Anna Giulia Balducci

Brain distribution of ribavirin after intranasal administration

Ribavirin has proved to be effective in vitro against several RNA viruses responsible for encephalitis in humans and animals. However, the in vivo efficacy towards the cerebral viral load seems to be limited by the blood-brain barrier. Since the nose-to-brain pathway has been indicated for delivering drugs to the brain, we investigated here the distribution of ribavirin in the central nervous system (CNS) after intranasal administration. We first tested in vitro ribavirin diffusion from an aqueous solution across a biological membrane, using Franz cells and rabbit nasal mucosa. About 35% of ribavirin permeated in 4 h across the mucosa, after reaching steady-state flux in less than 30 min. In the first in vivo experiment, ribavirin aqueous solution was administered intranasally to Sprague Dawley rats (10 mg/kg). Animals were sacrificed at 10, 20 or 30 min after administration to collect brain areas (cerebellum, olfactory bulb, cerebral cortex, basal ganglia and hippocampus) and biological fluids (cerebrospinal fluid and plasma). Ribavirin, quantified by LC-MS/MS spectrometry, was detected at each time point in all compartments with the highest concentration in olfactory bulb and decreasing in rostro-caudal direction. Two subsequent in vivo experiments compared the nasal route (ribavirin solution) with the intravenous one and the nasal administration of ribavirin solution with ribavirin powder (10 mg/kg). It was found that 20 min after administration, ribavirin concentration in olfactory bulb was similar after intravenous or nasal administration of the ribavirin solution, whereas the powder led to significantly higher levels. Ribavirin was also present in deeper compartments, such as basal ganglia and hippocampus. Even if the mechanisms involved in ribavirin nose-to-brain transport are not clear, these results suggest a rapid extracellular diffusive flux from the nasal epithelium to the olfactory bulb and different CNS areas.

Liposome sensing and monitoring by organic electrochemical transistors integrated in microfluidics

BACKGROUND Organic electrochemical transistors (OECTs), which are becoming more and more promising devices for applications in bioelectronics and nanomedicine, are proposed here as ideally suitable for sensing and real time monitoring of liposome-based structures. This is quite relevant since, currently, the techniques used to investigate liposomal structures, their stability in different environments as well as drug loading and delivery mechanisms, operate basically off-line and/or with pre-prepared sampling. METHODS OECTs, based on the PEDOT:PSS conductive polymer, have been employed as sensors of liposome-based nanoparticles in electrolyte solutions to assess sensitivity and monitoring capabilities based on ion-to-electron amplified transduction. RESULTS We demonstrate that OECTs are very efficient, reliable and sensitive devices for detecting liposome-based nanoparticles on a wide dynamic range down to 10(-5)mg/ml (with a lowest detection limit, assessed in real-time monitoring, of 10(-7)mg/ml), thus matching the needs of typical drug loading/drug delivery conditions. They are hence particularly well suited for real-time monitoring of liposomes in solution. Furthermore, OECTs are shown to sense and discriminate successive injection of different liposomes, so that they could be good candidates in quality-control assays or in the pharmaceutical industry. GENERAL SIGNIFICANCE Drug loading and delivery by liposome-based structures is a fast growing and very promising field that will strongly benefit from real-time, highly sensitive and low cost monitoring of their dynamics in different pharma and biomedical environments, with a particular reference to the pharmaceutical and production processes, where a major issue is monitoring and measuring the formation and concentration of liposomes and the relative drug load. The demonstrated ability to sense and monitor complex bio-structures, such as liposomes, paves the way for very promising developments in biosensing and nanomedicine. This article is part of a Special Issue entitled Organic Bioelectronics-Novel Applications in Biomedicine.

Spray dried amikacin powder for inhalation in cystic fibrosis patients: A quality by design approach for product construction

An amikacin product for convenient and compliant inhalation in cystic fibrosis patients was constructed by spray-drying in order to produce powders of pure drug having high respirability and flowability. An experimental design was applied as a statistical tool for the characterization of amikacin spray drying process, through the establishment of mathematical relationships between six Critical Quality Attributes (CQAs) of the finished product and five Critical Process Parameters (CPPs). The surface-active excipient, PEG-32 stearate, studied for particle engineering, in general did not benefit the CQAs of the spray dried powders for inhalation. The spray drying feed solution required the inclusion of 10% (v/v) ethanol in order to reach the desired aerodynamic performance of powders. All desirable function solutions indicated that the favourable concentration of amikacin in the feed solution had to be kept at 1% w/v level. It was found that when the feed rate of the sprayed solution was raised, an increase in the drying temperature to the maximum value (160 °C) was required to maintain good powder respirability. Finally, the increase in drying temperature always led to an evident increase in emitted dose (ED) without affecting the desirable fine particle dose (FPD) values. The application of the experimental design enabled us to obtain amikacin powders with both ED and FPD, well above the regulatory and scientific references. The finished product contained only the active ingredient, which keeps low the mass to inhale for dose requirement.

