Impaired catabolism of free oligosaccharides due to MAN2C1 variants causes a neurodevelopmental disorder.

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

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.

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.

Predicting factors of neurodevelopmental performance in children with phenylketonuria.

In phenylketonuria (PKU), high phenylalanine (Phe) levels hamper neurodevelopment impairing executive function later in life. While the second has been more studied, fewer data exist on predictors of PKU patients' development in specific populations. To contribute to the field, we performed a retrospective analysis of predictors of neurodevelopment in PKU patients in a Portuguese cohort. We analyzed the retrospective data on the metabolic control of 89 patients, as their health and familial features. Griffith's Mental Development Scale performance at age 6 (GMDS6) was used to assess neurodevelopment. Our cohort included 14 GMDS6low and 75 GMDS6high patients. In a multivariate analysis, the better predictors of neurodevelopment were the metabolic control at age 3 and year of birth (n = 87, ß0  = -121, ß1  = -1.77, ß2  = 0.06, LRchi2(2) = 13.61, Prob > chi2 = 0.001, Pseudo R2 = 0.1773). With this model, it was possible to define a safety cut-off of 7.8 mg/dL for the Phe level at age 3 (sensitivity = 72.6%, specificity = 78.6%), confirming the safety of the cut-off of 6 mg/dL already used in the clinical practice. Our study supports the relevance of metabolic control to predict the neurodevelopment of PKU patients, in the historical context of the disease management.

Diagnostic accuracy and the first genotype-phenotype correlation in glycogen storage disease type V.

BACKGROUND: Glycogen storage disease type V (GSDV) is an autosomal recessive metabolic condition caused by pathogenic PYGM variants. This is an underdiagnosed condition as it presents with exercise intolerance in children. We reviewed the GSDV cases of a tertiary hospital center to assess diagnostic timing/accuracy, as well as potential clinical/analytical predictors of such factors. METHODS: We retrospectively reviewed all GSDV cases with follow-up in both Pediatric and Adult Metabolic Diseases consultations. We included 28 cases and assessed their hospital record for clinical information. RESULTS: Over 90% of our cases had late diagnoses, with more than 50% being diagnosed in adulthood despite symptom onset in preschool (very late diagnosis). Diagnostic age was lower in patients exhibiting myoglobinuria. Interestingly, patients with a positive family history of GSDV had similar rates of very late diagnoses, likely since the index case was already detected very late in life. Finally, we observe that the R50* variant is associated with increased myoglobinuria and CK elevation, in a dosage-dependent manner. CONCLUSION: We concluded that GSDV is severely underdiagnosed, and that some clinical and analytical aspects of the condition can be more indicative of this diagnosis. Furthermore, we propose for the first time a genotype-phenotype correlation in GSDV. IMPACT: GSDV is a pediatric-onset metabolic disorder that is mostly diagnosed late in the adult age and commonly misdiagnosed. We observed the first genotype-phenotype correlation in GSDV, regarding the common R50* variant. Awareness of GSDV for pediatricians and the overall medical community is vital.

An SPG7 mutation as a novel cause of monogenic progressive muscular atrophy.

BACKGROUND: Progressive muscular atrophy (PMA) is a rare adult-onset neurological disease that is characterized by isolated lower motor neuron degeneration. While it is still disputable whether PMA is a subtype of amyotrophic lateral sclerosis (ALS) or an isolated disorder, it is well-established as a clinically defined entity. About 5% of PMA cases are monogenic, and the implicated genes largely overlap with those causing monogenic ALS. CASE DESCRIPTION: Here we describe a 68-year-old female patient with progressive and asymmetric upper-limb weakness throughout an 18-month period, with muscle atrophy, dysphagia and slurring of speech. The lower limbs were unaffected, and there was no sign of upper motor neuron dysfunction. Comprehensive genetic testing for single nucleotide and copy-number variants revealed a pathogenic monoallelic variant c.1529C>T, p.(Ala510Val) in the SPG7 gene. DISCUSSION: Pathogenic biallelic SPG7 variants have been originally associated with hereditary spastic paraplegia, but other phenotypes are nowadays known to be linked to these variants, such as ALS. However, there is no report of this (or any) other SPG7 variant in association with PMA, whether it progressed to ALS or not. In conclusion, we present the first known case of PMA associated with a monoallelic SPG7 mutation.

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.

Deciphering specific miRNAs in brain tumors: a 5-miRNA signature in glioblastoma.

