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Kerstin J Frank,1,3 Ulrich Westedt,2 Karin M Rosenblatt,2 Peter Hölig,2 Jörg Rosenberg,2 Markus Mägerlein,2 Gert Fricker,3 Martin Brandl11Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark; 2Abbott GmbH and Co. KG, Ludwigshafen, Germany; 3Department of Pharmaceutical Technology, University of Heidelberg, Heidelberg, GermanyAbstract: Amorphous solid dispersions (ASDs) are a promising formulation approach for poorly soluble active pharmaceutical ingredients (APIs), because they ideally enhance both dissolution rate and solubility. However, the mechanism behind this is not understood in detail. In the present study, we investigated the supramolecular and the nano/microparticulate structures that emerge spontaneously upon dispersion of an ASD in aqueous medium and elucidated their influence on solubility. The ASD, prepared by hot melt extrusion, contained the poorly soluble ABT-102 (solubility in buffer, 0.05 µg/mL), a hydrophilic polymer, and three surfactants. The apparent solubility of ABT-102 from the ASD-formulation was enhanced up to 200 times in comparison to crystalline ABT-102. At the same time, the molecular solubility, as assessed by inverse equilibrium dialysis, was enhanced two times. Asymmetrical flow field-flow fractionation in combination with a multiangle light-scattering detector, an ultraviolet detector, and a refractometer enabled us to separate and identify the various supramolecular assemblies that were present in the aqueous dispersions of the API-free ASD (placebo) and of binary/ternary blends of the ingredients. Thus, the supramolecular assemblies with a molar mass between 20,000 and 90,000 could be assigned to the polyvinylpyrrolidone/vinyl acetate 64, while two other kinds of assemblies were assigned to different surfactant assemblies (micelles). The amount of ABT-102 remaining associated with each of the assemblies upon fractionation was quantified offline with high-performance liquid chromatography–ultraviolet-visible. The polymeric and the micellar fraction contributed to the substantial increase in apparent solubility of ABT-102. Furthermore, a microparticulate fraction was isolated by centrifugation and analyzed by scanning electron microscopy, X-ray scattering, and infrared spectroscopy. The microparticles were found to be amorphous and to contain two of the surfactants besides ABT-102 as the main component. The amorphous microparticles are assumed to be the origin of the observed increase in molecular solubility ("true" supersaturation).Keywords: solid dispersion, AFlFFF, micelles, solubility, supersaturation

Haemoglobin (Hb) was isolated from human erythrocytes under conditions which maintained NADH-cytochrome-b5 reductase activity and suppressed oxidation of Hb during storage at 4 degrees C (methaemoglobin values < 3% after 29 days). Hb was entrapped into liposomes composed of hydrogenated egg phosphatidylcholine and equimolar cholesterol according to the dehydration/rehydration procedure of Kirby and Gregoriadis ((1984) Biotechnology 2, 979). However, encapsulation of Hb in its intact form was poor (< 5%) as a result of its oxidation and denaturation during freeze-drying. The addition of cryoprotectants and the use of both, higher initial concentrations of Hb and very small void vesicles resulted in Hb-rich dehydration/rehydration vesicles (phospholipid/Hb molar ratio of about 200:1) of the preferred size of 110 nm (mean). Highly homogeneous and small void vesicles as starting material were prepared using the one-step method of Brandl et al. ((1990) Drug Dev. Ind. Pharm. 16, 2167). The cryoprotectants were chosen with respect to their sufficient protection of Hb without affecting its loading into vesicles during freeze-drying and rehydration. 51Cr-labelling of Hb was used for the in vivo monitoring of the fate of Hb-containing vesicles rather than 125I-labelling since the latter induced strong interactions of Hb with liposomes. Upon intravenous administration into rats, liposomal 51Cr-Hb showed greater blood levels and prolonged circulation times in the blood compared to free Hb. The present approach provides high yield entrapment of labile molecules into vesicles of small size known to exhibit long circulation time.

