Spectroscopic and solution properties characterization of quaternary ammonium ion containing polycations complexed with fluorescent rhodamine sulfonic acid dyes.

Based on the growing range of applications for polycations in research and commercial materials, a continuing need exists to advance the fundamental knowledge and understanding of this class of materials. Spectroscopic and solution properties characterizations of noncovalently labeled, fluorescent Alexa Fluor® dye complexes of two commercial polycations, poly(2-(trimethylamino) ethyl methacrylate) monocation and poly[bis[2-chloroethyl] ether-alt-1,3-bis[3-(dimethylamino) propyl] urea] dication are reported to help address this need. A variety of fluorescence spectroscopic methods are used with a special emphasis on fluorescence correlation spectroscopy (FCS) which is applied to characterize the Stokes radius (RS) and equilibrium dissociation constants (Kd) of dye-polycation complexes at nanomolar dye concentrations. Resulting RS values indicate dye binding to individual polycation chains. Measured Kd values in the sub-micromolar range are consistent with strong dye binding. Increasing solution ionic strength with sodium chloride addition inhibits dye binding and decreases the RS of dye-polycation complexes due to size collapse of polycation chains. The complexes differ in their solution stability to ionic strength changes suggesting that both electrostatic and hydrophobic binding interactions influence dye binding. This study establishes the viability of noncovalent dye-polycation complexation in concert with FCS characterization as a general approach for investigating the properties of quaternary ammonium ion containing polycations in aqueous solution.

Modeling hemoglobin levels of patients chronically transfused with red blood cells.

BACKGROUND: Good models of chronic red blood cell (RBC) transfusion can improve care by accurately estimating time-dependent hemoglobin (Hb) levels and clinically relevant transfusion parameters. STUDY DESIGN AND METHODS: A previously derived mathematical model based on overall Hb mass balance (HMB model) with input parameters of transfused units, transfusion efficiency, RBC lifespan, endogenous Hb, and transfusion interval was applied to three separate clinical studies spanning six transfusion conditions across patient populations with myelodysplastic syndrome (MDS) or thalassemia. RESULTS: The HMB model accurately predicted mean pretransfusion Hb levels for each ensemble cohort of patients with thalassemia or MDS. Dynamic changes in Hb levels were modeled as a function of changes in key input parameters. Improving the 24-h post-transfusion RBC survival from 72% to 86% can be used to either (1) reduce RBC usage by 15%-20% through longer transfusion intervals or (2) increase pretransfusion [Hb] by 8%-11% while maintaining a constant transfusion interval. DISCUSSION: The endogenous Hb level is introduced in the HMB model which represents the patient's self-contribution to overall Hb levels via the autologous RBC lifecycle and was estimated to be 5.0 g/dL for patients with MDS or thalassemia. Transfusion therapy approaches and complementary therapeutics can target multiple, unique model inputs while monitoring net, overall impact on transfusion efficacy. Applying the HMB model to fit individual patient Hb fluctuations will be explored in the future.

Modelling haemoglobin incremental loss on chronic red blood cell transfusions.

BACKGROUND AND OBJECTIVES: Understanding the impact of red blood cell (RBC) lifespan, initial RBC removal, and transfusion intervals on patient haemoglobin (Hb) levels and total iron exposure is not accessible for chronic transfusion scenarios. This article introduces the first model to help clinicians optimize chronic transfusion intervals to minimize transfusion frequency. MATERIALS AND METHODS: Hb levels and iron exposure from multiple transfusions were calculated from Weibull residual lifespan distributions, the fraction effete RBC removed within 24-h (Xe ) and the nominal Hb increment. Two-unit transfusions of RBCs initiated at patient [Hb] = 7 g/dl were modelled for different RBC lifespans and transfusion intervals from 18 to 90 days, and Xe from 0.1 to 0.5. RESULTS: Increased Xe requires shorter transfusion intervals to achieve steady-state [Hb] of 9 g/dl as follows: 30 days between transfusions at Xe  = 0.5, 36 days at Xe  = 0.4, 42 days at Xe  = 0.3, 48 days at Xe  = 0.2 and 54 days at Xe  = 0.1. The same transfusion interval/Xe pairs result in a steady-state [Hb] = 8 g/dl when the RBC lifespan was halved. By reducing transfused RBC increment loss from 30% to 10%, annual transfusions were decreased by 22% with iron addition decreased by 24%. Acute dosing of iron occurs at the higher values of Xe on the day after a transfusion event. CONCLUSION: Systematic trends in fractional Hb incremental loss Xe have been modelled and have a significant and calculatable impact on transfusion intervals and associated introduction of iron.