MicroRNAs are endogenous non-coding RNAs with a marked impact on the development and progression of brain tumors. However, they commonly share different expression patterns in other types of tumors, thereby exhibiting lack of tissue specificity. Here, an integrative holistic analysis of microarray data is established for deciphering dysregulated miRNAs in glioblastoma, distinguishing them from eight other CNS tumors. The identification of dysregulated miRNAs was performed in a pool of 176 patients, 118 of which diagnosed with glioblastoma. Dysregulated miRNAs commonly expressed in glioblastoma were then discriminated from those co-expressed in other CNS tumors and further characterized. Overall, 21 miRNAs were found to be commonly dysregulated in glioblastoma. Notwithstanding, 16 miRNAs also exhibited a differential expression in at least one other CNS tumor. The remaining 5, specifically, hsa-miR-21-3p, hsa-miR-338-5p, hsa-miR-485-5p, hsa-miR-491-5p and hsa-miR-1290, were solely associated to glioblastoma. This signature is in-depth characterized, with the spotlight on tumor progression, invasion and patient survival. These five endogenous molecules, differentially expressed in glioblastoma, are thus suggested as potential therapeutic targets, modulating several genes involved in major signalling pathways, including MAPK/ERK, calcium, PI3K/AKT, mTOR and Wnt. In summary, these findings lay a foundation for further research on the expression and function of specific patterns of miRNAs expression in glioblastoma, providing reference for potential novel targets.

A new tool to screen patients with severe obstructive sleep apnea in the primary care setting: a prospective multicenter study.

BACKGROUND: The coordination between different levels of care is essential for the management of obstructive sleep apnea (OSA). The objective of this multicenter project was to develop a screening model for OSA in the primary care setting. METHODS: Anthropometric data, clinical history, and symptoms of OSA were recorded in randomly selected primary care patients, who also underwent a home sleep apnea test (HSAT). Respiratory polygraphy or polysomnography were performed at the sleep unit to establish definite indication for continuous positive airway pressure (CPAP). By means of cross-validation, a logistic regression model (CPAP yes/no) was designed, and with the clinical variables included in the model, a scoring system was established using the ß coefficients (PASHOS Test). In a second stage, results of HSAT were added, and the final accuracy of the model was assessed. RESULTS: 194 patients completed the study. The clinical test included the body mass index, neck circumference and observed apneas during sleep (AUC 0.824, 95% CI 0.763-0.886, P < 0.001). In a second stage, the oxygen desaturation index (ODI) of 3% (ODI3% ≥ 15%) from the HSAT was added (AUC 0.911, 95% CI 0.863-0.960, P < 0.001), with a sensitivity of 85.5% (95% CI 74.7-92.1) and specificity of 67.8% (95% CI 55.1-78.3). CONCLUSIONS: The use of this model would prevent referral to the sleep unit for 55.1% of the patients. The two-stage PASHOS model is a useful and practical screening tool for OSA in primary care for detecting candidates for CPAP treatment. Clinical Trial Registration Registry: ClinicalTrials.gov; Name: PASHOS Project: Advanced Platform for Sleep Apnea Syndrome Assessment; URL: https://clinicaltrials.gov/ct2/show/NCT02591979 ; Identifier: NCT02591979. Date of registration: October 30, 2015.

Congenital Disorders of Glycosylation in Portugal-Two Decades of Experience.

OBJECTIVE: To describe the clinical, biochemical, and genetic features of both new and previously reported patients with congenital disorders of glycosylation (CDGs) diagnosed in Portugal over the last 20 years. STUDY DESIGN: The cohort includes patients with an unexplained multisystem or single organ involvement, with or without psychomotor disability. Serum sialotransferrin isoforms and, whenever necessary, apolipoprotein CIII isoforms and glycan structures were analyzed. Additional studies included measurement of phosphomannomutase (PMM) activity and analysis of lipid-linked oligosaccharides in fibroblasts. Sanger sequencing and massive parallel sequencing were used to identify causal variants or the affected gene, respectively. RESULTS: Sixty-three individuals were diagnosed covering 14 distinct CDGs; 43 patients diagnosed postnatally revealed a type 1, 14 a type 2, and 2 a normal pattern on serum transferrin isoelectrofocusing. The latter patients were identified by whole exome sequencing. Nine of them presented also a hypoglycosylation pattern on apolipoprotein CIII isoelectrofocusing, pointing to an associated O-glycosylation defect. Most of the patients (62%) are PMM2-CDG and the remaining carry pathogenic variants in ALG1, ATP6AP1, ATP6AP2, ATP6V0A2, CCDC115, COG1, COG4, DPAGT1, MAN1B1, SLC35A2, SRD5A3, RFT1, or PGM1. CONCLUSIONS: Portuguese patients with CDGs are presented in this report, some of them showing unique clinical phenotypes. Among the 14 genes mutated in Portuguese individuals, 8 are shared with a previously reported Spanish cohort. However, regarding the mutational spectrum of PMM2-CDG, the most frequent CDG, a striking similarity between the 2 populations was found, as only 1 mutated allele found in the Portuguese group has not been reported in Spain.

