Dextran Mass Ratio Controls Particle Drying Dynamics in a Thermally Stable Dry Powder Vaccine for Pulmonary Delivery.

PURPOSE: Thermally stable, spray dried vaccines targeting respiratory diseases are promising candidates for pulmonary delivery, requiring careful excipient formulation to effectively encapsulate and protect labile biologics. This study investigates the impact of dextran mass ratio and molecular weight on activity retention, thermal stability and aerosol behaviour of a labile adenoviral vector (AdHu5) encapsulated within a spray dried mannitol-dextran blend. METHODS: Comparing formulations using 40 kDa or 500 kDa dextran at mass ratios of 1:3 and 3:1 mannitol to dextran, in vitro quantification of activity losses and powder flowability was used to assess suitability for inhalation. RESULTS: Incorporating mannitol in a 1:3 ratio with 500 kDa dextran reduced viral titre processing losses below 0.5 log and displayed strong thermal stability under accelerated aging conditions. Moisture absorption and agglomeration was higher in dextran-rich formulations, but under low humidity the 1:3 ratio with 500 kDa dextran powder had the lowest mass median aerodynamic diameter (4.4 µm) and 84% emitted dose from an intratracheal dosator, indicating strong aerosol performance. CONCLUSIONS: Overall, dextran-rich formulations increased viscosity during drying which slowed self-diffusion and favorably hindered viral partitioning at the particle surface. Reducing mannitol content also minimized AdHu5 exclusion from crystalline regions that can force the vector to air-solid interfaces where deactivation occurs. Although increased dextran molecular weight improved activity retention at the 1:3 ratio, it was less influential than the ratio parameter. Improving encapsulation ultimately allows inhalable vaccines to be prepared at higher potency, requiring less powder mass per inhaled dose and higher delivery efficiency.

Pulmonary hypertension is attenuated and ventilation-perfusion matching is maintained during chronic hypoxia in deer mice native to high altitude.

Hypoxia at high altitude can constrain metabolism and performance and can elicit physiological adjustments that are deleterious to health and fitness. Hypoxic pulmonary hypertension is a particularly serious and maladaptive response to chronic hypoxia, which results from vasoconstriction and pathological remodeling of pulmonary arteries, and can lead to pulmonary edema and right ventricle hypertrophy. We investigated whether deer mice (Peromyscus maniculatus) native to high altitude have attenuated this maladaptive response to chronic hypoxia and whether evolved changes or hypoxia-induced plasticity in pulmonary vasculature might impact ventilation-perfusion (V-Q) matching in chronic hypoxia. Deer mouse populations from both high and low altitudes were born and raised to adulthood in captivity at sea level, and various aspects of lung function were measured before and after exposure to chronic hypoxia (12 kPa O2, simulating the O2 pressure at 4,300 m) for 6-8 wk. In lowlanders, chronic hypoxia increased right ventricle systolic pressure (RVSP) from 14 to 19 mmHg (P = 0.001), in association with thickening of smooth muscle in pulmonary arteries and right ventricle hypertrophy. Chronic hypoxia also impaired V-Q matching in lowlanders (measured at rest using SPECT-CT imaging), as reflected by increased log SD of the perfusion distribution (log SDQ) from 0.55 to 0.86 (P = 0.031). In highlanders, chronic hypoxia had attenuated effects on RVSP and no effects on smooth muscle thickness, right ventricle mass, or V-Q matching. Therefore, evolved changes in lung function help attenuate maladaptive plasticity and contribute to hypoxia tolerance in high-altitude deer mice.

Impact of inflammation, emphysema, and smoking cessation on V/Q in mouse models of lung obstruction.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is known to greatly affect ventilation (V) and perfusion (Q) of the lung through pathologies such as inflammation and emphysema. However, there is little direct evidence regarding how these pathologies contribute to the V/Q mismatch observed in COPD and models thereof. Also, little is known regarding how smoking cessation affects V/Q relationships after inflammation and airspace enlargement have become established. To this end, we have quantified V/Q on a per-voxel basis using single photon emission computed tomography (SPECT) in mouse models of COPD and lung obstruction. METHODS: Three distinct murine models were used to investigate the impact of different pathologies on V/Q, as measured by SPECT. Lipopolysaccharide (LPS) was used to produce neutrophilic inflammation, porcine pancreatic elastase (PPE) was used to produce emphysema, and long-term cigarette smoke (CS) exposure and cessation were used to investigate the combination of these pathologies. RESULTS: CS exposure resulted in an increase in mononuclear cells and neutrophils, an increase in airspace enlargement, and an increase in V/Q mismatching. The inflammation produced by LPS was more robust and predominantly neutrophilic, compared to that of cigarette smoke; nevertheless, inflammation alone caused V/Q mismatching similar to that seen with long-term CS exposure. The emphysematous lesions caused by PPE administration were also capable of causing V/Q mismatch in the absence of inflammation. Following CS cessation, inflammatory cell levels returned to those of controls and, similarly, V/Q measures returned to normal despite evidence of persistent mild airspace enlargement. CONCLUSIONS: Both robust inflammation and extensive airspace enlargement, on their own, were capable of producing V/Q mismatch. As CS cessation resulted in a return of V/Q mismatching and inflammatory cell counts to control levels, lung inflammation is likely a major contributor to V/Q mismatch observed in the cigarette smoke exposure model as well as in COPD patients. This return of V/Q mismatching to control values also took place in the presence of mild airspace enlargement, indicating that emphysematous lesions must be of a larger volume before affecting the lung significantly. Early smoking cessation is therefore critical before emphysema has an irreversible impact on gas exchange.

