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Background: Technetium-99 m-labelled macroaggregated human serum albumin ([99mTc]Tc-MAA) is commonly used for lung perfusion scintigraphy. The European Pharmacopoeia (Eu.Ph.) specifies thin-layer chromatography (TLC) as the only method to assess its radiochemical purity (RCP). Similarly, TLC is the sole method reported in the literature to evaluate the RCP of Gallium-68-labelled MAA [68 Ga]Ga-MAA, recently introduced for lung perfusion PET/CT imaging. Since [68 Ga]Ga-MAA is prepared from commercial kits originally designed for the preparation of [99mTc]Tc-MAA, it is essential to optimize and validate the preparation methods for [68 Ga]Ga-MAA. Results: We tested a novel, simplified method for the preparation of [68 Ga]Ga-MAA that does not require organic solvents, prewash or final purification steps to remove radioactive impurities. We assessed the final product using radio-TLC, radio-UV-HPLC, and radio SDS-PAGE. Overall, our quality control (QC) method successfully detected [68 Ga]Ga-MAA along with all potential impurities, including free Ga-68, [68 Ga]Ga-HSA, unlabeled HSA, which may occur during labelling process and HEPES residual, a non-toxic but non-human-approved contaminant, used as buffer solution. We then applied our QC system to [68 Ga]Ga-MAA prepared under different conditions (25°–40°–75°–95 °C), thus defining the optimal temperature for labelling. Scanning Electron Microscopy (SEM) analysis of the products obtained through our novel method confirmed that most [68 Ga]Ga-MAA particles preserved the morphological structure and size distribution of unlabeled MAA, with a particle diameter range of 25–50 μm, assuring diagnostic efficacy. Conclusions: We optimized a novel method to prepare [68 Ga]Ga-MAA through a QC system capable of monitoring all impurities of the final products. [ABSTRACT FROM AUTHOR]

Imiquimod (IMQ) has been successfully formulated to date mainly as semi-solid lipophilic formulations for topical application. In this study, we investigated the solubility of IMQ in solvents suitable for developing innovative formulations in the form of powder obtained, for instance, by spray drying; thus, water, ethanol, methanol, acetone, acetonitrile, and dimethyl sulfoxide were tested at different temperatures. Temperature variations, stirring intensity, and the contact time between IMQ and the solvent greatly affected the evaluation of IMQ equilibrium solubility. The attainment of the solid–liquid equilibrium requires 13 days starting from solid IMQ and 2 days from a cooled-down supersaturated IMQ solution. A correlation between IMQ solubility and the solubility parameters of solvents was not found. IMQ solutions in water, ethanol, methanol, acetonitrile, and dimethyl sulfoxide were neither ideal nor regular. The Scatchard–Hildebrand equation does not apply to IMQ solutions because of association phenomena due to intermolecular hydrogen bonds and/or π-stacking, as supported by the hyperchromic effect that was very pronounced in highly polar solvents, such as water, with the increase in temperature. Finally, IMQ solubility values measured in acetone cannot be considered reliable due to the reaction with the solvent, leading to the formation of new molecules. [ABSTRACT FROM AUTHOR]

The nebulization of alpha-1 antitrypsin (AAT) for its administration to the lung could be an interesting alternative to parenteral infusion for patients suffering from AAT genetic deficiency (AATD). In the case of protein therapeutics, the effect of the nebulization mode and rate on protein conformation and activity must be carefully considered. In this paper two types of nebulizers, i.e., a jet and a mesh vibrating system, were used to nebulize a commercial preparation of AAT for infusion and compared. The aerosolization performance, in terms of mass distribution, respirable fraction, and drug delivery efficiency, as well as the activity and aggregation state of AAT upon in vitro nebulization were investigated. The two nebulizers demonstrated equivalent aerosolization performances, but the mesh nebulizer provided a higher efficiency in the delivery of the dose. The activity of the protein was acceptably preserved by both nebulizers and no aggregation or changes in its conformation were identified. This suggests that nebulization of AAT represents a suitable administration strategy ready to be translated to the clinical practice for delivering the protein directly to the lungs in AATD patients, either as a support therapy to parenteral administration or for subjects with a precocious diagnosis, to prevent the onset of pulmonary symptoms. [ABSTRACT FROM AUTHOR]

This work illustrates the development of a dry inhalation powder of cyclosporine-A for the prevention of rejection after lung transplantation and for the treatment of COVID-19. The influence of excipients on the spray-dried powder's critical quality attributes was explored. The best-performing powder in terms of dissolution time and respirability was obtained starting from a concentration of ethanol of 45% (v/v) in the feedstock solution and 20% (w/w) of mannitol. This powder showed a faster dissolution profile (Weibull dissolution time of 59.5 min) than the poorly soluble raw material (169.0 min). The powder exhibited a fine particle fraction of 66.5% and an MMAD of 2.97 µm. The inhalable powder, when tested on A549 and THP-1, did not show cytotoxic effects up to a concentration of 10 µg/mL. Furthermore, the CsA inhalation powder showed efficiency in reducing IL-6 when tested on A549/THP-1 co-culture. A reduction in the replication of SARS-CoV-2 on Vero E6 cells was observed when the CsA powder was tested adopting the post-infection or simultaneous treatment. This formulation could represent a therapeutic strategy for the prevention of lung rejection, but is also a viable approach for the inhibition of SARS-CoV-2 replication and the COVID-19 pulmonary inflammatory process. [ABSTRACT FROM AUTHOR]

Rifaximin is a locally acting antibiotic practically insoluble in water. It presents several crystal phases characterized by different degrees of hydration. The aim of this work is to investigate the dissolution behaviour of rifaximin α, β, and amorphous forms in relation to their relative thermodynamic stability to contribute to clarifying possible solvent- or humidity-mediated conversion patterns. Kinetic and intrinsic solubility were investigated along with particle size distribution, specific surface area, and external morphology. The solution and moisture mediated conversion from metastable α and amorphous forms to stable β form were elucidated by coupling intrinsic dissolution test with chemometric analysis as well as by dynamic vapour sorption measurements. The dissolution behaviour of the α form stems mainly from the transition to β form that occurs upon exposition to relative humidity higher than 40%. The α form converted more rapidly than the amorphous form due to the smaller supersaturation ratio. It can be concluded that, due to its marked tendency to transform into β form, the dissolution test for the α form, even if conducted according to compendial procedures, needs to be accompanied by a panel of further tests that allow to uniquely identify the solid phase under investigation. [ABSTRACT FROM AUTHOR]