Effect of Flow Rate on In Vitro Aerodynamic Performance of NEXThaler® in Comparison with Diskus® and Turbohaler® Dry Powder Inhalers

Abstract Background: European and United States Pharmacopoeia compendial procedures for assessing the in vitro emitted dose and aerodynamic size distribution of a dry powder inhaler require that 4.0 L of air at a pressure drop of 4 kPa be drawn through the inhaler. However, the product performance should be investigated using conditions more representative of what is achievable by the patient population. This work compares the delivered dose and the drug deposition profile at different flow rates (30, 40, 60, and 90 L/min) of Foster NEXThaler® (beclomethasone dipropionate/formoterol fumarate), Seretide® Diskus® (fluticasone propionate/salmeterol xinafoate), and Symbicort® Turbohaler® (budesonide/formoterol fumarate). Methods: The delivered dose uniformity was tested using a dose unit sampling apparatus (DUSA) at inhalation volumes either 2.0 or 4.0 L and flow rates 30, 40, 60, or 90 L/min. The aerodynamic assessment was carried out using a Next Generation Impactor by discharging each inhaler at 30, 40, 60, or 90 L/min for a time sufficient to obtain an air volume of 4 L. Results: Foster® NEXThaler® and Seretide® Diskus® showed a consistent dose delivery for both the drugs included in the formulation, independently of the applied flow rate. Contrary, Symbicort® Turbohaler® showed a high decrease of the emitted dose for both budesonide and formoterol fumarate when the device was operated at airflow rate lower that 60 L/min. The aerosolizing performance of NEXThaler® and Diskus® was unaffected by the flow rate applied. Turbohaler® proved to be the inhaler most sensitive to changes in flow rate in terms of fine particle fraction (FPF) for both components. Among the combinations tested, Foster NEXThaler® was the only one capable to deliver around 50% of extra-fine particles relative to delivered dose. Conclusions: NEXThaler® and Diskus® were substantially unaffected by flow rate through the inhaler in terms of both delivered dose and fine particle mass.

Pure insulin highly respirable powders for inhalation.

The aim of the present research was to investigate the possibility to obtain by spray drying an insulin pulmonary powder respirable and stable at room temperature without the use of excipients. Several insulin spray-dried powders were prepared with or without the addition of excipients (mannitol, bovine serum albumin, aspartic acid) from water dispersions or from acidic aqueous solutions. Each formulation was characterized using laser diffraction, scanning electron microscopy and in vitro aerosol performance with a Turbospin DPI device. Stability was assessed by the quantification of impurities with a molecular mass greater than that of insulin (HMWP) and related proteins (A21+ORP). Insulin powders prepared without excipients from an acid solution showed a shrivelled, raisin-like shape of non-aggregated microparticles and a high respirability (FPF>65%). The optimal result with respect to respirability and stability was reached when the pH of the insulin acetic acid solution to spray dry was adjusted at pH 3.6 with ammonium hydroxide. The median volume diameter of the obtained powder was 4.04 μm, insulin content 95%, emitted dose of 89.5%, MMAD 1.79 μm and fine particle fraction of 83.6%. This powder was stable at room temperature over a period of eighteen months with respect to the content of A21+ORP. As far as the HMWP content was concerned, the powder complied with the specification limits for a period of five months. The insulin acetic powder opens up the possibility of a more effective pulmonary therapy less dependent on refrigerated storage.

Spray-dried amikacin sulphate powder for inhalation in cystic fibrosis patients: The role of ethanol in particle formation.

A Central Composite Design (CCD) was applied in order to identify positive combinations of the production parameters of amikacin sulphate spray-dried powders for inhalation, with the intent to expand the experimental space defined in a previous half-fractional factorial design. Three factors, namely drying temperature, feed rate and ethanol proportion, have been selected out of the initial five. In addition, the levels of these factors were increased from two to three and their effect on amikacin respirability was evaluated. In particular, focus was given on the role of ethanol presence on the formation of the microparticles for inhalation. The overall outcome of the CCD was that amikacin respirability was not substantially improved, as the optimum region coincided with areas already explored with the fractional factorial design. However, expanding the design space towards smaller ethanol levels, including its complete absence, revealed the crucial role of this solvent on the morphology of the produced particles. Peclet number and drug solubility in the spraying solution helped to understand the formation mechanism of these amikacin sulphate spray-dried particles.

