Novel Alaninamide Derivatives with Drug-like Potential for Development as Antiseizure and Antinociceptive Therapies─In Vitro and In Vivo Characterization.

In the present study, a series of original alaninamide derivatives have been designed applying a combinatorial chemistry approach, synthesized, and characterized in the in vivo and in vitro assays. The obtained molecules showed potent and broad-spectrum activity in basic seizure models, namely, the maximal electroshock (MES) test, the 6 Hz (32 mA) seizure model, and notably, the 6 Hz (44 mA) model of pharmacoresistant seizures. Most potent compounds 26 and 28 displayed the following pharmacological values: ED50 = 64.3 mg/kg (MES), ED50 = 15.6 mg/kg (6 Hz, 32 mA), ED50 = 29.9 mg/kg (6 Hz, 44 mA), and ED50 = 34.9 mg/kg (MES), ED50 = 12.1 mg/kg (6 Hz, 32 mA), ED50 = 29.5 mg/kg (6 Hz, 44 mA), respectively. Additionally, 26 and 28 were effective in the ivPTZ seizure threshold test and had no influence on the grip strength. Moreover, lead compound 28 was tested in the PTZ-induced kindling model, and then, its influence on glutamate and GABA levels in the hippocampus and cortex was evaluated by the high-performance liquid chromatography (HPLC) method. In addition, 28 revealed potent efficacy in formalin-induced tonic pain, capsaicin-induced pain, and oxaliplatin- and streptozotocin-induced peripheral neuropathy. Pharmacokinetic studies and in vitro ADME-Tox data proved favorable drug-like properties of 28. The patch-clamp recordings in rat cortical neurons showed that 28 at a concentration of 10 µM significantly inhibited fast sodium currents. Therefore, 28 seems to be an interesting candidate for future preclinical development in epilepsy and pain indications.

The Influence of Topinambur and Inulin Preventive Supplementation on Microbiota, Anxious Behavior, Cognitive Functions and Neurogenesis in Mice Exposed to the Chronic Unpredictable Mild Stress.

Daily living and functioning under stress can lead to mental health problems such as anxiety or depression. Over the past decades, a number of studies have been conducted to determine the relationship between the central nervous system (CNS), intestinal flora and bidirectional communication along the gut brain axis (GBA) in the maintaining of homeostasis. One of the most important factors regulating GBA functioning in exposure to stress may be a proper diet enriched in the supplementation with pre-, pro-and synbiotics. In the present study, we examined whether a 10-week oral preventive supplementation with natural prebiotics: topinambur powder (TPB) and chicory root inulin (INU) influenced an anxiety, depressive behavior and cognition in mice exposed to the chronic unpredictable mild stress (CUMS). Additionally, a fluoxetine (FLU) has been used as a reference antidepressive drug. Furthermore, we assessed the effect of TPB, INU and FLU administration on neurogenesis in mice exposed to CUMS and finally analyzed fecal microbiota for possible changes after TPB and INU supplementation in CUMS induced mice. Results obtained from the behavioral studies (elevated plaze maze, forced swim and Morris water maze test) indicated, that 10 week supplementation with TPB (250 mg/kg) and INU (66 mg/kg), similarly to FLU (12 mg/kg), significantly mitigated an anxiety and stress as well as protected learning and memory functions in the CUMS induced mice compared to the control stressed group. Additionally, TPB and INU CUMS mice showed significantly higher level of neurogenesis in comparison to control CUMS group. Interestingly, results obtained from the fecal microbiota analysis showed a beneficial effect of TPB and INU supplementation against CUMS-induced intestinal dysbiosis in mice. In conclusion, the obtained results showed that a long-term, preventive supplementation with TPB or INU alleviates the negative effects such as anxiety, cognitive disorders or dysbiosis in mice exposed to chronic unpredictable stress.

In Vivo and In Vitro Characterization of Close Analogs of Compound KA-11, a New Antiseizure Drug Candidate.

