A comprehensive assessment of palmatine as anticonvulsant agent - In vivo and in silico studies.

Previously, we demonstrated that palmatine (PALM) - an isoquinoline alkaloid from Berberis sibrica radix, exerted antiseizure activity in the pentylenetetrazole (PTZ)-induced seizure assay in larval zebrafish. The aim of the present study was to more precisely characterize PALM as a potential anticonvulsant drug candidate. A range of zebrafish and mouse seizure/epilepsy models were applied in the investigation. Immunostaining analysis was conducted to assess the changes in mouse brains, while in silico molecular modelling was performed to determine potential targets for PALM. Accordingly, PALM had anticonvulsant effect in ethyl 2-ketopent-4-enoate (EKP)-induced seizure assay in zebrafish larvae as well as in the 6 Hz-induced psychomotor seizure threshold and timed infusion PTZ tests in mice. The protective effect in the EKP-induced seizure assay was confirmed in the local field potential recordings. PALM did not affect seizures in the gabra1a knockout line of zebrafish larvae. In the scn1Lab-/- zebrafish line, pretreatment with PALM potentiated seizure-like behaviour of larvae. Repetitive treatment with PALM, however, did not reduce development of PTZ-induced seizure activity nor prevent the loss of parvalbumin-interneurons in the hippocampus of the PTZ kindled mice. In silico molecular modelling revealed that the noted anticonvulsant effect of PALM in EKP-induced seizure assay might result from its interactions with glutamic acid decarboxylase and/or via AMPA receptor non-competitive antagonism. Our study has demonstrated the anticonvulsant activity of PALM in some experimental models of seizures, including a model of pharmacoresistant seizures induced by EKP. These results indicate that PALM might be a suitable new drug candidate but the precise mechanism of its anticonvulsant activity has to be determined.

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.

Discovery of (R)-N-Benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide [(R)-AS-1], a Novel Orally Bioavailable EAAT2 Modulator with Drug-like Properties and Potent Antiseizure Activity In Vivo.

(R)-7 [(R)-AS-1] showed broad-spectrum antiseizure activity across in vivo mouse seizure models: maximal electroshock (MES), 6 Hz (32/44 mA), acute pentylenetetrazol (PTZ), and PTZ-kindling. A remarkable separation between antiseizure activity and CNS-related adverse effects was also observed. In vitro studies with primary glia cultures and COS-7 cells expressing the glutamate transporter EAAT2 showed enhancement of glutamate uptake, revealing a stereoselective positive allosteric modulator (PAM) effect, further supported by molecular docking simulations. (R)-7 [(R)-AS-1] was not active in EAAT1 and EAAT3 assays and did not show significant off-target activity, including interactions with targets reported for marketed antiseizure drugs, indicative of a novel and unprecedented mechanism of action. Both in vivo pharmacokinetic and in vitro absorption, distribution, metabolism, excretion, toxicity (ADME-Tox) profiles confirmed the favorable drug-like potential of the compound. Thus, (R)-7 [(R)-AS-1] may be considered as the first-in-class small-molecule PAM of EAAT2 with potential for further preclinical and clinical development in epilepsy and possibly other CNS disorders.

New Phenylglycinamide Derivatives with Hybrid Structure as Candidates for New Broad-Spectrum Anticonvulsants.

In the present study, a focused combinatorial chemistry approach was applied to merge structural fragments of well-known TRPV1 antagonists with a potent anticonvulsant lead compound, KA-104, that was previously discovered by our group. Consequently, a series of 22 original compounds has been designed, synthesized, and characterized in the in vivo and in vitro assays. The obtained compounds showed robust in vivo antiseizure activity in the maximal electroshock (MES) test and in the 6 Hz seizure model (using both 32 and 44 mA current intensities). The most potent compounds 53 and 60 displayed the following pharmacological profile: ED50 = 89.7 mg/kg (MES), ED50 = 29.9 mg/kg (6 Hz, 32 mA), ED50 = 68.0 mg/kg (6 Hz, 44 mA), and ED50 = 73.6 mg/kg (MES), ED50 = 24.6 mg/kg (6 Hz, 32 mA), and ED50 = 56.3 mg/kg (6 Hz, 44 mA), respectively. Additionally, 53 and 60 were effective in the ivPTZ seizure threshold and had no influence on the grip strength and body temperature in mice. The in vitro binding and functional assays indicated a multimodal mechanism of action for 53 and 60. These molecules, beyond TRPV1 antagonism, inhibited calcium currents and fast sodium currents in patch-clamp assays. Further studies proved beneficial in vitro ADME-Tox properties for 53 and 60 (i.e., high metabolic stability, weak influence on CYPs, no neurotoxicity, etc.). Overall, 53 and 60 seem to be interesting candidates for future preclinical development in epilepsy and pain indications due to their interaction with the TRPV1 channel.

