The blood brain barrier dysfunction in healthy aging and dementia: why, how, what for?

The blood brain barrier (BBB) is an indispensable structure that maintains the central nervous system (CNS) microenvironment for a correct neuronal function. It is composed by high specialized microvessels, surrounded by astrocytes, pericytes, neurons and microglia cells, which tightly control the influx and efflux of substances to the brain parenchyma. During aging, the BBB becomes impaired, and it may contribute to the development of neurodegenerative and neurological disorders including Alzheimer's disease and other dementias. Restoring BBB can be a strategy to prevent disease onset and development, reducing the symptoms of these conditions. This work critically reviews the major mechanisms underlying BBB breakdown in healthy and unhealthy aging, as well as biomarkers and methodologies that accurately assess its impairment. Complementarily, potential therapeutic targets are discussed as new strategies to restore the normal function of the BBB in aging.

Development and validation of an HPLC-DAD method for the quantification of cannabigerol, cannabidiol, cannabinol and cannabichromene in human plasma and mouse matrices.

Cannabigerol, cannabidiol, cannabinol and cannabichromene are non-psychoactive phytocannabinoids, highly present in Cannabis sativa, for which numerous therapeutical applications have been described. However, additional pre-clinical and clinical data, including toxicopharmacokinetic and pharmacodynamic studies, remain required to support their use in clinical practice and new therapeutic applications. To support these studies, a new high performance liquid chromatography technique (HPLC) with diode-array detection (DAD) was developed and validated to quantify these cannabinoids in human plasma and mouse matrices. Sample extraction was accomplished by protein precipitation and double liquid-liquid extraction. Simvastatin and perampanel were used as internal standards in human and mouse matrices, respectively. Chromatographic separation was achieved in 16 min on an InfinityLab Poroshell® 120 C18 column (4.6 mm × 100 mm, 2.7 µm) at 40 °C. A mobile phase composed of water/acetonitrile was pumped with a gradient elution program at 1.0 mL min-1. The technique revealed linearity in the defined concentration ranges with a determination coefficient of over 0.99. Intra and inter-day accuracy and precision values ranged from -14.83 to 13.97% and 1.08 to 13.74%, respectively. Sample stability was assessed to ensure that handling and storage conditions did not compromise analyte concentrations in different matrices. Carry-over was absent and recoveries were over 77.31%. This technique was successfully applied for the therapeutic monitoring of cannabidiol and preliminary pre-clinical studies with cannabigerol and cannabidiol. All samples were within calibration ranges, with the exception of cannabigerol after intraperitoneal administration. This is the first HPLC-DAD technique that simultaneously quantifies cannabinoids in these biological matrices, supporting future pre-clinical and clinical investigations.

Restoring the epigenome in Alzheimer's disease: advancing HDAC inhibitors as therapeutic agents.

Current treatment options for Alzheimer's disease (AD) focus on symptom relief rather than halting disease progression. In this context, targeting histone deacetylation emerges as a promising therapeutic alternative. Dysregulation of histone deacetylase (HDAC) activity is present in AD, contributing to cognitive decline. Pharmacological HDAC inhibition has shown benefits in preclinical models, namely reduced amyloid beta plaque formation, lower phosphorylation and aggregation of tau protein, greater microtubule stability, less neuroinflammation, and improved metabolic homeostasis and cell survival. Nonetheless, clinical trials evidenced limitations such as insufficient selectivity or blood-brain barrier penetration. Hence, future innovative strategies are required to enhance their efficacy/safety.

Krüppel-like factors: potential roles in blood-brain barrier dysfunction and epileptogenesis.