The growing demand for personalized medicine requires innovation in drug manufacturing to combine versatility with automation. Here, three-dimensional (3D) printing was explored for the production of chitosan (CH)/alginate (ALG)-based hydrogels intended as active dressings for wound healing. ALG hydrogels were loaded with 0.75% w/v silver sulfadiazine (SSD), selected as a drug model commonly used for the therapeutic treatment of infected burn wounds, and four different 3D CH/ALG architectures were designed to modulate the release of this active compound. CH/ALG constructs were characterized by their water content, elasticity and porosity. ALG hydrogels (Young's modulus 0.582 ± 0.019 Mpa) were statistically different in terms of elasticity compared to CH (Young's modulus 0.365 ± 0.015 Mpa) but very similar in terms of swelling properties (water content in ALG: 93.18 ± 0.88% and in CH: 92.76 ± 1.17%). In vitro SSD release tests were performed by using vertical diffusion Franz cells, and statistically significant different behaviors in terms of the amount and kinetics of drugs released were observed as a function of the construct. Moreover, strong antimicrobial potency (100% of growth inhibition) against Staphylococcus aureus and Pseudomonas aeruginosa was demonstrated depending on the type of construct, offering a proof of concept that 3D printing techniques could be efficiently applied to the production of hydrogels for controlled drug delivery. [ABSTRACT FROM AUTHOR]

Alpha-1 antitrypsin (AAT) deficiency is a genetic disorder associated with pulmonary emphysema and bronchiectasis. Its management currently consists of weekly infusions of plasma-purified human AAT, which poses several issues regarding plasma supplies, possible pathogen transmission, purification costs, and parenteral administration. Here, we investigated an alternative administration strategy for augmentation therapy by combining recombinant expression of AAT in bacteria and the production of a respirable powder by spray drying. The same formulation approach was then applied to plasma-derived AAT for comparison. Purified, active, and endotoxin-free recombinant AAT was produced at high yields and formulated using L-leucine and mannitol as excipients after identifying compromise conditions for protein activity and good aerodynamic performances. An oxygen-free atmosphere, both during formulation and powder storage, slowed down methionine-specific oxidation and AAT inactivation. This work is the first peer-reviewed report of AAT formulated as a dry powder, which could represent an alternative to current treatments. [ABSTRACT FROM AUTHOR]

Polysaccharide scaffolds have been successfully employed to reconstruct environments that sustain skin tissue regeneration after injuries. Three‐dimensional (3D) advanced additive manufacturing technologies allow creating scaffolds with controlled and reproducible macro‐ and micro‐structure that improve the quality of the restored tissue to favor spontaneous repair. However, when persistent inflammation occurs, the physiological tissue healing capacity is reduced, like in the presence of pathologies like diabetes, vascular diseases, chronic infection, and others. In these circumstances, the bioavailability of therapeutic adjuncts like the growth factors in addition to the standard treatments represents undoubtedly a promising strategy to accelerate the healing of skin lesions. Precisely designed polysaccharide scaffolds obtained by 3D printing represent a robust platform that can be further implemented with the controlled delivery of bioactive adjuncts. Human elastin‐like polypeptides (HELPs) are stimuli‐responsive biopolymers. Their structure allows the integration of domains endowed with biological functionality, making them attractive compounds to prepare composites with smart properties. In the present study, 3D‐printed alginate and chitosan scaffolds were combined with the HELP components. The HELP biopolymer was fused to the epidermal growth factor (EGF) as the bioactive domain. Different constructs were prepared and the stimuli‐responsive behavior as well as the biological activity were evaluated, suggesting that these smart bioactive composites are suitable to realize multifunctional dressings that sustain the local release of therapeutic adjuncts. [ABSTRACT FROM AUTHOR]

Due to the release of active pharmaceutical compounds in wastewater and their persistence in the environment, dangerous consequences can develop in the aquatic and terrestrial organisms. Chitosan/Ag/TiO2 3D printed scaffolds, at different Ag nanoparticle concentrations (10, 100, 1000 ppm) are investigated here as promising materials for photocatalytic degradation under the UV–Vis irradiation of pharmaceutical compounds in wastewater. As target drugs, amoxicillin, paracetamol and their 1:1 mix were selected. Ag nanoparticles increase the photocatalytic efficiency of the system based on titanium dioxide embedded in the chitosan scaffold: in the presence of Chitosan/Ag100/TiO2, the selected pharmaceuticals (PhCs), monitored by UV–Vis spectroscopy, are completely removed in about 2 h. The photodegradation products of the PhCs were identified by Liquid Chromatography–Mass Spectroscopy and assessed for their toxicological impact on six different bacterial strains: no antibacterial activity was found towards the tested strains. This new system based on Ag/TiO2 supported on 3D chitosan scaffolds may represent an effective strategy to reduce wastewater pollution by emerging contaminants. [ABSTRACT FROM AUTHOR]

The combination of a chitosan 3D-printed scaffold with a hydrogel matrix containing an elastin-like polypeptide functionalized with the epidermal growth factor (HEGF) was evaluated as a possible strategy to obtain a bioactive platform with stimuli-responsive properties. We designed a chitosan/HEGF hybrid scaffold and examined the physico-chemical properties and the in vitro behavior when in contact with simulated biological fluids. Primary human dermal fibroblasts (hDFs) were used to test the in vitro cytocompatibility. Overall, these data provide first insights into the integration of HEGF-based hydrogel with 3D-printed scaffolds, contributing towards the rational design of a new smart functional wound dressing. [ABSTRACT FROM AUTHOR]