Human hemoglobin is encapsulated in unilamellar liposomes by French press extrusion in order to form hemoglobin liposomes (HBL). The mean diameter of the HBL is 50 nm by TEM. Therefore the preparation can be passed through microbial retentive filters (200 nm). The encapsulation efficiency is 11.5% and the hemoglobin content 0.52 g/100 ml for a 5.75% (v/v) dispersion. The HBL are able to bind 3.8% (v/v) 02 (32% (v/v) dispersion) and show the same oxygen dissociation characteristics as the hemoglobin solution. The lipid mixture consists of hydrogenated soy lecithin, which is inexpensive and stable against oxidation, and cholesterol in a molar ratio 1:l. The French press extrusion process is suitable for a scaling up.

Escherichia coli are an indicator organism for fecal contamination and can represent a serious health hazard for humans. Common water quality tests are time consuming routine analysis. Our aim was the development of an automated biosensor for a fast identification and quantification of E. coli contaminations in ground-, surface- and drinking water. We applied an electrochemical assay to detect E. coli using their β-galactosidase activity. First, the enzyme production was stimulated in the cells and further, specifically released through a T2 bacteriophage mediated cell lysis. The enzyme concentration was monitored by potentiometric measurements. By using this methodological approach we were able to specifically detect at least 2 colony-forming units (CFU) of E. coli within 8 hours. The incorporation of this method into an automatic biosensor device would ensure the quality of our water. Furthermore, a continuous monitoring could be achieved and online data transfer ensures fast counteractions in water treatment plants. For future applications we developed a low-cost potentiostat with an easy-to-use PC software for environmental measurements and evaluated it with 3 different devices.

Wireless data acquisition and data fusion in medical and environmental applications is mainly based on additional information like the position of the sensor device. An accurate and easy to implement method for wireless position estimation of RFID devices is described in this paper. For RFID communication and data transmission, the tags operate within the 13.56 MHz ISM-band with a standardized protocol defined in ISO 14443. The RFID-tag position estimation is done by transmitting linear chirp signals from the RFID tag to base stations on RF frequencies. The chirp signals are generated by SAW devices which are triggered by a low power consumption pulse generator based on an avalanche transistor. The performance of position estimation under AWGN conditions was simulated and compared with common methods. An improvement in the mean square position error in the range of the chirp compression gain was found.

The Spectral Absorption Coefficient (SAC) is an easy detectable sum parameter, giving information about the organic load in wastewater. The SAC 254 measurement is done at 254 nm, implying parameters like COD (chemical oxygen demand), BOD (biochemical oxygen demand) and TOC (total organic carbon). Evaluation of the current state of technique revealed that SAC 254 probes, primarily used for wastewater determination, show significant defects in terms of mobility, accuracy of measurement and costs. Thus a reliable in-situ SAC 254 measurement system, including a remote-transmission of the measured data, was developed. Additional problems within the scope of optical measurement of parameters in wastewater, like contamination of the optical path, calibration or maintenance, have been successfully solved by a newly developed procedure.

Nowadays, RFID tags are under use in numerous applications. The communication distance and therefore the ranging distance between base station and RFID tags is strongly limited by the maximum transmission power and the receiver sensitivity. A method based on passive SAW compressive receivers is presented to enhance the transmission distance for RFID tag ranging. The position estimation was done by active transmission of chirp pulses by the RFID tag and ToA and PoA measurements at the receivers.

