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A Rifapentine-Containing Inhaled Triple Antibiotic Formulation for Rapid Treatment of Tubercular Infection
Abstract
The potential for rifapentine-containing oral therapeutic regimens to significantly shorten the current six-month anti-tubercular treatment regimen is confounded by high plasma protein binding of rifapentine. Inhaled aerosol delivery of rifapentine, a more potent anti-tubercular antibiotic drug, in combination with other first-line antibiotics may overcome this limitation to deliver a high drug dose at the pulmonary site of infection.
A formulation consisting of rifapentine, moxifloxacin and pyrazinamide, with and without leucine, was prepared by spray-drying. This formulation was assessed for its physico-chemical properties, in vitro aerosol performance and antimicrobial activity.
The antibiotic powders, with and without leucine, had similar median aerodynamic diameters of 2.58 ± 0.08 μm and 2.51 ± 0.06 μm, with a relatively high fine particle fraction of 55.5 ± 1.9% and 63.6 ± 2.0%, respectively. Although the powders were mostly amorphous, some crystalline peaks associated with the δ polymorph for the spray-dried crystalline pyrazinamide were identified.
Stabilisation of the powder with 10% w/w leucine and protection from moisture ingress was found to be necessary to prevent overt crystallisation of pyrazinamide after long-term storage. In vitro biological assays indicated antimicrobial activity was retained after spray-drying. Murine pharmacokinetic studies are currently underway.
... Leucine is able to recrystallize on the surface of the particles, forming a shell to reduce inter-particle interaction and enhance the flow and dispersibility of the powder [54,56]. Chan et al. reported that leucine could prevent water-induced recrystallization of hygroscopic pyrazinamide in an inhalable formulation containing rifapentine, moxifloxacin and pyrazinamide [57]. Due to the low water solubility of leucine (24 g/L), this surface coating was also shown to delay the dissolution rate of the drug particles [58]. ...
... Over the last decade, inhalable dry powder formulations consisting of first-line, second-line or a combination of both classes of anti-TB drugs have been studied to reflect current treatment regimens for the treatment of MDR and XDR cases [57,[61][62][63][64]. There are also few studies reported on spray dried BCG and novel vaccine candidates [39,65,66]. ...
... For multi-drug formulations, the combination ratios were determined based on the recommended oral dose [57,64]. Chan et al. produced an excipient-free dry powder consisting of pyrazinamide, rifampicin and isoniazid at a ratio of 5:2:1 (w/w) following the oral doses of 25 mg/kg, 10 mg/kg and 5 mg/kg, respectively [64]. ...
... While the dried state may confer inherent stabilization based upon the reduced molecular mobility of the solid state, sorption of moisture during storage may lead to potential increases in molecular mobility and subsequent chemical interactions. Chemical degradation was noted upon storage of triple-combination inhalable particles consisting of rifampicin, isoniazid, and pyrazinamide, in which the rifampicin and isoniazid components were noted to react to form an isonicotinyl hydrazine adduct, the formation of which was catalyzed by pyrazinamide [67,68]. This was alleviated through the replacement of isoniazid with moxifloxacin and through incorporation of the hydrophobic amino acid leucine to reduce moisture uptake during storage. ...
... Physical changes in the combination of drug powders may also result in deviations in aerosol deposition of the individual drugs in the combination, thereby resulting in sub-or supra-therapeutic drug levels, loss of synergistic effect, and potential development of bacterial resistance or unexpected drug toxicities. For example, the storage of co-spray dried rifapentine/moxifloxamine/pyrazinamide composite particles at elevated humidity (60%RH) for three months resulted in changes in the cascade impactor deposition profile for pyrazinamide, the deposition of which was found to shift from the lower size cutoff stages to the throat and higher cutoff stages [68]. This shift was contributed to the increased crystallization of the pyrazinamide component upon storage, which did exhibit partial crystallinity at baseline. ...
Introduction
Respiratory infections are a major cause of morbidity and mortality. As an alternative to systemic drug administration, inhaled drug delivery can produce high drug concentrations in the lung tissue to overcome resistant bacteria. The development of inhaled fixed-dose combination powders (I-FDCs) is promising next step in this field, as it would enable simultaneous drug-drug or drug-adjuvant delivery at the site of infection, thereby promoting synergistic activity and improving patient compliance.
Areas covered
This review covers the clinical and pharmaceutical rationales for the development of I-FDCs for the treatment of respiratory infections, relevant technologies for particle and powder generation, and obstacles which must be addressed to achieve regulatory approval.
Expert opinion
I-FDCs have been widely successful in the treatment of asthma and chronic obstructive pulmonary disease; however, application of I-FDCs towards the treatment of respiratory infections carries additional challenges related to the high dose requirements and physicochemical characteristics of anti-infective drugs. At present, co-spray drying is an especially promising approach for the development of composite fixed-dose anti-infective particles for inhalation. Though the majority of fixed-dose research has thus far focused on the combination of multiple antibiotics, future work may shift to the additional inclusion of immunomodulatory agents or repurposed non-antibiotics.
... In a recent report, Chan et al. found that inclusion of 10% (w/w) LL in the spray-dried rifapentine-moxiffloxacin-pyrazinamide formulation also kept the consistent inhalation performance after storage at 60% RH for three months. In contrast, the fine particle fraction (FPF) of the formulation containing no LL was decreased from 58.4% ± 1.3 to 43.5 ± 1.6% [18]. However, there are knowledge gaps in these studies to be filled. ...
... 1% w/w) was used [7]. Although Aquino et al. and Chan et al. investigated the effect of humidity on in vitro aerosolization, it was performed at 60% ± 5% RH, which resulted in only moderate water uptake of about 8.9% (w/w) for the pure gentamicin formulation and 4.2% (w/w) for the rifapentine-moxiffloxacin-pyrazinamide formulation, respectively [9,18]. It was unknown if LL would be sufficient in preventing the effect of higher humidity (e.g. ...
l-Leucine (LL) has been widely used to enhance the dispersion performance of powders for inhalation. LL can also protect powders against moisture, but this effect is much less studied. The aim of this study was to investigate whether LL could prevent moisture-induced deterioration in in vitro aerosolization performances of highly hygroscopic spray-dried powders. Disodium cromoglycate (DSCG) was chosen as a model drug and different amounts of LL (2–40% w/w) were added to the formulation, with the aim to explore the relationship between powder dispersion, moisture protection and physicochemical properties of the powders. The powder formulations were prepared by spray drying of aqueous solutions containing known concentrations of DSCG and LL. The particle sizes were measured by laser diffraction. The physicochemical properties of fine particles were characterized by X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and dynamic vapor sorption (DVS). The surface morphology and chemistry of fine particles were analyzed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In vitro aerosolization performances were evaluated by a next generation impactor (NGI) after the powders were stored at 60% or 75% relative humidity (RH), and 25 °C for 24 h. Spray-dried (SD) DSCG powders were amorphous and absorbed 30–45% (w/w) water at 70–80% RH, resulting in deterioration in the aerosolization performance of the powders. LL did not decrease the water uptake of DSCG powders, but it could significantly reduce the effect of moisture on aerosolization performances. This is due to enrichment of crystalline LL on the surface of the composite particles. The effect was directly related to the percentage of LL coverage on the surface of particles. Formulations having 61–73% (molar percent) of LL on the particle surface (which correspond to 10–20% (w/w) of LL in the bulk powders) could minimize moisture-induced deterioration in the aerosol performance. In conclusion, particle surface coverage of LL can offer short-term protection against moisture on dispersion of hygroscopic powders.
... The major advantage of AFREZZA over other pulmonary insulin formulations is its superior bioavailability (approximately 37%) and faster onset of action. An average of 39.5% of the initial loaded dose deposited in the lungs but the Fig. 9. Relative size of (a) Exubera [157] and (b) AFREZZA [158] insulin delivery systems. variation of lung deposition and blood glucose reduction amongst a larger population of patients is unclear [155]. ...
... We have already begun to see a wider application of inhaled dry powders beyond its current mainstay use in treating chronic diseases. Many drugs have shown to be suitable for lung delivery in dry powder form, ranging from antibiotics [158][159][160], antiviral [161], immunosuppressant [162], anticancer [163,164], nucleic acids [165], and vaccines [166]. Some are already commercialised for aerosol delivery to the lungs, whilst others are in animal studies and human clinical trials [159,167,168]. ...
The efficacy of pharmaceutical aerosols relates to its deposition in the clinically relevant regions of the lungs, which can be assessed by in vivo lung deposition studies. Dry powder formulations are popular as devices are portable and aerosolisation does not require a propellant. Over the years, key advancements in dry powder formulation, device design and our understanding on the mechanics of inhaled pharmaceutical aerosol have opened up new opportunities in treatment of diseases through pulmonary drug delivery. This review covers these advancements and future directions for inhaled dry powder aerosols.
... As shown above, inhaled antibiotics (particularly DPIs) have been used extensively and have shown great success in treating pulmonary infections by increasing local concentration, improving patient adherence and decrease systemic effects. This has opened a window of opportunity to treat other infectious diseases, especially tuberculosis, where multidrug resistance is common due to poor treatment efficacy and patient adherence [151]. ...
... The powder formulation with and without leucine had similar median aerodynamic diameters of 2.58 ± 0.08 μm and 2.51 ± 0.06 μm, with a relatively high FPF of 55.5 ± 1.9% and 63.6 ± 2.0%, respectively. Reprint from ref. [151] with kind permission from Springer. ...
Novel inhaled therapeutics including antibiotics, vaccines and anti-hypertensives, have led to innovations in designing suitable delivery systems. These emerging design technologies are in urgent demand to ensure high aerosolisation performance, consistent efficacy and satisfactory patient adherence. Recent vibrating-mesh and software technologies have resulted in nebulisers that have remarkably accurate dosing and portability. Alternatively, dry powder inhalers (DPIs) have become highly favourable for delivering high-dose and single-dose drugs with the aid of advanced particle engineering. In contrast, innovations are needed to overcome the technical constrains in drug-propellant incompatibility and delivering high-dose drugs with pressurised metered dose inhalers (pMDIs). This review discusses recent and emerging trends in pulmonary drug delivery systems.
... It is also used alone for tuberculosis preventive treatment. Due to its poor solubility and low stability, high doses of rifampicin are administered orally, which increase the side effects such as gastrointestinal disturbance, allergic reaction, and hepatotoxicity [2]. ...
The poor solubility and low stability of rifampicin limited its therapeutic efficacy. One of the common strategies is to form an inclusion complex to address the problems. In this study, acyclic cucurbit[n]uril was used for encapsulation of rifampicin to form an inclusion complex. The inclusion complex was prepared by the kneading method and characterized by DSC, SEM, FT-IR, NMR, PXRD, and UV-Vis spectroscopy. The results confirmed the formation of 1:1 inclusion complex. The drug content was calculated to be 27.35 ± 0.54%. The aqueous solubility of rifampicin was increased 20-fold by complexation with acyclic cucurbit[n]uril. Moreover, the thermo-stability and photo-stability of free rifampicin and inclusion complex were investigated by HPLC. Rifampicin was stabilized by encapsulating the piperazine ring in the cavity of acyclic cucurbit[n]uril. In addition, the MIC of free RIF and RIF‧ACB were determined by using in vitro experiments. It was found that rifampicin complexed by acyclic cucurbit[n]uril exhibited similar antibacterial activities against strain H37Rv to rifampicin alone.
Graphic abstract
•Acyclic cucurbit[n]uril was used to encapsulate rifampicin to form an inclusion complex.
•The aqueous solubility of rifampicin was increased 20-fold by encapsulation with acyclic cucurbit[n]uril.
•The thermo-stability and photo-stability of rifampicin was improved by formation of inclusion complex.
•The antibiotic potency of rifampicin complexed by acyclic cucurbit[n]uril was close to that of rifampicin alone.
... No degradation peaks were observed during HPLC analysis indicating the chemical stability of the stored powder. Previously it has been reported that L-leucine prevents moisture-induced deterioration in aerosolization and protects powder stability [65,66]. While no significant changes in aerosolization, morphology and crystallinity of SD-BL were observed during one month's storage, it is not unlikely to have some changes during a longer-term storage (more than a month). ...
Bedaquiline is a newly developed anti-tuberculosis drug, conditionally approved by the United States Food and Drug Administration (USFDA) for treating drug-resistant tuberculosis in adults. Oral delivery of bedaquiline causes severe side effects such as increased hepatic aminotransferase levels and cardiac arrhythmias (prolongation of QT-interval). This study aimed to develop inhalable dry powder particles of bedaquiline with high aerosolization efficiency to reduce the side-effects of oral bedaquiline. Bedaquiline (with or without l-leucine) powders were prepared using a Buchi Mini Spray-dryer. The powders were characterized for physicochemical properties and for their in vitro aerosolization efficiency using a next-generation impactor (NGI). The formulation with maximum aerosolization efficiency was investigated for physicochemical and aerosolization stability after one-month storage at 20 ± 2 °C/30 ± 2% relative humidity (RH) and 25 ± 2 °C/75% RH in an open Petri dish. The cytotoxicity of the powders on A549 and Calu-3 cell-lines was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The powders were also evaluated for antimicrobial activity against Mycobacterium tuberculosis. The aerodynamic diameter of the l-leucine-containing powder was 2.4 µm, and the powder was amorphous in nature. The aerosolization efficiency (fine-particle fraction) of l-leucine-containing powder (fine-particle fraction (FPF): 74.4%) was higher than the bedaquiline-only powder (FPF: 31.3%). l-leucine containing powder particles were plate-shaped with rough surfaces, but the bedaquiline-only powder was spherical and smooth. The optimized powder was stable at both storage conditions during one-month storage and non-toxic (up to 50 µg/mL) to the respiratory cell-lines. Bedaquiline powders were effective against Mycobacterium tuberculosis and had a minimal inhibitory concentration (MIC) value of 0.1 µg/mL. Improved aerosolization may help to combat pulmonary tuberculosis by potentially reducing the side-effects of oral bedaquiline. Further research is required to understand the safety of the optimized inhalable powder in animal models.
... 16 Combinations of first-line agents with small quantities of excipient have been reported. 17, 18 We are not aware of reports of combinations of pure drug in spray-dried particles for inhalation. ...
Tuberculosis (TB) remains the single most serious infectious disease attributable to a single-causative organism. A variety of drugs have been evaluated for pulmonary delivery as dry powders: capreomycin sulfate has shown efficacy and was safely delivered by inhalation at high doses to human volunteers, while CPZEN-45 is a new drug that has also been shown to kill resistant TB. The studies here combine these drugs — acting by different mechanisms — as components of single particles by spray drying, yielding a new combination drug therapy. The spray dried combination powder was prepared in an aerodynamic particle size range suitable for pulmonary delivery. Physicochemical storage stability was demonstrated for a period of 6 months. The spray-dried combination powders of capreomycin and CPZEN-45 have only moderate affinity for mucin, indicating that delivered drug will not be bound by these mucins in the lung and available for microbicidal effects. The pharmacokinetics of disposition in guinea pigs demonstrated high local concentrations of drug following direct administration to the lungs and subsequent systemic bioavailability. Further studies are required to demonstrate the in vivo efficacy of the combination to confirm the therapeutic potential of this novel combination.
... A significant part of the current research on new formulation strategies for the treatment of lung infections including MDR-TB is oriented toward the elimination of the use of excipients, with the aim to maximize the amount of drug delivered and the drug concentrations at the site of action [49][50][51]. ...
