Nasal administration of influenza vaccine has the potential to facilitate influenza control and prevention. However, when administered intranasally (i.n.), commercially available inactivated vaccines only generate systemic and mucosal immune responses if strong adjuvants are used, which are often associated with safety problems. We describe the successful use of a safe adjuvant Gram-positive enhancer matrix (GEM) particles derived from the food-grade bacterium Lactococcus lactis for i.n. vaccination with subunit influenza vaccine in mice. It is shown that simple admixing of the vaccine with the GEM particles results in a strongly enhanced immune response. Already after one booster, the i.n. delivered GEM subunit vaccine resulted in hemagglutination inhibition titers in serum at a level equal to the conventional intramuscular (i.m.) route. Moreover, i.n. immunization with GEM subunit vaccine elicited superior mucosal and Th1 skewed immune responses compared to those induced by i.m. and i.n. administered subunit vaccine alone. In conclusion, GEM particles act as a potent adjuvant for i.n. influenza immunization.

Two force fields, the GROMOS53A5/53A6 (united atom) and the AMBER95 (all atom) parameter sets, coupled with partial atomic charges derived from quantum mechanical calculations were evaluated for their ability to reproduce the known crystalline forms of the polyols mannitol and sorbitol. The force fields were evaluated using molecular dynamics simulations at 10 K (which is akin to potential energy minimization) with the simulation cell lengths and angles free to evolve. Both force fields performed relatively poorly, not being able to simultaneously reproduce all of the crystal structures within a 5% deviation level. The parameter sets were then systematically optimized using sensitivity analysis, and a revised AMBER95 set was found to reproduce the crystal structures with less than 5% deviation from experiment. The stability of the various crystalline forms for each of the parameter sets (original and revised) was then assessed in extended MD simulations at 298 K and 1 bar covering 1 ns simulation time. The AMBER95 parameter sets (original and revised) were found to be effective in reproducing the crystal structures in these more stringent tests. Remarkably, the performance of the original AMBER95 parameter set was found to be slightly better than that of the revised set in these simulations at 298 K. The results of this study suggest that, whenever feasible, one should include molecular simulations at elevated temperatures when optimizing parameters.

Tetracycline (Tc) antibiotics have been put to new uses in the construction of artificial gene regulation systems, where they bind to the Tet repressor protein (TetR) and modulate its affinity for DNA. Many Tc variants have been produced, both to overcome bacterial resistance and to achieve a broad range of binding strengths. To better understand TetR-Tc binding, we investigate a library of 16 tetracyclines, using fluorescence experiments and molecular dynamics free energy simulations (MDFE). The relative TetR binding free energies are computed by reversibly transforming one Tc variant into another during the simulation, with no adjustable parameters. The chemical variations involve polar and nonpolar substitutions along one entire edge of the elongated Tc structure, which provides many of the protein-ligand contacts. The binding constants span five orders of magnitude. The simulations reproduce the experimental binding free energies, when available, within the uncertainty of either method (+/-0.5 kcal/mol), and reveal many additional details. Contributions of individual Tc substituents are evaluated, along with their additivity and transferability among different positions on the Tc scaffold; differences between D- and B-class repressors are quantified. With increasing computer power, the MDFE approach provides an attractive complement to experiment and should play an increasing role in the understanding and engineering of protein-ligand recognition.

TMC240 is a very poorly soluble and poorly permeating HIV protease inhibitor. In order to enhance its oral bioavailability, a fast dissolving inulin based solid dispersion tablet was developed. During the dissolution test in water (0.5 or 1.0% SLS), this tablet released at least 80% of TMC240 within 30 min, while the physical mixture tablet of the same composition released only 6% after 2 h. In a subsequent single-dose study in dogs (200 mg of TMC240), plasma concentrations of TMC240 remained below the lower limit of quantification (<1.00 ng/mL) in all animals (n=3 per tested formulation), except in one dog receiving the inulin solid dispersion tablet (C(max)=1.8 ng/mL, AUC(0-7h)=3.0 ng.h/mL). In the latter treatment group, ritonavir co-administration (10 mg/kg b.i.d.) increased TMC240 exposure more than 30-fold (mean AUC(0-7h)=108 ng.h/mL; F(rel)=3588%). Exposure was also 16-fold higher than after TMC240 administration as PEG400 suspension in presence of ritonavir (AUC(0-7h)=6.7 ng.h/mL). The current data demonstrate that a solid dispersion of TMC240 in an inulin matrix allows considerable improvement in the release of poorly water-soluble TMC240, both in vitro in presence of a surfactant and in vivo upon oral administration.

The crucial event in the development of transmissible spongiform encephalopathies (TSEs) is the conformational change of a host-encoded membrane protein - the cellular PrP(C)- into a disease associated, fibril-forming isoform PrP(Sc). This conformational transition from the alpha-helix-rich cellular form into the mainly beta-sheet containing counterpart initiates an 'autocatalytic' reaction which leads to the accumulation of amyloid fibrils in the central nervous system (CNS) and to neurodegeneration, a hallmark of TSEs. The exact molecular mechanisms which lead to the conformational change are still unknown. It also remains to be brought to light how a polypeptide chain can adopt at least two stable conformations. This review focuses on structural aspects of the prion protein with regard to protein-protein interactions and the initiation of prion protein misfolding. It therefore highlights parts of the protein which might play a notable role in the conformational transition from PrP(C) to PrP(Sc) and consequently in inducing a fatal chain reaction of protein misfolding. Furthermore, features of different proteins, which are able to adopt insoluble fibrillar states under certain circumstances, are compared to PrP in an attempt to understand the unique characteristics of prion diseases.

