N-Benzyl-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) with Hybrid Structure as a Candidate for a Broad-Spectrum Antiepileptic Drug.

In our recent studies, we identified compound N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) as a broad-spectrum hybrid anticonvulsant which showed potent protection across the most important animal acute seizure models such as the maximal electroshock (MES) test, the subcutaneous pentylenetetrazole (s.c. PTZ) test, and the 6-Hz (32 mA) test in mice. Therefore, AS-1 may be recognized as a candidate for new anticonvulsant effective in different types of human epilepsy with a favorable safety margin profile determined in the rotarod test in mice. In the aim of further pharmacological evaluation of AS-1, in the current study, we examined its activity in the 6-Hz (44 mA) test, which is known as the model of drug-resistant epilepsy. Furthermore, we determined also the antiseizure activity in the kindling model of epilepsy induced by repeated injection of pentylenetetrazole (PTZ) in mice. As a result, AS-1 revealed relatively potent protection in the 6-Hz (44 mA) test, as well as delayed the progression of kindling induced by repeated injection of PTZ in mice at doses of 15 mg/kg, 30 mg/kg, and 60 mg/kg. Importantly, the isobolographic analysis showed that a combination of AS-1 and valproic acid (VPA) at the fixed ratio of 1:1 displayed a supra-additive (synergistic) interaction against PTZ-induced seizures in mice. Thus, AS-1 may be potentially used in an add-on therapy with VPA. Moreover, incubation of zebrafish larvae with AS-1 substantially decreased the number, cumulative but not the mean duration of epileptiform-like events in electroencephalographic assay. Finally, the in vitro ADME-Tox studies revealed that AS-1 is characterized by a very good permeability in the parallel artificial membrane permeability assay test, excellent metabolic stability on human liver microsomes (HLMs), no significant influence on CYP3A4/CYP2D6 activity, and moderate inhibition of CYP2C9 in a concentration of 10 µM, as well as no hepatotoxic properties in HepG2 cells (concentration of 10 µM).

Molecular Dynamic in Ethosuximide Glass Forming Pharmaceutical as Studied by Dielectric Relaxation Spectroscopy.

Polymorphism and molecular dynamics of ethosuximide with molecules of left- and right-handed chirality have been studied in detail using dielectric spectroscopy. Density functional theory calculations of molecular conformations and dimer formation were performed to aid the interpretation of measurements. Moving window correlation analysis of the imaginary part of dielectric permittivity spectra allowed us to complete the monotropic system of phases found by the differential scanning calorimetry method. Extra transition connected with freezing-in/activation of slow molecular motions was identified in partially ordered crystal CrI phase. In high-temperature orientationally disordered CrIh and in low-temperature conformationally disordered CrIl phases, 2 relaxation processes were detected at frequency range below 105 Hz. In glass of CONDIS CrIl, ß-relaxation was identified.

Microextraction combined with microderivatization for drug monitoring and protein modification analysis from limited blood volume using mass spectrometry.

In the clinic, ethosuximide is commonly used to treat generalized absence seizures but has recently been repurposed for other diseases. Because of adverse effects and drug interactions, high-throughput therapeutic drug monitoring of ethosuximide is necessary. Microextraction is a simple, effective, rapid, and low consumption of organic solvents method for sample preparation. In this study, microderivatization-increased detection (MDID)-combined microextraction was used to detect ethosuximide by mass spectrometry. Ethosuximide is a difficult to retain and ionize compound in the C18 nano-flow column and ionization interface, respectively. Hence, we developed a fast method for detecting ethosuximide in human plasma by using the MDID strategy (within 2 min). Chemical microderivatization parameters were studied and optimized to increase the sensitivity of ethosuximide detection at trace levels. The linear range for the analysis of ethosuximide in 10 µL plasma was 5-500 µg/mL with a coefficient of determination (r2) ≥ 0.995. The precision and accuracy of intraday and interday analyses of ethosuximide were below 13.0%. Furthermore, modifications of major proteins in plasma and blood cells, induced by ethosuximide, were identified. The proposed method effectively utilizes microliter samples to detect drug plasma concentrations under suitable microextraction procedures toward the eco-friendly goal of low consumption of organic solvents. Graphical abstract ᅟ.

Flow- and voltage-dependent blocking effect of ethosuximide on the inward rectifier K⁺ (Kir2.1) channel.

