JAC Advance Access originally published online on September 13, 2006
Journal of Antimicrobial Chemotherapy 2006 58(5):980-986; doi:10.1093/jac/dkl369
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Evaluation of dosing regimen of respirable rifampicin biodegradable microspheres in the treatment of tuberculosis in the guinea pig
School of Pharmacy, University of North Carolina Chapel Hill, NC 27599, USA
*Corresponding author. Tel: +1-919-962-0223; Fax: +1-919-962-0197; E-mail: ahickey{at}unc.edu
Received 1 March 2006; returned 5 April 2006; revised 17 July 2006; accepted 15 August 2006
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Abstract |
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Objectives: The efficacy of rifampicin-loaded polymeric microspheres (RPLGA) delivered to guinea pigs infected with Mycobacterium tuberculosis (H37Rv) was compared with a daily dose of nebulized rifampicin suspension.
Methods: Aerosol-infected animals were subjected to multiple dose or single dose treatment with RPLGA, PLGA microspheres or micronized rifampicin suspension aerosols. For comparison with treatment with suspensions of microspheres, additional groups received daily doses of rifampicin-only suspensions for 20 (20-RIF) and 10 (10-RIF) days.
Results: Drug and polymer treated multiple dose groups exhibited significantly lower wet lung weights than untreated animals. Spleen wet weights and viable bacterial counts (VBCs) were much lower for drug microsphere treated animals than for all other groups. In multiple dose studies with rifampicin-only suspensions, wet lung weights for 10-RIF and 20-RIF treated animals were much smaller than controls. Likewise, wet spleen weights of 10-RIF and 20-RIF treated animals were much smaller than controls, consistent with reduced inflammation. Spleen VBC of 20-RIF treated animals was much smaller than controls. No statistical differences were observed in the lung VBC among single dose groups. However, a trend similar to that of the wet weights was observed.
Conclusions: Aerosolized RPLGA reduced most measures of tuberculosis (TB) infection. These studies are further evidence for the potential of inhaled aerosol therapy for the treatment of TB. However, additional studies are required to elucidate underlying mechanisms of action and optimize this route of drug delivery.
Keywords: aerosols , nebulized suspensions , PLGA , lung deposition
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Introduction |
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Mycobacterium tuberculosis is the world's largest cause of death from a single microorganism.1 It is estimated that between 2000 and 2020, nearly 1 billion people will be newly infected with tuberculosis (TB) and 35 million will die from TB if control is not further strengthened.2 Current treatment involves prolonged systemic administration of high-dose oral combinations of antibiotics.3 For most patients, regimens that contain rifampicin and isoniazid can be completed in 6 months if pyrazinamide is included. When pyrazinamide is omitted, therapies are completed in 9 months, and when neither isoniazid nor rifampicin can be used, therapy is continued for a minimum of 12 months for up to 24 months.4 The extended use of these antibiotics frequently produces unwanted side effects in the patients due to intrinsic drug toxicity, often leading to non-compliance, which in turn may also lead to drug-resistant microorganisms. Among these ‘first-line’ antibiotics for TB treatment, rifampicin appears to have the largest number of side effects, notably hepatotoxicity, enzyme induction and interactions with several drug classes. Other common adverse effects such as nausea and the ‘flu-like’ syndrome sometimes associated with thrombocytopenia and acute renal failure often result in discontinuation of the treatment.5,6
Targeted delivery of drug directly to the primary site of infection may increase local drug concentrations while decreasing systemic side effects. Thus, targeting the lungs with antitubercular drugs as supplement to the oral therapy may provide improved efficacy to current existing treatments. Moreover, delivery of drugs encapsulated into microparticles to the lungs will target alveolar macrophages (AMs),7–10 the primary site of M. tuberculosis in infected lungs. The efficacy of aerosolized rifampicin-loaded poly (lactide-co-glycolide) microspheres (RPLGA) for the treatment of pulmonary TB in guinea pigs has been evaluated previously.11,12 A single dose of aerosolized RPLGA administered 24 h before infection significantly reduced the number of viable bacteria, markers of inflammation and lung damage compared with untreated controls and animals treated with blank microspheres or rifampicin alone after 28 days of infection.
Drug delivery systems prepared with PLGA have been shown to provide drug release over extended periods of time, the length determined by the proportion of lactic and glycolic acids in the polymer, its molecular weight, the particle size of the delivery system and the site of delivery.13–15 Thus, an additional advantage of delivering antitubercular agents encapsulated into PLGA microparticles to the lungs may be the reduction in the number and size of doses needed to be administered for treatment. The purpose of the present studies was to extend previous reports by evaluating the efficacy of a daily nebulized dose of micronized rifampicin suspension to treat TB in guinea pigs previously infected by the pulmonary route and compare the effect of treatment with a single dose of RPLGA with that of multiple nebulized doses of micronized rifampicin suspensions.