Active dressings acting on multiple fronts are requested in the field of care for chronic skin ulcers in order to ameliorate patient compliance and tissue restoration. Currently, three-dimensional polymeric hydrogels are widely investigated; however, no prototypes aiming to control oxidative stress and bacterial proliferation in the wound bed have been developed up until now. The present work describes the formulation of a novel chitosan-based printable material containing α-tocopherol at stable dosages to obtain reproducible 3D scaffolds possessing antioxidant and antimicrobial activity without the use of organic solvents. Stability assays mimicking the manufacturing process and storage conditions reveal no significant drug loss. Chemico-physical characterizations including porosity and behavior after dehydration/hydration demonstrate that the dressings are highly porous, can be dehydrated up to 80%, and can recover more than 90% of water upon 1 h of rehydration. Elasticity determined by stress/strain tests was higher than human skin and was sufficiently resistant for potential clinical manipulation. Footage of fibroblasts in in vitro cultures demonstrated the biocompatibility of the constructs over 28 days. Finally, scaffolds loaded with α-tocopherol showed dose-dependent antioxidant activity (up to 80% in less than 1 h), while antimicrobial action versus multi-drug resistant strains of Pseudomonas aeruginosa and Staphilococcus aureus was assessed by inhibition rings obtained through the Kirby–Bauer technique. The proposed hydrogels can be useful as dressings for the treatment of chronically infected wounds. [ABSTRACT FROM AUTHOR]

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This study aimed to investigate the preservability and viability of the rabbit spermatozoa diluted in a new semen extender Formula®in comparison with Lepus®at 17 °C of storage. The main characteristic of the new extender formulation is the use of an enzymatic agent associated to a polysaccharide as energy source precursor, added with gentamycin. During eight trials, ejaculates from 70 bucks were collected and diluted at 1:10 ratio with both the extenders, after 24 h of storage the semen doses were used for the artificial insemination (AI). Aliquots of the semen doses for each trial were stored at 17 °C, the total and progressive motility were checked at 0, 4, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108 h of storage. A total of 1267 and 1525 does were inseminated, respectively with Formula®and Lepus®. During storage the mean total and progressive motility (77.23% and 72.854%, respectively) were significantly higher for Formula®(p < .01) and the progressive motility at almost 70% was maintained for at least 60 h vs the 24 h of storage for Lepus®with significant differences after 12 h of storage (p < .05). The new extender reported a higher pregnancy rate (p < .05) and an average of 9.25 rabbits born per litter vs 8.83 for the traditional extender (p < .05), while the mean of the newborn alive was 9.08 using Formula®vs 8.51 with Lepus®(p < .05). In conclusion, the use of Formula®is recommended for rabbit semen AI programmes. [ABSTRACT FROM AUTHOR]

RATIONALE: Drug development efforts involving therapeutic peptides or proteins strongly lead optimization of drug delivery, drug stability, solubility and functionality. The key feature of controlled drug delivery is the use of biocompatible polymers able to interact via non-covalent bonds with an active principle through multiple functional groups. Here amide hydrogen/deuterium exchange (HDX) mass spectrometry was employed to localize insulin dynamics induced by interactions with three natural polysaccharides, i.e. chitosan (CH), sodium alginate (ALG) and chondroitin sulfate (CS). METHODS: LTQ-Orbitap continuous-labelling mass spectra were collected by diluting insulin stock solution (10 mM in 0.1% formic acid) to a final concentration of 0.1 mM in D2O containing 1 mM deuterated ammonium acetate (final pH .6) (insulin:polysaccharide ratio 1:2, w/w). For peptide mapping, deuterated samples were quenched after 0.5, 30, 60, 120 minutes exchange by adding HCl (pH) and digested with pepsin before LC-MS/MS analysis. RESULTS: Differences in the insulin backbone dynamics in the presence of the three polysaccharides were highlighted by monitoring peptic peptides at different time points. No significant differences were observed in the presence of CH, whereas the negatively charged ALG and CS were able to induce significant conformational variations at the B-chain level resulting in more protection against H/D exchange. The A-chain interacted only with CS reducing the protein mobility on a long time scale (120 min). HDX data evidenced heterogeneous insulin dynamics in the presence of ALG and CS. CONCLUSIONS: The studies reported here demonstrated the capabilities of mass spectrometry techniques and HDX methods to obtain useful information toward the flexibility and the behavior of native insulin in the presence of natural polysaccharides, and could provide insights to study the behavior of pharmaceutical formulations. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this work was to evaluate the effect of two different dry powder inhalers, of the NGI induction port and Alberta throat and of the actual inspiratory profiles of asthmatic patients on in-vitro drug inhalation performances. Methods: The two devices considered were a reservoir multidose and a capsule-based inhaler. The formulation used to test the inhalers was a combination of formoterol fumarate and beclomethasone dipropionate. A breath simulator was used to mimic inhalatory patterns previously determined in vivo. A multivariate approach was adopted to estimate the significance of the effect of the investigated variables in the explored domain. Results: Breath simulator was a useful tool to mimic in vitro the in vivo inspiratory profiles of asthmatic patients. The type of throat coupled with the impactor did not affect the aerodynamic distribution of the investigated formulation. However, the type of inhaler and inspiratory profiles affected the respirable dose of drugs. Conclusions: The multivariate statistical approach demonstrated that the multidose inhaler, released efficiently a high fine particle mass independently from the inspiratory profiles adopted. Differently, the single dose capsule inhaler, showed a significant decrease of fine particle mass of both drugs when the device was activated using the minimum inspiratory volume (592 mL). [ABSTRACT FROM AUTHOR]

Release modules of amoxicillin and clarithromycin combined in a single dosage form designed to float in the gastric content and to sustain the intra-gastric concentrations of these two antibiotics used for the eradication ofHelicobacter pylorihave been studied. The modules having a disc shape with curved bases were formulated as hydrophilic matrices. Two modules of clarithromycin were assembled by sticking the concave base of one module to the concave base of the other, creating an internal void chamber. The final dosage form was a floating assembly of three modules of clarithromycin and two of amoxicillin in which the drug release mechanism did not interfere with the floatation mechanism. The assembled system showed immediatein vitrofloatation at pH 1.2, lasting 5 h. Thein vitroantibiotics release profiles from individual modules and assembled systems exhibited linear release rate during buoyancy for at least 8 h. The predicted antibiotic concentrations in the stomach maintained for long time levels significantly higher than the respective minimum inhibitory concentrations (MIC). In addition, anin vivoabsorption study performed on beagle dogs confirmed the slow release of clarithromycin and amoxicillin from the assembled system during the assembly’s permanence in the stomach for at least 4 h. [ABSTRACT FROM AUTHOR]

