Dry-powder inhalers: evaluation of testing methodology and effect of inhaler design.

We have previously developed a spray dried formulation of a model protein (beta-galactosidase) of a size suitable for evaluation in dry powder inhaler devices. In this study, we wished to evaluate the roles of various methods available for the laboratory testing of dry powders for inhalation (cascade impactor, twin impinger, aerodynamic time of flight and image analysis). Secondly we wished to compare different inhaler devices using formulations with and without a carrier. Both the cascade impactor and twin impinger were appropriate methods for the testing of dry powder inhalers, and gave comparable estimates of respirable fraction. Image analysis and aerodynamic time of flight were suitable methods for determining the particle size of the dry powders, although the former was considerably more time consuming than the latter. The four inhalers evaluated differed greatly in terms of in vitro deposition properties. The presence of a carrier significantly improved respirable fraction with the poorer inhalers, but was less critical to the performance of the more efficient devices.

The effect of process and formulation variables on the properties of spray-dried beta-galactosidase.

The objective of this study was to evaluate the joint effects of various processing and formulation variables on the properties of spray-dried beta-galactosidase using statistically designed experiments. The key response variables evaluated were product yield, residual enzymatic activity, moisture content and particle size and appearance. The residual enzymatic activity and product yield were significantly affected by the processing variables. The highest product yields were obtained when the drier outlet temperature was relatively high, resulting in extensive protein denaturation. Subsequent experiments, therefore, compared the relative effectiveness of four stabilizers (mannitol, sucrose, arginine hydrochloride and trehalose) in terms of their ability to preserve enzymatic activity during the spray-drying process and during long-term storage. Trehalose was the most suitable stabilizer. The effect of a number of other formulation variables (total solids level, ratio of stabilizer to protein, presence of surfactant and presence of buffer) was also investigated. A final formulation consisting of 6% beta-galactosidase and 10% trehalose in deionized water was selected. Spray-drying at inlet and outlet temperatures of 140 and 95 degrees C, respectively, results in greater than 70% yields of a fully active product with a moisture content of 2-5% and a mean particle size of 2-4 microns.

Reversal of colchicine-induced mitotic arrest in Chinese hamster cells with a colchicine-specific monoclonal antibody.

The ability of a high-affinity colchicine-binding monoclonal antibody to reverse the effects of colchicine on Chinese hamster ovary cells was investigated. Using flow cytometry, a complete mitotic blockade was demonstrated after 16 hours with 2.5 x 10(-7) mol/l (molar) colchicine. Colchicine-induced changes were reversible when equimolar antibody was added simultaneously with or up to 6 hours after colchicine. With further delay in addition of antibody, a progressive irreversible increase in mitotic blockade and increase in mean cell size was observed. Prolonged colchicine exposure, without antibody reversal, led to polyploidy and structural chromosome breakage. Early antibody reversal restored cells to the diploid state, whereas delayed reversal resulted in a time-dependent increase in polyploidy. Colchicine-induced polyploidy and chromosomal aberrations may be the basis for both colchicine toxicity and the time-dependent increase in irreversibility of colchicine effects.

Specific, high affinity colchicine binding monoclonal antibodies: development and characterization of the antibodies.

Nine colchicine specific monoclonal antibodies have been developed by immunizing BALB/c mice with a colchicine-keyhole limpet hemocyanin (Col-KLH) conjugate prepared using a bishydroxysuccinimide coupling reagent. Of four immunization procedures examined, intraperitoneal injection of the antigen attached to acid treated E. coli resulted in the maximum antigen specific antibody titers. A colchicine bovine serum albumin (Col-BSA) conjugate, prepared using a water soluble carbodiimide coupling technique, formed the basis of an enzyme linked immunosorbent assay used for screening hybridomas for colchicine specific antibody secretion and for determining the relative affinity and specificity profile of the monoclonal antibodies. All antibodies demonstrated high affinity, saturable binding to colchicine and low cross-reactivity with a panel of compounds structurally related to colchicine. The IC50 for the highest affinity antibody, C44, was 3.6 +/- 0.84 nM colchicine in the competitive enzyme immunoassay. The affinity of this antibody determined from Scatchard analysis of antibody binding to tritiated colchicine was 0.66 +/- 0.11 nM. Antibody C44 has the level of specificity and affinity suitable for a sensitive and selective immunoassay of colchicine for monitoring therapeutic drug levels. In addition, this antibody provides a specific pharmacologic antagonist for studies of colchicine's therapeutic mechanism and has the potential to reverse colchicine toxicity.