Stability assessment of extracts obtained from Arbutus unedo L. fruits in powder and solution systems using machine-learning methodologies.

Arbutus unedo L. (strawberry tree) has showed considerable content in phenolic compounds, especially flavan-3-ols (catechin, gallocatechin, among others). The interest of flavan-3-ols has increased due their bioactive actions, namely antioxidant and antimicrobial activities, and by association of their consumption to diverse health benefits including the prevention of obesity, cardiovascular diseases or cancer. These compounds, mainly catechin, have been showed potential for use as natural preservative in foodstuffs; however, their degradation is increased by pH and temperature of processing and storage, which can limit their use by food industry. To model the degradation kinetics of these compounds under different conditions of storage, three kinds of machine learning models were developed: i) random forest, ii) support vector machine and iii) artificial neural network. The selected models can be used to track the kinetics of the different compounds and properties under study without the prior knowledge requirement of the reaction system.

An 1H NMR study of the cytarabine degradation in clinical conditions to avoid drug waste, decrease therapy costs and improve patient compliance in acute leukemia.

Cytarabine, the 4-amino-1-(ß-D-arabinofuranosyl)-2(1H)-pyrimidinone, (ARA-C) is an antimetabolite cytidine analogue used worldwide as key drug in the management of leukaemia. As specified in the manufacturers' instructions, once the components-sterile water and cytarabine powder-are unpackaged and mixed, the solution begins to degrade after 6 hours at room temperature and 12 hours at 4°C. To evaluate how to avoid wasting the drug in short-term, low-dose treatment regimens, the reconstituted samples, stored at 25°C and 4°C, were analyzed every day of the test week by reversed-phase HPLC and high-field NMR spectroscopy. All the samples remained unchanged for the entire week, which corresponds to the time required to administer the entire commercial drug package during low-dose therapeutic regimens. The drug solution was stored in a glass container at 4°C in an ordinary freezer and drawn with sterile plastic syringes; during this period, no bacterial or fungal contamination was observed. Our findings show that an cytarabine solution prepared and stored in the original vials retains its efficacy and safety and can, therefore, be divided into small doses to be administered over more days, thus avoiding unnecessary expensive and harmful waste of the drug preparation. Moreover, patients who require daily administration of the drug could undergo the infusion at home without need to go to hospital. The stability of the aliquots would help decrease hospitalization costs.

Synthesis and in-vivo taste assessment of meloxicam pivalate.

Meloxicam (MX), a nonsteroidal anti-inflammatory drug, widely used to treat arthritis, has a very bitter taste. Chemical modification of the bitter functionality was achieved by synthesis of a prodrug, meloxicam pivalate (MXP). Taste improvement was evaluated using single bottle-test rat model. It was found that palatability of MXP solution improved significantly as compared to MX.

Short-time dynamics of lysozyme solutions with competing short-range attraction and long-range repulsion: Experiment and theory.

Recently, atypical static features of microstructural ordering in low-salinity lysozyme protein solutions have been extensively explored experimentally and explained theoretically based on a short-range attractive plus long-range repulsive (SALR) interaction potential. However, the protein dynamics and the relationship to the atypical SALR structure remain to be demonstrated. Here, the applicability of semi-analytic theoretical methods predicting diffusion properties and viscosity in isotropic particle suspensions to low-salinity lysozyme protein solutions is tested. Using the interaction potential parameters previously obtained from static structure factor measurements, our results of Monte Carlo simulations representing seven experimental lysoyzme samples indicate that they exist either in dispersed fluid or random percolated states. The self-consistent Zerah-Hansen scheme is used to describe the static structure factor, S(q), which is the input to our calculation schemes for the short-time hydrodynamic function, H(q), and the zero-frequency viscosity η. The schemes account for hydrodynamic interactions included on an approximate level. Theoretical predictions for H(q) as a function of the wavenumber q quantitatively agree with experimental results at small protein concentrations obtained using neutron spin echo measurements. At higher concentrations, qualitative agreement is preserved although the calculated hydrodynamic functions are overestimated. We attribute the differences for higher concentrations and lower temperatures to translational-rotational diffusion coupling induced by the shape and interaction anisotropy of particles and clusters, patchiness of the lysozyme particle surfaces, and the intra-cluster dynamics, features not included in our simple globular particle model. The theoretical results for the solution viscosity, η, are in qualitative agreement with our experimental data even at higher concentrations. We demonstrate that semi-quantitative predictions of diffusion properties and viscosity of solutions of globular proteins are possible given only the equilibrium structure factor of proteins. Furthermore, we explore the effects of changing the attraction strength on H(q) and η.

