Innovative first-order UV and fluorescence spectroscopic methods for the quantification of rufinamide in the presence of its toxic degradant: Application of AGREE tool.

Rufinamide (RUF) is a 3rd generation antiseizure medication (ASM) with a triazole ring that blocks voltage-gated sodium channels (VGSCs) and is most commonly used to treat Lennox-Gastaut syndrome. Thus, the present study examined the effect of RUF on EEG activity, behavioural testing, oxidative stress, and mRNA expression in PTZ-kindled mice. Male BALB/c mice were administered rufinamide (30, 60, and 90 mg/kg) for 21 days along with 11 injections of PTZ (40 mg/kg) given every other day. EEG recordings were monitored and a series of behavioural tests after RUF treatment were done to assess the post-kindling associated anxiety and memory impairment. We also assessed the oxidative alterations, variation in real-time BDNF/TrkB mRNA expression as well as neuroinflammatory markers in isolated mice brains. Analysis of results showed that RUF at the dose of 90 mg/kg maximally suppressed the progression of full-bloom seizures and decreased cortical epileptic spike discharge. Moreover, RUF showed significant anxiolytic action and prevented PTZ-induced cognitive decline in a dose-dependent manner. Because of its anti-inflammatory and antioxidant properties, RUF decreased lipid peroxidation, AChE activity, raised glutathione and superoxide dismutase levels in the mice brain. RUF suppressed the PTZ-induced upregulation of BDNF/TrkB signalling and significantly reduced pro-inflammatory cytokines. It is possible that the effects of RUF that have been seen are the consequence of decreased oxidative stress, BDNF/TrkB downregulation, and reduced expression of neuroinflammatory markers, which in turn reduce ictogenesis and improve the neuropsychiatric consequences associated with epilepsy.

Pharmacoresistance remains intractable in epilepsy, necessitating in-depth mechanism investigations. Cumulative data have pointed to active neuroinflammation in pharmacoresistant epilepsy, but the process between neuroinflammation and pharmacoresistance remains unknown. In this study we investigated how severe neuroinflammation altered anti-seizure drugs (ASMs) pharmacology. Hippocampal kindling or kainic acid-induced temporal lobe epilepsy (TLE) models were established in mice that had received intra-hippocampal LPS injection. Acute hippocampal slices were prepared; current-clamp recording was made in hippocampalpyramidal neurons to assess the impact of ASMs on neuronal excitability and sodium channels. We showed that intra-hippocampal LPS injection resulted in higher inflammatory cytokine levels in the hippocampus. LPS induced-neuroinflammation significantly decreased the antiseizure efficacy of phenytoin (PHT), carbamazepine (CBZ) and rufinamide (RUF), all the ASMs tested were unable to alleviate the seizure severities. We observed the "off-target" phenomena of ASMs, i.e. ASMs' loss of ability to suppress the firing of action potentials and the amplitudes of sodium currents in hippocampal pyramidal neurons from LPS-treated mice. We demonstrated that LPS induced-neuroinflammation promoted the degradation of spermine, an essential polyamine linked with ASM performance on sodium channels, through upregulating the catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT). Intra-hippocampal injection of SSAT agonist DENSPM mimicked LPS-induced "off-target" phenomena of ASMs, whereas injection of SSAT antagonist diminazene aceturate into hippocampus reversed the "off-target" phenomenon of ASMs in LPS-treated mice. Finally, intrahippocampal injection of spermine restored the efficacy of ASMs on action potential firings and sodium currents, resulting in the reversal of pharmacoresistance in LPS-treated TLE models. These results provide new evidence that neuroinflammation causes pharmacoresistance in TLE via promoting spermine degradation, and highlight spermine supplementation as a promising therapy for pharmacoresistant TLE.