The advances in pharmaceutical development and drug discovery impose the availability of reliable high-throughput screening methods for the rapid evaluation of drug metabolism and pharmacokinetic (PK) in biological samples. Here, a desorption electrospray mass spectrometry (DESI-MS) method has been developed and validated for the PK profiling of esomeprazole and its metabolites (5-hydroxyomeprazole and omeprazole sulfone) in rat plasma. Rats were treated with an esomeprazole solution (2.5 mg/mL) for endovenous administration and the analyte levels were profiled over 2 h after liquid-liquid extraction from plasma. MS and tandem mass spectrometry (MS/MS) experiments were performed by using a DESI-LTQ-Orbitrap XL instrument and an on-spot fixed time analysis on PMMA surfaces. Validation was performed for the esomeprazole. The DESI-MS/MS method exhibited for the esomepazole excellent sensitivity (limit of detection (LOD)=60 ng/mL), linearity (0.2-20 µg/mL concentration range; y=23848(±361)X, n=15; r2=0.987) and precision (RSD<9%) by using an internal standard method. The PK results were discussed in terms of Area Under the Curve, Cmax and Tmax. Data reliability was demonstrated by comparison with a liquid chromatography-tandem mass spectrometry method (p>0.05). The data achieved demonstrated that the DESI-MS method is suitable for sensitive and fast profiling of a drug and its metabolites at the therapeutic concentration levels. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A novel, simple and reliable reversed-phase liquid chromatography (LC)-spectrophotometric UV stability-indicating method was developed and validated for the simultaneous assay of marbofloxacin, clotrimazole and dexamethasone acetate in the presence of their impurities and degradation products in a pharmaceutical formulation for veterinary use. A C18 (75 × 4.6 mm, 4 µm) column was used with an acetonitrile-ammonium acetate mixture as mobile phase delivered with gradient elution. A diode-array detection was used in the 200-400 nm range and the detection wavelength was set at 260 nm. Validation carried out on the pharmaceutical dosage form, according to Veterinary International Conference on Harmonization guidelines, demonstrated excellent specificity, linearity, precision, accuracy and robustness. Excellent specificity with respect to vehicle and degradation products obtained after forced degradation (i.e., oxidation, acid, alkaline and thermal degradation) was demonstrated. As for linearity, the LC-UV assay method is applicable in the 0.180-0.420 mg mL concentration range for marbofloxacin ( r = 0.99), 0.060-0.140 mg mL for dexamethasone acetate ( r = 0.97) and 0.600-1.400 mg mL for clotrimazole ( r = 0.98). Very good repeatability (RSD < 0.8 %) and inter-day precision (RSD < 2.5 %) were observed for all analytes. Accuracy was in the 93-104 %, 98-111 % and 99-108 % confidence interval (95 %) for marbofloxacin, dexamethasone acetate and clotrimazole, respectively. The variations (±20 %) of mobile phase flow rate and pH, and oven column temperature did not exhibit an impact on the analyte content accuracy, demonstrating the robustness of the method. The LC-UV method here developed and validated may be used routinely for quality control. [ABSTRACT FROM AUTHOR]

Hydrophobic materials, in particular hydrogenated vegetable oils, HVO, are extensively used as coating materials in food and pharmaceutical systems. Correct application of these coatings requires an evaluation of their behaviour as a function of various parameters such as melting temperature, solubility, concentration and/or pH. The purpose of this study was to assess the physico-chemical properties of an HVO in terms of composition, crystallisation, phase transition and polymorphism using a variety of analytical techniques, such as electrospray mass spectrometry (ESI-MS), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). High-resolution ESI-MS allowed establishment of the HVO main composition of long-chain triglycerides (average molecular weight 1183 Da). DSC results showed that thermal history determines the formation of at least two polymorphs of HVO, namely two different crystal forms, assigned as form α, melting point (m.p.) 48 °C, and form β′, m.p. 60 °C. A third polymorph, the more thermodynamically stable β-form, having a melting point at 62 °C, is obtained by solution-mediated re-crystallisation. Phase transformation paths were investigated by isothermal DSC experiments, which evidenced that the α-form is kinetically stable at temperatures lower than 25 °C. These data are of particular interest in practical applications such as spray freezing or pan coating where significant heat transfer phenomena are involved. [ABSTRACT FROM PUBLISHER]

RATIONALE The control of drug release involves gaining an understanding of the complex interaction networks among drug-excipients-matrix-biological fluids. Thus, novel analytical methods that will lead to a better understanding of these interaction networks are urgently required. METHODS Desorption electrospray ionization high-resolution mass spectrometry (DESI-HRMS) was used to evaluate the behaviour of four biocompatible polysaccharides (chondroitin sulfate, chitosan, sodium alginate and λ-carrageenan) in the release of atenolol (ATN) from drug tablets. An aqueous solution at three different pH values (pH 7.4, 4.5 and 1.2) was electrosprayed onto the tablets, allowing direct, fast, sensitive detection of atenolol as the protonated molecule in positive ion mode. Information about the desorption mechanism was obtained by analyzing the ATN [M+H]+ ion signal as a function of time. ATN-polymer interactions in the drug/polymer mixtures were also studied by Horizontal Attenuated Total Reflectance (HATR) Fourier transform infrared (FTIR) spectroscopy. RESULTS The DESI-MS results revealed statistically different ATN desorption trends as a function of the polysaccharide investigated and the pH of the desorbing solution. Different release kinetics were ascribed to the drug-polymer interactions, and to the diffusion process of the drug through the hydrated polymer mesh. In particular, the alginate and λ-carrageenan matrices were able to sustain drug release from the tablet even for a highly soluble drug. The HATR results confirmed the presence of ATN-polymer interactions that, depending on the polymer-drug-solvent combination used, might affect ATN diffusion. CONCLUSIONS These results suggest that DESI-MS has a potential role for the micro-environmental analysis of drug diffusion and surface distribution in polymeric matrices. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The aims of the study were to identify microbial flora in boar semen under field conditions in northern Italy, to investigate antibiotic resistance and sensitivity of isolated bacteria, and to evaluate elimination of bacteria after storage in two types of extenders added with different antibiotics (amikacin vs gentamicin). A total of 60 boars were collected in 13 pig farms. Bacteriological and mycological investigations were performed immediately on raw semen samples, then at 48 and 120 h of storage on semen diluted randomly in a new short-term modified extender (ME-S) or in a commercial one (CRONOSTM). Bacterial contamination was found in 63% of raw semen samples and different bacterial species were isolated: E.coli, Serratia marcescens, Staphylococcus epidermidis and aureus, Proteus spp., Streptococcus spp. and Pseudomonas aeruginosa. E. coli was the most isolated contaminant (53%); Pseudomonas aeruginosa was found only in one semen sample. The analysis of variance of factors affecting contamination levels was significant for the farm of origin (P<0.05) and not significant for the breed. Antibiotic resistance of these bacteria was assessed using different antibiotics. Significant differences (P<0.05) between observed and expected frequencies of bacterial isolates resistant or not to the antibiotics contained in the extenders were found. At 48 h of storage a reduction of aerobic contamination was found after ME-S dilution by 85.3% and after CRONOSTM by 63.8%. This paper proved the presence of pathogenic bacteria in semen. We thus believe it is highly advisable to perform periodic microbiological screening of boar semen in the swine industry to avoid the use of low sperm quality. [ABSTRACT FROM AUTHOR]