Epilepsy is a chronic and debilitating neurological disorder, known for the occurrence of spontaneous and recurrent seizures. Despite the availability of antiseizure drugs, 30% of people with epilepsy experience uncontrolled seizures and drug resistance, evidencing that new therapeutic options are required. The process of epileptogenesis involves the development and expansion of tissue capable of generating spontaneous recurrent seizures, during which numerous events take place, namely blood-brain barrier (BBB) dysfunction, and neuroinflammation. The consequent cerebrovascular dysfunction results in a lower seizure threshold, seizure recurrence, and chronic epilepsy. This suggests that improving cerebrovascular health may interrupt the pathological cycle responsible for disease development and progression. Krüppel-like factors (KLFs) are a family of zinc-finger transcription factors, encountered in brain endothelial cells, glial cells, and neurons. KLFs are known to regulate vascular function and changes in their expression are associated with neuroinflammation and human diseases, including epilepsy. Hence, KLFs have demonstrated various roles in cerebrovascular dysfunction and epileptogenesis. This review critically discusses the purpose of KLFs in epileptogenic mechanisms and BBB dysfunction, as well as the potential of their pharmacological modulation as therapeutic approach for epilepsy treatment.

Intranasal delivery of paroxetine: A preclinical study on pharmacokinetics, depressive-like behaviour, and neurochemical sex differences.

Treatment of major depressive disorder remains a major unmet clinical need. Given the advantages of intranasal administration for targeted brain delivery, the present study aimed at investigating the pharmacokinetics of paroxetine, after its intranasal instillation and assessing its potential therapeutic effect on female and male mice subjected to unpredictable chronic mild stress (UCMS) protocol. IN administration revealed direct nose-to-brain paroxetine delivery but dose- and sex-dependent differences. Pharmacokinetics was nonlinear and paroxetine concentrations were consistently higher in plasma and brain of male mice. Additionally, UCMS decreased animal preference for sucrose in both male and female mice following acute (p < 0.01) and chronic stress (p < 0.05), suggesting anhedonia. Both male and female mice exhibited depressive-like behavior in the forced swimming test. UCMS females displayed a significantly longer immobility time and shorter climbing time than the control group (p < 0.05), while no differences were found between male mice. Two weeks of paroxetine intranasal administration reduced immobility time and lengthened climbing and swimming time, approaching values similar to those observed in the healthy control group. The therapeutic effect was stronger on female mice. Importantly, melatonin plasma levels were significantly decreased in female mice following UCMS (p < 0.05), while males exhibited heightened corticosterone levels. On the other hand, treatment with IN paroxetine significantly increased corticosterone and melatonin levels in both sexes compared to healthy mice (p < 0.05). Intranasal paroxetine delivery undoubtedly ameliorated the behavioral despair, characteristic of depressive-like animals. Despite its efficiency in male and female mice subjected to UCMS, females were more prone to this novel therapeutic strategy.

Intranasal administration of sertraline ensures sustained brain delivery and antidepressant effect in a mouse model of depression.

The pursuit of more potent and efficacious antidepressant therapies is of utmost significance. Herein, the intranasal (IN) route was investigated for sertraline brain delivery, encompassing a comparative pharmacokinetic study after a single-dose administration to mice by IN, intravenous (IV) (4.87 mg/kg) and oral (10 mg/kg) routes, and an efficacy/toxicity study to explore the therapeutic effect in mice subjected to the unpredictable chronic mild stress (UCMS) protocol. Neurotransmitters and melatonin were quantified in prefrontal cortex and plasma, respectively. A different drug biodistribution behavior was unveiled for a CNS-acting drug administered by means of the IN route. For the first time, IN administration of sertraline exhibited heightened systemic exposure (bioavailability = 166 %), and a sustained drug release into the brain, in opposition to IV and oral routes, avoiding drug fluctuation. The lower lung exposition (given by normalized area under the curve) observed after IN instillation envisions the reduction of sertraline pulmonary side effects and similarly other peripheral side effects. IN sertraline treatment displayed significant efficacy in ameliorating anhedonia after one week of administration while the 14-day IN treatment regimen translated into decreased immobility time and increased swimming time in the forced swimming test, suggesting an improvement of the depressive-like behavior displayed by the animal depressive-model. Remarkably, these effects were absent with oral sertraline, despite the higher used dose. Noteworthy neurotransmitter alterations were observed, with IN sertraline markedly reducing adrenaline in the prefrontal cortex, while serotonin and melatonin increased following both administration routes. With its sustained brain delivery and serotonin- and melatonin-enhancing potential, the innovative strategy of IN sertraline holds the potential not only to effectively address depressive symptoms but also to mitigate challenges inherent to classic treatments.