Tuberculosis resistant cases have been estimated to grow every year. Besides Mycobacterium tuberculosis, other mycobacterial species are responsible for an increasing number of difficult-to-treat infections. To increase efficacy of pulmonary treatment of mycobacterial infections an inhalable antibiotic powder targeting infected alveolar macrophages (AMs) and including an efflux pump inhibitor was developed. Low molecular weight sodium hyaluronate sub-micron particles were efficiently loaded with rifampicin, isoniazid and verapamil, and transformed in highly respirable microparticles (mean volume diameter: 1 μm) by spray drying. These particles were able to regenerate their original size upon contact with aqueous environment with mechanical stirring or sonication. The in vitro drugs release profile from the powder was characterized by a slow release rate, favorable to maintain a high drug level inside AMs. In vitro antimicrobial activity and ex vivo macrophage infection assays employing susceptible and drug resistant strains were carried out. No significant differences were observed when the powder, which did not compromise the AMs viability after a five-day exposure, was compared to the same formulation without verapamil. However, both preparations achieved more than 80% reduction in bacterial viability irrespective of the drug resistance profile. This approach can be considered appropriate to treat mycobacterial respiratory infections, regardless the level of drug resistance.
... The difference in crystallinity of the combination powder than the previous reports could be due to the differences in the spray-drying conditions or physicochemical properties of the drugs. Partial crystallinity in L-leucine containing combination powder is supported by the previous reports [39,40]. Figures 3 and 4 show the thermal analysis of the supplied and spray-dried samples by DSC and TGA. ...
This study aimed to develop a combination dry powder formulation of ethionamide and moxifloxacin HCl as this combination is synergistic against drug-resistant Mycobacterium tuberculosis (Mtb). L-leucine (20% w/w) was added in the formulations to maximize the process yield.
Moxifloxacin HCl and/or ethionamide powders with/without L-leucine were produced using a Buchi Mini Spray-dryer. A next generation impactor (NGI) was used to determine the in vitro aerosolization efficiency. The powders were also characterized for other physicochemical properties and cytotoxicity. All the spray-dried powders were within the aerodynamic size range of <5.0 µm except ethionamide-only powder (6.0 µm). The combination powders with L-leucine aerosolized better (%FPF: 61.3 and 61.1 for ethionamide and moxifloxacin, respectively) than ethionamide-only (%FPF: 9.0) and moxifloxacin-only (%FPF: 30.8) powders. The combination powder particles were collapsed with wrinkled surfaces whereas moxifloxacin-only powders were spherical and smooth and ethionamide-only powders were angular-shaped flakes. The combination powders had low water content (<2.0%). All the powders were physically stable at 15% RH and 25 ± 2 °C during one-month storage and tolerated by bronchial epithelial cell-lines up to 100 µg/mL.
The improved aerosolization of the combination formulation may be helpful for the effective treatment of drug-resistant tuberculosis. Further studies are required to understand the mechanisms for improved aerosolization and test the synergistic activity of the combination powder.
... Moxifloxacin is a second line anti-TB drug belonging to the fluoroquinolone class. Among the fluoroquinolones, moxifloxacin has the highest bactericidal activity against the growing mycobacteria and its combination with a first line anti-TB drug has the greater potential to shorten the TB treatment period without relapse (Nuermberger et al., T 2004a;Nuermberger et al., 2004b;Rustomjee et al., 2008;Chan et al., 2014). Moreover, its activity against slow growing persister mycobacteria is higher than the first line drug isoniazid and other fluoroquinolones (Hu et al., 2003). ...
This study aimed to develop a high payload dry powder inhalation formulation containing a combination of the first line anti-TB drug, pyrazinamide, and the second line drug, moxifloxacin HCl. Individual powders of pyrazinamide (PSD) and moxifloxacin (MSD) and combination powders of the two drugs without (PM) and with 10% L-leucine (PML) and 10% DPPC (PMLD) were produced by spray drying. PSDcontained >10 μm crystalline particles and showed poor aerosolization behaviour with a fine particle fraction (FPF) of 18.7 ± 3.4%. PM produced spherical hollow particles with aerodynamic diameter < 5 μm and PML showed improved aerosolization with a high FPF of ~70%. However, PMLD showed a significantly reduced FPF (p > 0.05) compared to PML.Solid state studies and surface elemental analysis by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry confirmed the surface coating of particles contained amorphous M and both L-leucine and DPPC over crystalline P. Furthermore, pyrazinamide, moxifloxacin, PML and PMLD were found to display low toxicity to both A549 and Calu-3 cell lines even at a concentration of 100 μg/mL. In conclusion, a combination powder formulation of PML has the potential to deliver a high drug dose to the site of infection resulting in efficient treatment.
... Since then, many different formulation approaches have been investigated. Micro [10,11], nanoparticles [12,13], liposomes [14], in situ forming liposomes [15], proliposomes [16,17] and liposphere [18], with single [19,20] and combination drugs [21,22], have all been manufactured with different degrees of success, although mostly limited to data collected in vitro or from preclinical studies where, for the majority, formulations were found effective in reducing bacterial counts. Nevertheless, there is no completed clinical study and it is difficult to identify which system would be the most advantageous to take further. ...
... High systemic exposure, side effects and lengthy treatment time are problems with current TB treatment (Chan et al., 2014a). In addition, sub-therapeutic levels of drugs in the infected site (i.e. ...
Background:
Kanamycin, an injectable agent, is currently used to treat drug-resistant tuberculosis (TB). Parenteral kanamycin causes high systemic toxicity which could be avoided by direct delivery to the lungs. This study focused on producing a highly aerosolizable dry-powder of hygroscopic kanamycin by spray-drying with l-leucine.
Methods:
Kanamycin powders were prepared with different concentrations (0, 5, 10, 15 and 20% w/w) of l-leucine using the Buchi B-290 Mini Spray-Dryer. In vitro aerosolization efficiency, particle size, morphology, crystallinity, surface composition, drug-excipient interaction and moisture content of the powders were characterized by a Next Generation Impactor (NGI), laser diffraction, scanning electron microscopy, X-ray diffractometry, XPS, ATR-FTIR and thermogravimetric analysis. The physicochemical and aerosolization stability of the powders were investigated after one-month storage at 25±2°C/15% RH and 25±2°C/75% RH. The cytotoxicity on Calu-3 and A549 cells of the kanamycin powders was evaluated by MTT assay.
Results:
The spray-dried powder particles were in the inhalable size range (<6.1μm). The powders with l-leucine were wrinkled in shape, amorphous in nature and had low moisture content (<5.0%). Kanamycin with 5% (w/w) of l-leucine showed the best aerosolization efficiency of 73.0±2.5%. The powders remained stable during storage at 25±2°C/15% RH and tolerated by respiratory cell lines.
Conclusion:
l-leucine improved the aerosolization of kanamycin by surface modification, which may be helpful for the effective treatment of drug-resistant tuberculosis.
... In another work, the formulation of a rifapentine, moxifloxacin and pyrazinamide ternary dry powder combination has been investigated 101 . The formulation obtained by co-spray-drying of the drug mixture could enhance effectiveness by also preventing the drug-drug interactions sometimes recorded upon systemic administration of multiple antibiotics. ...
AbstractNot all of the issues impacting the success of Tuberculosis (TB) treatment arise from pathogen-related disease characteristics. Nowadays, there is an increasing awareness that antibiotic treatment is not the only answer to the TB problem, promoting the search for alternative administration strategies and host-directed therapies. Among all the administration routes, being the lungs the main TB focus, inhalation is conceptually a logical solution to enhance treatment effectiveness and compliance. Nevertheless, research efforts and funding are almost entirely conveyed to conventional approaches.This review will critically evaluate the reasons constraining research in this field, providing some future perspectives. The most recent advances in inhalation approaches for TB will be discussed, either at the preclinical or clinical phase, illustrating the risk of failure and chances of success.
... This observation suggests an incomplete deagglomeration of the powder, and this could be due to a larger fraction of the powder exiting the device before a maximum level of turbulence and impaction was generated in the swirling chamber (19,20). Studies have reported that during spray drying, L-leucine is coated onto the surface of the drug particles to improve the flowability of the powder (39,40). This may aid the premature emptying of the tobramycin powder prior to the maximum flow and impaction development. ...
The routine of loading multiple capsules for delivery of high-dose antibiotics is time consuming, which may reduce patient adherence to inhaled treatment. To overcome this limitation, an investigation was carried out using four modified versions of the Aerolizer® that accommodate a size 0 capsule for delivery of high payload formulations. In some prototypes, four piercing pins of 0.6 mm each were replaced with a single centrally located 1.2-mm pin and one-third reduced air inlet of the original design. The performance of these inhalers was evaluated using spray-dried antibiotic powders with distinct morphologies: spherical particles with a highly corrugated surface (colistin and tobramycin) and needle-like particles (rifapentine). The inhalers were tested at capsule loadings of 50 mg (colistin), 30 mg (rifapentine) and 100 mg (tobramycin) using a multistage liquid impinger (MSLI) operating at 60 L/min. The device with a single pin and reduced air inlet showed a superior performance than the other prototypes in dispersing colistin and rifapentine powders, with a fine particle fraction (FPF wt% <5 μm in the aerosol) between 62 and 68%. Subsequently, an Aerolizer® with the same configuration (single pin and one-third air inlet) that accommodates a size 00 capsule was designed to increase the payload of colistin and rifapentine. The performance of the device at various inspiratory flow rates and air volumes achievable by most cystic fibrosis (CF) patients was examined at the maximum capsule loading of 100 mg. The device showed optimal performance at 45 L/min with an air volume of 1.5-2.0 L for colistin and 60 L/min with an air volume of 2.0 L for rifapentine. In conclusion, the modified size 00 Aerolizer® inhaler as a low-cost generic device demonstrated promising results for delivery of various high-dose formulations for treatment of lung infections.
... An inhalable drug combination powder allows deposition of multiple drugs in the pulmonary region in a single inhalation [33,36]. However, formulating drugs with different aqueous solubility poses a significant challenge. ...
Thioridazine is an orally administered antipsychotic drug with potential for treatment of drug-resistant tuberculosis (TB). However, drug-induced adverse cardiac effects have been reported when thioridazine was used at an efficacious oral dose of 200 mg/day to treat TB. Pulmonary delivery of thioridazine could be a rational approach to reduce dose-related side effects while enabling high drug concentrations at the primary site of infection. The present study compares in vitro aerosol performance, storage stability, and in vitro antimicrobial activity and cytotoxicity of two inhalable powders composed of thioridazine and a first-line anti-TB drug, rifapentine. Formulation 1 is a combination of amorphous thioridazine and crystalline rifapentine, while Formulation 2 consisted of both drugs as amorphous forms. Both thioridazine-rifapentine formulations were found suitable for inhalation with a total fine particle fraction (< 5 μm) of 68 - 76%. The two powders had similar MIC90 to rifapentine alone, being 0.000625 μg/mL and 0.005 μg/ml against M. tuberculosis H37Ra and M. tuberculosis H37Rv, respectively. In contrast, thioridazine alone had a MIC90 of 12.5 μg/mL and 500 μg/mL, against M. tuberculosis H37Ra and M. tuberculosis H37Rv, respectively, demonstrating no synergistic anti-TB activity. However, thioridazine and rifapentine in a ratio of 1:3 enhanced the killing of M. tuberculosis H37Ra within the human monocyte-derived macrophages (THP-1) compared to the single drug treatments. Both powders showed an acceptable half maximal inhibitory concentration (IC50) of 31.25 μg/mL on both THP-1 and human lung epithelial (A549) cells. However, Formulation 1 showed greater chemical stability than Formulation 2 after three months of storage under low humidity (vacuum) at 20 ± 3 °C. In conclusion, we have demonstrated a novel inhalable powder consisted of amorphous thioridazine and crystalline rifapentine (Formulation 1) with a good aerosol performance, potent anti-TB activity and storage stability, which deserves further in vivo investigations.
... Drug delivery to the lung, once reserved for the symptomatic treatment of asthma and chronic obstructive pulmonary disease (COPD) [1], nowadays lends itself to innovative drug formulations containing not only 2 adrenergic agonists, anticholinergics and corticosteroids, but also drugs belonging to different therapeutic classes (anti-inflammatory agents, antibiotics, mucolytics) [2][3][4]. ...
Antibiotic therapy for a direct administration to the lung in cystic fibrosis patients has to provide suitable availability, possibly in the lower respiratory tract, characterized by the presence of thick secretions. One of the crucial steps in the therapeutic management of the respiratory disease could be the drug solubilization directly in this site of action. The aim of the study was to prepare respirable powders of clarithromycin, while improving drug aqueous solubility. With this aim, several batches of micronized particles were prepared by spray drying different feed solutions, varying the solvent composition (water/isopropyl alcohol ratio), the drug concentration and pH of the liquid feeds. Particle size distribution of raw materials and engineered particles was determined using a light-scattering laser granulometer while particle morphology was assessed by scanning electron microscopy. The in vitro deposition of the micronized clarithromycin powders was evaluated by means of a Single-Stage Glass Impinger using the RS01 model7 by Plastiape® as device for the aerosolization. Solubility measurements of raw and spray-dried (SD) drug were carried out at 37 °C in phosphate buffer (0.05 M, pH 6.8). Results indicate that morphology and aerodynamic properties of SD particles were strongly influenced by organic solvent concentration and pH of the liquid feeds processed, both modifying drug solubility. Spherical particles and crystals were obtained at higher pH and lower organic solvent content, while wrinkled particles with very interesting aerodynamic properties and higher drug solubility were obtained at lower pH values. Thanks to a fine tuning of the process parameters and liquid feed composition, we produced SD powders with good aerodynamic properties, without using any excipients. Furthermore, SD powders of clarithromycin hydrochloric salt showed higher activity against Pseudomonas aeruginosa growth, compared to clarithromycin raw material.
... New approaches to drug delivery involving the inhalation of antibiotics [11] and anti-TB drugs could offer more effective treatment regimens for drug-resistant disease and latent infection [12]. Despite numerous studies reporting anti-TB drug inhalation, there are (to date) no respirable products on the market [13][14][15]. Pneumonia is often a category of health care-associated infection (HCAI) (also referred to as nosocomial infection). ...
A large range of microorganisms can be associated with primary and secondary lung infections such as the bacteria associated with the aetiology of tuberculosis and pneumonia or the periodic complications that occur in patients suffering from cystic fibrosis or chronic obstructive pulmonary disease. The different classes of available antibiotics, based on the chemical structure and mechanisms of action to treat this conditions that might be delivered by the pulmonary route are discussed. In particular, both solutions for nebulisation and dry-powder formulations for inhalation have either been developed or are under current study. The likely posology and instructions for the user for such formulations are important considerations. Improvements in nebulizer design and recent developments in dry-powder devices have enabled localised pulmonary antibiotics to become a viable option but several issues remain to be addressed so as to improve the efficiency of delivery. The main goal for nebulisers has been to develop a strategy that seeks to reduce the time of administration whilst maintaining a high respirable dose. The success of employing dry-powder inhaler (DPI) formulations depends upon the success of device design and particle engineering techniques aimed at increasing the drug deposition in the lung whilst reducing the total amount of inhaled powder that is required. These innovative strategies, intended to treat the infections directly at the target site of the disease, are reviewed. The use of antibiotics not currently delivered via the airways, using possible novel methods, is anticipated in the near future.
