Article pubs.acs.org/molecularpharmaceutics
Novel Inhaled Combination Powder Containing Amorphous Colistin and Crystalline Rifapentine with Enhanced Antimicrobial Activities against Planktonic Cells and Biofilm of Pseudomonas aeruginosa for Respiratory Infections Qi (Tony) Zhou,† Si-Ping Sun,† John Gar Yan Chan,†,‡ Ping Wang,† Nicolas Barraud,§ Scott A. Rice,§,∥ Jiping Wang,⊥ Jian Li,⊥ and Hak-Kim Chan*,† †
Advanced Drug Delivery Group, Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia JHL Biotech, Inc., Zhubei City, Hsinchu County 302, Taiwan, R.O.C. § Centre for Marine Bio-Innovation and School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia ∥ Singapore Centre on Environmental Life Sciences Engineering, and the School of Biological Sciences, Nanyang Technological University, Singapore, Singapore ⊥ Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia ‡
S Supporting Information *
ABSTRACT: Colistin has been increasingly used for the treatment of respiratory infections caused by Gram-negative bacteria. Unfortunately parenteral administration of colistin can cause severe adverse effects. This study aimed to develop an inhaled combination dry powder formulation of colistin and rifapentine for the treatment of respiratory infections. The combination formulation was produced by spray-drying rifapentine particles suspended in an aqueous colistin solution. The combination dry powder had enhanced antimicrobial activities against planktonic cells and biofilm cultures of Pseudomonas aeruginosa, with both minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) values (2 and 4 mg/L, respectively) being half that of pure colistin (MIC 4 mg/L and MBIC 8 mg/L) and 1/16th that of pure rifapentine (MIC 32 mg/L and MBIC 64 mg/L). High aerosol performance, as measured via an Aerolizer device, was observed with emitted doses >89% and fine particle fraction (FPF) total >76%. The proportion of submicron particles of rifapentine particles was minimized by the attachment of colistin, which increased the overall particle mass and aerodynamic size distribution. Using the spray-drying method described here, stable particles of amorphous colistin and crystalline rifapentine were distributed homogeneously in each stage of the impinger. Unlike the colistin alone formulation, no deterioration in aerosol performance was found for the combination powder when exposed to a high relative humidity of 75%. In our previous study, surface coating by rifampicin contributed to the moisture protection of colistin. Here, a novel approach with a new mechanism was proposed whereby moisture protection was attributed to the carrier effect of elongated crystalline rifapentine particles, which minimized contact between hygroscopic colistin particles. This inhaled combination antibiotic formulation with enhanced aerosol dispersion efficiency and in vitro efficacy could become a superior treatment for respiratory infections. KEYWORDS: inhalable powder formulation, respiratory infection, combination antibiotics, colistin, rifapentine, elongated carrier, antibiofilm, moisture protection
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INTRODUCTION
Antibiotics have saved billions of lives since their discovery. However, the past decades have seen a remarkable increase of multidrug-resistant (MDR) bacteria with only a limited number of new antibiotics in the pipeline.1 Indeed, no new antibiotics have reached advanced stages of development for infections caused by MDR Gram-negative “superbugs”, including those © XXXX American Chemical Society
Special Issue: Advances in Respiratory and Nasal Drug Delivery Received: September 1, 2014 Revised: November 4, 2014 Accepted: November 25, 2014
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DOI: 10.1021/mp500586p Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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the amorphous form,27 in this study a novel combination powder formulation was developed that contained amorphous colistin and crystalline rifapentine such that both drugs exist in their stable forms under ambient conditions and exhibited strong synergistic activity in vitro against P. aeruginosa planktonic cells and biofilm.
