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14 February 2020 Inhalation of the nose-only port. 7, 8 To provide assurance that the delivery device and methodology achieve the target inhaled dose, we routinely insert a glass fiber filter in one exposure port to assay the deposited drug dose (drawn at a flow rate to mimic the rodent's exposure). In these stud- ies, the concentration of aerosol in the exposure chamber is calculated based on the amount of drug collected on the filter and compared to the expected theoretical target aerosol concentration. In addition, to provide absolute measurements of the deposited dose in the rodent's lung, we sacrifice a subset of the rats immediately after inha- lation exposure, homogenize their lungs and assay the amount of deposited drug in the lungs. An MDI study at a CRO We have relationships with CROs and have trained their personnel to conduct nose-only animal inhalation expo- sure studies in mice, rats and guinea pigs. We typically conduct site visits every 6 to 12 months to discuss any questions or issues with our programs, evaluate their inhalation exposure equipment and provide refresher training. Recently, we asked one CRO to conduct an inhalation exposure and PK study in rats using novel metered dose inhaler (MDI) formulations of INS1009, a drug with which they have experience in both nebulized and dry powder inhaler (DPI) formats. We planned to evaluate two different MDI formulations, one formu- lated with propellant HFA-227 and one with propel- lant HFA-134a. e differences in density and vapor pressure of these two propellants, and their interactions with the drug and excipients in the canisters, would be expected to result in changes in aerosol emitted dose, particle size distribution and rates of evaporation. 9 In the initial study with the first MDI formulation, the chamber efficiency was low; ~15% based on the recov- ered dose from the filter. e amount of drug recov- ered from the rat lungs immediately post-exposure was measured and found to be consistent with the dose cal- e common link between these two "standard curve" examples is that by having our chemistry, analytical and bioanalysis teams together in a single facility, the flow of communication was streamlined and we were able to expedite trouble-shooting efforts and resolution of the issues. Aerosol exposure studies To assess the pharmacokinetics in animal studies of a drug intended for deposition in the lung, some of the first questions are which animal model to use and how to deliver the drug. 5 e larger the animal species, the greater the amount of drug required to achieve a target dose relative to the body weight or lung weight. is can be mitigated somewhat by conducting the initial studies in mice or rats. When drug supplies are limited, intratracheal (IT) instillation or insufflation is often the chosen administration method. With IT adminis- tration, one has the added benefit of knowing the exact dose delivered to the rodent. However, a major down- side to instillation is that "flooding" the lung with the formulation does not replicate the deposition pattern from inhalation of aerosols. In contrast, the use of nose-only inhalation cham- bers to deliver aerosols to rodents can better simulate deposition of aerosols in the lung but has the down- side of requiring much higher quantities of drug than IT administration. Typically, each rodent may inhale less than 1% of the dose loaded in the nebulizer(s). 5 In addition, most of the inhaled dose deposits in the nasal regions, leading to a lower and less consistent dose to the lung. e regulatory agencies typically utilize assumptions of a 10% deposition fraction (i.e., 10% of the inhaled dose deposits in the lung) for mice and rats. 6 Furthermore, it is important to recognize that differences still exist when trying to bridge to humans. Rodents are obligate nose breathers and the nasal and respiratory tract dimensions in rodents are smaller, leading to greater upper airway deposition. erefore, the use of smaller particle aerosols can improve lung deposition efficiencies in rodents. 5 In many of our initial PK and preclinical inhalation effi- cacy studies, we utilize a nose-only inhalation chamber (Figure 2). In order to estimate the dose delivered to the rodent, the theoretical aerosol concentration can be cal- culated based on the total drug amount aerosolized, the air flow rate provided to the system and the time of aero- solization. As a confirmation, the actual aerosol concen- tration at the port can be determined by collecting the aerosol delivered to the chamber on a filter for a specified period of time and quantifying the drug deposited on that filter. e ratio of the measured aerosol concentra- tion to the theoretical aerosol concentration is an indica- tor of the chamber efficiency. Based on the design of the nose-only chamber and the properties of formulation, the chamber efficiency can vary widely from one set-up to another but never reaches 100%, due to aerosol depo- sition that occurs in transit prior to reaching the entrance Rodent restraint tubes Rodent restraint tubes Tangential air supply MDI canister actuator Marple Cascade Impactor Exhaust vacuum Figure 2 A schematic of an MDI nose-only exposure system (In some experiments, a collection filter is used instead of, or in addition to, a Marple Cascade Impactor.)