Aspects of pulmonary drug delivery strategies for

Review

1.

Introduction

3.

Latest developments in inhaled antibiotic treatment for CF

4.

Novel concepts in inhaled antibiotic treatment of

Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Nandini Loganathan on 02/09/15 For personal use only.

CF-associated respiratory

Aspects of pulmonary drug delivery strategies for infections in cystic fibrosis -- where do we stand? Mareike Klinger-Strobel, Christian Lautenschla¨ger, Dagmar Fischer, Jochen G Mainz, Tony Bruns, Lorena Tuchscherr, Mathias W Pletz & Oliwia Makarewicz† †

Jena University Hospital, Center for Infectious Diseases and Infection Control, Jena, Germany

infections 5.

Future perspectives

6.

Expert opinion

Introduction: Cystic fibrosis (CF) is the most common life-shortening hereditary disease among Caucasians and is associated with severe pulmonary damage because of decreased mucociliary clearance and subsequent chronic bacterial infections. Approximately 90% of CF patients die from lung destruction, promoted by pathogens such as Pseudomonas aeruginosa. Consequently, antibiotic treatment is a cornerstone of CF therapy, preventing chronic infection and reducing bacterial load, exacerbation rates and loss of pulmonary function. Many drugs are administered by inhalation to achieve high pulmonary concentration and to lower systemic side effects. However, pulmonary deposition of inhaled drugs is substantially limited by bronchial obstruction with viscous mucus and restrained by intrapulmonary bacterial biofilms. Areas covered: This review describes challenges in the therapy of CF-associated infections by inhaled antibiotics and summarizes the current state of microtechnology and nanotechnology-based pulmonary antibiotic delivery strategies. Recent and ongoing clinical trials as well as experimental approaches for microparticle/nanoparticle-based antibiotics are presented and their advantages and disadvantages are discussed. Expert opinion: Rapidly increasing antimicrobial resistance accompanied by the lack of novel antibiotics force targeted and more efficient use of the available drugs. Encapsulation of antimicrobials in nanoparticles or microparticles of organic polymers may have great potential for use in CF therapy. Keywords: amikacin, aztreonam lysine, biofilms, Burkholderia cepacia complex, colistin, nanoparticles, poly(lactic-co-glycolic acid), Pseudomonas aeruginosa, Staphylococcus aureus, tobramycin Expert Opin. Drug Deliv. [Early Online]

1.

Introduction

Cystic fibrosis-related airway pathology Cystic fibrosis (CF) is the most frequent life-shortening genetic disease within the Caucasian population [1,2]. It encodes an ion channel in the apical membrane of exocrine glands in secretory cells of paranasal sinuses, lungs, pancreas, gut, liver and the reproductive system. Impaired transport of chloride ions and increased absorption by epithelial sodium channels result in abnormally viscous mucosal secretions (detailed review on CF can be found elsewhere [1]). In the respiratory tract, altered ion composition and increased viscosity of the secretions impair mucociliary clearance and promote bacterial colonization of the airways. The most abundant bacterial species in the lungs of patients causing premature death with CF are Staphylococcus aureus and Haemophilus influenzae, both 1.1

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M. Klinger et al.

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Cystic fibrosis (CF) is the most frequent life-shortening inherent disease within the Caucasian population and the pulmonary therapy for CF is multimodal and exhaustive for the patients, including an aggressive and long-term antibiotic treatment. Chronic infections by Pseudomonas aeruginosa are characteristic of CF and the proportion of multidrugresistant pathogens increases during the course of the disease. Inhalation of anti-pseudomonal antibiotics (tobramycin, colistin and aztreonam lysine) combined with further oral or intravenous antibiotics are a cornerstone to prevent or control P. aeruginosa infections, but formation of recalcitrant and highly resistant biofilms in CF mucus challenges prolonged therapy success. Nanoparticle- or microparticle-based pulmonary delivery systems for antibiotics might control many CF-related complications of inhaled therapy, such as the adherent viscous mucus obstructing the airways and protecting the bacterial biofilms. For controlled pulmonary release of drugs in CF treatment, lipid-based systems, such as liposomes and solid lipid microparticles, are most intensively investigated, with liposomal amikacin being the most advanced application that is supposed to be shortly approved for anti-pseudomonal therapy. Advances in inhaled particle-based formulations of other antibiotics, like anti-staphylococcal vancomycin, are currently at basic stages of research; mainly in in vitro models but some already in animal models. Biodegradable organic polymers, such as PLGA and PEG, as carriers offer some benefits for inhaled application since they can be easily modified to improve the drug encapsulation efficacy, aerosolization and mucus and biofilm penetration or to optimize the targeting and drug release.

This box summarizes key points contained in the article.

mainly found early during the course of disease, progressively followed by Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Achromobacter xylosoxidans [2]. Less frequent but even more problematic are infections by other opportunistic species that often exhibit multidrug-resistant phenotypes. Such infections progressively increase with patients’ age; an example is the Burkholderia cepacia complex (Bcc), which is associated with high morbidity and mortality [3]. Concomitantly, CF transmembrane conductance regulator (CFTR)-deficient epithelial cells release proinflammatory cytokines, including IL-1b, IL-6, IL-8 and TNF-a, whereas levels of anti-inflammatory cytokines, such as IL-10, are decreased (Figure 1) [1]. The disease is characterized by a vicious circle of decreased mucociliary clearance that leads to chronic bacterial infection and increased inflammation, which in turn results in bronchopulmonary destruction (i.e., emphysema and bronchiectasis) and a further decrease in mucociliary clearance (Figure 2). The decline in patient health can be 2

accelerated by exacerbations that may occur up to several times per year and are associated with a high mortality. As a consequence, persistent infection and enhanced but ineffective inflammatory host responses progressively destroy the lung parenchyma leading to pulmonary insufficiency, which is the main cause of premature death in 90% of the patients [2]. Current standard of care for CF-related pulmonary infections