causing respiratory infections, and such pathogens are the most problematic.2,3 Pseudomonas aeruginosa is one of the most common pulmonary infecting bacteria, which are responsible for both acute pneumonia and chronic respiratory infections in cystic fibrosis (CF) patients.4 P. aeruginosa can rapidly acquire resistance to first-line antibiotics5 and in many cases form biofilm communities, which comprise bacteria with highly heterogeneous gene expression profiles and phenotypes encased in a matrix of self-produced polymeric substances. Colistin (also known as polymyxin E) is often the only effective antibiotic against many MDR strains of P. aeruginosa.1 It has been shown to selectively kill the subpopulation of dormantlike persister cells that are abundant in P. aeruginosa biofilms and unaffected by other antibiotics such as ciprofloxacin, tetracycline, or tobramycin.6,7 Colistin (multiple components with molecular mass 1155− 1170 g/mol, freely soluble in water) is an old polypeptide antibiotic. High systemic exposure from intravenous administration of colistin can lead to nephrotoxicity in up to 60% of patients, which is a dose-limiting factor.8 However, high doses are necessary to treat respiratory infections as only a small proportion of colistin can reach the pulmonary sites of infection after systemic administration.9 Pulmonary delivery of antibiotics directly to the respiratory tract is advantageous in achieving higher drug concentrations at the target sites with a minimal systemic exposure10 and side effects. A pharmacokinetic study of nebulized colistin methanesulfonate solution (4 million IU) in CF patients demonstrated significantly high colistin concentrations in the sputum (Cmax 4−16 mg/L) for a prolonged period (colistin concentrations >3 mg/L for 12 h) and negligible systemic drug exposure (Cmax < 0.5 mg/L).11 Inhaled antibiotic products12,13 and formulations14,15 in development have been recently reviewed. Although development of bacterial resistance to colistin is currently limited, emergence of colistin resistance has been reported.4,16 The growing use of colistin as monotherapy has raised concerns of accelerated resistance development. Administration of synergistic combination antibiotics is an effective strategy to minimize colistin resistance.1 Rifampicin has been used clinically with colistin against MDR P. aeruginosa17 and demonstrated both in vitro18,19 and in vivo synergistic antibacterial activities.20,21 In our earlier study, an inhaled powder formulation containing colistin and rifampicin was developed by cospray-drying in a cosolvent, with the combined advantageous characteristics of high aerosol efficiency, synergistic antimicrobial activity, and moisture protection.22 The moisture protection effect was attributed to the surface coating with rifampicin. However, while inhaled rifampicin is rapidly cleared from lungs within 4 h of pulmonary delivery,23 concentrations of colistin administered via the pulmonary route are reported to remain above the minimum inhibitory concentration (MIC) in the airways for more than 12 h.11,24 Encapsulation of rifampicin in polymeric particles (such as poly(lactic-co-glycolic acid), PLGA) has been extensively investigated to achieve sustained-release profiles.25,26 However, it is extremely challenging to produce sustained-release formulations of combinations with satisfactory encapsulation efficiency and release profile for both drugs.14 To address this discrepancy in pulmonary drug residence times and maximize synergistic antibacterial killing, rifapentine (molecular mass 877 g/mol, insoluble in water), a hydrophobic derivative of rifampicin, could be a better candidate to be used together with colistin as an inhaled therapy. As rifapentine is unstable in
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MATERIALS AND METHODS Chemicals. Colistin sulfate was supplied by Zhejiang Shenghua Biology Co., Ltd. (Hangzhou, Zhejiang, China), and rifapentine from Hangzhou ICH Imp and Exp Co. Ltd. (Hangzhou, Zhejiang, China). High-performance liquid chromatography (HPLC) grade acetonitrile, ethanol, and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and trifluoroacetic acid from Sigma-Aldrich (Castle Hill, New South Wales, Australia). Two forms of colistin are available: colistin sulfate (referred to as colistin below) and colistin methanesulfonate. The latter is an inactive pro-drug of colistin16 that requires conversion to active colistin in vivo.28 Therefore, colistin sulfate was selected for the current study. Bacterial Strain. P. aeruginosa PAO1 was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in tryptone soya broth (TSB; Oxoid Australia, Adelaide, SA, Australia) with 20% glycerol at −80 °C. Production of Spray-Drying Feed Solution/Suspension. A colistin solution (16 mg/mL) was prepared by dissolving the drug in purified water. A rifapentine suspension was generated by a previously reported method.27 Briefly, rifapentine (16 mg/mL) was dissolved in acetone and deionized water. The solution was heated to 67 °C to remove