Silymarin as a flavonoid-type P-glycoprotein inhibitor with impact on the pharmacokinetics of carbamazepine, oxcarbazepine and phenytoin in rats.

P-glycoprotein (P-gp) is an efflux transporter involved in drug-resistant epilepsy and some flavonoids have been targeted as effective P-gp inhibitors. Herein, we assessed the impact of silymarin on the pharmacokinetics of three antiepileptic drugs (AEDs) in rats. Animals were pretreated with silymarin, verapamil (positive control) or vehicle (negative control) 1 h before AEDs administration (carbamazepine (25 mg/kg), oxcarbazepine (OXC) (50 mg/kg), or phenytoin (100 mg/kg)). Multiple blood samples were collected after AED dosing, and a non-compartmental analysis was performed. An independent study was also conducted to investigate the effects of silymarin on the OXC plasma-to-brain distribution. Silymarin altered the pharmacokinetics of OXC, increasing its peak plasma concentration by 50% and its extent of systemic exposure by 41%, which had also impact on brain drug concentrations. These findings support that the co-administration of silymarin and OXC should continue to be explored as a strategy to reverse the pharmacoresistance in epilepsy.

Integrating Whole-Genome Sequencing in Clinical Genetics: A Novel Disruptive Structural Rearrangement Identified in the Dystrophin Gene (DMD).

While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a ~8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics.

Repairing blood-CNS barriers: Future therapeutic approaches for neuropsychiatric disorders.

Central nervous system (CNS) drug development faces significant difficulties that translate into high rates of failure and lack of innovation. The pathophysiology of neurological and psychiatric disorders often results in the breakdown of blood-CNS barriers, disturbing the CNS microenvironment and worsening disease progression. Therefore, restoring the integrity of blood-CNS barriers may have a beneficial influence in several CNS disorders and improve treatment outcomes. In this review, pathways that may be modulated to protect blood-CNS barriers from neuroinflammatory and oxidative insults are featured. First, the participation of the brain endothelium and glial cells in disruption processes is discussed. Then, the relevance of regulatory systems is analysed, specifically the hypothalamic-pituitary axis, the renin-angiotensin system, sleep and circadian rhythms, and glutamate neurotransmission. Lastly, compounds of endogenous and exogenous origin that are known to mediate the repair of blood-CNS barriers are presented. We believe that enhancing the protection of blood-CNS barriers is a promising therapeutic strategy to pursue in the future.

Cystic fibrosis: Physiopathology and the latest pharmacological treatments.

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease, caused by a mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which primarily affects the lungs and digestive system. This gene encodes the CFTR protein, a distinctive membrane transporter of the ATP-binding cassette (ABC) superfamily. It functions as a chloride channel, allowing the balance and transport of chloride through the apical membrane of epithelial cells. Due to its ubiquitous location, mutations in the CFTR gene trigger multiple changes in ion transport and metabolic pathways, affecting various organs, as it will be herein explained. Pulmonary impairment is the most characteristic comorbidity of CF and respiratory failure is the main cause of death. This review presents the importance of an early diagnosis of CF to establish, as soon as possible, a primary therapy for symptomatic prevention and relief. It also mentions new therapeutic approaches that include CFTR modulators. They are correctors and/or potentiators of the deficient CFTR channel. In an attempt to overcome the disadvantages of CFTR modulators, the application of biotechnology techniques is addressed, such as gene therapy, gene editing, RNA therapy and therapeutic microRNAs. The potential of the intranasal administration route is another presented aspect.

Impact of direct oral anticoagulant off-label doses on clinical outcomes of atrial fibrillation patients: A systematic review.