Absence seizures are manifestations of abnormal thalamocortical oscillations characterized by spike-and-wave complexes in EEG. Ethosuximide (ETX) is one of the principal medications against absence seizures. We investigate the effect of ETX on the Kir2.1 channel, a prototypical inward rectifier K(+) channel possibly playing an important role in the setting of neuronal membrane potential. We demonstrate that the outward currents of Kir2.1 channels are significantly inhibited by intracellular ETX. We further show that the movement of neutral molecule ETX in the Kir2.1 channel is accompanied by ∼1.2 K(+), giving rise to the vivid voltage dependence of ETX unbinding rate. Moreover, the apparent affinity (K d ) of ETX in the channels are decreased by single-point mutations involving M183, E224, and S165, and especially by double mutations involving T141/S165, which always also disrupt the flux-coupling feature of ETX block. Molecular dynamics simulation demonstrates narrowing of the pore at ∼D172 by binding of ETX to S165 or T141. ETX block of the Kir2.1 channels may cause a modest but critical depolarization of the relevant neurons, decreasing available T-type Ca(2+) channels and consequently lessening pathological thalamocortical burst discharges.

Induction of neuronal differentiation of rat muscle-derived stem cells in vitro using basic fibroblast growth factor and ethosuximide.

Several studies have demonstrated that basic fibroblast growth factor (bFGF) can induce neural differentiation of mesenchymal stem cells. In this study, we investigated the neural differentiation of muscle-derived stem cells (MDSCs) following treatment with bFGF and ethosuximide, a small molecule used as an anticonvulsant in humans. Stem cells isolated from rat skeletal muscle (rMDSCs) were pre-induced by culturing with 25 ng/mL bFGF for 24 h and then were transferred to a medium supplemented with or without 4 mM ethosuximide. Neuronal differentiation was assessed by immunocytochemical and western blotting analyses of marker expression. Immunocytochemistry of rMDSCs treated with bFGF and ethosuximide identified abundant cells expressing neuronal markers (TuJ1, neuron-specific class III ß-tubulin; NeuN, neuronal nuclear antigen; and NF-MH; neurofilament M and H). Olig2 (oligodendrocyte transcription factor 2)-positive cells were also observed, indicating the presence of oligodendrocyte lineage cells. These findings were substantiated by western blotting analysis of marker proteins. In particular, the expression of NeuN and TuJ1 was significantly higher in rMDSCs treated with ethosuximide and bFGF than in cells stimulated with bFGF alone (NeuN, p < 0.05 and TuJ1, p < 0.001). Expression of the astrocyte marker GFAP (glial fibrillary acidic protein) was not detected in this study. Collectively, the results showed that treatment with bFGF and ethosuximide induced effective transdifferentiation of rMDSCs into cells with a neural-like phenotype. Notably, rMDSCs treated with a combination of bFGF plus ethosuximide showed enhanced differentiation compared with cells treated with bFGF alone, implying that ethosuximide may stimulate neuronal differentiation.

How did phenobarbital's chemical structure affect the development of subsequent antiepileptic drugs (AEDs)?

Phenobarbital has been in clinical use as an antiepileptic drug (AED) since 1912. The initial clinical success of phenobarbital and other barbiturates affected the design of subsequent AEDs (e.g., phenytoin, primidone, ethosuximide), developed between 1938 and 1962, the chemical structures of which resemble that of phenobarbital. However, the empirical discovery of carbamazepine (1962) and the serendipitous discovery of valproic acid (1967) led to subsequent AEDs having chemical structures that are diverse and completely different from that of phenobarbital. Sixteen AEDs were introduced between 1990 and 2012. Most of these AEDs were developed empirically, using mechanism-unbiased anticonvulsant animal models. The empirical nature of the discovery of these AEDs, coupled with their multiple mechanisms of action, explains their diverse chemical structures. The antiepileptic market is therefore crowded. Future design of new AEDs must have a potential for treating nonepileptic central nervous system (CNS) disorders (e.g., bipolar disorder, neuropathic pain, migraine prophylaxis, or restless legs syndrome). The barbiturates were once used as sedative-hypnotic drugs, but have been largely replaced in this role by the much safer benzodiazepines. In contrast, phenobarbital is still used worldwide in epilepsy. Nevertheless, the development of nonsedating phenobarbital derivatives will answer a clinical unmet need and might make this old AED more attractive.

Design and characterization of a novel amphiphilic chitosan nanocapsule-based thermo-gelling biogel with sustained in vivo release of the hydrophilic anti-epilepsy drug ethosuximide.

