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- MATERIALS AND METHODS
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.
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- MATERIALS AND METHODS
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.