Epilepsy is a neurological disorder involving a number of disease syndromes with a complex etiology. A properly matched antiseizure drug (ASD) gives remission in up to 70% of patients. Nevertheless, there is still a group of about 30% of patients suffering from drug-resistant epilepsy. Consequently, the development of new more effective and/or safer ASDs is still an unmet clinical need. Thus, our current studies were focused on the structural optimization/modifications of one of the leading compounds, KA-11, aiming at the improvement of its antiseizure activity. As a result, we designed and synthesized two close analogs with highly pronounced drug-like physicochemical properties according to in silico predictions, namely KA-228 and KA-232, which were subsequently tested in a panel of animal seizure models, i.e., MES, 6 Hz (32 mA), scPTZ and ivPTZ. Among these compounds, KA-232, which was designed as a water-soluble salt, was distinctly more effective than KA-228 and assured similar antiseizure protection as its chemical prototype KA-11. With the aim of a more detailed characterization of both new molecules, in vitro binding tests were performed to evaluate the potential mechanisms of action. Furthermore, KA-232 was also evaluated in several ADME-Tox studies, and the results obtained strongly supported its drug-like potential. The proposed chemical modification of KA-11 enabled the identification of new pharmacologically active chemotypes, particularly water-soluble KA-232, which, despite the lack of better efficacy than the leading compound, may be used as a chemical prototype for the development of new ASDs, as well as substances potentially active in other neurological or neurodegenerative conditions.

The Effect of a Diet Enriched with Jerusalem artichoke, Inulin, and Fluoxetine on Cognitive Functions, Neurogenesis, and the Composition of the Intestinal Microbiota in Mice.

The aim of the study was to assess the effect of long-term administration of natural prebiotics: Jerusalem artichoke (topinambur, TPB) and inulin (INU) as well as one of the most popular antidepressants, fluoxetine (FLU), on the proliferation of neural stem cells, learning and memory functions, and the composition of the intestinal microbiota in mice. Cognitive functions were assessed using the Morris Water Maze (MWM)Test. Cells were counted using a confocal microscope and ImageJ software. We performed 16S rRNA sequencing to assess changes in the gut microbiome of the mice. The obtained results showed that the 10-week supplementation with TPB (250 mg/kg) and INU (66 mg/kg) stimulates the growth of probiotic bacteria, does not affect the learning and memory process, and does not disturb the proliferation of neural stem cells in the tested animals. Based on this data, we can assume that both TPB and INU seem to be safe for the proper course of neurogenesis. However, 2-week administration of FLU confirmed an inhibitory impact on Lactobacillus growth and negatively affected behavioral function and neurogenesis in healthy animals. The above studies suggest that the natural prebiotics TPB and INU, as natural supplements, may have the potential to enrich the diversity of intestinal microbiota, which may be beneficial for the BGM axis, cognitive functions, and neurogenesis.

Influence of Umbelliferone on the Anticonvulsant and Neuroprotective Activity of Selected Antiepileptic Drugs: An In Vivo and In Vitro Study.

Umbelliferone (7-hydroxycoumarin; UMB) is a coumarin with many biological properties, including antiepileptic activity. This study evaluated the effect of UMB on the ability of classical and novel antiepileptic drugs (e.g., lacosamide (LCM), levetiracetam (LEV), phenobarbital (PB) and valproate (VPA)) to prevent seizures evoked by the 6-Hz corneal-stimulation-induced seizure model. The study also evaluated the influence of this coumarin on the neuroprotective properties of these drugs in two in vitro models of neurodegeneration, including trophic stress and excitotoxicity. The results indicate that UMB (100 mg/kg, i.p.) significantly enhanced the anticonvulsant action of PB (p < 0.01) and VPA (p < 0.05), but not that of LCM orLEV, in the 6-Hz test. Whether alone or in combination with other anticonvulsant drugs (at their ED50 values from the 6-Hz test), UMB (100 mg/kg) did not affect motor coordination; skeletal muscular strength and long-term memory, as determined in the chimney; grip strength; or passive avoidance tests, respectively. Pharmacokinetic characterization revealed that UMB had no impact on total brain concentrations of PB or VPA in mice. The in vitro study indicated that UMB has neuroprotective properties. Administration of UMB (1 µg/mL), together with antiepileptic drugs, mitigated their negative impact on neuronal viability. Under trophic stress (serum deprivation) conditions, UMB enhanced the neurotrophic abilities of all the drugs used. Moreover, this coumarin statistically enhanced the neuroprotective effects of PB (p < 0.05) and VPA (p < 0.001) in the excitotoxicity model of neurodegeneration. The obtained results clearly indicate a positive effect of UMB on the anticonvulsant and neuroprotective properties of the selected drugs.