1.2

Whereas no cure exists for this disease, new treatments for CF target the secondary effects of CFTR protein dysfunction. Thereby, for CF patients with the rare mutation G551D [4,5], CFTR modulators such as ivacaftor have recently been approved, and for the most frequent mutation F508del, worldwide trials assessing combined modulators are presently on the way [6]. Meanwhile, the multimodal pulmonary therapy for CF includes improvement of mucociliary clearance (e.g., by inhalation of DNAse or hyperosmolar sodium chloride combined with physiotherapy), suppression of chronic inflammation by anti-inflammatory drugs and symptomatic treatment with bronchodilators. Thereby, systemic (i.e., orally and/or intravenously administered) and inhaled antibiotic treatment of chronic bacterial infections is a cornerstone of CF therapy (Figure 3), relevantly contributing to the substantially increased life expectancy of patients. Because S. aureus is the dominating bacterial pathogen in the early phase of CF, some outpatient healthcare centers administer anti-staphylococcal antimicrobials, such as flucloxacillin (up to 100 mg/kg/day), orally or intravenously to CF patients after diagnosis [7]. Some centers combine this therapy with a second anti-staphylococcal antibiotic (sodium fusidate or rifampicin). However, this treatment has been suggested to favor earlier airway colonization by P. aeruginosa [1]. Pseudomonas aeruginosa infects about 70 -- 80% of CF adults and is responsible for most of premature death of patients. Antibiotic treatment of CF airway colonization and infection with P. aeruginosa can be subdivided into four principles: i) antibiotic prophylaxis for P. aeruginosa colonization; ii) initial antibiotic eradication therapy after first time detection of P. aeruginosa; iii) treatment of infective exacerbations; and iv) antibiotic therapy for treatment of chronic P. aeruginosa infections (chronic suppressive therapy) [8,9]. Prevention or postponement of chronic pulmonary colonization by P. aeruginosa, currently, is among the principal aims of early CF treatment [10]. The agrressive treatments combine oral, intravenous and inhaled antibiotics. According to a Cochrane Review [11], a pooled estimation of the level of benefit from introduction of inhaled antibiotics as a standard for CF patients chronically colonized with P. aeruginosa is not possible from the present literature. However, introduction of this treatment clearly was shown to decrease the rate of exacerbations and the decline of lung function, as well as to reduce sputum bacterial load compared to placebo treatment [12]. Whereas patients born in the 1960s usually died

Expert Opin. Drug Deliv. (2015) 12(5)

1–5 μm

Aspects of pulmonary drug delivery strategies for infections in cystic fibrosis -- where do we stand?

2. Targeted drug release LPS ROS

Ciliated cells

Proteases

Pseudomonas aeruginosa

3. Destruction of bacteria and biofilms

10 μm 60 μm


Mucus height in healthy

Inhaled antibiotic particles

1. Mucus penetration

Bacteria-mediated response

1.5 μm

Mucus height in cystic fibrosis

A multitude of normal mucus

Airway

Degradation and clearance of bacterial remains by activated macrophages and mucociliary clearance

Chronic infection and inflammation IL-6 IL-8 IL-1β GM-CSF TNF-α

Abatement and improvement of clinical symptoms of CF

Figure 1. Illustration of the chronic infection and inflammation of human bronchi and the challenges of the respiratory therapy of cystic fibrosis-associated biofilms by inhaled aerosol antibiotic particles: 1) mucus adhesion/penetration; 2) targeted drug release (time-, pH- or bacteria-dependent); and 3) efficient destruction of bacteria and biofilms by mucussoluble antibiotic agents. LPS: Lipopolysaccharides; ROS: Reactive oxygene species.

Inhaled DNAse, Inhalaed hyperosmolar NaCl Physiotherapy Decreased Bronchodilators mucociliary clearance

Destruction of bornchioli (i.e., bronchiectasis)

Long-term inhaled antibiotics, repeated courses of i.v. antibiotics Chronic bacterial infection

Inflammation Anti-inflammatory drugs (e.g., long-term macrolides)

Figure 2. The vicious circle of chronic infections in cystic fibrosis is shown. i.v.: Intravenous.

before reaching school age, median life expectancy of German CF patients meanwhile rose to 42 years [13]. This was achieved by rigorous reduction of the bacterial load as well as of the immunological stimuli by intermittent or continuous inhalation of antibiotics, including colistin, tobramycin or aztreonam lysine, combined with frequent parenteral antibiotic courses [2,10]. A therapy applied in a series of European CF centers [14], for example, consists of 14-day courses of systemically applied and highly dosed antibiotic combinations with anti-pseudomonal activity every 3 months and continuous treatment with azithromycin as an anti-inflammatory agent [10]. Other countries predominantly administer systemic antibiotics for initial eradication and exacerbation only.

While prolonging life of CF patients, these therapeutic regimens favor the development of bacterial resistance [15], leading to difficulties in treatment and in prevention of crosscolonization [2]. Multiresistant bacteria such as P. aeruginosa, Burkholderia cenocepacia or A. xylosoxidans are increasingly found in CF patients. In addition, the regional burden of community-acquired methicillin-resistant S. aureus (MRSA) that is frequent in the US but rare in Europe may also contribute to this difference. The rates of MRSA strongly vary between the countries and correlate with the general prevalence of MRSA of each country: with highest prevalence in Argentina (25.9%) [16] and US (26.5%) [2] and the lowest prevalence in Netherlands (reported for 2007 as 0%) [17].


3

Nebulizer

Colistin • ColiFin® • Colistin® CF • Promixin® Tobramycin • Bramitob® • Gernebcin® • Tobi® Aztreonam • Cayston®

Particular distribution


• Autosomal recessive genetic disorder of CFTR gene • Clogging of airways due to mucus accumulation, decreased mucociliary clearance and inflammation • Chronic lung infections casued by S. aureus, P. aeruginosa, H. influenza • Macrobial biofilms are resistant to immune response and antibiotic treatment

Colistin • Colobreathe® Tobramycin • Tobi® Podhaler

Cystic fibrosis

Powder inhaler

M. Klinger et al.

y lung Health

Disea

sed lu

ng

Figure 3. Overview of CF characteristics and the administered inhaled antibiotics. CF: Cystic fibrosis.

Challenges in the therapy of CF-associated infections by inhaled antibiotics

2

A therapeutic benefit of antibiotic treatment by inhalation is the possibility of achieving highest local antibiotic concentrations while limiting systemic side effects, which are achieved by direct drug delivery to the site of disease, combined with reduced dosage. Currently, inhaled antibiotics are a cornerstone of guideline-based treatment of CF and have tremendously improved the quality of life of affected patients [10]. However, efficient and targeted drug delivery by inhalation remains challenging in these patients and important obstacles to inhaled antibiotics have to be considered. Efficiency of inhaled therapies The efficiency of inhalation therapy depends on primary aerosol particle size suitable for pulmonary deposition, inhalation device (i.e., nebulizers, metered-dose inhalers [MDIs] or dry powder inhalers [DPIs]) and, particularly for DPIs, the capacity of the patient to generate an adequate inspiratory air flow and the inhalation technique of the patient. The estimated efficiencies to deposit the drug dose in the lung are ~ 10 -- 20% [18]. Nebulizers (jet or ultrasonic) and MDIs generate inhalable aerosols of small liquid droplets (primary diameter 1 -- 5 µm) of the drug solubilized in solvents, propellants and/or excipients. They do not require high inspiratory air flows (usually 6 -- 8 l/min for nebulizers and 18 -- 30 l/min for MDIs) and thus are also suitable for infants [18]. DPIs do not need any solvents or propellants to release individual doses of the powder drug capsules. The loose powder particle size is generally too large due to agglomeration and/or large-size excipients 2.1