Licochalcone A: A Potential Multitarget Drug for Alzheimer's Disease Treatment.

Licochalcone A (Lico-A) is a flavonoid compound derived from the root of the Glycyrrhiza species, a plant commonly used in traditional Chinese medicine. While the Glycyrrhiza species has shown promise in treating various diseases such as cancer, obesity, and skin diseases due to its active compounds, the investigation of Licochalcone A's effects on the central nervous system and its potential application in Alzheimer's disease (AD) treatment have garnered significant interest. Studies have reported the neuroprotective effects of Lico-A, suggesting its potential as a multitarget compound. Lico-A acts as a PTP1B inhibitor, enhancing cognitive activity through the BDNF-TrkB pathway and exhibiting inhibitory effects on microglia activation, which enables mitigation of neuroinflammation. Moreover, Lico-A inhibits c-Jun N-terminal kinase 1, a key enzyme involved in tau phosphorylation, and modulates the brain insulin receptor, which plays a role in cognitive processes. Lico-A also acts as an acetylcholinesterase inhibitor, leading to increased levels of the neurotransmitter acetylcholine (Ach) in the brain. This mechanism enhances cognitive capacity in individuals with AD. Finally, Lico-A has shown the ability to reduce amyloid plaques, a hallmark of AD, and exhibits antioxidant properties by activating the nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of antioxidant defense mechanisms. In the present review, we discuss the available findings analyzing the potential of Lico-A as a neuroprotective agent. Continued research on Lico-A holds promise for the development of novel treatments for cognitive disorders and neurodegenerative diseases, including AD. Further investigations into its multitarget action and elucidation of underlying mechanisms will contribute to our understanding of its therapeutic potential.

Nasal-PAMPA: A novel non-cell-based high throughput screening assay for prediction of nasal drug permeability.

In nasal drug product development, screening studies are vital to select promising compounds or formulations. The Parallel Artificial Membrane Permeability Assay (PAMPA), a high throughput screening tool, has been applied to evaluate drug permeability across several barriers such as the skin or blood-brain barrier. Herein, a new nasal-PAMPA model was optimized to predict nasal permeability, using a biorelevant donor medium containing mucin. The apparent permeability (Papp) of 15 reference compounds was assessed in six different experimental conditions, and the most discriminating and predictive model was applied to a test drug (piroxicam) and mucoadhesive powder formulations loading the same drug. The model with 0.5% (w/v) mucin in the donor compartment and 2% (w/v) phosphatidylcholine in the lipid membrane accurately distinguished high and low permeable compounds. Additionally, it exhibited the highest correlation with permeation across human nasal epithelial cells, RPMI 2650 (R2 = 0.93). When applied to powder formulations, this model was sensitive to the presence of mucoadhesive excipients and the drug solid state. Overall, the nasal-PAMPA model was more rapid than cell-based assays, without requiring specialized training or equipment, showing to be a promising in vitro tool that can be applied in drug and formulation screening for nasal delivery.

Under the umbrella of depression and Alzheimer's disease physiopathology: Can cannabinoids be a dual-pleiotropic therapy?

Depression and Alzheimer´s disease (AD) are two disorders highly prevalent worldwide. Depression affects more than 300 million people worldwide while AD affects 60-80% of the 55 million cases of dementia. Both diseases are affected by aging with high prevalence in elderly and share not only the main brain affected areas but also several physiopathological mechanisms. Depression disease is already ascribed as a risk factor to the development of AD. Despite the wide diversity of pharmacological treatments currently available in clinical practice for depression management, they remain associated to a slow recovery process and to treatment-resistant depression. On the other hand, AD treatment is essentially based in symptomatology relieve. Thus, the need for new multi-target treatments arises. Herein, we discuss the current state-of-art regarding the contribution of the endocannabinoid system (ECS) in synaptic transmission processes, synapses plasticity and neurogenesis and consequently the use of exogenous cannabinoids in the treatment of depression and on delaying the progression of AD. Besides the well-known imbalance of neurotransmitter levels, including serotonin, noradrenaline, dopamine and glutamate, recent scientific evidence highlights aberrant spine density, neuroinflammation, dysregulation of neurotrophic factor levels and formation of amyloid beta (Aß) peptides, as the main physiopathological mechanisms compromised in depression and AD. The contribution of the ECS in these mechanisms is herein specified as well as the pleiotropic effects of phytocannabinoids. At the end, it became evident that Cannabinol, Cannabidiol, Cannabigerol, Cannabidivarin and Cannabichromene may act in novel therapeutic targets, presenting high potential in the pharmacotherapy of both diseases.

