Preclinical Comparison of Mechanistically Different Antiseizure, Antinociceptive, and/or Antidepressant Drugs in a Battery of Rodent Models of Nociceptive and Neuropathic Pain.

The series of experiments herein evaluated prototype drugs representing different mechanisms of antiseizure, antinociceptive or antidepressant action in a battery of preclinical pain models in adult male CF#1 mice (formalin, writhing, and tail flick) and Sprague Dawley rats partial sciatic nerve ligation (PSNL). In the formalin assay, phenytoin (PHT, 6 mg/kg), sodium valproate (VPA, 300 mg/kg), amitriptyline (AMI, 7.5 and 15 mg/kg), gabapentin (GBP, 30 and 70 mg/kg), tiagabine (TGB, 5 and 15 mg/kg), and acetominophen (APAP, 250 and 500 mg/kg) reduced both phases of the formalin response to ≤ 25% of vehicle-treated mice. In the acetic acid induced writhing assay, VPA (300 mg/kg), ethosuximide (ETX, 300 mg/kg), morphine (MOR, 5 & 10 mg/kg), GBP (10, 30, and 60 mg/kg), TGB (15 mg/kg), levetiracetam (LEV, 300 mg/kg), felbamate (FBM, 80 mg/kg) and APAP (250 mg/kg) reduced writhing to ≤ 25% of vehicle-treated mice. In the tail flick test, MOR (1.25-5 mg/kg), AMI (15 mg/kg) and TGB (5 mg/kg) demonstrated significant antinociceptive effects. Finally, carbamazepine (CBZ, 20 and 50 mg/kg), VPA, MOR (2 and 4 mg/kg), AMI (12 mg/kg), TPM (100 mg/kg), lamotrigine (LTG, 40 mg/kg), GBP (60 mg/kg), TGB (15 mg/kg), FBM (35 mg/kg), and APAP (250 mg/kg) were effective in the PSNL model. Thus, TGB was the only prototype compound with significant analgesic effects in each of the four models, while AMI, GBP, APAP, and MOR each improved three of the four pain phenotypes. This study highlights the importance evaluating novel targets in a variety of pain phenotypes.

Validation of a Preclinical Drug Screening Platform for Pharmacoresistant Epilepsy.

The successful identification of promising investigational therapies for the treatment of epilepsy can be credited to the use of numerous animal models of seizure and epilepsy for over 80 years. In this time, the maximal electroshock test in mice and rats, the subcutaneous pentylenetetrazol test in mice and rats, and more recently the 6 Hz assay in mice, have been utilized as primary models of electrically or chemically-evoked seizures in neurologically intact rodents. In addition, rodent kindling models, in which chronic network hyperexcitability has developed, have been used to identify new agents. It is clear that this traditional screening approach has greatly expanded the number of marketed drugs available to manage the symptomatic seizures associated with epilepsy. In spite of the numerous antiseizure drugs (ASDs) on the market today, the fact remains that nearly 30% of patients are resistant to these currently available medications. To address this unmet medical need, the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) revised its approach to the early evaluation of investigational agents for the treatment of epilepsy in 2015 to include a focus on preclinical approaches to model pharmacoresistant seizures. This present report highlights the in vivo and in vitro findings associated with the initial pharmacological validation of this testing approach using a number of mechanistically diverse, commercially available antiseizure drugs, as well as several probe compounds that are of potential mechanistic interest to the clinical management of epilepsy.

Acute treatment with minocycline, but not valproic acid, improves long-term behavioral outcomes in the Theiler's virus model of temporal lobe epilepsy.

OBJECTIVE: Infection with Theiler's murine encephalomyelitis virus (TMEV) in C57Bl/6J mice induces acute seizures and development of spontaneous recurrent seizures and behavioral comorbidities weeks later. The present studies sought to determine whether acute therapeutic intervention with an anti-inflammatory-based approach could prevent or modify development of TMEV-induced long-term behavioral comorbidities. Valproic acid (VPA), in addition to its prototypical anticonvulsant properties, inhibits histone deacetylase (HDAC) activity, which may alter expression of the inflammasome. Minocycline (MIN) has previously demonstrated an antiseizure effect in the TMEV model via direct anti-inflammatory mechanisms, but the long-term effect of MIN treatment on the development of chronic behavioral comorbidities is unknown. METHODS: Mice infected with TMEV were acutely administered MIN (50 mg/kg, b.i.d. and q.d.) or VPA (100 mg/kg, q.d.) during the 7-day viral infection period. Animals were evaluated for acute seizure severity and subsequent development of chronic behavioral comorbidities and seizure threshold. RESULTS: Administration of VPA reduced the proportion of mice with seizures, delayed onset of symptomatic seizures, and reduced seizure burden during the acute infection. This was in contrast to the effects of administration of once-daily MIN, which did not affect the proportion of mice with seizures or delay onset of acute symptomatic seizures. However, VPA-treated mice were no different from vehicle (VEH)-treated mice in long-term behavioral outcomes, including open field activity and seizure threshold. Once-daily MIN treatment, despite no effect on the maximum observed Racine stage seizure severity, was associated with improved long-term behavioral outcomes and normalized seizure threshold. SIGNIFICANCE: Acute seizure control alone is insufficient to modify chronic disease comorbidities in the TMEV model. This work further supports the role of an inflammatory response in the development of chronic behavioral comorbidities and further highlights the utility of this platform for the development of mechanistically novel pharmacotherapies for epilepsy.