Abstract In this study, a simple, rapid, and sensitive capillary electrophoresis (CE) method was developed that allows simultaneous analysis of zonisamide (ZNS), rufinamide (RFN), and lamotrigine (LMT) in pharmaceutical preparations. This study introduces the first application of CE for the simultaneous determination of ZNS, RFN, and LMT in pharmaceutical dosage forms. For the comparison a High-Performance Liquid Chromatography (HPLC) method was developed. In CE method, detection was achieved using a photodiode array detector (DAD) at a wavelength of 210 nm. A capillary with an internal diameter of 75 µm and an effective length of 40 cm was used for separation. The optimum conditions were achieved using 35 mM SDS, 6 mM borate, 10 mM phosphate buffer (pH 9.00) containing 5% isopropyl alcohol (IPA), and applying a potential of 15 kV. Under these conditions, the migration times of ZNS, RFN and LMT were observed to be 5.99 min, 6.77 min and 8.46 min, respectively. In HPLC method, detection was achieved using a photodiode array detector at a wavelength of 210 nm. A C18 column (4.6 × 100.0 mm, 3.5 µm i.d.) was used for separation, with a mobile phase consisting of acetonitrile and pH 4.0 50 mM phosphate buffer (18:82, v/v) at a flow rate of 1.0 mL min−1. In this method, the retention times of ZNS, RFN and LMT were observed to be 6.41 min, 7.29 min and 4.95 min, respectively. The validity of the developed methods was examined through parameters such as linearity, precision, accuracy, sensitivity, stability, and robustness. In CE method, the limit of detection (LOD) values for ZNS, RFN and LMT were calculated as 0.035 μg mL−1, 0.016 μg mL−1 and 0.007 μg mL−1, respectively. For HPLC method, the LOD values were determined as 0.003 μg mL−1, 0.002 μg mL−1 for RFN and 0.001 μg mL−1 for LMT. Both developed methods were successfully applied to pharmaceutical dosage forms and shown to be compliant with United States Pharmacopeia (USP 47 - NF 42). In conclusion, both methods developed in our study yielded comparable results in terms of robustness and analysis time. HPLC demonstrated higher sensitivity compared to CE and is preferred for analyses at very low concentrations, while CE is ideal for green chemistry applications due to its minimal solvent and sample consumption.

ABSTRACT Background Rufinamide (RUF) is an antiepileptic drug recently introduced for managing seizures in Lennox-Gastaut syndrome (LGS), but its adverse reactions are not well understood. This study aims to evaluate RUF’s safety profile using data from the FDA Adverse Event Reporting System (FAERS). Methods Disproportionality analysis was conducted to assess RUF-associated adverse drug events (ADEs), using reporting odds ratio (ROR), proportional reporting ratio (PRR), Bayesian confidence propagation neural network (BCPNN), and multi-item gamma-Poisson shrinker (MGPS). Results We collected 338 ADE reports related to RUF. Nervous system disorders were the most frequently reported signals, and several new ADEs were detected, including atonic seizures, sudden unexplained death in epilepsy, seizure clusters, multi-drug resistance, and Stevens-Johnson syndrome. Nearly half of the ADEs in pediatric patients were psychological or neurological. Disproportionality analysis within 4 weeks of treatment showed high RORs for QT shortening, sudden death, and atonic seizures. Conclusions Our study revealed prospective signals of new ADEs linked to RUF as well as revealed that both prescribers and patients were more conscious of the risks involved in its clinical use.

Pentylenetetrazole- (PTZ)-induced kindling is a broadly used experimental model to evaluate the impact of antiseizure drugs and their novel combination on seizure progression. The current study aimed to evaluate the anti-kindling effects of ivermectin (IVM) and rufinamide (RUFI) alone and their combination with vitamin E. The mice were administered 11 injections of PTZ (40 mg/kg) followed by assessment for anxiety-like behavior and cognitive abilities in a series of behavior tests with subsequent brain isolation for biochemical and histopathological evaluation. The outcomes showed a marked protection by IVM + RUFI (P<0.001) from kindling progression, anxiety-like behavior and cognitive deficit. However, additional supplementation with vitamin E worked superior to duo therapy as these mice were noted to be most fearless to visiting open, illuminated and elevated zones of open field, light/dark and elevated-plus maze (P<0.0001). Further, they showed marked remembrance of the familiar milieu in y-maze (P<0.01) and novel objection recognition (P<0.05) tests. Additionally, their recollection of aversive stimuli in passive avoidance and spatial memory in Morris water maze were evident (P<0.0001), in comparison to kindled mice. The IVM + RUFI duo therapy and its co-administration with vitamin E prevented kindling-triggered oxidative stress in brains and neuronal damage in hippocampus. We conclude that the benefits of the co-administration of vitamin E might be the results of antioxidant and anti-inflammatory effects of vitamin E which might be potentiating the antiseizure effects of RUFI and GABA-A modulating potential by ivermectin.

