Antitubercular inhaled therapy: opportunities, progress and challenges

Rajesh Pandey and G. K. Khuller*

Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh—160 012, India


* Corresponding author. Tel: +91-172-2747585, ext. 5174-75; Fax: +91-172-2744401/2745078; Email: gkkhuller{at}yahoo.co.in


    Abstract
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Pulmonary tuberculosis remains the commonest form of this disease and the development of methods for delivering antitubercular drugs directly to the lungs via the respiratory route is a rational therapeutic goal. The obvious advantages of inhaled therapy include direct drug delivery to the diseased organ, targeting to alveolar macrophages harbouring the mycobacteria, reduced risk of systemic toxicity and improved patient compliance. Research efforts have demonstrated the feasibility of various drug delivery systems employing liposomes, polymeric microparticles and nanoparticles to serve as inhalable antitubercular drug carriers. In particular, nanoparticles have emerged as a remarkably useful tool for this purpose. While some researchers have preferred dry powder inhalers, others have emphasized nebulization. Beginning with the respiratory delivery of a single antitubercular drug, it is now possible to deliver multiple drugs simultaneously with a greater therapeutic efficacy. More experience and expertise have been observed with synthetic polymers, nevertheless, the possibility of using natural polymers for inhaled therapy has yet to be explored. Several key issues such as patient education, cost of treatment, stability and large scale production of drug formulations, etc. need to be addressed before antitubercular inhaled therapy finds its way from theory to clinical reality.

Keywords: tuberculosis , liposomes, polymers , nebulization , drug delivery


    Introduction
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A Greek pharmacist, Pedanus Discorides, introduced the concept of inhaled fumigation during the first century. Antiseptic aerosol therapy, e.g. boiling tar vapours, became a popular antitubercular medication in the middle of the 20th century, although it hardly had any therapeutic value.1 Since then, antitubercular inhaled therapy has come a long way to a stage of experimental reality with potential clinical applications. The importance of the subject stems from the fact that tuberculosis (TB) continues to be a leading killer disease causing 3 million deaths annually2 and has emerged as an occupational disease in the health care system.3 Oral therapy using the currently employed antitubercular drugs (ATDs) is very effective, but is still associated with a number of significant drawbacks. More than 80% of TB cases are of pulmonary TB alone and high drug doses are required to be administered because only a small fraction of the total dose reaches the lungs after oral administration. Even this small fraction is cleared in a matter of a few hours thus explaining the necessity to administer multiple ATDs on a regular basis, a regimen which the majority of TB patients find difficult to adhere to. Clearly, ATD delivery systems which can be administered via the pulmonary route and can avoid the daily dosing, would be a vast improvement because they would help in: (i) direct drug delivery to the diseased organ; (ii) targeting to alveolar macrophages which are used by the mycobacteria as a safe site for their prolonged survival; (iii) reduced systemic toxicity of the drugs; and (iv) improved patient compliance. The present review highlights the progress made in antitubercular inhaled therapy especially with the ATDs formulated into suitable delivery systems.


    Modes of respiratory drug delivery
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A convenient way of delivering drugs to the lungs is the aerosolization of the drugs as fine powders with the aid of dry powder inhalers (DPIs). Alternatively, the drug may be first solubilized/suspended in an aqueous medium and subsequently aerosolized (liquid aerosolization or nebulization) through a nebulizer. A nebulizer requires a dispersing force (either a jet of gas or ultrasonic waves) for aerosolization.4 A drug may also be delivered to the lungs directly, i.e. without prior aerosolization, using a device called an insufflator. Compared with a nebulizer, a DPI is more efficient in terms of drug delivery and less time consuming.5 However, nebulizers can be designed to make the best use of a patient's breathing pattern, the so-called ‘breath-assisted nebulizers’.6 Further, with jet nebulizers, adjustments in drug dosing are easier to achieve.7 Although nebulization is the most common method of aerosol delivery of antibiotics, other factors such as nebulizer technology, breath holding patterns, degree of airway disease, pulmonary function as well as the aerodynamics of the pharmaceutical aerosol, are all known to affect the efficiency of drug delivery.8,9 An important aerodynamic parameter is the mass median aerodynamic diameter (MMAD), the diameter above and below which 50% of the mass of aerosolized particles are contained. The smaller the diameter, the better are the chances that particle deposition would occur in the deeper parts of the lungs, i.e. the alveoli. The optimum range is defined as 0.5–5.0 µm (the respirable range) because particles < 0.5 µm are usually exhaled whereas particles > 5.0 µm are impacted in the oropharynx.10