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Materials and methods |
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Materials and characterization of formulations
Rifampicin (Sigma, St Louis, MO, USA) was micronized with a modified Trost GEM-T jet mill (Garlock Inc., Newton, PA, USA) operated at an inlet pressure of 60 psig and outlet pressure of 40 psig. The morphology of the micronized particles was evaluated by scanning electron microscopy (SEM; Model 6300 JEOL, Peabody, NY, USA). Projected area diameter (Dp) of micronized particles was determined by Sigma Scan® software by counting 
250 particles in a minimum of 10 fields of view. The Hatch-Choate conversion was used to calculate the mass median diameter (MMD) and geometric standard deviation (GSD) from the Dp.16
RPLGA and PLGA microspheres were prepared using rifampicin and PLGA (75:25, mol. wt = 85 200) by the emulsion/solvent evaporation method described previously.17 Morphology of microspheres was initially characterized by SEM. Volume diameter (Dv) of the powders was obtained by laser diffraction (HELOS particle size analysis, H0838, Sympatec, GMBH, Germany) following shear dispersion (Rodos, Sympatec, GMBH, Germany).
Characterization of nebulizer output
The aerosol droplets produced by the Acorn II nebulizer (Marquest Medical Products, Inc. Englewood, CO, USA) were characterized by cascade impaction, using 0.01% sodium fluorescein solutions. Aerosols were sampled at 28.3 L/min by an Andersen 1ACFM NonViable Ambient Particle Sizing Sampler (Thermo Electron, Waltham, MA, USA). The T-piece of the nebulizer was connected to a USP throat18 of the cascade impactor and the solutions were nebulized at 40 psig for 10 min. All runs were performed in quintuplicate (n = 5).
Animals
All animal procedures were approved by the UNC-CH Institutional Animal Care and Use Committee. Specific pathogen free male Dunkin–Hartley guinea pigs (Hilltop, Scottsdale, PA, USA) weighing 297–443 g were housed individually in a bio-safety level 3 (BL-3) containment area with a 12 h light/dark cycle. Animals were allowed free access to water and food (Prolab guinea pig 5P18, PMI feeds, Inc., St Louis, MO, USA) at all times.
Respiratory infection
Animals were infected via the respiratory route with a small inoculum (2 x 105 cfu/mL) of M. tuberculosis strain H37Rv.19 Animals were placed randomly in an exposure chamber and aerosols were generated by pumping compressed air through a modified MRE-type 3 jet collision nebulizer (Waltham, MA, USA) containing 5 mL of bacterial suspension. This concentration was expected to result in the inhalation and retention of 
10–15 viable, virulent organisms per guinea pig.19,20
Treatments
Animals receiving treatment with RPLGA were divided into two groups (Table 1): multiple dose (MD, dosed at days 3, 7, 14 and 21 after infection) and single dose (SD, dosed 5 days after infection). Each group had four subgroups: animals treated with RPLGA (40 mg, MD-RPLGA and SD-RPLGA) with three control treatments, blank PLGA microspheres (32 mg, MD-PLGA and SD-PLGA), rifampicin alone (corresponding to the 30% loading in RPLGA = 8 mg, MD-RIF and SD-RIF) and saline controls (MD-SAL and SD-SAL). The respective dose was suspended in 5 mL of 0.05% Tween 80 saline solution and nebulized at 40 psig for 10 min, five times to animals in a nose-only inhalation chamber (ADG Developments, Ltd, Herts, UK).
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For comparison with treatment with once a week microsphere suspensions, additional experiments were performed for the last 20 (20-RIF) and 10 days (10-RIF) of a 28 day TB infection cycle which involved the daily administration of suspensions of micronized rifampicin, as well as saline to control animals (untreated controls, 10-SAL and 20-SAL). Animals were placed into the ports of the nose-only inhalation chamber for treatment. Rifampicin (13.1 mg) was suspended in 5 mL of a 0.05% Tween 80 saline solution and nebulized as described above (total daily dose = 65.5 mg of rifampicin,
145–215 mg/kg). Treatments were given to animals starting at 8 days (20-RIF) and 18 days (10-RIF) after infection. Necropsy procedure and assessment of the number of viable bacteria
Animals were euthanized by an intraperitoneal lethal dose of sodium pentobarbital 28 days after infection. The peritoneal and chest cavities were exposed and lungs, spleen and liver were removed, weighed and inspected to determine the degree of inflammation and primary lesions. The lower left lobe of the lung and a portion of the spleen and a portion of the liver were resected using separate sterile instruments and stored in 10% neutral buffered formalin for histopathological analysis. The lower right lobe and a portion of the spleen were homogenized separately in sterile saline solution. After proper dilutions, aliquots of 0.1 mL were inoculated in M7H10 agar plates. The plates were incubated at 37°C for 21 days and the number of viable bacteria were counted (cfu).
