Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

The overall goal of this procedure is to generate aerosolized drug samples for their characterization and uniform deposition into the lungs of rodents. This method can help answer key questions in the inhalation, pharmacology, and toxicology field about the safety and efficacy of specific doses of inhaled drugs. The main advantage of this technique is that it allows the uniform and simultaneous distribution of different doses of the same drug into the lungs of small animals.

Generally, innovators, new to this method will struggle because generating, categorizing, and measuring the efficacy of aerosols in animal models of respiratory disease requires a careful calculation, and technique. For dry powder aerosol generation first use a jet mill to micronize the dry drug powder, taking care that the micronized particle size distribution contains particles of respirable size. Blend the potent test compounds that require dilution with micronized lactose.

Then use a manual hydraulic press at approximately 1, 000 pounds per square inch to pack the micronized drug into the cylindrical reservoir of a Wright dust feed dry powder aerosol generator to produce compacted cakes of powder. Next, screw the cylindrical reservoir onto the Wright dust feed, advancing the reservoir until the scraper blade is in contact with the drug cake. Connect the outlet of the Wright dust feed to a cyclond and the inlet to a compressed air source.

Set the feed rate control to 0.7 revolutions per minute, and turn on the Wright dust feed aerosol generator. Then, connect the outlet of the cyclond to the central aerosol plenum of the inhalation unit. To generate a nebulized liquid aerosol, dissolve the drug of interest in 100 milliliters of water, and load a 100 milliliter syringe with the resulting drug solution.

Place the syringe into a syringe pump with the flow rate set at one milliliter per minute, and connect the pump to the jet nebulizer. Purge the air from the feed line leading to the nebulizer, and connect the pressurized air source to the jet nebulizer, then set the air flow meter to ten liters per minute with the jet nebulizer installed into the pre-separator, which connects the nebulizer to the central aerosol plenum of the inhalation unit. First, transfer a pre-weighed filter into the filter holder, and assemble the holder.

Connect the inlet of the absolute filter holder to a central aerosol plenum sample port, and the outlet to a vacuum source. Next, assemble the cascade impactor to collect the aerosol, by placing pre-weighed filters onto each of the seven stages of the cascade impactor, and assemble the Mercer cascade impactor. Proper assembly of the cascade impactor is critical.

The stages must be loaded in the correct order, and orientation with the largest stage closest to the inlet. Connect the inlet of the cascade impactor to a central aerosol plenum sample port, and the outlet to a vacuum source. To monitor the aerosol content of the inhalation unit, connect the inlet of the real-time aerosol monitor to a central aerosol plenum sample port, and the outlet to a vacuum source.

Set the process control parameters to the appropriate values according to the number of animals to be connected to the inhalation unit and begin the delivery of the aerosolized drug of interest. Plug the delivery points of the inhalation unit with stoppers, and activate the aerosol generator, the compressed air flow controller, and the inhalation unit vacuum pump from within the process control software. Now, load the experimental mice into the nose-only restrainers, angling the restraining tubes up towards the ceiling, as the animals tend to run upward during the loading process, with the noses in the pointed ends of the tubes.

Fix the variable position plungers into the back ends of the restrainers so that the animals can rotate, but not turn head to tail. Loading the mice into the restraining tubes takes patience and coordination. Please handle the animals gently.

When the readings from the real-time aerosol monitor demonstrate that the aerosol concentration has reached equilibrium, begin removing the stoppers, and inserting the nose-only restraining tubes into the inhalation unit. When all of the experimental animals are connected to the exposure unit, turn on the vacuum sampling pumps connected to the absolute filter, and the cascade impactor. Deliver the drug to the animals for the appropriate time periods.

When all of the exposures are complete, turn off the aerosol generator, and remove the mice from the inhalation unit. Then, collect the cascade impactor, and absolute filters for analysis. In this experiment, after aerosol delivery of the bronchodilator ipratropium bromide, to 24, 8 week-old, C57BL/6 mice, the aerosol deposited on the cascade impactor, and absolute filters during the 45 minutes of aerosol exposure was dissolved in 50%acetonitrile, and the mass of the ipratropium was quantified by high-pressure liquid chromatography.

The mass median aerodynamic diameter times the geometric standard deviation of the ipratropium aerosol was then calculated, and a deposition fraction of 0.037 for the mice was used for an aerosol, with the mass median aerodynamic diameter of 1.7 micrometers. Nebulized methacoline treatment induced bronchoconstriction in the mice within seconds of administration. Pre-treatment with inhaled ipratropium, however, inhibited the nebulized methacoline-induced bronchoconstriction, in a dose-dependent manner.

The respiratory system resistance of the control group, however, demonstrated a nearly 170%increase in respiratory system resistance, peaking 70 seconds after the methacoline aerosol administration. While attempting this procedure, it's important to remember to continuously monitor the mice for distress when they are in the restraining tubes. Following this procedure, other methods like measuring pulmonary mechanics, inflammatory cells in lavage fluid, or lung histology can be performed to determine the efficacy or safety of experimental drugs of interest.