Crystal structures of polypeptide deformylase (PDF) of Escherichia coli with nickel(II) replacing the native iron(II) have been solved with chloride and formate as metal ligands. The chloro complex is a model for the correct protonation state of the hydrolytic hydroxo ligand and the protonated status of the Glu133 side chain as part of the hydrolytic mechanism. The ambiguity that recently some PDFs have been identified with Zn(2+) ion as the active-site centre whereas others are only active with Fe(2+)(or Co(2+), Ni(2+)) is discussed with respect to Lewis acid criteria of the metal ion and substrate activation by the CD loop.

In a previous study we have developed a novel process to produce drug nanocrystals. This process,"controlled crystallization during freeze-drying" has shown to be a successful method to increase the dissolution rate of poorly water soluble drugs [de Waard et al., 2008. J. Control. Release 128, 179]. This process consisted of two steps: a solution of a matrix material (mannitol) in water was mixed with a solution of a drug (fenofibrate) in tertiary butyl-alcohol (TBA). This mixture was frozen and subsequently freeze-dried at relatively high temperature (-25 degrees C). Since the solution of matrix and drug in the water-TBA mixture is thermodynamically unstable, it had to be frozen immediately and fast after preparation to prevent premature crystallization of the drug resulting in the formation too large drug crystals. Therefore, small quantities were manually mixed in a vial and this vial was immersed in liquid nitrogen. To make this process ready for large scale production, the modification of this batch process to a semi-continuous process by the application of a 3-way nozzle was studied. With this nozzle, the aqueous and TBA-solutions were pumped into the nozzle via two separate channels and mixed just at the moment they left the nozzle. Thorough mixing was facilitated by the atomizing air, supplied via the third channel. Since the mixture was sprayed immediately into liquid nitrogen, premature crystallization was prevented. A further advantage was that the atomizing air generated small droplets which were directly immersed into liquid nitrogen. Consequently, the mixture was frozen even faster than in the batch process. This resulted in a reduced size of the drug crystals and hence a higher dissolution rate. Therefore, using the semi-continuous process does not only result in successfully making this process suitable for large scale production of the controlled crystallized dispersions, but it also results in a better product.

Two mutants of the toxic extracellular zinc endopeptidase AsaP1 (AsaP1_E294Q and AsaP1_E294A) of Aeromonas salmonicida subsp. achromogenes were expressed in Escherichia coli and crystallized by the vapour-diffusion method. Crystals were obtained using several precipitants and different protein concentrations. Protein crystals were found in a monoclinic (C2) as well as an orthorhombic (P2(1)2(1)2(1)) space group. The crystals belonging to the monoclinic space group C2 had unit-cell parameters a = 103.4, b = 70.9, c = 54.9 A, beta = 109.3 degrees for AsaP1_E294A, and a = 98.5, b = 74.5, c = 54.7 A, beta = 112.4 degrees for AsaP1_E294Q. The unit-cell parameters of the orthorhombic crystal obtained for AsaP1_E294A were a = 57.9, b = 60.2, c = 183.6 A. The crystals of the two different mutants diffracted X-rays beyond 2.0 A resolution.

A formulation and process development study was performed to formulate recombinant human deoxyribonuclease I as a powder for inhalation. First, excipient compatibility (with bovine DNase as a model substance) was examined with a stability study at stressed conditions (60 and 85 degrees C) while monitoring for occurrence of the Maillard reaction. Next, powders for inhalation were prepared by spray drying and spray freeze drying. We found that spray drying with inulin as stabilizer resulted in the best powder for inhalation. Finally, an ex-vivo test with the spray dried rhDNase I/inulin powder significantly decreased elastic and viscous moduli of sputum from five cystic fibrosis patients.

In this study it was shown that the incorporation of superdisintegrants in solid dispersion tablets containing a high drug load can strongly enhance the dissolution rate of the highly lipophilic drug fenofibrate. In addition, the dissolution rate was more increased when the superdisintegrant was incorporated in the drug containing solid dispersions than when it was physically mixed with the solid dispersions. The dissolution rate enhancement strongly depended on the type of superdisintegrant and increased in the order: Polyplasdone((R)) XL-10 < Polyplasdone((R)) XL << Ac-Di-Sol((R)) approximately Primojel((R)). The dissolution behavior also depended on the type of hydrophilic carrier. Solid dispersion tablets based on inulin 4 kDa, polyethylene glycol 20K and polyvinylpyrrolidone K30 showed a much faster dissolution than those based on mannitol and hydroxypropyl-beta-cyclodextrin. Finally, inulin 4 kDa based solid dispersion tablets showed an excellent storage stability while polyethylene glycol 20K and polyvinylpyrrolidone K30 based solid dispersion tablets did not.