Automated system for kinetic analysis of particle size distributions for pharmaceutically relevant systems.

Detailing the kinetics of particle formation for pharmaceutically relevant solutions is challenging, especially when considering the combination of formulations, containers, and timescales of clinical importance. This paper describes a method for using commercial software Automate with a stream-selector valve capable of sampling container solutions from within an environmental chamber. The tool was built to monitor changes in particle size distributions via instrumental particle counters but can be adapted to other solution-based sensors. The tool and methodology were demonstrated to be highly effective for measuring dynamic changes in emulsion globule distributions as a function of storage and mixing conditions important for parenteral nutrition. Higher levels of agitation induced the fastest growth of large globules (≥5 µm) while the gentler conditions actually showed a decrease in the number of these large globules. The same methodology recorded calcium phosphate precipitation kinetics as a function of [Ca(2+)] and pH. This automated system is readily adaptable to a wide range of pharmaceutically relevant systems where the particle size is expected to vary with time. This instrumentation can dramatically reduce the time and resources needed to probe complex formulation issues while providing new insights for monitoring the kinetics as a function of key variables.

Probability-based compatibility curves for calcium and phosphates in parenteral nutrition formulations.

BACKGROUND: The information content of the calcium phosphate compatibility curves for adult parenteral nutrition (PN) solutions may benefit from a more sophisticated statistical treatment. Binary logistic regression analyses were evaluated as part of an alternate method for generating formulation compatibility curves. MATERIALS AND METHODS: A commercial PN solution was challenged with a systematic array of calcium and phosphate concentrations. These formulations were then characterized for particulates by visual inspection, light obscuration, and filtration followed by optical microscopy. Logistic regression analyses of the data were compared with traditional treatments for generating compatibility curves. RESULTS: Assay-dependent differences were observed in the compatibility curves and associated probability contours; the microscopic method of precipitate detection generated the most robust results. Calcium and phosphate compatibility data generated from small-volume glass containers reasonably predicted the observed compatibility of clinically relevant flexible containers. CONCLUSIONS: The published methods for creating calcium and phosphate compatibility curves via connecting the highest passing or lowest failing calcium concentrations should be augmented or replaced by probability contours of the entire experimental design to determine zones of formulation incompatibilities. We recommend researchers evaluate their data with logistic regression analysis to help build a more comprehensive probabilistic database of compatibility information.

Interactions between Parenteral Lipid Emulsions and Container Surfaces.

OBJECTIVE: To evaluate the relationship between changes in emulsion globule size distributions and container uptake of lipid emulsions in total nutrient admixtures. METHODS: A total nutrient admixture was prepared from a commercial lipid emulsion, 20% ClinOleic®, separated into glass (borosilicate) and ethylene vinyl acetate (EVA) plastic containers, and then stored at ambient conditions for approximately 24 h. The large globule size distribution was monitored continuously for both containers, and the quantity of triglycerides associated with both containers was measured by liquid chromatography. The changes in mass of the EVA containers were also measured gravimetrically. RESULTS: The volume percent of globules greater than 5 microns in diameter (PFAT5) levels for an emulsion admixture in EVA containers showed a 75% reduction compared to a marginal decrease of PFAT5 when in the glass container. Extraction of the containers showed that the quantity of triglycerides associated with the EVA surfaces steadily increased with emulsion exposure time, while the glass showed a significantly lower triglyceride content compared to the EVA. Gravimetric measurements confirmed that the EVA containers gained significant mass during exposure to the emulsion admixture. CONCLUSION: A time-dependent decrease in PFAT5 values for an emulsion admixture was associated with container triglyceride absorption where EVA containers had a greater uptake than glass containers. The larger globules appear to absorb preferentially, and the admixture globule size distribution fraction represented by PFAT5 accounts for 15-20% of the total triglyceride adsorption to the container. LAY ABSTRACT: The goal of this work is to evaluate how emulsions in total nutrition admixtures are affected by the containers within which they are stored. Specifically, the study examines how the emulsion globule size distribution in different containers is related to adsorption or absorption of the lipids onto or into the container. The admixtures were prepared from a commercial lipid emulsion, 20% ClinOleic®, and the containers were either glass (borosilicate) or plastic (ethylene vinyl acetate, EVA). The large globule size distribution was monitored continuously for both containers over the course of 24 h, and the quantity of triglycerides taken up by both containers was measured by liquid chromatography. The lipid uptake by the EVA containers was also monitored by gravimetric methods. Briefly, the percent of fat globules greater than 5 micrometers (PFAT5) in EVA containers showed a 75% reduction compared to a marginal decrease of PFAT5 when in the glass container. Extraction of the lipids from the containers showed that the quantity of triglycerides associated with the EVA surfaces steadily increased with admixture exposure time, while the glass showed a significantly lower triglyceride content. Gravimetric measurements confirmed that the EVA containers gained measurable mass during exposure to the emulsion admixture.