Antinociceptive and Antiallodynic Activity of Some 3-(3-Methylthiophen-2-yl)pyrrolidine-2,5-dione Derivatives in Mouse Models of Tonic and Neuropathic Pain.

Antiseizure drugs (ASDs) are commonly used to treat a wide range of nonepileptic conditions, including pain. In this context, the analgesic effect of four pyrrolidine-2,5-dione derivatives (compounds 3, 4, 6, and 9), with previously confirmed anticonvulsant and preliminary antinociceptive activity, was assessed in established pain models. Consequently, antinociceptive activity was examined in a mouse model of tonic pain (the formalin test). In turn, antiallodynic and antihyperalgesic activity were examined in the oxaliplatin-induced model of peripheral neuropathy as well as in the streptozotocin-induced model of painful diabetic neuropathy in mice. In order to assess potential sedative properties (drug safety evaluation), the influence on locomotor activity was also investigated. As a result, three compounds, namely 3, 6, and 9, demonstrated a significant antinociceptive effect in the formalin-induced model of tonic pain. Furthermore, these substances also revealed antiallodynic properties in the model of oxaliplatin-induced peripheral neuropathy, while compound 3 attenuated tactile allodynia in the model of diabetic streptozotocin-induced peripheral neuropathy. Apart from favorable analgesic properties, the most active compound 3 did not induce any sedative effects at the active dose of 30 mg/kg after intraperitoneal (i.p.) injection.

Identification of New Compounds with Anticonvulsant and Antinociceptive Properties in a Group of 3-substituted (2,5-dioxo-pyrrolidin-1-yl)(phenyl)-Acetamides.

We report herein a series of water-soluble analogues of previously described anticonvulsants and their detailed in vivo and in vitro characterization. The majority of these compounds demonstrated broad-spectrum anticonvulsant properties in animal seizure models, including the maximal electroshock (MES) test, the pentylenetetrazole-induced seizure model (scPTZ), and the psychomotor 6 Hz (32 mA) seizure model in mice. Compound 14 showed the most robust anticonvulsant activity (ED50 MES = 49.6 mg/kg, ED50 6 Hz (32 mA) = 31.3 mg/kg, ED50scPTZ = 67.4 mg/kg). Notably, it was also effective in the 6 Hz (44 mA) model of drug-resistant epilepsy (ED50 = 63.2 mg/kg). Apart from favorable anticonvulsant properties, compound 14 revealed a high efficacy against pain responses in the formalin-induced tonic pain, the capsaicin-induced neurogenic pain, as well as in the oxaliplatin-induced neuropathic pain in mice. Moreover, compound 14 showed distinct anti-inflammatory activity in the model of carrageenan-induced aseptic inflammation. The mechanism of action of compound 14 is likely complex and may result from the inhibition of peripheral and central sodium and calcium currents, as well as the TRPV1 receptor antagonism as observed in the in vitro studies. This lead compound also revealed beneficial in vitro ADME-Tox properties and an in vivo pharmacokinetic profile, making it a potential candidate for future preclinical development. Interestingly, the in vitro studies also showed a favorable induction effect of compound 14 on the viability of neuroblastoma SH-SY5Y cells.

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.

Synthesis, Anticonvulsant, and Antinociceptive Activity of New 3-(2-Chlorophenyl)- and 3-(3-Chlorophenyl)-2,5-dioxo-pyrrolidin-1-yl-acetamides.

