Liquid chromatographic methods for determination of the new antiepileptic drugs stiripentol, retigabine, rufinamide and perampanel: A comprehensive and critical review

The new antiepileptic drugs perampanel,retigabine,rufinamide and stiripentol have been recently approved for different epilepsy types.Being them an innovation in the antiepileptics armamentarium,a lot of investigations regarding their pharmacological properties are yet to be performed.Besides,considering their broad anticonvulsant activities,an extension of their therapeutic indications may be worthy of investigation,especially regarding other seizure types as well as other central nervous system disorders.Although different liquid chromatographic(LC)methods coupled with ultraviolet,fluorescence,mass or tandem-mass spectrometry detection have already been developed for the determination of perampanel,retigabine,rufinamide and stiripentol,new and more cost-effective methods are yet required.Therefore,this review summarizes the main analytical aspects regarding the liquid chromatographic methods developed for the analysis of perampanel,retigabine(and its main active metabolite),rufinamide and stiripentol in biological samples and pharmaceutical dosage forms.Furthermore,the physicochemical and stability properties of the target compounds will also be addressed.Thus,this review gathers,for the first time,important background information on LC methods that have been developed and applied for the determination of perampanel,retigabine,rufinamide and stiripentol,which should be considered as a starting point if new(bio)analytical techniques are aimed to be implemented for these drugs.

Retigabine alleviates chronic restraint stress -induced memory retrieval impairment in male mice

Aim To investigate whether Kv7 channels opener retigabine could alleviate memory impairment induced by chronic restraint stress （CRS） and the underlying neuroprotective mechanisms. Methods Adult male Kunming （KM） mice, weighing 20 - 25 g, were restrained in well-ventilated Plexiglass tubes for 6 h daily beginning from 10 ： 00 to 16 ： 00 for 21 consecutive days. Mice were injected with retigabine （ 10 mg · kg^-1） or vehicle （ 10% DM- SO） 30 rain before restraint stress for 21 days. After stressor cessation, the spatial learning and memory was deter- mined by Morris water maze test, the levels of p-Akt, p-GSK-3β and p-Erkl/2 of hippocampal tissues were exam- ined by western blot. Results Compared with control group, CRS mice exhibited significantly longer escape laten- cies on day 2, 3 and 4 （P 〈 0.05, P 〈 0. 01, P 〈 0.01 ） respectively, but retigabine （ 10 mg · kg^-1） treatment had no influences on escape latencies compared with CRS group. During the probe test, CRS mice spent significant less time in target quadrant than control group （P 〈 0.01 ）. Compared with CRS group, retigabine （ 10 mg · kg^-1 ） treatment increased the time spent in target quadrant （P 〈 0.01 ）. Additionally, the swimming speed showed no significant differences among groups. Western blot results showed that the levels of p-Akt, p-GSK-3β and p-Erkl/ 2 in the hippocampus of CRS mice were significantly decreased compared with control group. Compared with CRS group, retigabine （ 10 mg· kg^-1） treatment strongly prevented the reduction of p-Akt and p-GSK-3 β （P 〈 0.01 ）, but had no effect on the reduction of p-Erkl/2. Conclusion Retigabine protected against CRS-induced spatial memory retrieval impairment partly via activation of Akt/GSK-3β signaling pathway.

Development and validation of a stability-indicating HPLC method for the determination of retigabine and its related substances in drug substances

A stability-indicating high-performance liquid chromatography（HPLC） method has been developed and validated for the separation and determination of Retigabine and its related substances. The chromatographic separation was achieved on Agilent Eclipse Plus C18 column（4.6 mm×150 mm, 5 μm）. The mobile phase was constituted of triethylamine-phosphate buffer as A and acetonitrile as B. The analysates were then eluted under the gradient conditions as description in this paper. The forced degradation study validated that the newly developed method was specific and selective to the degraded products. The performance of the method was verified according to the present International Conference on Harmonisation（ICH） guidelines for specificity, linearity, accuracy, precision and robustness. The correlation coefficients for Retigabine and its six impurities were greater than 0.999, which was shown in the regression analysis. Limits of detection（LOD） of these impurities were in the range of 0.0092%–0.0103%, indicating the high sensitivity of the newly developed method. Accuracy of the method was determined on the basis of recoveries to be between 96.49% and 118.35% for all impurities. Relative standard derivation（RSD） receiving in the repeatability and intermediate precision experiment, was less than 1.0%. The method can be successfully applied to routine quantify and stability testing Retigabine and its related substances in bulk drugs.

