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Objectives Cannabidiolic acid (CBDa) is pharmacologically unique from cannabidiol (CBD), but its chemical instability poses challenges for potential clinical utility. Here, we used magnesium ions to stabilize two cannabidiolic acid-enriched hemp extracts (Mg-CBDa and Chylobinoid, the latter of which also contains minor cannabinoid constituents) and compared their anticonvulsant activities with CBD in the maximal electroshock seizure test (MES) in rats. Methods Sprague-Dawley rats received intraperitoneal (i.p.) injections of Chylobinoid, Mg-CBDa, or CBD at varying doses at discrete time points. Rats were challenged with a 0.2 s, 60 Hz, 150 mA corneal stimulation and evaluated for resultant hindlimb tonic extension. Dose-response relationships were calculated using Probit analysis and statistical significance was assessed with a two-sample z-test. Results Median effective doses (ED50) and 95% confidence intervals were calculated for each compound and adjusted according to percentage of CBDa (w/w): Chylobinoid: 76.7 (51.7 - 109.2) mg/kg. Mg-CBDa: 115.4 (98.8 - 140.9) mg/kg. CBD: 68.8 (56.6 - 80.0) mg/kg. Significance CBDa-enriched hemp extracts exhibited dose-dependent protection in the MES model at doses comparable, but not more effective than, CBD. Chylobinoid was more effective than Mg-CBDa despite lower CBDa content. Test compounds should be compared by sub-chronic dosing in the MES test in order to assess safety and pharmacokinetic profiles. CBDa should be evaluated in pharmacoresistant and chronic animal models of epilepsy.

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.

The potential clinical utility of galanin peptidic analogs has been hindered by poor metabolic stability, lack of brain penetration, and hyperglycemia. In addition to possessing potent anticonvulsant efficacy, galanin analogs are analgesic in various assays. The purpose of these studies was to evaluate the lead galanin receptor type 2 (GalR2)-preferring analog, NAX 810-2, in various pain assays, as well as determine any potential for insulin inhibition, growth hormone stimulation, and cognitive impairment. NAX 810-2 was evaluated in mouse (carrageenan, formalin, tail flick, plantar incision) and rat pain models (partial sciatic nerve ligation). NAX 810-2 dose-dependently increased paw withdrawal latency following plantar administration of carrageenan (ED50 4.7 mg/kg). At a dose of 8 mg/kg, NAX 810-2 significantly attenuated nociceptive behaviors following plantar administration of formalin, and this was observed for both phase I (acute) and phase II (inflammatory) components of the formalin behavioral response. NAX-810-2 was active at higher doses in the mouse tail flick model (ED50 20.2 mg/kg) and similarly, reduced mechanical allodynia following plantar incision in mice at a dose of 24 mg/kg. NAX 810-2 also reduced mechanical allodynia in the partial sciatic nerve ligation model at a dose of 4 mg/kg. In addition, NAX 810-2 did not impair insulin secretion at doses of 2.5 and 8 mg/kg (acutely) or at a dose of 8 mg/kg given daily for 5 days. Similarly, 8 mg/kg (twice daily, 5 days) of NAX 810-2 did not increase growth hormone levels. These results demonstrate that NAX 810-2 possesses a favorable pre-clinical profile as a novel and first-in-class analgesic.