The new series of 3-(2-chlorophenyl)- and 3-(3-chlorophenyl)-pyrrolidine-2,5-dione-acetamide derivatives as potential anticonvulsant and analgesic agents was synthesized. The compounds obtained were evaluated in the following acute models of epilepsy: maximal electroshock (MES), psychomotor (6 Hz, 32 mA), and subcutaneous pentylenetetrazole (scPTZ) seizure tests. The most active substance-3-(2-chlorophenyl)-1-{2-[4-(4-fluorophenyl)piperazin-1-yl]-2-oxoethyl}-pyrrolidine-2,5-dione (6) showed more beneficial ED50 and protective index values than the reference drug-valproic acid (68.30 mg/kg vs. 252.74 mg/kg in the MES test and 28.20 mg/kg vs. 130.64 mg/kg in the 6 Hz (32 mA) test, respectively). Since anticonvulsant drugs are often effective in neuropathic pain management, the antinociceptive activity for two the promising compounds-namely, 6 and 19-was also investigated in the formalin model of tonic pain. Additionally, for the aforementioned compounds, the affinity for the voltage-gated sodium and calcium channels, as well as GABAA and TRPV1 receptors, was determined. As a result, the most probable molecular mechanism of action for the most active compound 6 relies on interaction with neuronal voltage-sensitive sodium (site 2) and L-type calcium channels. Compounds 6 and 19 were also tested for their neurotoxic and hepatotoxic properties and showed no significant cytotoxic effect.

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.

Asymmetric synthesis and in vivo/in vitro characterization of new hybrid anticonvulsants derived from (2,5-dioxopyrrolidin-1-yl)phenylacetamides.

In the current studies we carried out an optimized multistep asymmetric synthesis of R-enantiomers (eutomers) for a previously identified series of racemic hybrid anticonvulsants. The spatial structure of selected enantiomers was solved by the use of crystallographic methods. The compound (R)-16 was identified as a lead, which revealed broad-spectrum protective activity in a range of epilepsy models with the following ED50 values: the maximal electroshock (MES) test (36.0 mg/kg), the 6 Hz (32 mA) seizure model (39.2 mg/kg), and the pentylenetetrazole-induced seizure model (scPTZ) (54.8 mg/kg). Furthermore, (R)-16 displayed a low potency for the induction of motor impairment in the rotarod test (TD50 = 468.5 mg/kg), resulting in potentially very beneficial therapeutic window. Finally, (R)-16 showed satisfying ADME-Tox properties in the in vitro assays. Therefore, the data obtained in the current studies justify the further preclinical development of (R)-16 as candidate for potentially broad-spectrum and safe anticonvulsant.

Design, Synthesis and Biological Activity of New Amides Derived from 3-Benzhydryl and 3-sec-Butyl-2,5-dioxo-pyrrolidin-1-yl-acetic Acid.

The aim of this study was to design and synthesize two new series of pyrrolidine-2,5-dione-acetamides with a benzhydryl or sec-butyl group at position 3 as potential anticonvulsants. Their anticonvulsant activity was evaluated in standard animal models of epilepsy: the maximal electroshock (MES), the 6 Hz, and the subcutaneous pentylenetetrazole (scPTZ) tests. The in vivo studies revealed the most potent anticonvulsant activity for 15 (3-(sec-butyl)-1-(2-(4-(3-trifluoromethylphenyl)piperazin-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione), with ED50 values of 80.38 mg/kg (MES) and 108.80 mg/kg (6 Hz). The plausible mechanism of action was assessed in in vitro binding assays, in which 15 interacted effectively with voltage-gated sodium (site 2) and L-type calcium channels at a concentration of 100 µM. Subsequently, the antinociceptive activity of compounds 7 and 15 was observed in the hot plate test of acute pain. Moreover, compounds 7, 11 and 15 demonstrated an analgesic effect in the formalin test of tonic pain. The hepatotoxic properties of the most effective compounds (7, 11 and 15) in HepG2 cells were also investigated.

Synthesis, anticonvulsant, and antinociceptive activity of new 3-(3-methyl-2,5-dioxo-3-phenylpyrrolidin-1-yl)propanamides and 3-phenyl-butanamides.