Interactions of hyaluronan with oppositely charged surfactants in very diluted solutions in water.

The phase behavior of aqueous systems containing hyaluronan, at concentrations between 2 and 100 mg/L, and oppositely charged surfactants was investigated. A fluorescence probe technique revealed the formation of micellar structures on the hyaluronan in homogeneous systems well below the surfactant standard, critical, micellar concentration. Moreover, regions of gel-phase separation were revealed. A detailed phase diagram was, thus, constructed in the very diluted region and the hyaluronan concentration was found to be the main parameter controlling the phase behavior, in contrast to the charge ratio. The stability of hyaluronan-surfactant aggregates in the homogeneous systems while in storage at 4 °C (up to three months), against dilution, salt addition and on heating-cooling (between 10 and 50 °C) was also investigated. The aggregates were stable while in storage or upon increasing and decreasing the temperature. The dilution of hyaluronan-surfactant complexes or the addition of 0.15 M NaCl led to their disintegration. Finally, systems prepared in a 0.15 M NaCl solution showed that interactions are suppressed and no aggregation below the standard critical micellar concentration was observed.

Albumin-Assisted Method Allows Assessment of Release of Hydrophobic Drugs From Nanocarriers.

Polymeric nanoparticles have been extensively studied as drug delivery vehicles both in vitro and in vivo for the last two decades. In vitro methods to assess drug release profiles usually utilize degradation of nanoparticles in aqueous medium, followed by the measurement of the concentration of the released drug. This method, however, is difficult to use for drugs that are poorly water soluble. In this study, a protocol for measuring drug release kinetic using albumin solution as the medium is described. Albumin is a major blood transport protein, which mediates transport of many lipid soluble compounds including fatty acids, hormones, and bilirubin. The use of a dialysis-based system utilizing albumin dialysate solution allows hydrophobic drug release from a diverse set of drug delivery modalities is demonstrated. The method using liposomes and PLGA nanoparticles as drug carriers, and two model hydrophobic drugs, 17ß-estradiol, and dexamethasone is validated.

A suspending-droplet mode paper-based microfluidic platform for low-cost, rapid, and convenient detection of lead(II) ions in liquid solution.

A paper-based microfluidic device based on unconventional principle was developed and used to detect lead ions through a two-step process including heated incubation and subsequent mixing. The device was made by generating a superhydrophobic pattern, which defines channel and reservoir barriers, on a water-impermeable paper substrate, followed by loading and drying the reagents in the defined reservoirs. Different from the conventional paper-based devices that are made of water-permeable paper, the as-prepared device holds water drops in discrete reservoirs, and the water drops will not move unless the device is titled along the direction of the predefined channels. In this way, the liquid samples applied onto the device are handled as individual drops and could be stored, transported, and mixed on demand. Different from the conventional paper-based devices that use capillary force to drive liquid, our new device uses wetting and gravity as driving force. We name this operation principle suspending-droplet mode paper-based device (SD-µPAD). The use of a Teflon contact-printing stamp makes the production of such devices rapid, cost efficient, and mass productive. Utilizing a G-quadruplex-based luminescence switch-on assay, we demonstrated rapid, convenient, highly sensitive, and low cost detection of lead(II) ions in water samples, using a custom made battery-powered portable device, and a smart phone as the detector.

