The aerodynamic properties and particle distribution of the powder were determined with standard methodologies by using an eight stage Andersen nonviable 1ACFM cascade impactor and hand held, breath activated, capsule based dry powder inhaler device. Fostamatinib R788 The morphology of the dry particles was evaluated using a 982 field emission scanning electron microscope after coating powder samples with a platinum/palladium layer. The PA 824 content of the spray dried powder was determined by a reversephase high performance liquid chromatography method using an Agilent Technologies 1100 series HPLC system. The mobile phase was run on a linear gradient from 20% acetonitrile and 80% water to 60% acetonitrile and 40% water over 30 min with 5 min of equilibration time. Analysis was performed on a 50 l injection volume at a flow rate of 1. 5 ml/min through an Agilent Zorbax Eclipse XDB C18 column, and absorbance was recorded at 330 nm.
An Agilent Zorbax Eclipse XDB C18 analytical guard column was also used. Respiratory infection. All animal procedures were approved by the University of North Carolina Chapel Hill Institutional Animal Care and Use Committee. Specific Cellular differentiation pathogen free male Dunkin Hartley guinea pigs weighing 369 45 g were housed individually in a biosafety level 3 containment area with a 12 h light/dark cycle. Animals were allowed free access to water and food at all times. Animals were infected via the respiratory route with a small inoculum of M. tuberculosis strain H37Rv. Animals were placed randomly in an exposure chamber, and aerosols were generated by pumping compressed air through a modified MRE type 3 jet Collison nebulizer containing 5 ml of bacterial suspension.
Validation of this procedure indicated Flupirtine that it results in the inhalation and retention of 10 to 15 viable, virulent organisms per guinea pig. Animals remained untreated for 4 weeks following infection, when the bacterial burden is known to plateau. Body weights of each animal were recorded throughout the study, as were changes in behavior or any other signs of toxicity. Determination of PA 824 inhaled dose. A custom designed dry powder dosing chamber was used to deliver aerosol powders to the animals. The dosing chamber was initially loaded in separate studies with two amounts of PA 824 powders, referred to as high and low nominal doses, in order to achieve the actual dose inhaled by each animal. These initial loading doses of powder were sufficient to deliver doses to the animal based on efficiency of delivery to the port of the dosing chamber and the proportion of this dose inhaled by the animal.
The doses delivered to the port were measured experimentally, and the respirable dose was calculated. The efficiency of delivery to the port of the dosing chamber from which the animal inhaled was approximately 6% of the nominal doses.