4

(e.g., lactose) but become broken up to smaller inhalable primary particles of median diameters between 1 and 2 µm by the turbulent airflow inside the powder container [18]. PDIs show increased deposition efficiency (up to 70% [19]) that, however, strongly depends on some factors, like the humidity and sufficient inspiratory flow (30 -- 60 l/min) [20]. Interestingly, there even are considerable divergences among different DPIs. Whereas the Turbohaler (AstraZeneca) (for dry powder inhalation of bronchodilators and topical steroids) requires higher inspiratory flow rates, dry powder inhalation of antibiotics using the TOBI Podhaler (Novartis Pharmaceuticals) (tobramycin) or Colobreathe (Forest Laboratories, Inc.) (colistimethate) requires a lower continuous inspiratory flow. Nevertheless some patients with severely limited lung function may not be able to empty the capsules to achieve lung deposition [21]. Ventilation disorders causing decreased airflow Obstructed bronchi, hyperinflation and emphysema result in obstructive and restrictive ventilation disorders causing severely limited air flow, incapable of generating sufficient inspiratory flow for DPIs. The inspiratory air flow can be measured by spirometry and depends on sex, age and body size. Important clinical parameters for the choice of an adequate inhaler are the inspiratory flow and the ability to effectively perform inhalational therapy. Nebulizers and pressurized MDIs with and without a valve holding chamber or spacer are suitable for CF patients with an inspiratory flow of < 30 l/min; whereas, DPIs and MDIs with a breath triggering are available for CF patients with an inspiratory flow of > 30 l/min. 2.2



A.

Pulmonal route of dispersed particles

Separation of dispersed particles

Particles diameter

Trachea Particular influx Primary bronchi Secondary bronchi


Tertiary bronchi Bronchioles

Alveoli

B.

Impaction (inertia)

> 3 μm

Sedimentation (gravity)

> 0.5 − 3 μm

Brownian motion (diffusion)

< 0.5 μm

Distribution of inhaled aerosol particles

Nasal 6 − 9 μm Pharynx 4 − 6 μm

lung lthy Hea Uniform distribution with normal FEV1

Disea

sed lu

ng

Non-uniform central distribution with redused FEV1

Figure 4. Pulmonary deposition of inhaled particles and their way of separation in healthy lung-dependent on the particle size (A) is shown. Distribution forms of inhaled particles (3 µm) in healthy and diseased lungs (B) are shown. FEV1: Forced expiratory pressure in 1 s.

Parameters such as particle size, density and geometry play an important role in particle deposition [22]. Independent of the drug delivery system, the choice of an adequate aerosol particle size is elementary for the targeted pulmonary drug release. In healthy humans, inhaled particles with a diameter > 5 µm deposit in the nose and throat, whereas particles sized between 1 and 5 µm deposit in the primary and secondary bronchi via impaction (inertia). Particles of a size < 1 µm reach tertiary bronchi and bronchioles via sedimentation (gravity) and particles < 0.5 µm diffuse and deposit via Brownian motion into the alveoli. Even smaller aerosol particles do not deposit during inhalation but are exhaled [23]. Consequently, therapeutic dry powder aerosols have been made with particle mass densities of ~ 1 ± 0.5 g/cm3 and mean geometric diameters of < 5 µm. As paradox, powder drug carriers have to be small

enough to distribute in the deep airways, but large enough to prevent particle aggregation and increased phagocytosis by macrophages (Figure 4). Particles with a low density of ~ 0.4 g/cm3 and diameters > 5 µm have been shown to be more efficiently aerosolized than smaller nonporous particles and avoid phagocytic clearance [24]. This is reflected by the so-called aerodynamic equivalent diameter that is the diameter of an idealized spherical particle with density 1 g/cm3 that has the same terminal settling velocity under gravity as the particle of interest, taking shape and density of the particle into account. The respiratory deposition of aerosol particles verifiably differs in patients with CF compared to healthy individuals. Bronchial obstruction enhances the central airway deposition with a highly non-uniform particle distribution in the lung.


5

M. Klinger et al.

Additionally, particle deposition within the lung inversely correlates with the forced expiratory volume in 1 s (FEV1), resulting in an increased ratio of central-to-peripheral deposition at a low FEV1 [25]. Abnormally thick and sticky mucus layer Mucus is a natural protective layer of epithelial cells in trachea, bronchus and bronchioles. In healthy individuals, inhaled pathogens as well as small particles, such as dust, pollutants and allergens, are cleared by mucociliary transport from pulmonary periphery to the pharynx at a velocity of 2 -- 5 mm/min [26] within 24 to 48 h. The clearance of particles and pathogens that have reached the alveoli is accomplished solely by alveolar macrophages. The efficiency of mucociliary clearance depends on: i) an adequate volume, depth and composition of the periciliary fluid layer, that is, its hydration; ii) the lubrication of the underlying airway epithelium; and iii) a certain adhesiveness that allows a mucus flow opposed to gravity in the respiratory bronchioles but is low enough to mobilize the mucus by airflow during coughing [27]. In CF, the periciliary fluid layer and respiratory mucus fluid are dehydrated due to dysregulated sodium absorption and chloride secretion. Adherent viscous mucus forms plugs in the trachea, bronchi and bronchioles, thereby obstructing the airways. The resulting slow and defective mucus clearance of inhaled bacteria facilitates local establishment of pathogens and the development of recalcitrant microbial biofilms (Figure 2) [28]. Inhaled antibiotics must be capable of penetrating this sticky mucus to target the enclosed bacteria and to reach peripheral airways by overcoming mucosal plugs, which are the main obstacles. Moreover, mucus plugs at the central bronchi and bronchioles inhibit the deposition of inhaled antibiotics in peripheral regions. Thus, under these conditions, inhaled antibiotics may not reach the regions with highest bacterial loads and therefore show limited efficacy. On the other hand, the residence time of deposited drug carriers in CF is increased by the presence of abnormal mucus and diminished mucociliary clearance [29]. In conclusion, alterations in mucosal transports in CF confer largely unpredictable accumulation, deposition and clearance [30].


2.3

Latest developments in inhaled antibiotic treatment for CF 3.

The development of inhaled formulations for the treatment of bacterial infections primarily focuses on anti-pseudomonal antibiotics. The most recommended inhaled antibiotic classes for treatment of P. aeruginosa infections are aminoglycosides (tobramycin) and colistin. More recently, inhaled fluoroquinolones (ciprofloxacin (CIP) 2007, liposomal CIP 2009; levofloxacin 2008), aztreonam lysine (2004) and amikacin (2006) have been licensed by the European Medicines Agency (EMA) for the use in CF patients (http://www.ema.europa. eu/). Inhaled anti-staphylococcal antibiotic in the most 6

advanced stage of clinical development is vancomycin. All these classes of antibiotics except aztreonam lysine have a concentration-dependent killing effect on bacteria (Cmax or AUC above MIC); thus, high dosage is needed for sufficient eradication. Dosing of parenterally applied aminoglycosides or colistin is, however, limited, for example, by nephrotoxicity. Hence, inhaled applications are favorable as local, targeted alternatives. This section describes the approved standard antibiotic formulations and the most advanced experimental efforts to improve the antimicrobial therapy in CF.

