[Assessing adverse reactions in clinical trials]. / Erfassung von Nebenwirkungen in der klinischen Forschung.

The intake of each drug represents an intervention into the complex human organism. In addition to the desired therapeutic effect, adverse reactions (AR) can occur. Package inserts should inform patients about the safety profile of drugs, but how reliable is this information and how are side effects determined? To explore this question, we reviewed data related to the ascertainment of adverse reactions in clinical research from 1977 to 2011. This article sums up the results of our literature research as well as own experiences in clinical research. It reveals very different methods of assessing side effects, discusses the obvious problems with inconsistent data collection and presents possible solutions. To create valid and comparable side effect profiles of drugs and thus ensure the safe use of medicine, a common European standard for the structured assessment of Adverse Reactions (AR) in clinical research, that does not exist yet, is required.

Differences in the coalescence kinetics of fat emulsions in dependence on the amount of fat and age.

The destabilizing effect of calcium ions on emulsions was studied as a function of the age of the emulsions and the degree of emulsion dilution (2%, 0.2% an 0.02% fat). Particle size measurements were performed both in the Coulter counter and a laser diffraction device equipped with PIDS technology. The data of both instruments showed a good correlation. zeta-Potential was determined by laser doppler anemometrie. The physical stability of the emulsions in 6 mmolar Calcium Chloride decreased with increasing dilution--despite the diminished rate of collision in diluted systems. In addition, and increased electrolyte sensitivity was observed with increasing age of the emulsions--despite enhanced electrostatic stabilization by an increased zeta-potential. Both effects were attributed to an increased binding of calcium ions per surface area of the droplets, i.e. increased ratio calcium ions to surface by dilution and increased binding by the increased charge of aged emulsions.

'Stealth' corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption.

Nanoparticles possessing poly(ethylene glycol) (PEG) chains on their surface have been described as blood persistent drug delivery system with potential applications for intravenous drug administration. Considering the importance of protein interactions with injected colloidal dug carriers with regard to their in vivo fate, we analysed plasma protein adsorption onto biodegradable PEG-coated poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA) and poly(varepsilon-caprolactone) (PCL) nanoparticles employing two-dimensional gel electrophoresis (2-D PAGE). A series of corona/core nanoparticles of sizes 160-270 nm were prepared from diblock PEG-PLA, PEG-PLGA and PEG-PCL and from PEG-PLA:PLA blends. The PEG Mw was varied from 2000-20000 g/mole and the particles were prepared using different PEG contents. It was thus possible to study the influence of the PEG corona thickness and density, as well as the influence of the nature of the core (PLA, PLGA or PCL), on the competitive plasma protein adsorption, zeta potential and particle uptake by polymorphonuclear (PMN) cells. 2-D PAGE studies showed that plasma protein adsorption on PEG-coated PLA nanospheres strongly depends on the PEG molecular weight (Mw) (i.e. PEG chain length at the particle surface) as well as on the PEG content in the particles (i.e. PEG chain density at the surface of the particles). Whatever the thickness or the density of the corona, the qualitative composition of the plasma protein adsorption patterns was very similar, showing that adsorption was governed by interaction with a PLA surface protected more or less by PEG chains. The main spots on the gels were albumin, fibrinogen, IgG, Ig light chains, and the apolipoproteins apoA-I and apoE. For particles made of PEG-PLA45K with different PEG Mw, a maximal reduction in protein adsorption was found for a PEG Mw of 5000 g/mole. For nanospheres differing in their PEG content from 0.5 to 20 wt %, a PEG content between 2 and 5 wt % was determined as a threshold value for optimal protein resistance. When increasing the PEG content in the nanoparticles above 5 wt % no further reduction in protein adsorption was achieved. Phagocytosis by PMN studied using chemiluminescence and zeta potential data agreed well with these findings: the same PEG surface density threshold was found to ensure simultaneously efficient steric stabilization and to avoid the uptake by PMN cells. Supposing all the PEG chains migrate to the surface, this would correspond to a distance of about 1.5 nm between two terminally attached PEG chains in the covering 'brush'. Particles from PEG5K-PLA45K, PEG5K-PLGA45K and PEG5K-PCL45K copolymers enabled to study the influence of the core on plasma protein adsorption, all other parameters (corona thickness and density) being kept constant. Adsorption patterns were in good qualitative agreement with each other. Only a few protein species were exclusively present just on one type of nanoparticle. However, the extent of proteins adsorbed differed in a large extent from one particle to another. In vivo studies could help elucidating the role of the type and amount of proteins adsorbed on the fate of the nanoparticles after intraveinous administration, as a function of the nature of their core. These results could be useful in the design of long circulating intravenously injectable biodegradable drug carriers endowed with protein resistant properties and low phagocytic uptake.

The influence of alkali fatty acids on the properties and the stability of parenteral O/W emulsions modified with solutol HS 15.

Arachis oil based parenteral O/W emulsions were prepared using soya bean phosphatidylcholine (SPC) and different combinations of co-emulsifiers containing polyethylene glycol fatty acid esters (Solutol HS 15) and alkali fatty acids (sodium laurate, sodium stearate). The parameters measured were droplet size (both by photon correlation spectroscopy and laser diffractometry), pH and zeta potential. All emulsions were subjected to autoclaving. The addition of polyethylene glycol 12-hydroxy stearate (Solutol HS 15) led to a significant decrease of mean oil droplet size. For long-term stability the amount added turned out to be the most important factor. With increased amounts of Solutol HS 15 the packing density of the emulsifier layer and the zeta potential decreased leading to instability. The optimum load of Solutol HS 15 was found to be 15 micromol/ml. Alkali fatty acids markedly improved the physical stability of the emulsions. Improved stability properties conferred to emulsions by alkali fatty acids could be attributed to the zeta potential increase even in the presence of Solutol HS 15. Consequently a mixed emulsifier film was established in which the ionized fatty acids determined the interface charge. In addition to this a strengthening of the molecular interactions occurring between phospholipid and Solutol HS 15 emulsifier in the presence of ionized fatty acids at the O/W interface can be assumed (L. Rydhag, The importance of the phase behaviour of phospholipids for emulsion stability, Fette Seifen Anstrichm. 81 (1979) 168-173). Different co-emulsifier mixtures were shown to have a pronounced impact on the plasma protein adsorption onto emulsion droplets.