acetone and to provide a primary aqueous suspension of crystalline rifapentine. The suspension was homogenized at 10,000 rpm for 2 min (Silverson L4RT High Shear Mixer, Silverson, Chesham, United Kingdom) to reduce particle size. The colistin solution and rifapentine suspension were then combined in a mass ratio of 1:1 by magnetic stirring (500 rpm) to give an aqueous mixture of solubilized colistin (8 mg/mL) and rifapentine particles (8 mg/mL). Spray Drying. A B-290 mini spray-dryer (Büchi Labortechnik AG, Falwil, Switzerland) was used under the following operating conditions: inlet temperature 60 °C; atomizer setting 700 NL/h, aspirator 40 m3/h, and feed rate 2 mL/min.25 The aqueous antibiotic mixture was stirred at a speed of 500 rpm throughout the spray-drying process. The collected spray-dried antibiotic dry powder was stored in a desiccator containing silica gel at 20 ± 3 °C until used. Particle Sizing. Particle size distribution of the powder formulations was measured by laser diffraction with a Scirocco dry powder dispersion unit (Mastersizer 2000, Malvern Instruments, Worcestershire, U.K.). Powder formulations were poured into the dispersion channel, dispersed by compressed air with a pressure of 4 bar and drawn through a laser measurement zone by vacuum. D10 (diameter at 10% undersize), D50 (diameter at 50% undersize), and D90 (diameter at 90% undersize) were calculated from the size distribution data. Measurements for each sample were carried out in triplicate. Particle Morphology. Particle morphology of the powder formulations was evaluated by scanning electron microscopy (SEM, Carl Zeiss SMT AG, Oberkochen, Germany). Powder was spread on a carbon sticky tape and mounted on an SEM stub, followed by sputter coating with gold (15 nm thick) using B
DOI: 10.1021/mp500586p Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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dried formulations was examined by storing the formulations at 75% RH. The powder formulations were spread in a thin layer in an open-top plastic container (3 cm in diameter) and placed in the environment-controlled chamber of DVS unit for 24 h at 75 ± 2% RH and 20 °C. On the basis of the DVS data, 24 h of storage is more than sufficient to reach water sorption equilibrium. After storage, the aerosolization performance of each powder was evaluated as described earlier but at an RH of 75% ± 2%. Minimal Inhibitory Concentration (MIC) against P. aeruginosa. P. aeruginosa can cause both acute and chronic respiratory infections4 and therefore was selected to test the antibacterial activity of the formulations. P. aeruginosa cultures were grown overnight in 3 mL of Luria−Bertani medium with 10 g/L NaCl (LB10) incubated at 37 °C with shaking. Broth microdilution method was employed to measure the MICs against P. aeruginosa. An aliquot of an overnight culture was diluted 200-fold to an OD600 of 0.005 in M9 minimal medium (containing 48 mM Na2HPO4, 22 mM KH2PO4, 9 mM NaCl, 19 mM NH4Cl, 2 mM MgSO4, 20 mM glucose, and 100 μM CaCl2, pH 7.0) giving approximately 106 cfu/mL for the inoculum.7 Drug concentrations of 0.5, 1, 2, 4, 8, 16, 32, and 64 mg/L were obtained by diluting the stock drug solution (5.12 mg/mL) with fresh M9 medium. Microplates were incubated at 37 °C in a humidified incubator for approximately 20 h. MICs were determined (n = 4) by measuring OD600 of supernatant directly from the wells in 96-well plates using a microplate reader (Wallac Victor2, PerkinElmer, Waltham MA, USA). MIC values were determined as the minimum concentration that inhibited growth by more than 90% after 24 h of incubation.7 Minimal Biofilm Inhibitory Concentration (MBIC) against P. aeruginosa. To determine the MBIC, P. aeruginosa cultures were grown in microplates as described above. Instead of determining planktonic growth by measuring the OD600 of the supernatant, the plates were analyzed for biofilm growth using a crystal violet staining procedure. Briefly, P. aeruginosa overnight cultures were diluted to an OD600 of 0.005 in fresh M9 medium (with 20 mM glucose).33 Final drug concentrations ranged from 0.5 to 64 mg/L. Plates were incubated for 24 h at 37 °C. The wells were then washed with 200 μL of phosphate-buffered saline (PBS, Sigma-Aldrich, Castle Hill, New South Wales, Australia) solution to remove planktonic bacterial cells. The remaining biofilms on the walls of each well were stained with 0.03% crystal violet for 20 min. Crystal violet solution was removed by washing the wells twice with 200 μL of PBS. Pure ethanol (200 μL) was added to dissolve the crystal violet that remained bound to the biofilm on the well surface, which was then quantified (biofilm biomass) by measuring the OD550 of the homogenized suspension with a microplate reader. Measurements of each sample were performed in four replicates. Statistical Analysis. One-way analysis of variance (ANOVA) with a Tukey post hoc analysis was employed for statistical analysis with probability values of less than 0.05 considered as a statistical significant difference.