... This could be due to the presence of L-leucine, which has been reported to prevent water-induced recrystallization of amorphous pyrazinamide in an inhalable antibiotic formulation consisting of rifapentine, moxifloxacin, and pyrazinamide. 44 Furthermore, a similar reversible sorption profile was observed in Vrp_Rpt_Leu with an additional sigmoid profile after 60% RH. The presence of a sigmoid profile is attributable to the crystalline rifapentine, which forms a monohydrate followed by a dihydrate. ...
Recent studies have demonstrated that drug efflux pumps of Mycobacterium tuberculosis (M. tb) provide a crucial mechanism in the development of drug resistant to anti-mycobacterial drugs. Drugs that inhibit these efflux pumps, such as verapamil have shown the potential in enhancing the treatment success. We therefore hypothesized that the combined inhaled administration of verapamil and a first-line rifamycin antibiotic will further improve the treatment efficacy. An inhalable dry powder consisting of amorphous verapamil and crystalline rifapentine with L-leucine as an excipient was produced by spray drying. The in vitro aerosol characteristic of the powder, its microbiological activity and stability were assessed. When the powder was dispersed by an Osmohaler®, the total fine particle fraction (FPFtotal, wt. % of particles in aerosol < 5 µm) of verapamil and rifapentine was 77.4 ± 1.1 % and 71.5 ± 2.0 %, respectively. The combination drug formulation showed a minimum inhibitory concentration (MIC90) similar to that of rifapentine alone when tested against both M. tb H37Ra and M. tb H37Rv strains. Importantly, the combination resulted in increased killing of M. tb H37Ra within the infected macrophage cells compared to either verapamil or rifapentine alone. In assessing cellular toxicity, the combination exhibited an acceptable half maximal inhibitory concentration (IC50) values (62.5 µg/mL) on both human monocytic (THP-1) and lung alveolar basal epithelial (A549) cell lines. Finally, the powder was stable after 3 months storage in 0 % relative humidity at 20 ± 3°C.
... Similar finding was also reported by Fiegel et al., where dry powder aerosol formulation of capreomycin and leucine was found to possess good aerosolization property and physico-chemical stability up to 3 months when stored at room temperature while decrease in aerosolization performance was observed when powders were stored at 40°C and 75% RH. [27] Also, Chan et al. showed that addition of 10% w/w leucine in the triple antibiotic formulation prevented overt crystallization of pyrazinamide after long-term storage by reducing the ingress of the moisture as well as improved the aerosolization performance of the developed formulation. [28] 3.4 In vitro drug deposition and diffusion using air-interface Calu-2 model ...
Objective:
To develop and characterize a highly respirable dry powder inhalable formulation of voriconazole (VRZ).
Methods:
Powders were prepared by spray drying aqueous/alcohol solutions. Formulations were characterized in terms of particle size, morphology, thermal, moisture responses and aerosolization performance. Optimized powder was deposited onto an air-interface Calu-3 model to assess their uptake across Calu-3 lung epithelia. Optimized formulation was evaluated for stability (drug content and aerosol performance) for 3 months. Additionally, Calu-3 cell viability, lung bioavailability and tissue distribution of optimized formulation were evaluated.
Results:
Particle size and aerosol performance of dry powder containing 80% w/w VRZ and 20% w/w leucine was appropriate for inhalation therapy. Optimized formulation showed irregular morphology, crystalline nature, low moisture sensitivity and was stable for 3 months at room temperature. Leucine did not alter the transport kinetics of VRZ, as evaluated by air-interface Calu-3 model. Formulation was non-cytotoxic to pulmonary epithelial cells. Moreover, lung bioavailability and tissue distribution studies in murine model clearly showed that VRZ dry powder inhalable formulation has potential to enhance therapeutic efficacy at the pulmonary infection site whilst minimizing systemic exposure and related toxicity.
Conclusion:
This study supports the potential of inhaled dry powder VRZ for the treatment of fungal infections.
... where 'd' is the particle diameter distribution, 'ρ' is the packing fraction distribution (volume of particles/volume of aggregates) and 'W' is the work of adhesion or cohesion distribution of particles. Isoniazid +Rifabutin PLA microparticles High drug payload over 10 days drug concentration in macrophages 20-fold larger than with solutions [2,65] Rifapentine + Moxifloxacin + Pyrazinamide Spray dried with or without leucine High fine particle fraction, 10% leucine was essential for stability [66] According to this equation, the de-agglomeration can be improved by increasing particle size, decreasing packing fraction or by decreasing work of cohesion. Since the particle size of drugs needs to be <5µm for deep lung deposition, increasing particle size to promote de-agglomeration is not a feasible approach. ...
Tuberculosis is the second leading cause of death from infectious diseases. Although anti-tubercular drugs have been traditionally administered orally, there is a growing interest in delivering drugs via the pulmonary route using nebulisers or dry powder inhalers. Drugs in dry powder inhalers (DPI) are stable and DPI are user-friendly compared to nebulisation which is time consuming, inconvenient and inefficient and requires special equipment. For tuberculosis treatment, drugs should target alveolar macrophages that harbour microorganisms and/or maintain high drug concentration at the infection site in the lung. Drug particles include micro-particles or nanoparticles. Powders can be engineered by micronisation, crystallisation, spray drying, freeze drying and particle coating approaches. The formulation may contain single or combination drugs. This paper will provide an update on current status of TB, its pathogenesis, current treatment strategies, shortcomings of current oral or parenteral delivery strategies, pulmonary delivery devices, advantages of pulmonary delivery of powder formulations, formulation approaches and pharmacokinetic studies of pulmonary delivery of powders for inhalation.
Introduction:
This work aimed to develop chitosan-coated cubosomal nanoparticles intended for nose-to-brain delivery of paliperidone palmitate. They were compared with standard and cationic cubosomal nanoparticles. This comparison relies on numerous classical in vitro tests and powder deposition within a 3D-printed nasal cast.
Methods:
Cubosomal nanoparticles were prepared by a Bottom-up method followed by a spray drying process. We evaluated their particle size, polydispersity index, zeta-potential, encapsulation efficiency, drug loading, mucoaffinity properties and morphology. The RPMI 2650 cell line was used to assess the cytotoxicity and cellular permeation. An in vitro deposition test within a nasal cast completed these measurements.
Results:
The selected chitosan-coated cubosomal nanoparticles loaded with paliperidone palmitate had a size of 305.7 ± 22.54 nm, their polydispersity index was 0.166 ± 0.022 and their zeta potential was +42.4 ± 0.2 mV. This formulation had a drug loading of 70% and an encapsulation efficiency of 99.7 ± 0.1%. Its affinity with mucins was characterized by a ΔZP of 20.93 ± 0.31. Its apparent permeability coefficient thought the RPMI 2650 cell line was 3.00E-05 ± 0.24E-05 cm/s. After instillation in a 3D-printed nasal cast, the fraction of the injected powder deposited in the olfactory region reached 51.47 ± 9.30% in the right nostril and 41.20 ± 4.59% in the left nostril, respectively.
Conclusion:
The chitosan coated cubosomal formulation seems to be the most promising formulation for nose-to-brain delivery. Indeed, it has a high mucoaffinity and a significantly higher apparent permeability coefficient than the two other formulations. Finally, it reaches well the olfactory region.
Objective
Physicochemical characterization and assessment of aerosol dispersion performance of anti-TB proliposome dry powders for inhalation (DPIs) prepared using a single-step jet-milling (JM) approach.
Significance
Conventional tuberculosis (TB) treatment involves isoniazid and rifampicin as first-line agents in extended oral multi-drug regimes. Liposomal DPIs are emerging as promising alternatives for targeted delivery of anti-TB agents to alveolar macrophages harboring Mycobacterium tuberculosis. However, traditional approaches for liposomal DPI preparation are tedious, time consuming and require sophisticated/expensive equipment. The proposed JM technique for preparation of proliposome DPIs could obviate these limitations and facilitate use of these drugs for more effective and safer treatment.
Methods
Proliposome DPIs containing isoniazid and/or rifampicin, cholesterol and cholesterol sulfate were successfully prepared via JM (injection pressure, 7.4 bar; milling pressure, 3.68 bar). Their physicochemical, content uniformity, and in vitro aerosol dispersion performance were assessed using scanning electron microscopy/energy-dispersive X-ray spectroscopy, transmission electron microscopy, dynamic light scattering/Zeta potential, X-ray diffraction spectroscopy, thermogravimetric analysis, high-performance liquid chromatography, and the Next-Generation Impactor.
Results
The DPIs exhibited consistent, spherically shaped, smooth particles. Drug particles were evenly distributed with acceptable content uniformity. Drug crystallinity was not significantly affected by milling and the formulations had minimal (<2.0%) water content. After reconstitution of the DPIs, the hydrodynamic size was about 370.9–556.2 nm and charge was –12.3 to –47.3 mV. Furthermore, the proliposome DPIs presented emitted dose (69.04–89.03%), fine particle fraction, <4.4 µm (13.7 − 57.8%), and mass median aerodynamic diameter (<3.0 µm), which satisfied the requirements for deep lung delivery.
Conclusion
The proposed approach was suitable for preparation of proliposome DPIs that could be deployed for local targeting of the lower respiratory tract for the treatment of TB.
Inhaled drug delivery is a promising approach to achieving high lung drug concentrations to facilitate efficient treatment of tuberculosis (TB) and to reduce the overall duration of treatment. Rifampicin is a good candidate for delivery via the pulmonary route. There have been no clinical studies yet at relevant inhaled doses despite the numerous studies investigating its formulation and preclinical properties for pulmonary delivery. This review discusses the clinical implications of pulmonary drug delivery in TB treatment, the drug delivery systems reported for pulmonary delivery of rifampicin, animal models, and the animal studies on inhaled rifampicin formulations, and the research gaps hindering the transition from preclinical development to clinical investigation. A review of reports in the literature suggested there have been minimal attempts to test inhaled formulations of rifampicin in laboratory animals at relevant high doses and there is a lack of appropriate studies in animal models. Published studies have reported testing only low doses (≤ 20 mg/kg) of rifampicin, and none of the studies has investigated the safety of inhaled rifampicin after repeated administration. Preclinical evaluations of inhaled anti-TB drugs, such as rifampicin, should include high-dose formulations in preclinical models, determined based on allometric conversions, for relevant high-dose anti-TB therapy in humans.
Graphical abstract
Despite advancements in the medical and pharmaceutical fields, tuberculosis remains a major health problem globally. Patients do not widely accept the conventional approach to treating tuberculosis (TB) due to prolonged treatment periods with multiple high doses of drugs and associated side effects. A pulmonary route is a non-invasive approach to delivering drugs, hormones, nucleic acid, steroids, proteins, and peptides directly to the lungs, improving the efficacy of the treatment and consequently decreasing the adverse effect of the treatment. This route has been successfully developed for the treatment of various respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), tuberculosis (TB), lung cancer, and other pulmonary infections. The major approaches of inhalation delivery systems include nebulizers, metered-dose inhalers (MDIs), and dry powder inhalers (DPIs). However, dry powder inhalers (DPIs) are more advantageous due to their stability and ability to deliver a high dose of the drug to the lungs. The present review analyzes the modern therapeutic approach of inhaled dry powders, with a special focus on novel drug delivery system (NDDS) based DPIs for the treatment of TB. The article also discussed the challenges of preparing inhalable dry powder formulations for the treatment of TB. The clinical development of inhalable anti-TB drugs is also reviewed.
Drug-resistant tuberculosis (TB) can be considered the man-made result of interrupted, erratic or inadequate TB therapy. As reported in WHO data, resistant Mycobacterium tuberculosis (Mtb) strains continue to constitute a public health crisis. Mtb is naturally able to survive host defence mechanisms and to resist most antibiotics currently available. Prolonged treatment regimens using the available first-line drugs give rise to poor patient compliance and a rapid evolution of strains resistant to rifampicin only or to both rifampicin and isoniazid (multi drug-resistant, MDR-TB). The accumulation of mutations may give rise to extensively drug-resistant strains (XDR-TB), i.e. strains with resistance also to fluoroquinolones and to the injectable aminoglycoside which represent the second-line drugs. Direct lung delivery of anti-tubercular drugs, as an adjunct to conventional routes, provides high concentrations within the lungs, which are the intended target site of drug delivery, representing an interesting strategy to prevent or reduce the development of drug-resistant strains. The purpose of this paper is to describe and critically analyse the most recent and advanced results in the formulation development of WHO second-line drug inhalation products, with particular focus on dry powder formulation. Although some of these formulations have been developed for other lung infectious diseases (pseudomonas aeruginosa, nontuberculous mycobacteria), they could be valuable to treat MDR-TB and XDR-TB.
Drug-resistant tuberculosis (DR-TB) is an emerging health problem, challenging the effective control of global TB. Current treatment of DR-TB includes administration of multiple anti-TB drugs via oral and parenteral routes for a duration of 20–28 months. High systemic exposure, side effects and lengthy treatment time are problems affecting current treatment. The success rate of current lengthy treatment regimens is generally <50%. Bedaquiline, a new anti-TB drug is synergistic with pyrazinamide and in combination with moxifloxacin accelerates sputum-culture conversion. Therefore, a triple combination of these drugs may have the potential to shorten the treatment time and improve treatment success. Additionally, inhalation of these drugs in combination may be advantageous due to the direct delivery to the lungs, possibly reducing systemic exposure. This study aimed to develop an inhalable triple combination powder of bedaquiline, moxifloxacin and pyrazinamide and study their physicochemical properties and safety.
An inhalable (aerodynamic diameter: ≤2.4 µm) triple combination powder of bedaquiline, moxifloxacin and pyrazinamide with 20% w/w of L-leucine was prepared using a Buchi Mini Spray-Dryer. Combination powder consisted of spherical and porous particles. In vitro aerosolization (fine particle fraction, FPF) determined using a next generation impactor (NGI) showed improved FPF as a combination powder (>75.0%) when compared to single drug-only formulations (<45.0%). The powder was non-toxic to A549 and Calu-3 cells up to 100 µg/mL and stable at 30 ± 2% RH and ambient room temperature during one-month storage.
This is the first study reporting the development of inhalable triple combination powder of bedaquiline, moxifloxacin and pyrazinamide with high aerosolization efficiency. The improved aerosolization may help to deliver a high dose of these drugs to treat drug-resistant tuberculosis.
The purpose of this study was to investigate the influence of storage humidity on in vitro aerosolization and physicochemical properties of co-spray dried powders of kanamycin with rifampicin.
The powders were stored for one-month in an open Petri dish at different relative humidities (RHs) (15%, 43% and 75%) and 25 ± 2 °C. The in vitro aerosolization (fine particle fraction, FPF) of the powders was determined by a next generation impactor (NGI). The moisture content, particle morphology and crystallinity of the powders were determined by Karl Fischer titration, scanning electron microscopy and X-ray powder diffractometry, respectively.
At all RH, the FPF of hydrophobic rifampicin-only powder was unaffected but the FPF of hygroscopic kanamycin-only powder significantly decreased even at 43% RH. The kanamycin-only particles fused together, crystallized and formed hard cakes at 75% RH. The aerosolization of kanamycin and rifampicin in the combination powders remained unaffected at 15% and 43% RH, but aerosolization significantly decreased at 75% RH. Enrichment of the surface of the particles with hydrophobic rifampicin did not protect the combination powders from moisture uptake but it prevented particle agglomeration up to 43% RH. At 75% RH, the moisture uptake led to agglomeration of the particles of the combination powder particles and consequently an increase in aerodynamic diameter. Further studies are required to investigate how rifampicin enrichment prevents particle agglomeration, the possible mechanisms (e.g. particle interactions due to capillary forces or electrostatic forces) for the changes in the aerosolization and changes in surface composition during storage.
