Galanin is an endogenous neuropeptide that modulates seizures in the brain. Because this neuropeptide does not penetrate the blood-brain barrier, we designed truncated galanin analogues in which nonessential amino acid residues were replaced by cationic and/or lipoamino acid residues. The analogues prevented seizures in the 6 Hz mouse model of epilepsy following intraperitoneal administration. The most active analogue, Gal-B2 (NAX 5055), contained the -Lys-Lys-Lys(palmitoyl)-Lys-NH(2) motif and exhibited high affinity for galanin receptors (K(i) = 3.5 nM and 51.5 nM for GalR1 and GalR2, respectively), logD = 1.24, minimal helical conformation and improved metabolic stability. Structure-activity-relationship analysis suggested that cationization combined with position-specific lipidization was critical for improving the systemic activity of the analogues. Because the anticonvulsant activity of galanin is mediated by the receptors located in hippocampus and other limbic brain structures, our data suggest that these analogues penetrate into the brain. Gal-B2 may lead to development of first-in-class antiepileptic drugs.

The endogenous neuropeptide galanin and its associated receptors GalR1 and GalR2 are highly localized in the brain limbic structures and play an important role in the control of seizures in animal epilepsy models. As such, galanin receptors provide an attractive target for the development of novel anticonvulsant drugs. Our efforts to engineer galanin analogs that can penetrate the blood-brain-barrier and suppress seizures, yielded NAX 5055 (Gal-B2), a systemically-active analog that maintains low nanomolar affinity for galanin receptors and displays a potent anticonvulsant activity. In this report, we show that NAX 5055 is active in three models of epilepsy; i.e., the Frings audiogenic seizure-susceptible mouse, the mouse corneal kindling model of partial epilepsy and the 6 Hz model of pharmacoresistant epilepsy. NAX 5055 was not active in the traditional maximal electroshock and subcutaneous pentylenetetrazol seizure models. Unlike most antiepileptic drugs (AEDs), NAX 5055 showed high potency in the 6 Hz model of epilepsy across all three different stimulation currents; i.e., 22, 32 and 44 mA, suggesting a potential use in the treatment of pharmacoresistant epilepsy. Furthermore, NAX 5055 was found to be biologically active following intravenous, intraperitoneal and subcutaneous administration and efficacy was associated with a linear pharmacokinetic profile. The results of the present investigation suggest that NAX 5055 is a first-in-class neurotherapeutic for the treatment of epilepsy in patients refractory to currently approved AEDs.

Introduction of lipoamino acid (LAA), Lys-palmitoyl, and cationization into a series of galanin analogs yielded systemically-active anticonvulsant compounds. To study the relationship between the LAA structure and anticonvulsant activity, orthogonally protected LAAs were synthesized in which the Lys side chain was coupled to fatty acids varying in length from C 8 to C 18 , or to a monodispersed polyethylene glycol, PEG 4 . Galanin receptor affinity, serum stability, lipophilicity (logD) and activity in the 6 Hz mouse model of epilepsy of each of the newly synthesized analogs was determined following systemic administration. The presence of various LAAs or Lys(MPEG 4 ) did not affect the receptor binding properties of the modified peptides, but their anticonvulsant activities varied substantially, and were generally correlated with their lipophilicity. Our results suggest that varying the length or polarity of the LAA residue adjacent to positively-charged amino acid residues may effectively modulate the antiepileptic activity of the galanin analogs.

The neuropeptides galanin (GAL), neuropeptide Y (NPY) or neurotensin (NT) exhibit anticonvulsant activities mediated by their respective receptors in the brain. To transform these peptides into potential neurotherapeutics, their systemic bioavailability and metabolic stability must be improved. Our recent studies with GAL analogs suggested that an introduction of lipoamino acids in the context of oligo-Lys residues (lipidization-cationization motif) significantly increases their penetration into the brain, yielding potent antiepileptic compounds. Here, we describe an extension of this strategy to NPY and NT. Rationally designed analogs of NPY and NT containing the lipidization-cationization motif were chemically synthesized and their physicochemical and pharmacological properties were characterized. The analogs NPY-BBB2 and NT-BBB1 exhibited increased serum stability, possessed log D > 1.1, retained high affinities toward their native receptors and produced potent antiseizure activities in animal models of epilepsy following intraperitoneal administration. Our results suggest that the combination of lipidization and cationization may be an effective strategy for improving systemic bioavailability and metabolic stability of various neuroactive peptides.

Objective Potential clinical utility of galanin or peptidic analogs has been hindered by poor metabolic stability, lack of brain penetration, and hyperglycemia due to GalR1 receptor activation. NAX 810-2, a GalR2-preferring galanin analog, possesses 15-fold greater affinity for GalR2 over GalR1 and protects against seizures in the mouse 6 Hz, corneal kindling, and Frings audiogenic seizure models. The purpose of these studies was to further evaluate the pre-clinical efficacy and pharmacokinetics of NAX 810-2 in mice. Methods NAX 810-2 was administered by intravenous (IV; tail vein, bolus) injection to fully kindled (corneal kindling assay) or naïve CF-1 mice (6 Hz assay and pharmacokinetic studies). Plasma NAX 810-2 levels were determined from trunk blood samples. NAX 810-2 was also added to human plasma at various concentrations for determination of plasma protein binding. Results In the mouse corneal kindling model, NAX 810-2 dose-dependently blocked seizures following IV administration (ED50 0.5 mg/kg). In the mouse 6 Hz (32mA) seizure model demonstrated that NAX 810-2 dose-dependently blocked seizures following IV bolus administration (0.375 – 1.5 mg/kg, IV; ED50 0.7 mg/kg), with a time-to-peak effect of 0.5 h post-treatment. Motor impairment was observed at 1.5 mg/kg IV whereas one-half of this dose, 0.75 mg/kg IV, was maximally effective in the 6 Hz test. Plasma levels of NAX 810-2 show linear pharmacokinetics following IV administration and a half-life of 1.2 h. Functional agonist activity studies demonstrate that NAX 810-2 effectively activates GalR2 receptors at therapeutic concentrations. Significance These studies further suggest the potential utility of NAX 810-2 as a novel therapy for epilepsy.