Airways delivery of rifampicin microparticles for the treatment of tuberculosis

Sandra Suareza, Patrick O'Haraa, Masha Kazantsevaa, Christian E. Newcomerb, Roy Hopferc, David N. McMurrayd and Anthony J. Hickeya,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A Mycobacterium tuberculosis (H37Rv)-infected guinea pig model was used to screen for targeted delivery to the lungs by insufflation (with lactose excipient) or nebulization, of either rifampicin alone, rifampicin within poly(lactide-co-glycolide) microspheres (R-PLGA) or polymer microparticles alone (PLGA). Animals treated with single and double doses of R-PLGA microspheres exhibited significantly reduced numbers of viable bacteria, inflammation and lung damage compared with lactose-, PLGA- or rifampicin-treated animals 28 days post-infection (P < 0.05). Two doses of R-PLGA resulted in reduced splenic enlargement. These studies support the potential of R-PLGA delivered to the lung to treat pulmonary tuberculosis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Pulmonary Mycobacterium tuberculosis infection (TB) is characterized by alveolar macrophages (AMs) containing large numbers of bacilli. Current treatment of pulmonary TB involves prolonged oral administration of large systemic doses of combined antibiotics, which are associated with unwanted side effects and poor patient compliance.1 Targeting the drug to AMs would be a rational addition to current therapy, potentially enhancing efficacy and reducing toxicity. Microsphere formulations have been developed for intravenous and oral delivery of rifampicin, targeting host macrophages outside the lung.2 Thus, a microparticle drug delivery system3 targeted to the AMs might be effective, but has yet to be evaluated by direct administration to the lungs.

A low-level respiratory challenge ‘rational animal model’4 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


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chemicals and reagents

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 (45–125 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 Dunkin–Hartley guinea pigs (150–200 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 (7–9 mg/L, 2–3 h after administration).8 Assuming AMs are spherical with a cell diameter of 10 µm, and that there are 106–107 cells/lung, the total dose required for efficacy is c. 5–50 ng [10 µg x no. cells (106–107) x volume of cell (500 nL)]. This is less than the dose delivered to the lungs (rifampicin 1.0–1.7 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 = 6–10) 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 = 3–5) 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 21–28 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 haematoxylin–eosin.

Statistical analysis

Data were analysed using Scheffe's or Duncan's multiple comparison statistical tests.10


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Single dose administered by insufflation

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 1aGo).



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Figure 1. Number of viable bacteria (cfu/mL) in lung ({blacksquare}) and spleen () tissues following (a) insufflation of a single dose of R-PLGA (12.0 mg/kg), PLGA or rifampicin (12 mg/kg). (b) Double doses: insufflation (12.0 mg/kg) and nebulization of R-PLGA (equivalent to 5 mg/kg of rifampicin), PLGA or rifampicin (17 mg/kg). Bars represent mean ± S.D. for n = 3–5. *P < 0.05 (level of significance for both lung and spleen samples).

 
Double dose administered by insufflation followed by nebulization

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)Go (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 2dGo). The control, rifampicin and PLGA treatments resulted in multiple large sheets of epithelioid macrophages and caseous granulomas (Figure 2a–cGo). 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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Figure 2. Lung histopathology in guinea pigs treated by insufflation followed by nebulization and infected for 4–5 weeks with virulent M. tuberculosis. (a) Control animal with some degree of histopathological change. Arrows indicate a subpleural parenchymal zone with patchy histiocytic interstitial pneumonia. (b) Blank microsphere-treated animal. Epithelioid sheets cuff many bronchovascular bundles. Arrows indicate a large epithelioid sheet. (c) Double dose rifampicin-treated animal (equivalent to rifampicin 17 mg/kg). Arrows indicate large sheets of epithelioid macrophages. (d) Double R-PLGA microspheres (equivalent to rifampicin 17 mg/kg)-treated animal with minimal pathology. Small granulomas (arrows) appeared to be associated with the resorption of residual microparticles. Samples were stained with haematoxylin–eosin. Magnification 25x.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Rifampicin was incorporated in microspheres because of the compatability of its solubility with manufacturing methods and the perceived benefit of extending its usefulness as a first-line anti-tubercular agent. Low oral bioavailability and variability in delivery from various dosage forms11 may be avoided by direct delivery to the lungs. Slow release microsphere aerosols (1–5 µm) offer several potential advantages, including: (i) higher patient compliance; (ii) drug targeting to AMs; and (iii) reduction of systemic side effects by decreasing the total dose and frequency of drug administration compared with conventional preparations. These attributes make microspheres a promising alternative for the treatment of mycobacterial infections, especially pulmonary tuberculosis.

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 1bGo), while a single dose was only effective in reducing the bacterial burden in the lungs (Figure 1aGo). 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The assistance of Ms Susan Phalen (Texas A&M University), Professor Bernd Muller and Dr Peter Holzner (University of Kiel) is gratefully acknowledged. This study was supported by the National Heart, Lung and Blood Institute (NIH grant no. HL 55789).


    Notes
 
* Corresponding author. Tel: +1-919-962-0223; Fax: +1-919-962-0197; E-mail: ahickey{at}unc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Schreiber, J., Zissel, G., Greinert, U., Schlaak, M. & Muller-Quernheim, J. (1999). Lymphocyte transformation test for the evaluation of adverse effects of antituberculous drugs. European Journal of Medical Research 4, 67–71.[Medline]

2 . Quenelle, D. C., Stass, J. K., Winchester, G. Z., Barrow, E. L. & Barrow, W. W. (1999). Efficacy of microencapsulated rifampin in Mycobacterium tuberculosis-infected mice. Antimicrobial Agents and Chemotherapy 43, 1144–51.[Abstract/Free Full Text]

3 . Gupta, P. K. & Hickey, A. J. (1991). Contemporary approaches in aerosolized drug delivery to the lung. Journal of Controlled Release 17, 129–48.

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6 . O'Hara, P. & Hickey, A. J. (2000). Respirable PLGA microspheres containing rifampicin for the treatment of tuberculosis: manufacture and characterization. Pharmaceutical Research 17, 955–61.[ISI][Medline]

7 . Thadepalli, H. (1984). Lower respiratory tract. In Antimicrobial Therapy, (Ristuccia, A. M. & Cunha, B. A., Eds), pp. 439–51. Raven Press, New York, NY.

8 . Kucers, A., Bennet, N. M. & Kemp, R. J. (1988). The Use of Antibiotics, 4th edn. J. B. Lippincot Company, London.

9 . Wiegeshaus, E. H., McMurray, D. N., Grover, A. A., Harding, G. E. & Smith, D. W. (1970). Host–parasite relationships in experimental airborne tuberculosis. III. Relevance of microbial enumeration to acquired resistance in guinea pigs. American Review of Respiratory Disease 102, 422–8.[ISI][Medline]

10 . SAS/STAT User's Guide, Version 6. (1989). 4th edn, vol. 2. The SAS Institute Inc., Cary, NC.

11 . Ellard, G. A. & Fourie, P. B. (1999). Rifampicin bioavailability: a review of its pharmacology and the chemotherapeutic necessity for ensuring optimal absorption. International Journal of Tuberculosis and Lung Disease 3, S301–8.[ISI][Medline]

12 . Suarez, S., O'Hara, P. & Hickey, A. J. (1998). Uptake and disposition of rhodamine-labeled rifampicin-loaded poly(lactideco-glycolide) microspheres following powder insufflation. AAPS PharmSci 1, S208.

Received 19 January 2001; returned 9 May 2001; revised 4 June 2001; accepted 18 June 2001