Inhaled liquid antibiotics Two inhaled liquid antibiotic formulations are FDA-approved and are in clinical use for years for treatment of P. aeruginosa infections in CF patients: tobramycin inhalation solution (TIS) (TOBI by Novartis, approved 1997) and aztreonam lysine inhalation solution (AZLI) (Cayston by Gilead Sciences, approved 2010). Both are subjects of further clinical trials in Phase III and Phase IV to optimize the treatment regimens and to increase clinical benefits. For example, a randomized, double-blinded, placebo-controlled multicenter Phase III trial (NCT01641822, started in 2012) investigates whether a continuously alternating therapy regimen of two antibiotics with different modes of action (TIS/AZLI) reduces acute pulmonary exacerbations, maintains lung function, controls respiratory symptoms of CF patients and minimizes the risk of emergence of antibiotic-resistant P. aeruginosa strains. Besides, other pharmaceutical companies also provide approved inhalants of tobramycin (e.g., Bramitob by Chiesi Farmaceutici S.p.A., Inc.); noninferiority in comparison to TIS has been demonstrated (NCT00885365) [31]. Two Phase III trials on the inhaled fluoroquinolone levofloxacin solution (Aeroquin by Aptalis Pharma/ Mpex Pharmaceuticals) were completed in 2012 (NCT01180634) and in 2013 (NCT01270347). Despite the fact that the primary end point, that is, time to exacerbation, was not achieved under levofloxacin treatment, the bacterial load of P. aeruginosa in sputum could be decreased and lung functions (measured as changes in FEV1) were improved (NCT01180634). The noninferiority regarding clinical outcome comparing to TIS could be also demonstrated (NCT01270347). These data were released by Aptalis Pharma, Inc. [32]. Considering these results, levofloxacin might be an alternative for treatment of aminoglycosideresistant P. aeruginosa triggered by the TIS administration. Recently, a combination of fosfomycin and tobramycin for inhalation (FTI) was assessed in CF patients with P. aeruginosa colonization in two-dose combination design (28 days of FIT followed by 28 days of AZLI) [33]. A statistically significant increase in FEV1 and decrease in mean P. aeruginosa sputum density was shown for this FTI formulation that contained reduced tobramycin concentrations compared to TIS (NCT00794586). 3.1




The only anti-staphylococcal antibiotic assessed in clinical trials for treatment of CF-associated infections is vancomycin. Two clinical trials in Phase I (NCT01509339) and Phase II (NCT01594827) currently test inhaled vancomycin solutions regarding safety, antimicrobial efficacy and pharmacokinetics. However, results have not been published yet. The rising emergence of multidrug-resistant Bcc that is intrinsically resistant to most CF-related antibiotics, such as aminoglycosides, b-lactames and colistin currently leads to increased interest on altered treatment options. Therefore of little surprise is that in a clinical trial, neither TIS nor AZLI improved the lung function (FEV1) or reduced the bacterial load in sputum of CF patients with diagnosed Bcc infections (NCT01059565). An interesting approach, a combination therapy of inhaled amiloride and TIS to eradicate the difficult-to-treat Bcc, was described 2005 [34] and was followed by a Phase I clinical trial (NCT00547053). As amiloride is not an antibiotic but blocks ion channels and ion antiporters, it was believed to synergistically support the aminoglycoside antibiotic. In the pilot study, sputum cultures were negative for Bcc in three of four patients even after 2 years of follow up. However, no further data have been published; thus, the therapeutic potential of this combination therapy remains unclear. Inhaled dry-powder antibiotics A dry powder formulation of solid porous microparticles (based on the PulmoSphere technology) made of tobramycin and the lipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) has recently been approved by the FDA in 2013. This therapy has already been included in the ‘Best Practice Guidelines’ for Cystic Fibrosis [10] as an alternative treatment of P. aeruginosa infections. A placebo-controlled trial of the tobramycin inhalation powder (TIP) demonstrated increased FEV1 and decreased P. aeruginosa loads in sputum at day 28 (NCT00918957). A trial with TIS as a comparator showed similar results for FEV1, bacterial loads as well as for adverse events. However, a significantly reduced administration time (5.6 vs 19.7 min, p < 0.0001) was described for TIP because of higher dosage per inhalation [35]. Several Phase IV trials on TIP are currently conducted to optimize the dosing (NCT02015663) and handling (NCT02178540) to analyze the long-term safety (NCT01775137, NCT01519661) and to simplify the use and to identify prevalence of inhaler contaminations (NCT01844778). A lipid-based (DSPC) dry powder formulation of CIP (PulmoSphere technology) was recently tested in a clinical Phase II trial in CF patients and non-CF patients (NCT00930982) with contrary results concerning the reduction of colonization by P. aeruginosa (Table 1). Whereas reduction of ~ 3 log units in bacterial load was achieved in patients with non-CF bronchiectasis (p = 0.001), no significant changes were observed in CF patients (p = 0.076). This illustrates the extraordinary obstacles to reach effective antibiotic concentrations in the mucus under CF conditions. 3.2

Nevertheless, inhaled CIP powder is a promising treatment of pulmonary infections, which is currently tested in further Phase III trials to evaluate the safety and pharmacokinetics in patients with bacterial pneumonia (NCT01561794) and in non-CF patients (NCT01764841 and NCT02106832). An approval can be expected within the next few years. However, whether this formulation will be approved for CF is unclear and further clinical trials are clearly necessary. Colistin DPI (Colobreathe, Forest Laboratories, Inc.) is an antibiotic that has been approved by the EMA in 2012 for treatment of chronic P. aeruginosa infection in CF patients and has already been included in the ‘Best Practice Guidelines’ for CF [10] as an alternative treatment of such infections. Colistin dry powder contains only colistimethate sodium, the inactive prodrug, which undergoes spontaneous hydrolysis in aqueous environment to form bioactive colistin (a mixture of colistin A and colistin B). Colistimethate has only been FDA-approved for intravenous (i.v.) or intramuscular injection. Aqueous solutions of colistimethate for inhalation are common but controversial due to the instability of the prodrug and the spontaneous hydrolysis that increase the concentration of nephrotoxic colistin A [36]. In clinical trials, colistin, administered as colistimethate sodium dry powder was well tolerable and exhibited similar effects on reduction of FEV1 and P. aeruginosa load when compared to TIS [37,38]. Prophylactic anti-staphylococcal therapy is not supported by the ‘Best Practice Guidelines’ [10]. However, MRSA is increasingly diagnosed in CF patients. The CF Foundation Patients Registry noted 26.5% of the CF patients positive for MRSA; in contrast to 2002 where only 9.2% were noted to be MRSA-positive [2]. Some anti-staphylococcal oral or i.v. antibiotics, such as fusidic acid/rifampin combinations, linezolid or teicoplanin have been described for MRSA decolonization of CF patients (recommended review [39]), but only vancomycin is under investigation for inhaled application. The dry powder formulation of vancomycin (AeroVanco by Savara, Inc.) was tested in a Phase I trial in healthy volunteers and in CF patients; results regarding safety and pharmacokinetics have been posted (NCT01537666). In total, four healthy volunteer groups (6 participants each) were treated with different single doses of vancomycin dry powder (16, 32 and 80 mg) or with 250 mg vancomycin i.v. In addition, two CF patient groups (six participants each) were treated with 32 or 80 mg vancomycin dry powder. In all groups, treatment-emergent adverse events have been observed. Most of them were not interpreted to be typical side effects of vancomycin (e.g., dizziness and headaches). However, in the CF group treated with 80 mg, three of six patients reported ‘respiratory tract congestion’. This was neither observed in healthy volunteers regardless of the concentration used nor in CF patients treated with 32 mg. Therefore, this may indicate a relevant side effect limiting treatment with higher concentrations of vancomycin dry powder.