Xanthotoxin enhances the anticonvulsant potency of levetiracetam and valproate in the 6-Hz corneal stimulation model in mice.

Xanthotoxin (8-methoxypsoralen; XANT) is a furanocoumarin that has many biological properties, including antiepileptic activity. This study evaluated the effect of XANT on the ability of classical and novel antiepileptic drugs to prevent seizures evoked by the 6-Hz corneal stimulation-induced seizure model, which is thought to be an experimental model of psychomotor (limbic) seizures in humans. XANT (50 mg/kg, administered i.p.) significantly potentiated the anticonvulsant activity of levetiracetam and valproate, decreasing their median effective dose (ED50 ) values from 19.37 to 2.83 mg/kg (P < 0.01) for levetiracetam and from 92.89 to 44.44 mg/kg (P < 0.05) for valproate. Neither XANT (50 mg/kg) alone nor its combination with the anticonvulsant drugs (at their ED50 values from the 6-Hz test) affected motor coordination; skeletal muscular strength and long-term memory, as determined in the chimney; and grip strength and passive avoidance tests, respectively. Measurement of total brain antiepileptic drug concentrations revealed that XANT (50 mg/kg) had no impact on levetiracetam total brain concentrations, indicating the pharmacodynamic nature of interaction between these antiepileptic drugs in the mouse 6-Hz model. However, XANT (50 mg/kg, i.p.) significantly increased total brain concentrations of valproate (P < 0.01), indicating the pharmacokinetic nature of interactions between drugs. XANT in combination with levetiracetam exerts beneficial anticonvulsant pharmacodynamic interactions in the 6-Hz mouse psychomotor seizure model.

Effect of Lacosamide and Ethosuximide Chronic Treatment on Neural Precursor Cells and Cognitive Functions after Pilocarpine Induced Status Epilepticus in Mice.

Seizures in about 40% of patients with epilepsy fail to respond to anti-seizure medication (ASM) and may lead to uncontrolled and prolonged seizures often inducing status epilepticus (SE). The aim of the study was to evaluate the impact of a long-term treatment with two different generation ASMs: ethosuximide (ETS, a classic ASM) and lacosamide (LCM, a 3rd generation ASM) on neural stem cells' (NSCs') proliferation and learning and memory functions after pilocarpine (PILO)-induced SE in mice. The following drugs were used: LCM (10 mg/kg), ETS (20 mg/kg), and PILO (300 mg/kg). Cell counting was done using confocal microscope and ImageJ software. Cognitive functions were evaluated with the Morris water maze (MWM) test. The level of several selected neurometabolites was measured with magnetic resonance spectroscopy (MRS). Obtained results indicated no significant impact of ETS treatment on the neurogenesis process in PILO mice. Interestingly, LCM significantly decreased the total amount of newborn neurons. The MWM test indicated no significant changes in the time and distance traveled by the ETS and LCM groups compared to PILO control mice, although all measured parameters were more favorable for the PILO mice treated with ASM. Conclusions: The presented results show that long term treatment with LCM and ETS seems to be safe for the cognitive functions and the proper course of neurogenesis in the mouse PILO-induced SE model, although one should remember that LCM administered chronically may act to reduce new neurons' formation.