Predicting Protein-Protein Interactions of Concentrated Antibody Solutions Using Dilute Solution Data and Coarse-Grained Molecular Models.

Protein-protein interactions for solutions of an IgG1 molecule were quantified using static light scattering (SLS) measurements from low to high protein concentrations (c2). SLS was used to determine second osmotic virial coefficients (B22) at low c2, and excess Rayleigh profiles (Rex/K vs. c2) and zero-q structure factors (Sq=0) as a function of c2 at higher c2 for a series of conditions (pH, sucrose concentration, and total ionic strength [TIS]). Repulsive (attractive) interactions were observed at low TIS (high TIS) for pH 5 and 6.5, with increasing repulsions when 5% w/w sucrose was also present. Previously developed and refined coarse-grained antibody models were used to fit model parameters from B22 versus TIS data. The resulting parameters from low-c2 conditions were used as the sole input to multiprotein Monte Carlo simulations to predict high-c2Rex/K and Sq=0 behavior up to 150 g/L. Experimental results at high-c2 conditions were quantitatively predicted by the simulations for the coarse-grained models that treated antibody molecules as either 6 or 12 (sub) domains, which preserved the basic shape of a monoclonal antibody. Finally, preferential accumulation of sucrose around the protein surface was identified via high-precision density measurements, which self-consistently explained the simulation and experimental SLS results.

An accurate coarse-grained model for chitosan polysaccharides in aqueous solution.

Computational models can provide detailed information about molecular conformations and interactions in solution, which is currently inaccessible by other means in many cases. Here we describe an efficient and precise coarse-grained model for long polysaccharides in aqueous solution at different physico-chemical conditions such as pH and ionic strength. The Model is carefully constructed based on all-atom simulations of small saccharides and metadynamics sampling of the dihedral angles in the glycosidic links, which represent the most flexible degrees of freedom of the polysaccharides. The model is validated against experimental data for Chitosan molecules in solution with various degree of deacetylation, and is shown to closely reproduce the available experimental data. For long polymers, subtle differences of the free energy maps of the glycosidic links are found to significantly affect the measurable polymer properties. Therefore, for titratable monomers the free energy maps of the corresponding links are updated according to the current charge of the monomers. We then characterize the microscopic and mesoscopic structural properties of large chitosan polysaccharides in solution for a wide range of solvent pH and ionic strength, and investigate the effect of polymer length and degree and pattern of deacetylation on the polymer properties.

Direct electron irradiation of DNA in a fully aqueous environment. Damage determination in combination with Monte Carlo simulations.

We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), was determined by gel electrophoresis. The median lethal dose of D1/2 = 1.7 ± 0.3 Gy was found to be much smaller as compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of the DSBs to SSBs was found to be 1 : 12 as compared to 1 : 88 found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, co-solutes) for an electron energy range which is difficult to probe by standard methods.

Preparation of polymer-blended quinine nanocomposite particles by spray drying and assessment of their instrumental bitterness-masking effect using a taste sensor.

CONTEXT: The development of taste-masking technologies for foods and drugs is essential because it would enable people to consume and receive healthy and therapeutic effect without distress. OBJECTIVE: In the current study, in order to develop a novel method to prepare nanocomposite particles (microparticles containing bitter nanoparticles) in only one step, by using spray drying, a two-solution mixing nozzle-equipped spray dryer that we previously reported was used. The nanocomposite particles with or without poorly water-soluble polymers prepared using our spray-drying technique were characterized. METHODS: (1) The organic solution containing quinine, a model of bitter compound and poorly water-soluble polymers and (2) sugar alcohol (mannitol) aqueous solution were separately flown in tubes and two solutions were spray dried through two-solution type spray nozzle to prepare polymer-blended quinine nanocomposite particles. Mean diameters of nanoparticles, taste-masking effect and dissolution rate of quinine were evaluated. RESULTS: The results of taste masking by taste sensor suggested that the polymer (Eudragit EPO, Eudragit S100 or Ethyl cellulose)-blended quinine nanocomposite particles exhibited marked masking of instrumental quinine bitterness compared with the quinine nanocomposite particles alone. Quinine nanocomposite formulations altered the quinine dissolution rate, indicating that they can control intestinal absorption of quinine. CONCLUSION: These results suggest that polymer-blended quinine composite particles prepared using our spray-drying technique are useful for masking bitter tastes in the field of food and pharmaceutical industry.