7

Vancomycin solution

FTI

Levofloxacin solution (Aeroquin)

NCT01404234

NCT01641822

III

III

NCT01180634

NCT00547053

I

III

NCT01509339

NCT01594827

I

II

Time to an exacerbation

Proportion of patients with P. aeruginosa -negative cultures Percentage of subjects who have discontinued the study drug due to safety or tolerability reasons by day 168 Rate of protocol-defined exacerbations from baseline through week 24 Safety and efficacy of 2 dose combination FTI and run-in course of AZLI (reduced FEV1) Percentage of patients MRSA-free by induced sputum respiratory tract culture after 1 month treatment Area under curve (pharmacokinetic analysis) Eradication of Burkholderia dolosa

Relative change in lung function from baseline (FEV1) at day 28 Relative change in lung function from baseline (FEV1) at day 28 Relative change in FEV1% predicted from baseline to week 24

Proportion of patients Pseudomonas aeruginosafree at 29 days Evaluation of bacterial load decrease

Primary outcome

n.s.

P. aeruginosa

Infection

Randomized, doubleblinded, parallel versus placebo

Open label, single group


Randomized, doubleblinded, parallel versus placebo and oral ABx

Randomized, doubleblinded, parallel AZLI versus placebo Randomized, doubleblinded, parallel FLI versus placebo

Single group, open label

Single group, open label

Randomized, doubleblinded, crossover, AZLI versus placebo

10

P. aeruginosa

330

Burkholderia dolosa 25

S. aureus

40

120

P. aeruginosa

Staphylococcus aureus

250

61

105

100

268

120

10

50

Patients*

P. aeruginosa

P. aeruginosa

P. aeruginosa

Bcc

Randomized, doubleP. aeruginosa blinded parallel, FTI/AZLI versus placebo/ AZLI Randomized, openP. aeruginosa labeled, parallel versus TIS

Randomized, doubleblinded, crossover TIS versus placebo Non-randomized, openlabel, single group

Design/comparator

Completed 2011/no results posted or published Completed 2012/no results posted or published

Active, not recruiting

Recruiting, estimated completion March 2015

Active, not recruiting, estimated completion 2015 Completed 2010, significant improvements in FEV1 % (p = 0.002)

Completed 2012/no results posted or published Completed 2010/no results posted or published Completed 2010/ significantly increased FEV1 (p = 0.0001) Completed 2012/no significant improvement of lung functions an in Bcc counts in sputum Completed 2013/no results posted or published Competed 2013/no subject discontinued

Recruiting until September 2015

Status/results

*Number of patients intend for enrolment, if trial is in recruiting phase, or number of eligible patients if study completed. ABx: Antibiotics; AE: Adverse event; AZLI: Aztreonam lysine for inhalation (solution or powder); Bcc: Burkholderia cepacia complex; CF: Cystic fibrosis; CFU: Colony forming units; CIP: Ciprofloxacin; FEV1: Forced expiratory pressure in 1 s; FTI: Fosfomycin/ tobramycin for inhalation; MRSA: Methicillin resistant Staphylococcus aureus; n.s.: Not specified; SDI: Simpson Diversity Index; TIS: Tobramycin inhalation solution; Vh: Vancomycin hydrochloride.

Mpex Pharmaceuticals

NCT01375049

II

NCT00794586

NCT01059565

III

II

NCT00757237

III

NCT01608555

NCT01082367

NCT-number

NCT00794586

Phase

II

IV

TIS (Bramitob)

AZLI

III

TIS (TOBI )

Drug

Case Western Reserve University Children’s Hospi- Amiloride/ TSI tal Boston

Michael Boyle Johns Hopkins University

Gilead Sciences

University of Milan

Novartis Pharmaceuticals

Sponsor

Table 1. Clinical trials investigating inhaled antibiotic therapy in CF patients (last update June 2014) since year 2010 (for more details, see ClinicalTrials.gov).


TIP (TBM100) PulmoSpheres

Novartis Pharmaceuticals

NCT01746095

NCT01400750

NCT00918957

NCT00982930

NCT01069705

II

IV

III

III

III

Randomized, doubleblinded, parallel versus placebo

Randomized, open-label, parallel versus Vh

First extension to bridging Open label, single group study CTBM100C2303 Safety of TIP for the treatment of infections with P. aeruginosa in subjects suffering from CF Second extension to bridging Open label, single group study CTBM100C2303 Safety of TIP for the treatment of infections with P. aeruginosa in patients suffering from CF

Randomized, open label parallel oral CIP + inhaled colistin versus TIS Randomized, double Relative change in lung function from baseline (FEV1) blinded, crossover TIP versus placebo

Change from baseline at day 29 of the dosing period in the number of MRSA CFU in sputum culture P. aeruginosa eradication at end of the treatment

Number of participants with treatment-emergent AEs

Infection

P. aeruginosa

49

57

62

P. aeruginosa

P. aeruginosa

61

80

P. aeruginosa

S. aureus

25

288

P. aeruginosa

S. aureus

19

267

Patients*

P. aeruginosa

Randomized, doubleP. aeruginosa blinded, parallel versus TIS

Design/comparator

Safety and tolerability of Open label, single group inhaled CIP given as single inhalation dose to pediatric CF patients, aged 6 -- 12 years Randomized, doubleRelative change in lung function from baseline (FEV1) blinded, parallel versus placebo

Safety and efficacy (reduced FEV1)

Primary outcome

Completed 2012/no results posted or published

Completed 2011/ significantly increased FEV1 versus placebo (no statistics provided) Completed 2011/no results posted or published

Completed 2010/2011; no results posted

Completed 2011/no significant effects: FEV1 (P 0.076), P. aeruginosa in sputum (P 0.068) Completed 2012/drugrelated AE: 50% in volunteer group, > 60% in CF group, detailed pharmacokinetics Recruiting until September 2014

Completed 2013/no results posted or published Completed 2010/no results posted or published

Status/results


Colistimethate (Colobreathe)

Forest Laboratories, Inc.

