Ivana Aleksic

Molecular and immunological characterization of Mus a 5 allergen from banana fruit

SCOPE Banana fruit has become an important cause of fruit allergy in the recent years. Among the five registered IUIS allergens, Mus a 1 and Mus a 2 have been characterized in detail. In this study, molecular characterization and evaluation of the allergenic properties of β-1,3-glucanase from banana (Musa acuminata), denoted as Mus a 5, were performed. METHODS AND RESULTS The gene of Mus a 5 was cloned and sequenced. The obtained cDNA revealed a novel Mus a 5 isoform with an open reading frame encoding a protein of 340 amino acids comprising a putative signal peptide of 28 amino acid residues. By MALDI-TOF analysis Mus a 5 isolated from banana fruit revealed a molecular mass of 33451±67 Da. Two Mus a 5 isoforms (pI 7.7 and 8.0) were detected by 2D immunoblot with an identical N-terminal sequence. By mass fingerprint, 76 and 83% of the primary structure was confirmed for the two mature Mus a 5 isoforms, respectively. IgE reactivity to Mus a 5 was found in 74% of patients sensitized to banana fruit. Upregulation of basophil activation markers CD63 and CD203c was achieved with Mus a 5 in a concentration-dependent manner. CONCLUSION Mus a 5 is a functional allergen and a candidate for the component-resolved allergy diagnosis of banana allergy.

In silico modeling of in situ fluidized bed melt granulation.

Fluidized bed melt granulation has recently been recognized as a promising technique with numerous advantages over conventional granulation techniques. The aim of this study was to evaluate the possibility of using response surface methodology and artificial neural networks for optimizing in situ fluidized bed melt granulation and to compare them with regard to modeling ability and predictability. The experiments were organized in line with the Box-Behnken design. The influence of binder content, binder particle size, and granulation time on granule properties was evaluated. In addition to the response surface analysis, a multilayer perceptron neural network was applied for data modeling. It was found that in situ fluidized bed melt granulation can be used for production of spherical granules with good flowability. Binder particle size had the most pronounced influence on granule size and shape, suggesting the importance of this parameter in achieving desired granule properties. It was found that binder content can be a critical factor for the width of granule size distribution and yield when immersion and layering is the dominant agglomeration mechanism. The results obtained indicate that both in silico techniques can be useful tools in defining the design space and optimization of in situ fluidized bed melt granulation.

Active actinidin retains function upon gastro‐intestinal digestion and is more thermostable than the E‐64‐inhibited counterpart

BACKGROUND Actinidin is a cysteine protease and major allergen from kiwi fruit. When purified under specific native conditions, actinidin preparations from fresh kiwi fruit contain both an active and inactive form of this enzyme. In this study, biochemical and immunological properties upon simulated gastro-intestinal digestion, as well as thermal stability, were investigated for both active and E-64-inhibited actinidin. RESULTS Active actinidin retained its primary structure and proteolytic activity after 2 h of simulated gastric digestion, followed by 2 h of intestinal digestion, as assessed by SDS-PAGE, zymography and mass spectroscopy. Immunological reactivity of active actinidin was also preserved, as tested by immunoelectrophoresis. The E-64 inhibited actinidin was fully degraded after 1 h of pepsin treatment. Differential scanning calorimetry showed that active actinidin has one transition maximum temperature (Tm ) at 73.9°C, whereas in the E-64-actinidin complex the two actinidin domains unfolded independently, with the first domain having a Tm value of only 61°C. CONCLUSION Active actinidin is capable of reaching the intestinal mucosa in a proteolytically active and immunogenic state. Inhibitor binding induces changes in the actinidin molecule that go beyond inhibition of proteolytic activity, also influencing the digestion stability and Tm values of actinidin, features important in the characterisation of food allergens.

An investigation into the usefulness of different empirical modeling techniques for better control of spray-on fluidized bed melt granulation.