Population Pharmacokinetic Analysis of Perampanel in Portuguese Patients Diagnosed with Refractory Epilepsy.

Perampanel is a promising antiepileptic drug (AED) for refractory epilepsy treatment due to its innovative mechanism of action. This study aimed to develop a population pharmacokinetic (PopPK) model to be further used in initial dose optimization of perampanel in patients diagnosed with refractory epilepsy. A total of seventy-two plasma concentrations of perampanel obtained from forty-four patients were analyzed through a population pharmacokinetic approach by means of nonlinear mixed effects modeling (NONMEM). A one-compartment model with first-order elimination best described the pharmacokinetic profiles of perampanel. Interpatient variability (IPV) was entered on clearance (CL), while the residual error (RE) was modeled as proportional. The presence of enzyme-inducing AEDs (EIAEDs) and body mass index (BMI) were found as significant covariates for CL and volume of distribution (V), respectively. The mean (relative standard error) estimates for CL and V of the final model were 0.419 L/h (5.56%) and 29.50 (6.41%), respectively. IPV was 30.84% and the proportional RE was 6.44%. Internal validation demonstrated an acceptable predictive performance of the final model. A reliable population pharmacokinetic model was successfully developed, and it is the first enrolling real-life adults diagnosed with refractory epilepsy.

Therapeutic Drug Monitoring of Amikacin in Neutropenic Oncology Patients.

Amikacin is the antibiotic of choice for the treatment of Gram-negative infections, namely, those in neutropenic oncology patients. No populational pharmacokinetic studies are currently available reporting amikacin pharmacokinetics in neutropenic oncology patients despite their specific pathophysiological features and treatments. A large-scale retrospective study was herein conducted to specifically investigate the effects that tumor diseases have on the pharmacokinetic parameters of amikacin and identify whether chemotherapy, the lag time between administration of chemotherapy and amikacin, age and renal function contribute to amikacin pharmacokinetics in neutropenic cancer patients. A total of 1180 pharmacokinetic analysis from 629 neutropenic patients were enrolled. The daily dose administered to oncology patients was higher than that administered to non-oncology patients (p < 0.0001). No statistical differences were found in amikacin concentrations, probably because drug clearance was increased in cancer patients (p < 0.0001). Chemotherapy influenced amikacin pharmacokinetics and drug clearance decreased as the lag time enhanced. The elderly group revealed no statistical differences between the doses administered to both the oncology groups, suggesting that the impact of ageing is stronger than chemotherapy. Our research suggests that cancer patients require higher initial doses of amikacin, as well as when chemotherapy is received less than 30 days before amikacin treatment has started.

Air-liquid interface (ALI) impact on different respiratory cell cultures.

The intranasal route has been receiving greater attention from the scientific community not only for systemic drug delivery but also for the treatment of pulmonary and neurological diseases. Along with it, drug transport and permeability studies across the nasal mucosa have exponentially increased. Nevertheless, the translation of data from in vitro cell lines to in vivo studies is not always reliable, due to the difficulty in generating an in vitro model that resembles respiratory human physiology. Among all currently available methodologies, the air-liquid interface (ALI) method is advantageous to promote cell differentiation and optimize the morphological and histological characteristics of airway epithelium cells. Cells grown under ALI conditions, in alternative to submerged conditions, appear to provide relevant input for inhalation and pulmonary toxicology and complement in vivo experiments. Different methodologies and a variety of materials have been used to induce ALI conditions in primary cells and numerous cell lines. Until this day, with only exploratory results, no consensus has been reached regarding the validation of the ALI method, hampering data comparison. The present review describes the most adequate cell models of airway epithelium and how these models are differently affected by ALI conditions. It includes the evaluation of cellular features before and after ALI, and the application of the method in primary cell cultures, commercial 3D primary cells, cell lines and stem-cell derived models. A variety of these models have been recently applied for pharmacological studies against severe acute respiratory syndrome-coronavirus(-2) SARS-CoV(-2), namely primary cultures with alveolar type II epithelium cells and organotypic 3D models. The herein compiled data suggest that ALI conditions must be optimized bearing in mind the type of cells (nasal, bronchial, alveolar), their origin and the objective of the study.