Rat Palatability Study for Taste Assessment of Caffeine Citrate Formulation Prepared via Hot-Melt Extrusion Technology.

Developing a pediatric oral formulation with an age-appropriate dosage form and taste masking of naturally bitter active pharmaceutical ingredients (APIs) are key challenges for formulation scientists. Several techniques are used for taste masking of bitter APIs to improve formulation palatability; however, not all the techniques are applicable to pediatric dosage forms because of the limitations on the kind and concentration of the excipients that can be used. Hot-melt extrusion (HME) technology is used successfully for taste masking of bitter APIs and overcomes some of the limitations of the existing taste-masking techniques. Likewise, analytical taste assessment is an important quality control parameter evaluated by several in vivo and in vitro methods, such as the human taste panel, electrophysiological methods, electronic sensor, and animal preference tests to aid in selecting a taste-masked formulation. However, the most appropriate in vivo method to assess the taste-masking efficacy of pediatric formulations remains unknown because it is not known to what extent the human taste panel/electronic tongue can predict the palatability in the pediatric patients. The purpose of this study was to develop taste-masked caffeine citrate extrudates via HME and to demonstrate the wide applicability of a single bottle-test rat model to record and compare the volume consumed of the taste-masked solutions to that of the pure API. Thus, this rat model can be considered as a low-cost alternative taste-assessment method to the most commonly used expensive human taste panel/electronic tongue method for pediatric formulations.

Homogenization Theory for the Prediction of Obstructed Solute Diffusivity in Macromolecular Solutions.

The study of diffusion in macromolecular solutions is important in many biomedical applications such as separations, drug delivery, and cell encapsulation, and key for many biological processes such as protein assembly and interstitial transport. Not surprisingly, multiple models for the a-priori prediction of diffusion in macromolecular environments have been proposed. However, most models include parameters that are not readily measurable, are specific to the polymer-solute-solvent system, or are fitted and do not have a physical meaning. Here, for the first time, we develop a homogenization theory framework for the prediction of effective solute diffusivity in macromolecular environments based on physical parameters that are easily measurable and not specific to the macromolecule-solute-solvent system. Homogenization theory is useful for situations where knowledge of fine-scale parameters is used to predict bulk system behavior. As a first approximation, we focus on a model where the solute is subjected to obstructed diffusion via stationary spherical obstacles. We find that the homogenization theory results agree well with computationally more expensive Monte Carlo simulations. Moreover, the homogenization theory agrees with effective diffusivities of a solute in dilute and semi-dilute polymer solutions measured using fluorescence correlation spectroscopy. Lastly, we provide a mathematical formula for the effective diffusivity in terms of a non-dimensional and easily measurable geometric system parameter.

Thermoseparating aqueous two-phase systems: Recent trends and mechanisms.

Having the benefits of being environmentally friendly, providing a mild environment for bioseparation, and scalability, aqueous two-phase systems (ATPSs) have increasingly caught the attention of industry and researchers for their application in the isolation and recovery of bioproducts. The limitations of conventional ATPSs give rise to the development of temperature-induced ATPSs that have distinctive thermoseparating properties and easy recyclability. This review starts with a brief introduction to thermoseparating ATPSs, including its history, unique characteristics and advantages, and lastly, key factors that influence partitioning. The underlying mechanism of temperature-induced ATPSs is covered together with a summary of recent applications. Thermoseparating ATPSs have been proven as a solution to the demand for economically favorable and environmentally friendly industrial-scale bioextraction and purification techniques.

