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Keywords:

  • Microparticle;
  • Controlled release;
  • Epilepsy;
  • Muscarinic;
  • Prophylaxis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Summary:  Purpose: To investigate the efficacy of in situ lipid–protein–sugar particles (LPSPs) in mitigating the epileptogenic and histologic effects of intrahippocampal pilocarpine in rats.

Methods: LPSPs with and without muscimol were produced by spray-drying, sized by Coulter counter, and muscimol content determined by high-pressure liquid chromatography (HLPC). Particles, free muscimol or saline, were injected into the hippocampi of Sprague–Dawley rats before 40 mM pilocarpine, and seizure activity was scored. The trajectories of behavioral scores between groups were compared with two-way repeated measures analysis of variance. Animals were killed after 2 weeks. Brain sections were stained (Timm and thionin) and scored.

Results: LPSPs were 4 to 5 μm in diameter, and contained 0 or 2% (wt/wt) muscimol. In vitro, muscimol was released over a 5-day period. Intrahippocampal injections of normal saline and blank LPSPs did not deter seizure activity from pilocarpine. The rise of the trajectory in behavior scores in animals given LPSPs containing 5 μg muscimol was significantly slower than in those receiving saline, blank particles, or 5 μg of unencapsulated muscimol. There was less apparent neuronal injury and CA3 and supragranular mossy fiber sprouting in hippocampi of animals receiving muscimol-containing particles than in animals that did not receive muscimol. Hippocampi of animals that received 5 μg of encapsulated muscimol showed less supragranular sprouting than did those receiving 5 μg of unencapsulated muscimol, but showed no difference in cell loss or CA3 sprouting.

Conclusions: Focally delivered biodegradable microparticles loaded with muscimol are effective in reducing seizure activity from pilocarpine in animals and mitigate the histologic effects.

Oral pharmacotherapy is the cornerstone of the treatment of chronic seizure disorders. Antiepileptic drugs (AEDs) are typically administered multiple times daily; the dosage and frequency of administration are determined by the pharmacokinetic characteristics of the drugs and their systemic side effects (1,2). The dose of systemically delivered drug required to achieve a brain concentration sufficient to control seizures may result in unacceptable side effects (3,4). This is particularly true in some forms of epilepsy (e.g., epilepsia partialis continua), in which seizure activity can be unrelenting. The sequelae of the disorder and the treatment (barbiturate coma, neurosurgery) can be severe. A drug-delivery system that could directly target the epileptic region in the brain would offer enormous advantages, especially because ∼60% of seizures are partial (5,6). Furthermore, status epilepticus is most likely to occur in patients with partial seizures (7).

The effectiveness of focally delivered AEDs in treating experimental models has been demonstrated (8). Automated systems using a catheter at the epileptogenic focus have been devised that are effective in terminating induced seizures (9). Relatively large implants impregnated with various agents (10–12) also are effective in animal studies.

We examined whether intrahippocampal injection of biodegradable and biocompatible lipid–protein–sugar particles (LPSPs) loaded with an AED can prevent seizures in a rat model. These microparticles are generally several microns in diameter, suspend readily in physiological carrier fluids (13), and can be injected stereotactically through a small-gauge catheter or needle (14). Such particles have been used for drug delivery to the peripheral (13) and central (14) nervous systems. They can be engineered to contain a wide variety of drugs and excipients and to provide varying rates of drug release. They are biocompatible in the epineurium in the rat peripheral nervous system (15), and the murine brain (14).

Our model of epilepsy is hippocampal injection of pilocarpine (16,17), a nonselective muscarinic agonist. This model has been shown to bear histologic similarities to temporal lobe epilepsy in humans (18). Muscimol, a potent γ-aminobutyric acid (GABA)A-receptor agonist anticonvulsant (19), was the AED used.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Animal care

Sprague–Dawley rats (Charles River Laboratories, Cambridge, MA, U.S.A.) (n = 21), weighing 200–250 g at surgery, were maintained in a 12-h light/dark cycle. Animals had access to food and water ad libitum. All procedures were approved by the Animal Care Committee of Children's Hospital and were in accordance with guidelines set by the National Institutes of Health.

