Ex-vivo perfusion bioassay : an excellent technique to measure the bioactivity of inhalable insulin coated microcrystals

Purpose: To measure the bioactivity of inhalable insulin coated microcrystals using a perfusion bioassay that measures its vasodilatory effect on smooth muscle arterial tissue. Methods: The bioactivity of an insulin protein coated microcrystal (PCMC), a potential candidate for pulmonary drug delivery and commercial insulin was determined on a Danish Myo Tech P110 pressure myograph system. 12 week old Mesenteric resistance arteries from Male Wistar rats were isolated and immersed in a physiological salt solution (PSS) and attached to 2 opposing hollow glass micro-cannula (outer diameter 80 microns). The PSS was gradually warmed to 37°C (at a pressure less than 5mm Hg) for 1hr. Subsequently the pressure was increased up to 40mm Hg over a period 15 minutes and equilibrated for a further 15 minutes after gassing with 95%O2 / 5%CO2 to achieve a pH of 7.4 at 37°C. After normalisation by two washes of 123mM KCl and exposure to 1-10mM noradrenaline the arteries were exposed intraluminally to each insulin preparation by gradual infusion directly into the lumen via a fetal microcannulae inserted to the tip of the glass mounting cannula, at a constant pressure. Results: The preliminary results (full cummulative response curve yet to be determined) demonstrate insulin mediated relaxation to noradrenaline preconstriction. The level of constriction drops from 100% to 42% as the concentration of insulin increases from -11 to -9 Log M for the PCMC compared with a drop from 100 % to 65% for the commercial insulin preparation. However the more potent vasodilatory effect found for the insulin PCMC is more likely to be a result of variance introduced in each dilution step than a real increase in potency. Conclusion: The perfusion bioassay technique provides an excellent method of measuring insulin bioactivity and indicates the insulin loaded on the microcrystal support is fully active

Dry powder therapeutic mAb formulations with enhanced temperature stability

Purpose. There is an increasing demand for differentiated strategies for formulating and delivering mAb in particulate form. The aim of this study was to investigate methods for optimising protein-coated microcrystal (PCMC) formulations of a human monoclonal antibody. PCMC technology provides a novel method of stabilising these important biopharmaceuticals in the form of dry powders. Methods. Human monoclonal antibody coated microcrystals were prepared by coprecipitation of an aqueous mixture of PBS buffered human monoclonal antibody and concentrated glycine into propan-2-ol. The standard formulation contained mAb buffer salts and glycine and the effect of including potential precipitation stabilizing additives (PSA) was investigated e.g. Glu, Arg. Following coprecipitation, the PCMC particles were filtered and air-dried to form free-flowing dry powders. Protein integrity was assessed by comparing optical clarity of the reconstituted formulations, protein concentration by UV spectroscopy and monomer content by size-exclusion chromatography, using a Tosoh TSKgel G3000 SWXL 7.8 mm ID x 30 cm column. Results. The measured protein loadings were found to be within 5% of the target protein loading for all formulations. However, the optical clarity of reconstituted PCMC prepared with optimal PSA was significantly better. Protein particulates could be observed in reconstituted standard samples, whilst PCMC utilising PSA, such as Glu, Arg, were optically clear and free of particulates. Consistent with this, measurement of the degree of monomer conservation following coprecipitation showed that material produced without PSA generally had monomer retention of less than 90% whilst optimised samples incorporating PSA resulted in >98% of monomer conservation. Conclusion. Human monoclonal antibodies can be readily formulated using PCMC technology by incorporating precipitation stabilizing additives (PSA). PCMC coprecipitation leads to finely-divided dry powders, which can be rapidly reconstituted back into aqueous, to release the monoclonal antibody in monomeric form

Precipitation stabilising compositions

The present invention relates to maintaining bioactive molecules in their native or substantially near-native form and preventing or reduce aggregation. In particular, the present invention relates to precipitation-protective or stabilising additives and a method of using said precipitation-protective/stabilising additives to protect and maintain the bioactive molecules in a native or substantially near-native form and to prevent or reduce aggregation during or following precipitation to form particles

Slow release compositions

The present invention relates to the provision of micron or sub-micron sized particles formed from one or more water-soluble crystals comprising a surface coating comprising one or more bioactive molecules wherein the particles are prepared such that in use the release of the bioactive molecule(s) is/are delayed and/or continually released over a period of time. Processes for the preparation of said particles, as well as the particles themselves are described, as well as uses of the particles

Tuning of the product spectrum of vanillyl-alcohol oxidase by medium engineering

The flavoenzyme vanillyl-alcohol oxidase (VAO) catalyzes the conversion of 4-alkylphenols through the initial formation of p-quinone methide intermediates. These electrophilic species are stereospecifically attacked by water to yield (R)-1-(4'- hydroxyphenyl) alcohols or rearranged in a competing reaction to 1-(4'-hydroxyphenyl)alkenes. Here, we show that the product spectrum of VAO can be controlled by medium engineering. When the enzymatic conversion of 4-propylphenol was performed in organic solvent, the concentration of the alcohol decreased and the concentration of the cis-alkene, but not the trans-alkene, increased. This change in selectivity occurred in both toluene and acetonitrile and was dependent on the water activity of the reaction medium. A similar shift in alcohol/cis-alkene product ratio was observed when the VAO-mediated conversion of 4- propylphenol was performed in the presence of monovalent anions that bind specifically near the enzyme active site. (C) 2001 Federation of European Biochemical Societies