Evaluating an etiologically relevant platform for therapy development for temporal lobe epilepsy: effects of carbamazepine and valproic acid on acute seizures and chronic behavioral comorbidities in the Theiler's murine encephalomyelitis virus mouse model.

Central nervous system infections can underlie the development of epilepsy, and Theiler's murine encephalomyelitis virus (TMEV) infection in C57BL/6J mice provides a novel model of infection-induced epilepsy. Approximately 50-65% of infected mice develop acute, handling-induced seizures during the infection. Brains display acute neuropathology, and a high number of mice develop spontaneous, recurrent seizures and behavioral comorbidities weeks later. This study characterized the utility of this model for drug testing by assessing whether antiseizure drug treatment during the acute infection period attenuates handling-induced seizures, and whether such treatment modifies associated comorbidities. Male C57BL/6J mice infected with TMEV received twice-daily valproic acid (VPA; 200 mg/kg), carbamazepine (CBZ; 20 mg/kg), or vehicle during the infection (days 0-7). Mice were assessed twice daily during the infection period for handling-induced seizures. Relative to vehicle-treated mice, more CBZ-treated mice presented with acute seizures; VPA conferred no change. In mice displaying seizures, VPA, but not CBZ, reduced seizure burden. Animals were then randomly assigned to acute and long-term follow-up. VPA was associated with significant elevations in acute (day 8) glial fibrillary acidic protein (astrocytes) immunoreactivity, but did not affect NeuN (neurons) immunoreactivity. Additionally, VPA-treated mice showed improved motor performance 15 days postinfection (DPI). At 36 DPI, CBZ-treated mice traveled significantly less distance through the center of an open field, indicative of anxiety-like behavior. CBZ-treated mice also presented with significant astrogliosis 36 DPI. Neither CBZ nor VPA prevented long-term reductions in NeuN immunoreactivity. The TMEV model thus provides an etiologically relevant platform to evaluate potential treatments for acute seizures and disease modification.

A role of SCN9A in human epilepsies, as a cause of febrile seizures and as a potential modifier of Dravet syndrome.

A follow-up study of a large Utah family with significant linkage to chromosome 2q24 led us to identify a new febrile seizure (FS) gene, SCN9A encoding Na(v)1.7. In 21 affected members, we uncovered a potential mutation in a highly conserved amino acid, p.N641Y, in the large cytoplasmic loop between transmembrane domains I and II that was absent from 586 ethnically matched population control chromosomes. To establish a functional role for this mutation in seizure susceptibility, we introduced the orthologous mutation into the murine Scn9a ortholog using targeted homologous recombination. Compared to wild-type mice, homozygous Scn9a(N641Y/N641Y) knockin mice exhibit significantly reduced thresholds to electrically induced clonic and tonic-clonic seizures, and increased corneal kindling acquisition rates. Together, these data strongly support the SCN9A p.N641Y mutation as disease-causing in this family. To confirm the role of SCN9A in FS, we analyzed a collection of 92 unrelated FS patients and identified additional highly conserved Na(v)1.7 missense variants in 5% of the patients. After one of these children with FS later developed Dravet syndrome (severe myoclonic epilepsy of infancy), we sequenced the SCN1A gene, a gene known to be associated with Dravet syndrome, and identified a heterozygous frameshift mutation. Subsequent analysis of 109 Dravet syndrome patients yielded nine Na(v)1.7 missense variants (8% of the patients), all in highly conserved amino acids. Six of these Dravet syndrome patients with SCN9A missense variants also harbored either missense or splice site SCN1A mutations and three had no SCN1A mutations. This study provides evidence for a role of SCN9A in human epilepsies, both as a cause of FS and as a partner with SCN1A mutations.

Introduction of lipidization-cationization motifs affords systemically bioavailable neuropeptide Y and neurotensin analogs with anticonvulsant activities.

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.

Electroconvulsive seizure thresholds and kindling acquisition rates are altered in mouse models of human KCNQ2 and KCNQ3 mutations for benign familial neonatal convulsions.