Preparation of lipid-protein-sugar particles

Dipalmitoylphosphatidyl-choline (DPPC; Avanti Polar Lipids, Alabaster, AL, U.S.A.) was dissolved in ethanol; albumin, lactose, and muscimol (all from Sigma Chemical Co., St. Louis, MO, U.S.A.) were dissolved in water. The two solutions were mixed [so the final proportion (wt/wt) of solutes was DPPC, 59.3; albumin, 19.3; lactose, 19.3; muscimol, 2], and spray-dried by using a model 190 bench top spray drier (Büchi Co., Switzerland) as described (13). Blank particles were produced in an identical manner, except that muscimol was not included, and the amounts of the inactive excipients were increased accordingly.

Particle size and shape determination

Particle size was determined with a Coulter Multisizer (Coulter Electronics Ltd., Luton, U.K.), using a 30-μm orifice. Surface characteristics of particles were determined by scanning electron microscopy on an AMR-1000 (Amray Inc., Bedford, MA, U.S.A.). Samples were mounted on stubs, given a gold–palladium conductive coating, and scanned at 10 kV.

Muscimol content of particles

The actual muscimol content of particles was determined by dissolving a known quantity of particles in 1 ml methanol, adding HPLC running buffer (see later) to a total volume of 5 ml, centrifuging the solution at 14,000 rpm for 10 min, and measuring the muscimol concentration in the supernatant, by using a standard curve.

Muscimol release

Twenty-five milligrams of particles containing 500 μg muscimol were suspended in phosphate-buffered saline (PBS), pH 7.4, and placed in a dialysis tube with an 8,000 MW cut-off (Spectra/Por 1.1 Biotech Dispo-dialyzer). The tube was then submerged in 12 ml of PBS and incubated at 37°C. At predetermined intervals, the external PBS was removed for analysis by HPLC (see later), and replaced with fresh PBS.

HPLC

HPLC assays were performed on an HP 1100 HPLC system. Samples in 50-μl volume were injected onto a 4.6-mm (ID) × 25 cm (L) Spherisorb ODS-2 column (Column Engineering, Ontario, CA, U.S.A.). The column was eluted with an aqueous solution of 0.5% vol/vol HBTA (heptafluorobutyric acid, Fluka) at 1 ml/min. Muscimol was detected by a UV detector with absorbance wavelength set at 230 nm.

Induction of seizures

The method is similar to that described by Cavalheiro et al. (20). Animals were anesthetized with pentobarbital (PTB; 40 mg/kg), and an electrode-cannula guide (Plastic one, U.S.A.) was stereotactically implanted in the CA3 region of the dorsal hippocampus [lateral from midline 3.5 mm, posterior from bregma 3.8 mm, and 3.8 mm deep from skull; Fig. 35 in (21)]. The electrode-cannula guide was fixed to skull by using dental cement and skull screws. After surgery, a dummy cannula was placed into the guide cannula to prevent occlusion. One week later, the rats received 30 μl of one AED or control treatment (encapsulated or free) through the cannula by using a microinfusion pump (Baxter, model AS40A) for 15 min at a rate of 0.12 ml/h. Thirty minutes after completion of these treatments, 40 mM pilocarpine was infused for 50 min at a rate of 0.12 ml/h. Subsequently, continuous EEGs were recorded from the electrode-cannulae, and the animals were videotaped for a minimum of 2 h. Animals were then kept in the laboratory for 6 h under close visual observation and were returned to the vivarium only after the animal was seizure free for an hour. Behavior was coded every 10 min by using the scale in Table 1. All procedures and observations were done by a single observer between 6 a.m. and 6 p.m. Four animals were used in each treatment group, except as specified in the text.

