Luis Sojo

The assessment of the efficacy of antiseizure medications (ASMs) in animal models of acute seizures has played a critical role in these drugs’ success in clinical trials for human epilepsy. One of the most widely used animal models for this purpose is the maximal electroshock seizure (MES) model. While there are numerous published reports on the efficacy of conventional ASMs in MES models, there is a need to expand the understanding on the brain concentrations that are needed to achieve optimal levels of efficacy in this model. We assessed the pharmacokinetic/pharmacodynamic (PK/PD) profiles of six ASMs, namely carbamazepine (CBZ), phenytoin (PHT), valproic acid (VPA), lacosamide (LSM), cenobamate (CNB), and retigabine (RTG), using MES models in mice and rats. EC₅₀ values for plasma and the brain were generally higher in mice than rats, with fold differences ranging from 1.3- to 8.6-fold for plasma and from 1.2- to 11.5-fold for brain. Phenytoin showed the largest interspecies divergence. These results suggest that rats may exhibit greater sensitivity to seizure protection in the MES model, likely reflecting species differences in metabolism and brain penetration. These findings highlight the value of considering concentration–response variations and species-specific differences when assessing the efficacy of both conventional ASMs and novel compounds exhibiting anticonvulsant activity.

Background/Objective: The assessment of the efficacy of antiseizure medications (ASMs) in animal models of acute seizures has played a critical role in these drugs’ success in clinical trials for human epilepsy. One of the most widely used animal models for this purpose is the maximal electroshock seizure (MES) model. While there are numerous published reports on the efficacy of conventional ASMs in MES models, there is a need to expand the understanding on the brain concentrations that are needed to achieve optimal levels of efficacy in this model. Methods: We evaluated six clinically available ASMs in both a mouse and a rat MES model by analyzing dose-response and concentration-response for brain and plasma exposure levels: carbamazepine (CBZ), phenytoin (PHT), valproic acid (VPA), lacosa-mide (LSM), cenobamate (CNB), and retigabine (RTG). Results: Our data indicate that these ASMs tend to be more effective in reducing seizures in the rat MES, with notable differences in drug metabolism and brain penetrance be-tween mice and rats. Conclusion: These findings highlight the value of considering concentration-response variations and species-specific differences when assessing the efficacy of both conventional ASMs and novel compounds exhibiting anticonvulsant activity.

Rationale: Na _v 1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage‐gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development. Methods: Unique peptides for Na _v 1.1, Na _v 1.2, and Na _v 1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography–electrospray ionization tandem mass spectrometry. Results: Na _v 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/μg membrane protein, respectively. In brains, Na _v 1.2 showed the highest expression, with approximately three times higher ( P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/μg in human brain. Both Na _v 1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Na _v 1.1 than rodent Na _v 1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Na _v 1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/μg membrane proteins. Conclusions: Multiple reaction monitoring was used to quantify sodium channels Na _v 1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Na _v expression ranking among the three species was Na _v 1.2 ≫ Na _v 1.1 > Na _v 1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.

Rationale Na _v 1.1, 1.2 and 1.6 are transmembrane proteins acting as voltage gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development. Methods Unique peptides for Na _v 1.1, Na _v 1.2 and Na _v 1.6 were selected as quantotropic peptides for each protein and used for their quantification in plasma membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry. Results Na _v 1.1, 1.2 and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1±0.2, 6.4±1.2 and 4.0±0.6 fmole/µg membrane protein respectively. Na _v 1.2 showed the highest expression, with approximately 3 times higher (p> Na _v 1.1> Na _v 1.6, with the human brain expressing much lower concentrations overall in comparison to rodents.

Nonselective antagonists of voltage-gated sodium (NaV) channels have been long used for the treatment of epilepsies. The efficacy of these drugs is thought to be due to the block of sodium channels on excitatory neurons, primarily NaV1.6 and NaV1.2. However, these currently marketed drugs require high drug exposure and suffer from narrow therapeutic indices. Selective inhibition of NaV1.6, while sparing NaV1.1, is anticipated to provide a more effective and better tolerated treatment for epilepsies. In addition, block of NaV1.2 may complement the anticonvulsant activity of NaV1.6 inhibition. We discovered a novel series of aryl sulfonamides as CNS penetrant, isoform-selective NaV1.6 inhibitors, which also displayed potent block of NaV1.2. Optimization focused on increasing selectivity over NaV1.1, improving metabolic stability, reducing active efflux, and addressing a PXR (pregnane X-receptor) liability. We obtained compounds 30-32, which produced potent anticonvulsant activity in mouse seizure models, including a direct current maximal electroshock (DC-MES) seizure assay.