NCT01537666

I

NCT00645788

II

Vancomycin powder AeroVanco

NCT00910351

CIP (BAYQ3939) I PulmoSpheres

NCT-number NCT01270347

Phase

III

Drug

Savara, Inc.

Bayer

Sponsor

Table 1. Clinical trials investigating inhaled antibiotic therapy in CF patients (last update June 2014) since year 2010 (for more details, see ClinicalTrials.gov) (continued).


Liposomal Amikacin Arikace

Drug

NCT01316276

NCT01315678

NCT01315691

III

III

NCT02212587

I

III

NCT02113397

Intervention

NCT00777296

NCT01519661

IV

I/II

NCT02015663

IV

NCT00558844

NCT01844778

IV

I/II

NCT01775137

NCT-number

IV

Phase

Incidence of treatmentemergent AEs, relative change in lung function from baseline (FEV1) Relative change in lung function from baseline (FEV1) on day 168 Time to an exacerbation

Safety and tolerability of nebulized Arikace by changes in FEV1, SaO2, chemistry and hematology lab tests, vital signs and clinical exam, assess treatment-emergent AEs

Incidence of treatmentemergent AEs in 2 years Total time for administration of TIP with the Podhaler compared to the total time for administration of TIS or colistimethate with nebulizers Relative change in lung function from baseline (FEV1) Incidence of treatmentemergent AEs Mean effects of continuous alternating therapy compared to cyclic therapy on SDI averaged at month 6 Change in sputum density of Bcc in CFUs Safety and tolerability of dosing of 560 mg/day Arikace versus placebo for 28 days

Primary outcome

P. aeruginosa

301

Randomized, openP. aeruginosa labeled, parallel versus TIS Randomized, doubleblinded, parallel versus placebo

250

P. aeruginosa


300

66

P. aeruginosa


46

20

30

P. aeruginosa

Bcc

n.s.

157

200

67

86

Patients*

Randomized, doubleblinded, parallel Arikace versus placebo

open label, single group

P. aeruginosa

Randomized, open-label, parallel, dosage Randomized, open label, single group Non-randomized, openlabel parallel oral inhaled colistin versus TIP P. aeruginosa

P. aeruginosa

P. aeruginosa

Infection

Randomized, openlabeled, crossover


Design/comparator

Active, not recruiting/no results posted or published Not yet recruiting

Not yet opened for recruitment Completed 2009/results pooled with NCT00777296 for publication (32) (summary see once below) Completed 2010/ significantly increased FEV1 at day 28 (p = 0.032) and 28 days post-treatment (p = 0.033), and reduction of CFU > 1 log of the 580 mg group versus placebo Recruiting

Recruiting, estimated completion 2016

Recruiting until December 2015 Active, not recruiting

Recruiting until November 2014 Recruiting until December 2014

Status/results


Insmed

Sponsor

Table 1. Clinical trials investigating inhaled antibiotic therapy in CF patients (last update June 2014) since year 2010 (for more details, see ClinicalTrials.gov) (continued).




3.3

Liposomal antibiotic formulations

Because of the encapsulation of the drugs in a lipid layer which is related to natural membranes, liposomes exhibit high biocompatibility and further benefits. Detailed description and benefits of lipid-based delivery systems is given in Section 4.1. Currently, two innovative liposomal drug delivery systems, encapsulated amikacin (Arikace, by Insmed) and CIP (Lipoquin and Pulmaquin, by Aradigm Corp.) are under investigation in clinical studies for usage in CF patients (Table 2). Both antibiotics exhibit a broad spectrum activity but are mainly active against P. aeruginosa. A Phase I/IIa trial (NCT01090908) of Lipoquin/ Pulmaquin in CF patients has been withdrawn prior to enrolment of patients for unspecified reasons. However, liposomal CIP has successfully been tested in non-CF patients in a Phase II trial (NCT00889967) with 4 log units reduction of bacterial load compared to placebo (p = 0.002) [40]. Further Phase III studies are presently recruiting non-CF patients (NCT01515007 and NCT02104245) indicating that further clinical trials in CF patients can be expected in near future. In contrast, clinical studies regarding nebulized amikacin (Arikace, Insmed) are in advanced stages: two ongoing and one completed Phase III trials in CF patients show promising features. The liposomal particles of 0.3 µm diameter with uncharged surface can penetrate into mucus and into biofilms [41]. Patients receiving 560 mg Arikace exhibited increased FEV1 values of 8.1 versus 1.1% for placebo (p = 0.033; liposomes in 1.5% saline), decreased P. aeruginosa density in sputum, reduced pulmonary exacerbation and hospitalization rates and a durable improvement of lung function as well as increased quality of life [42,43]. Furthermore, the liposomal encapsulation of amikacin provided a prolonged pulmonary deposition half-life. The overall mean retention was 60.4% of the initial dose deposited at 24 h and 38.3% at 48 h [44].

Novel concepts in inhaled antibiotic treatment of CF-associated respiratory infections

4.

The lack of promising new antibiotic classes under advanced clinical development have prompted the search for alternative strategies to improve the antimicrobial efficacy, prevent bacterial resistance mechanisms and extend the targeting or prolonged action of already existing drugs. A plethora of drug delivery systems has been extensively investigated for the pulmonary application of antibiotics in numerous preclinical studies in vitro in cell cultures and in vivo in animal studies; however, only a limited number made their way to the clinics or the market. Even less was reported with a direct reference to the challenging conditions of CF. As outlined above, the complex biopharmaceutical and pharmacological requirements for an effective inhaled antibiotic therapy of CF-associated infections are the major