Histopathological analysis
Lung and spleen tissues preserved in formalin solution (10% w/v) were then embedded in paraffin and sectioned with a microtome (Leica Microsystems, Bannockburn, IL, USA) at 5 µm. The sections were mounted on a glass slide and stained with haematoxylin–eosin for microscopic evaluation.
Statistical analysis
Wet lung weights and the log-transformed number of viable bacteria [viable bacterial count (VBC)] were assumed to be normally distributed and analysed by ANOVA (SAS/STAT, Cary, NC, USA). The difference between treatments was determined by Tukey's HSD and Scheffe's multiple range comparisons tests.
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Results |
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Preparation and characterization of rifampicin formulations
The SEM evaluation of micronized rifampicin revealed polyhedral drug particles with a mean Dp of 2.83 µm and a GSD of 1.5. SEM micrographs also showed that RPLGA and PLGA microspheres had spherical shape with MMD = 3.2 µm and GSD = 1.76. The Dv of the powder by laser diffraction was 63.4 µm with a skewed distribution (16% = 9.67 µm and 84% = 112.69 µm). Thus the difference between Dp and Dv indicated that the powder was aggregated.
Characterization of nebulizer output
The cascade impaction method revealed that the Acorn II nebulizer used in these studies produced aerosol droplets with a mass median aerodynamic diameter (MMAD) of 1.5 µm and a GSD of 2.9.
Wet organ weights
Wet tissue weight has been used in many studies as an indirect marker of inflammation21–26 or sign of acute toxicity.27–31 In the present studies, lung and spleen wet weights of infected animals after the respective treatments were used as an indicator of the degree of inflammation of each organ. The larger organ weight would then indicate the greater extent of inflammation due to TB infection. The wet tissue weights of animal groups treated with microspheres are shown in Table 2. Wet lung weights of animals receiving MD-RIF and MD-PLGA were (12% and 15%, respectively) significantly lower than untreated animals (MD-SAL). Spleen wet weights of animals receiving MD-RPLGA aerosols were also 27% lower than those receiving MD-RIF or untreated controls (MD-SAL). Likewise, wet lung weights of animals dosed with SD-RIF were 20% significantly lower than those of untreated controls (SD-SAL) and there was no significant difference between SD-RPLGA and SD-RIF groups. Wet spleen weights of animals receiving SD-RPLGA were also 50% significantly lower than those of SD-PLGA or untreated controls (SD-SAL, Table 2).
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The wet tissue weights of animal groups receiving multiple nebulizations of rifampicin suspensions are shown in Table 3. Wet lung weights and spleen weights of animals treated with nebulized rifampicin (10-RIF and 20-RIF) were significantly lower than those of animals exposed to aerosolized saline (TB controls, 10-SAL and 20-SAL).
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Bacteriology
The number of viable bacteria [log cfu/mL, or VBC] remaining in the lungs and spleens of animals receiving treatments with microspheres are shown in Figure 1. Large variability was observed in the VBC of affected organs among animals treated with microspheres (MD-RLPGA, SD-RPLGA, MD-PLGA and SD-PLGA), which heavily influenced the statistical interpretation of the data. The VBC in the spleens of animals receiving MD-RPLGA was significantly smaller, by 17%, than that of untreated controls (MD-SAL). The VBC in the spleens of animals receiving SD-RPLGA was significantly smaller, by 13%, than that of SD-RIF and 22% smaller than that of untreated controls (SD-SAL).
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The VBCs in lungs and spleens of animals receiving aerosolized saline for 10 and 20 days (10-SAL and 20-SAL) were statistically similar and thus were grouped in a single control group for the purposes of comparison with animals receiving rifampicin suspensions (Figure 2). Among animals receiving aerosolized rifampicin-only suspensions, animals treated for 20 days (20-RIF) had significantly smaller VBCs in spleen than those receiving saline (10- and 20-SAL). Although no significant differences were observed in the VBC of lungs among the different treatments, the VBC in the lungs of animals treated for 20 days (20-RIF) with aerosolized rifampicin suspensions were slightly smaller than those of animals treated for 10 days (10-RIF) and smaller than those of animals receiving saline.