a K550X sputter coater (Quorum Emitech, Kent, U.K.). The images were captured at 5 kV. Crystallinity. X-ray powder diffraction (XRPD) (Shimadzu XRD-6000, Shimadzu Corporation, Kyoto, Japan) was employed to evaluate powder crystallinity. Cu−Kα radiation at a voltage of 40 kV and a current of 30 mA was employed. Data were collected by the 2θ method at a scan speed of 2° per min in the range of 5−50°. Dynamic Water Vapor Sorption. Moisture sorption behavior was measured using a dynamic vapor sorption system (DVS-1, Surface Measurement Systems Ltd., London, U.K.). Each formulation was analyzed in the measurement chamber at the relative humidity (RH) values ranging from 0 to 90%. The environmental RH was increased at 10% RH increments from 0 to 90% for the sorption cycle and then decreased from 90 to 0% for the desorption cycle. Equilibrium moisture content at each testing RH was determined by a dm/dt of 0.002% per minute.29 In Vitro Aerosol Performance. In vitro aerosolization performance was evaluated by a multistage liquid impinger (Apparatus C, British Pharmacopeia 2012, Copley, Nottingham, U.K.) with a USP induction port (USP throat). Each powder formulation was stored in 60% RH at 20 °C for 24 h prior to dispersion. Each capsule (size 3 hydroxypropyl methylcellulose capsules, Capsugel, West Ryde, Australia) was loaded with 10 ± 1 mg of powder prior to aerosolization and dispersed through an Aerolizer in a controlled environment cabinet: temperature, 20 ± 3 °C, and relative humidity, 60 ± 3%. A standard pharmacopeial dispersion procedure was used, whereby 4 L of air was passed through the inhaler at an airflow of 100 L/min for 2.4 s, with a pressure drop of approximately 4 kPa across the device.30,31 The cutoff diameters of stages 1−4 of the liquid impinger at 100 L/min were 10.4, 4.9, 2.4, and 1.2 μm, respectively.30 The Aerolizer was used here as a model device because it is a commercially available inhaler with a low resistance and patients can comfortably generate sufficient flow rates to disperse powder formulations.29,32 Three replicates were carried out for each formulation. Drug particles deposited on capsule, inhaler, USP throat, stages 1−4, and filter were collected using a cosolvent (acetonitrile/water, 1:1). Emitted dose (ED) was calculated as a percentage of drug released from the capsules and inhaler device. The recovered dose included the drug collected from the capsule, inhaler, USP throat, stages 1−4, and filter. Fine particle fraction total (FPFtotal) was calculated as the percentage mass of drug particles with an aerodynamic diameter smaller than 5 μm relative to the total recovered drug. FPF emitted was calculated as the fine particles relative to the ED. Drug Quantification. Concentrations of colistin and rifapentine were determined using an established HPLC method at a wavelength of 214 nm.22 The HPLC system consisted of a Shimadzu CBM-20A controller, LC-20AT pump, SIL-20A HT autosampler, SPD-20A UV/vis detector (Shimadzu, Kyoto, Japan), and a PhenoSphere-Next 5 μm C18 150 × 4.60 mm column (Phenomenex, Torrance, CA). The mobile phase consisted of 0.05% trifluoroacetic acid in Milli-Q water (A) and methanol (B). The gradient program was set as 30% B to 80% B in 15 min, then 80% B to 30% B in 5 min with a flow rate of 1 mL/min. A calibration curve (0.01−1 mg/mL) was prepared for colistin and rifapentine in a cosolvent (acetonitrile/water, volume ratio 1:1) and was linear in the required concentration range (r2 > 0.999). Effect of Elevated Relative Humidity on Aerosolization. The effect of humidity on aerosolization of the spray-