In this work we investigated the moving boundaries and the associated drug release kinetics in matrix tablets prepared with two complexes between λ-carrageenan and two solublemodel drugs, namely, diltiazemHCl and metoprolol tartrate aiming at clarifying the role played by drug/polymer interaction on the water uptake, swelling, drug dissolution, and drug release performance of the matrix. The two studied complexes released the drug with different mechanism indicating two different drug/polymer interaction strengths. The comparison between the drug release behaviour of the complexes and the relevant physical mixtures indicates that diltiazem gave rise to a less soluble andmore stable complex with carrageenan thanmetoprolol. The less stable metoprolol complex afforded an erodible matrix, whereas the stronger interaction between diltiazem and carrageenan resulted in a poorly soluble, slowly dissolving matrix. It was concluded that the different stability of the studied complexes affords two distinct drug delivery systems: in the case of MTP, the dissociation of the complex, as a consequence of the interaction with water, affords a classical soluble matrix type delivery system; in the case of DTZ, the dissolving/diffusing species is the complex itself because of the very strong interaction between the drug and the polymer. [ABSTRACT FROM AUTHOR]

Two simple and fast C18 and HILIC liquid chromatography–electrospray mass spectrometry methods for the determination of hyaluronic acid (HA) in a mucoadhesive chitosan-based formulation were developed and validated. The performances of both methods were compared in terms of validation parameters and matrix effect. A simple sample preparation method based on sulphuric acid-based degradation was optimized for the detection of HA fragments (i.e. m/z 380 2-mer, m/z 759 4-mer, m/z 1,138 8-mer and m/z 1,518 16-mer). By operating under selected ion-monitoring mode, excellent selectivity towards chitosan fragments was obtained. For validation, good linearity, detection limits (<4 μg mL −1) and precision (RSD % < 16 %) were generally obtained on matrix with both columns. However, HILIC column exhibited improved performances in terms of HA fragment separation and selectivity. By analyzing on the C18 column the chitosan-based formulation and sample extracts from pig mucosa treated with the formulation, matrix effects exhibited a dependence of signal suppression degree (ranging from 37 to 83 %) as a function of the HA fragment dimension. The HILIC column afforded instead a significantly reduced suppression degree (ranging from 1 to 16 %) and a better separation. These findings demonstrated the improved performances of the HILIC column with respect to conventional C18 mechanism for the analysis of HA fragments in complex matrices. [ABSTRACT FROM AUTHOR]

Artemisinin, a poorly water-soluble antimalarial drug, presents a low and erratic bioavailability upon oral administration. The aim of this work was to study an agglomerated powder dosage form for oral administration of artemisinin based on the artemisinin/β-cyclodextrin primary microparticles. These primary microparticles were prepared by spray-drying a water-methanol solution of artemisinin/β-cyclodextrin. β-Cyclodextrin in spray-dried microparticles increased artemisinin water apparent solubility approximately sixfold. The thermal analysis evidenced a reduction in the enthalpy value associated with drug melting, due to the decrease in drug crystallinity. The latter was also evidenced by powder X-ray diffraction analysis, while C-NMR analysis indicated the partial complexation with β-cyclodextrin. Agglomerates obtained by sieve vibration of spray-dried artemisinin/β-cyclodextrin primary microparticles exhibited free flowing and close packing properties compared with the non-flowing microparticulate powder. The in vitro dissolution rate determination of artemisinin from the agglomerates showed that in 10 min about 70% of drug was released from the agglomerates, whereas less than 10% of artemisinin was dissolved from raw material powder. Oral administration of agglomerates in rats yielded higher artemisinin plasma levels compared to those of pure drug. In the case of the agglomerated powder, a 3.2-fold increase in drug fraction absorbed was obtained. [ABSTRACT FROM AUTHOR]

The chemical composition of the essential oil of Citrus medica L. cv. Diamante peel obtained by hydrodistillation, cold-pressing and supercritical carbon dioxide extraction techniques was determined by GC/MS analysis. Forty-six components were fully characterised. Limonene and γ-terpinene were the major components of the oils obtained by hydrodistillation (HD) and cold-pressing (CP), while citropten was the major constituent in the oil obtained by supercritical carbon dioxide extraction (SFE). Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were evaluated. The essential oil obtained by hydrodistillation exerted the highest inhibitory activity against BChE (IC50 value of 154.6 µg mL-1) and AChE (IC50 value of 171.3 µg mL-1). Interestingly, the oil obtained by cold-pressing exhibited a selective inhibitory activity against AChE. The essential oils have also been evaluated for the inhibition of NO production in LPS induced RAW 264.7 macrophages. The oil obtained by hydrodistillation exerted a significant inhibition of NO production with an IC50 value of 17 µg mL-1 (IC50 of positive control 53 µg mL-1). [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the physico-chemical characteristics of sucralfate humid gel dried by microwaves, in relation to the residual water content. Differential scanning calorimetry (DSC) allowed for the determination of the water state in sucralfate samples. Fourier-transform infrared (FT-IR) spectroscopy was used to monitor the changes in sucralfate gel structure induced by the microwave drying. A boundary value of total water content for sucralfate gel samples was found at 42% (w/w). Below this value only bound water was present, whereas above this value, the increase in total water was due to free water. In the physical form of gel, the strength of the coordination between sulfate anions and the positively charged aluminum hydroxide was dependent on the residual water content. The study of the sedimentation behavior of water suspensions prepared with dried sucralfate allowed for the evaluation of the retention of gel properties. We found that the microwave drying process affected the sedimentation of sucralfate dried gel suspensions independent of the residual water content: when suspensions were prepared from sucralfate dried gel powders containing more than 42% (w/w) of residual water, the sedimentation ratio was higher than 0.9. The non-gel powder suspension showed a sedimentation ratio of 0.68 ± 0.02, whereas the sucralfate humid gel suspension did not sediment. [ABSTRACT FROM AUTHOR]