A focused library of new 3-(3-methyl-2,5-dioxo-3-phenylpyrrolidin-1-yl)propanamides and their nonimide analogs were synthesized and tested for anticonvulsant activity. These compounds were obtained through the coupling reaction of the starting carboxylic acids with appropriate amines. The initial anticonvulsant screening was performed in mice (intraperitoneal administration) using the maximal electroshock seizure (MES) and the subcutaneous pentylenetetrazole (scPTZ) seizure models. The most promising compound 6 showed more potent protection in the MES and scPTZ tests than valproic acid, which is still recognized as one of the most relevant first-line anticonvulsants. The structure-activity relationship analysis revealed that the presence of the pyrrolidine-2,5-dione ring is important but not indispensable to retain anticonvulsant activity. Additionally, compound 6 showed potent antinociceptive properties in the oxaliplatin-induced neuropathic pain model in mice. The most plausible mechanism of action for compound 6 may result from its influence on the neuronal sodium channel (Site 2) and the high-voltage-activated L-type calcium channel.

The Search for New Anticonvulsants in a Group of (2,5-Dioxopyrrolidin-1-yl)(phenyl)Acetamides with Hybrid Structure-Synthesis and In Vivo/In Vitro Studies.

Epilepsy belongs to the most common and debilitating neurological disorders with multifactorial pathophysiology and a high level of drug resistance. Therefore, with the aim of searching for new, more effective, and/or safer therapeutics, we discovered a focused series of original hybrid pyrrolidine-2,5-dione derivatives with potent anticonvulsant properties. We applied an optimized coupling reaction yielding several hybrid compounds that showed broad-spectrum activity in widely accepted animal seizure models, namely, the maximal electroshock (MES) test and the psychomotor 6 Hz (32 mA) seizure model in mice. The most potent anticonvulsant activity and favorable safety profile was demonstrated for compound 30 (median effective dose (ED50) MES = 45.6 mg/kg, ED50 6 Hz (32 mA) = 39.5 mg/kg, median toxic dose (TD50) (rotarod test) = 162.4 mg/kg). Anticonvulsant drugs often show activity in pain models, and compound 30 was also proven effective in the formalin test of tonic pain, the capsaicin-induced pain model, and the oxaliplatin (OXPT)-induced neuropathic pain model in mice. Our studies showed that the most plausible mechanism of action of 30 involves inhibition of calcium currents mediated by Cav1.2 (L-type) channels. Importantly, 30 revealed high metabolic stability on human liver microsomes, negligible hepatotoxicity, and relatively weak inhibition of CYP3A4, CYP2D6, and CYP2C9 isoforms of cytochrome P450, compared to reference compounds. The promising in vivo activity profile and drug-like properties of compound 30 make it an interesting candidate for further preclinical development.

KA-104, a new multitargeted anticonvulsant with potent antinociceptive activity in preclinical models.

OBJECTIVE: The main objective of the present work was to assess the utility of KA-104 as potential therapy for drug-resistant seizures and neuropathic pain, and to characterize its druglike properties in a series of absorption, distribution, metabolism, excretion and toxicity (ADME-Tox) studies. We also aimed to establish its mechanism of action in electrophysiological studies. METHODS: The activity of KA-104 against drug-resistant seizures was tested in the mouse 6-Hz (44-mA) model, whereas the antinociceptive activity was assessed with the capsaicin- and oxaliplatin-induced pain models in mice. The patch-clamp technique was used to study the influence of KA-104 on fast voltage-gated sodium currents in rat prefrontal cortex pyramidal neurons. The pharmacokinetic profile was determined after intraperitoneal (ip) injection in mice. The in vitro ADME-Tox properties were studied by applying routine testing procedures. RESULTS: KA-104 was effective in the 6-Hz (44-mA) model (median effective dose [ED50 ] = 73.2 mg/kg) and revealed high efficacy in capsaicin-induced neurogenic pain as well as in oxaliplatin-induced neuropathic pain in mice. Patch-clamp technique showed that KA-104 reversibly inhibits voltage-gated sodium currents. KA-104 was rapidly absorbed after the ip injection and showed relatively good penetration through the blood-brain barrier. This molecule was also characterized by high passive permeability, moderate influence on CYP2C9, and negligible hepatotoxicity on HepG2 cells. SIGNIFICANCE: The results reported herein indicate that KA-104 is a new wide-spectrum multitargeted anticonvulsant with favorable in vitro ADME-Tox properties. Importantly, this compound may also prove to become an interesting and hopefully more effective therapeutic option for treatment of neuropathic pain.