Clinical Application of Pharmacokinetics to Appraise Adherence to Levetiracetam in Portuguese Epileptic Patients.

Adherence to antiseizure drug treatment determines its effectiveness and safety, and consequently affects patients' quality of life. Herein, we assessed adherence to levetiracetam in Portuguese patients with refractory epilepsy (n = 115), with resort to a pharmacokinetic drug monitoring approach. The pharmacokinetic parameters of levetiracetam in each patient were determined in steady-state while admitted to the hospital. Then, adherence was assessed by comparing the plasma concentration of the drug observed on the first day of hospitalization with the predicted plasma concentration, considering previously determined pharmacokinetic parameters. The rate of adherence was assessed according to gender, age, diagnosis, and antiseizure drug regimen. Among 115 enrolled patients, 49 (42.6%) were identified as non-adherent, 30 (26.1%) classified as under-consumers, and 19 (16.5%) as over-consumers. A relationship between adherence, daily dose and plasma concentrations was herein reported for the first time. Adherent patients received higher daily doses of levetiracetam [2500 (2000-3000) mg] than non-adherent over-consumers [1500 (1000-2000) mg] and non-adherent under-consumers [2000 (1500-3000) mg]. Higher average steady-state plasma concentrations of levetiracetam were found in non-adherent under-consumers [27.28 (15.33-36.36) mg/L], followed by adherent patients [22.05 (16.62-29.81) mg/L] and non-adherent over-consumers [17.50 (10.69-24.37) mg/L]. This study demonstrates that adherence (or lack thereof) influences the plasma concentrations of levetiracetam in steady-state and its pharmacological effects. Moreover, it emphasizes the importance of educating patients to encourage adherence to therapy. Otherwise, the risk of developing toxic and subtherapeutic concentrations is undeniable, compromising the therapeutic effect and safety of treatment.

Targeting brain Renin-Angiotensin System for the prevention and treatment of Alzheimer's disease: Past, present and future.

Alzheimer's disease (AD) is a well-known neurodegenerative disease characterized by the presence of two main hallmarks - Tau hyperphosphorylation and Aß deposits. Notwithstanding, in the last few years the scientific evidence about the drivers of AD have been changing and nowadays age-related vascular alterations and several cardiovascular risk factors have been shown to trigger the development of AD. In this context, drugs targeting the Renin Angiotensin System (RAS), commonly used for the treatment of hypertension, are evidencing a high potential to delay AD development due to their action on brain RAS. Indeed, the ACE 1/Ang II/AT1R axis is believed to be upregulated in AD and to be responsible for deleterious effects such as increased oxidative stress, neuroinflammation, blood-brain barrier (BBB) hyperpermeability, astrocytes dysfunction and a decrease in cerebral blood flow. In contrast, the alternative axis - ACE 1/Ang II/AT2R; ACE 2/Ang (1-7)/MasR; Ang IV/ AT4R(IRAP) - seems to counterbalance the deleterious effects of the principal axis and to exert beneficial effects on memory and cognition. Accordingly, retrospective studies demonstrate a reduced risk of developing AD among people taking RAS medication as well as several in vitro and in vivo pre-clinical studies as it is herein critically reviewed. In this review, we first revise, at a glance, the pathophysiology of AD focused on its classic hallmarks. Secondly, an overview about the impact of the RAS on the pathophysiology of AD is also provided, focused on their four essential axes ACE 1/Ang II/AT2R; ACE 2/Ang (1-7)/MasR; Ang IV/ AT4R(IRAP) and ACE 1/Ang II/AT1R. Finally, the therapeutic potential of available drugs targeting RAS on AD, namely angiotensin II receptor blockers (ARBs) and angiotensin converting enzyme inhibitors (ACEIs), is highlighted and data supporting this hope will be presented, from in vitro and in vivo pre-clinical to clinical studies.

