Mixing Time Effects on the Dispersion Performance of Adhesive Mixtures for Inhalation

This paper deals with the effects of mixing time on the homogeneity and dispersion performance of adhesive mixtures for inhalation. Interactions between these effects and the carrier size fraction, the type of drug and the inhalation flow rate were studied. Furthermore, it was examined whether or not changes in the dispersion performance as a result of prolonged mixing can be explained with a balance of three processes that occur during mixing, knowing drug redistribution over the lactose carrier; (de-) agglomeration of the drug (and fine lactose) particles; and compression of the drug particles onto the carrier surface. For this purpose, mixtures containing salmeterol xinafoate or fluticasone propionate were mixed for different periods of time with a fine or coarse crystalline lactose carrier in a Turbula mixer. Drug detachment experiments were performed using a classifier based inhaler at different flow rates. Scanning electron microscopy and laser diffraction techniques were used to measure drug distribution and agglomeration, whereas changes in the apparent solubility were measured as a means to monitor the degree of mechanical stress imparted on the drug particles. No clear trend between mixing time and content uniformity was observed. Quantitative and qualitative interactions between the effect of mixing time on drug detachment and the type of drug, the carrier size fraction and the flow rate were measured, which could be explained with the three processes mentioned. Generally, prolonged mixing caused drug detachment to decrease, with the strongest decline occurring in the first 120 minutes of mixing. For the most cohesive drug (salmeterol) and the coarse carrier, agglomerate formation seemed to dominate the overall effect of mixing time at a low inhalation flow rate, causing drug detachment to increase with prolonged mixing. The optimal mixing time will thus depend on the formulation purpose and the choice for other, interacting variables.     

A New Role of Fine Excipient Materials in Carrier-Based Dry Powder Inhalation Mixtures: Effect on Deagglomeration of Drug Particles During Mixing Revealed

The potential of fine excipient materials to improve the performance of carrier-based dry powder inhalation mixtures is well acknowledged. The mechanisms underlying this potential are, however, open to question till date. Elaborate understanding of these mechanisms is a requisite for rational rather than empirical development of ternary dry powder inhalation mixtures. While effects of fine excipient materials on drug adhesion to and detachment from surfaces of carrier particle have been extensively investigated, effects on other processes, such as carrier–drug mixing, capsule/blister/device filling, or aerosolization in inhaler devices, have received little attention. We investigated the influence of fine excipient materials on the outcome of the carrier–drug mixing process. We studied the dispersibility of micronized fluticasone propionate particles after mixing with α-lactose monohydrate blends comprising different fine particle concentrations. Increasing the fine (D < 10.0 μm) excipient fraction from 1.84 to 8.70% v/v increased the respirable drug fraction in the excipient–drug mixture from 56.42 to 67.80% v/v (p < 0.05). The results suggest that low concentrations of fine excipient particles bind to active sites on and fill deep crevices in coarse carrier particles. As the concentration of fine excipient particles increases beyond that saturating active sites, they fill the spaces between and adhere to the surfaces of coarse carrier particles, creating projections and micropores. They thereby promote deagglomeration of drug particles during carrier–drug mixing. The findings pave the way for a comprehensive understanding of contributions of fine excipient materials to the performance of carrier-based dry powder inhalation mixtures.     

Design of experiments applications in bioprocessing: Chromatography process development using split design of experiments

Development of a chromatographic step in a time and resource efficient manner remains a serious bottleneck in protein purification. Chromatographic performance typically depends on raw material attributes, feed material attributes, process factors, and their interactions. Design of experiments (DOE) based process development is often chosen for this purpose. A challenge is, however, in performing a DOE with such a large number of process factors. A split DOE approach based on process knowledge in order to reduce the number of experiments is proposed. The first DOE targets optimizing factors that are likely to significantly impact the process and their effect on process performance is unknown. The second DOE aims to fine-tune another set of interacting process factors, impact of whom on process performance is known from process understanding. Furthermore, modeling of a large set of output response variables has been achieved by fitting the output responses to an empirical equation and then using the parametric constants of the equation as output response variables for regression modeling. Two case studies involving hydrophobic interaction chromatography for removal of aggregates and cation exchange chromatography for separation of charge variants and aggregates have been utilized to illustrate the proposed approach. Proposed methodology reduced total number of experiments by 25% and 72% compared to a single DOE based on central composite design and full factorial design, respectively. The proposed approach is likely to result in a significant reduction in resources required as well as time taken during process development. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2730, 2019     