Purpose
Dissolution behavior of dry powder inhaler (DPI) antibiotic formulations in the airways may affect their efficacy especially for poorly-soluble antibiotics such as azithromycin. The main objective of this study was to understand the effects of surface composition on the dissolution of spray dried azithromycin powders by itself and in combination with colistin.
Methods
Composite formulations of azithromycin (a poorly water-soluble molecule) and colistin (a water-soluble molecule) were produced by spray drying. The resultant formulations were characterized for particle size, morphology, surface composition, solid-state properties, solubility and dissolution.
Results
The results demonstrate that surfaces composition has critical impacts on the dissolution of composite formulations. Colistin was shown to increase the solubility of azithromycin. For composite formulations with no surface colistin, azithromycin released at a similar dissolution rate as the spray-dried azithromycin alone. An increase in surface colistin concentration was shown to accelerate the dissolution of azithromycin. The dissolution of colistin from the composite formulations was significantly slower than the spray-dried pure colistin. In addition, FTIR spectrum showed intermolecular interactions between azithromycin and colistin in the composite formulations, which could contribute to the enhanced solubility and dissolution of azithromycin.
Conclusions
Our study provides fundamental understanding of the effects of surface concentration of colistin on azithromycin dissolution of co-spray-dried composite powder formulations.
Tuberculosis (TB) is a major global health burden. The emergence of the human immunodeficiency virus (HIV) epidemic and drug resistance has complicated global TB control. Pulmonary delivery of drugs using dry powder inhalers (DPI) is an emerging approach to treat TB. In comparison with the conventional pulmonary delivery for asthma and chronic obstructive pulmonary disease (COPD), TB requires high dose delivery to the lung. However, high dose delivery depends on the successful design of the inhaler device and the formulation of highly aerosolizable powders. Particle engineering techniques play an important role in the development of high dose dry powder formulations. This review focuses on the development of high dose dry powder formulations for TB treatment with background information on the challenges of the current treatment of TB and the potential for pulmonary delivery. Particle engineering techniques with a particular focus on the spray drying and a summary of the developed dry powder formulations using different techniques are also discussed.
High outputs of respirable solid-phase aerosols were generated from viscous solutions or suspensions of low- and high-molecular weight polyvinylpyrrolidone (PVP) solutions, 10% (w/v) albumin and, gamma globulin solutions as well as 10.3% (w/v) surfactant suspensions. A central fluid flow was aerosolized by coaxial converging compressed air. The water was evaporated from the droplets using warm dilution air and infrared radiation. The resulting aerosol particles were concentrated using a virtual impactor. The aerosols were generated at fluid flow rates between 1 and 3 ml/min and delivered at a flow rate of 44 l/min as 2.6–3.6 μm MMAD aerosols with geometric standard deviations between 1.5 and 2. Increases in viscosity over the range of 4–39 cSt caused a modest increase in MMAD. Increases in aerosol exit orifice diameter were associated with a decrease in aerosol diameter. Increases in compressed air pressure caused a decrease in aerosol diameter. Increases in fluid flow rate resulted in modest increases in MMAD together with proportional increases in output mass. Aerosolizing 10% 8 kDa PVP at 3 ml/min resulted in the delivery of 193 mg/min of PVP at 64% efficiency enabling 1.2 g to be collected in 7 min. Aerosolizing 10.3% surfactant suspensions at 3 ml/min resulted in the delivery of up to 163 mg/min with 59% efficiency. The surface tension of the surfactant was not changed by these processes. SEM showed dimpled particles of PVP, albumin, and gamma globulin indicating that their aerodynamic diameter was less than their morphometric diameter.
Copyright © 2018 American Association for Aerosol Research
The repurposing of drug delivery by the pulmonary route has been applied to treatment and prophylaxis of an increasingly wide range of respiratory diseases. Repurposing has been most successful for the delivery of inhaled bronchodilators and corticosteroids in patients with asthma and chronic obstructive pulmonary disease (COPD). Repurposing utilizes the advantages that the pulmonary route offers in terms of more targeted delivery to the site of action, the use of smaller doses, and a lower incidence of side-effects. Success has been more variable for other drugs and treatment indications. Pulmonary delivery is now well established for delivery of inhaled antibiotics in cystic fibrosis (CF), and in the treatment of pulmonary arterial hypertension (PAH). Other inhaled treatments such as those for idiopathic pulmonary fibrosis (IPF), lung transplant rejection or tuberculosis may also become routine. Repurposing has progressed in parallel with the development of new drugs, inhaler devices and formulations.
Pharmacotherapy of tuberculosis is potentially more efficient when delivered by the inhaled route than by the current oral and/or parenteral routes due to the higher concentration of drug reaching the primary region of infection in the lungs. This study investigated the influence of the amino acid L-leucine alone and in combination with the phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), on the aerosolization behaviour of the anti-TB drugs, pyrazinamide and moxifloxacin HCl. Spray dried powders of pyrazinamide (P), moxifloxacin (M) alone and in combination with 10% L-leucine (PL and ML) and 10% DPPC (PLD and MLD) were produced. The particle sizes of all powders except P were in the inhalable size range (< 5 µm) but differ in their morphology in presence of the excipients. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed the migration of surface active L-leucine and DPPC onto the surface of the particles during the spray drying process. The aerosolization from a dry powder inhaler, Aerolizer®, using a next generation impactor revealed fine particle fraction (FPF) values for P, PL and PLD of 18.7 ± 3.4%, 53.0 ± 3.2% and 74.5 ± 5.3% respectively while FPF values for M, ML and MLD were 55.6 ± 3.3%, 74.7 ± 4.7% and 74.1 ± 1.3% respectively. In conclusion, the differences in the aerosolization behaviours of the pyrazinamide and moxifloxacin spray dried powders with and without excipients was a combination of difference in the surface morphology and surface composition.
In the midst of Tuberculosis (TB) pandemic, a research about new tuberculosis drug that results in more rapid resolution of tubercular infection is important. It will play a crucial role in accelerating the reductions in tuberculosis incidence that is occurring worldwide. The effectiveness of rifapentine has been assessed and it has been proven to be the most effective antibiotics for TB. A frequent administration and dose of rifapentine resulted in more rapid resolution of tubercular infection. However, based on former research, high exposure levels for treatment shortening may be unachievable with oral administration and might instead be achieved by direct aerosol delivery of rifapentine to the pulmonary site of infection. Therefore, with the growing interest in the effectiveness of rifapentine in frequent administration and dose, this research integrates an inhalable form of crystalline rifapentine prepared using a batch process. Moreover, this research investigates the effect of seed loading, supersaturation ratio, and residence time on the characterization of crystalline rifapentine in order to form a crystalline rifapentine in an inhalable size. The research was carried out by using anti-solvent crystallization method with acetone as a solvent and distilled water as an anti-solvent. Based on the assessment of various operating variables, it can be concluded that the optimum result was obtained at the unseeded experiment with supersaturation ratio = 1.26. Unseeded experiments are preferred because the ideal size for therapeutic aerosol was achieved in unseeded experiments.
Tuberculosis (TB) is a life threatening pulmonary infection caused by Mycobacterium tuberculosis (MTB). Current treatments are complex, lengthy and associated with severe side effects that decrease patient compliance and increase the probability of the emergence of drug resistant strains. Thus, more effective drugs with little to no side effects are needed to diversify the armamentarium against the global TB epidemic. SHetA2, an anticancer compound with null toxicity at doses much higher than the effective dose, was recently discovered to be active against MTB. In the present study, a dry powder formulation of SHetA2 for pulmonary delivery was developed to overcome its poor aqueous solubility and to maximize its concentration in the lungs, the main site of TB infection. Using quality by design methodology, three different formulations of SHetA2 microparticles (MPs) were designed, manufactured and optimized: SHetA2 alone, SHetA2 PLGA and SHetA2 mannitol MPs, to maximize the drug dose, target alveolar macrophages and increase drug solubility, respectively. The resulting three SHetA2 MP formulations had spherical shape with particle size ranging from 1 to 3 µm and a narrow size distribution, suitable for uniform delivery to the alveolar region of the lungs. Upon dispersion with the Aerolizer® dry powder inhaler (DPI), all three SHetA2 MP formulations had aerodynamic diameters smaller than 3.3 µm and fine particle fractions (FPF4.46) greater than 77%. SHetA2 remained chemically stable after MP manufacture by spray drying, but the drug transformed from the crystalline to the amorphous form, which significantly enhanced the solubility of SHetA2. Using a custom-made dissolution apparatus, the FPF4.46 of SHetA2 MP dissolved much faster and to a greater extent (21.19 ± 4.40%) than the unprocessed drug (3.51 ± 0.9%). Thus, the physicochemical characteristics, in vitro aerosol performance and dissolution rate of the optimized SHetA2 MPs appear to be suitable to achieve therapeutic concentrations in the lungs.
This work aims to identify a suitable formulation for the pulmonary delivery of combinations of inhalational drugs using highly branched cyclic dextrin (HBCD) macromolecules. We compared the effectiveness between powders prepared from HBCD with those prepared from five alternative excipients (lactose, maltose, sucrose, β-cyclodextrin and methyl β-cyclodextrin) in the pulmonary delivery of a single-dosage form of two anti-tuberculosis drugs (isoniazid and rifampicin).
Fine particles of untreated active pharmaceutical ingredients (APIs) and combination products using excipients were prepared by spray drying. Rifampicin, a hydrophobic compound, was dissolved in ethanol, whereas isoniazid, a hydrophilic compound combined with either HBCD or an alternative excipient was dissolved in water. This was followed by the preparation of the spray-dried particle formulations (SDPs). The SDPs were characterised in terms of particle size, surface morphology, drug content, specific surface area, powder X-ray diffraction and inhalational properties.
The addition of either an excipient or HBCD decreased API particle sizes, producing submicron-size particles. Surface morphology examination using scanning electron microscopy revealed API SDPs to be cylindrical and non-wrinkled. However, API–excipient SDPs were wrinkled and rough. Only HBCD SDPs were porous and non-aggregating, thereby suggesting superior aerodynamic properties and suitability for pulmonary delivery of these particles. HBCD formulations had the highest drug content in terms of both isoniazid (97.5%) and rifampicin (92.3%). Larger surface areas were obtained for SDPs of HBCD than those of other sugars. Regarding inhalational properties, HBCD formulations had higher emitted dose and fine-particle fractions than formulations of all other sugars tested. Our results confirm the feasibility of the formulation of hydrophilic and hydrophobic drug substances into a single-dosage preparation for pulmonary delivery using HBCD as an excipient.
Inhaled drug formulations are designed with one of two primary objectives: (a) total replacement of a conventional method of administration to achieve comparable blood levels, or (b) targeting lung macrophages and parenchyma without regard to blood levels. As monotherapy is contra-indicated in the treatment of tuberculosis (TB), treatment regimens must include multiple drugs, hitting a variety of drug targets. Inhaled therapies proposed for TB include both single- and multiple-drug formulations. Formulations containing either first-line or second-line anti-TB agents are being studied by a number of research groups as either as stand-alone interventions or as adjunct therapies. Combinations of two or more first-line drugs; first-line drugs with experimental agents such as fluoroquinolones or rifamycins other than rifampicin; and with second- line drugs have been proposed. Drugs that inhibit resistance mechanisms of the pathogen, such as drug efflux pumps, are also proposed as candidates for inclusion in combination therapy of multiple-drug-resistant (MDR) TB.
This chapter considers the respiratory delivery of drugs used in the treatment of tuberculosis (TB), an infectious disease caused by the bacterium Mycobacterium tuberculosis. It provides a rationale for respiratory delivery of anti-tubercular drugs to the lungs, including the targeting of alveolar macrophages and granulomas. The doses of anti-tubercular drugs are high and most formulations will be carrier free containing drug and limited amounts of excipient. The aerosolization of high dose drugs existing in a cohesive matrix will be related to de-agglomeration processes. The chapter outlines the studies that have occurred in this area using particle engineering approaches. Formulations of particles or engineered particles which possess a suitable size for respiratory delivery can be considered as cohesive matrices. The particles in the matrix will interact due to the various interactive forces that exist between particles, including contact potential forces, Coulombic forces, intermolecular forces, capillary force and solid bridging.
This chapter gives an overview of the spray drying process, including details about the hardware, process parameters, and key theories. It highlights the recent technological advances that could facilitate the development of innovative therapeutics for tuberculosis (TB), and evaluates the anti-TB formulations produced by spray drying. Spray drying technology is well placed to meet the challenge of developing targeted drug delivery systems for TB. First, it is a unique processing method that allows control over critical fine-particle design features. Secondly, it is an established technique that continues to be attractive for its relative simplicity, cost-effectiveness, and scalability for commercial production. The expansive list of patents recently registered with the US Patent Office exemplifies that there is continuous worldwide interest in spray drying technologies for various pharmaceutical applications. Spray drying has been largely used to repurpose therapeutics as superior inhalation products with improved safety, efficacy, and convenience for patients.
This conclusion presents some closing thoughts on key concepts discussed in the preceding chapters of this book. The book demonstrates a dynamic and progressively expanding portfolio in the field of drug delivery system development for tuberculosis (TB). In all cases presented in the book, the objectives are to enhance efficacy and possibly reduce occurrence of adverse events by optimising drug and vaccine delivery options. Technologies already exist for formulation and manufacturing of drugs, for example, nano- and micro-particle constructs that may be used as drug and vaccine carriers for targeted delivery to cells using appropriate delivery devices. The potential clinical benefits of these developments can be demonstrated in a growing array of applicable animal models, and to a large extent have already informed study designs for clinical trials. Regulatory experience with inhaled and other alternative means of administration of therapies and vaccines for TB is almost non-existent.
L-Leucine is used as the most common force control agent used in the inhaled dry powder formulations. In this study, the effect of L-leucine on the surface morphology, surface energy and the Young’s modulus of the composite spray dried particles was studied. In addition, how L-leucine modify the interaction of particles with simulated pulmonary surfactant is also studied. Voriconazole (VRZ) was spray dried with different concentration of L-leucine from hydroalcoholic solutions. Formulations were found to possess irregular morphology. Surface concentration of L-leucine was increased with increasing feed concentration and plateaued at about 20% w/w L-leucine. Atomic force microscopy (AFM) coupled with drug colloid probe enabled measurement of cohesion forces between the prepared formulations and found cohesion to be reduced significantly (p<0.05) with the increase of L-leucine concentration. Peak Force tapping enabled characterization of nanomechanical properties (elasticity and deformation) of formulations. Co-spray drying L-leucine with VRZ does not seem to have any influence on the Young’s modulus of the formulations. Lastly, AFM revealed that surface chemistry of the drug particle and pulmonary surfactant, as well as the contact geometry of the interacting surfaces, plays an important role in determining nature and extent of interaction between inhaled drug particles and pulmonary surfactant.
