Practical Considerations in Dose Extrapolation from Animals to Humans.

Animal studies are an important component of drug product development and the regulatory review process since modern practices have been in place, for almost a century. A variety of experimental systems are available to generate aerosols for delivery to animals in both liquid and solid forms. The extrapolation of deposited dose in the lungs from laboratory animals to humans is challenging because of genetic, anatomical, physiological, pharmacological, and other biological differences between species. Inhaled drug delivery extrapolation requires scrutiny as the aerodynamic behavior, and its role in lung deposition is influenced not only by the properties of the drug aerosol but also by the anatomy and pulmonary function of the species in which it is being evaluated. Sources of variability between species include the formulation, delivery system, and species-specific biological factors. It is important to acknowledge the underlying variables that contribute to estimates of dose scaling between species.

Inhalation Pharmacodynamics.

Pharmacodynamics (PD) is discussed in relation to inhalation exposure to inhaled pharmaceutical and toxic agents. Clearly PD is closely related to pharmacokinetics, and this relation is illustrated with reference to inhaled insulin. PD can be related to pharmacologic responses, and some examples are cited. However, PD can also be thought of as the improvement or deterioration in lung disease state. Some of the major PD endpoints, including histopathology, pulmonary function, and bronchoalveolar lavage are reviewed. Brief reference is also given to other specialty biomarkers of PD response.

Perspectives on Lung Dose and Inhaled Biomolecules.

Dose is highly important to studies of inhaled agents because there must be an understanding of the dose delivered to humans, the dose delivered to animals in toxicology studies, and an ability to interpret and compare both sets of information relative to safety. Unlike oral or intravenous administrations, total delivered or inhaled dose is not easy to determine following inhalation exposure and is also not necessarily the most important determinant of toxicity. A review of dose distribution throughout the respiratory tract as well as total inhaled dose is provided. The implications of regional deposition for biologics are reviewed and specific examples over a range of different molecular weights are provided. Biologics are generally large enough that absorption from ciliated epithelia is low. Thus, deposition of biologics in head airways and tracheobronchial regions is unlikely to be of high importance unless there are interactions with specific receptors at these sites. Therefore, it is the dose of proteins or biologics deposited in the alveolar region that are generally of most interest.

Comparison of inhalation toxicity studies of gentamicin in rats and dogs.

Nebulized gentamicin solution was administered to rats (nose-only) and dogs (face mask) for 14 days with a 14-day recovery period. Control groups of each were exposed to saline aerosols. Mean estimated inhaled lung doses of gentamicin were 39, 123 and 245 mg/kg for rats (deposited doses 6, 17 and 34 mg/kg) over 30, 90 and 180 min, respectively. Since dogs do not tolerate exposures as long as rats, inhaled lung doses were limited to 7, 14 and 41 mg/kg (deposited doses of 1, 3 and 8 mg/kg) over 15, 30 and 60 min. Comparable doses were achieved at the low dose for rats and high dose for dogs. Serum AUCs (14 ± 2 µg/mL h (mean ± SD) at 6 mg/kg in rats and 11 ± 7 µg/mL h at 8 mg/kg in dogs) showed comparable exposure between the two, implying similar absorbed doses and confirming similar deposited lung doses. Rat exposures resulted in dose-related lung pathology (including low dose) manifested as upper respiratory tract irritant reactions with alveolar histiocytosis, inflammation, airway epithelial metaplasia and lymphomegaly in lung tissue. This was associated with high lung tissue gentamicin levels 24 h post-dose on Day 14 (375 ± 33 µg/g at deposited dose of 6 mg/kg). Dose-related kidney tubular necrosis (a well-known toxicity of parenteral gentamicin) was observed, but no test-article related effects on lung histopathology in dogs (even at highest deposited dose of 8 mg/kg) and low levels of lung tissue gentamicin (42 ± 11 µg/g) 24 h post-dose on Day 14.

The relevance of animal models for aerosol studies.

