A consensus research agenda for optimising nasal drug delivery

Nasal drug delivery has specific challenges which are distinct from oral inhalation, alongside which it is often considered. The next generation of nasal products will be required to deliver new classes of molecule, e.g. vaccines, biologics and drugs with action in the brain or sinuses, to local and systemic therapeutic targets. Innovations and new tools/knowledge are required to design products to deliver these therapeutic agents to the right target at the right time in the right patients. We report the outcomes of an expert meeting convened to consider gaps in knowledge and unmet research needs in terms of (i) formulation and devices, (ii) meaningful product characterization and modeling, (iii) opportunities to modify absorption and clearance. Important research questions were identified in the areas of device and formulation innovation, critical quality attributes for different nasal products, development of nasal casts for drug deposition studies, improved experimental models, the use of simulations and nasal delivery in special populations. We offer these questions as a stimulus to research and suggest that they might be addressed most effectively by collaborative research endeavors

pH Dependence of Tryptophan Synthase Catalytic Mechanism: I. The first stage, the beta-elimination reaction

The pyridoxal 5′-phosphate-dependent β-subunit of the tryptophan synthase α2β2 complex catalyzes the condensation of L-serine with indole to form L-tryptophan. The first stage of the reaction is a β-elimination that involves a very fast interconversion of the internal aldimine in a highly fluorescent L-serine external aldimine that decays, via the α-carbon proton removal and β-hydroxyl group release, to the α-aminoacrylate Schiff base. This reaction is influenced by protons, monovalent cations, and α-subunit ligands that modulate the distribution between open and closed conformations. In order to identify the ionizable residues that might assist catalysis, we have investigated the pH dependence of the rate of the external aldimine decay by rapid scanning UV-visible absorption and single wavelength fluorescence stopped flow. In the pH range 6-9, the reaction was found to be biphasic with the first phase (rate constants k1) accounting for more than 70% of the signal change. In the absence of monovalent cations or in the presence of sodium and potassium ions, the pH dependence of k1 exhibits a bell shaped profile characterized by a pKa1 of about 6 and a pKa2 of about 9, whereas in the presence of cesium ions, the pH dependence exhibits a saturation profile characterized by a single pKa of 9. The presence of the allosteric effector indole acetylglycine increases the rate of reaction without altering the pH profile and pKa values. By combining structural information for the internal aldimine, the external aldimine, and the α-aminoacrylate with kinetic data on the wild type enzyme and β-active site mutants, we have tentatively assigned pKa1 to βAsp-305 and pKa2 to βLys-87. The loss of pKa1 in the presence of cesium ions might be due to a shift to lower values, caused by the selective stabilization of a closed form of the β-subunit

Snapshots of the cystine lyase C-DES during catalysis

The cystine lyase (C-DES) of Synechocystis is a pyridoxal-5′-phosphate-dependent enzyme distantly related to the family of NifS-like proteins. The crystal structure of an N-terminal modified variant has recently been determined. Herein, the reactivity of this enzyme variant was investigated spectroscopically in solution and in the crystalline state to follow the course of the reaction and to determine the catalytic mechanism on a molecular level. Using the stopped-flow technique, the reaction with the preferred substrate cystine was found to follow biphasic kinetics leading to the formation of absorbing species at 338 and 470 nm, attributed to the external aldimine and the α-aminoacrylate; the reaction with cysteine also exhibited biphasic behavior but only the external aldimine accumulated. The same reaction intermediates were formed in crystals as seen by polarized absorption microspectrophotometry, thus indicating that C-DES is catalytically competent in the crystalline state. The three-dimensional structure of the catalytically inactive mutant C-DESK223A in the presence of cystine showed the formation of an external aldimine species, in which two alternate conformations of the substrate were observed. The combined results allow a catalytic mechanism to be proposed involving interactions between cystine and the active site residues Arg-360, Arg-369, and Trp-251*; these residues reorient during the β-elimination reaction, leading to the formation of a hydrophobic pocket that stabilizes the enolimine tautomer of the aminoacrylate and the cysteine persulfide product

Sequential \u201casymmetric\u201d D-optimal designs: a practical solution in case of limited resources and not equally expensive experiments

When the cost of an experiment depends on the values of the variables not all the experiments are equally expensive. Since in all the standard Designs of Experiments the experiments are symmetrically distributed, in case of finite resources the number of experiments is limited by the cost of the most expensive ones. The approach here shown produces asymmetrical designs, with an increase of the number of the cheapest experiments and a decrease of the number of the expensive ones. Compared to the standard designs having the same total cost, the information obtained by this strategy is slightly worse in the region corresponding to the most expensive experiments, but much better in the region corresponding to the cheapest experiments. This approach was successfully applied to the micronization of an Active Pharmaceutical Ingredient