Since the 1990s the rising incidence of multiple drug resistant TB particularly in the context of human immunodeficiency virus co-infected patients has threatened global TB control. At that time funding agencies began to support formal investigation of aerosol therapy which until then had been the subject of case reports of individual investigators. Over the last decade proponents of aerosol therapy have increased in number within the TB research community as the incidence of multiple and extremely drug resistant TB has increased dramatically around the world. Aerosol therapy offers the potential to deliver drug at target concentrations directly into the lungs, use the alveolar-capillary interface to achieve systemic levels, while reducing the risk of systemic toxicity seen with parentally administered doses. In addition, there are insufficient new drugs in the pipeline to anticipate the appearance of a new regimen in time to assure future control of drug resistance. Consequently, alternative strategies are critical to achieving global TB control, and inhaled therapies should be considered as one such strategy.
Lung infections may be bacterial, viral or fungal and they are typically treated with oral or parenteral antibiotics. Inhaled dry powder formulations offer unique opportunities for treating lung infections with enhanced effectiveness and stability. Since drug delivery to the lungs requires chronic and repeated administration of larger amounts of therapeutics, dry powder formulations are attractive alternatives to deliver drugs directly to the lungs as they are not limited by solubility issues and they are regarded as being more stable than liquid dosage forms. This state-of-the-art review presents the use of inhaled formulations for tuberculosis as a main focus, but also for other diseases such as bronchiectasis, chronic obstructive pulmonary disease (COPD), pneumonia and respiratory infections occurring as complications during lung transplants. Opportunities for the use of inhaled therapies and other respiratory diseases or as prevention or antidotes against warfare agents are offered. Typical and novel inhaled formulations that have been used as well as preclinical and clinical studies and evaluation of these inhaled therapies are discussed for each disease state. Lastly, the use of inhaled therapies as an alternative to end the emergence of drug resistant strains is discussed along with the risks of increasing these resistant strains if the inhaled therapy is not designed based on dosing regimens established by well-planned pharmacokinetic and pharmacodynamic studies.
Numerous studies have reported low concentrations of antituberculosis drugs in tuberculosis (TB) patients, but few studies
have examined whether low drug concentrations affect TB treatment response. We examined steady-state plasma concentrations
of isoniazid, rifampin, and pyrazinamide at 2 h after the administration of drugs (C2 h) among 181 patients with pulmonary tuberculosis in Indonesia and related these to bacteriological response during treatment.
C2 h values below reference values for either isoniazid, rifampin, or pyrazinamide were found in 91% of patients; 60% had at least
two low C2 h concentrations. The isoniazid C2 h was noticeably lower in fast versus slow acetylators (0.9 mg/liter versus 2.2 mg/liter, P < 0.001). At the end of treatment, 82% of the patients were cured, whereas 30 patients (17%) had dropped out during the study,
and 2 patients (1%) failed treatment. No association was found between C2 h concentrations and sputum culture results at 8 weeks of treatment. Post hoc analysis showed that patients with low pyrazinamide C2 h (P = 0.01) and patients with large extensive lung lesions (P = 0.01) were at risk of at least one positive culture at week 4, 8, or 24/32. Antituberculosis drug concentrations were often
low, but treatment response was nevertheless good. No association was found between drug concentrations and 8 weeks culture
conversion, but low pyrazinamide drug concentrations may be associated with a less favorable bacteriological response. The
use of higher doses of pyrazinamide may warrant further investigation.
A new approach for enhancing the dispersibility of respirable powders containing poorly soluble pharmaceutical actives is demonstrated. Budesonide was spray-dried with leucine from an ethanol–water cosolvent feedstock. For near-azeotropic cosolvent ratios, leucine was enriched on the surface of the particles relative to the bulk composition, and the particles showed a low-density morphology. Partial encapsulation of budesonide was accomplished. The dispersibility and manufacturability of the powder were greatly improved relative to powders made from budesonide alone. Fine particle fractions from a passive dry powder inhaler exceeded 80%. The particle size was in a suitable range for respiratory drug delivery (mass mean aerodynamic diameter between 2 and 3 μm). These results demonstrate that leucine can be used in ethanol–water cosolvent systems to design respirable particles with high dispersibility and good manufacturability.
Tuberculosis (TB) is a major killer worldwide. The World Health Organization estimates that in 2011, there were 8.7 million incident cases of TB, 1.4 million deaths from TB including 0.43 million deaths from HIV-associated TB. In recent years, after a century of stagnation, new diagnostic technologies have been developed, and they are already being scaled up in some high-burden countries. Likewise, new drugs and regimens to treat TB are being evaluated, and the development of new vaccines is also progressing. However, despite the hope for reduced transmission with earlier detection and effective treatment, new cases continue to emerge from latently infected individuals.
Background
Multidrug resistant tuberculosis threatens global TB control. The lengthy treatment includes one of the injectable drugs: kanamycin, amikacin, or capreomycin, usually for the first six months. These drugs have potentially serious toxicities, and when given as intramuscular injections, dosing can be painful. Advances in particulate drug delivery have led to the formulation of capreomycin as the first anti-tuberculosis drug available as a microparticle dry powder for inhalation and clinical study. Delivery by aerosol may result in successful treatment with lower doses. Here we report on a Phase I, single dose, dose-escalating study aimed at demonstrating safety and tolerability in normal subjects and measuring PK parameters.Methods and FindingsTwenty healthy adults (n=5 per group) were recruited to self administer a single dose of inhaled dry powder capreomycin (25 mg, 75 mg, 150 mg, or 300 mg nominal dose) using a simple, handheld delivery device. Inhalations were well-tolerated by all subjects. The most common adverse event was mild to moderate transient cough, in five subjects. There were no changes in lung function, audiometry, or laboratory parameters. Capreomycin was rapidly absorbed after inhalation. Systemic concentrations were detected in each dose group within 20 minutes. Peak and mean plasma concentrations of capreomycin were dose proportional. Serum concentrations exceeded 2 μg/mL (MIC for Mycobacterium tuberculosis) following the highest dose; t1/2 was 4.8 ± 1.0 hours.ConclusionsA novel inhaled microparticle dry powder formulation of capreomycin was well-tolerated. A single 300 mg dose rapidly achieved serum drug concentrations above the MIC for Mycobacterium tuberculosis, suggesting the potential of inhaled therapy as part of an MDR-TB treatment regimen.
In previous experiments, replacing the 10-mg/kg of body weight daily dose of rifampin with 7.5 to 10 mg/kg of rifapentine
in combinations containing isoniazid and pyrazinamide reduced the duration of treatment needed to cure tuberculosis in BALB/c
mice by approximately 50% due to rifapentine's more potent activity and greater drug exposures obtained. In the present study,
we performed dose-ranging comparisons of the bactericidal and sterilizing activities of rifampin and rifapentine, alone and
in combination with isoniazid and pyrazinamide with or without ethambutol, in BALB/c mice and in C3HeB/FeJ mice, which develop
necrotic lung granulomas after infection with Mycobacterium tuberculosis. Each rifamycin demonstrated a significant increase in sterilizing activity with increasing dose. Rifapentine was roughly
4 times more potent in both mouse strains. These results reinforce the rationale for ongoing clinical trials to ascertain
the highest well-tolerated doses of rifampin and rifapentine. This study also provides an important benchmark for the efficacy
of the first-line regimen in C3HeB/FeJ mice, a strain in which the lung lesions observed after M. tuberculosis infection may better represent the pathology of human tuberculosis.
We described the population pharmacokinetics of moxifloxacin and the effect of high-dose intermittent rifapentine in patients
with pulmonary tuberculosis who were randomized to a continuation-phase regimen of 400 mg moxifloxacin and 900 mg rifapentine
twice weekly or 400 mg moxifloxacin and 1,200 mg rifapentine once weekly. A two-compartment model with transit absorption
best described moxifloxacin pharmacokinetics. Although rifapentine increased the clearance of moxifloxacin by 8% during antituberculosis
treatment compared to that after treatment completion without rifapentine, it did not result in a clinically significant change
in moxifloxacin exposure.
Rifamycins are key sterilizing drugs in the current treatment of active tuberculosis (TB). Daily dosing of rifapentine (P), a potent rifamycin with high intracellular accumulation, in place of rifampin (R) in the standard antitubercular regimen significantly shortens the duration of treatment needed to prevent relapse in a murine model of active TB. We undertook the current study to compare directly the activities of human-equivalent doses of P and R in a guinea pig model of chronic TB, in which bacilli are predominantly extracellular within human-like necrotic granulomas. Hartley strain guinea pigs were aerosol infected with ~200 bacilli of Mycobacterium tuberculosis H37Rv, and treatment given 5 days/week was initiated 6 weeks later. R at 100 mg/kg of body weight and P at 100 mg/kg were given orally alone or in combination with isoniazid (H) at 60 mg/kg and pyrazinamide (Z) at 300 mg/kg. Culture-positive relapse was assessed in subgroups of guinea pigs after completion of 1 and 2 months of treatment. Human-equivalent doses of R and P showed equivalent bactericidal activity when used alone and in combination therapy. In guinea pigs treated with rifampin, isoniazid, and pyrazinamide (RHZ) or PHZ, microbiological relapse occurred in the lungs of 8/10 animals treated for 1 month and in 0/10 animals treated for 2 months. Substitution of P for R in the standard antitubercular regimen did not shorten the time to cure in this guinea pig model of chronic TB. Data from ongoing clinical trials comparing the activity of these two drugs are awaited to determine the relevance of the guinea pig TB model in preclinical drug screening.
Coadministration of moxifloxacin and rifampin was evaluated in a murine model of Mycobacterium tuberculosis pulmonary infection to determine whether the finding of antagonism documented in a hollow-fiber infection model could be
recapitulated in vivo. Colony counts were followed in a no-treatment control group, groups administered moxifloxacin or rifampin monotherapy, and
a group administered a combination of the two agents. Following 18 days of once-daily oral administration to mice infected
with M. tuberculosis, there was a reduction in the plasma exposure to rifampin that decreased further when rifampin was coadministered with moxifloxacin.
Pharmacodynamic analysis demonstrated a mild antagonistic interaction between moxifloxacin and rifampin with respect to cell
kill in the mouse model for tuberculosis (TB). No emergence of resistance was noted over 28 days of therapy, even with monotherapy.
This was true even though one of the agents in the combination (moxifloxacin) induces error-prone replication. The previously
noted antagonism with respect to cell kill shown in the hollow-fiber infection model was recapitulated in the murine TB lung
model, although to a lesser extent.
To evaluate clinical studies on the role of moxifloxacin in the treatment of pulmonary tuberculosis (TB).
A literature search was conducted using MEDLINE (1960-July 2011). Key search terms were moxifloxacin, BAY 12-8039, fluoroquinolones, and TB. References of review articles and of a Cochrane Review were also evaluated.
All articles published in English were evaluated. Prospective studies that evaluated the use of moxifloxacin in addition to or in substitution for first-line TB therapies were reviewed and included. Case series and retrospective studies were excluded.
Four 8-week clinical trials were included. All had comparator regimens containing standard doses of rifampin, isoniazid, ethambutol, and pyrazinamide. Two evaluated the substitution of moxifloxacin for ethambutol, 1 evaluated the substitution of moxifloxacin for isoniazid, and 1 studied the addition of moxifloxacin to a standard regimen. The dose of moxifloxacin was 400 mg daily in all studies, but dosing frequency differed from 3 to 7 days per week. All used culture conversion at 8 weeks as the primary end point, although they collected cultures at different times. Only 1 study found a significant difference in time to culture conversion at 8 weeks. One study found no significant difference at any point during the study. The other 2 studies found a significant difference in time to culture conversion at 4 and 6 weeks. In all trials, moxifloxacin was well tolerated, with nausea being the only significant adverse effect reported compared to the other arms of the studies.
Although it cannot be stated definitively, available evidence suggests that moxifloxacin appears to be as effective as ethambutol and is possibly as effective as isoniazid in the treatment of pulmonary TB. Given the generally poor second-line options for the treatment of TB, moxifloxacin is an attractive option as an alternative drug in TB treatment.
The 600-mg once daily dose of rifampicin plays a key role in tuberculosis treatment. The evidence underpinning this dose is
scant. A review of the historical literature identified 3 strands of reasoning. The first is the pharmacokinetic argument:
The 600-mg dose yields serum drug concentrations well above the minimum inhibitory concentration of rifampicin against Mycobacterium tuberculosis. The second is the argument that adverse events may be dose related. The third is the economic argument: Rifampicin was prohibitively
expensive at the time of its introduction. Recent in vitro, animal, and early bactericidal activity studies suggest that the
600-mg once daily dose is at the lower end of the dose-response curve, refuting the pharmacokinetic argument. The reduced
cost and the lack of evidence of toxicity at higher daily doses remove the other arguments. To optimize tuberculosis treatment,
the clinical value of higher doses of rifampicin should be tested in clinical trials.
The lengthy treatment regimen for tuberculosis is necessary to eradicate a small sub-population of M. tuberculosis that persists in certain host locations under drug pressure. Limited information is available on persisting bacilli and their location within the lung during disease progression and after drug treatment. Here we provide a comprehensive histopathological and microscopic evaluation to elucidate the location of bacterial populations in animal models for TB drug development.To detect bacilli in tissues, a new combination staining method was optimized using auramine O and rhodamine B for staining acid-fast bacilli, hematoxylin QS for staining tissue and DAPI for staining nuclei. Bacillary location was studied in three animal models used in-house for TB drug evaluations: C57BL/6 mice, immunocompromised GKO mice and guinea pigs. In both mouse models, the bacilli were found primarily intracellularly in inflammatory lesions at most stages of disease, except for late stage GKO mice, which showed significant necrosis and extracellular bacilli after 25 days of infection. This is also the time when hypoxia was initially visualized in GKO mice by 2-piminidazole. In guinea pigs, the majority of bacteria in lungs are extracellular organisms in necrotic lesions and only few, if any, were ever visualized in inflammatory lesions. Following drug treatment in mice a homogenous bacillary reduction across lung granulomas was observed, whereas in guinea pigs the remaining extracellular bacilli persisted in lesions with residual necrosis. In summary, differences in pathogenesis between animal models infected with M. tuberculosis result in various granulomatous lesion types, which affect the location, environment and state of bacilli. The majority of M. tuberculosis bacilli in an advanced disease state were found to be extracellular in necrotic lesions with an acellular rim of residual necrosis. Drug development should be designed to target this bacillary population and should evaluate drug regimens in the appropriate animal models.
Daily rifapentine plus isoniazid-pyrazinamide in mice infected with Mycobacterium tuberculosis produces cure in 3 months. Whether cure corresponds to latent infection contained by host immunity or true tissue sterilization is unknown.
To determine the length of treatment with rifapentine-isoniazid-pyrazinamide or rifampin-isoniazid-pyrazinamide needed to prevent relapse in immune-deficient mice.
Aerosol-infected BALB/c and nude mice were treated 5 days per week with either 2 months of the rifapentine-based regimen followed by rifapentine-isoniazid up to 12 months or the same regimen with rifampin instead of rifapentine. Cultures of lung homogenates were performed during the first 3 months and then every 3 months. Relapse rates were assessed after 3, 6, 9, and 12 months of treatment in BALB/c (± 1 mo of cortisone) and nude mice.
All rifapentine-treated mice were lung culture-negative at 3 months but 13% of BALB/c that received cortisone and 73% of nude mice relapsed. After 6, 9, and 12 months of treatment no mouse relapsed. Rifampin-treated BALB/c mice remained culture positive at 3 months. All were culture negative at 6, 9, and 12 months. None, including those receiving cortisone, relapsed. Rifampin-treated nude mice harbored more than 4 log(10) lung cfu at Month 2 and approximately 6 log(10) cfu with isoniazid resistance at Month 3. A supplementary experiment demonstrated that 7 days a week treatment did not prevent isoniazid resistance, whereas addition of ethambutol did.