Predicting and Analyzing Interactions between Mycobacterium tuberculosis and Its Human Host

The outcome of infection by Mycobacterium tuberculosis (Mtb) depends greatly on how the host responds to the bacteria and how the bacteria manipulates the host, which is facilitated by protein–protein interactions. Thus, to understand this process, there is a need for elucidating protein interactions between human and Mtb, which may enable us to characterize specific molecular mechanisms allowing the bacteria to persist and survive under different environmental conditions. In this work, we used the interologs method based on experimentally verified intra-species and inter-species interactions to predict human-Mtb functional interactions. These interactions were further filtered using known human-Mtb interactions and genes that are differentially expressed during infection, producing 190 interactions. Further analysis of the subcellular location of proteins involved in these human-Mtb interactions confirms feasibility of these interactions. We also conducted functional analysis of human and Mtb proteins involved in these interactions, checking whether these proteins play a role in infection and/or disease, and enriching Mtb proteins in a previously predicted list of drug targets. We found that the biological processes of the human interacting proteins suggested their involvement in apoptosis and production of nitric oxide, whereas those of the Mtb interacting proteins were relevant to the intracellular environment of Mtb in the host. Mapping these proteins onto KEGG pathways highlighted proteins belonging to the tuberculosis pathway and also suggested that Mtb proteins might use the host to acquire nutrients, which is in agreement with the intracellular lifestyle of Mtb. This indicates that these interactions can shed light on the interplay between Mtb and its human host and thus, contribute to the process of designing novel drugs with new biological mechanisms of action.     

Presence of anionic phospholipids rules the membrane localization of phenothiazine type multidrug resistance modulator

Substances able to modulate multidrug resistance (MDR), including antipsychotic phenothiazine derivatives, are mainly cationic amphiphiles. The molecular mechanism of their action can involve interactions with transporter proteins as well as with membrane lipids. The interactions between anionic phospholipids and MDR modulators can be crucial for their action. In present work we study interactions of 2-trifluoromethyl-10-(4-[methanesulfonylamid]buthyl)-phenothiazine (FPhMS) with neutral (PC) and anionic lipids (PG and PS). Using microcalorimetry, steady-state and time-resolved fluorescence spectroscopy we show that FPhMS interacts with all lipids studied and drug location in membrane depends on lipid type. The electrostatic attraction between drug and lipid headgroups presumably keeps phenothiazine derivative molecules closer to surface of negatively charged membranes with respect to neutral ones. FPhMS effects on bilayer properties are not proportional to phosphatidylserine content in lipid mixtures. Behavior of equimolar PC:PS mixtures is similar to pure PS bilayers, while 2:1 or 1:2 (mole:mole) PC:PS mixtures resemble pure PC ones.     

Fluoroquinolone-drug interactions: An overview

It is apparent that the fluoroquinolones represent an important advancement in infectious disease therapy. However, like other anti-microbial compounds, they are not without side effects. An understanding and appreciation of fluoroquinolone-drug interactions is essential for their optimal clinical use. Many questions concerning the potential of the fluoroquinolones with regard to drug interactions still remain.In addition, the majority of research on fluoroquinolone-drug interactions has been done in the research center environment using normal, healthy adult volunteers. The clinical significance of fluoroquinolone-drug interactions needs to be based upon observations in various patient populations, where feasible. Many of the aforementioned interactions could be influenced by race, dosage regimen design, smoking status of the patient, diet composition, and underlying organ function. Future research needs to assess the multiple factors of the clinical setting that might have an influence on patient outcome as a result of combining a fluoroquinolone with another interacting drug. Because norfloxacin and ciprofloxacin are commercially available in the United States, an active surveillance program in the community and institutional settings will help to identify and clarify many of the issues related to flouroquinolone-drug interactions.     

