a School of Pharmacy, and Departments of b Pathology and Laboratory Animal Medicine, and c Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; d Department of Medical Microbiology and Immunology, College of Medicine, Texas A&M University, System Health Science Center, College Station, TX 77843, USA
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Abstract |
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Introduction |
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A low-level respiratory challenge rational animal model4 was used to screen the ability of rifampicin alone or encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres (R-PLGA), to prevent bacterial growth and tissue damage. Low-dose M. tuberculosis aerosol delivery to guinea pigs results in infection characterized by slow initial growth followed by logarithmic growth, bacillaemia and a positive purified protein derivative (PPD) skin test, similar to that in humans.5
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Materials and methods |
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R-PLGA and PLGA (75:25, mol. wt 85.2 kDa) microspheres and rifampicin were prepared by spray drying.6 Volume median diameters (VMD) and geometric standard deviations (S.D.) for microparticles were [VMD (µm)/geometric S.D.]: R-PLGA, 2.76/1.57; PLGA, 2.87/1.45; and rifampicin alone, 3.83/1.75. The dissolution rate up to 24 h for 30% w/w loaded R-PLGA microspheres was 75% and 52% at pH 7.4 and pH 5.3, respectively, at 37°C. The terminal rate constant at both pHs was 0.46%/h½. Lactose (45125 µm; Mallinckrodt, Paris, KY, USA) was used as a carrier. Sodium pentobarbital (Sigma, St Louis, MO, USA) was used for killing.
Animals
Procedures were approved by the UNC-CH Institutional Animal Care and Use Committee. Specific-pathogen-free male DunkinHartley guinea pigs (150200 g) (Hilltop, Scottsdale, PA, USA) housed individually (biosafety level three containment, 12 h light/dark cycle) with free access to water and food (Prolab guinea pig 5P18; PMI feeds, Inc., St Louis, MO, USA), but fasted overnight before experiments.
Dose selection
The rifampicin concentration in AMs from 600 mg oral doses is twice that in serum7 (79 mg/L, 23 h after administration).8 Assuming AMs are spherical with a cell diameter of 10 µm, and that there are 106107 cells/lung, the total dose required for efficacy is c. 550 ng [10 µg x no. cells (106107) x volume of cell (500 nL)]. This is less than the dose delivered to the lungs (rifampicin 1.01.7 mg/kg) in the following studies. However, drug release dictates the instantaneous dose, making large drug loads a necessity to achieve an equivalent effect to an oral dose in the AM. Practically, the doses were limited by the maximum drug load (30% R-PLGA) in a lactose blend (90:10 rifampicin: lactose), which was delivered in a powder bolus (10 mg) from the insufflator.
R-PLGA microspheres administered by insufflation and nebulization
Guinea pigs were anaesthetized with ketamine 50 mg/kg, xylazine 5 mg/kg and acepromazine 2 mg/kg by ip injection, intubated endotracheally, and the powder (10 mg) was insufflated using 3 mL of air (Insufflator; PennCentury, Philadelphia, PA, USA). The treatment groups (n = 610) were: rifampicin/lactose (rifampicin 12 mg/kg), R-PLGA/ lactose (rifampicin 12 mg/kg), PLGA/lactose and lactose. Animals recovered for 24 h before infection with M. tuberculosis. Ten days after infection, half of each treatment group (n = 35) was exposed to a second dose of drug or control material by nebulization (inlet air at 40 psig; Acorn II, Marquest, Englewood, CO, USA): (i) R-PLGA (rifampicin 5 mg/kg); (ii) rifampicin 5 mg/kg; and (iii) 40 mg PLGA samples were prepared in 5 mL of 0.05% Tween 80 saline solution, or (iv) solution control.
Experimental infection
M. tuberculosis (H37Rv, 5 mL, 2 x 105 cfu/mL; ATCC, Rockville, MD, USA) suspensions were nebulized (modified MRE-3 Collison; BGI Inc., Waltham, MA, USA) to animals in an exposure chamber.9
Assessment of the number of viable bacteria
Twenty-eight days after the infection the caudal right lung lobe and approximately three-quarters of spleen tissue were homogenized in sterile saline. The caudal left lung lobe and approximately one-quarter of spleen tissue were placed in 10% neutral buffered formalin for histopathology. Duplicate M7H10 agar plates (Hardy Diagnostics, Santa Maria, CA, USA) were inoculated with 0.1 mL diluted homogenates and incubated at 37°C for 2128 days. Colony counts were expressed as log10, meeting the assumption of parametric statistics.