    Inhaled therapy with conventional or unformulated ATDs
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Many patients continue to remain sputum smear-positive for Mycobacterium tuberculosis despite ongoing chemotherapy, which is mainly attributable to (other than drug resistance) extensive cavitary lesions where the antimycobacterial drugs fail to reach when administered orally.11 Sacks et al.12 selected such patients of pulmonary TB who were sputum smear-positive after at least 2 months of conventional treatment. The patients were treated with gentamicin or kanamycin via nebulization as adjunctive therapy while the conventional drugs were maintained in parallel. The frequency of nebulization was thrice daily whereas the duration was dictated by practical considerations and smear conversion times which ranged from 9 to 122 days. It was observed that 86% (6 out of 7) of the patients with drug-susceptible TB and 58% (7 out of 12) of the patients with drug-resistant TB underwent smear conversion during the study period, suggesting that residual aminoglycosides in sputum expectorated from pulmonary cavities could inhibit intracavitary bacillary growth and prevent transmission, though not necessarily affecting the bacteria inside the macrophages. Nevertheless, the study did document the supportive role of inhaled aminoglycosides in patients with refractory TB.

Aerosol administration of interferon gamma (IFN-{gamma}), a key cytokine in the immunological response against mycobacteria, has also been attempted. The initial studies were inconclusive as the patients receiving adjunctive aerosol IFN-{gamma} became smear-negative after 1 month but continued to be culture-positive and the smear response was not sustained.13 However, when the aerosolized IFN-{gamma} therapy was continued for 6 months (thrice weekly), most of the patients showed a definite radiological improvement and a reduction in the size of the cavitary lesions.14 It appears that merely aerosolizing an antimycobacterial compound may be inadequate; for efficient bacterial killing, drugs need to be formulated into suitable delivery systems thereby ensuring their rapid uptake into macrophages which harbour the tubercle bacilli. The dictum holds true for the majority of intracellular infections, and liposomes as well as micro/nanoparticles have emerged as useful drug carriers (Table 1) in this context.15,16 Hence, it is not surprising that these carriers have established their potential for antitubercular inhaled therapy (Table 2).


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Table 1. Salient features of important drug carriers

 

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Table 2. Salient features of inhalable ATD delivery systems

 

    Pulmonary delivery of liposome-encapsulated ATDs
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Liposome-encapsulated drugs are especially effective against intracellular pathogens and their demonstrated advantages include:17 (i) the ability to formulate biologically active molecules; (ii) the ability to encapsulate hydrophilic compounds; (iii) reduction in toxicity of the active agent; (iv) increased therapeutic index; (v) increased stability of labile drugs; (vi) improved pharmacokinetics; (vii) increased delivery to target tissues; and (viii) the feasibility of nebulization. Liposomes are more popular as intravenous ATD carriers.18 However, keeping in mind that liposomes have been successfully nebulized to treat intracellular pulmonary infections,19 conventional (phosphatidylcholine/cholesterol) liposomes encapsulating rifampicin and isoniazid were prepared in our laboratory for nebulization. The loading of rifampicin was better compared with isoniazid.20 The inhalable distearoylphosphatidylcholine/cholesterol liposomes encapsulating ATDs, as prepared by Justo & Moraes,21 also showed a satisfactory drug loading for isoniazid as well as pyrazinamide. In their case, however, the encapsulation of rifampicin, streptomycin and ethionamide was low. Aerodynamic characterization of our formulation showed 94% of generated aerosol to be respirable, with an MMAD of 0.96 ±0.06 µm.20 A single nebulization of liposomal ATDs to guinea pigs could maintain therapeutic drug concentrations in the plasma for 48 h whereas free/unencapsulated drugs were cleared by 24 h. Liposomal drugs were present in the lungs and more importantly in the alveolar macrophages till day 5 post-nebulization, suggesting that liposome-based controlled ATD release may obviate the need for daily drug dosing. Our findings and predictions are supported by the results of Kurunov et al.,22 who reported an equivalent therapeutic efficacy of twice weekly nebulized liposomal rifampicin and daily conventional rifampicin in a murine TB model. The authors suggested that the liposomal formulation helps in the persistence of rifampicin in the lung tissue.