Efficacy, tolerability and safety of add-on third-generation antiseizure medications in treating focal seizures worldwide: a network meta-analysis of randomised, placebo-controlled trials

Antiepileptic drugs (AEDs) have been used to control epilepsy. More than 17 new AEDs, including gabapentin (GPN), lacosamide (LCM), perampanel (PER), pregabalin (PRG), rufinamide (RFM), and vigabatrin (VGB) have been approved and marketed since 1989. Accurate measurement of serum concentration of the antiepileptic drugs is crucial to achieve optimal efficacy and avoid adverse events. We describe an accurate and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of GPN, LCM, PER, PRG, RFM, and VGB in serum. The method requires a small volume of sample (10μL) and has a total chromatographic run time of 4min for simultaneous measurement of these drugs. The method showed good accuracy with a bias of -0.2-5%. The intra- and inter-day imprecision were less than 5.0% for all the analytes. The linear assay ranges were 0.3-26μg/mL for GPN, 0.15-24μg/mL for LCM, 7.4-1881ng/mL for PER, 0.03-13μg/mL for PRG, 0.78-90μg/mL for RFM, and 0.3-43μg/mL for VGB.

Rufinamide (RFM) is a clinically utilized antiepileptic drug that, as a triazole derivative, has a unique structure. The extent to which this drug affects membrane ionic currents remains incompletely understood. With the aid of patch clamp technology, we investigated the effects of RFM on the amplitude, gating, and hysteresis of ionic currents from pituitary GH3 lactotrophs. RFM increased the amplitude of Ca2+-activated K+ currents (IK(Ca)) in pituitary GH3 lactotrophs, and the increase was attenuated by the further addition of iberiotoxin or paxilline. The addition of RFM to the cytosolic surface of the detached patch of membrane resulted in the enhanced activity of large-conductance Ca2+-activated K+ channels (BKCa channels), and paxilline reversed this activity. RFM increased the strength of the hysteresis exhibited by the BKCa channels and induced by an inverted isosceles-triangular ramp pulse. The peak and late voltage-gated Na+ current (INa) evoked by rapid step depolarizations were differentially suppressed by RFM. The molecular docking approach suggested that RFM bound to the intracellular domain of KCa1.1 channels with amino acid residues, thereby functionally affecting BKCa channels' activity. This study is the first to present evidence that, in addition to inhibiting the INa, RFM effectively modifies the IK(Ca), which suggests that it has an impact on neuronal function and excitability.

Two optimized and validated high performance liquid chromatography (HPLC) and spectrophotometric methods are proposed. The developed methods were quantified with high sensitivity, accuracy, and precision at low concentrations to determine rufinamide (RUF) in active pharmaceutical ingredients (API) and pharmaceutical preparations. HPLC method was developed using a base deactivated silica Hypersil C18 column and a combination of methanol: acetonitrile: water (15: 10: 75, v/v/v) as the mobile phase and detected at 210 nm. A reaction of RUF with sodium nitrite and hydrochloric acid occurred, absorbed maximally at 385 nm was extended to develop a ultraviolet (UV)-visible spectrophotometric method to determine RUF in API and pharmaceutical preparations. Different analytical validation parameters, including specificity, linearity, accuracy, precision, the limit of detection, quantification, ruggedness, and robustness, were determined as per International Conference on Harmonization guidelines. The linearity range of RUF was 0.15-3.5 and 10-100 μg/mL for HPLC and spectrophotometric methods, respectively. The proposed investigations were valuable for drug monitoring and regular analysis of RUF in quality control and research laboratories. Moreover, the accuracy and precision obtained with the UV-visible spectrophotometer implied that it could be a cheap, easy, and alternative method, while HPLC could be sensitive to determine RUF at low concentration levels.

