Quantitative detection of drug dose and spatial distribution in the lung revealed by Cryoslicing Imaging.

Administration of drugs via inhalation is an attractive route for pulmonary and systemic drug delivery. The therapeutic outcome of inhalation therapy depends not only on the dose of the lung-delivered drug, but also on its bioactivity and regional distribution. Fluorescence imaging has the potential to monitor these aspects already during preclinical development of inhaled drugs, but quantitative methods of analysis are lacking. In this proof-of-concept study, we demonstrate that Cryoslicing Imaging allows for 3D quantitative fluorescence imaging on ex vivo murine lungs. Known amounts of fluorescent substance (nanoparticles or fluorophore-drug conjugate) were instilled in the lungs of mice. The excised lungs were measured by Cryoslicing Imaging. Herein, white light and fluorescence images are obtained from the face of a gradually sliced frozen organ block. A quantitative representation of the fluorescence intensity throughout the lung was inferred from the images by accounting for instrument noise, tissue autofluorescence and out-of-plane fluorescence. Importantly, the out-of-plane fluorescence correction is based on the experimentally determined effective light attenuation coefficient of frozen murine lung tissue (10.0 ± 0.6 cm(-1) at 716 nm). The linear correlation between pulmonary total fluorescence intensity and pulmonary fluorophore dose indicates the validity of this method and allows direct fluorophore dose assessment. The pulmonary dose of a fluorescence-labeled drug (FcγR-Alexa750) could be assessed with an estimated accuracy of 9% and the limit of detection in ng regime. Hence, Cryoslicing Imaging can be used for quantitative assessment of dose and 3D distribution of fluorescence-labeled drugs or drug carriers in the lungs of mice.

Protein stability in pulmonary drug delivery via nebulization.

Protein inhalation is a delivery route which offers high potential for direct local lung application of proteins. Liquid formulations are usually available in early stages of biopharmaceutical development and nebulizers are the device of choice for atomization avoiding additional process steps like drying and enabling fast progression to clinical trials. While some proteins were proven to remain stable throughout aerosolization e.g. DNase, many biopharmaceuticals are more susceptible towards the stresses encountered during nebulization. The main reason for protein instability is unfolding and aggregation at the air-liquid interface, a problem which is of particular challenge in the case of ultrasound and jet nebulizers due to recirculation of much of the generated droplets. Surfactants are an important formulation component to protect the sensitive biomolecules. A second important challenge is warming of ultrasound and vibrating mesh devices, which can be overcome by overfilling, precooled solutions or cooling of the reservoir. Ultimately, formulation development has to go hand in hand with device evaluation.

Prediction of protein degradation during vibrating mesh nebulization via a high throughput screening method.

Maintaining the integrity of biopharmaceuticals is a major requirement for successful pulmonary delivery by nebulization. Sparing laborious nebulization tests, this study aimed to demonstrate the feasibility of a high throughput, material saving surrogate method to predict protein stability after nebulization. Detrimental conditions during nebulization with a PARI eFlow® vibrating mesh nebulizer were mimicked by vigorous agitation at elevated temperatures. Comparing the effect of several different excipients on the stability of the protein SM101 after nebulization and after the surrogate method revealed an excellent correlation regarding SM101 aggregation (R(2)=0.97). Design of experiment was used to develop an inhalable formulation of SM101 based entirely on the new surrogate method. Two lead formulation candidates were selected based on their predicted stability profile. The conservation of full SM101 stability and activity after nebulization was confirmed for an AKITA(2) vibrating mesh nebulizer. This study demonstrated that biopharmaceutical formulation development for nebulization is feasible by means of imitating nebulizer related detrimental factors, allowing an accelerated and more economic formulation development.

That's cool!--Nebulization of thermolabile proteins with a cooled vibrating mesh nebulizer.

Despite contrary reports, heating inside the medication reservoir was observed for several vibrating mesh nebulizers, which may be detrimental when nebulizing biopharmaceuticals. In this study we evaluated different strategies to reduce reservoir heating during nebulization with a PARI eFlow® regarding cooling efficiency, impact on nebulizer performance and on protein stability after nebulization. Passive cooling was achieved by solution pre-cooling, overcharging of the reservoir with 1 mL additional solution or intermittent nebulization. Active cooling was realized with a micro Peltier element attached to the nebulizer reservoir. Passive cooling was most effective when the reservoir was overcharged with pre-cooled solution reducing the average reservoir temperature (TRES AVG) by 8.4°C. Active cooling enabled nebulization at a constant reservoir temperature (TRES) as low as 15°C. TRES manipulation had a linear impact on nebulizer performance. While the output rate decreased with decreasing TRES, the inhalable fraction increased resulting in an inhalable aerosol rate constant over a large TRES range. The effect on protein stability depended on the susceptibility to thermal stress and was predicted by Tm values. For lactic dehydrogenase and SM101, both exhibiting a Tm below 60°C, cooling was protective in increasing the residual activity and reducing protein aggregation. A more thermostable IgG1 did not benefit from cooled nebulization. Nebulizer cooling is a prerequisite to retain the activity and stability of thermolabile proteins during vibrating mesh nebulization. It is best achieved by micro Peltier based active cooling or by simple passive cooling strategies.