Differences in physical chemistry and dissolution rate of solid particle aerosols from solution pressurised inhalers.

Solution composition alters the dynamics of beclomethasone diproprionate (BDP) particle formation from droplets emitted by pressurised metered dose inhalers (pMDIs). The hypothesis that differences in inhaler solutions result in different solid particle physical chemistry was tested using a suite of complementary calorimetric techniques. The atomisation of BDP-ethanol solutions from commercial HFA-pMDI produced aerodynamically-equivalent solid particle aerosols. However, differences in particle physico-chemistry (morphology and solvate/clathrate formation) were detected by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and supported by hot stage microscopy (HSM). Increasing the ethanol content of the formulation from 8 to 12% (w/w), which retards the evaporation of propellant and slows the increase in droplet surface viscosity, enhanced the likelihood of particles drying with a smooth surface. The dissolution rate of BDP from the 12% (w/w) ethanol formulation-derived particles (63% dissolved over 120 min) was reduced compared to the 8% (w/w) ethanol formulation-derived particles (86% dissolved over 120 min). The addition of 0.01% (w/w) formoterol fumarate or 1.3% (w/w) glycerol to the inhaler solution modified the particles and reduced the BDP dissolution rate further to 34% and 16% dissolved in 120 min, respectively. These data provide evidence that therapeutic aerosols from apparently similar inhaler products, including those with similar aerodynamic performance, may behave non-equivalently after deposition in the lungs.

Polymeric films loaded with vitamin E and aloe vera for topical application in the treatment of burn wounds.

Burns are serious traumas related to skin damage, causing extreme pain and possibly death. Natural drugs such as Aloe vera and vitamin E have been demonstrated to be beneficial in formulations for wound healing. The aim of this work is to develop and evaluate polymeric films containing Aloe vera and vitamin E to treat wounds caused by burns. Polymeric films containing different quantities of sodium alginate and polyvinyl alcohol (PVA) were characterized for their mechanical properties and drug release. The polymeric films, which were produced, were thin, flexible, resistant, and suitable for application on damaged skin, such as in burn wounds. Around 30% of vitamin E acetate was released from the polymeric films within 12 hours. The in vivo experiments with tape stripping indicated an effective accumulation in the stratum corneum when compared to a commercial cream containing the same quantity of vitamin E acetate. Vitamin E acetate was found in higher quantities in the deep layers of the stratum corneum when the film formulation was applied. The results obtained show that the bioadhesive films containing vitamin E acetate and Aloe vera could be an innovative therapeutic system for the treatment of burns.

Layered lipid microcapsules for mesalazine delayed-release in children

The goal was to make available a delayed-release dosage form of mesalazine to be dispersed in water to facilitate swallowing in adults and children. Mesalazine microparticles containing carnauba wax were prepared by spray-congealing technique. A second step of spray-congealing of carnauba microparticles dispersed in liquefied stearic acid gave rise to mesalazine lipid microcapsules in which several carnauba microparticles remained embedded as cores in a reservoir structure. In order to favor their water dispersion, the lipid microcapsules were dry coated by tumbling them with different ratios of mannitol/lecithin microparticles prepared by spray-drying. Release rate measurements showed a delayed-release behavior, in particular a pH-dependence with less than 10% of drug released in acidic medium and complete release in phosphate buffer pH 7.4 in 4-5h. The layering with hydrophilic excipient microparticles allowed manufacturing of a pH-dependent dosage form suitable for extemporaneous oral use in adults and children.

Hyaluronate nanoparticles included in polymer films for the prolonged release of vitamin E for the management of skin wounds

Lecithin and hyaluronic acid were used for the preparation of polysaccharide decorated nanoparticles loaded with vitamin E using the cationic lipid dioctadecyldimethylammonium bromide (DODMA). Nanoparticles showed mean particle size in the range 130-350 nm and narrow size distribution. Vitamin E encapsulation efficiency was higher than 99%. These nanoparticles were incorporated in polymeric films containing Aloe vera extract, hyaluronic acid, sodium alginate, polyethyleneoxide (PEO) and polyvinylalcohol (PVA) as an innovative treatment in skin wounds. Films were thin, flexible, resistant and suitable for application on burn wounds. Additionally, in vitro occlusion study highlighted the dependence of the occlusive effect on the presence of nanoparticles. The results obtained show that the bioadhesive films containing vitamin E acetate and Aloe vera could be an innovative therapeutic system for the treatment of skin wounds, such as burns. The controlled release of the vitamin along with a reduction in water loss through damaged skin provided by the nanoparticle-loaded polymer film are considered important features for an improvement in wound healing and skin regeneration.