In nude mice, sterilization of tuberculosis is obtained with rifapentine-containing treatment, whereas failure with development of isoniazid resistance is obtained with rifampin-containing treatment.
Recent in vitro pharmacokinetic data suggest that the currently recommended dose of pyrazinamide may be suboptimal for killing intracellular
bacilli in humans. We evaluated a range of pyrazinamide doses against intracellular and extracellular Mycobacterium tuberculosis in chronically infected mice and guinea pigs, respectively. Antibiotics were given five times weekly for 4 weeks beginning
28 days after infection. Human-equivalent doses of isoniazid reduced lung bacterial counts 10-fold in each species. Pyrazinamide
given at 1/4 and 1/2 the human-equivalent dose was minimally active, while human-equivalent doses reduced lung bacterial counts
by ∼1.0 log10 in each species. Doubling the human-equivalent dose of pyrazinamide reduced the lung bacillary burden by 1.7 and 3.0 log10 in mice and guinea pigs, respectively. As in humans and mice, pyrazinamide showed significant synergy with rifampin in guinea
pigs. Clinical studies are warranted to investigate the sterilizing activity and tolerability of higher doses of pyrazinamide
in combination tuberculosis regimens.
The main objectives of tuberculosis therapy are to cure the patients and to minimize the possibility of transmission of the bacillus to healthy subjects. Adverse effects of antituberculosis drugs or drug interactions (among antituberculosis drugs or between antituberculosis drugs and other drugs) can make it necessary to modify or discontinue treatment. We describe the general mechanism of action, absorption, metabolization, and excretion of the drugs used to treat multidrug resistant tuberculosis (aminoglycosides, fluoroquinolones, cycloserine/terizidone, ethionamide, capreomycin, and para-aminosalicylic acid). We describe adverse drug reactions and interactions (with other drugs, food, and antacids), as well as the most appropriate approach to special situations, such as pregnancy, breastfeeding, liver failure, and kidney failure.
Moxifloxacin is under development for expanded use against Mycobacterium tuberculosis . Rifampin is a mainstay of therapy. We examined the interaction of moxifloxacin plus rifampin for log-phase and nonreplicating persister (NRP) organisms. For this evaluation, we employed our hollow-fiber infection model, in which organisms are exposed to clinically relevant drug concentration-time profiles and the impact on bacterial cell kill and resistant subpopulation amplification is determined. In log phase, resistance emergence was observed in all monotherapy regimens and in no combination therapy regimen. No difference was seen in time to a 3-log reduction in the bacterial burden; there was a significant difference in time to resistance emergence ( P = 0.0006). In the NRP experiment, no resistance emergence was seen. There was a significant difference between the monotherapy and combination therapy regimens in time to a 3-log reduction in the bacterial burden ( P = 0.042). The combination is efficacious for suppressing resistant organisms but is antagonistic for cell kill.
IMPORTANCE M. tuberculosis infects one-third of the world’s population. Multiresistant organisms have become more frequent, threatening our ability to provide adequate chemotherapy. Moxifloxacin has been seen as an important new agent with the potential to supplant isoniazid or add to the rifampin/isoniazid combination. M. tuberculosis also exists in different physiological states, including the NRP phenotype. We examined the moxifloxacin/rifampin combination in a new in vitro system to allow judgment of how moxifloxacin would interact with rifampin and allow its performance in clinical trials to be placed into perspective. Importantly, the combination suppressed resistance emergence, but at the price of slightly slowing bacterial cell kill. This new combination is a welcome addition to the physician’s armamentarium.
Antimicrobial pharmacokinetic-pharmacodynamic studies suggest that pyrazinamide doses higher than those currently recommended may be more efficacious. However, high pyrazinamide doses are believed to be hepatotoxic. Searches for clinical trials in MEDLINE, EBSCOHOST, and the Cochrane Controlled Trial Register were made. Studies that employed pyrazinamide dose scheduling and pharmacokinetic analysis design were examined. Population pharmacokinetic modeling methods were utilized to identify parameters associated with toxicity. At an equivalent area under the concentration-time curve, the time that concentration persisted above some thresholds was associated with overall adverse events (P = 0.032), arthralgia (P = 0.089), and an elevated serum aspartate aminotransferase level at 3 months (P = 0.067). Next, a meta-analysis was utilized to compare rates of adverse events (i) between different pyrazinamide doses, (ii) between different dosing schedules, and (iii) between pyrazinamide-containing and non-pyrazinamide-containing antituberculosis regimens. The 29 studies selected were heterogeneous (Cochrane Q statistic P value of <0.001; I(2) of >95%). For the once-a-day dosing schedule, arthralgia was dose dependent (r(2) = 0.996). However, arthralgia was less common with intermittent dosing, consistent with the time concentration persisted above the threshold. Arthralgia was generally clinically inconsequential. The frequencies of hepatotoxicity were 0.057 (95% confidence interval [CI], 0.021 to 0.141) for pyrazinamide monotherapy, 0.044 (CI, 0.033 to 0.059) for pyrazinamide-containing combination regimens, and 0.040 (CI, 0.023 to 0.040) for non-pyrazinamide-containing combination regimens. The frequencies of hepatotoxicity were 0.042 (CI, 0.026 to 0.067) for 30 mg/kg of body weight, 0.055 (CI, 0.031 to 0.094) at 40 mg/kg, and 0.098 (CI, 0.047 to 0.193) at 60 mg/kg of pyrazinamide. Thus, high-dose pyrazinamide did not significantly increase hepatotoxicity. This suggests that a considerable portion of hepatotoxicity rates may be idiosyncratic.
Tuberculosis (TB) is the second most deadly infectious disease. Despite potentially curative pharmacotherapies being available for over 50 years, the length of the treatment and the pill burden can hamper patient lifestyle. Thus, low compliance and adherence to administration schedules remain the main reasons for therapeutic failure and contribute to the development of multi-drug-resistant (MDR) strains. Pediatric patients constitute a high risk population. Most of the first-line drugs are not commercially available in pediatric form. The design of novel antibiotics attempts to overcome drug resistance, to shorten the treatment course and to reduce drug interactions with antiretroviral therapies. On the other hand, the existing anti-TB drugs are still effective. Overcoming technological drawbacks of these therapeutic agents as well as improving the effectiveness of the drug by targeting the infection reservoirs remains the central aims of Pharmaceutical Technology. In this framework, nanotechnologies appear as one of the most promising approaches for the development of more effective and compliant medicines. The present review thoroughly overviews the state-of-the-art in the development of nano-based drug delivery systems for encapsulation and release of anti-TB drugs and discusses the challenges that are faced in the development of a more effective, compliant and also affordable TB pharmacotherapy.
Macrophage responses to infection with Mycobacterium tuberculosis (MTB) and treatment with soluble isoniazid (INH) plus rifabutin (RFB) versus microparticles containing equivalent amounts of drugs were compared. It was investigated whether macrophages driven to alternative activation upon infection with MTB could be rescued to display the classical activation phenotype. It was established that microparticles sustain high levels of drugs in cytosol of macrophages for longer period as compared to soluble drugs. Microparticles co-localized with intracellular bacteria, and induced a variety of innate bactericidal responses, including induction of free radicals, alteration of mitochondrial membrane potential and apoptosis. The data strongly suggest that additional benefit may be derived from the nature of the drug delivery system, which fulfils Koch's dictum 'stimulate the phagocyte' for curing tuberculosis.
To investigate the antagonism between isoniazid (INH) and rifampin (rifampicin) (RIF)-pyrazinamide (PZA) combination observed
in Mycobacterium tuberculosis-infected mice, extensive pharmacokinetic studies of INH were performed and followed by experiments to assess the impact of
increasing doses of INH on the antimicrobial activity of RIF-PZA combination. INH at 6.25 mg/kg of body weight produced a
maximum concentration of drug in serum (Cmax) value of 4 μg/ml and an area under the concentration-time curve from 0 to 24 h (AUC0-24) value of 4.9 μg·h/ml, the former being close to the Cmax value observed after the standard 5-mg/kg dose in humans. INH at 25 mg/kg produced a Cmax value of 22 μg/ml and an AUC0-24 value of 29 μg·h/ml, the latter being close to the AUC observed after a 5-mg/kg dose of INH in humans with the slow acetylation
phenotype. Beginning 2 weeks after aerosol infection with M. tuberculosis, mice were treated for 8 weeks with INH at twofold-increasing doses, ranging from 1.56 to 50 mg/kg, either alone or in combination
with RIF-PZA. Given alone, INH exhibited dose-dependent activity. Combined with RIF-PZA, INH exhibited dose-dependent antagonism
of RIF-PZA activity. To determine the individual components of RIF-PZA combination with which INH was antagonistic, mice were
treated for 8 weeks with RIF alone, PZA alone, RIF-PZA, and INH at 3.125, 12.5, or 50 mg/kg either alone or combined with
RIF or PZA. Addition of INH to RIF had additive activity, whereas addition of INH to PZA resulted in a negative interaction.
Finally, a 10-mg/kg dose of INH in mice may best represent the 5-mg/kg dose in humans and decrease the antagonism of INH with
RIF-PZA.
There are currently renewed efforts to develop drugs that could shorten the duration of antituberculosis therapy. This is best achieved by optimizing the sterilizing effect. However, the current pathway for the development of new molecules with the potential to have a sterilizing effect is inefficient. We designed an in vitro pharmacokinetic-pharmacodynamic model in which Mycobacterium tuberculosis replicating slowly at pH 5.8 was exposed to pyrazinamide by use of the concentration-time profiles encountered in patients. The sterilizing effect rates and the time to the emergence of drug resistance were examined. Daily pyrazinamide dosing for 28 days accurately achieved (i) the pyrazinamide pharmacokinetic parameters, (ii) the lack of early bactericidal activity, (iii) a sterilizing effect rate of 0.10 log(10) CFU/ml per day starting on day 6 of therapy, and (iv) a time to the emergence of resistance of the from 2 to 3 weeks of monotherapy encountered in patients with tuberculosis. Next, dose-scheduling studies were performed. The sterilizing effect was linked to the pyrazinamide ratio of the area under the concentration-time curve from 0 to 24 h (AUC(0-24)) to the MIC (r(2) = 0.80 to 0.90), with 90% of the maximal effect being achieved by an AUC(0-24)/MIC of 209.08. Resistance suppression was associated with the percentage of time that the concentration persisted above the MIC (r(2) = 0.73 to 0.91). Monte Carlo simulations of 10,000 patients demonstrated that the currently recommended pyrazinamide doses (15 to 30 mg/kg of body weight/day) achieved the AUC(0-24)/MIC of 209.08 in the epithelial lining fluid of only 15.1 to 53.3% of patients. Doses of >60 mg/kg per day performed better. Our vitro model for the sterilizing effect, together with Monte Carlo simulations, can be used for the faster identification of the clinical doses that are needed to achieve a sterilizing effect and that can then be studied in clinical trials.
Moxifloxacin has potent activity against Mycobacterium tuberculosis in vitro and in a mouse model of antituberculosis (TB) chemotherapy, but data regarding its activity in humans are limited.
Our objective was to compare the antimicrobial activity and safety of moxifloxacin versus isoniazid during the first 8 weeks of combination therapy for pulmonary TB.
Adults with sputum smear-positive pulmonary TB were randomly assigned to receive either moxifloxacin 400 mg plus isoniazid placebo, or isoniazid 300 mg plus moxifloxacin placebo, administered 5 days/week for 8 weeks, in addition to rifampin, pyrazinamide, and ethambutol. All doses were directly observed. Sputum was collected for culture every 2 weeks. The primary outcome was negative sputum culture at completion of 8 weeks of treatment.
Of 433 participants enrolled, 328 were eligible for the primary efficacy analysis. Of these, 35 (11%) were HIV positive, 248 (76%) had cavitation on baseline chest radiograph, and 213 (65%) were enrolled at African sites. Negative cultures at Week 8 were observed in 90/164 (54.9%) participants in the isoniazid arm, and 99/164 (60.4%) in the moxifloxacin arm (P = 0.37). In multivariate analysis, cavitation and enrollment at an African site were associated with lower likelihood of Week-8 culture negativity. The proportion of participants who discontinued assigned treatment was 31/214 (14.5%) for the moxifloxacin group versus 22/205 (10.7%) for the isoniazid group (RR, 1.35; 95% CI, 0.81, 2.25).
Substitution of moxifloxacin for isoniazid resulted in a small but statistically nonsignificant increase in Week-8 culture negativity.
New treatments are needed to shorten the time required to cure tuberculosis and to treat drug-resistant strains. The fluoroquinolone moxifloxacin is a promising new agent that might have additive activity to existing antituberculosis agents. We assessed the activity and safety of moxifloxacin in the initial stage of tuberculosis treatment.
We undertook a phase II, double-blind, randomised controlled trial of a regimen that included moxifloxacin in adults with sputum smear-positive tuberculosis at one hospital in Rio de Janeiro, Brazil. 170 participants received isoniazid, rifampicin, and pyrazinamide at standard doses and were assigned by permuted block randomisation to receive either moxifloxacin (400 mg) with an ethambutol placebo (n=85) or ethambutol (15-20 mg/kg) plus moxifloxacin placebo (n=85) 5 days per week for 8 weeks. The primary endpoint was the proportion of patients whose sputum culture had converted to negative by week 8. Analysis was by modified intention to treat (ITT); patients whose baseline cultures were negative, contaminated, or contained drug-resistant Mycobacterium tuberculosis were excluded from the analysis. Additionally, all missing 8-week results were deemed treatment failures. This study is registered with ClinicalTrials.gov, number NCT00082173.
74 patients assigned to the moxifloxacin group and 72 in the ethambutol group were included in the modified ITT population. 125 patients had 8-week data (moxifloxacin n=64, ethambutol n=61); the main reason for absence of data was culture contamination. At 8 weeks, culture conversion to negative had occurred in 59 (80%) of 74 patients in the moxifloxacin group compared with 45 (63%) of 72 in the ethambutol group (difference 17.2%, 95% CI 2.8-31.7; p=0.03). There were 16 adverse events (eight in each group) in 12 patients. Only one event was judged related to study drug (grade 3 cutaneous reaction in the ethambutol group).
Moxifloxacin improved culture conversion in the initial phase of tuberculosis treatment. Trials to assess whether moxifloxacin can be used to shorten the duration of tuberculosis treatment are justified.
We formulated PA-824, a nitroimidazopyran with promise for the treatment of tuberculosis, for efficient aerosol delivery to the lungs in a dry powder porous particle form. The objectives of this study were to prepare and characterize a particulate form of PA-824, assess the stability of this aerosol formulation under different environmental conditions, and determine the pharmacokinetic parameters for the powder after pulmonary administration. The drug was spray dried into porous particles containing a high drug load and possessing desirable aerosol properties for efficient deposition in the lungs. The physical, aerodynamic, and chemical properties of the dry powder were stable at room temperature for 6 months and under refrigerated conditions for at least 1 year. Pharmacokinetic parameters were determined in guinea pigs after the pulmonary administration of the PA-824 powder formulation at three doses (20, 40, and 60 mg/kg of body weight) and compared to those after the intravenous (20 mg/kg) and oral (40 mg/kg) delivery of the drug. Oral and inhaled delivery of PA-824 achieved equivalent systemic delivery at the same body dose within the first 12 h of dosing. However, animals dosed by the pulmonary route showed drug loads that remained locally in the lungs for 32 h postexposure, whereas those given the drug orally cleared the drug more rapidly. Therefore, we expect from these pharmacokinetic data that pulmonary delivery may achieve the same efficacy as oral delivery at the same body dose, with a potential improvement in efficacy related to pulmonary infection. This may translate into the ability to deliver lower body doses of this drug for the treatment of tuberculosis by aerosol.