Objective We previously demonstrated that positive allosteric modulators (PAMs) of metabotropic glutamate subtype 2 (mGlu2 ) receptors have potential synergistic interactions with the antiseizure drug levetiracetam (LEV). The present study utilizes isobolographic analysis to evaluate the combined administration of JNJ-46356479, a selective and potent mGlu2 PAM, with LEV as well as sodium valproate (VPA) and lamotrigine (LTG). Methods The anticonvulsant efficacy of JNJ-46356479 was evaluated in the 6-Hz model of psychomotor seizures in mice. JNJ-46356479 was administered in combination with LEV using 3 fixed dose-ratio treatment groups in the mouse 6-Hz (44-mA) seizure test. The combination of JNJ-46356479 with LEV was also evaluated in the mouse corneal kindling model. The potential interactions of JNJ-46356479 with the antiseizure drugs VPA and LTG were also evaluated using fixed dose-ratio combinations. Plasma levels were obtained for analysis of potential pharmacokinetic interactions for each combination studied in the mouse 6-Hz model. Results JNJ-46356479 was active in the 6-Hz model at both 32-mA and 44-mA stimulus intensities (median effective dose = 2.8 and 10.2 mg/kg, respectively). Using 1:1, 1:3, and 3:1 fixed dose-ratio combinations (LEV:JNJ-46356479), coadministration was significantly more potent than predicted for additive effects, and plasma levels suggest this synergism was not due to pharmacokinetic interactions. Studies in kindled mice further demonstrate the positive pharmacodynamic interaction of LEV with JNJ-46356479. Using 1:1 dose-ratio combinations of JNJ-46356479 with either VPA or LTG, there were no significant differences observed for coadministration. Significance These studies demonstrate a synergistic interaction of JNJ-46356479 with LEV, whereas no such effect occurred for JNJ-46356479 with either VPA or LTG. The synergy seems therefore to be specific to LEV, and the combination LEV/mGlu2 PAM has the potential to result in a rational polypharmacy approach to treat patients with refractory epilepsy, once it has been confirmed in clinical studies.

Hydrocarbon stapling is an effective strategy to stabilize the helical conformation of bioactive peptides. Here we describe application of stapling to anticonvulsant neuropeptides, galanin (GAL) and neuropeptide Y (NPY), that are implicated in modulating seizures in the brain. Dicarba bridges were rationally introduced into minimized analogs of GAL and NPY resulting in increased α-helical content, in vitro metabolic stability and n -octanol/water partitioning coefficient (log D ). The stapled analogs retained agonist activities towards their respective receptors and suppressed seizures in a mouse model of epilepsy.

Chronic pain is a multifactorial disease comprised of both inflammatory and neuropathic components that affect ∼20% of the world's population. sec-Butylpropylacetamide (SPD) is a novel amide analogue of valproic acid (VPA) previously shown to possess a broad spectrum of anticonvulsant activity. In this study, we defined the pharmacokinetic parameters of SPD in rat and mouse, and then evaluated its antinociceptive potential in neuropathic and acute inflammatory pain models. In the sciatic nerve ligation (SNL) model of neuropathic pain, SPD was equipotent to gabapentin and more potent than its parent compound VPA. SPD also showed either higher or equal potency to VPA in the formalin, carrageenan, and writhing tests of inflammatory pain. SPD showed no effects on compound action potential properties in a sciatic nerve preparation, suggesting that its mechanism of action is distinct from local anesthetics and membrane stabilizing drugs. SPD's activity in both neuropathic and inflammatory pain warrants its development as a potential broad-spectrum anti-nociceptive drug.

Conantokins are short peptides derived from the venoms of marine cone snails that act as antagonists of the N-methyl-D-aspartate (NMDA) receptor family of excitatory glutamate receptors. These peptides contain γ-carboxyglutamic acid residues typically spaced at i,i+4 and/or i,i+7 intervals, which by chelating divalent cations induce and stabilize helical conformation of the peptide. Introduction of a dicarba bridge (or a staple) can covalently stabilize peptide helicity and improve its pharmacological properties. To test the hypothesis that stapling can effectively replace γ-carboxyglutamic acid residues in stabilizing the helical conformation of conantokins, we designed, synthesized, and characterized several stapled analogs of conantokin G (conG), with varying connectivities in terms of staple length and location along the face of the α-helix. NMR studies confirmed that the ring-closing metathesis reaction yielded a single product with the Z configuration of the olefinic bond. Based on circular dichroism and molecular modeling, the stapled analogs exhibited significantly enhanced helicity compared with the native peptide in a metal-free environment. Stapling i,i+4 was benign with respect to effects on in vitro and in vivo pharmacological properties. One analog, namely conG[11-15,S(i,i+4)S(8)], blocked NR2B-containing NMDA receptors with IC(50) = 0.7 μm and provided significant protection in the 6-Hz psychomotor model of pharmacoresistant epilepsy in mice. Remarkably, unlike native conG, conG[11-15,S(i,i+4)S(8)] produced no behavioral motor toxicity. Our results extend the applications of peptide stapling to helical peptides with extracellular targets and provide a means for engineering conantokins with improved pharmacological properties.