Melt granulation in fluid bed processors is an emerging technique, but literature data regarding the modeling of this granulation method are lacking. In the present study different techniques (response surface analysis, multilayer perceptron neural network, and partial least squares method) were applied for modeling of spray-on fluidized bed melt granulation. Experiments were organized in line with central composite design. The effect of binder content and spray air pressure on granule properties was evaluated. The results obtained indicate that binder content can be identified as a critical factor controlling the granule size and size distribution. It was found that agglomeration mechanism involved, i.e., granule shape, can be greatly influenced by binder properties. The spray air pressure was identified as critical process parameter affecting granule flowability. The results presented indicate that application of in silico tools enables enhanced understanding and better control of novel pharmaceutical processes, such as melt granulation in fluidized bed. The artificial neural networks and partial least squares method were found to be superior to response surface methodology in prediction of granule properties. According to the results obtained, application of more advanced empirical modeling techniques complementary to design of experiments can be a suitable approach in defining the design space and optimization of spray-on fluidized bed melt granulation.

In vitro and in vivo investigation of taste-masking effectiveness of Eudragit E PO as drug particle coating agent in orally disintegrating tablets

Abstract Context: Considering that bitter taste of drugs incorporated in orally disintegrating tablets (ODTs) can be the main reason for avoiding drug therapy, it is of the utmost importance to achieve successful taste-masking. The evaluation of taste-masking effectiveness is still a major challenge. Objective: The objective of this study was to mask bitter taste of the selected model drugs by drug particle coating with Eudragit® E PO, as well as to evaluate taste-masking effectiveness of prepared ODTs using compendial dissolution testing, dissolution in the small-volume shake-flask assembly and trained human taste panel. Materials and methods: Model drugs were coated in fluidized bed. Disintequik™ ODT was used as a novel co-processed excipient for ODT preparation. Selected formulations were investigated in vitro and in vivo using techniques for taste-masking assessment. Results and discussion: Significantly slower drug dissolution was observed from tablets with coated drug particles during the first 3 min of investigation. Results of in vivo taste-masking assessment demonstrated significant improvement in drug bitterness suppression in formulations with coated drug. Strong correlation between the results of drug dissolution in the small-volume shake-flask assembly and in vivo evaluation data was established (R ≥ 0.970). Conclusion: Drug particle coating with Eudragit® E PO can be a suitable approach for bitter taste-masking. Strong correlation between in vivo and in vitro results implicate that small-volume dissolution method may be used as surrogate for human panel taste-masking assessment, in the case of physical taste-masking approach application.

Copper(II) complexes with aromatic nitrogen-containing heterocycles as effective inhibitors of quorum sensing activity in Pseudomonas aeruginosa

Five copper(II) complexes 1–5 with aromatic nitrogen-containing heterocycles, pyrimidine (pm, 1), pyrazine (pz, 2), quinazoline (qz, 3 and 4) and phthalazine (phtz, 5) have been synthesized and structurally characterized by spectroscopic and single-crystal X-ray diffraction techniques. The crystallographic results show that, dependent on the ligand structure, complexes 1–5 are of different nuclearity. The antimicrobial efficiency of complexes 1–5 has been evaluated against three clinically relevant microorganisms and none of the complexes showed significant growth inhibiting activity, with values of minimum inhibitory concentrations (MIC) in the mM range. Since in many bacteria, pathogenicity and virulence are regulated by intercellular communication processes, quorum sensing (QS), the effect of the copper(II) complexes on bacterial QS has also been examined. The results indicate that the investigated complexes inhibit violacein production in Chromobacterium violaceum CV026, suggesting an anti-QS activity. In order to differentiate, which of the QS pathways was affected by the copper(II) complexes, three biosensor strains were used: the PAO1 ΔrhlIpKD-rhlA and the PA14-R3ΔlasIPrsaI lux strain to directly measure the levels of C4-HSL (N-butanoyl-homoserine lactone) and 3OC12-HSL (N-3-oxo-dodecanoyl-homoserine lactone), respectively, and PAO1 ΔpqsA mini-CTX luxPpqsA for the detection of AHQs (2-alkyl-4-quinolones). Complexes 1–5 were shown to be efficient inhibitors of biofilm formation of the human opportunistic pathogen Pseudomonas aeruginosa PAO1, with the qz-containing complex 3 being the most active. Finally, the most anti-QS-active complexes 1 and 3 showed synergistic activity against a multi-drug resistant clinical isolate of P. aeruginosa, when supplied in combination with the known antibiotics piperacillin and ceftazidime.

