Delivery of aerosolized liposomal amikacin as a novel approach for the treatment of nontuberculous mycobacteria in an experimental model of pulmonary infection

Abstract

Pulmonary infections caused by nontuberculous mycobacteria (NTM) are an increasing problem in individuals with chronic lung conditions and current therapies are lacking. We investigated the activity of liposomal amikacin for inhalation (LAI) against NTM in vitro as well as in a murine model of respiratory infection. Macrophage monolayers were infected with three strains of Mycobacterium avium, two strains of Mycobacterium abscessus, and exposed to LAI or free amikacin for 4 days before enumerating bacterial survival. Respiratory infection was established in mice by intranasal inoculation with M. avium and allowing three weeks for the infection to progress. Three different regimens of inhaled LAI were compared to inhaled saline and parenterally administered free amikacin over a 28 day period. Bacteria recovered from the mice were analyzed for acquired resistance to amikacin. In vitro, liposomal amikacin for inhalation was more effective than free amikacin in eliminating both intracellular M. avium and M. abscessus. In vivo, inhaled LAI demonstrated similar effectiveness to a ∼25% higher total dose of parenterally administered amikacin at reducing M. avium in the lungs when compared to inhaled saline. Additionally, there was no acquired resistance to amikacin observed after the treatment regimen. The data suggest that LAI has the potential to be an effective therapy against NTM respiratory infections in humans.

Figure 1. Efficacy of LAI against intracellular mycobacteria in vitro.

LAI, a liposome-encapsulated amikacin compound, was tested against intracellular M. avium subsp. hominissuis (MAH) and M. abscessus (Ma) in vitro. THP-1 human cells (that were first differentiated and adhered with PMA) were infected with a 10∶1 MOI of MAH strains 104 and A5, which are blood isolates (A and B, respectfully), MAH 3388, a lung isolate (C), and Ma strains 26 and 36, which are also lung isolates (D and E, respectfully) for 1 hour. Extracellular bacteria were removed and cells containing internalized bacteria were incubated at 37°C for 24 hours. Wells were either plated for CFU (day 0 control) or treated daily with HBSS (day 4 control), empty liposome, 10 µg/ml amikacin sulfate, or between 10 and 1 µg/ml of LAI for 4 days. Cells were disrupted, diluted, and plated to obtain CFUs of surviving bacteria. Bars represent means of CFU counts and error bars represent standard deviation. Statistical comparisons: 1 = p<0.05 compared to day 0 control; 2 = p<0.05 compared to day 4 control; 3 = p<0.05 compared to empty liposome; 4 = p<0.05 compared to free amikacin.

Figure 2. Efficacy of inhaled LAI compared to parenterally administered free amikacin in murine respiratory infection.

Six groups of 12 mice were intranasally infected with MAH 104 and a respiratory infection was allowed to establish for three weeks. At this point, an initial group of 12 mice were euthanized to determine the pre-treatment burden of MAH 104 in the respiratory tract. The other 5 groups received either saline, LAI, or amikacin sulfate by various delivery routes and treatment regimens, as listed in the figure. Twenty-eight days after the initiation of treatment, all mice were euthanized and lungs were processed to determine the post-treatment MAH 104 burden. Dots resting on the x-axis are animals that had no culturable MAH in their lungs. Abbreviations: QID×28d = treatment every day for 28d; Q1D×14d = treatment every other day for 14d, then no treatment for 14d; Q2D×28d = treatment every other day for 28d. Horizontal lines represent the mean and SEM. Statistical comparisons: * = p<0.05 compared with inhaled saline control.