Short-term and long-term efficacy and safety of antiseizure medications in Lennox Gastaut syndrome: A network meta-analysis

Rufinamide (RUF) is a structurally unique anti-epileptic drug, but its protective mechanism against brain injury remains unclear. In the present study, we validated how the RUF protected mice with kainic acid (KA)-induced neuronal damage. To achieve that, a mouse epilepsy model was established by KA intraperitoneal injection. After Nissl staining, although there was a significant reduction in Nissl bodies in mice treated with KA, 40, 80, and 120 mg/kg, RUF significantly reduced KA-induced neuronal damage, in a dose-dependent manner. Among them, 120 mg/kg RUF was most pronounced. Immunohistochemistry (IHC) and western blot analysis showed that RUF inhibited the IBA-1 overexpression caused by KA to block microglia cell overactivation. Further, RUF treatment partially reversed neuroinflammatory cytokine (IL-1β, TNFα, HMGB1, and NLRP3) overexpression in mRNA and protein levels in KA mice. Moreover, although KA stimulation inhibited the expression of tight junctions, RUF treatment significantly upregulated expression of tight junction proteins (occludin and claudin 5) in both mRNA and protein levels in the brain tissues of KA mice. RUF inhibited the overactivation of microglia, suppressed the neuroinflammatory response, and reduced the destruction of blood–brain barrier, thereby alleviating the excitatory nerve damage of the KA-mice.

Cognitive, adaptive, and behavioral effects of adjunctive rufinamide in Lennox–Gastaut syndrome: A prospective observational clinical study

Rufinamide is a triazole derivative that is structurally dissimilar to other marketed antiepileptic drugs, has been assumed a marketing authorization, by the European Union and FDA, for use as a complementary therapy for seizures associated with Lennox-Gastaut syndrome. This work is concerned with development of two methods for determination of rufinamide (RUF) in presence of 1-[(2,6-difluorophenyl)methyl]-1H-1,2,3-triazole-4 carboxylic acid as its alkaline degradation product in dosage form. The first method was capable of determing RUF in the presence of its alkaline degradation product and in dosage form. Kromasil C8 column and mobile phase consisting of acetonitrile-water (50:50, v/v) were used and UV detection at 210 nm. In the second method, first derivative ratio spectrophotometry, RUF was determined by measuring peak amplitude at 269.5 nm over 5-30 μg/mL. The linearity range of RUF was 10-90 μg/mL for HPLC method covering its therapeutic range with r2 = 0.9999. Forced degradation under alkaline conditions was carried out, the degradation product was isolated and its structure was confirmed. Both methods were validated in accordance to ICH guidelines. Statistical analysis revealed no significant difference between obtained results and reported ones. The present study is useful for therapeutic drug monitoring and routine analysis of RUF in quality control laboratories. Kinetics of the alkaline degradation of RUF was studied by following the concentration of the remaining drug until complete degradation was achieved.

Lennox-Gastaut syndrome (LGS) is a severe, childhood-onset, developmental epileptic encephalopathy, with different etiologies and co-morbidities. Seizure treatment in LGS represents a major challenge; new antiepileptic drugs (AEDs) are developed to especially address seizures resulting in high morbidity and mortality, such as drop seizures. Rufinamide (RFN) is one of the latest AEDs licensed for patients with LGS. Its mechanism of action involves sodium channels in a way that is unrelated to other AEDs. Here we discuss the use of adjunctive RFN in children and adolescents with LGS and its efficacy and safety profile, based on a systematic literature review. RFN shows a very favorable profile in terms of adverse events and drug-interactions in children. It is particularly effective on tonic-atonic seizures and spasms, impacting on the quality of life of the patients. Further studies are needed to clarify the interaction profile with the newest AEDs for LGS and to assess correlations between the etiology of LGS and drug response to individualize treatment and maximize efficacy.