Acceptance Probability (P a) Analysis for Process Validation Lifecycle Stages.

This paper introduces an innovative statistical approach towards understanding how variation impacts the acceptance criteria of quality attributes. Because of more complex stage-wise acceptance criteria, traditional process capability measures are inadequate for general application in the pharmaceutical industry. The probability of acceptance concept provides a clear measure, derived from specific acceptance criteria for each quality attribute. In line with the 2011 FDA Guidance, this approach systematically evaluates data and scientifically establishes evidence that a process is capable of consistently delivering quality product. The probability of acceptance provides a direct and readily understandable indication of product risk. As with traditional capability indices, the acceptance probability approach assumes that underlying data distributions are normal. The computational solutions for dosage uniformity and dissolution acceptance criteria are readily applicable. For dosage uniformity, the expected AV range may be determined using the s lo and s hi values along with the worst case estimates of the mean. This approach permits a risk-based assessment of future batch performance of the critical quality attributes. The concept is also readily applicable to sterile/non sterile liquid dose products. Quality attributes such as deliverable volume and assay per spray have stage-wise acceptance that can be converted into an acceptance probability. Accepted statistical guidelines indicate processes with C pk > 1.33 as performing well within statistical control and those with C pk < 1.0 as "incapable" (1). A C pk > 1.33 is associated with a centered process that will statistically produce less than 63 defective units per million. This is equivalent to an acceptance probability of >99.99%.

Thermodynamics of amyloid formation and the role of intersheet interactions.

The self-assembly of proteins into ß-sheet-rich amyloid fibrils has been observed to occur with sigmoidal kinetics, indicating that the system initially is trapped in a metastable state. Here, we use a minimal lattice-based model to explore the thermodynamic forces driving amyloid formation in a finite canonical (NVT) system. By means of generalized-ensemble Monte Carlo techniques and a semi-analytical method, the thermodynamic properties of this model are investigated for different sets of intersheet interaction parameters. When the interactions support lateral growth into multi-layered fibrillar structures, an evaporation/condensation transition is observed, between a supersaturated solution state and a thermodynamically distinct state where small and large fibril-like species exist in equilibrium. Intermediate-size aggregates are statistically suppressed. These properties do not hold if aggregate growth is one-dimensional.

Optimizing the affinity and specificity of ligand binding with the inclusion of solvation effect.

Solvation effect is an important factor for protein-ligand binding in aqueous water. Previous scoring function of protein-ligand interactions rarely incorporates the solvation model into the quantification of protein-ligand interactions, mainly due to the immense computational cost, especially in the structure-based virtual screening, and nontransferable application of independently optimized atomic solvation parameters. In order to overcome these barriers, we effectively combine knowledge-based atom-pair potentials and the atomic solvation energy of charge-independent implicit solvent model in the optimization of binding affinity and specificity. The resulting scoring functions with optimized atomic solvation parameters is named as specificity and affinity with solvation effect (SPA-SE). The performance of SPA-SE is evaluated and compared to 20 other scoring functions, as well as SPA. The comparative results show that SPA-SE outperforms all other scoring functions in binding affinity prediction and "native" pose identification. Our optimization validates that solvation effect is an important regulator to the stability and specificity of protein-ligand binding. The development strategy of SPA-SE sets an example for other scoring function to account for the solvation effect in biomolecular recognitions.

Enniatin-containing solutions for oromucosal use: Quality-by-design ex-vivo transmucosal risk assessment of composition variability.