Thermo-gelling injectable nanogels, with no burst release of loaded drug, were prepared by a simple route by combining self assembled nanocapsules of amphiphilically modified chitosan with glycerophosphate di-sodium salt and glycerol. The potential as a depot drug delivery system was demonstrated in vivo through the therapeutic effect of ethosuximide (ESM) loaded nanogels, suppressing spike wave discharges (SWDs) in Long Evan rat model. Simultaneously clearance of gels from the site of administration was monitored non-invasively using MRI. The gel structure was characterized using TEM and SEM, confirming the gels to be an assembly of nanocapsules and using two-photon microscopy to visualize the network structure. In vitro drug release studies using ESM revealed that the nanogels exhibited extended, mostly Fickian release. Finally, all investigated formulations displayed excellent cytotoxicity data determined by MTT assay using human retinal pigmented epithelium cells. All presented properties are highly desirable for injectable depot gels for drug delivery.

Ethosuximide and phenobarbital promote wound healing via enhancing collagenization.

The fact that ethosuximide (ETO), phenobarbital (PHO), and barbituric acid (BARB) share structural and pharmacophoric homologies with phenytoin and allantoin, both known to have significant wound-healing properties, prompted us to evaluate them as wound-healing agents. Accordingly, ETO-, PHO-, and BARB-containing ointments were applied onto full-thickness excision and incision wounds created on the dorso-lumbar region of experimental rats. ETO-and PHO-treated incision wounds illustrated significant enhancement in breaking strengths (1380 ± 61 and 1240 ± 42 g, respectively) compared to vehicle controls (1070 ± 18 g) and BARB (1080 ± 45 g). Moreover, biochemical analyses revealed significant increase in hydroxyproline contents in ETO- and PHO-treated wounds compared to vehicle controls. Histological evaluation revealed that both ETO and PHO promoted collagen synthesis and deposition. This is the first time to describe the significant wound-healing merits of ETO and PHO as potential clinical agents for treatment of chronic wounds.

Quantum mechanical and spectroscopic (FT-IR, FT-Raman, 13C, 1H and UV) investigations of antiepileptic drug Ethosuximide.

The Fourier Transform Infrared (FT-IR) and Fourier Transform Raman (FT-Raman) spectra of antiepileptic drug Ethosuximide (ETX) have been recorded and analyzed. In addition, the IR spectra in CCl(4) at various concentrations of ETX are also recorded. The equilibrium geometry, bonding features and harmonic vibrational frequencies have been investigated with the help of Density Functional Theory (DFT) method. The (1)H and (13)C nuclear magnetic resonance (NMR) chemical shifts of the molecule were calculated by the Gauge Including Atomic Orbital (GIAO) method. Stability of the molecule arising from hyperconjugative interactions and charge delocalization has been analyzed using natural bond orbital (NBO) analysis. The results show that charge in electron density (ED) in the σ* and π* antibonding orbitals and second order delocalization energies E(2) confirms the occurrence of intramolecular charge transfer (ICT) within the molecule. UV-vis spectrum of the compound was recorded and the electronic properties, such as HOMO and LUMO energies, were performed by Time-Dependent Density Functional Theory (TD-DFT) approach. Finally the calculation results were applied to simulate infrared and Raman spectra of the title compound which showed good agreement with observed spectra.

Development of a high-throughput method for the determination of ethosuximide in human plasma by liquid chromatography mass spectrometry.

A simple, rapid, sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of ethosuximide in human plasma is described. Analyte was chromatographed on a Hypersil Gold C18 column (100 mm x 2.1 mm, i.d., 1.9 microm) with isocratic elution at a flow rate of 0.250 mL/min and pravastatin was used as the internal standard. The assay involves a simple solid-phase extraction procedure of 0.25 mL human plasma and the analysis was performed on a triple-quadrupole tandem mass spectrometer by MRM mode via electrospray ionization (ESI). The method was linear in the concentration range of 0.25-60.0 microg/mL. The lower limit of quantification (LLOQ) was 0.25 microg/mL. The within- and between-day precision and accuracy of the quality control samples were within 10.0%. The recovery was 95.1% and 94.4% for ethosuximide and pravastatin, respectively. The analysis time for each sample was 1.8 min. The method was highly reproducible and gave peaks with excellent chromatography properties.

A flexible drug delivery chip for the magnetically-controlled release of anti-epileptic drugs.

