Therapeutic drug monitoring (TDM) of antiseizure medications (ASMs) represents a valuable tool to establish an appropriate patient therapy, to collect important information about drugs’ interactions and to evaluate patient’s metabolic capabilities. In recent years, a new volumetric absorptive microsampling technique using VAMS^® technology and Mitra^® devices, consisting of a sampling technique for the collection of fixed-volume capillary blood, was developed. These new devices provide a new home-sampling technique for whole blood that has been spread out to simplify sample collection from finger-pricks. This review is aimed to compare published articles concerning the application of VAMS^® in epilepsy and to identify the strengths and improvement points for the TDM of antiseizure medications. VAMS^® allowed a minimally invasive blood sampling even in the absence of trained personnel. Good stability data have indicated that storage and delivery can be facilitated only for specific ASMs. Trueness and precision parameters have been evaluated, and the hematocrit (HCT) effect was minimized.

Objective: Cenobamate (CNB), a recently approved antiseizure medication by the European Medicines Agency (EMA), serves as an adjunctive therapy for focal‐onset seizures in adult patients unresponsive to at least two other treatments. Administered in polytherapy, CNB can potentially interact with co‐administered drugs in epilepsy patients, necessitating dose adjustments and the need for effective therapeutic drug monitoring (TDM).Methods: In this study, we introduce a novel LC–MS/MS method for precise CNB quantification using Volumetric Absorptive Microsampling (VAMS), following validation according to ICH guidelines M10. VAMS samples are efficiently extracted with 200 μL of methanol, with chromatographic separation achieved using an Acquity UPLC HSS PFP column. The method's efficacy was confirmed through its application to real samples from adult CNB‐treated patients.Results: Our results demonstrate that the method exhibits linearity within the range of 0.05–30 mg/L, with intra‐ and inter‐run precision ranging from 1% to 8% and accuracy from 1% to 10% based on 30 μL of sample. Furthermore, CNB stability in VAMS is confirmed for up to 15 days at 25°C and −20°C. Importantly, no significant difference was observed between CNB concentrations in VAMS samples and those in plasma obtained from venous blood.Significance: This VAMS‐based LC–MS/MS method presents a robust alternative for TDM in CNB‐treated patients. Future investigations should explore CNB concentrations in capillary blood and assess their correlation with plasma levels to further enhance its clinical utility.Plain Language Summary: Cenobamate is an antiepileptic drug and used for treatment of focal‐onset seizures in adult patients (≥18 age). TDM can prevent drug interactions and minimize drug toxicity. The aim of this work is to evaluate volumetric absorptive microsampling (VAMS) from capillary blood as an alternative strategy for TDM in patients treated with the newly antiepileptic drug. Our method is suitable for TDM, and this study suggests that VAMS allows monitoring of cenobamate concentration and can offer valuable support for personalized therapy in refractory epilepsy.

Levodopa (L-DOPA) treatment, combined with the administration of dopa-decarboxylase inhibitors (DDCIs), is still the most effective symptomatic treatment of Parkinson’s disease (PD). Although its efficacy in the early stage of the disease has been confirmed, its complex pharmacokinetics (PK) increases the variability of the intra-individual motor response, thus amplifying the risk of motor/non-motor fluctuations and dyskinesia. Moreover, it has been demonstrated that L-DOPA PK is strongly influenced by several clinical, therapeutic, and lifestyle variables (e.g., dietary proteins). L-DOPA therapeutic monitoring is therefore crucial to provide personalized therapy, hence improving drug efficacy and safety. To this aim, we have developed and validated an ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method to quantify L-DOPA, levodopa methyl ester (LDME), and the DDCI carbidopa in human plasma. The compounds were extracted by protein precipitation and samples were analyzed with a triple quadrupole mass spectrometer. The method showed good selectivity and specificity for all compounds. No carryover was observed, and dilution integrity was demonstrated. No matrix effect could be retrieved; intra-day and inter-day precision and accuracy values met the acceptance criteria. Reinjection reproducibility was assessed. The described method was successfully applied to a 45-year-old male patient to compare the pharmacokinetic behavior of an L-DOPA-based medical treatment involving commercially available Mucuna pruriens extracts and an LDME/carbidopa (100/25 mg) formulation.