Recent studies have demonstrated that combined substitutions of rifapentine for rifampin and moxifloxacin for isoniazid in the standard, daily, short-course regimen of rifampin, isoniazid, and pyrazinamide produces stable cure in 12 weeks or less. This study was designed to more precisely evaluate the contribution of moxifloxacin and isoniazid to rifapentine-based regimens.
We compared bactericidal activity and treatment-shortening potential between regimens consisting of isoniazid or moxifloxacin plus rifapentine and pyrazinamide administered either thrice-weekly or daily.
Using a mouse model of tuberculosis, we assessed bactericidal activity by performing quantitative cultures of lung homogenates over the first 12 weeks of treatment. Relapse rates were assessed after completing 8, 10, and 12 weeks of treatment to determine the duration of treatment necessary for stable cure.
After 4 weeks of treatment, daily and thrice-weekly therapy with rifapentine, moxifloxacin, and pyrazinamide was significantly more active than treatment with rifapentine, isoniazid, and pyrazinamide. By 8 weeks of treatment, all mice receiving the moxifloxacin-containing regimens were lung culture negative, whereas those mice receiving the isoniazid-containing regimens continued to be lung culture positive. However, the duration of treatment necessary to achieve stable cure was 10 weeks for daily regimens and 12 weeks for thrice-weekly regimens, regardless of whether isoniazid or moxifloxacin was used. All mice receiving standard daily therapy with rifampin, isoniazid, and pyrazinamide relapsed after 12 weeks of treatment.
These results suggest that regimens consisting of isoniazid or moxifloxacin plus rifapentine and pyrazinamide may dramatically shorten the duration of treatment needed to cure human tuberculosis.
Mice that had been inoculated intravenously with 6.30 log10 Mycobacterium tuberculosis H37Rv 14 days earlier were administered one of three combinations of drugs, i.e., isoniazid (INH)-rifampin (RMP)-pyrazinamide (PZA), INH-RMP, and RMP-PZA, during an initial 2-month period to mimic the initial phase of chemotherapy for human tuberculosis and during a later 4-month period to mimic the continuation phase of chemotherapy. At the end of the initial phase, all three combined regimens were found to have been highly effective in terms of the number of CFUs in the spleens of infected mice. The bactericidal activities of INH-RMP-PZA and INH-RMP were similar, whereas that of RMP-PZA was significantly greater. The spleens of all of the mice that had been treated initially with INH-RMP-PZA were culture negative by the end of 6 months of treatment, regardless of the regimen employed during the continuation phase. However, after an additional period of 6 months without treatment, the proportion of spleen culture positivity, or relapse rate, was significantly smaller in the subgroup treated with RMP-PZA during the continuation phase than in the subgroups treated with INH-RMP-PZA or INH-RMP; the relapse rate did not differ significantly between the latter two subgroups. These results suggest that antagonism occurs between INH and the combination RMP-PZA during both the initial and continuation phases of chemotherapy, compromising the benefit conferred by the addition of PZA to the combined regimen. The preliminary pharmacokinetic analysis suggested that the pharmacological interaction between INH and RMP was very likely to be involved in the mechanism of antagonism, as concomitant treatment with INH had significantly reduced the peak serum level and the area under the serum concentration-time curve of RMP in mice.
Moxifloxacin (BAY12-8039) is a new 8-methoxyquinolone shown to be active against Mycobacterium tuberculosis in vitro. We tested moxifloxacin for activity in mice against M. tuberculosis CSU93, a highly virulent, recently isolated clinical strain. The MIC of moxifloxacin for the CSU93 strain was 0.25 microg/ml. The serum moxifloxacin concentration after oral administration in mice peaked within 0.25 h, reaching 7.8 microg/ml with doses of 100 mg/kg of body weight; the maximum concentration and the analysis of the area under the concentration-time curve revealed dose dependency. When mice were infected with a sublethal inoculum of mycobacteria and then treated with moxifloxacin at 100 mg/kg per day for 8 weeks, the log10 CFU counts in the organs of treated mice were significantly lower than those for the control group (0.6 +/- 0.2 versus 5.6 +/- 0. 3 in the lungs and 1.5 +/- 0.7 versus 4.9 +/- 0.5 in the spleens, respectively; P < 0.001 in both organs). The effectiveness of moxifloxacin monotherapy was comparable to that seen in mice receiving isoniazid alone. Combination therapy with moxifloxacin plus isoniazid was superior to that with moxifloxacin or with isoniazid alone in reducing bacillary counts in the organs studied. Using a sensitive broth-passage subculture method, we demonstrated that 8 weeks of treatment with moxifloxacin (100 mg/kg per day) or with moxifloxacin plus isoniazid (100 mg/kg and 25 mg/kg, respectively, per day) sterilized the lungs in seven of eight and in eight of eight mice, respectively. Among surviving bacilli isolated from animals infected with a high-titer inoculum and treated for 7 weeks with low-dose moxifloxacin (20 mg/kg per day), breakthrough resistance to moxifloxacin was not observed. These results indicate that moxifloxacin is highly effective in reducing M. tuberculosis infection in mice and has activity comparable to that of isoniazid. Combination therapy with moxifloxacin and isoniazid was highly effective, suggesting that moxifloxacin may be useful in multiple-drug regimens for human tuberculosis.
The rifamycin antibacterials, rifampicin (rifampin), rifabutin and rifapentine, are uniquely potent in the treatment of patients with tuberculosis and chronic staphylococcal infections.
Absorption is variably affected by food; the maximal concentration of rifampicin is decreased by food, whereas rifapentine absorption is increased in the presence of food. The rifamycins are well-known inducers of enzyme systems involved in the metabolism of many drugs, most notably those metabolised by cytochrome P45O (CYP) 3A. The relative potency of the rifamycins as CYP3A inducers is rifampin > rifapentine > rifabutin; rifabutin is also a CYP3A substrate.
The antituberculosis activity of rifampicin is decreased by a modest dose reduction from 600 to 450mg. This somewhat surprising finding may be due to the binding of rifampicin to serum proteins, limiting free, active concentrations of the drug. However, increasing the administration interval (after the first 2 to 8 weeks of therapy) has little effect on the sterilising activity of rifampicin, suggesting that relatively brief exposures to a critical concentration of rifampicin are sufficient to kill intermittently metabolising mycobacterial populations. The high protein binding of rifapentine (97%) may explain the suboptimal efficacy of the currently recommended dose of this drug.
The toxicity of rifampicin is related to dose and administration interval, with increasing rates of presumed hypersensitivity with higher doses combined with administration frequency of once weekly or less. Rifabutin toxicity is related to dose and concomitant use of CYP3A inhibitors.
The rifamycins illustrate the complexity of predicting the pharmacodynamics of treatment of an intracellular pathogen with the capacity for dormancy.
Drug therapy of tuberculosis (TB) requires long-term oral administration of multiple drugs for curing as well as preventing and/ or combating multi-drug resistance. Persistent, high blood levels of antitubercular drugs resulting from prolonged oral administration of anti-TB drugs may be neither necessary nor sufficient to kill mycobacteria residing in macrophages (M4). Inhalable biodegradable microparticles containing two of the first-line anti-TB drugs, isoniazid (H), and rifampicin (R), were prepared and tested for (i) phagocytosis by mouse Mphi. (ii) administration as a dry powder inhalation to rats, and (iii) targeting alveolar Mphi with high drug doses when administered to rats.
poly(D-L lactic acid) microparticles were prepared by emulsion methods and their drug content and size distribution determined. These were tested for uptake by murine Mphi in culture and resultant intracellular drug concentrations determined by high performance thin-layer chromatography (HPTLC). Rats were administered an inhalation of microparticles using an inhalation chamber developed in the lab. The extent of microparticle delivery in vivo was examined by flow-cytometry. Drug concentrations in the blood and in alveolar Mphi were estimated by high-performance liquid chromatography after oral, vascular. intratracheal, and inhalation administration.
Inhalable microparticles could be prepared and were taken up by cultured Mphi. Large numbers of particles could be delivered to the bronchiopulmonary system through a 2-min exposure to fluidized particles. The intracellular drug concentrations resulting from vascular delivery of soluble drugs were found to be lower than those resulting from particle inhalation.
Inhalable microparticles containing multiple anti-TB drugs offer promises of dose and dosing-frequency reduction, toxicity alleviation, and targeting Mphi-resident persistent mycobacteria.
Pyrazinamide (PZA) is an important front-line anti-tuberculosis drug that is active only at acid pH. However, acid pH causes significant difficulty for PZA susceptibility testing. A common problem in PZA testing is false resistance caused by large bacterial inocula. This study investigated the relationship of false resistance to numbers of bacilli, pH and other factors that potentially affect susceptibility to PZA. Large inocula (10(7-8) bacilli/ml) of M. tuberculosis H37Ra caused significant increase in medium pH from 5.5 towards neutrality, and thus produced false resistance results. The increase in medium pH was determined to be a function of live bacilli; heat-killed bacilli had little or no effect. Susceptibility to PZA and its active derivative pyrazinoic acid (POA) was comparable on 7H11 agar medium, but POA was less active than PZA in liquid medium containing bovine serum albumin (BSA), suggesting that susceptibility to PZA or POA was reduced in the presence of BSA, because of its neutralising effect on medium pH and significant POA binding. A 3-month-old H37Ra culture was shown to be more susceptible to PZA exposure than a 4-day log-phase culture, suggesting that PZA is more active for non-growing bacilli. Finally, reserpine, an inhibitor of POA efflux pump, increased susceptibility to PZA even near neutral pH 6.8, with an MIC of 400 mg/L compared with 1,000 mg/L without reserpine. These findings should have implications for understanding the mode of action of PZA and for PZA susceptibility testing.
The development of lesions in pulmonary tuberculosis, the most common form of tuberculosis, is the result of the conflict between the invader, Mycobacterium tuberculosis, and the host. This is a standard for all infectious diseases. Tuberculosis, however, is the paradigm for diseases with multiplication of the responsible organism within macrophages and monocytes (2) and control of the infection by cell-mediated immunity (CMI) (5) orchestrated by T-cell-derived lymphokines and carried out by the effector cells, activated macrophages (31). In fact, the CMI generated in tuberculosis is so potent that on average, 90% of the immunocompetent humans infected with M. tuberculosis are able to contain the infection and avoid progression to clinical disease during their lifetimes (8, 33, 34). This potency of CMI may be demonstrated experimentally: guinea pigs previously infected with M. tuberculosis react against cutaneous reinfection with live bacilli by rapidly forming a necrotic skin lesion that subsequently resolves spontaneously (31). This reaction, now known as the “Koch phenomenon,” suggests that the immune reaction mounted by infected animals is also particularly potent. In leprosy, a related disease, the responsible organism, M. leprae, is an obligate intracellular organism. It ensures its intracellular multiplication by inducing a specific immune paralysis, best demonstrated in the case of lepromatous leprosy, in which patients fail to develop delayed-type hypersensitivity (DTH) to the organism. In tuberculosis, while there may be immune dysregulation, specific DTH to purified protein derivative and granulomatous inflammation are prominent hallmarks of the disease. This contrast between M. leprae and M. tuberculosis begs the critical question of how M. tuberculosis avoids killing as a result of CMI despite its intracellular location. It is not simply an academic issue because it determines where antituberculous agents must encounter and kill M. tuberculosis during preventive and curative chemotherapy. To address this issue it is appropriate to review the remarkable but largely ignored work done in the past by Opie and Aronson (25), Long (15), Canetti (1), Lurie (18), and Dannenberg and Rook (7) and to recall the main steps of the pathogenesis of tuberculosis in humans, with emphasis on the events that take place after the development of acquired CMI.
Patients in whom acid-fast bacilli smear-positive pulmonary tuberculosis was newly diagnosed were randomized to receive 400 mg moxifloxacin, 300 mg isonaizid, or 600 mg rifampin daily for 5 days. Sixteen-hour overnight sputa collections were made for the 2 days before and for 5 days of monotherapy. Bactericidal activity was estimated by the time taken to kill 50% of viable bacilli (vt50) and the fall in sputum viable count during the first 2 days designated as the early bactericidal activity (EBA). The mean vt50 of moxifloxacin was 0.88 days (95% confidence interval [CI], 0.43-1.33 days) and the mean EBA was 0.53 (95% CI 0.28-0.79). For the isoniazid group, the mean vt50 was 0.46 days (95% CI, 0.31-0.61 days) and the mean EBA was 0.77 (95% CI, 0.54-1.00). For rifampin, the mean vt50 was 0.71 days (95% CI, 0.48-0.95 days) and the mean EBA was 0.28 (95% CI, 0.15-0.41). Using the EBA method, isoniazid was significantly more active than rifampin (p < 0.01) but not moxifloxacin. Using the vt50 method, isoniazid was more active than both rifampin and moxifloxacin (p = 0.03). Moxifloxacin has an activity similar to rifampin in human subjects with pulmonary tuberculosis, suggesting that it should undergo further assessment as part of a short course regimen for the treatment of drug-susceptible tuberculosis.
Amorphous states, ubiquitous in pharmaceutical products, possess higher tendency for chemical degradation in comparison to crystalline materials. This instability can be further enhanced by water, which is present even in nominally dry systems. It has been increasingly recognized that in addition to the plasticizing effect of lowering the glass transition temperature, water could influence the degradation rates through medium effects (e.g., through change in solvation of the reactants and the transition state) as well as by direct participation in solid-state hydrolytic degradation processes. In the current review, the impact of water on the chemical stability of small molecules is examined, with emphasis on hydrolysis reactions in freeze-dried materials remaining in the glassy state. Quantitative relationships between water content and stability are discussed, including molecular mobility (global and local) and solution-like mechanisms, using the medium effects concept that has been developed for liquid-state reactions. Further progress in this field requires the development of quantitative and mechanistic understanding of the relationship between local mobility and chemical reactivity in amorphous solids, as well as incorporating the learning from solution chemistry on the role of reaction media in chemical processes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
Tuberculosis continues to be a major cause of morbidity and mortality in the world. The expansion of tuberculosis control programs has been limited by the lengthy and cumbersome nature of current chemotherapeutic regimens. A new drug that improves the sterilizing activity of current regimens would reduce the duration of therapy without sacrificing efficacy, thereby enhancing treatment completion rates and preserving precious public health resources. The new 8-methoxyfluoroquinolone moxifloxacin has potent activity against both actively multiplying and nonactively multiplying tubercle bacilli. Using a murine model that is representative of chemotherapy for human tuberculosis, we show that the combination of moxifloxacin, rifampin, and pyrazinamide reduced the time needed to eradicate Mycobacterium tuberculosis from the lungs of infected mice by up to 2 months when compared with the standard regimen of isoniazid, rifampin, and pyrazinamide. The findings suggest that this regimen has the potential to substantially shorten the duration of therapy needed to cure human tuberculosis.