Table 1.  Seizure-scoring system
ScoreBehavior
0Normal behavior
1Motionless, staring
2Chewing
3Forelimb clonus
4Bilateral forelimb clonus
5Bilateral forelimb clonus with rearing
6Tonic posturing

Histology

Two weeks after induction of seizure, animals were killed and prepared for routine histologic examination and Timm histochemistry. After deep anesthesia with sodium PTB (100 mg/kg), rats were perfused transcardially with 300 ml sodium sulfide medium (2.295 g Na2S, 2.975 g NaH2PO4/H2O in 500 ml H2O) followed by 300 ml 4% paraformaldehyde. Brains were postfixed in 4% paraformaldehyde for 24 h and then placed in a 30% sucrose solution until they sank to the bottom of the vial. Coronal sections through the entire extent of the hippocampus were cut at 40 μm on a freezing microtome, and sections were stored in PBS. Every fourth section was stained for mossy fibers by using Timm stain as follows. The sections were developed in the dark for 40–45 min in a solution of 50% arabic gum (120 ml), 10 ml of citric acid (51 g/100 ml H2O), 10 ml sodium citrate (47 g/100 ml H2O), 3.47 g hydroquinone in 60 ml, and 212.25 mg AgNO3. After washing, the slides were dehydrated in alcohol, cleared in xylene, and mounted on slides with Permount (Fisher Scientific, Pittsburgh, PA, U.S.A.). Timm staining was analyzed by using a scoring system (0–5) for terminal sprouting in the CA3 and supragranular regions (22,23). In addition, another series of sections were stained with thionin to assess cell number and architecture (24). Slides were analyzed for cell loss in the CA3, CA1, and hilar region by using a semiquantitative visual scoring system (0–5) (24). Cell loss was assessed both ipsilateral and contralateral to the injection site. Five brain slices per rat (10 hippocampal sections in total) were examined, and these scores were added to obtain a total score for each region. This score was divided by 10 to come to a mean score per hippocampus per rat. All scoring was done by an investigator blinded to treatment group (G.L.H.).

Statistical analysis

The Kolmogorov–Smirnov goodness-of-fit test was used to assess normality (gaussian-shaped distribution) for all continuous variables. Two-way repeated-measures analysis of variance (ANOVA) was used to compare behavior scores between normal saline, free muscimol, and encapsulated muscimol groups with the F test for interaction used to assess differences in trajectories over the 120-min time course after pilocarpine injection (25). A Bonferroni-adjusted value of p < 0.017 (0.05/3) was considered statistically significant to account for multiple group comparisons. Factorial ANOVA with post hoc multiple comparisons by Fisher's least significant difference (LSD) procedure was used to evaluate total cell loss and Timm scores (CA3 and supragranular regions) between muscimol, LPSP, and normal saline treatment groups (26). Cell loss and sprouting of Timm fibers were compared between pilocarpine-induced status epilepticus and nonseizure rats by unpaired Student t tests. Data are presented in terms of the mean and standard deviation (SD). Statistical analysis was performed by using the SAS software package (version 6.12; SAS Institute, Cary, NC, U.S.A.). All reported p values are two-tailed.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Characteristics of muscimol-containing and blank particles

LPSPs were produced as a fine white dry powder. By electron microscopy, blank and muscimol-loaded LPSPs were generally spherical (Fig. 1). The median volume-weighted diameter was 4–5 μm [i.e., even though the majority of particles were smaller (∼1 μm), the larger particles contributed proportionally more to the total volume of material]. Typical yields from each production run were 30–40% of total solute. Actual loading of particles was verified by HPLC and was found to be equal to the theoretical loading [20 μg of muscimol per mg of particle, or 2% (wt/wt)]. Muscimol release from samples of particles was measured as per Methods (Fig. 2), with complete release occurring over 5 days.

image

Figure 1. Scanning electron micrograph of lipid–protein–sugar particles loaded 0.2% (wt/wt) with muscimol.

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image

Figure 2. In vitro release of muscimol from 0.2% (wt/wt) muscimol particles. Data points are medians with standard deviations.