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Histopathology
A semi-quantitative histopathological evaluation was undertaken, examining the degree of involvement of lung lobes and of splenic white pulp. Overall, the findings reported for wet weights were supported by the histopathological analysis, with less extensive granulomatous involvement of lung lobes, smaller granulomata, less severe necrosis and less lymphocytic infiltration typically being seen in treated animals compared with controls. Likewise, treated animals exhibited a decreased granulomatous involvement with minimal caseous necrosis in the splenic white pulp. Animals receiving RPLGA (MD and SD) showed improvement in both lung and spleen histology, while rifampicin (SD and MD) treatment resulted in greater histological improvement in lung than spleen. For animals receiving daily rifampicin treatment by nebulization, only animals in the 20-RIF group showed a marked histological improvement.
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Discussion |
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The potential therapeutic benefit of the pulmonary administration of RLPGA 24 h before infection was demonstrated in screening studies with the guinea pig model of TB.11,12 However, the true efficacy of particles administered in this manner for the treatment of TB requires demonstration in a realistic experimental design of delivery, after infection has occurred. The present studies were designed to demonstrate the efficacy of pulmonary administration of RPLGA after infection and to compare the effect of a single RPLGA administration, where it is anticipated that the drug would be released slowly over an extended period of time, with that of multiple administrations of rifampicin alone.
Although histopathological analysis of lung and spleen tissues in all treated animals indicated a favourable effect of the aerosolized treatment with RPLGA, quantitative measures such as wet tissue weight and VBC did not reflect that trend in all cases. RPLGA (MD and SD) had a significant effect in the spleen but not in the lungs of treated animals compared with untreated controls. Significantly lower wet weights and VBC were observed in the spleens of animals treated with RPLGA. Lower wet spleen weights were an indirect indication of a smaller degree of inflammation,21–34 possibly due to a smaller microbial dissemination to this organ by M. tuberculosis. Unlike the screening studies reported previously, pulmonary administration of RPLGA (MD and SD) did not have a measurable effect on the reduction of VBC in the lungs of treated animals. The differences between the screening studies and those reported here may be explained by the difference in the time of infection for each group of animals. In screening studies RPLGA were administered before the animals were infected with M. tuberculosis, and in the present study RPLGA were administered after infection. When RPLGA were delivered prior to infection, AMs in the lungs would phagocytose these particles.35 Subsequently, when animals inhale M. tuberculosis, AMs already containing RPLGA and potentially some AMs not containing RPLGA will phagocytose the bacteria. In AMs that contain RPLGA, the rifampicin released from the microspheres will kill local bacteria. This may impact on the number of bacteria colonizing the lungs causing a lag time in bacterial replication and bacillaemia leading to delayed distribution to other organs. Thus, at 28 days post-infection, a difference in the bacterial count in the lungs between treated and untreated animals was observed. The same effect was not observed in spleen, since dissemination of M. tuberculosis from lung to spleen takes 10–14 days.36 In contrast, when animals are infected and then treated, it is likely that RPLGA did not affect the initial number of bacteria colonizing the lungs. However, upon release of rifampicin from RPLGA, drug available in the lungs may have decreased the number of bacteria translocating from the lungs to the spleen, so that at 28 days a measurable difference would be anticipated between spleens of treated animals and untreated controls. This hypothesis may be more plausible than a reduction in the number of bacteria in the spleen due to significant systemic levels of rifampicin after inhalation treatments, since very low plasma concentrations (0.7% of the corresponding intravenous dose) were obtained after nebulization of micronized rifampicin suspensions to guinea pigs for 50 min.37
Based on the outcome after treatment of animals with SD-RPLGA, it was anticipated that treatment with MD-RPLGA would have further reduced the VBC in the spleen of animals in that group. Contrary to expectations, MD-RPLGA treatment had approximately the same effect (0.9 log reduction) as that after SD-RPLGA (1.2 log reduction) on VBC in the spleen of treated animals. The efficiency of RPLGA aerosolization and lung delivery may have influenced these results. In general only 15–20% by mass of nebulized formulations reaches the alveolar region of lungs.38 The mean volume diameter of suspended RPLGA (63.4 µm) was much larger than the aerodynamic diameter of nebulized droplets from the Acorn II nebulizer (MMAD = 1.5 µm, GSD = 2.9), as determined by inertial impaction of nebulized fluorescein solutions. The large size of the RPLGA aggregates would likely limit their incorporation into the smaller droplets, thus decreasing the concentration of RPLGA in the aerosol delivered. A surfactant (Tween 80) was added to the suspension to decrease possible aggregation caused by electrostatic effects, but would have had no effect on aggregates formed by physical bridging during particle manufacture. Therefore, the lack of effect in the MD-RLPGA group may be explained by the low efficiency of RPLGA delivery by nebulization and low local rifampicin concentrations as previously determined in pharmacokinetic studies.37