Encapsulated Escitalopram and Paroxetine Intranasal Co-Administration: In Vitro/In Vivo Evaluation.

Depression is a common mental disorder. Its treatment with selective serotonin reuptake inhibitors (SSRIs) is effective only in a fraction of patients, and pharmacoresistance is increasing steadily. Intranasal (IN) drug delivery to the brain stands out as a promising strategy to improve current therapeutic approaches by operating as a shuttle to overcome the blood-brain barrier. This work aimed to simultaneously administer escitalopram and paroxetine by IN route to mice. For this purpose, three nanostructured lipid carriers (NLC1, NLC2, and BorNLC) and one nanoemulsion (NE) were tested for drug loading. After their characterization, investigation of their impact on nasal cell viability and SSRI permeability assays were performed, using a human nasal RPMI 2650 cell line in air-liquid interface. In vitro assays demonstrated that NLCs, including borneol (BorNLC), significantly increased escitalopram permeability (p < 0.01) and paroxetine recovery values (p < 0.05) in relation to the other formulations and non-encapsulated drugs. IN and intravenous (IV) pharmacokinetic studies performed in vivo with a single dose of 2.38 mg/kg demonstrated similar results for escitalopram brain-to-plasma ratios. IN administrations delayed escitalopram peak concentrations in the brain for 15-60 min and no direct nose-to-brain delivery was detected. However, encapsulation with BorNLC considerably decreased escitalopram exposure in the lungs (124 µg min/g) compared with free escitalopram by IN (168 µg min/g) and IV (321 µg min/g) routes. Surprisingly, BorNLC IN instillation increased concentration levels of paroxetine in the brain by five times and accelerated brain drug delivery. Once again, lung exposure was considerably lower with BorNLC (AUCt = 0.433 µg min/g) than that with IV administration (AUCt = 1.01 µg min/g) and non-encapsulated IN formulation (AUCt = 2.82 µg min/g). Direct nose-to-brain delivery was observed for paroxetine IN administration with a direct transport percentage (DTP) of 56.9%. If encapsulated, it increases to 74.2%. These results clearly emphasize that nose-to-brain delivery and lung exposure depend on the formulation and on the characteristics of the drug under investigation. NLCs seem to be an advantageous strategy for nose-to-brain delivery of lipophilic molecules, since they reduce systemic and lung exposure, thereby decreasing adverse effects. For hydrophilic compounds, NLCs are particularly important to decrease lung exposure after IN administration.

Antidepressants and Circadian Rhythm: Exploring Their Bidirectional Interaction for the Treatment of Depression.

Scientific evidence that circadian rhythms affect pharmacokinetics and pharmacodynamics has highlighted the importance of drug dosing-time. Circadian oscillations alter drug absorption, distribution, metabolism, and excretion (ADME) as well as intracellular signaling systems, target molecules (e.g., receptors, transporters, and enzymes), and gene transcription. Although several antidepressant drugs are clinically available, less than 50% of depressed patients respond to first-line pharmacological treatments. Chronotherapeutic approaches to enhance the effectiveness of antidepressants are not completely known. Even so, experimental results found until this day suggest a positive influence of drug dosing-time on the efficacy of depression therapy. On the other hand, antidepressants have also demonstrated to modulate circadian rhythmicity and sleep-wake cycles. This review aims to evidence the potential of chronotherapy to improve the efficacy and/or safety of antidepressants. It includes pre-clinical and clinical studies that demonstrate the relevance of determining the most appropriate time of administration for antidepressant drugs. In parallel, their positive influence on the resynchronization of disrupted circadian rhythms is also herein discussed. It is expected that this review will promote the investigation of chronotherapy for the treatment of depression, contribute to a better understanding of the relationship between antidepressants and circadian rhythms, and consequently promote the development of new therapeutics.

Pharmacology of lacosamide: From its molecular mechanisms and pharmacokinetics to future therapeutic applications.