The specific targeting of liposomes towards the alveolar macrophages can be achieved by coating the liposomes with alveolar macrophage-specific ligands such as O-stearyl amylopectin (O-SAP) and maleylated bovine serum albumin (MBSA). The therapeutic efficacy of O-SAP-coated liposomal ATDs was recently reported, however, the intravenous route was employed for liposomal administration.23 Vyas et al.24 prepared O-SAP- and MBSA-appended inhalable liposomes entrapping rifampicin. In vivo studies in albino rats demonstrated a higher pulmonary delivery and better localization of ligand-appended liposomes to alveolar macrophages compared with conventional liposomes or free rifampicin, from 30 min to 24 h post-nebulization. Subsequently, the alveolar macrophages were isolated, spread as a monolayer and infected with Mycobacterium smegmatis. The percentage viability of the bacilli was significantly reduced to 10.9% in the case of MBSA- and 7.1% in the case of O-SAP-coated liposomes, compared with 69% and 31% for control macrophages and conventional liposome-treated macrophages, respectively. The results were based on a single nebulization of liposomal rifampicin and the authors speculated that an ideal situation of 0% viability may be obtained by repeated dosing. It is therefore clear that nebulization of liposomal ATDs, coupled to the use of alveolar macrophage-specific ligands, may improve the chemotherapy of pulmonary TB especially in view of the fact that liposomes are known to be safe when administered via the respiratory route.25 However, with the use of biodegradable polymers in the arena of drug delivery, more emphasis began to be laid on the use of polymeric systems for antitubercular inhaled therapy.


    Pulmonary delivery of microparticle-encapsulated ATDs
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The use of polymeric microparticles to deliver ATDs by different routes (injectable, oral and aerosol) has been reported by several investigators.26 Because of its biodegradability and biocompatibility, poly (lactide-co-glycolide) (PLG; a synthetic polymer) has been a popular choice as a drug carrier.27 By employing solvent evaporation as well as spray drying methods, PLG microparticles encapsulating rifampicin were prepared.28 The former technique resulted in spherical particles with 20% drug loading and 3.45 µm volume median diameter whereas the latter technique produced shrivelled particles with 30% drug loading and 2.76 µm diameter. The microspheres were administered via insufflation or nebulization to guinea pigs, 24 h before aerosol infection with M. tuberculosis H37Rv. The model was adopted as a post-treatment screening method for antimicrobial efficacy.29 The assessment of colony forming units (cfu) 28 days post-infection showed a dose–effect relationship, i.e. lower cfu with higher doses of microspheres. The cfu count was significantly reduced compared with free rifampicin. With a similar experimental approach, the authors next evaluated the effect of repeated dosing of the microspheres. At 10 days post-infection, half of the treatment group received a second dose of the microspheres. There was a significant reduction in cfu in lungs (but not in spleens) in the case of animals receiving a single dose of the formulation, whereas two doses resulted in a significant decrease in cfu in lungs as well as in spleens.30 It was realized that besides the methodology involved in microparticle preparation, the surface characteristics of dry powders also play a key role in predicting particle dispersion and pulmonary deposition.31

Although the results with rifampicin-loaded microspheres proved to be encouraging, it was necessary to incorporate other ATDs because the disease requires multidrug therapy for its cure. Hence, other investigators encapsulated isoniazid with rifampicin in polylactide microparticles for dry powder inhalation to rats.32 Drug concentrations inside the alveolar macrophages were found to be higher than that resulting from systemic delivery of free drugs, an indication of the rapid phagocytic uptake and cytosolic localization of the drug-loaded microparticles. The authors discussed that since alveolar macrophages migrate to secondary lymphoid organs, loading these cells with microparticles might lead to transport of drugs to those very sites where macrophages migrate (mimicking the course of spread of mycobacteria). That is to say, pulmonary delivery of microparticle-encapsulated ATDs has the potential to reach extrapulmonary sites of infection as well. Unfortunately, chemotherapeutic studies were not carried out by the authors.