hurdles that seriously hamper their therapeutic utility so far. Nevertheless, some interesting delivery concepts to increase the efficacy of antibiotics against P. aeruginosa and S. aureus, the main pathogens found in CF, are experimentally investigated. The major goals for the development of advanced pulmonary delivery systems for CF are: i) drug protection during storage and from the environment (e.g., mucociliary clearance and phagocytosis); ii) nebulization in units suitable for alveolar deposition; iii), controlled release keeping constant drug levels in the lung for prolonged periods of time especially in the presence of the thick and sticky CF mucus; iv) improved mucosal penetration to guide the drug to the site of action; and v) simple treatment regimes to improve the patient compliance [45]. Additionally, for repeated and long-term treatments, biodegradability, biocompatibility and nonimmunogenicity need to be ensured. In this context, certain lipid-based formulations, organic and inorganic polymers as well as magnetic particles have been exploited as carrier systems for pulmonary antibiotic applications. The most promising approaches will be discussed in the following. Overviews of other nanoparticle (NP)-based applications, such as anti-inflammatory treatment and gene therapy, are not addressed here, but have been published elsewhere [22,46]. Lipid-based delivery systems The term ‘lipid-based delivery systems’ subsummarizes liposomes, solid lipid NPs (SLN) and solid lipid microparticles (SLM), the latter especially found in DPIs as described above (see Section 3.2). Liposomes are artificially prepared, monoor multilaminar vesicles of bilayered membrane structures consisting of natural or synthetic amphiphilic lipids, whereas SLN are mostly made of solid lipids, such as triglycerides, fatty acids, steroids or waxes. Some of these components are inherent in the lung. Liposomes are by far the most advanced pulmonary delivery systems, in particular, because of the possibility of encapsulation of poorly water-soluble and hydrophilic drugs, which is a major advantage compared to many polymer-based delivery systems. Hydrophobic drugs can be directly dissolved into the membrane, whereas hydrophilic agents are encapsulated in the aqueous core inside the liposome inhibiting the passage through the membrane. Improvements focus on: i) increasing the stability of liposomes during storage, drying and inhalation; ii) enhancing the antimicrobial efficacy by improving the cell permeation of liposomes especially in P. aeruginosa; and iii) enhancing the antimicrobial efficacy by coadministration of antibiotics with other antimicrobial agents such as metals. Lipid-based drug delivery systems possess several advantages including favorable safety profiles and easy-to-modify surfaces [47,48]. Liposomes consisting of cholesterol-stabilized lipid formulations prevailed for studies related to inhaled antibiotic applications. One innovative delivery system for administration of amikacin (Arikace, see below) already 4.1


11

12


Tobramycin

Tobramycin

Tobramycin

Tobramycin

Gentamicin

Gentamicin

Tobramycin Gentamicin Amikacin Amikacin

Amikacin

1991

1994

1995

1996

2006

2005

2006/2007

2008

DPPC/CH

DPPC/CH

DMPC/CH DPPC/CH DSPC/CH

DMPC/CH

DPPC/DMPG DSPC/ DMPC

PC/PA/CH

DPPC/DMPG

CH/Ph90H

DSPC/DMPG

Formulation

In vivo (rat) intratracheal instillation

In vitro

In vitro

In vitro

0.4 -- 1 µm

~ 430 nm 408 -- 418 nm

~ 217 nm ~ 196 nm ~ 201 nm 375 nm

~ 300 nm

In vivo (rat) intravenous and endotracheal

0.7 -- 1 µm

In vitro In vivo (rat) Inhaled

In vitro

In vivo (rat) intratracheal instillation

In vitro

~ 1.2 -- 1.4 µm n.s.

In vitro

Study design

n.s.

Particle diameter Liposome-enclosed ticarcillin and tobramycin showed increased efficacy against Pseudomonas aeruginosa Encapsulation efficiency strongly depends on trapping technique High encapsulation efficacy and suitable flow ability, no results on drug efficacy on bacteria or release in CF-fluids Liposome-encapsulated tobramycin resulted in high and sustained levels of tobramycin in rats lungs but animals showed no reduction of infection compared to free tobramycin Endotracheal delivery of antibiotic increased retention and improved distribution in the lung due to reduced systemic absorption Cured infection by mucoid P. aeruginosa Bactericidal efficacy/antibiotic release depends on the liposomal composition (best results for DPPC:DMPC ratios 10:1 and 15:1) that are accompanied by increased fluidity Liposome-encapsulated gentamicin decreased the MIC against P. aeruginosa and indicated a prolonged antimicrobial activity at higher dosage Significantly reduced MIC even for highly gentamicinresultant mucoid and non-mucoid P. aeruginosa isolates No significant changes of antibiotic efficacy due to liposome composition Strongly reduced MIC and increased cytoplasmic concentration in P. aeruginosa and Burkholderia cepacia when compared to free antibiotic Liposomal amikacin formulation (Arikace) that is being developed as an inhaled treatment for Gram negative infections was aerosolized with an eFlow nebulizer, reclaimed from the various stages of an Andersen Cascade Impactor (ACI) Liposomes penetrated P. aeruginosa biofilms and mucus and amikacin release was increased in these media. Compared to free antibiotic, inhaled liposomal amikacin was released slower and sustained in healthy rat lung but was evidently more efficacious in infected lungs

Results

CF: Cystic fibrosis; CH: Cholesterol; CMS: Colistin methanesulfonate; DCP: Dicetyl phosphate; DMPC: 1,2-Dimyristoyl-sn-glycero-3-phosphocholine; DMPG: 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol; DOPC: 1,2-Dioleoyl-sn-glycero-3-phosphocholine; DPPC: 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; DSPC: 1,2-Distearoyl-sn-glycero-3-phosphocholine; DSPE: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine; EPC: Egg phosphatidylcholine; HSPC: Hydrogenated soybean phosphatidylcholine; n.s.: Not specified; PA: Phosphatidic acid; PC: Phosphatidylcholine; Ph90H: Phospholipon 90H; SA: Stearylamine.

2008

Ticarcillin Tobramycin

Antibiotic

1991

Year

Table 2. Lipid-based antibiotic delivery systems under experimental investigations.


[41]

[116]

[53,56]

[55]

[115]

[52]

[114]

[113]

[112]

[54]

Ref.

M. Klinger et al.


Moxifloxacin chitosan-moxifloxacin

Polymyxin B

Polymyxin B

Colistin CMS

2008

1999

1993

2012

EPC (neutral) EPC/DCP (negative) EPC/SA (positive) EPC (neutral) EPC/DCP (negative) EPC/SA (positive) DOPC/CH

DPPC

DSPE/PEG

DSPE/PEG

HSPC/CH/ DCP

DMPG/lactose

CH

DPPC/CH

Formulation

In vivo (rat) Inhaled In vitro

In vitro

In vitro

In vitro

2.5 -- 6 µm 5 -- 6 µm 1 -- 3 µm ~ 180 nm


100 nm

100 nm 400 nm 1 µm 2 µm 1 µm


In vitro (rat) Inhaled In vitro

~ 164 nm < 600 nm

In vivo (male)

Study design

1.6 µm

Particle diameter

Negatively charged liposomes incorporated higher contents of the polycationic antibiotic polymyxin B leading to a reduced Tm for phase transmission Degradation of CMS to form colistin led to charge reversal and colloidal instability in CMS-loaded liposomes as well as colistin-loaded liposomes

Prolonged retention of amikacin-loaded liposomes in the lungs of healthy volunteers Higher concentrations in lungs and lower concentrations in kidney compared to free amikacin High encapsulation efficacy in characteristic pulmonary fluids Best performance of ciprofloxacin-liposomes with 1 µm diameter regarding drug release properties (AUC/ MIC90) efficiency, uptake by epithelia cells, prevention against phagocytosis Delivery efficiency and pharmacokinetics of drug release after pulmonary administration of 100 nm to 1 µm ciprofloxacin-liposomes increased with increased particle diameter Optimized performance by PEGylation of 100 nm ciprofloxacin-liposomes regarding the sustained drug release, prevention against phagocytosis Crosslinking of chitosan and moxifloxacin improved the encapsulation efficacy and performance in relation of sustained drug release Liposomally encapsulated polymyxin B showed similar efficacy to free antibiotic against P. aeruginosa

Results

CF: Cystic fibrosis; CH: Cholesterol; CMS: Colistin methanesulfonate; DCP: Dicetyl phosphate; DMPC: 1,2-Dimyristoyl-sn-glycero-3-phosphocholine; DMPG: 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol; DOPC: 1,2-Dioleoyl-sn-glycero-3-phosphocholine; DPPC: 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; DSPC: 1,2-Distearoyl-sn-glycero-3-phosphocholine; DSPE: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine; EPC: Egg phosphatidylcholine; HSPC: Hydrogenated soybean phosphatidylcholine; n.s.: Not specified; PA: Phosphatidic acid; PC: Phosphatidylcholine; Ph90H: Phospholipon 90H; SA: Stearylamine.