Multitargeted Compounds Derived from (2,5-Dioxopyrrolidin-1-yl)(phenyl)-Acetamides as Candidates for Effective Anticonvulsant and Antinociceptive Agents.

We developed a focused set of original hybrid pyrrolidine-2,5-dione derivatives with potent anticonvulsant and antinociceptive properties. These hybrid compounds demonstrated broad-spectrum protective activity in a range of mouse models, such as the maximal electroshock (MES) test, the pentylenetetrazole-induced seizures (scPTZ), and the 6 Hz (32 mA) seizures. Compound 22 showed the most potent anticonvulsant activity (ED50 MES = 23.7 mg/kg, ED50 6 Hz (32 mA) = 22.4 mg/kg, ED50scPTZ = 59.4 mg/kg). In addition, 22 revealed potent efficacy in the formalin-induced tonic pain. These in vivo activities of 22 are likely mediated by several targets and may result from the inhibition of central sodium/calcium currents and transient receptor potential vanilloid 1 (TRPV1) receptor antagonism. Finally, the lead compound 22 revealed drug-like absorption, distribution, metabolism, excretion, toxicity (ADME-Tox) properties in the in vitro assays, making it a potential candidate for further development in epilepsy and neuropathic pain indications.

Evaluation of the impact of compound C11 a new anticonvulsant candidate on cognitive functions and hippocampal neurogenesis in mouse brain.

Searching for the new and effective anticonvulsants in our previous study we developed a new hybrid compound C-11 derived from 2-(2,5-dioxopyrrolidin-1-yl) propanamide. C11 revealed high efficacy in acute animal seizure models such as the maximal electroshock model (MES), the pentylenetetrazole model (PTZ) and the 6 Hz (6 Hz, 32 mA) seizure model, as well as in the kindling model of epilepsy induced by repeated injection of PTZ in mice. In the aim of further in vivo C11 characterization, in the current studies we evaluated its influence on cognitive functions, neurodegeneration and neurogenesis process in mice after chronical treatment. All experiments were performed on 6 weeks old male C57/BL mice. The following drugs were used: C11, levetiracetam (LEV), ethosuximide (ETS) and lacosamide (LCM). We analyzed proliferation, migration and differentiation of newborn cells as well as neurodegenerative changes in a mouse brain after long-term treatment with aforementioned AEDs. Additionally, we evaluated changes in learning and memory functions in response to chronic C11, LEV, LCM and ETS treatment. C11 as well as LEV and ETS did not disturb the proliferation of newborn cells compared to the control mice, whereas LCM treatment significantly decreased it. Chronic AEDs therapy did not induce significant neurodegenerative changes. Behavioral studies with using Morris Water Maze test did not indicate any disturbances in the spatial learning and memory after C11 as well as LEV and ETS treatment in comparison to the control group except LCM mice where significant dysfunctions in time, distance and direct swim to the platform were observed. Interestingly, results obtained from in vivo MRI spectroscopy showed a statistically significant increase of one of the neurometabolites- N-acetyloaspartate (NAA) for LCM and LEV mice. A new hybrid compound C11 in contrast to LCM has no negative impact on the process of neurogenesis and neurodegeneration in the mouse hippocampus. Furthermore, chronic treatment with C11 turned out to have no negative impact on cognitive functions of treated mice, which, is certainly of great importance for further more advanced preclinical and especially clinical trials.

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).

Chirality as an Important Factor for the Development of New Antiepileptic Drugs.