The rising incidence of multidrug-resistant TB (MDR-TB) is a matter of great concern because the treatment involves the use of second-line ATDs, which are more costly and toxic compared with the first-line drugs used to treat drug-susceptible TB. Furthermore, the treatment schedule is more prolonged with a greater risk of patient non-compliance.33 Some of the second-line drugs, e.g. para-aminosalicylic acid (PAS), need to be administered in very large amounts (up to 12 g daily), which is inconvenient to the patient. In order to reduce the drug dosage, investigators have formulated an inhalable microparticulate system for PAS, based on dipalmitoylglycero-3-phosphocholine.34 The microparticles were produced by spray drying, possessed a 95% drug loading and were administered to rats via insufflation. The drug was maintained at therapeutic concentrations in the lung tissue for at least 3 h (the authors did not monitor the drug levels further) following a single dose of just 5 mg of the dried formulation. Accelerated stability studies indicated that the formulation was stable for up to 4 weeks and the authors suggested that the technology could be extended to include other drugs such as rifampicin, aminoglycosides as well as fluoroquinolones.

Despite the satisfactory results obtained with microparticles, the quest for better drug delivery systems ushered in the era of nanoparticles. The design and development of polymeric nanoparticles for experimental antitubercular inhaled therapy have been the recent focus of interest in our laboratory.


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Nanoparticles range in size from 10 to 1000 nm whereas microparticles lie in the size range of 1 and 1000 µm.15 The difference between microparticles and nanoparticles lies not merely in the size, but also in the ability of nanoparticles to achieve a high drug loading, minimize the consumption of polymers, cross permeability barriers and elicit a better therapeutic response.26,35 Furthermore, inhalable nanoparticles stand better chances of mucosal adherence, particle(s) delivery and hence net drug delivery to the lungs.36 For the reasons discussed in the case of microparticles, PLG is the most intensively studied nanoparticulate drug carrier.37 We prepared PLG-nanoparticles according to the double emulsion/solvent evaporation technique,38 co-encapsulating rifampicin, isoniazid and pyrazinamide. The particle size ranged from 186 to 290 nm. Upon aerosolization, the MMAD (as determined on a 7-stage Andersen Cascade Impactor) was found to be 1.88 µm and thus suitable for deep lung delivery. It is known that high surface hydrophobicity can result in particle aggregation during nebulization especially on a jet nebulizer. Because the PLG-nanoparticles were stabilized by polyvinyl alcohol thereby imparting hydrophilicity to the formulation, particle aggregation was not a problem.

A single nebulization of the formulation to guinea pigs was able to maintain a therapeutic drug concentration in the plasma for 6–8 days and in the lungs for 9–11 days. There was a striking improvement in the half-life, mean residence time and relative/absolute bioavailability of encapsulated drugs compared with free drugs. It may be asked that if one is aiming at pulmonary deposition of ATDs, how the improvement in systemic bioavailability would be advantageous following inhaled therapy? The argument was that the enhanced bioavailability would lead to more of the drugs reaching the lungs by way of the circulation, i.e. the systemic spillover could not be considered as a drug wastage.39 Repeated administration of the formulation failed to elicit hepatotoxicity as assessed on a biochemical basis. In M. tuberculosis H37Rv infected guinea pigs, five nebulized doses of the formulation spaced 10 days apart, resulted in undetectable cfu in the lungs replacing 46 conventional doses. This was the first report of PLG-nanoparticles as an inhalable ATD carrier.39 The advantage of the system over inhalable microspheres was clear cut; firstly, it was possible to co-administer multiple ATDs encapsulated in nanoparticles and secondly, a better therapeutic response was elicited in the case of nanoparticles.

The formulation was further refined and improved by coupling it to lectin (wheat germ agglutinin, a commonly occurring plant glycoprotein). With the knowledge that lectin receptors are widely distributed in the respiratory tract,40 it was worthwhile to evaluate the chemotherapeutic potential of lectin-functionalized PLG-nanoparticles,41 a somewhat similar approach to ligand-appended liposomes.24 Upon nebulization to guinea pigs, therapeutic drug concentrations were maintained in the plasma/organs for 6–15 days. Most of the pharmacokinetic parameters were upgraded compared with uncoated PLG-nanoparticles. Most importantly, when nebulized to TB-infected guinea pigs every fortnight, three doses of the formulation produced undetectable cfu in the lungs as well as spleens.41 The series of experiments proved that 46 conventional doses could be reduced to five nebulized doses of PLG-nanoparticles and further to just three doses with lectin-PLG-nanoparticles.