In the second part of the study, treatment of animals with micronized rifampicin for 10 and 20 consecutive days had a positive effect on the degree of inflammation of lungs and spleen of these animals as indicated by wet tissue weights (a surrogate marker of the degree of inflammation)21–26 and histopathological analysis. Although no statistical difference was observed among VBC in the lungs of treated animals compared with controls, a trend showing decreased VBC in lungs of treated animals versus untreated controls was observed, correlating with lower lung weights. Unlike the VBC in spleens of animals treated for 10 days with micronized rifampicin suspensions, the VBC in spleens of animals treated for 20 days was significantly smaller than that of untreated controls. The delay in the bacterial growth in the spleen was longer for the animals treated for 20 days. As discussed for the effects of RPLGA on VBC, it is likely that a longer treatment with rifampicin suspensions would have decreased the number of bacteria translocating from the lungs to the spleen due to both a larger total dose received (1310 mg) and treatment starting before bacteria would translocate to the spleen (8 days after infection). In contrast, when animals were treated for 10 days, the total dose (655 mg) may not have been sufficient to have an effect on VBC in the spleen.
Most importantly, the decrease in VBC in the spleen after treatment of animals with micronized rifampicin suspensions for 20 days (0.8 log) was comparable to the decrease in VBC in the spleen observed after the administration of a single dose of RLPGA (1.2 log), which was also supported by histopathological analysis. This effect is most likely due to a controlled release of rifampicin from RPLGA and targeting of AMs and lung environment, where M. tuberculosis resides after infection. Thus, it is possible that a therapeutic regimen using RPLGA, in addition to other necessary antibiotics, may require less frequent rifampicin dosing, thus decreasing side effects associated with this drug. In addition, the modest effect observed in the present studies with inhaled RPLGA (approximately 1 log reduction in VBC) may also be attributed to treatment with monotherapy. It has been reported that oral administration of a high-concentration rifampicin suspension to infected guinea pigs for 4 weeks was not as effective as a combination of a low dose rifampicin suspension and a low dose of isoniazid (1.7 versus 3.2 log reduction in lung VBC, respectively) in the treatment of tuberculosis.39
Other antitubercular drugs have been encapsulated in different types of particles for inhalation, including large porous particles,40,41 PLGA microparticles42 and nanoparticles.43–45 These particles have been evaluated in rats41,42 and guinea pigs43–45 in terms of drug pharmacokinetics41,42,44 and efficacy.43,45 Treatment with nanoparticles was reported to be effective in decreasing VBC in the lungs of infected animals. However, unlike the studies presented here in which particles contained rifampicin only, nanoparticles employed in those studies contained three of the front-line antitubercular agents, isoniazid, rifampicin and pyrazinamide.
In summary, treatment with RPLGA had a positive effect in protecting the spleen of treated animals from infection as indicated by the reduced inflammation and smaller VBCs compared with untreated controls. Treatment of infected animals for 20 consecutive days with rifampicin suspensions also had the same positive effects on the spleen of these animals. Furthermore, the effect of treatment with a single dose of RLPGA was comparable to that after treatment with rifampicin suspensions for 20 consecutive days, supporting the potential use and advantages of treatment with RPLGA over conventional treatments for pulmonary TB. More studies employing particles with higher drug loads, optimized particle size and aerosolization properties, and extended dosing periods (>20 days) are required to establish an appropriate dosing regimen for the treatment of TB with RPLGA.
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There is no interest or conflict (financial or non-financial) by any of the authors that may have influenced the views expressed in this paper.
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Acknowledgements |
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This work was supported by the National Heart, Lung and Blood Institute (NIH Grant HL55789).
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  L. Garcia-Contreras, J. Fiegel, M. J. Telko, K. Elbert, A. Hawi, M. Thomas, J. VerBerkmoes, W. A. Germishuizen, P. B. Fourie, A. J. Hickey, et al. Inhaled Large Porous Particles of Capreomycin for Treatment of Tuberculosis in a Guinea Pig Model Antimicrob. Agents Chemother., August 1, 2007; 51(8): 2830 - 2836. [Abstract] [Full Text] [PDF]
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