Metformin attenuates adhesion between cancer and endothelial cells in chronic hyperglycemia by recovery of the endothelial glycocalyx barrier

BackgroundEpidemiologic studies suggest that diabetes is associated with an increased risk of cancer. Concurrently, clinical trials have shown that metformin, which is a first-line antidiabetic drug, displays anticancer activity. The underlying mechanisms for these effects are, however, still not well recognized.MethodsMethods based on atomic force microscopy (AFM) were used to directly evaluate the influence of metformin on the nanomechanical and adhesive properties of endothelial and cancer cells in chronic hyperglycemia. AFM single-cell force spectroscopy (SCFS) was used to measure the total adhesion force and the work of detachment between EA.hy926 endothelial cells and A549 lung carcinoma cells. Nanoindentation with a spherical AFM probe provided information about the nanomechanical properties of cells, particularly the length and grafting density of the glycocalyx layer. Fluorescence imaging was used for glycocalyx visualization and monitoring of E-selectin and ICAM-1 expression.ResultsSCFS demonstrated that metformin attenuates adhesive interactions between EA.hy926 endothelial cells and A549 lung carcinoma cells in chronic hyperglycemia. Nanoindentation experiments, confirmed by confocal microscopy imaging, revealed metformin-induced recovery of endothelial glycocalyx length and density. The recovery of endothelial glycocalyx was correlated with a decrease in the surface expression of E-selectin and ICAM-1.ConclusionOur results identify metformin-induced endothelial glycocalyx restoration as a key factor responsible for the attenuation of adhesion between EA.hy926 endothelial cells and A549 lung carcinoma cells.General significanceMetformin-induced glycocalyx restoration and the resulting attenuation of adhesive interactions between the endothelium and cancer cells may account for the antimetastatic properties of this drug.     

Thermal structural evolutions of DMPC-water biomimetic systems investigated by Raman Spectroscopy

Many cell membranes of living organisms can be represented as phospholipid bilayers immersed into a water environment. The physical-chemical interactions at the membranes/water interface are responsible for the stabilization of the membranes. In addition, the drug efficiency, the pharmaceutical mechanism and the improvement of the drug design can be addressed to the interactions between the membranes-water interface with the drug and to the membrane-drug interface. In this framework, it is important to find membranes models able to simulate and simultaneously simplify the biological systems to better understand both physical and chemical interactions at the interface level. Dimyristoylphosphatidylcholine (DMPC) is a synthetic phospholipid used in order to make Multilamellar Vesicle (MLV), Large Unilamellar Vesicle (LUV) and Giant Unilamellar Vesicle (GUV). In order to understand the mechanisms of vesicle formation, we have analyzed mixtures of DMPC and water by micro-Raman spectroscopy at different temperatures in the range between 10 and 35 °C. Particularly, we analyzed the temperature dependence of the CN vibrational frequency, which appears well correlated to the order degree of the various phases. These investigations, beyond the determination of phospholipid hydrocarbon chains order, provide information about the conformation of the lipid membranes.We have identified the mixture of DMPC/water that is best suited for Raman studies and can be used as an in-vitro model for biological systems.A peculiar frequency shift across the transition gel-ripple-liquid crystalline phases has been proposed as a useful diagnostic marker to detect the “order degree” and subsequently the phases of biomimetic membranes made by DMPC.     