Reduction of artifacts in fluorescence correlation spectroscopy due to sample adsorption on optical glass surfaces.

The preparation of glass cell surfaces that are chemically functionalized with poly(ethylene glycol) (PEG) chains to reduce sample adsorption and their use in fluorescence correlation spectroscopy (FCS) is described. Optical glass coverslips were acid etched and reacted with either 750 Mr PEG (PEG-750) or 5000 Mr PEG (PEG-5000) to produce adsorption-resistant optical surfaces. FCS data for Nile red-loaded Triton X-100 micelles (NR-TX-100) and Alexa Fluor 555-labeled proteins, bovine serum albumin (BSA-A555), lipidized BSA (lipid-BSA-A555), and three low molecular weight dyes deposited on PEGylated coverslips were evaluated. Measurement artifacts due to sample adsorption on the PEG-5000 functionalized coverslips were reduced significantly for the majority of test materials. Calculations of translational diffusion coefficients and Stokes radii confirmed the effectiveness of this approach. PEG-5000 functionalized coverslips were demonstrated as more effective in inhibiting adsorption than PEG-750 functionalized coverslips. Neither of the functionalized coverslips inhibited the adsorption of one test compound, rhodamine B, a dye that adsorbs strongly on glass surfaces. The use of longer PEG chains in conjunction with chemical cross-linking is proposed for producing a denser, less porous PEG layer for the prevention of strongly glass-adsorbing fluorophores that do not interact with the PEG layer.

Antimicrobial testing for surface-immobilized agents with a surface-separated live-dead staining method.

Modification of a traditional live-dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface-immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3-(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si-QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si-QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si-QAC surfaces showed no kill after removing all Si-QAC elutables via rinsing procedures. The semi-quantitative surface-separated live-dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents.

Forces between insulin microspheres and polymers surfaces for a dry powder inhaler.

Here we demonstrate the use of a colloidal probe atomic force microscope (AFM) to compare the interactions between a model protein microsphere (insulin) and a set of common device polymers (polytetrafluoroethylene, polyethylene and polypropylene) with and without antistatic additive. For inhalation-based delivery devices the solid protein microspheres will interact with the device surfaces under ambient atmospheric conditions, and as such we studied the particle device interaction at a range of relative humidities. The results clearly discriminate between the five different polymer choices, and the impact of the antistatic additive. Although the mechanistic understanding is incomplete, it is evident that the polypropylene with antistatic additive gives consistent and relatively small interaction forces over the entire humidity range. The other polymer surfaces have humidity ranges where the pull-off forces are substantially greater. At 80% relative humidity, the insulin-polymer adhesion forces were similar for all the polymers probably due to the dominance of static charge mitigation and surface hydration effects. Overall, direct measurement of adhesion forces between pharmaceutical microspheres and container substrates can help direct rational choice of plastics/coatings for medical devices.

Anion effects on electrostatic charging of sterically stabilized, water insoluble drug particles.

Water-insoluble suspensions of itraconazole and budesonide were sterically stabilized using nonionic polymers (poloxamer 188 or polysorbate 80) and probed for polymer-anion interactions by measuring changes in particle zeta potential. Anions comprising a range of functionalities and aqueous solubilities were examined. Results showed that the more hydrophobic anions partitioned to the particle interface, and a simple model for predicting anion adsorption was developed from their calculated properties. Anions with a calculated Klopman water solubility less than 10 microM or a calculated log P>3.5 were adsorbed to the particle-polymer interface effectively increasing the overall particle charge. Anions of similar hydrophobicities with sulfonate or sulfate functionalities showed a much higher degree of particle charging compared with their carboxylate and phosphonate analogs at pH 9.5. In addition, the electrostatic charging of particles occurred at lower solution concentrations of sulfonate derivatives. These results suggest that the relative basicity of the oxoanion functionality may influence protonation or ion-pairing phenomena of the anions when adsorbed at the particle-polymer interface. Cetyltetramethylammonium bromide (CTAB) produced a positively charged particle consistent with the model developed for anion adsorption. Particle charging of sterically stabilized drug suspensions appeared largely independent of drug and polymer type. Anion hydrophobicity (solubility) and headgroup functionality dictated the observed charging behavior.