The surface morphology of α-lactose monohydrate particles was modified by a new wet-smoothing process performed in a high-shear mixer using solvents. Successive steps of wetting and drying of lactose powders during rolling in the mixer's cylindrical bowl were performed. Smoothed particles were tested for size distribution, flow, and packing. The wet-smoothing process flattened the surface and rounded the edges of lactose particles. In comparison with original lactose, an improvement of powder packing and flow properties was evidenced. When the process was performed in the presence of a ternary agent such as magnesium stearate, the smoothing was improved. The evolution of rugosity during the smoothing process was assessed through a fractal descriptor of SEM picture. Atomic force microscopy and surface area measurements quantified the surface rugosity. A very significant reduction of the rugosity, more remarkable in the presence of magnesium stearate, was measured. This new process of powder wet-smoothing allows the preparation of lactose particles with different degrees of smoothed surface for the control of flow and packing properties and particle-particle interactions. [ABSTRACT FROM AUTHOR]

This work was aimed at investigating the preparation ofβ-cyclodextrin-microcrystalline cellulose pellets by means of a high-shear mixer, both in the absence or in the presence of ibuprofen as model drug. Drug loading of pellets was accomplished by means of two alternative techniques: 1) solution layering or 2) powder layering. The prepared pellets were characterised in terms of size distribution, shape factor, friability and dissolution rate. The interaction between ibuprofen andβ-cyclodextrin was monitored by Differential Scanning Calorimetry(DSC). Micro Fourier Transform Infrared spectroscopy(MicroFTIR) was applied to determine the distribution of components within each pellet on a micro scale. Pellets with narrow size distribution and containing up to about 90% of BCD were prepared using water as binder. The process yield resulted around 84 and 63% for drug-free and medicate pellets respectively. Drug loaded pellets with favourable technological and biopharmaceutical characteristics can be obtained both by powder or solution layering techniques. The latter proved to be more suitable for producing pellets with high drug contents, reduced friability and high drug dissolution rates. [ABSTRACT FROM AUTHOR]

The aim of this paper was to study the effect of the granulate properties and tablet compression force on disintegrating force behavior in order to investigate the capability of the disintegrating force to characterize tablets that have the same composition but were manufactured in different conditions. Several tablets containing spironolactone in the external or internal granulated mixture of calcium carbonate and maize starch differing in particle size distribution, were prepared at 3 compression levels. The force developed by tablets during water uptake and disintegration was measured and plotted versus time. The curves obtained were analyzed by the Weibull equation in order to calculate the parameters characterizing the tablet disintegration kinetics. The disintegrating force time parameter, the maximum force developed, and the area under the curve were determined. In general, the reduction of time parameter value and/or the increase in maximum force developed corresponded to an acceleration in tablet disintegration. In addition, the area under the force curve increased in stronger tablets, monitoring in a sensitive way the tablet structural changes introduced by compression force. The results showed that the disintegrating force measurement can detect small changes in the structure of the tablet that cannot be discriminated by pharmacopoeia tests. The effect of manufacturing, in particular compression force, on tablet properties was quantified by the parameters of disintegrating force kinetics. [ABSTRACT FROM AUTHOR]

The topical photochemotherapy of dermatoses with psoralens (PUVA therapy) requires an adequate drug level at the target site (basal epidermis) at the time of UVA radiation. The aim of this work was to enhance 5-methoxypsoralen transport to the basal epidermis, with the goal to shorten the delay between drug application and UVA irradiation. 5-Methoxypsoralen transport through rabbit skin was studied in vitro from topical formulations (water solution, gel, and emulsion). The results obtained show that the use of the emulsion increased the flux through rabbit ear skin, even if partitioning was not favorable. Additionally, the time lag was sensibly reduced, compared with the gel and solution. Furthermore, drug accumulation in human skin in vitro was determined using the thin slicing technique. Human skin accumulation profile was significantly higher for the emulsion, compared with the gel, indicating that the delay between psoralen application and UVA irradiation can be shortened. [ABSTRACT FROM AUTHOR]

The aim of this work was to measure the disintegrating force concomitantly with tablet disintegration, in order to evaluate the disintegration propensity of tablets. Disintegration and dissolution were measured on tablets containing two poorly soluble drugs (diclofenac sodium or ketoprofen), including different percentages of two disintegrants (Explotab® or Ac-Di-Sol®). Because of the experimental setup, the disintegrating force measured was the result of the force generated by disintegrant swelling and dissipated by tablet disintegration. The disintegrating force versus time curves had shapes ranging from a skewed distribution curve to a bell-shaped curve, depending on slow or rapid disintegration of tablets, respectively. Interestingly, the shape of the resulting curves was very sensitive to the composition of the tablet. When the disintegrant in the formula was increased, the force–time curve approached the bell-like shape. The disintegration propensity of the tablet can be evaluated by the disintegrating force development during disintegration. The disintegration improvement of the formula can be predicted. The disintegrating force curve allows for the clear identification of the optimal percentage of disintegrant to be used. [ABSTRACT FROM AUTHOR]

