Physical properties, lung deposition modeling, and bioactivity of recombinant GM-CSF aerosolised with a highly efficient nebulizer

doi:10.1016/j.pupt.2010.08.004

Pulmonary Pharmacology & Therapeutics

Volume 24, Issue 1, February 2011, Pages 123-127

https://doi.org/10.1016/j.pupt.2010.08.004 Get rights and content

Abstract

Pulmonary alveolar proteinosis (PAP) is a rare condition characterized by the accumulation of lipoproteinaceous material within air spaces. Although whole lung lavage is the current standard of care, recent advances in our understanding of PAP pathophysiology suggest that the disorder may benefit from inhalation of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). The aim of this study was to determine the physical properties and bioactivity of rGM-CSF aerosolised by the highly efficient AKITA² APIXNEB^® nebulizer system. The physical properties of aerosolised rGM-CSF were investigated in terms of droplet size, output and output rate by laser diffraction and gravimetrical analysis. Lung deposition was assessed using deposition modeling (ICRP). Molecular mass before and after aerosolisation was determined by SDS-PAGE, while the bioactivity of rGM-CSF was evaluated by measuring the GM-CSF-stimulated increase in pSTAT5 using mAM-hGM-R cells. Ninety-six % of the rGM-CSF filling dose was aerosolised with the Akita² Apixneb^® nebulizer system. Particle size was highly reproducible, and the amount deposited within the lung was 80.35% of the delivered dose. The aerosolisation did not alter the molecular structure of rGM-CSF, nor its ability to stimulate the pSTAT5, which increased by 99.5%, similar to values for rGM-CSF prior to aerosolisation. We conclude that the highly efficient AKITA² APIXNEB^® nebulizer system is likely to efficaciously deliver rGM-CSF to the airways of patients with autoimmune PAP.

Introduction

Pulmonary alveolar proteinosis (PAP) is a rare condition characterized by the accumulation of lipoproteinaceous material within air spaces, and occurs in different clinical forms [1]. The presence of autoantibodies (Abs) neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) [2]and the reproduction of the pathologic disease manifestations by passive transfer of human Abs to primates [3] has permitted the reclassification of this condition to “autoimmune PAP”, the most frequent clinical form of PAP; previously it was referred to as “idiopathic” or “acquired” PAP [1]. These findings have opened the way to novel therapeutic scenarios.

The current standard of care for PAP is whole lung lavage (WLL), which results in the complete and durable resolution of PAP lung abnormalities in a percentage ranging from 20 to 70 of lavaged patients [4], [5]. In addition, WLL is a highly invasive procedure, and even though complications are infrequent it requires prolonged anesthesia. The evidence that autoimmune PAP is caused by a disruption in GM-CSF signaling, has prompted investigations with recombinant GM-CSF (rGM-CSF) supplementation. A small series of PAP patients have been treated with rGM-CSF either subcutaneous (s.c.) or by inhalation, the overall response rate was slightly better by the latter route [6]. To date, no side effects, even for prolonged treatments, have been reported. Clearly, this therapeutic option, although very promising, requires further verification, in larger randomized trials.

A clinical trial, aimed at maximizing the therapeutic outcome in PAP patients, has been recently approved [7]. The objective of the trial is to demonstrate the superiority of WLL followed by inhaled rGM-CSF vs WLL alone to obtain sustained resolution of lung abnormalities in autoimmune PAP patients. To optimize drug delivery, we decided to utilize the AKITA² APIXNEB^® apparatus, a vibrating mesh nebulizer with controlled breathing and aerosol pulse technology. A series of experiments, preliminary to the trial, were therefore planned to investigate the particle size distribution and output. In addition, an important variable to be determined was the retention of rGM-CSF biological activity after aerosolisation. A series of experiments were therefore planned to investigate this aim. In this study we demonstrate the highly effective performance of the Akita² Apixneb^® nebulizer system with the rGM-CSF formulation. In addition, aerosolisation does not alter the molecular mass or the biological activity of rGM-CSF.