The stability of four polymorphs of pyrazinamide, α, β, γ, and δ, was studied under solvent-mediated crystallization, neat and liquid-assisted grinding, polymorph seeding, and ambient storage conditions. In contrast to a recent report that the δ polymorph is the most stable modification (Castro et al. Cryst. Growth Des. 2010, 10, 274), we find that the α polymorph is the thermodynamic form. β, γ, and δ transform to the α phase in the above-mentioned conditions as monitored by infrared, near-infrared, and Raman spectroscopy, differential scanning calorimetry, and X-ray powder diffraction. Transformation to the high temperature γ phase is monitored by thermogravimetric analysis-infrared (TG-IR) spectrometry. A semischematic energy−temperature diagram consistent with phase transformation experiments, thermal measurements, and crystal structure data gives the order α < δ < γ < β at 25 °C (α is the most stable form), whereas at 160 °C γ < α < δ < β (γ stable modification), but at absolute zero δ < α < β < γ (δ stable modification). Even though the δ polymorph has the lowest free energy at absolute zero temperature, the α polymorph is the thermodynamic form under the ambient conditions regime more relevant to crystallization and handling of pharmaceuticals. The intrinsic dissolution rate of the γ form is faster than α and δ polymorphs, but α is the preferred polymorph of pyrazinamide considering both stability and bioavailability criteria. We also report high quality X-ray crystal structures of all the four polymorphs of pyrazinamide (R = 0.0387, 0.0340, 0.0392, and 0.0372 for α, β, γ, and δ).
Treatment for tuberculosis (TB) using the standard oral antibiotic regimen is effective but inefficient, requiring high drug dosing and lengthy treatment times. Three concurrent first-line antibiotics recommended by the World Health Organization (WHO) guidelines are pyrazinamide, rifampicin and isoniazid. Combining these antibiotics in a novel formulation for dry powder inhalation (DPI) may facilitate rapid and efficient resolution of local and systemic infection. However, spray-dried individually, these antibiotics were found to be physically unstable. A solution of the three antibiotics, at the WHO-recommended ratio, was spray-dried. The collected powder was assessed by a series of in vitro methods to investigate aerosol performance, particle physico-chemical characteristics and dissolution profile. Particles obtained were spherical with a surface composed primarily of rifampicin, as identified by TOF-SIMS. A mass median aerodynamic diameter of 3.5±0.1μm and fine particle fraction (<5μm) of 45±3% indicated excellent aerosol performance. The combination powder was differentiated by the presence of rifampicin dihydrate and the delta polymorph of pyrazinamide. Quantitative analysis indicated individual particles contained the three antibiotics at the expected proportions (400:150:75 w/w). This excipient-free triple antibiotic DPI formulation could be used as a significant enhanced treatment for TB.
The aim of this research was to develop a novel carrier-free dry powder formulation of rifampicin for inhalation with controlled-release properties.
Rifampicin dihydrate (RFDH) microcrystals were prepared by a polymorphic transformation of rifampicin. The prepared RFDH microcrystals were coated with poly (DL-lactide-co-glycolide) or poly (DL-lactide), using a spray-dryer equipped with two different types of three-fluid (3F) spray nozzles. The physicochemical and aerodynamic properties of the coated RFDH microcrystals were compared with those of conventional matrix microparticles.
The coated RFDH powder, encapsulating 50% of rifampicin, was successfully prepared by simple in-situ coating methods using two different types of 3F nozzles and had mass median aerodynamic diameter values of 3.5-4.5 µM. The thin flaky morphology of RFDH powders, providing good aerosolization properties, was maintained after coating. The coated RFDH formulations showed relatively low initial rifampicin release, compared with the uncoated RFDH crystals, followed by slow rifampicin release (about 70%) over 8 h in phosphate-buffered saline media (pH 7.4). Significant chemical degradations were not observed from the crystalline-structured RFDH formulations, while the amorphous-structured matrix formulations showed chemical degradation in six months.
These polymer coated RFDH formulations may be a valuable alternative in the treatment of tuberculosis since the carrier-free formulation offers the benefit of delivering a maximum-potency formulation of the antibiotic directly to the site of infection, and long drug residence times may be achieved by the controlled release of the drug.
Rifapentine (RP T) is an antituberculosis drug that may shorten treatment duration when substituted for rifampin (RI F).The maximal tolerated daily dose of RP T and its potential for cytochrome 3A4 induction and autoinduction at clinically relevant doses are unknown. In this phase I, dose-escalation study among healthy volunteers, daily doses as high asa prespecified maximum of 20 mg/kg/day were well tolerated. Steady-state RP T concentrations increased with dose from 5 to 15 mg/kg, but area under the plasma concentration–time curve (AU C0–24) and maximum concentration (Cmax)were similar in the 15- and 20-mg/kg cohorts. Although RP T pharmacokinetics (PK) appeared to be time-dependent,accumulation occurred with daily dosing. The mean AU C0–12 of oral midazolam (MDZ), a cytochrome 3A (CYP 3A) probe drug, was reduced by 93% with the coadministration of RPT and by 74% with the coadministration of RIF (P < 0.01).Changes in the oral clearance of MDZ did not vary by RP T dose. In conclusion, RP T was tolerated at doses as high as20 mg/kg/day, its PK were less than dose-proportional, and its CYP 3A induction was robust.
Dry powder inhalations (DPI) of microparticles containing isoniazid (INH) and rifabutin (RFB) are under preclinical development for use in pulmonary tuberculosis. Microparticles containing 0.25, 2.5, or 25 mg of each drug were administered daily for 90 days to rhesus macaques (n = 4/group). Single inhalations or intravenous (i.v.) doses were administered to separate groups. Drugs in serum, alveolar macrophages, and organ homogenates were assayed by high-performance liquid chromatography (HPLC). The RFB/INH in the lungs (101.10 ± 12.90/101.07 ± 8.09 μg/g of tissue) was twice that of the liver concentrations (60.22 ± 04.97/52.08 ± 4.62 μg/g) and four times that of the kidneys (22.89 ± 05.22/30.25 ± 3.71 μg/g). Pharmacokinetic parameters indicated the operation of flip-flop kinetics. Thus, the elimination half-life (t(1/2)) of RFB and INH was calculated as 8.01 ± 0.5 and 2.49 ± 0.23 h, respectively, upon intravenous (iv) administration, and as 13.8 ± 0.8 and 10.43 ± 0.77 h following a single inhalation; or 13.36 ± 3.51 and 10.13 ± 3.01 at a presumed steady state (day 60 of dosing). Targeted and sustained drug delivery to nonhuman primate lungs and alveolar macrophages was demonstrated. Flip-flop serum pharmacokinetics was observed, and nonlinearity in some pharmacokinetic parameters at logarithmic dose increments was indicated. The results suggest that human patients would benefit through improvement in biodistribution following DPI.
The aim of this research was to investigate a novel dry powder formulation of rifampicin (RF) that presents an improved lung deposition profile by means of a polymorphic transformation into a flake-like crystal hydrate. Rifampicin dihydrate (RFDH) was prepared by recrystallization of RF in anhydrous ethanol. A control formulation, amorphous RF (RFAM) was prepared by spray drying. The physicochemical properties of the RFDH and the RFAM were characterized. Aerosol performances of RFDH and RFAM were studied with two dry powder inhalers (DPIs), an Aerolizer and a Handihaler, using a Next Generation Impactor (NGI). The RFDH powder was successfully prepared using simple recrystallization process and had a MMAD of 2.2 μm. The RFDH powders were characterized as having a very thin flaky structure; this unique morphology provided improved aerosolization properties with a decreased device dependency upon aerosolization. The flaky morphology of RFDH resulted in a reduced agglomeration tendency than that of spherical RFAM particles. The maximum fine particle fraction (FPF(TD)) of 68% for the RFDH was achieved with the Aerolizer device. Significant chemical degradation was not observed from the RFDH, while the RFAM showed significant chemical degradation at 9 months. The excipient-free formulation of the RFDH offers the benefit of delivering a maximum potency formulation, of the antibiotic, directly to the site of infection, the lung.
While we wait for improved new anti-tuberculosis drugs, the main aim for improving current treatment should be to optimize the use of the two current drugs, rifampicin and the pro-drug pyrazinamide, which are responsible to a similar extent for the entire sterilizing activity of current therapy. The rifamycin activity could be improved by increasing the dose size of rifampicin or by daily dosing with long acting rifapentine. Increasing the dose size of pyrazinamide is limited by toxicity but an alternative approach is to use inhalation with pyrazinoic acid, as an adjunct to standard oral therapy. This would acidify pulmonary lesions, thus increasing the bactericidal activity of the orally administered pyrazinamide. Because pyrazinoic acid is the active moiety, it should also increase overall pyrazinamide activity and, because most resistance arises in the pncA gene that converts pyrazinamide to pyrazinoic acid, it should act on most pyrazinamide resistant strains. Inhalation technology allows delivery of drug to lesions rapidly and without first pass toxicity. The properties of drug containing microparticles and nanoparticles during inhalation and storage are reviewed. Spray-dried larger Trojan particles in which the smaller encapsulated particles can reside should be able to improve localisation within alveoli and avoid some storage problems.
The controlled co-delivery of multiple agents to the lung offers potential benefits to patients. This study investigated the preparation and characterisation of highly respirable spray-dried powders displaying the sustained release of two chemically distinct therapeutic agents. Spray-dried powders were produced from 30% (v/v) aqueous ethanol formulations that contained hydrophilic (terbutaline sulphate) and hydrophobic (beclometasone dipropionate) model drugs, chitosan (as a drug release modifier) and leucine (aerosolisation enhancer). The influence of chitosan molecular weight on spray-drying thermal efficiency, aerosol performance and drug release profile was investigated. Resultant powders were physically characterised: with in vitro aerosolisation performance and drug release profile investigated by the Multi-Stage Liquid Impinger and modified USP II dissolution apparatus, respectively. It was found that increased chitosan molecular weight gave increased spray-drying thermal efficiency. The powders generated were of a suitable size for inhalation-with emitted doses over 90% and fine particle fractions up to 72% of the loaded dose. Sustained drug release profiles were observed in dissolution tests for both agents: increased chitosan molecular weight associated with increased duration of drug release. The controlled co-delivery of hydrophilic and hydrophobic entities underlines the capability of spray drying to produce respirable particles with sustained release for delivery to the lung.
The treatment of tuberculosis (TB) is a mature discipline, with more than 60 years of clinical experience accrued across the globe. The requisite Multi-drug treatment of drug-susceptible TB, however, lasts 6 months and has never been optimized according to current standards. Multi-drug resistant TB and TB in individuals coinfected with HIV present additional treatment challenges.
This article reviews the role that existing drugs and new compounds could have in shortening or improving treatment for TB. The key to treatment shortening seems to be sterilizing activity, or the ability of drugs to kill mycobacteria that persist after the initial days of multi-drug treatment.
Among existing anti-TB drugs, the rifamycins hold the greatest potential for shortening treatment and improving outcomes, in both HIV-infected and HIV-uninfected populations, without dramatic increases in toxicity. Clinical studies underway or being planned, are supported by in vitro , animal and human evidence of increased sterilizing activity--without significant increases in toxicity--at elevated daily doses. Fluoroquinolones also seem to have significant sterilizing activity. At present, at least two class members are being evaluated for treatment shortening with different combinations of first-line drugs. However, in light of apparent rapid selection for fluoroquinolone-resistant mutants, relative frequency of serious adverse events and a perceived need to 'reserve' fluoroquinolones for the treatment of drug-resistant TB, their exact role in TB treatment remains to be determined. Other possible improvements may come from inhaled delivery or split dosing (linezolid) of anti-TB drugs for which toxicity (ethionamide) or lack of absorption (aminoglycosides and polypeptides) precludes delivery of maximally effective, oral doses, once daily. New classes of drugs with novel mechanisms of action, nitroimidazopyrans and a diarylquinoline, among others, may soon provide opportunities for improving treatment of drug-resistant TB or shortening treatment of drug-susceptible TB.
More potential options for improved TB treatment currently exist than at any other time in the last 30 years. The challenge in TB pharmacotherapy is to devise well-tolerated, efficacious, short-duration regimens that can be used successfully against drug-resistant and drug-resistant TB in a heterogeneous population of patients.
In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.
Targeted delivery of rifampicin loaded microspheres to the alveolar macrophage, the host cell for Mycobacterium tuberculosis (MTB), may be an effective targeted approach to pulmonary tuberculosis therapy. A guinea pig infection model has been adopted as a post-treatment screening method for antimicrobial effect. Insufflation and nebulization methods of drug delivery were evaluated.
Rifampicin alone (RIF, 1.03-1.72 mg/kg), within poly(lactide-co-glycolide) microspheres (R-PLGA, equivalent to 1.03-1.72 mg/kg) or polymer microparticles alone (PLGA) were administered by insufflation or nebulization, 24 h before bacterial aerosol exposure. Animals were infected with an aerosol containing a small number (2 x 10(5) cfu/mL) of virulent H37Rv strain of MTB. Lung and spleen tissue samples were collected 28 days after infection for quantitative bacteriology and histopathological analysis.
There was a dose-effect relationship between insufflated R-PLGA and burden of bacteria in the lungs. In addition, guinea pigs treated with R-PLGA had a significantly smaller number of viable bacteria (P < 0.05), reduced inflammation and lung damage than lactose or saline control, PLGA or RIF treated animals.
These studies indicate the potential of R-PLGA, delivered by insufflation or nebulization directly to the lungs, to affect the early development of pulmonary TB.
Dry powder inhalers for eformoterol fumarate dihydrate, a long-acting beta-2 agonist for bronchodilation, are currently available as the Foradile Aerolizer and the Oxis Turbuhaler. The two products are different in the formulation, the aerosol production mechanism, and the device resistance to air flow. These disparities are likely to lead to different aerosol characteristics. Our objective was to compare the in vitro performance of these two inhalers in producing eformoterol aerosols. Emitted dose uniformity was measured using a sampling apparatus described in the British Pharmacopaeia. Ten individual doses (dose number 2, 3, 15, 16, 30, 31, 45, 46, 59, and 60) of the entire content (60 doses) were collected from the Aerolizer and the Turbuhaler (six inhalers each). Particle size distribution of the aerosols generated by the two inhalers were measured by a multiple stage liquid impinger at four different air flows (30-120 L/min). Eformoterol collected from the sampling devices was measured by HPLC. Fine particles are those of < or = 1.7-5.0 microm in size in the aerosols obtained by interpolation of the data at the specified air flow. The Aerolizer showed a slight dependence of the emitted dose on the air flow, with the average emitted dose increased from 80% (at 30 L/min) to 90% (at higher flows) of the 12-microg label claim as compared with 60% for the Turbuhaler. When the emitted dose was normalized by the average emitted dose value, the Aerolizer showed less variation in the normalized emitted dose uniformity than the Turbuhaler. At high air flows, 90 and 120 L/min, both inhalers produced similar amounts (4 microg) of fine particles in the aerosol per dose discharged. As the flow as decreased to 30 and 60 L/min, both inhalers produced significantly less fine particles (p < 0.05), with the Oxis Turbuhaler producing lesser amounts than the Foradile Aerolizer. However, due to the different device resistance, comparing the inhaler performance at the same inspiratory effort may be more appropriate. At a comfortable effort of 40 cm H2O, the Foradile Aerolizer would produce a significantly higher fine particle mass in the aerosols. We conclude that the two inhalers were dissimilar in the emitted dose uniformity. The fine particle mass of eformoterol produced by the two inhalers was equivalent at high but not at low air flows. The disparities may be due to the difference in the formulation and the aerosol generation mechanism of the inhalers.