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RESULTS Physico-Chemical Properties. Table 1 shows that all spray-dried powder formulations had fine physical sizes with D90 values 89% (Figure 4). There was no significant difference in emitted dose (p > 0.05) between the spray-dried rifapentine alone (90.1 ± 1.7%) and the combination (89.5 ± 1.7%) formulations. The spray-dried colistin alone formulation exhibited significantly higher emitted dose of 94.6 ± 0.5% (p < 0.05). FPFtotal values of all formulations were >76%. The combination formulation had an FPFtotal of 79.6 ± 2.8% and 78.5 ± 2.1% for the colistin and rifapentine components, respectively. There was no significant difference in FPFtotal and FPFemitted among all spray-dried formulations. For the combination formulation, no significant difference (p > 0.05) was found for the drug deposition in each impinger stage between colistin and rifapentine (Figure 5). Both the colistin alone and rifapentine alone formulations had higher drug deposition on the filter and lower drug deposition on Stage 4 than the combination formulation (p < 0.05). The rifapentine alone formulation possessed the highest filter deposition of 19.5 ± 2.3% and the lowest Stage 3 deposition of 24.7 ± 2.1%, demonstrating the rifapentine alone powder contained a considerable amount of submicron particles. Effect of Humidity on Aerosolization. Given the colistin alone formulation absorbed approximately 20% (w/w) of water at the RH of 75% during DVS measurements, it is not
Figure 1. SEM micrographs of (a) colistin alone formulation; (b) rifapentine alone formulation; and (c) the combination formulation.
surprising that substantial deterioration of FPFtotal (from 80.1 ± 1.3% to 54.7 ± 7.1%) was observed when the RH was increased from 60% to 75% (Figure 6a). No significant changes in emitted dose, FPFtotal, and FPFemitted were measured for the rifapentine alone and combination formulations when the RH increased (p > 0.05). Compared to results at 60% RH (p < 0.05), drug deposition of colistin alone formulation was significantly lower on Stages 3, 4, and filter, and was greater on Stages 1, 2, and throat at 75% RH (Figure 7a). In contrast, no changes in drug deposition D
DOI: 10.1021/mp500586p Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Figure 2. Powder X-ray diffraction patterns of the antibiotic powder formulations.
Figure 5. Drug deposition of the antibiotic powder formulations via the Aerolizer device at 60% RH (error bars represent standard deviation, n = 3).
Figure 3. Dynamic vapor sorption behavior of the antibiotic powder formulations.
profile were observed after increased RH for the rifapentine alone dry powder and for the colistin component in the combination powder (p > 0.05). Statistically, a significant change in the drug deposition of rifapentine in the combination was observed on only Stage 2 (p < 0.05). In general, humidity had negligible influence on aerosolization of the rifapentine alone and combination formulations. Antimicrobial Activities. The MIC of colistin alone formulation against planktonic cells P. aeruginosa PAO1 was 4 mg/L, which was lower than pure rifapentine (32 mg/L) (Table 2). The antibiotic combination formulation exhibited the lowest MIC of 2 mg/L. The MBIC value of each formulation was two-fold greater compared to the corresponding MIC. Similar to the MIC results, the combination antibiotic formulation showed the lowest MBIC value of 4 mg/L, which demonstrated enhanced antibacterial effects against both planktonic cells and biofilm of P. aeruginosa.
Figure 4. Aerosolization performance of the antibiotic powder formulations via the Aerolizer device at 60% RH (error bars represent standard deviation, n = 3).
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DOI: 10.1021/mp500586p Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Figure 6. Effect of humidity on the aerosolization performance of the antibiotic powder formulations: (a) colistin alone; (b) rifapentine alone; (c) colistin in combination; and (d) rifapentine in combination (error bars represent standard deviation, n = 3).