A flexible drug delivery device was designed and fabricated using electrophoretic deposition of drug-carrying magnetic core-shell Fe(3)O(4) at SiO(2) nanoparticles onto an electrically conductive flexible PET substrate. The PET substrate was first patterned to a desired layout and subjected to deposition. In doing so, a uniform and nanoporous membrane could be produced. After lamination of the patterned membranes, a final chip-like device of thickness less than 0.5mm is formed that is used for controlled delivery of an anti-epileptic drug, i.e., ethosuximide (ESM). The release of useful drugs can be controlled by directly modulating the magnetic field, and the chip is capable of demonstrating a variety of release profiles (i.e., slow release, sustained release, step-wise release and burst release profiles). These profiles can follow a wide spectrum of patterns ranging from zero to pulsatile release kinetics depending on the mode of magnetic operation. When the magnetic field was removed, the release behavior was instantly ceased, and vice versa. A preliminary in-vivo study using Long-Evans rat model has demonstrated a significant reduction in spike-wave discharge after the ESM was burst released from the chip under the same magnetic induction as in-vitro, indicating the potential application of the drug delivery chip. The flexible and membrane-like drug delivery chip utilizes drug-carrying magnetic nanoparticles as the building blocks that ensure a rapid and precise response to magnetic stimulus. Moreover, the flexible chip may offer advantages over conventional drug delivery devices by improvement of dosing precision, ease of operation, wider versatility of elution pattern, and better compliance.

Ethosuximide: from bench to bedside.

Ethosuximide, 2-ethyl-2-methylsuccinimide, has been used extensively for "petit mal" seizures and it is a valuable agent in studies of absence epilepsy. In the treatment of epilepsy, ethosuximide has a narrow therapeutic profile. It is the drug of choice in the monotherapy or combination therapy of children with generalized absence (petit mal) epilepsy. Commonly observed side effects of ethosuximide are dose dependent and involve the gastrointestinal tract and central nervous system. Ethosuximide has been associated with a wide variety of idiosyncratic reactions and with hematopoietic adverse effects. Typical absence seizures are generated as a result of complex interactions between the thalamus and the cerebral cortex. This thalamocortical circuitry is under the control of several specific inhibitory and excitatory systems arising from the forebrain and brainstem. Corticothalamic rhythms are believed to be involved in the generation of spike-and-wave discharges that are the characteristic electroencephalographic signs of absence seizures. The spontaneous pacemaker oscillatory activity of thalamocortical circuitry involves low threshold T-type Ca2+ currents in the thalamus, and ethosuximide is presumed to reduce these low threshold T-type Ca2+ currents in thalamic neurons. Ethosuximide also decreases the persistent Na+ and Ca2+ -activated K+ currents in thalamic and layer V cortical pyramidal neurons. In addition, there is evidence that in a genetic absence epilepsy rat model ethosuximide reduces cortical gamma-aminobutyric acid (GABA) levels. Also, elevated glutamate levels in the primary motor cortex of rats with absence epilepsy (but not in normal animals) are reduced by ethosuximide.

Estimation of pK(a) values using microchip capillary electrophoresis and indirect fluorescence detection.

Microchip capillary electrophoresis (CE), coupled with indirect fluorescence detection was investigated for estimating the pK(a) values of non-fluorescent compounds. The CE method is based on the differences in electrophoretic mobility of the analyte as a function of the pH of the running buffer. Nine compounds were tested, including several of pharmaceutical importance, with pK(a) values from 10.3 to 4.6. All buffers contained 5-TAMRA as the fluorescent probe for indirect detection. Calculated pK(a) values agreed well with literature values obtained by traditional methods, differing not more than 0.2 from the literature value. The current work on single lane chips demonstrates the principle of microchip CE with indirect detection as a viable method for estimating pK(a) values. However, increased throughput will be required using a multilane chip to enable the approach to be used practically.

Thermal and rheological study of lipophilic ethosuximide suppositories.

We have performed thermal (differential scanning calorimetry) and rheological studies in the preformulation phase of ethosuximide suppositories to determine the influence of the active ingredient and adjuvants on the melting characteristics and rheological performance of the suppositories. Both types of studies were performed on the fatty bases witepsol H-19 and suppocire AP, each mixed with 5% w/w polysorbate 80, 0.1% w/w docusate sodium and 3% w/w tetranyl AT-1/DP and on the suppository formulations obtained by the addition of 150 mg of ethosuximide to these excipients. Pure lipophilic excipients have two peaks in the DSC curve. The first one occurs at lower temperatures (27.35 degrees C for the suppocire AP and 34.3 degrees C for the witepsol H19) and the second peak at higher temperatures (36.27 and 35.95 degrees C for the suppocire AP and witepsol H19, respectively). Polysorbate 80, docusate sodium and tetranyl AT-1/DP cause the first melting peak to disappear and the second peak to occur at a lower temperature. The thermograms of the formulas show two melting peaks and the endothermic peak does not appear due to the melting of ethosuximide (47.8 degrees C). We attribute this behavior to the solubility of the drug in the melted excipient, thus originating a decrease of the melting temperature of the second peak in the DSC scan. The excipients and formulations behave as Newtonian fluids and the influence of ethosuximide and adjuvants can be noted in an increase in the viscosity of the suppository mass.