Kv7 channels a potential therapeutic target in fibromyalgia: A hypothesis

Fibromyalgia is characterized by the primary symptomsof persistent diffuse pain, fatigue, sleep disturbance and cognitive dysfunction. Persistent pain conditions, such as fibromyalgia, are often refractory to current available therapies. An involvement of K^+ channels in the pathophysiology of fibromyalgia is emerging and supported by drug treatments for this condition exhibiting action at these molecular processes. K^+ channels constitute potential novel target candidates for pain therapy offering peripheral and/or central actions. The Kv7 channel activators, flupirtine and retigabine, have exhibited pharmacological profiles compatible to the requirements needed for use as a therapeutic approach to fibromyalgia. Clinical trials to address the multidimensional challenges of fibromyalgia with flupirtine and retigabine will provide important insight to the role of K^+ channels in this condition.

KCNQ Channels in the Mesolimbic Reward Circuit Regulate Nociception in Chronic Pain in Mice

Mesocorticolimbic dopaminergic(DA) neurons have been implicated in regulating nociception in chronic pain, yet the mechanisms are barely understood. Here, we found that chronic constructive injury(CCI) in mice increased the firing activity and decreased the KCNQ channel-mediated M-currents in ventral tegmental area(VTA) DA neurons projecting to the nucleus accumbens(NAc). Chemogenetic inhibition of the VTA-to-NAc DA neurons alleviated CCI-induced thermal nociception.Opposite changes in the firing activity and M-currents were recorded in VTA DA neurons projecting to the medial prefrontal cortex(mPFC) but did not affect nociception. In addition, intra-VTA injection of retigabine, a KCNQ opener, while reversing the changes of the VTA-to-NAc DA neurons, alleviated CCI-induced nociception, and this was abolished by injecting exogenous BDNF into the NAc.Taken together, these findings highlight a vital role of KCNQ channel-mediated modulation of mesolimbic DA activity in regulating thermal nociception in the chronic pain state.

Targeting voltage-gated Kv7/KCNQ/M-channel for therapeutic potential of neuropsychiatric disorders

M-type potassium current （IM） was initially isolated from sympathetic neurons in 1980 and named as it was inhibited by muscarine. In 1998, the molecular identity of M-current was revealed to be heterotetramers of KCNQ2 and KCNQ3 subunits, whose mutations cause neonatal epilepsy. Reduction of voltage-gated KCNQ2/3 K＋ channel （M-channel） activity leads to neuronal byperexcitability that defines the fundamental mechanism of neurological disorders such as epilepsy and pain. Thus, suppression of neuronal hyperexcitability by activation of KCNQ2/3 channels serves the basis for development of the channel openers for treatment of epilepsy and pain. The well-known KCNQ opener is retigabine （Potiga） that was approved by FDA as an antiepileptic drug in 2011. Recent studies also provide evidence that KCNQ2/3 channel openers are effective in animal models of bipolar disorder, anxiety and schizophrenia, whereas KCNQ2/3 inhibitors, on the other hand, are indicated for improvement of learning and memory in animal models. We recently designed and validated a novel series of pyrazolo [1,5-a]pyrimidin-7（4H）-ones （PPOs） that selectively activate KCNQ2/3 and show antiepileptic and analgesic activity in vivo. Up to date, all the progress made enforces the view that targeting voltage-gated KCNQ/M-channel may provide therapeutic potential for treatment of neuropsychiatric disorders.