AIMS: Worldwide observational studies are evidencing discordance between guidelines and real-world practice regarding direct oral anticoagulant drug (DOAC) doses. This systematic review summarizes and evaluate DOACs use in real-world practice. METHODS: This review was performed following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines searching PubMed (MEDLINE) and Medscape databases. RESULTS: Data from 75 studies showed that most of the patients treated with DOACs for stroke prevention in atrial fibrillation received doses in accordance to the guidelines. However, a significant number of patients received off-label doses (25-50% in most of the studies evaluated). DOAC overdosing was associated with increased all-cause mortality and worse bleeding events while underdosing was associated with increased cardiovascular hospitalization and, particularly for apixaban, with a nearly 5-fold increased risk of stroke. CONCLUSION: Patients prescribed with off-label DOAC doses did not receive the full benefit of anticoagulation and presented an increased risk of stroke, bleeding and/or adverse effects.

Pre-Clinical Assessment of the Nose-to-Brain Delivery of Zonisamide After Intranasal Administration.

PURPOSE: Zonisamide clinical indications are expanding beyond the classic treatment of epileptic seizures to Parkinson's disease and other neurodegenerative diseases. However, the systemic safety profile of zonisamide may compromise its use as a first-line drug in any clinical condition. Since zonisamide is marketed as oral formulations, the present study aimed at exploring the potential of the intranasal route to centrally administer zonisamide, evaluating the systemic bioavailability of zonisamide and comparing its brain, lung and kidney pharmacokinetics after intranasal, oral and intravenous administrations. METHODS: In vitro cell studies demonstrated that zonisamide and proposed thermoreversible gels did not affect the viability of RPMI 2650 or Calu-3 cells. Thereafter, male CD-1 mice were randomly administered with zonisamide by oral (80 mg/kg), intranasal or intravenous (16.7 mg/kg) route. At predefined time points, animals were sacrificed and plasma and tissues were collected to quantify zonisamide and describe its pharmacokinetics. RESULTS: Intranasal route revealed a low absolute bioavailability (54.95%) but the highest value of the ratio between the area under the curve (AUC) between brain and plasma, suggesting lower systemic adverse events and non-inferior effects in central nervous system comparatively to intravenous and oral routes. Furthermore, drug targeting efficiency and direct transport percentage into the brain were 149.54% and 33.13%, respectively, corroborating that a significant fraction of zonisamide suffers direct nose-to-brain transport. Lung and kidney exposures obtained after intranasal administration were lower than those observed after intravenous injection. CONCLUSIONS: This pre-clinical investigation demonstrates a direct nose-to-brain delivery of zonisamide, which may be a promising strategy for the treatment of central diseases.

Salting-out assisted liquid-liquid extraction method optimized by design of experiments for the simultaneous high-performance liquid chromatography analysis of perampanel and stiripentol in mouse matrices.

We report a high-performance liquid chromatography method development able to simultaneously determine perampanel and stiripentol, two third-generation antiepileptics whose therapeutic spectrum can potentially be extended, in several mouse matrices. A salting-out assisted liquid-liquid extraction optimized by a design of experiments approach was adopted for sample preparations. Isopropanol and magnesium sulfate were the extraction solvent and salting-out agent, respectively. Both drugs and internal standard (terbinafine) were separated using a LiChroCART® Purospher Star column (C18 , 55 × 4 mm; 3 µm) isocratically pumped with mobile phase [1% triethylamine in water (pH 2.5) and acetonitrile (53:47, v/v)] at 1 mL/min. Stiripentol and terbinafine were detected by fluorescence at 254/372 nm and perampanel at 275/430 nm. Good linearity was demonstrated for perampanel at 1-500 ng/mL range in brain, 2-2000 ng/mL in liver and 1-2000 ng/mL in plasma and kidney (r2  ≥ 0.9922), and for stiripentol between 10 and 2000 ng/mL in brain and 10 and 20 000 ng/mL in the remaining matrices (r2  ≥ 0.9917). Precision (CV ≤ 15%) and accuracy (bias ±15%) were also verified, with obtained recovery values consistent with those predicted by the experimental design. This method was applied in preliminary pharmacokinetic studies to quantify perampanel or stiripentol after oral administration to mice, showing to be a promising bioanalytical tool to support future nonclinical in vivo pharmacokinetic studies.

ABC transporters in drug-resistant epilepsy: mechanisms of upregulation and therapeutic approaches.