Adsorption kinetics of plasma proteins on oil-in-water emulsions for parenteral nutrition.

The plasma protein adsorption patterns on colloidal drug carriers are regarded as an important factor for their in vivo fate. In this study the adsorption kinetics on oil-in-water emulsions were determined and compared to the adsorption kinetics on polystyrene particles. In addition, the adsorption kinetics on the same systems after surface-modification with a hydrophilic polymer were also investigated. The protein adsorption was determined by means of two dimensional polyacrylamide gel electrophoresis (2D-PAGE). The determination of the plasma protein adsorption kinetics was carried out using different concentrations of human plasma in the incubation medium to prolong the residence time of the more abundant plasma proteins on the surface. Proteins which are likely to be displaced in a split second are thus accessible to analysis. The oil-in-water emulsion showed a distinctly different adsorption behavior from the one previously described for solid surfaces, where initially adsorbed proteins are displaced by others, having a higher affinity to the surface ('Vroman effect'). No competitive protein adsorption could be observed on the emulsions. Moreover, the predominantly adsorbed apolipoproteins A-I, A-IV, C-II and C-III increase in amount with increasing plasma concentration. The knowledge of the adsorption kinetics of colloidal carriers might be helpful for a better understanding of the in vivo behavior of such systems and for the transfer of principles already known from other carrier systems to the controlled development of emulsions for site specific drug delivery.

Visualization of in vitro protein-rejecting properties of PEGylated stealth polycyanoacrylate nanoparticles.

The in vitro protein-rejecting properties of PEG-coated polyalkylcyanoacrylate (PACA) nanoparticles were for the first time visualized after freeze-fracture of the nanoparticles pre-incubated with fibrinogen as a model blood protein. The reduced protein association to the nanoparticles was evidenced also by two-dimensional PAGE after incubation of the nanoparticles with human plasma. In vivo experiments showed the 'stealth' long-circulating properties of the PEGylated nanoparticles after intravenous administration to mice. Thus, the images obtained after nanoparticle-protein incubation were predictive of the behavior observed in vivo. In conclusion, freeze-fracture analysis represents a novel and original qualitative approach to investigate the interactions between proteins and particulate systems.

Plasma protein adsorption patterns on emulsions for parenteral administration: establishment of a protocol for two-dimensional polyacrylamide electrophoresis.

The two-dimensional polyacrylamide electrophoresis (2-D PAGE) of the plasma protein adsorption pattern previously established for polymeric nanoparticles was modified and transferred to oil in water emulsions for intravenous administration. The emulsions were incubated with citrated plasma, and separation from excess plasma was performed by centrifugation under optimized conditions: 15000 g and three washing steps with 0.05 M phosphate buffer, pH 7.4. With this sample preparation, coalescence of droplets could be avoided and an unchanged surface area maintained, in addition the phosphate buffer minimized artificial IgG adsorption. Critical factors affecting sensitivity were contamination of the sample by oil residues and the use of thiourea in the immobilized pH gradients. Changes in the protein adsorption pattern caused by altered surface properties of the emulsion (i.e. adsorbed Poloxamer 407) were detectable when applying the optimized protocol. Knowledge of the protein adsorption patterns and their correlation to in vivo behavior opens the perspective for the development of intravenous emulsions for controlled drug delivery.

Identification of plasma proteins facilitated by enrichment on particulate surfaces: analysis by two-dimensional electrophoresis and N-terminal microsequencing.

Plasma protein adsorption on intravenously injectable drug carriers is regarded as an important factor for the fate of the particles in the body after their administration. Therefore, the plasma protein adsorption patterns on a number of different carrier systems were analyzed in vitro employing two-dimensional electrophoresis (2-DE). The particulate systems presented in this study were polystyrene (PS) model particles, PS nanoparticles surface-modified by adsorption of a surfactant, a commercial fat emulsion, and magnetic iron oxide particles used as contrast agents in magnetic resonance imaging. Most of the spots in the plasma protein adsorption patterns could be identified by matching the resulting 2-DE gels with a reference map of human plasma proteins. Several other proteins that indicated preferentially adsorbed proteins on the surface of the particles investigated have either not been identified on the reference map, or their identity was found to be ambiguous. The relevant proteins are all present in plasma in low abundance. Since these proteins were strongly enriched on the surface of the particles, the resulting spots on the 2-DE gels were successfully identified by N-terminal microsequencing. With this approach, two chains of spots, designated PLS:6 and PLS:8, were determined on a plasma reference map: inter-alpha-trypsin inhibitor family heavy chain-related protein (also named PK-120) and a dimer of fibrinogen gamma, respectively. Plasma gelsolin is presented in a 2-DE adsorption pattern of PS model particles. One of the main proteins adsorbed by droplets of a commercial fat emulsion was identified as apoliprotein H. Moreover, the positions of apolipoproteins apoC-II and apoC-III were also verified on the 2-DE protein map of human plasma. Thus, protein adsorption experiments of the kind presented in this study are increasing our insight into human plasma proteins.