Rationale: Na _v 1.6 is a transmembrane voltage gated sodium channel implicated in various forms of epilepsy. Modulation of its activity in epilepsy animal models can be accomplished using inhibitors which may result in changes in its expression. There is a need to generate reliable quantitative measurements of Na _v 1.6 expression in animal models. This research explores the feasibility of quantifying Na _v 1.6 expression in mouse brains using targeted multiple reaction monitoring (MRM) mass spectrometry. Methods: A combination of in silico tryptic Na _v 1.6 peptides and MRM transitions were used to select target peptides. This was followed by a simple proteomic work‐up including plasma membrane isolation, trypsin‐based proteolysis and ultra‐high‐performance/electrospray ionization tandem mass spectrometry (UHPLC/ESI‐MS/MS) to detect the presence of Na _v 1.6 in induced HEK293 cells. The unique Na _v 1.6 peptide, DSLFIPR, was selected as probe for quantifying Na _v 1.6 levels in brains from C57BL/6J wild‐type mice as well as two kinds of mutants including Scn8a ^N1768D/+ and heterozygous null Scn8a ^+/− mice using isotope dilution targeted mass spectrometry. Results: The feasibility of using targeted MRM for quantifying Na _v 1.6 expression in mice brains was demonstrated. Expression of Na _v 1.6 in brains (hippocampi) from wild‐type and mutant Scn8a ^N1768D/+ mice were found to be around 0.40 fmol/μg. Mutant null Scn8a ^+/− heterozygous mice, on the other hand, showed levels of 0.22 fmol/μg as expected based on this particular mutation which only generates 50% of the expression in wild‐type mice. Na _v 1.6‐overexpressed HEK293 cells showed 3.7 fmol/μg of Na _v 1.6 expression, suitable for screening new compounds for Na _v 1.6 blocking activity. Conclusions: The results of the present feasibility study support the use of DSLFIPIR for quantification of Nav1.6 in brain tissues using UHPL/ESI‐MS/MS.

RATIONALE: Catechols are an important class of analytes occurring in many natural and synthetic products. Electrospray ionization in negative mode is the preferred way of ion generation for these compounds; however, studies in positive ion mode can reveal their potential for in‐source oxidation and further structural changes, some of which may also occur in the solution phase. Therefore in‐source oxidation can provide a forward look into the potential for solution oxidation. METHODS: 1:1 Acetonitrile/water solutions of catechol (Cat), 4,5‐dichlorocatechol (4,5‐DCC), 3,4‐dichlorocatechol (3,4‐DCC) and tetrachlorocatechol (TCC) were analyzed by positive ion ultrahigh‐performance liquid chromatography (UHPLC/ESI‐MS) and UHPLC/ESI‐MS/MS under various emitter voltages to assess their liability towards in‐source oxidation. Structural information for in‐source generated compounds was obtained through the use of product ion scans. RESULTS: Using catechols as probe compounds, we have demonstrated that under the conditions used in many analytical laboratories in‐source oxidation can severely affect the sensitivity and response functions of an analyte. Under standard UHPLC conditions (300 μL/min flow rate), Cat, 3,4‐DCC, 4,5‐DCC and TCC can undergo in‐source oxidation. The extent of oxidation is dependent either on the instrument or on the characteristics of the emitter. This is evident by a change in the isotopic pattern of these compounds and the generation of ions at lower m/z values due to a loss of 1 and/or 2 hydrogens and electrons. In the case of catechol, the formation of a dimer resulting from in‐source oxidation reactions was observed. This dimer has the same fragmentation pattern as the dimer generated by oxidation in the solution phase. CONCLUSIONS: The present work demonstrates the potential of positive ion ESI for oxidizing electroactive compounds during regular analytical operation using commercially available mass spectrometers. Using Cat and some of its chlorinated analogues as probe compounds, we have demonstrated that under the conditions used in many analytical laboratories in‐source oxidation and dimerization can and does take place. Copyright © 2014 John Wiley & Sons, Ltd.