Detection of lung dysfunction using ventilation and perfusion SPECT in a mouse model of chronic cigarette smoke exposure.

UNLABELLED: Chronic obstructive pulmonary disease is a leading cause of morbidity and mortality worldwide. Exposure to cigarette smoke (CS) is a major risk factor for developing this chronic airflow impairment, but the early progression of disease is not well defined or understood. Ventilation/perfusion (V/Q) SPECT provides a noninvasive assessment of lung function to further our current understanding of how CS affects the lung. METHODS: BALB/c mice were imaged with V/Q SPECT and CT after 8 and 24 wk of whole-body exposure to mainstream CS. Bronchoalveolar lavage was collected and cell differentials produced to determine inflammatory patterns. Histologic lung sections were collected, and a semiautomated quantitative analysis of airspace enlargement was applied to whole histology slices. RESULTS: Exposure to CS induced an inflammatory response that included increases in the numbers of both mononuclear cells and neutrophils. Airspace enlargement was also significantly increased at 8 wk of CS exposure and was still more pronounced at 24 wk. Ventilation and perfusion correlation at the voxel level depicted a significant decrease in matching at 8 wk of CS exposure that was also apparent after 24 wk. The standard deviation (SD) of the log(V/Q) curve, a basic measure of heterogeneity, was increased from 0.44 ± 0.02 in age-matched controls to 0.62 ± 0.05 with CS exposure at 24 wk, indicating an increase in V/Q mismatching between 8 and 24 wk of CS exposure. CT, however, was not capable of discriminating control from CS-exposed animals at either time point, even with greater resolution and respiratory gating. CONCLUSION: This study demonstrated that, before CT detection of structural changes, V/Q imaging detected changes in gas-exchange potential. This functional impairment corresponded to increased lung inflammation and increased airspace enlargement. In vivo V/Q imaging can detect early changes to the lung caused by CS exposure and thus provides a noninvasive method of longitudinally studying lung dysfunction in preclinical models. In the future, these measures could be applied clinically to study and diagnose the early stages of chronic obstructive pulmonary disease.

Imaging lung function in mice using SPECT/CT and per-voxel analysis.

Chronic lung disease is a major worldwide health concern but better tools are required to understand the underlying pathologies. Ventilation/perfusion (V/Q) single photon emission computed tomography (SPECT) with per-voxel analysis allows for non-invasive measurement of regional lung function. A clinically adapted V/Q methodology was used in healthy mice to investigate V/Q relationships. Twelve week-old mice were imaged to describe normal lung function while 36 week-old mice were imaged to determine how age affects V/Q. Mice were ventilated with Technegas™ and injected with (99m)Tc-macroaggregated albumin to trace ventilation and perfusion, respectively. For both processes, SPECT and CT images were acquired, co-registered, and quantitatively analyzed. On a per-voxel basis, ventilation and perfusion were moderately correlated (R = 0.58±0.03) in 12 week old animals and a mean log(V/Q) ratio of -0.07±0.01 and standard deviation of 0.36±0.02 were found, defining the extent of V/Q matching. In contrast, 36 week old animals had significantly increased levels of V/Q mismatching throughout the periphery of the lung. Measures of V/Q were consistent across healthy animals and differences were observed with age demonstrating the capability of this technique in quantifying lung function. Per-voxel analysis and the ability to non-invasively assess lung function will aid in the investigation of chronic lung disease models and drug efficacy studies.

In vitro estimations of in vivo jet nebulizer efficiency using actual and simulated tidal breathing patterns.

In vivo aerosol delivery efficiency was estimated in vitro for two jet nebulizers using a breath monitor (Breathe!; Pari GmbH, Germany) and breath simulator (COMPAS; Pari GmbH) to reproduce subject tidal breathing patterns. The AeroEclipse (Trudell Medical International, Canada), a breath-actuated nebulizer, and the LC Star (Pari GmbH), a breath-enhanced nebulizer, were filled with levalbuterol HCl solution (Sepracor, USA) and operated with compressed O(2) at 8 lpm. Tidal breathing patterns of 20 adult subjects were digitally recorded with the Breathe! Breath Monitor. Subjects then breathed tidally from each nebulizer separately for 1 minute and to nebulizer dryness. Levalbuterol aerosol collected on filters placed between the nebulizer and mouth was chemically assayed to determine the inspired mass (IM), wasted mass (WM) and total emitted mass (TM). Measurements were repeated using the COMPAS Breath Simulator to simulate each subject's tidal breathing pattern. IM, WM, and TM measurements using actual versus simulated tidal breathing were highly comparable for each nebulizer, except the IM (p < 0.05) from LC Star measured at nebulizer dryness. Breath simulation was an inaccurate tool for estimating the time to nebulizer dryness as simulated measurements to nebulizer dryness took significantly longer than measurements preformed with actual tidal breathing (p < 0.001). While breath simulation provides an accurate in vitro tool for estimating in vivo aerosol delivery, it should not completely replace in vivo measurements until inherent limitations in simulator operation can be overcome to provide a more clinically realistic simulation.