Drug-resistant epilepsy (DRE) affects approximately one third of epileptic patients. Among various theories that try to explain multidrug resistance, the transporter hypothesis is the most extensively studied. Accordingly, the overexpression of efflux transporters in the blood-brain barrier (BBB), mainly from the ATP binding cassette (ABC) superfamily, may be responsible for hampering the access of antiepileptic drugs into the brain. P-glycoprotein and other efflux transporters are known to be upregulated in endothelial cells, astrocytes and neurons of the neurovascular unit, a functional barrier critically involved in the brain penetration of drugs. Inflammation and oxidative stress involved in the pathophysiology of epilepsy together with uncontrolled recurrent seizures, drug-associated induction and genetic polymorphisms are among the possible causes of ABC transporters overexpression in DRE. The aforementioned pathological mechanisms will be herein discussed together with the multiple strategies to overcome the activity of efflux transporters in the BBB - from direct transporters inhibition to down-regulation of gene expression resorting to RNA interference (RNAi), or by targeting key modulators of inflammation and seizure-mediated signalling.

First-time oral administration of resveratrol-loaded layer-by-layer nanoparticles to rats - a pharmacokinetics study.

trans-Resveratrol (RSV) is a plant-derived polyphenol endowed with a broad spectrum of promising therapeutic activities. The applicability of RSV in vivo has, however, had limited success so far, largely due to its inefficient systemic delivery resulting from its low water solubility. Layer-by-Layer (LbL) nanotechnology constitutes an innovative formulation strategy to address this concern, and is based on the design of tunable onion-like multilayered nanoarchitectures on the surface of low solubility drug nanocores, such as RSV. The purpose of this study was the investigation of the bioavailability of an LbL nanoformulation composed of 5.5 bilayers of polyallylamine hydrochloride (PAH) and dextran sulfate (DS) (LbL NPs) by pharmacokinetic studies following oral dosing to Wistar rats (20 mg kg-1). The systemic exposure of LbL NPs was compared to the respective nanoformulation without LbL coatings (RSV nanocores) and the free RSV suspension. The results demonstrated that both LbL NPs and RSV nanocores significantly enhanced, respectively, 1.76-fold and 2.74-fold the systemic exposure of RSV compared to the free RSV suspension, emphasizing their biopharmaceutical advantage. Surprisingly, besides the modified drug release potential of the LbL NPs, these exhibited a slightly lower systemic exposure (0.36-fold) in comparison with non-LbL modified RSV nanocores. These results were justified only by the electrostatic interactions composition of the LbL shell composition, requiring further research towards the application of stronger interactions. For this study, due to the key role of the bioanalytical method in the in vivo data acquisition, a rapid, selective, and sensitive HPLC-DAD method has been successfully optimized and fully validated to confidently quantify RSV levels in the rat plasma matrix, together with the optimization of the sample preparation procedure. Moreover, the chemical stability of RSV was evaluated for 24 h in simulated gastric and intestinal fluids with enzymes. Overall, our findings suggest that LbL NPs should be given great attention, representing a potential drug delivery system for RSV in view of the application of RSV not solely as a supplement but also as a therapeutic drug.

Development and full validation of an HPLC methodology to quantify atorvastatin and curcumin after their intranasal co-delivery to mice.

There is increasing interest in atorvastatin and curcumin owing to their potential anticancer activity. A new, accurate and sensitive HPLC method was developed, for the first time, to simultaneously quantify atorvastatin and curcumin in mouse plasma and brain, liver, lung and spleen tissues following protein precipitation sample preparation. The chromatographic separation was achieved in 13 min on a C18 column, at 35°C, using a mobile phase composed of acetonitrile-methanol-2% (v/v) acetic acid (37.5:2.5:60, v/v/v) at a flow rate of 1.0 mL/min. The detection of analytes and internal standard was carried out at 247, 425 and 250 nm, respectively. According to international guidelines, the method was shown to be selective, with lower limits of quantification ranging from 10 to 500 ng/mL for curcumin, and from 100 to 600 ng/mL for atorvastatin, linear over a wide concentration range (r2 ≥ 0.9971) and with acceptable accuracy (bias ± 12.29%) and precision (coefficient of variation ≤13.15%). The analytes were reproducibly recovered at a percentage >81.10% and demonstrated to be stable under various experimental conditions in all biological matrices. This method can be easily applied to in vivo biodistribution studies related to the intranasal administration of atorvastatin and curcumin, separately or simultaneously.