Inhalation of Calcium Phosphate nanoparticles (CaPs) has recently unmasked the potential of this nanomedicine for a respiratory lung-to-heart drug delivery targeting the myocardial cells. In this work, we investigated the development of a novel highly respirable dry powder embedding crystalline CaPs. Mannitol was selected as water soluble matrix excipient for constructing respirable dry microparticles by spray drying technique. A Quality by Design approach was applied for understanding the effect of the feed composition and spraying feed rate on typical quality attributes of inhalation powders. The in vitro aerodynamic behaviour of powders was evaluated using a medium resistance device. The inner structure and morphology of generated microparticles were also studied. The 1:4 ratio of CaPs/mannitol led to the generation of hollow microparticles, with the best aerodynamic performance. After microparticle dissolution, the released nanoparticles kept their original size. [ABSTRACT FROM AUTHOR]

The delivery of a dexamethasone formulation directly into the lung appears as an appropriate strategy to strengthen the systemic administration, reducing the dosage in the treatment of lung severe inflammations. For this purpose, a hyaluronic acid-dexamethasone formulation was developed, affording an inhalable reconstituted nanosuspension suitable to be aerosolized. The physico-chemical and biopharmaceutical properties of the formulation were tested: size, stability, loading of the spray-dried dry powder, reconstitution capability upon redispersion in aqueous media. Detailed structural insights on nanoparticles after reconstitution were obtained by light and X-ray scattering techniques. (1) The size of the nanoparticles, around 200 nm, is in the proper range for a possible engulfment by macrophages. (2) Their structure is of the core-shell type, hosting dexamethasone nanocrystals inside and carrying hyaluronic acid chains on the surface. This specific structure allows for nanosuspension stability and provides nanoparticles with muco-inert properties. (3) The nanosuspension can be efficiently aerosolized, allowing for a high drug fraction potentially reaching the deep lung. Thus, this formulation represents a promising tool for the lung administration via nebulization directly in the pipe of ventilators, to be used as such or as adjunct therapy for severe lung inflammation. [ABSTRACT FROM AUTHOR]

The search for best performing carriers for dry powder inhalers is getting a great deal of interest to overcome the limitations posed by lactose. The aerosolization of adhesive mixtures between a carrier and a micronized drug is strongly influenced by the carrier solid-state properties. This work aimed at crystallizing kinetically stable D-mannitol polymorphs and at investigating their aerosolization performance when used in adhesive mixtures with two model drugs (salbutamol sulphate, SS, and budesonide, BUD) using a median and median/high resistance inhaler. A further goal was to assess in vitro the cytocompatibility of the produced polymer-doped mannitol polymorphs toward two lung epithelial cell lines. Kinetically stable (up to 12 months under accelerate conditions) α, and δ mannitol forms were crystallized in the presence of 2% w/w PVA and 1% w/w PVP respectively. These solid phases were compared with the β form and lactose as references. The solid-state properties of crystallized mannitol significantly affected aerosolization behavior, with the δ form affording the worst fine particle fraction with both the hydrophilic (9.3 and 6.5%) and the lipophilic (19.6 and 32%) model drugs, while α and β forms behaved in the same manner (11–13% for SS; 53–58% for BUD) and better than lactose (8 and 13% for SS; 26 and 39% for BUD). Recrystallized mannitol, but also PVA and PVP, proved to be safe excipients toward lung cell lines. We concluded that, also for mannitol, the physicochemical properties stemming from different crystal structures represent a tool for modulating carrier-drug interaction and, in turn, aerosolization performance. [ABSTRACT FROM AUTHOR]

Purpose. The aim of the work was to study iontophoretic transdermal administration of salmon calcitonin (sCt) in rabbits, with particular attention to drug reservoir composition. A dry sCt disc, to be dissolved on the application site, was used for preparing the reservoir for transdermal iontophoresis. As a reference drug reservoir, a pad wetted with drug solution was used. Methods. Experiments were done in rabbits depositing 100 IU of salmon calcitonin on skin and applying anodal iontophoresis. Serum calcium concentration was measured during iontophoresis, passive diffusion and after i.v. administration. Parameters such as pH value and reservoir type were examined. Results. Transdermal iontophoresis of sCt elicited a decrease in the serum calcium level, whereas, in the absence of electric current, no significant fall was measured. Using the reservoir prepared from drug solution, anodal iontophoresis at pH 4.2 was more effective than at pH 7.4, probably due to higher sCt net positive charge. Using the reservoir prepared from dry disc, similar kinetics and extent of drug effect were observed at both pH values. The reservoir prepared from solid drug deposit concentrated sCt next to the skin. Conclusions. Anodal iontophoresis for transdermal calcitonin administration shows therapeutical applicability. The type of reservoir is an important parameter affecting sCt transdermal iontophoresis. [ABSTRACT FROM AUTHOR]

Purpose. Delivery of nasal powders of granulated β-cyclodextrin by insufflation was studied in order to find the relationship between powder properties and delivery behavior. Methods. Three nasal powder formulations, prepared by granulating β-cyclodextrin with different binders, were delivered from a powder insufflation device, in which the dose to be emitted was loaded in a gelatin capsule. The delivery sequence of powder was recorded and characterized using an image analysis program. Results. Particle size was the main parameter affecting nasal powder delivery, both as to the amount of dose sprayed and the aspect of cloud produced. Between 50-150 µm of particle size a substantial change in delivery behavior of powders was observed. Powder of around 100 µm in size showed useful insufflation characteristics for nasal delivery. Bioavailability of nasal formulations of progesterone/β-cyclodextrin powders was discussed in term of delivery behavior. Conclusions. The formulation approaches for improving nasal delivery of powders require the use of size optimized carriers. Insufflation of powders over 50 µm can favour the particle deposition by impaction, whereas for powders below 50 µm, deposition by sedimentation is moved. β-cyclodextrin is a suitable carrier for achieving high systemic availability following nasal administration of powder formulations. [ABSTRACT FROM AUTHOR]