Container effects on the physicochemical properties of parenteral lipid emulsions.

OBJECTIVE: We evaluated the effects of glass and plastic containers on the physicochemical properties of parenteral nutrition lipid emulsions and total nutrient admixtures with an emphasis on globule size distribution and colloidal stability. METHODS: A commercial lipid emulsion, 20% ClinOleic, was separated into glass (type II soda-lime-silica) and plastic (polypropylene multilayer) containers, sterilized, and then stored for 16 wk at 40 degrees C. Globule size distribution, pH, and zeta potential measurements were made every 4 wk. Admixtures derived from parent lipid emulsions were tested after admixing (t = 0), storage for 7 d at 5 degrees C plus 24 h at 25 degrees C (t = 7 + 1), and then after an additional 3 d at 25 degrees C (t = 7 + 4). RESULTS: The parent lipid emulsions in glass and plastic containers exhibited identical time-dependent behavior with respect to mean globule size, percentage of oil droplets >or=5 mum, pH, and zeta potential measurements. The percentages of oil droplets >or=5 mum of all test conditions remained well below the United States Pharmacopeia <729> limits of 0.05%. The total nutrient admixture time-dependent physicochemical characteristics were also found to be independent of the parent lipid emulsion container type. CONCLUSION: Plastic and glass containers were found to be suitable, safe, and indistinguishable with respect to physicochemical stability of a representative parenteral nutrition lipid emulsion and total nutrient admixtures derived from the parent lipid emulsion.

Physicochemical stability of phospholipid-dispersed suspensions of crystalline itraconazole.

The physicochemical stability of an aqueous, phospholipid-based dispersion of itraconazole microcrystals was studied as a model water-insoluble drug suspension. The particle size, phospholipid concentrations, free fatty acid (FFA) content, pH, and zeta potential of two test suspensions were followed over 63 days at 5 and 40 degrees C storage conditions. Hydrolysis of a control suspension containing Lipoid E80 led to rapid FFA formation, pH drop, and subsequent particle aggregation. In the second suspension, sodium oleate used in conjunction with Lipoid E80 significantly enhanced the suspension physicochemical stability. Oleate anions effectively (1) increased the anionic charge of the phospholipid surface layer, (2) buffered the suspension near pH 7, and (3) reduced the specific production of oleic acid as a phosphatidylcholine (PC) degradant. The observed hydrolysis rate constants k(obs) approximately 2 x 10(-7) (Lipoid only) and k(obs) approximately 5 x 10(-8) (Lipoid and oleate) were consistent with the pH dependent behavior reported for saturated soybean PC solutions. Mechanistically, FFA formed initially in the control suspension partitioned to the aqueous phase with limited influence on the phospholipid microenvironment at the itraconazole particle surface. Phospholipid stabilization of water-insoluble drugs was demonstrated with clear benefits from fatty acid anions as co-additives to influence the surface microenvironment, reduce hydrolysis kinetics, and enhance suspension physicochemical stability.

Physicochemical stability of lipid injectable emulsions: correlating changes in large globule distributions with phase separation behavior.

Single particle optical sensing (SPOS) and visual inspection were used to characterize a series of lipid injectable emulsions (n=21) featuring three lipid types, two electrolyte conditions, and three pH levels (7.0, 4.75, and 2.5). Seven of the twenty-one sample conditions exhibited phase separation instability by visual inspection within 98 h of emulsion preparation. The phase instability was driven by electrolyte type and pH, and "cracking" phenomena were independent of lipid type despite the base lipids ranging almost two orders of magnitude in PFAT5 levels. Logistic regression analysis showed that the PFAT5 level determined 1h after admixture preparation was not correlated with phase separation behavior. However, PFAT5 measured at later times showed much improved correlations with emulsion instability. PFAT5 was highly correlated with neighboring cumulative distributions termed PFATX where X=2-10 microm. Although the admixtures studied were not clinically relevant, the data demonstrate some limitations of developing empirical correlations between single-point SPOS measurements and emulsion instability. An alternative limit test for emulsion stability based on the rate of change in the large globule counts is proposed to mitigate inherent deficiencies in the current USP Chapter 729 limit test based on single-point determination of PFAT5 values.