Adhesive Interactions and the Patterning of Nerve Connections: An Experimental and Theoretical Approach

Adhesive interactions have long been proposed to play a centralrole in the patterning of neural structures and their interconnections.Many of these ideas are based upon experiments on the projectionfrom the eye to the optic tectum (the retinotectal projection)in lowervertebrates. In order to test the feasibility of suchproposals, a detailed model, based largely on adhesive interactionsbetween cells, has been developed. Computer simulations of themodelshow that simple adhesive interactions are sufficient toexplain much of the literature on the patterning of the amphibianretinotectal projection. Aspects of the model have been experimetallytested through the use of antibodies to known adhesive molecules.The results of these experiments appear consistent with therole of adhesion in the patterning of the connections and withthe predictions of the model. Although such experiments demonstratethe power of adhesive cell interactions in the patterning ofnerve connections, additional experiments and simulations demonstratethat some other non-adhesive processes may play a role. In particular,the addition of a process that is dependent on the activityof the neurons allows the model to better fit the literature.An activity-dependent competition between neurons for adhesivesites on the target cells appears to be sufficient to play thisrole.     

Modeling the influence of cyclodextrins on oral absorption of low‐solubility drugs: I. Model development

The ability to quantitatively predict the influence of a solubilization technology on oral absorption would be highly beneficial in rational selection of drug delivery technology and formulation design. Cyclodextrins (CDs) are cyclic oligosaccharides which form inclusion complexes with a large variety of compounds including drugs. There are many studies in the literature showing that complexation between CD and drug enhances oral bioavailability and some demonstrating failure of CD in bioavailability enhancement, but relatively little guidance regarding when CD can be used to enhance bioavailability. A model was developed based upon mass transport expressions for drug dissolution and absorption and a pseudo‐equilibrium assumption for the complexation reaction with CD. The model considers neutral compound delivered as a physical mixture with CD in both immediate release (IR) and controlled release (CR) formulations. Simulation results demonstrated that cyclodextrins can enhance, have no effect, or hurt drug absorption when delivered as a physical mixture with drug. The predicted influence depends on interacting parameter values, including solubility, drug absorption constant, binding constant, CD:drug molar ratio, dose, and assumed volume of the intestinal lumen. In general, the predicted positive influence of dosing as a physical mixture with CD was minimal, alluding to the significance of dosing as a preformed complex. The model developed enabled examination of which physical and chemical properties result in oral absorption enhancement for neutral drug administered as a physical mixture with CD, demonstrating the utility of modeling the influence of a drug delivery agent (e.g., CD) on absorption for rational dosage form design. Biotechnol. Bioeng. 2010; 105: 409–420. © 2009 Wiley Periodicals, Inc.     

Dry Powder Inhalers: Study of the Parameters Influencing Adhesion and Dispersion of Fluticasone Propionate

Interactions between particles are dependent on the physicochemical characteristics of the interacting particles but it is also important to consider the manufacturing process. Blending active pharmaceutical ingredient (API) with carrier is a critical stage that determines the blend homogeneity and is the first step towards obtaining the final quality of the powder blend. The aim of this work was to study parameters that influence the interactions between API and carrier in adhesive mixtures used in DPI and their effect on API dispersion. The study was done with fluticasone propionate blended with lactose ‘Lactohale 200’. The study was based on the influence of the operating conditions (speed, mixing time, resting steps during mixing), the size of the carrier and the storage conditions on the blend properties and on the API dispersion. The quality of the blends was examined by analysing the API content uniformity. Adhesion characteristics were evaluated by submitting mixtures to a sieving action by air depression with the Alpine air-jet sieve. Aerodynamic evaluation of fine particle fraction (FPF) was obtained using a Twin Stage Impinger; the FPF being defined as the mass percentage of API below 6.4 μm. For good dispersion and therefore good homogeneity of the API in the carrier particles, speed and powder blending time have to be sufficient, but not too long to prevent the appearance of static electricity, which is not favourable to homogeneity and stability. The FPF increases with the decrease in the carrier size. The storage conditions have also to be taken into consideration. Higher humidity favours the adhesion of API on the carrier and decreases the FPF.KEY WORDS: adhesion, DPI performance, fluticasone propionate, operating parameters     