Here, a formulation of silver nanoparticles (AgNPs) and two natural polymers such as alginate (ALG) and nanocrystalline cellulose (CNC) was developed for the 3D printing of scaffolds with large surface area, improved mechanical resistance and sustained capabilities to promote antimicrobial and cytotoxic effects. Mechanical resistance, water content, morphological characterization and silver distribution of the scaffolds were provided. As for applications, a comparable antimicrobial potency against S. aureus and P. aeruginosa was demonstrated by in vitro tests as function of the AgNP concentration in the scaffold (Minimal Inhibitory Concentration value: 10 mg/mL). By reusing the 3D system the antimicrobial efficacy was demonstrated over at least three applications. The cytotoxicity effects caused by administration of AgNPs to hepatocellular carcinoma (HepG2) cell culture through ALG and ALG/CNC scaffold were discussed as a function of time and dose. Finally, the liquid chromatography-mass spectrometry (LC-MS) technique was used for targeted analysis of pro-apoptotic initiation and executioner caspases, anti-apoptotic and proliferative proteins and the hepatocyte growth factor, and provided insights about molecular mechanisms involved in cell death induction. [ABSTRACT FROM AUTHOR]

Physico-chemical properties of lactose are key factors in adhesive mixtures used as dry powder inhaler (DPI). Despite the abundant literature on this topic, the effect of the polymorphism and pseudo-polymorphism of lactose has been seldom investigated and discussed although often lactose used in DPI is subjected to unit operations, which may alter its solid-state properties. Here, we studied the aerosolization performance of salbutamol sulphate (SS) or budesonide (BUD) formulations by investigating the effect of lactose pseudopolymorphism in ternary (coarse lactose/fine lactose/drug) and binary (coarse lactose/drug) mixtures. An improvement of the aerosolization performance of SS formulations with the increase of the amount of fine micronized lactose up to 30% (fine particle fraction (FPF) = 57%) was observed. Micronized lactose contained hygroscopic anhydrous α-lactose, which converted to α-lactose monohydrate at ambient conditions. This implied that the positive effect of fines on the aerosolization performance decreased and eventually disappeared with the formulation aging. Positive effect on SS deposition was observed also with binary mixtures with anhydrous lactose, whereas the opposite occurred with budesonide-containing formulations. The collected data demonstrated the crucial role of the carrier crystal form on the positive effect of fines on the deposition. [ABSTRACT FROM AUTHOR]

Ucuùba fat is fat obtained from a plant found in South America, mainly in Amazonian Brazil. Due to its biocompatibility and bioactivity, Ucuùba fat was used for the production of ketoconazole-loaded nanostructured lipid carriers (NLC) in view of an application for the treatment of onychomycosis and other persistent fungal infections. The development and optimization of Ucuùba fat-based NLC were performed using a Box-Behnken design of experiments. The independent variables were surfactant concentration (% w/v), liquid lipids concentration (% w/v), solid lipids concentration (% w/v), while the outputs of interest were particle size, polydispersity index (PDI) and drug encapsulation efficiency (EE). Ucuùba fat-based NLC were produced and the process was optimized by the development of a predictive mathematical model. Applying the model, two formulations with pre-determined particle size, i.e., 30 and 85 nm, were produced for further evaluation. The optimized formulations were characterized and showed particle size in agreement to the predicted value, i.e., 33.6 nm and 74.6 nm, respectively. The optimized formulations were also characterized using multiple techniques in order to investigate the solid state of drug and excipients (DSC and XRD), particle morphology (TEM), drug release and interactions between the formulation components (FTIR). Furthermore, particle size, surface charge and drug loading efficiency of the formulations were studied during a one-month stability study and did not show evidence of significant modification. [ABSTRACT FROM AUTHOR]

Tuberculosis resistant cases have been estimated to grow every year. Besides Mycobacterium tuberculosis, other mycobacterial species are responsible for an increasing number of difficult-to-treat infections. To increase efficacy of pulmonary treatment of mycobacterial infections an inhalable antibiotic powder targeting infected alveolar macrophages (AMs) and including an efflux pump inhibitor was developed. Low molecular weight sodium hyaluronate sub-micron particles were efficiently loaded with rifampicin, isoniazid and verapamil, and transformed in highly respirable microparticles (mean volume diameter: 1 μm) by spray drying. These particles were able to regenerate their original size upon contact with aqueous environment with mechanical stirring or sonication. The in vitro drugs release profile from the powder was characterized by a slow release rate, favorable to maintain a high drug level inside AMs. In vitro antimicrobial activity and ex vivo macrophage infection assays employing susceptible and drug resistant strains were carried out. No significant differences were observed when the powder, which did not compromise the AMs viability after a five-day exposure, was compared to the same formulation without verapamil. However, both preparations achieved more than 80% reduction in bacterial viability irrespective of the drug resistance profile. This approach can be considered appropriate to treat mycobacterial respiratory infections, regardless the level of drug resistance. [ABSTRACT FROM AUTHOR]

A decisive step in cell-biomaterial interaction is represented by the adsorption of proteins at the interface, whose fine control may be useful to trigger proper cell response. To this purpose, we can selectively control protein adsorption on biomaterials by means of aptamers. Aptamers selected to recognize fibronectin dramatically enhance chitosan ability to promote cell proliferation and adhesion, but the underlying biological mechanism remains unknown. We supposed that aptamers contributed to ameliorate the adsorption of fibronectin in an advantageous geometrical conformation for cells, thus regulating their morphology by the proper activation of the integrin-mediated pathway. We investigated this possibility by culturing epithelial cells on chitosan enriched with increasing doses of aptamers in the presence or in the absence of cytoskeleton pharmacological inhibitors. Our results showed that aptamers control cell morphology in a dose dependent manner (p < 0.0001). Simultaneously, when the inhibition of actin polymerization was induced, the control of cell morphology was attenuated (p < 0.0001), while no differences were detected when cells contractility was challenged (p > 0.05). Altogether, our data provide evidence that aptamers contribute to control fibronectin adsorption on biomaterials by preserving its conformation and thus function. Furthermore, our work provides a new insight into a new way to accurately tailor material surface bioactivity. [ABSTRACT FROM AUTHOR]

3D biomaterial manufacturing strategies show an extraordinary driving force for the development of innovative therapies in the tissue engineering field. Here, the behaviour of 3D printed chitosan (CH)-based scaffolds was explored as a function of the post-printing gelation process. To this purpose, gel forming properties of different media were tested on their capability to retain 3D structure, water content, mechanical resistance and surface/internal porosity. Three different gelation media (i.e. KOH 1.5 M, Na2CO3 1.5 M, ammonia vapours) were selected and the 3D CH scaffolds were tested in terms of biocompatibility toward fibroblast as skin associated human cell line. [ABSTRACT FROM AUTHOR]