Long-Chain 4-Aminoquinolines as Quorum Sensing Inhibitors in Serratia marcescens and Pseudomonas aeruginosa

Antibiotic resistance has become a serious global threat to public health; therefore, improved strategies and structurally novel antimicrobials are urgently needed to combat infectious diseases. Here we report a new type of highly potent 4-aminoquinoline derivatives as quorum sensing inhibitors in Serratia marcescens and Pseudomonas aeruginosa, exhibiting weak bactericidal activities (minimum inhibitory concentration (MIC) > 400 μM). Through detailed structure-activity study, we have identified 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines (5 and 10) as biofilm formation inhibitors with 50% biofilm inhibition at 69 μM and 63 μM in S. marcescens and P. aeruginosa, respectively. These two compounds, 5 and 10, are the first quinoline derivatives with anti-biofilm formation activity reported in S. marcescens. Quantitative structure-activity relationship (QSAR) analysis identified structural descriptors such as Wiener indices, hyper-distance-path index (HDPI), mean topological charge (MTC), topological charge index (TCI), and log D(o/w)exp as the most influential in biofilm inhibition in this bacterial species. Derivative 10 is one of the most potent quinoline type inhibitors of pyocyanin production described so far (IC50 = 2.5 μM). While we have demonstrated that 5 and 10 act as Pseudomonas quinolone system (PQS) antagonists, the mechanism of inhibition of S. marcescens biofilm formation with these compounds remains open since signaling similar to P. aeruginosa PQS system has not yet been described in Serratia and activity of these compounds on acylhomoserine lactone (AHL) signaling has not been detected. Our data show that 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines present the promising scaffolds for developing antivirulence and anti-biofilm formation agents against multidrug-resistant bacterial species.

Anti-biofilm Properties of Bacterial Di-Rhamnolipids and Their Semi-Synthetic Amide Derivatives

A new strain, namely Lysinibacillus sp. BV152.1 was isolated from the rhizosphere of ground ivy (Glechoma hederacea L.) producing metabolites with potent ability to inhibit biofilm formation of an important human pathogens Pseudomonas aeruginosa PAO1, Staphylococcus aureus, and Serratia marcescens. Structural characterization revealed di-rhamnolipids mixture containing rhamnose (Rha)-Rha-C10-C10, Rha-Rha-C8-C10, and Rha-Rha-C10-C12 in the ratio 7:2:1 as the active principle. Purified di-rhamnolipids, as well as commercially available di-rhamnolipids (Rha-Rha-C10-C10, 93%) were used as the substrate for the chemical derivatization for the first time, yielding three semi-synthetic amide derivatives, benzyl-, piperidine-, and morpholine. A comparative study of the anti-biofilm, antibacterial and cytotoxic properties revealed that di-Rha from Lysinibacillus sp. BV152.1 were more potent in biofilm inhibition, both cell adhesion and biofilm maturation, than commercial di-rhamnolipids inhibiting 50% of P. aeruginosa PAO1 biofilm formation at 50 μg mL-1 and 75 μg mL-1, respectively. None of the di-rhamnolipids exhibited antimicrobial properties at concentrations of up to 500 μg mL-1. Amide derivatization improved inhibition of biofilm formation and dispersion activities of di-rhamnolipids from both sources, with morpholine derivative being the most active causing more than 80% biofilm inhibition at concentrations 100 μg mL-1. Semi-synthetic amide derivatives showed increased antibacterial activity against S. aureus, and also showed higher cytotoxicity. Therefore, described di-rhamnolipids are potent anti-biofilm agents and the described approach can be seen as viable approach in reaching new rhamnolipid based derivatives with tailored biological properties.

