD within 30 minutes of SE prevented the hippocampal cell death in a majority of animals (7/15). It is interesting to note that SPD showed variability in its neuroprotective effects: 47% of animals showed complete neuroprotection in all areas of hippocampus. 40% of animals show neuroprotection in CA3 and DG, while showing some damage in CA1 area, while 93% of animals showed complete neuroprotection of dentate hilar neurons. Only 13% of animals showed significant damage in CA1, CA3 or DG (). 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.
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 naïve, and treated group of rats. (more ...)
3.5 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 hr. SPD was added to the slice cultures at 10 μM 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).
3.6 SPD blocks electrographic and convulsive seizures induced by soman in rats and guinea pigs
As discussed above, 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) () 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 (SEM) while the seizure termination latencies for the 40-min treatment delay time group averaged 31.3±2.7min (SEM). The seizure termination latencies of the 20-min treatment delay group were significantly shorter than those of the 40-min treatment delay group (). 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 = 71 mg/kg, p<0.05, and seizure termination latency = 389 sec (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%.
Anticonvulsant dose-response curve of SPD administered 20 min after the onset of soman-induced seizures in rats.
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 (more ...)
When SPD was tested in the guinea pig soman status model SPD administered at seizure onset was found to be effective with an ED50=67mg/kg; p=0.06; mean time seizure termination = 3.3 min from injection to spike termination. SPD administered at 40 min after seizure onset produced an anticonvulsant effect with an ED50= 92mg/kg; p=0.01; mean time to seizure termination equivalent to 5.8 min from injection to spike termination. These ED50 curves and seizure termination latencies are displayed in and . depicts the rapid anticonvulsant effect of SPD in the guinea pig model when given 40 min after SE onset.
Probit-derived dose-effect curves for the anticonvulsant action of SPD at the two different test times in the guinea pig model.
Figure 8 Latency for seizure control – the time from when SPD was administered to guinea pig until the last epileptiform event could be detected on the EEG record. Asterisks indicate a significantly (p < 0.001) shorter latency at the 0 min treatment (more ...)
Figure 9 An example of the rapid anticonvulsant effect of SPD in the guinea pig model. Shown is a compressed EEG file of one animal, with time of day on the x-axis. This animal was administered soman at 9:02:54 AM (left arrow); seizures began at 9:08:19 and were (more ...)
3.7 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 , brains from these animals showed that seizure control prevented neuropathology, while animals that had seizures continued to display extensive neuropathology.
Number of animals and degree of neuropathology (mean neuropathology score) as a function of soman induced seizure control by SPD in rats
3.8 SPD pharmacokinetics in rats
The pharmacokinetics (PK) of SPD was studied following intraperitoneal administration (60 mg/kg) to naïve rats (not treated with soman). SPD’s water solubility 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 it solubility was increased to 27 mg/mL. The plasma concentration–time plots of SPD are presented in Figure S1
. SPD PK parameters, calculated by non-compartmental analysis, are summarized in . SPD total clearance (CL) of 0.3 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 7-fold higher CL than VCD and due to its higher lipophilicity SPD volume of distribution (V) was 3-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 (Altman and Dittmer, 1974
). Assuming that SPD metabolism occurs primarily in the liver and that SPD blood-to-plasma ratio is about one, shows that SPD liver extraction ratio (E) is E=CL/Q=CLm/Q=20/65=0.3. 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.
3.8 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 min and 40 min is presented in and When SPD (100-132 mg/kg) was administered at 20 min post (soman-induced) seizure onset, the maximal effect was reached at 30 min and lasted for 4-6hr (). When SPD (145-158 mg/kg) was administered at 40 min post (soman-induced) seizure onset, the maximal effect was reached at 1h and lasted for 3-5hr. Assuming linear PK the PK-PD correlation depicted in and shows that SPD effective plasma levels ranged between 8-40 mg/L (20 min post seizure onset) and 12-50 mg/L (40 min post seizure onset). The time to peak effect (PD-tmax) occurred after the PK-tmax and may indicate slow distribution of SPD to the extra-plasmatic 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 20min post seizure onset lasted for 24h.
Figure 10 Pharmacokinetic-pharmacodynamic (PK-PD) correlation. Solid line indicates plasma concentrations following ip administration of SPD (60mg/kg) to rats. Dashed lines indicate % responders to SPD administered 20min after seizure onset, measured as the number (more ...)
Fig. 11 Pharmacokinetic-pharmacodynamic PK-PD correlation. Solid line indicates plasma concentrations following ip administration of SPD (60mg/kg) to rats. Dashed lines indicate % responders to SPD administered 40min after seizure onset, measured as the number (more ...)