The glycosylation of neuroactive peptides is a promising strategy to treat neurological and psychiatric disorders. Herein we investigated the effects of site-specific glycosylation of neurotensin (NT). The glycosylated analogues have low-nanomolar affinities and agonist activities toward NTS1, and suppress seizures with sub-picomolar potency. Our work points to a new research direction of exploring BBB-permeable NT analogues as potential first-in-class antiepileptic drugs. Neurotensin (NT) is an endogenous neuropeptide involved in a variety of central and peripheral neuromodulatory effects. Herein we show the effects of site-specific glycosylation on the in vitro and in vivo pharmacological properties of this neuropeptide. NT analogues containing O-linked disaccharides (β-melibiose and α-TF antigen) or β-lactose units linked by a PEG3 spacer were designed and chemically synthesized using Fmoc chemistry. For the latter analogue, Fmoc-Glu-(β-Lac-PEG3-amide) was prepared. Our results indicate that the addition of the disaccharides does not negatively affect the sub-nanomolar affinity or the low-nanomolar agonist potency for the neurotensin receptor subtype 1 (NTS1). Interestingly, three glycosylated analogues exhibited sub-picomolar potency in the 6 Hz limbic seizure mouse model of pharmacoresistant epilepsy following intracerebroventricular administration. Our results suggest for the first time that chemically modified NT analogues may lead to novel antiepileptic therapies.

Disruptions in GABAergic neurotransmission have been implicated in numerous CNS disorders, including epilepsy and neuropathic pain. Selective inhibition of neuronal and glial GABA transporter subtypes may offer unique therapeutic options for regaining balance between inhibitory and excitatory systems. The ability of two GABA transport inhibitors to modulate inhibitory tone via inhibition of mGAT1 (tiagabine) or mGAT2/BGT-1 (N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-4-(methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), also known as EF1502) was evaluated using an in vitro model of spontaneous interictal-like bursting (SB). SBs were recorded extracellularly in combined mEC-HC horizontal brain slices (400 microm; 31+/-1 degrees C) obtained from KA-treated rats. Slice recordings demonstrated that EF1502 exhibited a concentration-dependent reduction in SB frequency. EF1502 significantly reduced SB rate to 32% of control at the 30 microM concentration, while reducing the area and duration of SB activity to 60% and 46% of control, respectively, at the 10 microM concentration. In contrast, the GAT1 selective inhibitor tiagabine (3, 10, and 30 microM) was unable to significantly reduce the frequency of SB activity in the mEC, despite significantly reducing both the duration (51% of control) and area (58% of control) of the SB at concentrations as low as 3 microM. The ability of EF1502, but not tiagabine, to inhibit SBs in the mEC suggests that this in vitro model of pharmacoresistant SB activity is useful to differentiate between novel anticonvulsants with similar mechanisms of action and suggests a therapeutic potential for non-GAT1 transport inhibitors.

Hyperexcitability in the medial entorhinal cortex-hippocampal (mEC-HC) circuit in the initial weeks after prolonged seizure activity may contribute to the epileptogenic process in animal models of temporal lobe epilepsy (TLE). The present study examined combined mEC-HC slices (400 microm) using field potential recordings 1-2 weeks following the multiple administration, low-dose kainic acid (KA) model of TLE [Hellier, J.L., Patrylo, P.R., Buckmaster, P.S., Dudek, F.E., 1998. Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res. 31, 73-84]. Field potential recordings in slices from KA-treated rats demonstrated hallmarks of hyperexcitability in the mEC and in the CA1 and CA3 cell body regions of the HC. Spontaneous burst (SB) activity was observed under baseline recording conditions in the mEC of several slices from KA-treated rats, but not in the slices from saline-treated control rats. Elevating ACSF [K(+)](o) (6mM) in the presence of picrotoxin (50 microM) increased SB rates in all slices tested. However, there was a significantly shorter latency to onset of bursting and prolonged evoked response durations in layer II of the mEC of slices from KA-treated rats versus those from controls. Neither carbamazepine (CBZ) nor phenytoin (PHT) abolished SB activity in slices from KA-treated rats; whereas, SB activity in slices from control rats was dose-dependently reduced at 100 microM CBZ. In contrast, the novel anticonvulsant retigabine (RGB) dramatically reduced SB frequency in both control and KA-treated groups. The hyperexcitability observed in combined mEC-HC brain slices from KA-treated rats suggests that the mEC, as well as the HC, may contribute to the epileptogenic process after KA-induced seizure activity. This model may provide an efficient, flexible in vitro paradigm for differentiating novel AEDs in a model of pharmacoresistant bursting.