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Effect of particles against focal pilocarpine-induced seizures

In animals in which pilocarpine injection was preceded by administration of normal saline (i.e. no muscimol), seizure activity was stereotyped (Fig. 3). Around 10–15 min after pilocarpine injection, rats became immobile with minimal facial movements and staring. This was followed by chewing salivation, which then progressed into forelimb clonus, occurring either unilaterally or bilaterally. Eventually the animal had bilateral forelimb clonus with rearing. The final stage of the seizure was tonic posturing. After the tonic phase, the animals returned to early seizure stages. For example, tonic activity would be interspersed with forelimb clonus, chewing, and immobility.

image

Figure 3. Mean seizure scores, with standard deviations, of rats given pilocarpine after administration of various treatments (n = 4 in all groups).

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Repeated-measures ANOVA indicated a significant overall difference in mean behavior scores between animals receiving normal saline, 5 μg of unencapsulated (free) muscimol, and 5 μg of encapsulated muscimol before pilocarpine; F(2, 9) = 11.95, p < 0.001. Multiple comparisons revealed that the rise of the trajectory in behavior score over the 120-min time course after pilocarpine injection was significantly faster in the normal saline group compared with the free muscimol (F = 4.52, p < 0.001) and encapsulated muscimol (F = 7.39, p < 0.001) groups, indicating that muscimol reduces seizure activity as measured by behavior score. The increase in behavior score was significantly faster (steeper slope) for animals in the free muscimol compared with the encapsulated muscimol group (F = 2.68, p < 0.01), suggesting that encapsulation enhances the protective antiepileptic effect of muscimol. Blank particles did not exert an antiepileptic effect: there was no significant difference in the change in seizure scores over time between animals receiving blank LPSPs and normal saline over the 120 min after pilocarpine injection (F = 0.44, p = 0.69).

Animals receiving particles containing 10 μg (n = 4) and 20 μg (n = 1) before administration of pilocarpine did not experience seizures. We did not pursue additional experiments in these groups because of the high mortality in the unencapsulated comparison groups, presumably from the side effects of muscimol.

Histologic findings

The brains of animals from these experimental groups were analyzed as described earlier for cell-loss scores (from thionin-stained sections) and sprouting of Timm fibers (from Timm-stained sections; Figs. 4 and 5). Animals receiving pilocarpine-induced status epilepticus had apparent cell loss in CA3, CA1, and the hilus and sprouting of Timm fibers in the supragranular and CA3 hippocampal subfield. As expected, compared with nonseizure rats, animals that had seizures had significantly higher cell-loss scores (10.2 ± 3.1 vs. 4.5 ± 2.8; p < 0.001) and supragranular Timm scores (1.8 ± 1.0 vs. 0.9 ± 0.7; p = 0.03). CA3 Timm scores were also higher in the status epilepticus rats than in the nonseizure rats (1.5 ± 0.7 vs. 0.6 ± 0.4; p < 0.01).

image

Figure 4. Representative examples of cell loss in hippocampus (A, B, CA3 subfield; C, D, CA1 subfield of rats given pilocarpine after 5 μg of encapsulated (A, C) or unencapsulated (B, D) muscimol. Images A and C were from a rat that did not have a seizures, whereas images B and D were from a rat that did. Note cell loss as indicated by arrows. Brains were stained with thionin. (Calibration = 50 μm.)

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image

Figure 5. Representative examples of Timm staining in hippocampus (A, B, CA3 subfield; C–E, hilus). A and C are from rats given pilocarpine after 5 μg of encapsulated muscimol. The rat in B and D received unencapsulated muscimol, whereas the rat in E received normal saline before pilocarpine. Images A and C were from a rat that did not have a seizure, whereas images B, D, and E were from rats that did. Note sprouting (arrow) in the CA3 subfield in B. Sprouting of mossy fibers also was seen in the inner molecular layer (IML) of the dentate granule cells (DGC) in the two rats with seizures (arrows in D and E). Brains were stained with the Timm stain. (Calibration = 100 μm.)