PURPOSE: Benign familial neonatal convulsions (BFNC) is caused by mutations in the KCNQ2 and KCNQ3 genes, which encode subunits of the M-type potassium channel. The purpose of this study was to examine the effects of orthologous BFNC-causing mutations on seizure thresholds and the acquisition of corneal kindling in mice with heterozygous expression of the mutations. METHODS: The effects of the Kcnq2 gene A306T mutation and the Kcnq3 gene G311V mutation were determined for minimal clonic, minimal tonic hindlimb extension, and partial psychomotor seizures. The rate of corneal kindling acquisition was also determined for Kcnq2 A306T and Kcnq3 G311V mice. RESULTS: Seizure thresholds were significantly altered relative to wild-type animals in the minimal clonic, minimal tonic hindlimb extension, and partial psychomotor seizure models. Differences in seizure threshold were found to be dependent on the mutation expressed, the seizure testing paradigm, the genetic background strain, and the gender of the animal. Mutations in Kcnq2 and Kcnq3 were associated with an increased rate of corneal kindling. In the Kcnq2 A306T mice, an increased incidence of death occurred during and immediately following the conclusion of the kindling acquisition period. CONCLUSIONS: These results suggest that genetic alterations in the subunits that underlie the M-current and cause BFNC alter seizure susceptibility in a sex-, mouse strain-, and seizure-test dependent manner. Although the heterozygous mice do not appear to have spontaneous seizures, the increased seizure susceptibility and incidence of death during and after kindling suggests that these mutations lead to altered excitability in these animals.

Generating orally active galanin analogues with analgesic activities.

The endogenous neuropeptide galanin has anticonvulsant and analgesic properties mediated by galanin receptors expressed in the central and peripheral nervous systems. Our previous work showed that by combining truncation of the galanin peptide with N- and C-terminal modifications afforded analogues that suppress seizures or pain upon intraperitoneal (i.p.) administration. To generate orally active galanin analogues, the previously reported lead compound Gal-B2 (NAX 5055) was redesigned by 1) central truncation, (2) introduction of D-amino acids, and 3) addition of backbone spacers. Analogue D-Gal(7-Ahp)-B2, containing 7-aminoheptanoic acid as a backbone spacer and an oligo-D-lysine motif at the C terminus, exhibits anticonvulsant and analgesic activity post-i.p. administration. Oral administration of D-Gal(7-Ahp)-B2 demonstrates analgesic activity with decreases in both acute and inflammatory pain in the mouse formalin model of pain at doses as low as 8 mg kg(-1) .

Engineering galanin analogues that discriminate between GalR1 and GalR2 receptor subtypes and exhibit anticonvulsant activity following systemic delivery.

Galanin modulates seizures in the brain through two galanin receptor subtypes, GalR1 and GalR2. To generate systemically active galanin receptor ligands that discriminate between GalR1 and GalR2, the GalR1-preferring analogue Gal-B2 (or NAX 5055) was rationally redesigned to yield GalR2-preferring analogues. Systematic truncations of the N-terminal backbone led to [N-Me,des-Sar]Gal-B2, containing N-methyltryptophan. This analogue exhibited 18-fold preference in binding toward GalR2, maintained agonist activity, and exhibited potent anticonvulsant activity in mice following intraperitoneal administration.

Structural requirements for a lipoamino acid in modulating the anticonvulsant activities of systemically active galanin analogues.

Introduction of lipoamino acid (LAA), Lys-palmitoyl, and cationization into a series of galanin analogues 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 was coupled to a monodispersed polyethylene glycol, PEG(4). Galanin receptor affinity, serum stability, lipophilicity (log D), and activity in the 6 Hz mouse model of epilepsy of each of the newly synthesized analogues were 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 analogues.

Synthesis and applications of polyamine amino acid residues: improving the bioactivity of an analgesic neuropeptide, neurotensin.

Conjugated polyamines are potential carriers for biotherapeutics targeting the central nervous system. We describe an efficient synthesis of a polyamine-based amino acid, lysine-trimethylene(diNosyl)-spermine(triBoc) with Dde or Fmoc orthogonal protecting groups. This nonnatural amino acid was incorporated into a neurotensin analogue using standard Fmoc-based protocols. The analogue maintained high affinity and agonist potency for neurotensin receptors and exhibited dramatically improved analgesia in mice. Our work provides a basis for use of polyamine amino acids in polypeptides.

Developing novel antiepileptic drugs: characterization of NAX 5055, a systemically-active galanin analog, in epilepsy models.

The endogenous neuropeptide galanin and its associated receptors galanin receptor 1 and galanin receptor 2 are highly localized in 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: 1) the Frings audiogenic seizure-susceptible mouse, 2) the mouse corneal kindling model of partial epilepsy, and 3) 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, 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 after 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 antiepileptic drugs.

Design, synthesis, and characterization of high-affinity, systemically-active galanin analogues with potent anticonvulsant activities.

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.