Nasal Inhalation

Kooguchi K, Hashimoto S, Kobayashi A, Kitamura Y, Kudoh I, Wiener-Kronish J, Sawa,T.
Role of alveolar macrophages in initiation and regulation of inflammation in Pseudomonas aeruginosa pneumonia.
Infect. Immun. 1998 Jul;66(7):3164–9.

[toggle_content title=”Abstract”] To evaluate the role of alveolar macrophages (AMs) in acute Pseudomonas aeruginosa pneumonia in mice, AMs were depleted by aerosol inhalation of liposomes containing clodronate disodium. AM-depleted mice were then intratracheally infected with 5 x 10⁵ CFU of P. aeruginosa . In addition to monitoring neutrophil recruitment and chemokine releases, lung injury was evaluated soon after infection (8 h) and at a later time (48 h). At 8 h, depletion of AMs reduced neutrophil recruitment, chemokine release, and lung injury. At 48 h, however, depletion of AMs decreased bacterial clearance and resulted in delayed movement of neutrophils from the site of inflammation with aggravated lung injury. With instillation of 5 x 10⁷ CFU of bacteria, AM-depleted mice showed low mortality within 24 h of infection but high mortality at a later time, in contrast to non-AM-depleted mice. These results demonstrate that depletion of AMs has beneficial early effects but deleterious late effects on lung injury and survival in cases of P. aeruginosa pneumonia.
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Clodronate Concentration	Total Lipid Concentration	Lipid Composition	Lipid Mole %	Liposome Type	Control Liposomes
not stated	11 mg/ml	EPC/Chol	57/43	REV	none

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Animal Description	Clodronate Dose	Dosing Site/Method	Target Phagocytes	Systemic Dosing?	Systemic Results
CD-1, male, 35-37 g	not stated	aerosol / lungs	alveolar macrophages (AM)	no	not evaluated

[/custom_table] [/toggle_content] [toggle_content title=”Notes”]

1. Authors state that liposomes were prepared as in Publication 1, however lipid composition is different as is final volume. Encapsulated clodronate is not published. Lipid concentration calculated assuming 100% recovery.
2. As discussed for publication 1., passing liposomes through a syringe filter is not equivalent to extrusion.
3. Aerotech II nebulizer at 12 L/min. No further details published.

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1. >95% of the AM were depleted (BAL cell counts via light microscopy).
2. No neutrophils at t=0 post-innoculation, therefore neutrophilia is not observed in this model.
3. No control liposomes dosed; could chemokine levels be altered by liposomes alone? Not likely but important control.

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Hashimoto S, Pittet JF, Hong K, Folkesson H, Bagby G, Kobzik L, Frevert C, Wanatabe K, Tsurufuji S, Weiner-Kronish, J.
Depletion of alveolar macrophages decreases neutrophil chemotaxis to Pseudomonas airspace infections.
American Journal of Physiology – Lung Cellular and Molecular Physiology. 1996 May 1;270(5):L819 –L828.

[toggle_content title=”Abstract”] The mechanism for neutrophil (PMN) influx into infected airspaces of the lung is not known. To determine whether alveolar macrophage products are important in the initiation of chemotaxis, we depleted rats of alveolar macrophages by aerosolizing negatively charged oligolamellar liposomes complexed to clodronate disodium. Ninety-five percent of the alveolar macrophages were depleted, and lung injury and inflammation were minimized with this depletion technique. Rats depleted of alveolar macrophages were then anesthetized, and either 5 x 10⁶ colony-forming units (CFU) or 5 x 10⁷ CFU of Pseudomonas aeruginosa were instilled into the airspaces of these animals. When recombinant macrophage inflammatory protein (MIP-2) was intratracheally instilled into rats depleted of alveolar macrophages, PMN were recruited to their airspaces. Nonetheless, PMN numbers were decreased in the lavage fluids when moderate or large inoculums of bacteria were instilled into depleted rats, although the PMN response appeared dose dependent. Levels of bioactive tumor necrosis factor-α and immunoreactive proteins CINC/gro (cytokine-induced PMN chemoattractant) in the lavage fluids obtained from infected rats depleted of alveolar macrophages were significantly decreased compared with the levels in the lavage fluids obtained from normal infected rats. MIP-2 mRNA expression, as detected by Northern analysis, was also decreased in the infected lungs of depleted rats, and the lavage fluid from these rats had significantly decreased chemotactic activity. Therefore these results suggest that alveolar macrophage products play a direct role in the initial recruitment of PMN into infected lungs. [/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]

Clodronate Concentration	Total Lipid Concentration	Lipid Composition	Lipid Mole %	Liposome Type	Control Liposomes
7 mg/ml	33.4 mg/ml	EPC/BPS/Chol	55/9/36	REV	PBS(-)

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Animal Description	Clodronate Dose	Dosing Site/Method	Target Phagocytes	Systemic Dosing?	Systemic Results
Sprague Dawley rats, male, 300-375 g	0.05-0.25 µmoles	aerosol / lungs	alveolar macrophages (AM)	no	no change in peripheral moncytes or PMN detected

[/custom_table] [/toggle_content] [toggle_content title=”Notes”]

1. The authors state that liposomes were “extruded” through 0.2 µm syringe filters. A syringe filter is not a substitute for extrusion therefore the resulting size distribution is questionable. Liposomes >400 nm have been shown to be disrupted by aerosolization, therefore it’s likely that there was free clodronate in the aerosol.
2. Aerotech II nebulizer at 10 L/min. MMAD = 1.6 µm; GSD = 2.5. Fluorescent liposome aerosol delivery experiments indicated that 0.1-0.5% of the dose was delivered to the lung. This value was used to estimate the clodronate dose delivered to the lungs. The authors estimate that 26X more drug is delivered by this method than by liquid installation.

[/toggle_content] [toggle_content title=”Results”]

1. >95% of the AM were depleted (BAL cell counts via light microscopy).
2. The authors further state that clodronate liposome delivery by aerosol prevents neutrophilia often observed when direct instillation is used.

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