Section snippets

Methods

The rGM-CSF (Sargramostim, Leukine^®) used in all experiments was a lyophilized preparation (250 μg/vial preservative-free powder) purchased from Bayer HC (Seattle, WA), and kept at 4 °C until needed, and then dissolved in 1 ml saline. In all experiments, the AKITA² APIXNEB^® nebulizer handset (Activaero, Gemünden, Germany) was equipped with a Smart Card with the following configuration: inhalation volumes: 1500 ml; bolus width: 1250 ml; bolus depth: 1500 ml; flow rate: 15 L/min.

Particle size distribution, output and output rate

Particle size distribution, analyzed by laser diffraction, showed a volume median diameter (VMD) of 4.31μm ± 0.11 (mean ± SD; range 4.28–4.43 μm). The geometric standard deviation (GSD) of all measurements showed the mean value 1.58 ± 0.03. The output dose, determined by gravimetrical analysis, was estimated as 95.71% ± 0.018 for a filling dose of 1 mL. Finally, the continuous output rate observed was 1.043 mL/min. These data are reported in detail in Table 1, Table 2, Table 3.

Lung deposition modeling

For the detected

Discussion

The experiments described in this paper provide important information on nebulized GM-CSF. These data are essential for the design of a trial to provide local delivery of GM-CSF in patients with autoimmune PAP. In fact, GM-CSF supplementation in patients with PAP characterized by the presence of anti-GM-CSF Abs was first delivered s.c. with variable results. Over the last 14 years, approximately 50 PAP patients have been treated with rGM-CSF by this route [14], [15], [16], [17], with the

Authors contribution

ML and BCT designed the study and drafted the paper; WCZ, BM, JG, PK and MF designed, performed, and collected data on the aerosolised rGM-CSF physical characterization (particle sizing, output rate and lung deposition modeling); TS, ZK, IC, FM, FF and GR designed, performed, and collected data on the bioactivity experiments.

Acknowledgements

This study was supported by the AIFA (Italian Agency for Medicines) project FARM7MCPK4.

References (25)

Y. Shibata et al.
GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1
Immunity
(2001)
B.C. Trapnell et al.
Pulmonary alveolar proteinosis
N Engl J Med
(2003)
T. Kitamura et al.
Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor
J Exp Med
(1999)
T. Sakagami et al.
Human GM-CSF autoantibodies and pulmonary alveolar proteinosis
N Engl J Med
(2009)
J.F. Seymour et al.
Pulmonary alveolar proteinosis. Progress in the first 44 years
Am J Respir Crit Care Med
(2002)
M. Beccaria et al.
Long term durable benefit after whole lung lavage in pulmonary alveolar proteinosis
Eur Respir J
(2004)
Luisetti M, Trapnell BC. Pulmonary alveolar proteinosis. In: Interstitial lung disease 5th ed. Edited by Schwarz MI,...
International Commission on Radiological Protection
Human respiratory model for radiological protection
Ann ICRP
(1994)
R. Köbrich et al.
A mathematical model of mass deposition in man
Ann Occup Hyg
(1994)

J.S. Fleming et al.

Comparison of SPECT aerosol deposition data with a human respiratory tract model

J Aerosol Med

(2006)

T. Suzuki et al.

Familial pulmonary alveolar proteinosis caused by mutations in CSF2RA

J Exp Med

(2008)

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Physical properties, lung deposition modeling, and bioactivity of recombinant GM-CSF aerosolised with a highly efficient nebulizer

Abstract

Introduction

Section snippets

Methods

Particle size distribution, output and output rate

Lung deposition modeling

Discussion

Authors contribution

Acknowledgements

Immunity

Pulmonary alveolar proteinosis

N Engl J Med

Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor

J Exp Med

Human GM-CSF autoantibodies and pulmonary alveolar proteinosis

N Engl J Med

Pulmonary alveolar proteinosis. Progress in the first 44 years

Am J Respir Crit Care Med

Long term durable benefit after whole lung lavage in pulmonary alveolar proteinosis

Eur Respir J

Human respiratory model for radiological protection

Ann ICRP

A mathematical model of mass deposition in man

Ann Occup Hyg

Comparison of SPECT aerosol deposition data with a human respiratory tract model

J Aerosol Med

Familial pulmonary alveolar proteinosis caused by mutations in CSF2RA

J Exp Med