miRNA nanoencapsulation to regulate the programming of the blood-brain barrier permeability by hypoxia.

Central nervous system (CNS)-related diseases are difficult to treat as most therapeutic agents they cannot reach the brain tissue, mainly due to the blood-brain barrier (BBB), arguably the tightest barrier between the human body and cerebral parenchyma, which routinely excludes most xenobiotic therapeutics compounds. The BBB is a multicellular complex that structurally forms the neurovascular unit (NVU) and is organized by neuro-endothelial and glial cells. BBB breakdown and dysfunction from the cerebrovascular cells lead to leakages of systemic components from the blood into the CNS, contributing to neurological deficits. Understanding the molecular mechanisms that regulate BBB permeability and disruption is essential for establishing future therapeutic strategies to restore permeability and improve cerebrovascular health. MicroRNAs (miRNAs), a type of small non-coding RNAs, are emerging as an important regulator of BBB integrity by modulating gene expression by targeting mRNA transcripts. miRNAs is implicated in the development and progression of various illnesses. Conversely, nanoparticle carriers offer unprecedented opportunities for cell-specific controlled delivery of miRNAs for therapeutic purposes. In this sense, we present in this graphical review critical evidence in the regulation of cell junction expression mediated by miRNAs induced by hypoxia and for the use of nanoparticles for the delivery of miRNA-based therapeutics in the treatment of BBB permeability.

Formulation of Direct Compression Zidovudine Tablets to Correlate the SeDeM Diagram Expert System and the Rotary Press Simulator Styl'ONE Results.

The SeDeM diagram expert system has been applied to study Zidovudine and some excipients. From the obtained diagrams, a pharmaceutical formula has been designed. SeDeM diagram ascertains the critical parameters that are suitable for a direct compression. The formula is compressed using a rotary tablet press simulator which emulates rotary tablet press' compression profiles. From these compressions, we study the formula behavior under different industrial production conditions but saving a huge amount of material. The study is done at different compression forces and compression speeds and taking into account the influence of the pre-compression force. The differences observed between the compression profiles are hereby described. The results indicate that the formulation is able to be compressed adequately with the emulated compression profiles and no differences are observed between the final products. Therefore, we can assure that the SeDeM diagram expert system is accurate and robust. Moreover, its results are comparable with the compression results in a rotary tablet press, which has never been described in the pharmaceutical literature before. From the obtained results, it is possible to select the best rotary press to scale-up this formulation.

The Administration of Chitosan-Tripolyphosphate-DNA Nanoparticles to Express Exogenous SREBP1a Enhances Conversion of Dietary Carbohydrates into Lipids in the Liver of Sparus aurata.

In addition to being essential for the transcription of genes involved in cellular lipogenesis, increasing evidence associates sterol regulatory element binding proteins (SREBPs) with the transcriptional control of carbohydrate metabolism. The aim of this study was to assess the effect of overexpression SREBP1a, a potent activator of all SREBP-responsive genes, on the intermediary metabolism of Sparus aurata, a glucose-intolerant carnivorous fish. Administration of chitosan-tripolyphosphate nanoparticles complexed with a plasmid driving expression of the N-terminal transactivation domain of SREBP1a significantly increased SREBP1a mRNA and protein in the liver of S. aurata. Overexpression of SREBP1a enhanced the hepatic expression of key genes in glycolysis-gluconeogenesis (glucokinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), fatty acid synthesis (acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2), elongation (elongation of very long chain fatty acids protein 5) and desaturation (fatty acid desaturase 2) as well as reduced nicotinamide adenine dinucleotide phosphate production (glucose-6-phosphate 1-dehydrogenase) and cholesterol synthesis (3-hydroxy-3-methylglutaryl-coenzyme A reductase), leading to increased blood triglycerides and cholesterol levels. Beyond reporting the first study addressing in vivo effects of exogenous SREBP1a in a glucose-intolerant model, our findings support that SREBP1a overexpression caused multigenic effects that favoured hepatic glycolysis and lipogenesis and thus enabled protein sparing by improving dietary carbohydrate conversion into fatty acids and cholesterol.

Administration of chitosan-tripolyphosphate-DNA nanoparticles to knockdown glutamate dehydrogenase expression impairs transdeamination and gluconeogenesis in the liver.

Glutamate dehydrogenase (GDH) plays a major role in amino acid catabolism. To increase the current knowledge of GDH function, we analysed the effect of GDH silencing on liver intermediary metabolism from gilthead sea bream (Sparus aurata). Sequencing of GDH cDNA from S. aurata revealed high homology with its vertebrate orthologues and allowed us to design short hairpin RNAs (shRNAs) to knockdown GDH expression. Following validation of shRNA-dependent downregulation of S. aurata GDH in vitro, chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a selected shRNA (pCpG-sh2GDH) were produced to address the effect of GDH silencing on S. aurata liver metabolism. Seventy-two hours following intraperitoneal administration of chitosan-TPP-pCpG-sh2GDH, GDH mRNA levels and immunodetectable protein decreased in the liver, leading to reduced GDH activity in both oxidative and reductive reactions to about 53-55 % of control values. GDH silencing decreased glutamate, glutamine and aspartate aminotransferase activity, while increased 2-oxoglutarate content, 2-oxoglutarate dehydrogenase activity and 6-phosphofructo-1-kinase/fructose-1,6-bisphosphatase activity ratio. Our findings show for the first time that GDH silencing reduces transdeamination and gluconeogenesis in the liver, hindering the use of amino acids as gluconeogenic substrates and enabling protein sparing and metabolisation of dietary carbohydrates, which would reduce environmental impact and production costs of aquaculture.