Epilepsy is one of the most common brain disorders, affecting more than 50 million people worldwide. Although its treatment is currently symptomatic, the last generation of anti-seizure drugs is characterized by better pharmacokinetic profiles, efficacy, tolerability and safety. Lacosamide is a third-generation anti-seizure drug that stands out due to its good efficacy and safety profile. It is used with effectiveness in the treatment of partial-onset seizures with or without secondary generalization, primary generalized tonic-clonic seizures and off-label in status epilepticus. Despite scarcely performed until today, therapeutic drug monitoring of lacosamide is proving to be advantageous by allowing the control of inter and intra-individual variability and promoting a successful personalized therapy, particularly in special populations. Herein, the pharmacology, pharmacokinetics, and clinical data of lacosamide were reviewed, giving special emphasis to the latest molecular investigations underlying its mechanism of action and therapeutic applications in pathologies besides epilepsy. In addition, the pharmacokinetic characteristics of lacosamide were updated, as well as current literature concerning the high pharmacokinetic variability observed in special patient populations and that must be considered during treatment individualization.

HPLC method for the determination of antiepileptic drugs in human saliva and its application in therapeutic drug monitoring.

Epilepsy is one of the most prevalent neurological disorders, affecting approximately 1% of the world population. Despite the availability of dozens of antiepileptic drugs (AEDs) in clinical practice, the number of patients who do not respond to treatment and/or exhibit high pharmacokinetic variability remains significant, highlighting the importance of therapeutic drug monitoring (TDM). Plasma and serum are the main biological matrices applied for the TDM of AEDs, but the necessity of a specialized professional has been an obstacle to sample collection in ambulatory. Thus, drug quantification in saliva arises as a promising alternative. Herein, a novel highperformance liquid chromatographic (HPLC) technique with diode-array detection (DAD) was developed and fully validated, in order to simultaneously quantify carbamazepine, carbamazepine-10,11-epoxide, S-licarbazepine, lacosamide and levetiracetam in human saliva. The technique was linear in the following concentration ranges: 0.2-6 mg L-1 for carbamazepine and carbamazepine-10,11-epoxide; 0.3-9 mg L-1 for S- licarbazepine; 1-30 mg L-1 for lacosamide; and 0.8-24 mg L-1 for levetiracetam. The lower limits of the established calibration ranges are below therapeutic margins, attesting a sensitive drug quantification. Accuracy values ranged from -14.76 to 9.35 % and -12.87 and 11.18 % in intra-day and inter-day analysis, respectively. Intra-day values of precision varied between 3.45-10.76% and inter-day values ranged from 3.85 to 13.05 %. This method was subsequently applied to saliva samples of epileptic patients admitted to the Refractory Epilepsy Centre of Centro Hospitalar e Universitário de Coimbra (CHUC EPE, Coimbra). The results of saliva samples were correlated with drug concentrations in plasma from the same patients. Statistically significant correlations were observed (p < 0.05) for carbamazepine (r2 = 0.6887; r = 0.8299), carbamazepine-10,11-epoxide (r2 = 0.8633; r = 0.9291), S-licarbazepine (r2 = 0.5266; r = 0.7257) and levetiracetam (r2 = 0.7103; r = 0.8428). Our data support that this method can be used in TDM of AEDs using human saliva samples, constituting a new approach to establish individual therapeutic ranges and assess patient's adherence to treatment.

Development and application of an HPLC-DAD technique for human plasma concentration monitoring of perampanel and lamotrigine in drug-resistant epileptic patients.