Anti-quorum sensing activity, toxicity in zebrafish (Danio rerio) embryos and phytochemical characterization of Trapa natans leaf extracts

ETHNOPHARMACOLOGICAL RELEVANCE Trapa natans L. (water chestnut or water caltrop) is a widespread aquatic plant, which has been cultivated for food and traditional medicine since ancient times. Pharmacological studies showed that water chestnut exhibits the wide range of biological activities, such as antimicrobial, antioxidative, analgesic, anti-inflammatory, as well as antiulcer. AIM OF THE STUDY Evaluation of anti-virulence potential and toxicity of T. natans methanol (TnM), acetone (TnA) and ethyl acetate (TnEA) leaf extracts. MATERIALS AND METHODS The anti-quorum sensing activity of Tn extracts was addressed by measuring their effects on biofilm formation, swarming motility and pyocyanin and elastase production in Pseudomonas aeruginosa. Specific P. aeruginosa biosensors were used to identify which of the signaling pathways were affected. The lethal and developmental toxicity of extracts were addressed in vivo using the zebrafish (Danio rerio) model system. The phenolic composition of T. natans leafs extracts was analyzed by a linear ion trap-OrbiTrap hybrid mass spectrometer (LTQ OrbiTrapMS) and UHPLC system configured with a diode array detector (DAD) hyphenated with the triple quadrupole mass spectrometer. RESULTS Subinhibitory concentrations of Tn leaf extracts (0.2 MIC) inhibited pyocyanin and elastase production up to 50% and 60%, respectively, and reduced swarming zones, comparing to non-treated P. aeruginosa. TnA inhibited biofilm formation by 15%, TnM showed a stimulatory effect on biofilm formation up to 20%, while TnEA showed no effect. The bioactive concentrations of TnM and TnA were not toxic in the zebrafish model system. Twenty-two phenolic compounds were tentatively identified in TnM, where thirteen of them were identified in T. natans for the first time. Tn extracts, as well as their major components, ellagic and ferulic acids, demonstrated the ability to interfere with P. aeruginosa Las and PQS signaling pathways. CONCLUSIONS This study demonstrates anti-virulence potential of Tn leaf extracts against medically important pathogen P. aeruginosa and confirms the ethnopharmacological application of this plant against microbial infections.

Assessing the risk of alcohol-induced dose dumping from sustained-release oral dosage forms: in vitro–in silico approach

Abstract Consumption of alcoholic beverages with sustained-release oral dosage forms may pose a risk to patients due to potential alcohol-induced dose dumping (ADD). Regulatory guidances recommend in vitro dissolution testing to identify the risk of ADD, but the question remains whether currently proposed test conditions can be considered biopredictive. The purpose of this study was to evaluate different dissolution setups to assess ADD, and the potential of combined in vitro–in silico approach to predict drug absorption after concomitant alcohol intake for hydrophilic and lipophilic sustained-release tablets containing ibuprofen or diclofenac sodium. According to the obtained results, the impact of ethanol was predominantly governed by the influence on matrix integrity, with the increase in drug solubility being less significant. Hydrophilic matrix tablets were less susceptible to ADD than lipophilic matrices, although the conclusion on formulation ethanol-vulnerability depended on the employed experimental conditions. In silico predictions indicated that the observed changes in drug dissolution would not result in plasma concentrations beyond therapeutic window, but sustained-release characteristics of the formulations might be lost. Overall, the study demonstrated that in vitro–in silico approach may provide insight into the effect of ADD on drug clinical performance, and serve as a tool for ADD risk assessment.