Cenobamate (CNB) is a new anti-seizure medication (ASM) recently introduced in clinical practice after approval by the FDA and EMA for the add-on treatment of focal onset seizures in adult patients. Although its mechanism of action has not been fully understood, CNB showed promising clinical efficacy in patients treated with concomitant ASMs. The accessibility of CNB could pave a way for the treatment of refractory or drug-resistant epilepsies, which still affect at least one-third of the patients under pharmacological treatment. In this context, therapeutic drug monitoring (TDM) offers a massive opportunity for better management of epileptic patients, especially those undergoing combined therapy. Here, we describe the first fully validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method for the quantification of CNB and concomitant ASMs in human plasma, with samples extracted either manually or by means of a liquid handler. Our method was validated according to the most recent ICH International Guideline M10 for Bioanalytical Method Validation and Study Sample Analysis. The method proved to be selective for CNB and displayed a linear range from 0.8 to 80 mg/L; no matrix effect was found (98.2 ± 4.1%), while intra-day and inter-day accuracy and precision were within the acceptance range. Also, CNB short- and long-term stability in plasma under different conditions was assessed. Leftover human plasma samples were employed as study samples for method validation. Our method proved to be highly sensitive and selective to quantify CNB and concomitant ASMs in human plasma; therefore, this method can be employed for a routinely TDM-based approach to support physicians in the management of an epileptic patient.

Eleven calix[4]arene ligands, bearing zwitterionic α‐amino acids or charged ammonium or sulfonate/carboxylate groups, are synthesized and screened for the binding to the envelopes of three bacterial strain representatives of Gram‐positive, Gram‐negative, and mycobacteria. The binding is followed by on‐cell Saturation Transfer Difference Nuclear Magnetic Resonance (NMR) experiments directly on alive cells. While the anionic tetrasulfonatocalixarene does not bind to any bacterial strains significantly and the cationic calixarenes strongly bind to both Gram‐positive and Gram‐negative bacteria, the zwitterionic tetraprolino‐ and tetraphenylalaninocalix[4]arene show a remarkable selectivity for Gram‐negatives over Gram‐positives and mycobacteria. The tetraprolinocalixarene binds to the lipopolysaccharides extracted from two Gram‐negative bacteria (Pseudomonas putida or Escherichia coli), suggesting these biomacromolecules as possible targets in the recognition of their cell walls. The ligand binding epitope map demonstrates a deep involvement of the amino acids and calixarene aromatic nuclei in the interaction. In this study, for the first time, the ability of synthetic macrocycles to selectively recognize the envelope of Gram‐negative bacteria is highlighted, and the way to the chemical modifications of the ligand structure is paved to develop devices for the detection or treatment of bacterial infections, thus allowing to add another string to the bow for the fight against antimicrobial resistance.

BackgroundKRAS mutations are major oncogenic drivers, yet state-independent pan-KRAS inhibitors capable of targeting both active (GTP-bound) and inactive (GDP-bound) states remain extremely scarce.PurposeThe purpose of this work is to evaluate the non-covalent, state-independent pan-KRAS inhibitory potential of rosmarinic acid (RA) and botanical extracts from Rosmarinus officinalis and Salvia officinalis.Study designOur integrated multidisciplinary approach combines chemical characterization (qNMR and UPLC-ESI-HRMS/MS), biophysical binding assays (STD-NMR), computational analyses (molecular docking and molecular dynamics simulations), and in vitro functional assays on KRAS-dependent cancer cell lines, and Caco-2 cell monolayers as a non-proliferating epithelial model.MethodsMetabolites were profiled by NMR and UPLC-ESI-HRMS/MS and RA was quantified using qNMR. Direct interaction of RA and extracts with KRAS in GDP- and GMPPCP-bound states was characterized by STD-NMR, while molecular docking was used to investigate the predicted binding sites of RA. Antiproliferative activity and selectivity were assessed on KRAS-mutant cancer cells versus quiescent Caco-2 monolayers.ResultsqNMR provided accurate quantification of RA in both extracts. STD-NMR demonstrated direct, non-covalent, and state-independent binding of RA to KRAS in both inactive and active-like states, while docking analyses supported the proposed binding modes. Purified RA and botanical extracts inhibited the proliferation of KRAS-mutant cancer cells while showing limited cytotoxicity toward non-proliferating Caco-2 epithelial monolayers.ConclusionRosmarinic acid represents a promising non-covalent, state-independent pan-KRAS lead candidate, supporting further investigation of its potential for KRAS-targeted drug development.