A new concept in nanotechnology is that of solid lipid nanoparticles (SLNs), i.e. lipid nanocrystals in water possessing a solid core into which drugs are incorporated. The SLNs combine the virtues of more traditional drug carriers such as liposomes or polymeric nanoparticles while eliminating some of their disadvantages, e.g. the issues of burst release and long-term stability in the case of liposomes as well as the problems of residual solvents and bulk production in the case of polymeric nanoparticles.35,42 Furthermore, although PLG is completely biodegradable and biocompatible, the degradation rate is slow and repeated administration of the formulation carries a likelihood of accumulation of the polymer or its degradation products in the respiratory tract. The polymer is known to elicit a mild inflammatory response lasting 2–3 weeks,43 however, the implications for inhaled therapy and possible influence on lung function have yet to be evaluated.

Although the pulmonary delivery of SLNs is in its infancy,44 our experiments with inhalable ATD-loaded SLNs have produced encouraging results in a guinea pig TB model.45 Seven weekly inhaled doses of the formulation resulted in undetectable bacilli in the lungs of M. tuberculosis infected guinea pigs. Another aspect yet to be explored is that of natural polymer (e.g. alginate, chitosan) based ATD delivery systems. A recent report describing the pulmonary delivery of chitosan-loaded DNA encoding M. tuberculosis T cell epitopes46 might well serve as the starting point in this area. Work is in progress in our laboratory to encapsulate ATDs in chitosan-stabilized alginate nanoparticles for pulmonary delivery.


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Based on the experimental data, it is clear that respiratory drug delivery systems certainly have the potential for antitubercular inhaled therapy (Table 2). The requirements for fewer drug doses as well as a low dosing frequency are definite advantages. However, there are some key issues that still need to be addressed. The possibility of variable deposition of an inhaled formulation in the lungs needs to be considered and is a matter of concern because it could result in suboptimal drug concentrations in certain lung regions. If this does occur to a significant extent then treatment response could be impaired. However, delivery vehicles with good systemic bioavailability could overcome this potential problem. Indeed, whilst inhaled therapy would probably be most beneficial to patients with pulmonary TB, formulations with a good systemic bioavailability of ATDs (e.g. nanoparticles) might also be of benefit for patients with extra-pulmonary TB. Concerns regarding toxicity consequent on systemic absorption may be offset by the fact that these formulations are intended for intermittent therapy, with the net drug dose administered actually being reduced compared with oral therapy.

Patients suffering from endobronchial TB may be particularly suitable for inhaled therapy in future.47 MDR-TB not responding to conventional treatment is another scenario where inhaled therapy may come to have a significant future role. For patients who do not fit into the categories above, the future role of inhaled therapy is less clear. Potentially, a few inhaled doses at the start of treatment for uncomplicated pulmonary TB could help to significantly reduce the pulmonary bacterial burden and hence improve on the efficacy of conventional oral therapy. However, inhaled therapy will need to fit in with existing National TB programmes, and with initiatives such as the Directly Observed Treatment Shortcourse (DOTS) programme. Increased costs, together with the need for strict control of infection precautions to prevent device-associated cross-infections and/or risk to health personnel, may limit the extent to which such technologies come to be widely available, particularly in developing countries. The large-scale production of stable drug formulations at an affordable cost will be the fundamental and decisive obstacle which will need to be overcome before contemplating human trials. However, the rationale behind antitubercular inhaled therapy is persuasive. Hopefully, current and future research efforts will eventually result in this concept moving from the bench to the bedside.


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41 . Sharma, A., Sharma, S. & Khuller, G. K. (2004). Lectin-functionalized poly (lactide-co-glycolide) nanoparticles as oral/aerosolised antitubercular drug carriers for treatment of tuberculosis. Journal of Antimicrobial Chemotherapy 54, 761–6.[Abstract/Free Full Text]

42 . Dingler, A. & Gohla, S. (2002). Production of solid lipid nanoparticles (SLN): scaling up feasibilities. Journal of Microencapsulation 19, 11–6.[CrossRef][ISI][Medline]

43 . Grayson, A. C., Voskerician, G., Lynn, A. et al. (2004). Differential degradation rates in vivo and in vitro of biocompatible poly(lactic acid) and poly(glycolic acid) homo- and co-polymers for a polymeric drug-delivery microchip. Journal of Biomaterials Science, Polymer Edition 15, 1281–304.[CrossRef][ISI][Medline]

44 . Videira, M. A., Botelho, M. F., Santos, A. C. et al. (2002). Lymphatic uptake of pulmonary delivered radiolabelled solid lipid nanoparticles. Journal of Drug Targeting 10, 607–13.[CrossRef][ISI][Medline]

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