The aim of the current study was to investigate the ability of a fixed-angle routine photon correlation spectrometer (PCS) to resolve bimodal size distributions. The focus was on dispersions consisting of a majority of smaller and a minority of bigger particles. Monodisperse latex beads of sizes from 21 to 269 nm were measured first as single-size dispersions and then with various binary blends. For single-size dispersions, the mean diameters obtained were as indicated by the manufacturer, except for 21- and 34-nm particles, which were somewhat smaller. PCS analysis of blends of 21 + 102-nm and 34 + 102-nm particles resulted in bimodal distributions with particle diameters of the 2 peaks in the expected magnitude down to critical blending ratios of 0.002% and 0.08% of bigger particles, respectively. At these ratios, PCS results became inconsistent, and an increased number of monomodal results and/or high residuals were seen. For 21 + 102-nm blends, at even smaller ratios (0.001%), more consistent results were obtained again with predominantly monomodal distributions in the size range of the smaller particles (ie, the bigger particles were neglected). PCS analysis of blends of 21 + 269-nm particles yielded bimodal distributions with diameters within the expected magnitude as long as the content of bigger particles did not exceed 0.005%. Above this ratio, predominantly monomodal results with mean diameters in the magnitude of the bigger particles were obtained (ie, the smaller particles were neglected). In conclusion, a routine PCS instrument can resolve bimodal size distributions of colloidal dispersions only at certain ratios of the 2 subpopulations. Both low and high ratios lead to 1 of the 2 subpopulations being neglected.

Semisolid phospholipid dispersions of vesicular morphology, so-called vesicular phospholipid gels (VPGs), were prepared by high-pressure homogenisation and tested in vitro for their suitability as implantable sustained release system for the decapeptide cetrorelix, a potent LH-RH antagonist. The VPGs contained 300-500 mg/g egg phosphatidylcholine (E80) and 0.5-10 mg/g cetrorelix acetate (CXA). The in vitro release experiments showed a wide variability of the system in release, ranging from complete release within less than 24 h (0.5mg/g CXA; 400 mg/g E80) to a predicted 80% sustained release over 3 months (8.6 mg/g CXA; 280 mg/g E80). Erosion of the phospholipid matrix, i.e. release of phospholipid vesicles was found to be the main release mechanism, following zero order or first order kinetics depending on the composition of the VPG. CXA-concentration dependent drug-drug or drug-lipid interactions are assumed to be responsible for the change in release kinetics and the decrease of CXA release at high concentrations of the peptide. Multivariate analysis revealed that both lipid concentration and peptide concentration and also the interactions between the two factors are significant factors for the release rate of the peptide. In summary: based on the presented in vitro release data sustained release of therapeutically relevant CXA doses over up to 6 weeks appears feasible. VPGs are thus considered as a promising new approach for the sustained release of peptide hormones.

The purpose of this study was to establish a new experimental approach to determine the maximum amount of camptothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultracentrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubility of CPT in the buffer was improved by approximately a factor of 10 (from 2.7 to 15-50 microg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP), a maximum CPT-solubilization of 100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at 20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrinsic water solubility of the drug.

High performance liquid chromatography (HPLC) analysis of increasing amounts of the decapeptide Cetrorelix, a potent antagonist of the luteinising hormone-releasing hormone, in distilled water resulted in a poor and variable response when solutions of low concentration (0.2-4microg/ml) were analysed. Rinsing experiments revealed loss of analyte due to adsorption to the vial surfaces as the main reason for this. The adsorption of Cetrorelix was found to follow a Langmuir isotherm reaching a plateau at 0.4microg/cm(2) and to be influenced by both the dissolution medium and the type of vial used. The adsorption tendency of Cetrorelix was reduced by: (a) a more lipophilic solvent (ethanol), (b) a more acidic pH (acetic acid) inducing repulsive charges (c) a micellar solution of various tensides. With all of these media the HPLC response was higher (up to five times) and less variable. Adsorption of Cetrorelix to solid surfaces decreased in the rank order: glass > polypropylene = polyethylene > poly-(tetrafluoroethylene), with considerable differences between the glass vials of various suppliers.