Systematic discovery of drug interaction mechanisms

Drug combinations are increasingly important in disease treatments, for combating drug resistance, and for elucidating fundamental relationships in cell physiology. When drugs are combined, their individual effects on cells may be amplified or weakened. Such drug interactions are crucial for treatment efficacy, but their underlying mechanisms remain largely unknown. To uncover the causes of drug interactions, we developed a systematic approach based on precise quantification of the individual and joint effects of antibiotics on growth of genome‐wide Escherichia coli gene deletion strains. We found that drug interactions between antibiotics representing the main modes of action are highly robust to genetic perturbation. This robustness is encapsulated in a general principle of bacterial growth, which enables the quantitative prediction of mutant growth rates under drug combinations. Rare violations of this principle exposed recurring cellular functions controlling drug interactions. In particular, we found that polysaccharide and ATP synthesis control multiple drug interactions with previously unexplained mechanisms, and small molecule adjuvants targeting these functions synthetically reshape drug interactions in predictable ways. These results provide a new conceptual framework for the design of multidrug combinations and suggest that there are universal mechanisms at the heart of most drug interactions.     

A mechanism of dynamical interactions for two-person social dilemmas

We propose a new mechanism of interactions between game-theoretical agents in which the weights of the connections between interacting individuals are dynamical, payoff-dependent variables. Their evolution depends on the difference between the payoff of the agents from a given type of encounter and their average payoff. The mechanism is studied in the frame of two models: agents distributed on a random graph, and a mean field model. Symmetric and asymmetric connections between the agents are introduced. Long time behavior of both systems is discussed for the Prisoner's Dilemma and the Snow Drift games.     

Interactions of anticancer drugs with usual and mismatch base pairs - density functional theory studies

The antitumor activity of a drug is associated with its molecular properties as well as its interactions with target molecules. The molecular structures of usual, mismatch base pairs and their drug (Hydroxyurea and 5-Fluorouracil) interacting complexes were studied using density functional theory methods. The two and three-body interaction energies have been used to analyze the influence of a drug on the stability of base pairs. The sharing of electron density between the interacting molecules is shown through electron density difference maps. The Atoms in Molecules theory and Natural Bond Orbital analysis have been performed to study the hydrogen bonds in the drug interacting complexes.     

Food Webs in Relation to Variation in the Environment and Species Assemblage: A Multivariate Approach

The abiotic environment has strong influences on the growth, survival, behavior, and ecology of aquatic organisms. Biotic interactions and species life histories interact with abiotic factors to structure the food web. One measure of food-web structure is food-chain length. Several hypotheses predict a linear relationship between one environmental variable (e.g., disturbance or ecosystem size) and food-chain length. However, many abiotic and biotic variables interact in diverse ways to structure a community, and may affect other measures of food web structure besides food-chain length. This study took a multivariate approach to test the influence of several important environmental variables on four food-web characteristics measured in nine ponds along a hydroperiod gradient over two years. This approach allowed for testing the ecosystem size and dynamic constraints hypotheses while in context of other possibly interacting environmental variables. The relationship between amphibian and invertebrate communities and pond habitat variables was assessed to understand the underlying food-web structure. Hydroperiod and pond area had a strong influence on amphibian and invertebrate communities, trophic diversity and δ¹⁵N range. The range in δ¹³C values responded strongly to dissolved oxygen. Food-chain length responded to multiple environmental variables. Invertebrate and amphibian communities were structured by pond hydroperiod which in turn influenced the trophic diversity of the food web. The results of this study suggest food-chain length is influenced by environmental variation and species assemblage and that a multivariate approach may allow us to better understand the dynamics within and across aquatic food webs.     

KERA: analysis tool for multi-process,multi-state single-molecule data

Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA’s utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase.     