Development and validation of a simple high-performance liquid chromatography analytical method for simultaneous determination of phytosterols, cholesterol and squalene in parenteral lipid emulsions.

A simple analytical method for simultaneous determination of phytosterols, cholesterol and squalene in lipid emulsions was developed owing to increased interest in their clinical effects. Method development was based on commonly used stationary (C18 , C8 and phenyl) and mobile phases (mixtures of acetonitrile, methanol and water) under isocratic conditions. Differences in stationary phases resulted in peak overlapping or coelution of different peaks. The best separation of all analyzed compounds was achieved on Zorbax Eclipse XDB C8 (150 × 4.6 mm, 5 µm; Agilent) and ACN-H2 O-MeOH, 80:19.5:0.5 (v/v/v). In order to achieve a shorter time of analysis, the method was further optimized and gradient separation was established. The optimized analytical method was validated and tested for routine use in lipid emulsion analyses.

Chitosan-Mediated shRNA Knockdown of Cytosolic Alanine Aminotransferase Improves Hepatic Carbohydrate Metabolism.

Alanine aminotransferase (ALT) catalyses a transamination reaction that links carbohydrate and amino acid metabolism. In this study, we examined the effect of silencing cytosolic ALT (cALT) expression on the hepatic metabolism in Sparus aurata. A number of siRNA and shRNA designed to down-regulate cALT expression were validated in HEK-293 cells transfected with plasmids expressing S. aurata cALT or mitochondrial ALT (mALT) isoforms: ALT silencing significantly decreased the expression levels of S. aurata mRNA cALT1 to 62% (siRNA) and 48% (shRNA) of the values observed in control cells. The effect of cALT silencing was analysed in the liver of S. aurata 72 h after intraperitoneal injection of chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a shRNA to down-regulate cALT expression (pCpG-si1sh1). In fish fed diets with different ratio of protein to carbohydrate and treated with chitosan-TPP-pCpG-si1sh1, cALT1 and cALT2 mRNA levels significantly decreased irrespective of the diet. Consistently, ALT activity decreased in liver of treated animals. In the liver of S. aurata treated with chitosan-TPP-pCpG-si1sh1 nanoparticles, down-regulation of cALT expression increased the activity of key enzymes in glycolysis (6-phosphofructo-1-kinase and pyruvate kinase) and protein metabolism (glutamate dehydrogenase). Besides showing for the first time that administration of chitosan-TPP-pCpG-si1sh1 nanoparticles silences hepatic cALT expression in vivo, our data support that down-regulation of cALT could improve the use of dietary carbohydrates to obtain energy and spare protein catabolism.

Development and Validation of a Stability Indicating RP-HPLC Method for Hydrocortisone Acetate Active Ingredient, Propyl Parahydroxybenzoate and Methyl Parahydroxybenzoate Preservatives, Butylhydroxyanisole Antioxidant, and Their Degradation Products in a Rectal Gel Formulation.

A stability indicating method was established through a stress study, wherein different methods of degradation (oxidation, hydrolysis, photolysis, and temperature) were studied simultaneously to determine the active ingredient hydrocortisone acetate, preservatives propyl parahydroxybenzoate, and methyl parahydroxybenzoate, antioxidant butylhydroxyanisole (BHA), and their degradation products in a semisolid dosage gel form. The proposed method was suitably validated using a Zorbax SB-Phenyl column and gradient elution. The mobile phase consisted of a mixture of methanol, acetonitrile, and water in different proportions according to a planned program at a flow rate of 1.5 mL/min. The diode array detector was set at 240 nm for the active substance and two preservatives, and 290 nm for BHA. The validation study was conducted according to International Conference on Harmonization guidelines for specificity, linearity, repeatability, precision, and accuracy. The method was used for QC of hydrocortisone acetate gel and for the stability studies with the aim of quantifying the active substance, preservatives, antioxidant, and degradation products. It has proved to be suitable as a fast and reliable method for QC.

Determination of stress-induced degradation products of cetirizine dihydrochloride by a stability-indicating RP-HPLC method.