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DISCUSSION In a previous study, we prepared a combination powder of colistin and rifampicin by cospray-drying a feed solution of two drugs dissolved in a cosolvent of water and ethanol in a ratio of 1:1.22 However, the two molecules have different pharmacokinetic profiles after pulmonary delivery, whereby colistin concentrations in lungs after inhalation can last above MIC for at least 12 h at the doses of 0.41−1.49 mg/kg,11 while inhaled spray-dried rifampicin (2.5 mg/kg) can be rapidly cleared from lungs within 1 h.34 The resulting colistin monotherapy after 1 h would negate the effectiveness of synergistic activity. Rifapentine may be a viable substitute for rifampicin in this combination therapy. Data from our preliminary pharmacokinetic study have demonstrated that high rifapentine concentrations in the lung tissue of mice (approximately 50 μg/g) were maintained for at least 24 h after intratracheal administration of drug powder.35 Pulmonary delivery of the antibiotic combination (colistin and rifapentine) could therefore allow for “synchronization” of drug pulmonary pharmacokinetic profiles to maximize synergistic bacterial killing for extended periods greater than 12 h. Future pharmacokinetic study of the current combination formulations is warranted. Rifapentine, primarily indicated as an antitubercular drug, is not typically used in the treatment of respiratory infections caused by Gram-negative bacteria. It exhibited a high MIC of 32 mg/L against P. aeruginosa PAO1. However, the combination of rifapentine and colistin demonstrated enhanced antibacterial effects on both planktonic cells and biofilm of P.
aeruginosa. The in vitro MIC and MBIC values of the combination were much lower than those of colistin only and rifapentine only formulations. Such enhanced antimicrobial activities support the great potential of the combination formulation for treatment of respiratory infections. Bacterial biofilms are a serious challenge in the treatment of respiratory infections.36 Biofilms are a matrix of bacterial cells or microcolonies embedded in self-produced polymeric substances. Bacteria protected by the biofilm generally exhibit greater resistance to antibiotic treatment.37 Our antibiotic combination formulation shows enhanced antibiofilm activity, with a 2-fold and 16-fold reduction over that for colistin alone and rifapentine alone, respectively. A successful inhalable dry powder formulation of combination agents necessitates both drugs to be physically and chemically stable in the formulation. One of the major challenges in producing inhalable drug particles by spray drying is the amorphous powder generated by rapid solvent evaporation. The amorphous form of many drugs is unstable and inclined to recrystallize. In our previous study, we have shown that, unlike many small-molecule compounds, colistin is physically and chemically stable in the amorphous form at ambient conditions. In contrast, amorphous rifapentine was chemically unstable under ambient (60% RH) and dry (0% RH) conditions after storage for one month.27 Therefore, Chan et al. developed a novel inhalable powder formulation of rifapentine in crystalline form, which was stable for 3 months when stored in 60% RH.27 We further advanced the production F
DOI: 10.1021/mp500586p Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Figure 7. Effect of humidity on the drug deposition of the antibiotic powder formulations in the multistage liquid impinger: (a) colistin alone; (b) rifapentine alone; (c) colistin in combination; and (d) rifapentine in combination (error bars represent standard deviation, n = 3).
26.3−29.1% even with the addition of 2 mg of coarse lactose as a “sweeper”.40 For the DPI delivery of high-dose antibiotics, there is a demand to improve aerosol efficiency to minimize the total drug dose.14,41 Spray drying has increasingly been used to engineer inhalable drug powders and was employed for the commercial manufacture of inhaled insulin (Exubera), tobramycin (TOBI podhaler), and mannitol (Aridol).14,41 In the present study, inhalable powder formulations produced by spray-drying achieved high emitted doses of >89% and high FPFtotal of >76%. Consequently, we minimized the total dose of colistin that needs to be administered. More importantly, two drugs in the combination deposited uniformly in each stage of the impinger (Figure 5), indicating a homogeneous distribution of two active ingredients in the powder.42 This feature suggests two drugs can simultaneously reach the target infection sites in the respiratory tract at the intended ratio. Since the synergistic bacterial killing of combination antibiotics depends on the action of both drugs, simultaneous delivery of two drugs to the local infection sites in the respiratory tracts can maximize the bacterial-killing synergy. Submicron particles are undesirable for inhaled therapy, as they may be exhaled due to negligible inertial impaction and sedimentation.43,44 Figure 5 showed 20% of the drug dose from the rifapentine alone formulation deposited in the filter stage (