In recent years, chiral molecules (especially enantiomers) have occupied a significant place in pharmaceutical industry and have played a prominent role in the development of new drugs. Individual stereoisomers exhibit marked differences in pharmacodynamic, pharmacokinetic, and toxicological properties. Therefore, there is currently considerable interest in fully characterizing and examining both enantiomers in the early stages of new drug development. Despite the fact that epilepsy is a complex disease and that a given drug's mechanism of action may be multidirectional and not always fully understood, significant differences have been observed in the anticonvulsant activity of individual stereoisomers. Therefore, between 1996 and 2018, among 14 new antiepileptic drugs (AEDs) approved for the treatment of epilepsy, as many as seven are chiral and introduced to the market in the single-enantiomer (or diastereomer) form. This review provides an overview of the impact of chirality on the development and discovery of new AEDs that have entered into clinical trials or preclinical studies. These new AEDs were developed by applying the single-enantiomer approval strategy. Herein we focus our attention on the main synthetic pathways of stereoisomers, as well as on the influence of chirality on pharmacodynamic, pharmacokinetic, and/or toxicological properties.

KA-11, a Novel Pyrrolidine-2,5-dione Derived Broad-Spectrum Anticonvulsant: Its Antiepileptogenic, Antinociceptive Properties and in Vitro Characterization.

Recently, compound KA-11 was identified as a promising candidate for a new broad-spectrum anticonvulsant. This compound revealed wide protective activity across the most important animal models of seizures such as the maximal electroshock test (MES), the subcutaneous pentylenetetrazole test ( scPTZ), and the six-hertz test (6 Hz, 32 mA). Importantly, KA-11 was devoid of acute neurological activity, which was assessed by applying the chimney test (TD50 value higher than 1500 mg/kg). The preliminary in vivo results confirmed favorable anticonvulsant and safety properties of KA-11. With the aim of further biological characterization of KA-11, in the current studies we evaluated its antiepileptogenic activity in the kindling model of epilepsy induced by repeated injection of PTZ in mice. Furthermore, we assessed the antinociceptive activity of KA-11 in several animal pain models. As a result, KA-11 (at all doses applied: 25, 50, and 100 mg/kg) significantly delayed the progression of kindling induced by repeated injection of PTZ in mice. Additionally, KA-11 revealed potent antinociceptive activity in the formalin-induced tonic pain and, importantly, in the oxaliplatin-induced neuropathic pain model in mice. Moreover, KA-11 did not induce motor deficits in the rotarod test. Patch-clamp experiments revealed that one of the mechanisms of action of KA-11 is inhibition of voltage-gated sodium currents. Compound KA-11 appeared to be safe in relation to hepatotoxic properties as no phospholipidosis induction was determined in HepG2 cells at 50 µM, and a small, statistically significant decrease of cell viability was observed only at the highest used dose of 100 µM. Moreover, KA-11 did not affect the function of CYP2D6. The aforementioned hybrid substance proved to penetrate the biological membranes in the in vitro permeability assays.

Evaluation of anticonvulsant and analgesic activity of new hybrid compounds derived from N-phenyl-2-(2,5-dioxopyrrolidin-1-yl)-propanamides and -butanamides.

Epilepsy is a chronic neurological disorder that is associated with various types of recurrent seizures, which are drug-resistant in about one third of patients. Moreover, anticonvulsant drugs are used to treat a wide range of non-epileptic conditions, including chronic pain. Here, we investigated the anticonvulsant activity of six new hybrid compounds based on the pyrrolidine-2,5-dione scaffold in the 6 Hz corneal stimulation test with 44 mA stimulus intensity in mice, which is the model of pharmacoresistant seizures. We demonstrated that two molecules, DK-10 (11) and DK-14 (14) show higher anticonvulsant activity and similar safety profile in comparison with valproic acid and much higher in comparison with levetiracetam in the aforementioned test. The second aim of this study was to examine analgesic activity of these compounds. For this purpose, the hot plate test, the formalin test, and the oxaliplatin-induced peripheral neuropathy model were performed. Among tested agents DK-11 (12) revealed prominent antinociceptive activity at non-sedative doses in the second (inflammatory) phase of the formalin test, which is the model of tonic pain and antiallodynic activity in the oxaliplatin-induced neuropathic pain, the model of painful chemotherapy-induced peripheral neuropathy. No cytotoxic effect on hepatoma cells was observed. Compound DK-10 (11) had high affinity for voltage-gated sodium channels, whereas compound DK-11 (12) showed weak binding toward sodium and calcium voltage-gated channels and the NMDA receptor. As a result, hybrid compounds reported herein seem to be very promising broad spectrum anticonvulsant molecules with collateral analgesic activity.