Perampanel is a third-generation antiepileptic drug (AED), while lamotrigine is a second-generation AED. Both drugs are subject to extensive pharmacokinetic variability between different patients. Furthermore, it has been reported that perampanel and lamotrigine may be implied in pharmacokinetic drug-drug interactions with other AEDs such as carbamazepine or valproate, with consequent alterations of plasma concentrations. This emphasizes the relevance of therapeutic drug monitoring of perampanel and lamotrigine with appropriate bioanalytical methods. Herein, the development and validation of a bioanalytical techique for the simultaneous quantification of perampanel and lamotrigine in human plasma samples is described. The reported method is based on high-performance liquid chromatography coupled with diode-array detection (HPLC-DAD) and sample preparation consists of liquid-liquid extraction. Chromatographic separation of the analytes (lamotrigine and perampanel) and the internal standard (entacapone) was achieved in 12 min on a reversed-phase C18 column at 40 °C by applying a gradient elution program with a mobile phase composed of 0.1% ortho-phosphoric acid pH 2.79 (A) and acetonitrile (B) pumped at 1.0 mL/min. Perampanel was quantified at 320 nm while lamotrigine and the internal standard were monitored at 306 nm. Calibration curves were linear in the concentration range of 0.03-4.5 µg/mL (r2 = 0.9978) for perampanel and in the concentration range of 0.25-30 µg/mL (r2 = 0.9981) for lamotrigine. Overall precision did not exceed 14.3% and accuracy ranged from -6.08 to 12.66%. Some drugs potentially co-prescribed with perampanel and lamotrigine were tested and did not interfere with the retention times of the analytes and internal standard. The method was then successfully applied for the quantification of perampanel and lamotrigine in plasma samples obtained from 42 drug-resistant epileptic patients admitted to the Coimbra University Hospital Centre (CHUC.EPE, Coimbra, Portugal). In conclusion, it is a suitable method for the therapeutic drug monitoring of lamotrigine and perampanel in drug-resistant epileptic patients, as well as, for the assessment of drug-drug interactions. It can also be adopted by hospitals and laboratories, when HPLC with fluorescence and mass spectrometry detections are unavailable.

A combo-strategy to improve brain delivery of antiepileptic drugs: Focus on BCRP and intranasal administration.

The breast cancer resistance protein (BCRP) is an efflux transporter expressed at the apical surface of human brain endothelial cells of the blood-brain barrier (BBB). It was proposed as one of the transporters responsible for the development of drug resistance to several central nervous system (CNS) drugs, including antiepileptic drugs (AEDs). In this context, the present work aimed to characterize the interaction between new-generation AEDs, lacosamide, levetiracetam and zonisamide, and BCRP, in order to investigate whether intranasal administration can successfully avoid the impact of BCRP on brain drug distribution, preventing the development of refractory epilepsy. Firstly, BCRP substrates and/or inhibitors were identified resorting to intracellular accumulation and bidirectional transport assays on Madin-Darby canine kidney (MDCK) cells and the transfected cell line with human ABCG2 (MDCK-BCRP). Furthermore, in vivo pharmacokinetic studies were carried out for BCRP substrates with and without elacridar, a well-known P-gp and BCRP modulator, to assess the impact of efflux inhibition on brain drug distribution. The extent of drug equilibration between plasma and brain was compared after intravenous (IV) and intranasal administration to mice. Among the three tested AEDs, zonisamide was the only AED identified as BCRP substrate in vitro, as demonstrated by the net flux ratio of 2.73, which decreased 53.85 % in the presence of a BCRP inhibitor, Ko143. Lacosamide revealed to inhibit BCRP in all tested concentrations (2.5-75 µM), exhibiting a significant increase (p < 0.001) of the intracellular accumulation of a BCRP substrate (Hoechst 33342) in MDCK-BCRP cells. Levetiracetam did not behave as a BCRP substrate nor inhibitor. After IV administration, the plasma concentrations of zonisamide were unaffected by elacridar, but its extent of brain exposure increased three-fold (as assessed by AUCt, 674.12 vs 284.47 µg.min/mL). These results corroborate the previous in vitro findings, suggesting that BCRP is involved in the transport of zonisamide through the BBB. In opposition, no significant changes were found in plasma or brain concentrations after the administration of zonisamide by intranasal route, indicating that the influence of BCRP is less relevant than for IV route. In addition, direct nose-to-brain delivery of zonisamide, given by the direct transport percentage, was approximately 49 %. Altogether, these assays demonstrated that the impact of BCRP on the delivery of zonisamide to the brain is lower after intranasal administration, probably due to direct nose-to-brain transport. Therefore, the intranasal administration of AEDs may be a relevant strategy to avoid the impact of efflux transporters at the BBB and the development of drug resistance.