Neurotensin receptors have been studied as molecular targets for the treatment of pain, schizophrenia, addiction, or cancer. Neurotensin (NT) and Contulakin-G, a glycopeptide isolated from a predatory cone snail Conus geographus, share a sequence similarity at the C-terminus, which is critical for activation of neurotensin receptors. Both peptides are potent analgesics, although affinity and agonist potency of Contulakin-G toward neurotensin receptors are significantly lower, as compared to those for NT. In this work, we show that the weaker agonist properties of Contulakin-G result in inducing significantly less desensitization of neurotensin receptors and preserving their cell-surface density. Structure-activity relationship (SAR) studies suggested that both glycosylation and charged amino acid residues in Contulakin-G or NT played important roles in desensitizing neurotensin receptors. Computational modeling studies of human neurotensin receptor NTS1 and Contulakin-G confirmed the role of glycosylation in weakening interactions with the receptors. Based on available SAR data, we designed, synthesized, and characterized an analog of Contulakin-G in which the glycosylated amino acid residue, Gal-GalNAc-Thr10, was replaced by memantine-Glu10 residue. This analog exhibited comparable agonist potency and weaker desensitization properties as compared to that of Contulakin-G, while producing analgesia in the animal model of acute pain following systemic administration. We discuss our study in the context of feasibility and safety of developing NT therapeutic agents with improved penetration across the blood-brain barrier. Our work supports engineering peptide-based agonists with diverse abilities to desensitize G-protein coupled receptors and further emphasizes opportunities for conotoxins as novel pharmacological tools and drug candidates.

There are ongoing efforts to develop pain therapeutics with novel mechanisms of action that avoid common side effects associated with other analgesics. The anticonvulsant neuropeptide galanin is a potent regulator of neuronal excitability and has a well established role in pain modulation, making it a potential target for novel therapies. Our previous efforts focused on improving blood-brain-barrier penetration and enhancing the metabolic stability of galanin analogs to protect against seizures. More recently, we designed peripherally acting galanin analogs that reduce pain-related behaviors by acting in the periphery and exhibit preferential binding toward galanin receptor (GalR)2 over GalR1. In this study, we report preclinical studies of a monodisperse oligoethylene glycol–containing galanin analog, NAX 409-9 (previously reported as GalR2-dPEG24), in rodent analgesic and safety models. Results obtained with NAX 409-9 in these tests were compared with the representative analgesics gabapentin, ibuprofen, acetylsalicylic acid, acetaminophen, and morphine. In mice that received intraplantar carrageenan, NAX 409-9 increased paw withdrawal latency with an ED50 of 6.6 mg/kg i.p. NAX 409-9 also increased the paw withdrawal threshold to mechanical stimulation following partial sciatic nerve ligation in rats (2 mg/kg). Conversely, NAX 409-9 had no effect in the tail flick or hot plate assays (up to 24 mg/kg). Importantly, NAX 409-9 did not negatively affect gastrointestinal motility (4–20 mg/kg), respiratory rate (40–80 mg/kg), or bleed time (20 mg/kg). These studies illustrate that this nonbrain-penetrating galanin analog reduces pain behaviors in several models and does not produce some of the dose-limiting toxicities associated with other analgesics.