While the oral route of drug administration is very cost efficient and has high patient compliance, challenges that especially modern therapeutic protein and peptide drugs face are, among others, premature degradation and poor permeability over the intestinal barrier. The use of nanoparticulate drug carriers like liposomes (spherical vesicles surrounded by a phospholipid bilayer) is a promising way of overcoming these challenges. Advanced liposome formulations often contain enhancers to further stabilize the lipid bilayer, with archaeal tetraether lipids (TELs) being a fairly recently established enhancer. TELs can be found in hyperthermophilic Crenarchaeota, however their demanding growth requirements make the biotechnological production of TELs so far uneconomical. Yet unexplored sources of TELs are environmental Thaumarchaeota, which can be found in many environments including the ocean, soil, freshwater and also wastewater treatment plants (WWTP). To explore if indeed TELs from Thaumarchaeota can be used as enhancers of liposome stability, we collected biomass from different biological treatments at the Ejby Mølle WWTP (Odense) and subsequently optimized protocols for extracting lipids, settling on a modified Blight & Dyer protocol. These efforts were guided by a screening of the biological treatments of the WWTP for Archaea, indicating the presence of Thaumarchaeota, although often at low abundances. The initial results of these studies will be presented and discussed.

The purpose of this research was to adapt a colorimetric, phospholipase D-based serum-phospholipid assay for the quantification of phosphatidylcholine (PC) in liposomes using a microtitre plate reader. PC from natural egg PC liposomes was quantified reliably. In contrast, poor sensitivity was found for liposomes composed of saturated PCs (di-palmitoyl-phosphatidylcholine [DPPC], hydrogenated egg PC). Triton X-100 was then added to the liposomes followed by heating above the phase transition temperature. This modified sample preparation resulted in recoveries of 102.6% +/- 1.0%, 104.4% +/- 7.6%, and 109.4% +/- 3.2% for E80, E80-3/cholesterol, and DPPC liposomes, respectively. Absolute quantification of unknown PCs against a choline chloride standard is feasible, but relative measurements against the very same PC are recommended whenever possible. Validation experiments revealed an absolute quantification limit of 1.25 microg per assay, a good linearity in the range of 25 to 1000 microg/mL PC (r2or = 0.9990) and a quite high accuracy (99.8%-101.4% of theory) and precision (relative standard deviationor = 3.2%) for all 3 PCs studied. The method is thus regarded as suitable for sensitive, rapid, and reliable routine quantification of PCs in liposomes.

Supersaturated lipid-based drug delivery systems have recently been investigated for oral administration for a variety of lipophilic drugs and have shown either equivalent or superior oral bioavailability compared to conventional non-supersaturated lipid-based drug delivery systems. The aim of the present work was to explore supersaturated versus non-supersaturated lipid-based systems at equivalent lipid doses, on in vivo bioavailability in rats and on in vitro permeation across a biomimetic PermeapadⓇ membrane to establish a potential in vivo - in vitro correlation. A secondary objective was to investigate the influence of lipid composition on in vitro and in vivo performance of lipid systems. Results obtained indicated that increasing the celecoxib load in the lipid-based formulations by thermally-induced supersaturation resulted in increased bioavailability for medium and long chain mono-/di-glycerides systems relative to their non-supersaturated (i.e. 85%) reference formulations, albeit only significant for the medium chain systems. Long chain systems displayed higher celecoxib bioavailability than equivalent medium chain systems, both at supersaturated and non-supersaturated drug loads. In vitro passive permeation of celecoxib was studied using both steady-state and dynamic conditions and correlated well with in vivo pharmacokinetic results with respect to compositional effects. In contrast, permeation studies indicated that flux and percentage permeated of supersaturated systems, either at steady-state or under dynamic conditions, decreased or were unchanged relative to non-supersaturated systems. This study has shown that by using two cell-free PermeapadⓇ permeation models coupled with rat-adapted gastro-intestinal conditions, bio-predictive in vitro tools can be developed to be reflective of in vivo scenarios. With further optimization, such models could be successfully used in pharmaceutical industry settings to rapidly screen various prototype formulations prior to animal studies.