BACKGROUND: A new, simple and accurate stability-indicating reverse phase high performance liquid chromatography method was developed and validated during the early stage of drug development of an oral lyophilizate dosage form of cetirizine dihydrochloride. METHODS: For RP-HPLC analysis it was used an Eclipse XDB C8 column 150 mm × 4.6 mm, 5 µm (Agilent columns, Barcelona, Spain) as the stationary phase with a mobile phase consisted of a mixture of 0.2 M K2HPO4 pH 7.00 and acetonitrile (65:35, v/v) at a flow rate of 1 mL min (-1). Detection was performed at 230 nm using diode array detector. The method was validated in accordance with ICH guidelines with respect to linearity, accuracy, precision, specificity, limit of detection and quantification. RESULTS: The method results in excellent separation between the drug substance and its stress-induced degradation products. The peak purity factor is >950 for the drug substance after all types of stress, which confirms the complete separation of the drug substance peak from its stress induced degradation products. Regression analysis showed r(2) > 0.999 for cetirizine dihydrochloride in the concentration range of 650 µg mL (-1) to 350 µg mL(-1) for drug substance assay and a r(2) > 0.999 in the concentration range of 0.25 µg mL(-1) to 5 µg mL(-1) for degradation products. The method presents a limit of detection of 0.056 µg mL (-1) and a limit of quantification of 0.25 µg mL(-1). The obtained results for precision and accuracy for drug substance and degradation products are within the specifications established for the validation of the method. CONCLUSIONS: The proposed stability-indicating method developed in the early phase of drug development proved to be a simple, sensitive, accurate, precise, reproducible and therefore useful for the following stages of the cetirizine dihydrochloride oral lyophilizate dosage form development.

New classification of directly compressible (DC) excipients in function of the SeDeM Diagarm Expert System.

As a methodology for characterizing substances with regard to its viability in direct compression, the SeDeM Diagram Expert System may be considered a new tool in terms of the number of parameters applied and its optimization. The paper is based on the experimental SeDeM characterization study of 51 directly compressible (DC) excipients. After selecting the parameters, and comparing the corresponding results, the choices available within the SeDeM Expert System could be expanded. Through applied variants, the maximum and optimal values of the DC diluent excipient were precisely defined and the mathematical limits of the parameters, functions and parametric indices that define the level of direct compressibility were established. These studies have allowed us to propose a new classification of excipients CD based on its rheological and compressibility capability, resulting in a periodic table of CD excipients. It has been determined that the best excipient for direct compression should have an index of good compression (IGC) of 8.832.

DNA delivery via cationic solid lipid nanoparticles (SLNs).

In recent years the use of solid lipid nanoparticles (SLNs) as transport systems for the delivery of drugs and biomolecules has become particularly important. The use of cationic SLNs developed by the technique of microemulsion, which are complexed with DNA in order to study their application as non-viral vectors in gene therapy, is reported. The nanoparticles are characterized by scanning electron microscopy and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM) and differential scanning calorimetry (DSC). Furthermore, the process of lyophilization of the samples and their stability was studied. The nanoparticles obtained presented a particle size of 340 nm with a positive surface charge of 44 mV and the capability of forming lipoplexes with DNA plasmids was stated.

Optimization of parameters of the SeDeM Diagram Expert System: Hausner index (IH) and relative humidity (%RH).

As a methodology for characterizing substances in relation to their viability in direct compression, the SeDeM Diagram Expert System may be considered an open system in terms of the number of parameters applied and the optimization of these parameters. With the experience acquired from applying the SeDeM Diagram, in this study, we propose optimizing the parameters corresponding to the Hausner index (IH) and relative humidity (%HR) in order to simplify the mathematical calculation, so that it provides reliable data that can be extrapolated. The proposed optimization does not involve a conceptual change in the parameters considered nor a significant change in the results obtained compared with the previous calculation methodology initially established for the SeDeM Diagram Expert System, which means that the conclusions obtained by applying this method are equivalent.

Application of the SeDeM Diagram and a new mathematical equation in the design of direct compression tablet formulation.

Application of the new SeDeM Method is proposed for the study of the galenic properties of excipients in terms of the applicability of direct-compression technology. Through experimental studies of the parameters of the SeDeM Method and their subsequent mathematical treatment and graphical expression (SeDeM Diagram), six different DC diluents were analysed to determine whether they were suitable for direct compression (DC). Based on the properties of these diluents, a mathematical equation was established to identify the best DC diluent and the optimum amount to be used when defining a suitable formula for direct compression, depending on the SeDeM properties of the active pharmaceutical ingredient (API) to be used. The results obtained confirm that the SeDeM Method is an appropriate system, effective tool for determining a viable formulation for tablets prepared by direct compression, and can thus be used as the basis for the relevant pharmaceutical development.

A new expert systems (SeDeM diagram) for control batch powder formulation and preformulation drug products.

The new SeDeM Method is proposed for testing the batch-to-batch reproducibility of the same active pharmaceutical ingredient (API) in powder form. The procedure describes the study of the galenic properties of substances in powder form in terms of the applicability of direct compression technology. Through experimental determination of the SeDeM Method parameters, and their subsequent mathematical treatment and graphical expression (SeDeM Diagram), three batches of the same API were analysed to determine whether it was suitable for direct compression. Batch-to-batch reproducibility of the results was verified. It was concluded that the SeDeM Method is suitable for testing batch-to-batch reproducibility of characteristics in powdered APIs substances. The results obtained confirm that the SeDeM Method is a useful, effective tool for drug-preformulation studies providing the pharmacotechnical data required when formulating a drug in tablet form. In addition, the results were effective for defining the most appropriate manufacturing technology.