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The group of animals that received ≥5 μg of encapsulated muscimol before pilocarpine (n = 9) had significantly less apparent cell loss and Timm staining than the group comprising those that received blank LPSPs or normal saline (n = 7). Unpaired Student's t tests revealed that the mean total cell loss score in the encapsulated muscimol group was 4.42 ± 2.38 compared with 11.10 ± 2.37 for rats receiving blank LPSPs and normal saline (p < 0.001). The mean Timm-stain scores were also significantly lower in the encapsulated muscimol group compared with the blank LPSP and saline group in both the CA3 (0.60 ± 0.50 vs. 1.91 ± 0.65; p < 0.001) and supragranular regions (0.82 ± 0.48 vs. 1.62 ± 0.70; p = 0.02) of the dentate gyrus.

A further analysis focused on animals that received 5 μg of muscimol and their controls was done to allow comparison between free and encapsulated muscimol (Table 2). Groups of animals receiving 5 μg of either free or encapsulated muscimol had lower total cell-loss scores than did the groups that received saline or blank LPSPs. However, there was no significant difference between the protective effects of encapsulated and free muscimol. Animals treated with encapsulated muscimol showed less sprouting of Timm fibers in CA3 and supragranular regions than did the saline-treated animals. In the supragranular region, the Timm score for encapsulated muscimol was lower than that for free muscimol. Although there was no significant difference between encapsulated and free muscimol in Timm scores in CA3, the score for free muscimol was not lower than that for normal saline (whereas that for encapsulated muscimol was). Blank LPSPs did not have an effect on total cell-loss scores or CA3 Timm scores, but did have lower supragranular Timm scores than those in the normal saline group. LPSPs containing muscimol did not have lower Timm scores than blank LPSPs, although the former did have lower scores than saline in CA3, whereas the latter did not.

Table 2.  Cell loss and Timm scores according to treatment group
OutcomeEncapsulated muscimolFree muscimolLPSPNormal salinep Value
  • Total cell loss is based on thionin staining. Data expressed as mean ± SD. LPSP, lipid–protein–sugar particles. Dosage was 5 μg in both encapsulated and free muscimol groups. Sample sizes: for all groups, n = 4, except for LPSP, where n = 3. All p values were determined by ANOVA, followed by Fisher's least significant difference procedure for multiple post hoc comparisons, in which p < 0.05 was considered significant.

  • a

     Significantly lower in both encapsulated and free muscimol groups than in both LPSP and normal saline.

  • b

     Significantly lower in encapsulated muscimol group than in normal saline.

  • c

     Significantly lower in encapsulated muscimol compared with free muscimol and normal saline, and LPSP compared with normal saline.

Total cell loss5.38 ± 3.016.27 ± 0.5710.02 ± 3.7511.13 ± 1.36<0.05a
Timm scores     
 CA3 region0.75 ± 0.701.48 ± 0.631.55 ± 0.922.18 ± 0.24<0.05b
 Supragranular0.94 ± 0.431.75 ± 0.281.05 ± 0.881.97 ± 0.11<0.05c

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Controlled release LPSPs containing muscimol successfully mitigated the onset of seizures in pilocarpine-treated rats. The effectiveness of muscimol did not appear to be adversely affected by the spray-drying manufacture process, nor by coencapsulation with phospholipids, protein, or sugar. On the contrary, the encapsulated formulation showed enhanced anticonvulsant activity compared with the free drug, in terms of both seizure scores and histologic injury. This improved performance is unlikely to be due to a separate action of the putatively inert excipients on neurons or glia, or inactivation of pilocarpine by those excipients, because blank particles did not mitigate seizure scores. The latter finding also argues against the possibility that the injected particles—which were placed before pilocarpine—somehow acted as a barrier or sponge preventing pilocarpine from inducing seizures.