Histopathology
Formalin-fixed tissues embedded in paraffin wax and sectioned at 5 µm were mounted on glass slides and stained with haematoxylineosin.
Statistical analysis
Data were analysed using Scheffe's or Duncan's multiple comparison statistical tests.10
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Results |
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Single dose R-PLGA (12 mg/kg) insufflation significantly reduced the lung burden of bacteria (log cfu/mL = 3.7 ± 0.3) compared with rifampicin (log cfu/mL = 4.17 ± 0.1), PLGA (log cfu/mL = 4.4 ± 0.32) or the lactose group (log cfu/mL = 4.33 ± 0.16) (P < 0.05, Scheffe's) (Figure 1a).
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Double dose R-PLGA (12 mg/kg by insufflation followed by 5 mg/kg by nebulization) treatment significantly reduced the lung and spleen bacterial burden compared with an equivalent dose of rifampicin or PLGA, or untreated groups, respectively, as shown in Figure 1(b) (P < 0.05, Scheffe's).
Histopathological studies
Single or double doses of R-PLGA resulted in minimal patchy interstitial thickening and small areas of histiocytosis in the lungs (Figure 2d). The control, rifampicin and PLGA treatments resulted in multiple large sheets of epithelioid macrophages and caseous granulomas (Figure 2ac
). Double doses of R-PLGA resulted in less histiocytosis and purulent splenitis than control, and rifampicin- and PLGA-treated groups. Lung tissue wet weight ratios were significantly different (P < 0.05, Duncan's) for groups treated with both single (r = 0.78 ± 0.12) and double (r = 0.77 ± 0.04) doses of R-PLGA compared with single (r = 0.98 ± 0.12) and double (r = 0.95 ± 0.05) doses of rifampicin or PLGA (r = 0.92 ± 0.01). Double dosing resulted in significant differences (P < 0.05, Duncan's) in the spleen wet weight ratios of the R-PLGA-treated group (r = 0.6 ± 0.12) compared with the rifampicin-treated group (r = 1.04 ± 0.25) and the PLGA-treated group (r = 0.97 ± 0.17).
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Discussion |
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Rifampicin-loaded microspheres were administered directly into the lungs, potentially circumventing the toxicity (notably hepatitis) resulting from large oral doses (600 mg daily over 6 months). Delayed release of rifampicin from R-PLGA was contrasted with the immediate release from rifampicin by delivery of microparticles 24 h before infection. It is not suggested that this is a clinically relevant efficacy model, but it is a necessary screening tool to evaluate the influence of particle characteristics and targeting on action. The residence time of drug was enhanced since the lung burden of bacteria was reduced by the R-PLGA treatment compared with all other treatments, including rifampicin. In preliminary experiments, 20 µg of rifampicin was present in the airways, as determined by bronchoalveolar lavage 72 h after R-PLGA insufflation, compared with 6 µg after rifampicin insufflation. Rifampicin was undetectable in plasma at 72 h. Double doses of R-PLGA significantly reduced the bacterial burden in both lung and spleen compared with rifampicin, PLGA and lactose groups (Figure 1b), while a single dose was only effective in reducing the bacterial burden in the lungs (Figure 1a
). Since rifampicin did not inhibit bacterial growth, the AM uptake of the long residence time R-PLGA appears to be important for the action of the drug.12 These findings indicate a role for high local drug concentrations in decreasing the burden of bacteria in the lungs. However, the complex nature of the combination prophylactic/therapeutic double dosing regimen requires further study.
Animals treated with R-PLGA microspheres exhibited reduced histopathological changes indicative of lung damage compared with lactose-, PLGA- and rifampicin-treated animals. The use of R-PLGA microspheres as aerosol treatment for primary pulmonary tuberculosis is supported by these preliminary studies.
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Acknowledgements |
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Notes |
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References |
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2
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Received 19 January 2001; returned 9 May 2001; revised 4 June 2001; accepted 18 June 2001