Critical steps during the prilling process of molten lipids: Main stumbling blocks due to pharmaceutical excipient properties.

Prilling by ultrasonic jet break-up is an efficient process to produce perfectly spherical microparticles homogeneous in size. However, the material properties could affect the manufacturability and the final product properties especially with lipid-based excipients which often exhibit complex structural properties. This work presents the characterisation of six lipid-based excipients differing by their melting point and polymorphic behaviour which were used to produce microspheres using a pilot-scale prilling equipment. The experimental results were compared to theoretical calculations, especially the droplet solidification time which is a key-parameter for this process. This work highlighted that monotropic polymorphism of excipients and supercooling effect have a significant impact on process parameters which should be considered with care during formulation design.

Critical parameters involved in producing microspheres by prilling of molten lipids: from theoretical prediction of particle size to practice.

The aim of this work was to identify the key parameters which influence the running of the prilling process with lipid material from the initial melting to the formation of solid microspheres. The microsphere size would essentially result from break-up at the Rayleigh-Weber's wavelength which mostly depends on the liquid properties (mass density, surface tension and dynamic viscosity). After molten liquid extrusion through the nozzle, the cooling rate is very fast and the instantaneous temperature of the liquid jet decreases rapidly of 0.2-0.3 °C during the duration of the droplet formation (1-2 ms). This leads to no significant modification of the physical characteristics of the lipids and only a very slight change in the dynamic viscosity. Consequently, no significant effect on the optimal wavelength λ(W) and on droplet formation can occur. However, coalescence of liquid droplets has been observed during their fall, probably caused by turbulence into the air column, leading to a minor population of larger microspheres.

The crystal structure of a complex of p11 with the annexin II N-terminal peptide.

The aggregation and membrane fusion properties of annexin II are modulated by the association with a regulatory light chain called p11.p11 is a member of the S100 EF-hand protein family, which is unique in having lost its calcium-binding properties. We report the first structure of a complex between p11 and its cognate peptide, the N-terminus of annexin II, as well as that of p11 alone. The basic unit for p11 is a tight, non-covalent dimer. In the complex, each annexin II peptide forms hydrophobic interactions with both p11 monomers, thus providing a structural basis for high affinity interactions between an S100 protein and its target sequence. Finally, p11 forms a disulfide-linked tetramer in both types of crystals thus suggesting a model for an oxidized form of other S100 proteins that have been found in the extracellular milieu.

The interaction of metal ions with annexin V: a crystallographic study.

Three closely related rhombohedral crystal structures of human annexin V have been analysed and compared: a low-calcium, a high-calcium and an ytterbium-soaked crystal. The occupancy of the calcium sites increases at higher calcium concentrations, but the calcium is removed rather than replaced during soaking in the ytterbium solution. Instead, other sites are substituted at high calcium concentrations as well as in the presence of ytterbium. Furthermore, a new site is revealed in the ytterbium-soaked crystal which may give a clue to the mechanism of conformational change that takes place in the third domain of annexin V in the presence of very high calcium concentrations and of phospholipids.

The crystal structure of a new high-calcium form of annexin V.

Annexin V was crystallized in the presence of a high concentration of calcium and the structure refined at 1.9 A resolution. The crystals are triclinic (P1) with three molecules per asymmetric unit and pseudo-R3 symmetry, reflecting a tendency of annexin to form trimers. The overall structure of the protein is similar to that seen in other crystal forms. There are, however, significant changes in domain III, where a new calcium site is formed. The whole region surrounding this site is reorganized in our structure, rendering annexin V more symmetrical and more alike annexin I. The formation of the new calcium site causes the displacement of Trp187 from a buried to an exposed conformation, a change that has recently been demonstrated by fluorescence measurements. The affinity of the different potential calcium sites is modulated: there is no calcium bound in domains II and IV, while up to two secondary calcium ions sites (in domains I and III) can substitute, depending on the calcium concentration present. We suggest that annexin can act as a calcium buffer, binding or releasing calcium depending on its local concentration. Our results also show that annexin displays inherent mobility which, together with its capacity to modulate the calcium affinity of its sites, can be of importance for its function on the membrane surface.