A simple, rapid, cost effective and extractive UV-Vis spectrophotometric method has been developed for the determination of Rufinamide(RFM) in bulk drug and pharmaceutical formulation. It was based on UV-Vis spectrophotometric measurements in which the drug made a complex with ?-naphthol in alkaline medium and give stable pale yellow colored complex which exhibits absorption maximum at 540 nm. Beer’s law was obeyed in the concentration range of 2 - 24 ?g /ml. This method was tested and validated for various parameters according to ICH guidelines. The proposed method was successfully applied for the determination of RFM in oral formulation. The results demonstrated that the procedure is accurate, precise and reproducible (relative standard deviation &lt; 2 %). As it is simple, cheap and less time consuming, it can be suitably applied for the estimation of RFM in dosage forms in quality control labs.

Design of experiments-based RP – HPLC bioanalytical method development for estimation of Rufinamide in rat plasma and brain and its application in pharmacokinetic study

Rufinamide (RUF) is a structurally unique anti-epileptic drug, used in the treatment of seizure disorders such as Lennox-Gastaut syndrome. In the present study, we investigated whether RUF protected against excitotoxic neuronal damage in the mouse hippocampal CA3 region after intraperitoneal kainic acid (KA) injection. Treatment with 25, 50 and 100mg/kg RUF significantly decreased the KA-induced neuronal death in the hippocampal CA3 region in a dose-dependent manner. In addition, 100mg/kg RUF treatment reduced the KA-induced oxidative stress-related increase of MDA level and decrease of total SOD activity in the hippocampus. KA-induced increases of pro-inflammatory cytokines, TNF-α and IL-1β, levels as well as KA-induced microglial activation were also suppressed by RUF treatment. These results indicate that RUF displays a neuroprotective effect against KA-induced excitotoxic neuronal death in the mouse hippocampus through anti-oxidant and anti-inflammatory activities.

Objective: Lennox–Gastaut syndrome (LGS) is mainly treated with antiepileptic drugs (AEDs) but using one AED is not sufficient to relieve all or even most patients. A combination of agents is usually preferred. In the current study, an isocratic, selective, sensitive, precise, and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous determination of rufinamide (RUF), lamotrigine (LAM), clonazepam (CLO), valproic acid (VAL), and diazepam (DIA) which are commonly used in the management of LGS in their dosage forms using lacosamide as internal standard.Methods: The method depends on using RESTEK C18 column (5 μm, 250 mm × 4.6 mm) and a mobile phase composed of acetonitrile:water (55: 45, v/v), pH = 3.3 adjusted with phosphoric acid. The method was conducted in an isocratic mode with a flow rate of 1ml/min and ultraviolet detection at 210 nm.Results: The linearity range was 2–40 μg/ml for RUF and DIA, 0.5–40 μg/ml for LAM and CLO, and 36–180 μg/ml for VAL.Conclusion: Statistical analysis revealed no significant difference between the results obtained and the official or reported ones for each cited drug. The method is simple to be easily implemented in quality control studies of the mentioned drugs in their pharmaceutical preparations.

Rufinamide (RUF) was characterized in terms of cyclodextrin (CD) complexation in order to improve its aqueous solubility. Binary systems of RUF with three CDs—β-cyclodextrin (β-CD), randomly methylated-β-cyclodextrin (RAMEB) and sulfobutylether-β-cyclodextrin (SBE-β-CD)—were characterized with a wide variety of analytical techniques. Liquid state characterization was carried out by complementary techniques such as nuclear magnetic resonance spectroscopy (NMR), capillary electrophoresis (CE), mass spectrometry (MS) and phase solubility studies. The latter revealed that the stability of the complexes decreased in the order of RAMEB > β-CD > SBE-β-CD. AL-type diagrams were obtained in all cases, characteristic of 1:1 stoichiometry, with a maximum of over 15-fold increase in RUF solubility, when complexed with RAMEB. NMR Job plot and MS studies confirmed phase solubility results, regarding the binding stoichiometry. 1H NMR and 2D ROESY investigations revealed the inclusion of the triazole moiety of RUF, confirmed by molecular modeling. Solid state complexation in 1:1 molar ratio was carried out by kneading method and investigated by differential scanning calorimetry (DSC) and infrared spectroscopy (IR). Comparative dissolution studies indicated an over two-fold improvement in dissolution efficacy of the kneaded products, when compared to the pure drug. Results of the present study might pave the way for a drug formulation with improved bioavailability.