Animal models are essential for understanding the fates and effects of inhaled materials, because invasive methods are frequently necessary to provide the desired information. Because of the variability in humans of particle deposition, clearance, and effects, numerous animal models have been used in inhalation studies. Furthermore, humans are not typical mammals in some ways that affect inhalation phenomena. Humans have less fur, longer gestation and life times, simplified nasal structure, and symmetric bronchial branching in relation to other mammals. However, experience, plus the genetic similarity among mammals, underpins the use of animal models. Mammals are varied with respect to their inhaled particle deposition and clearance phenomena. Total inhaled aerosol deposition probability versus particle-size curves are qualitatively similar for various mammals of similar body mass, despite airway anatomy differences. However, more species variation is seen in regional particle deposition curves, complicating aerosol study design. The rates of clearance of deposited slowly dissolving particles are animal species dependent, apparently due to differences in gross, subgross, and cellular respiratory tract biology. Clearance rates for rapidly dissolving particles are not strongly species dependent. Inhalation toxicology studies require several animal species. Rodents are among the most frequently used, but for studies of lung development, diseases, exercise, etc., and for extrapolation to humans, larger mammals are also needed. Fortunately, the research database, and excellent monographs on inhalation phenomena provide ample guidance for study design.

Respiratory function in rats restrained for extended periods: assessment of the effects of bethanecol.

INTRODUCTION: The ICH guideline S7A recommends that the effects of drugs on the respiratory system are evaluated in laboratory mammals prior to administration in man. Previously, animals have been placed in plethysmography chambers for short durations. This study investigates the possibility of restraining animals in chambers for a longer duration to assess respiratory function over extended periods. METHODS: Respiratory function in conscious rats was assessed using plethysmography chambers where the rat body was enclosed in a sealed chamber while the head was free. Thoracic movements were measured by pressure transducers linked to a Buxco amplifier system and respiratory parameters were captured and analyzed by the Notocord HEM data acquisition system. Each animal was subjected to 5 acclimatization sessions of escalating duration (1, 2, 4, 5, and 6 hours (h)) over 5 days prior to testing, with a baseline recording session conducted the day prior to dosing. Animals (8 males/group) were dosed subcutaneously with saline or bethanecol (3, 10, or 30 mg/kg) and placed in the chambers for 6 h of continuous recording. Additionally, a recording session was conducted at 24 h post-dose. RESULTS: Subcutaneous administration of 30 mg/kg bethanecol decreased respiration rate by up to 33% during the first 1.5 h post-dose and increased tidal volume by up to 46% from 0.25 to 1.25 h post-dose when compared to vehicle group data. A decrease in minute volume of up to 33% was observed 0.25 h following administration of the 10 and 30 mg/kg doses. DISCUSSION: These data show a respiratory depression caused by the cholinergic agonist bethanecol, an effect partially compensated for by an increase in tidal volume. This also demonstrates the ability to continuously restrain and record respiratory parameters in conscious rats for up to 6 h without any negative impact on the quality of the data.

Evaluation of the survival of bacterial contaminants in an inhalable insulin powder.

Survival and growth of three model test bacterial species (Pseudomonas fluorescens, Staphylococcus epidermidis and Bacillus subtilis), present in the air and/or in the human respiratory tract, were tested in inhalable insulin-lactose powder under optimal relative humidity and temperature conditions (RH = 96% and optimal growth temperature for each bacterium of 26-37 degrees C) as well as representative indoor conditions (RH = 43% and T = 20 degrees C). The bacteria survived from 12 h to 7 days depending on the bacterial species and the test condition. P. fluorescens vegetative cells had the lowest and B. subtilis spores the highest survival rate. It was found that insulin-lactose powder does not support bacterial growth and that higher bacterial survival rate was found under representative indoor conditions. Selected experiments were performed with B. subtilis by adding sterile saliva into insulin-lactose powder to represent a typical condition for inhaler use. Furthermore, two other powders were tested with B. subtilis: one representing an inert powder without any nutrients (glass beads) and the other representing a powder with optimal nutrients (tryptic soy broth powder). The data indicate that the survival rate of B. subtilis did not change after the saliva was added and that the survival in insulin-lactose powder was similar to that in inert powder but lower than in powder with optimal nutrients. These results suggest that bacterial growth on residual powder in the inhaler under patient use conditions is unlikely and therefore the concern for patient safety is remote.