Selected flavonoids and their role in the treatment of epilepsy - a review of the latest reports from experimental studies.

Epilepsy is a chronic neurological disease characterized by recurrent seizures that affects about 70 million people worldwide. Antiepileptic drugs are the most commonly used medications in the treatment of epilepsy. They help control seizures in about 60­70% of people. The remaining percentage of patients suffer from drug­resistant epilepsy, prompting scientists to look for natural substances that would prevent seizures or support the effects of drugs in add­on therapy while reducing side effects. Currently, there is a lot of emphasis on natural product. Flavonoids are included in this group, and their use in the treatment of epilepsy could support the effect of other drugs. Due to very good results of preclinical studies, flavonoids are a promising candidate for epilepsy related clinical trials related. The article is an overview of literature reports from the past 10 years including mainly in vivo preclinical research on various models of experimental epilepsy with the use of selected flavonoids.

C-11, a New Antiepileptic Drug Candidate: Evaluation of the Physicochemical Properties and Impact on the Protective Action of Selected Antiepileptic Drugs in the Mouse Maximal Electroshock-Induced Seizure Model.

C-11 is a hybrid compound derived from 2-(2,5-dioxopyrrolidin-1-yl) propanamide, with a wide spectrum of anticonvulsant activity and low neurotoxicity. The aim of this study was to determine the effects of C-11 on the protective action of various antiepileptic drugs (i.e., carbamazepine CBZ, lacosamide LCM, lamotrigine LTG, and valproate VPA) against maximal electroshock-induced seizures (MES) in mice, as well as its neuroprotective and physicochemical/pharmacokinetic properties. Results indicate that C-11 (30 mg/kg, i.p.) significantly enhanced the anticonvulsant action of LCM (p < 0.001) and VPA (p < 0.05) but not that of CBZ and LTG in the MES test. Neither C-11 (30 mg/kg) alone nor its combination with other anticonvulsant drugs (at their ED50 values from the MES test) affected motor coordination; skeletal muscular strength and long-term memory, as determined in the chimney; grip strength and passive avoidance tests, respectively. Pharmacokinetic characterization revealed that C-11 had no impact on total brain concentrations of LCM or VPA in mice. Qualitative analysis of neuroprotective properties of C-11, after a single administration of pilocarpine, revealed no protective effect of this substance in the tested animals. Determination of physicochemical descriptors showed that C-11 meets the drug-likeness requirements resulting from Lipinski and Veber's rules and prediction of gastrointestinal absorption and brain penetration, which is extremely important for the CNS-active compounds.

Preclinical Assessment of a New Hybrid Compound C11 Efficacy on Neurogenesis and Cognitive Functions after Pilocarpine Induced Status Epilepticus in Mice.

Status epilepticus (SE) is a frequent medical emergency that can lead to a variety of neurological disorders, including cognitive impairment and abnormal neurogenesis. The aim of the presented study was the in vitro evaluation of potential neuroprotective properties of a new pyrrolidine-2,5-dione derivatives compound C11, as well as the in vivo assessment of the impact on the neurogenesis and cognitive functions of C11 and levetiracetam (LEV) after pilocarpine (PILO)-induced SE in mice. The in vitro results indicated a protective effect of C11 (500, 1000, and 2500 ng/mL) on astrocytes under trophic stress conditions in the MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) test. The results obtained from the in vivo studies, where mice 72 h after PILO SE were treated with C11 (20 mg/kg) and LEV (10 mg/kg), indicated markedly beneficial effects of C11 on the improvement of the neurogenesis compared to the PILO control and PILO LEV mice. Moreover, this beneficial effect was reflected in the Morris Water Maze test evaluating the cognitive functions in mice. The in vitro confirmed protective effect of C11 on astrocytes, as well as the in vivo demonstrated beneficial impact on neurogenesis and cognitive functions, strongly indicate the need for further advanced molecular research on this compound to determine the exact neuroprotective mechanism of action of C11.