Monte Carlo simulation of protein-induced lipid demixing in a membrane with interactions derived from experiment

Lipid domain formation induced by annexin was investigated in mixtures of phosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol (Chol), which were selected to mimic the inner leaflet of a eukaryotic plasma membrane. Annexins are ubiquitous and abundant cytoplasmic, peripheral proteins, which bind to membranes containing PS in the presence of calcium ions (Ca²⁺), but whose function is unknown. Prompted by indications of interplay between the presence of cholesterol in PS/PC mixtures and the binding of annexins, we used Monte Carlo simulations to investigate protein and lipid domain formation in these mixtures. The set of interaction parameters between lipids and proteins was assigned by matching experimental observables to corresponding variables in the calculations. In the case of monounsaturated phospholipids, the PS-PC and PC-Chol interactions are weakly repulsive. The interaction between protein and PS was determined based on experiments of annexin binding to PC/PS mixtures in the presence of Ca²⁺. Based on the proposal that PS and cholesterol form a complex in model membranes, a favorable PS-Chol interaction was postulated. Finally, protein-protein favorable interactions were also included, which are consistent with observations of large, two-dimensional, regular arrays of annexins on membranes. Those net interactions between pairs of lipids, proteins and lipids, and between proteins are all small, of the order of the average kinetic energy. We found that annexin a5 can induce formation of large PS domains, coincident with protein domains, but only if cholesterol is present.     

Geometry of nonbonded interactions involving planar groups in proteins

Although hydrophobic interaction is the main contributing factor to the stability of the protein fold, the specificity of the folding process depends on many directional interactions. An analysis has been carried out on the geometry of interaction between planar moieties of ten side chains (Phe, Tyr, Trp, His, Arg, Pro, Asp, Glu, Asn and Gln), the aromatic residues and the sulfide planes (of Met and cystine), and the aromatic residues and the peptide planes within the protein tertiary structures available in the Protein Data Bank. The occurrence of hydrogen bonds and other nonconventional interactions such as C-H...pi, C-H...O, electrophile-nucleophile interactions involving the planar moieties has been elucidated. The specific nature of the interactions constraints many of the residue pairs to occur with a fixed sequence difference, maintaining a sequential order, when located in secondary structural elements, such as alpha-helices and beta-turns. The importance of many of these interactions (for example, aromatic residues interacting with Pro or cystine sulfur atom) is revealed by the higher degree of conservation observed for them in protein structures and binding regions. The planar residues are well represented in the active sites, and the geometry of their interactions does not deviate from the general distribution. The geometrical relationship between interacting residues provides valuable insights into the process of protein folding and would be useful for the design of protein molecules and modulation of their binding properties.     

Quantitative considerations of the consequences of an interplay between ligand binding and reversible adsorption of a macromolecular solute

Explicit expressions are derived which determine the equilibrium composition of mixtures comprising a multivalent, insoluble matrix, a multivalent, macromolecular solute (acceptor) and a univalent ligand. With three-reactant mixtures of this type a range of combinations of interactions is possible wherein the ligand interacts with either the acceptor or the matrix, in either event perturbing the acceptor-matrix equilibria. Theory encompassing this range of possibilities is written in terms of a single site-binding constant for each type of interaction to account, in general terms, for both multiple binding and crosslinking effects. These explicit thermodynamic relationships are discussed, with the use of reported findings on several biological systems, in two frameworks. First, it is established that the theory is applicable to the quantitative interpretation of affinity chromatography experiments designed to elucidate the thermodynamic interaction parameters governing the various types of interacting system. Second, it is emphasized that the relationships are also relevant to metabolite-induced changes in the subcellular distribution of macromolecular species.     

Dangerous liaisons: the ecology of private interest and common good

Many ecological interactions that are called mutualistic are in fact mixtures of antagonistic and mutualistic aspects. For example, plasmids exploit their bacterial hosts but also protect them against external threats. In this study, we analyse the conditions for the evolution of what we call 'dangerous liaisons': interactions combining mutualistic and antagonistic aspects. Starting point of our analysis is a model that was proposed as early as 1934. In this model, partners have to form a complex (either temporary or long lasting) in order to interact. Using this model framework we then set out to define and tease apart private interests of the interacting partners from their common good. This dichotomy provides a unifying perspective to classify ecological interactions. We discuss some examples to illustrate how the outcome of the interaction may depend on densities or on other contextual variables. Finally, we note that having a common good is not a necessary condition for partners to have aligned interests. In a dangerous liaison partners may have interest to cooperate even when this does not bolster the common good.