Muscimol-loaded microparticles also mitigated the histologic changes from pilocarpine administration, but were only slightly more effective than free muscimol in doing so. It is possible that this effect will be accentuated in more chronic models of disease, and with formulations that have a more extended timeframe of drug release. The latter is certainly conceivable, as microspheres with drug-release durations lasting months are available clinically for other indications (27). In this regard it also is encouraging that other investigators, with more macroscopic devices, have shown prolonged effectiveness (11,12) (see later). Although blank particles did not mitigate seizure activity or cell loss, we cannot exclude the possibility that they had a mild intrinsic protective effect, given their mitigation of supragranular Timm sprouting.

The finding that particles loaded with muscimol prevented clinical seizure activity to a greater extent than did free muscimol was, in a way, counterintuitive. In general, one would expect a given amount of free drug to be more efficacious in the short term than the same amount of drug encapsulated, as it will cause higher drug levels initially. It is possible that the improved efficacy of the encapsulated drug stems from the design of the model used. The AED regimens were administered 80 min before the end of the pilocarpine infusion. Free muscimol may have largely diffused away from the site of injection during that interval, whereas the encapsulated form maintained an effective concentration for a longer time (∼80% of the encapsulated drug was released after 80 min).

These results demonstrate the potential utility of focally delivered drug-loaded microparticles in the treatment of clinical seizure activity. This is consistent with animals studies showing that a macroscopic implant releasing tetrodotoxin can prevent posttraumatic epileptogenesis (10), that a polymeric microdisk containing thyrotropin-releasing hormone can suppress kindling expression (11), and that a polymeric device containing phenytoin reduces experimental seizures (12). The microparticles described here are individually ∼100 times smaller than those devices and could easily be applied by stereotactic injection through a very fine needle, and, being composed of naturally occurring substances that are both biocompatible and completely biodegradable, would not present a long-term foreign body. It is likely that they would be safe for intracranial use. Similar particles injected into murine cerebral parenchyma did not cause any detectable tissue injury or inflammation. Furthermore, when injected into cerebral ventricles, they did not cause obstructive hydrocephalus, and when injected into the internal carotid artery, only had effects on cerebral blood flow when injected rapidly in great quantity (14).

Controlled release of AEDs at the focus of epileptic activity holds several theoretic advantages over systemic delivery by the oral or intravenous routes. Controlled-release technology can yield high local concentrations of drug with relatively low total drug release. Only the affected area of the brain will be treated, thereby minimizing neuropsychiatric effects of the drugs. Furthermore, intractable seizure activity treated in this manner might not require the generalized ablation of neural activity that a pentobarbital coma entails, with the associated respiratory depression and hypotension that always necessitate mechanical ventilation and routinely require vasoactive drugs. Microparticles could serve a diagnostic purpose, in helping to demarcate the extent of a seizure focus for eventual ablation. Local controlled release could markedly improve the therapeutic index of the drugs with respect to systemic effects (e.g., hepatotoxicity). Furthermore, because drugs given in this way should achieve much lower systemic levels for a given degree of effectiveness, there should be less induction of hepatic enzymes and other untoward drug interaction.

These particles are attractive vehicles for the delivery of therapeutics because the process by which they are produced—spray drying—is very flexible in terms of drugs and excipients that can be incorporated. Thus they can be made to contain a range of drugs or drug combinations, allowing exploration of the local effects of synergistic drug regimens. Similarly the excipients can readily be changed if they are undesirable for some reason (e.g., antigenicity of protein content). Varying the composition of the excipients could also potentially permit modulation of the duration of drug release (13), depending on which of several possible mechanisms (27) are relevant to the release of muscimol and/or other drugs. Such modifications will be important in optimizing the time span over which therapeutic effects can be extended. It also bears mentioning that particles similar to these (28) have been used for inhalational delivery of a variety of compounds. Presumably, therefore, such particles could be used for systemic delivery of AEDs, by inhalation.

Acknowledgment: We thank Dr. R. S. Fisher for helpful suggestions. This work was supported by grant NIH GM00684 (to D.S.K.), NIH GM26698 (to R.L.), NIH Mental Retardation Research Center 2P30HD18655, and NINDS NS27984 (to G.L.H.). This work is dedicated to Katelyn Gokey.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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