Fusafungine, a mixture of the cyclic hexadepsipeptides enniatins, is currently on the market for the treatment of upper respiratory tract diseases because of its bacteriostatic and anti-inflammatory effects. In this study, a quality-by-design risk assessment was performed with two objectives: (i) investigate whether enniatins are able to permeate the mucosa and reach blood circulation, as the summary of product characteristics indicates this is not the case, and if so, to quantify their transmucosal kinetics and (ii) study the influence of excipient concentration variability on mucosal permeation. First, the concentration of the two main excipients isopropyl myristate and ethanol, known penetration enhancers, in several marketed samples was determined using GC-FID. Then, the transmucosal kinetics of the enniatins were quantitatively evaluated for different dose solutions, using porcine buccal mucosa in an ex-vivo in-vitro Franz diffusion cell set-up, with UHPLC-MS/MS bioanalytics. This study demonstrated that enniatins are capable of permeating the mucosa. However, no risk of a significant different transmucosal permeability with varying excipient concentrations was detected.

Development of low-cost rotational rheometer.

Liquids with non-Newtonian properties are presented in many engineering areas, as for example in membrane bioreactors where active sludge exhibits shear thinning properties. Therefore, the ability to determine the rheology's dependence on shear is important when optimising systems with such liquids. However, rheometers capable of determining the viscosity are often expensive and so a cheaper alternative is constructed with this exact capability. Using the principle of rotating rheometers, a low-cost rheometer was built to determine the rheology of Newtonian and non-Newtonian liquids. The general principles and background assumptions and the physics are described. The rheometer was calibrated by comparison with measurements conducted on a Brookfield viscometer for Newtonian liquids. For validation measurements on non-Newtonian liquids, xanthan gum solutions were made and compared with measurements on the Brookfield viscometer and with values from other sources. Furthermore, the effect of excluding the different shear rates in the system is discussed and good practice hereto is given.

Scale-up of Sterilizing-grade Membrane Filters from Discs to Pleated Cartridges: Effects of Operating Parameters and Solution Properties.

UNLABELLED: For direct flow sterilizing-grade filtration, a linear scale-up between the performance of discs and pleated filter cartridges has traditionally been assumed. Linear scale-up assumes that the filtration performances, defined here as filter flux and capacity, scale linearly with the membrane area and remains independent of the selected device formats. However, experimental results show that the later assumption does not hold in all cases. In this article, we investigated the effect of solution properties and operating parameters on scale-up with both fouling and non-fouling feeds. For non-fouling solutions, such as buffers, the flux ratio, defined as α, between pleated filter cartridges and disc filters range from 0.5 to 0.85. For complex fouling feeds, such as protein or cell culture media solutions, the ratio of initial flux between pleated filter cartridges and discs was the same as the flux ratio, α. For fouling solutions, the ratio of filtration capacity between pleated cartridges and discs, referred to as capacity ratio, ß, was variable. We found that ß was sensitive to the particle size distribution of the challenge solution and the mode of filtration operation (constant pressure or constant flux), whereas it was less sensitive to the magnitude of the operating pressure or flux and concentration of the fouling species. For most conditions tested, ß among pleated cartridges and discs was within ±20% variation of unity. At the end, we present a modified standard model that accounts for both variations in flux ratio, α, as well as capacity ratios, ß, for estimating the requirement for membrane area at manufacturing scale with proteinacious fouling and non-protein/non-fouling feeds. The data show that for cases where filtration is capacity controlled, flux ratios between the pleated filter and disc are not critical. For such cases, the use of a high-area laid-over pleated cartridge construction allows for reducing the number of 10 inch pleated filter cartridges required to process the batch volume. LAY ABSTRACT: Scale-up remains at the core of a process development. For direct flow sterilizing-grade filtration, a linear scale-up between the performance of discs and pleated filter cartridges has traditionally been assumed. Linear scale-up assumes that the filtration performances, defined here as filter flux and capacity, scale linearly with the membrane area and remains independent of the selected device formats. However, experimental results show that the later assumption does not hold in all cases. We investigated the effect of solution properties and operating parameters on scale-up from membrane disc to pleated filter cartridges. Typical values of flux and capacity ratios and the guidelines on scale-up of direct flow filters from bench-scale to manufacturing-scale are presented. Specifically, we found that the flux ratio for pleated filter and small-scale disc range from 0.5 to 0.85, and capacity ratio for most cases is within ±20% variation of unity, with some exceptions.