Although propofol is most commonly known for its general anesthetic properties, at subanesthetic doses, propofol has been effectively used to suppress seizures during refractory status epilepticus, a mechanism, in part, attributed to the inhibition of neuronal sodium channels. In this study, we have designed and synthesized two novel analogs of propofol, HS245 [2-(3-ethyl-4-hydroxy-5-isopropyl-phenyl)-3,3,3-trifluoro-2-hydroxy-propionamide] and HS357 [2-hydroxy-8-(4-hydroxy-3,5-diisopropyl-phenyl)-2-trifluoromethyl-octanoic acid amide], and determined their effects on sodium currents recorded from cultured hippocampal neurons. HS357 had greater affinity for the inactivated state of the sodium channel than propofol and HS245 (0.22 versus 0.74 and 1.2 microM, respectively) and exhibited the greatest ratio of affinity for the resting over the inactivated state. HS357 also demonstrated greater use-dependent block and delayed recovery from inactivation in comparison with propofol and HS245. Under current-clamp conditions, action potentials from hippocampal CA1 neurons in slices were evoked by current injection, or following perfusion with a zero Mg(2+)/7 mM K(+) artificial cerebrospinal fluid solution. Propofol and HS357 reduced the number of current-induced action potentials; however, HS357 caused a greater reduction in the number of spontaneous action potentials. Consistent with these electrophysiology studies, propofol and HS357 protected mice against acute seizures in the 6-Hz (22-mA) partial psychomotor model. Efficacious doses of propofol were associated with an impairment of motor coordination as assessed in the rotorod toxicity assay. In contrast, HS357 demonstrated a 2-fold greater protective index than propofol. Thus, propofol analogs represent an important structural class from which not only effective, but also safer, anti-convulsants may be developed.

Delivery of neuropeptides into the central and/or peripheral nervous systems supports development of novel neurotherapeutics for the treatment of pain, epilepsy and other neurological diseases. Our previous work showed that the combination of lipidization and cationization applied to anticonvulsant neuropeptides galanin (GAL) and neuropeptide Y (NPY) improved their penetration across the blood-brain barrier yielding potent antiepileptic lead compounds, such as Gal-B2 (NAX 5055) or NPY-B2. To dissect peripheral and central actions of anticonvulsant neuropeptides, we rationally designed, synthesized and characterized GAL and NPY analogues containing monodisperse (discrete) oligoethyleneglycol-lysine (dPEG-Lys). The dPEGylated analogues Gal-B2-dPEG(24), Gal-R2-dPEG(24) and NPY-dPEG(24) displayed analgesic activities following systemic administration, while avoiding penetration into the brain. Gal-B2-dPEG(24) was synthesized by a stepwise deprotection of orthogonal 4-methoxytrityl and allyloxycarbonyl groups, and subsequent on-resin conjugations of dPEG(24) and palmitic acids, respectively. All the dPEGylated analogues exhibited substantially decreased hydrophobicity (expressed as logD values), increased in vitro serum stabilities and pronounced analgesia in the formalin and carrageenan inflammatory pain assays following systemic administration, while lacking apparent antiseizure activities. These results suggest that discrete PEGylation of neuropeptides offers an attractive strategy for developing neurotherapeutics with restricted penetration into the central nervous system.

Prosthesis of non-critical parts of a polypeptide backbone is an at- tractive strategy to simplify bioactive peptides. This approach was applied to an opi- oid neuropeptide, Met-enkephalin, in which two adjacent Gly2-Gly3 residues were replaced with a series of non-peptidic backbone spacers varying in length and/or physicochemical properties. The backbone spacers did not affect the overall struc- tural properties of the analogues, but they did dramatically reduce their affinities and agonist activities toward - and -opioid receptors. Molecular modeling sug- gested that the decrease of the affinity of Met-enkephalin to -opioid receptor could be accounted for by the loss of a single hydrogen bond. Remarkably, the ana- logues containing the most isostere spacers retained potent antinociceptive and anticonvulsant properties that were comparable to that of the endogenous pep- tide. This unexpected high in vivo potency could not be accounted for by an increase in metabolic stability. Moreover, the antiepileptic activity could not be reversed by opioid receptor antagonists. In summary, the results obtained with the analogues containing backbone spacers suggest a novel mechanism for seizure control in the brain that involves alternative non-opioid signaling.

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.