Ciprofloxacin

Ciprofloxacin

2006

2011

Ciprofloxacin

2005

Ciprofloxacin

Amikacin

2013

2008

Amikacin

Antibiotic

2009

Year

Table 2. Lipid-based antibiotic delivery systems under experimental investigations (continued).


[61]

[120]

[119]

[62]

[50]

[51]

[118]

[63]

[117]

[44]

Ref.


13


M. Klinger et al.

proved efficient for treatment of CF-related P. aeruginosa infections in Phase II clinical trials [41,44] (details see above). Liposomal bilayers have been shown to fuse with the plasma membranes of eukaryotic and bacterial cells and thus enable intracellular deposition of the encapsulated therapeutics [49]. Hence, liposomes are of particular interest for treatment of infections by intracellular bacteria, such as Mycobacterium spp. [50,51], but they might also be useful in targeting intracellularly hidden S. aureus variants or in improving the antibiotic deposition in metabolically inactive persister cells in bacterial biofilms. Most of the liposomal formulations have been investigated for anti-pseudomonal antibiotics. However, some of them exhibit a broad range of activities and thus might be suitable for the treatment of other infections. Experimental studies on lipid-based antibiotic delivery systems are summarized in Table 2; some interesting perspectives and outstanding issues are discussed below. In vivo experiments in rats where liposomally encapsulated aminoglycosides, amikacin and tobramycin with diameters of < 1 µm were intratracheal instilled demonstrated prolonged drug release and strongly increased efficacy against mucoidal and non-mucoidal P. aeruginosa strains [52] as well B. cenocepacia [53]. Further in vitro studies indicated that similar effects can be expected for other aminoglycosides [53-56]. However, few of these studies examined the impact of the mucus structure and composition on the penetration and efficacy of the lipid carriers. Liposomes < 500 nm were shown to overcome the mucus barrier and are small enough to diffuse through the mucus pores of infected lungs. To sufficiently deposit and release small liposomes of 300 nm diameter in deeper lung regions, aerosols were successfully used in a rat model [41]. These particles showed sustained release of amikacin and improved antimicrobial efficacy in vivo. In vitro experiments revealed adequate mucosal and biofilm penetration properties: as the surfactant properties of liposomes are similar to mucosal characteristics, drug release from the nanosystems increased in the mucus. Interestingly, this effect was also observed in biofilms [41]. The liposomal bilayer is impermeable for protons. Hence, encapsulation probably prevents the untimely pH-dependent inactivation of aminoglycosides in the acidic environment of the CF mucus [57]. This strongly contributes to the observed increased anti-pseudomonal activity compared to free antibiotics. Taken together, these features make liposomal aminoglycoside delivery systems promising therapeutic options in CF. The efficacy of drug release and intracellular deposition mainly depends on lipid composition and can, thus, be improved by additional liposomal surface modifications. To mention two representative examples: i) human leukocyte antigens, HLA-A, HLA-B and HLA-C, were successfully incorporated into liposomes, which improved binding to different clinical bacterial isolates [58]; and ii) immunoliposomes that contained mAbs against specific bacteria have been shown to specifically bind the targeted species [59]. 14

Encapsulation of antibiotics in and release from liposomal systems need to consider the nature and mode of action of the trapped antibiotic. For example, cationic polymyxins could be more efficiently incorporated into negatively charged liposomal formulations, but this did not increase the efficacy of the encapsulated antibiotic against P. aeruginosa. Amphiphilic polymyxins are known to interact with lipoid compounds and to induce instability and pore formation in bacterial membranes [60]. However, the fatty acid moiety of polymyxin is incorporated into the liposomal bilayer as well, thereby destabilizing the liposome and impeding controlled drug release. Accordingly, the attempt to encapsulate the prodrug colistimethate did not yield an improved delivery system [61]. The available data thus make successful application of inhaled liposome-polymyxin formulations for treatment of pulmonary infection unlikely. Possibly, SLN formulation of colistin or its prodrug would yield more suitable delivery systems, but studies are still missing. Besides aminoglycosides and polymyxins, two fluoroquinolones (CIP and moxifloxacin) were liposomally encapsulated. Similar to aminoglycosides, improved drug release and intracellular deposition of the antibiotic in epithelial cells were observed for liposomal CIP [50,51]. PEGylation of small liposomes (100 nm) improved the performance by prolonging CIP release and reducing the opsonization and uptake by alveolar macrophages [50]. Sustained antibiotic release could also be achieved by crosslinking of moxifloxacin with chitosan, a biodegradable carrier molecule, prior to liposomal encapsulation [62]. Both fluoroquinolones could be encapsulated with exceptionally high efficiencies in pulmonary fluids (up to 86%) [63] or with increased amount of crosslinked chitosan (0.5% w/v) (up to 95%) [62]. However, neither drug delivery nor bactericidal activities in mucosal compartments were determined in these studies. Thus, performance of these liposomes under conditions close to CF remains unclear. All delivery systems described above were primarily investigated with the aim to treat P. aeruginosa infections. Only few studies addressed liposomal formulations for the treatment of staphylococcal infections. An in vitro study on piperacillin indicated enhanced antimicrobial activity against S. aureus and liposomal encapsulation was assumed to prevent untimely degradation of the b-lactam antibiotic [64]. However, details regarding particle characteristics and in vivo studies are missing to draw any conclusions about inhaled pulmonary performance. A recent study demonstrated superior penetration of epithelial cells by chitosan-coated vancomycin liposomes, which leads to increased vancomycin concentrations in the tissue and reduced concentration in the bronchoalveolar lavage (BAL) compared to vancomycin dry powder [65]. But since chitosan has been shown to be highly mucus adhesive, chitosan-coated NPs are blocked by the CF mucus [66] and thus would hardly reach the mucus-embedded bacteria.



Summarizing, beside amikacin, liposomally encapsulated aminoglycosides might be the next anti-pseudomonal antibiotics that will find the way into clinical trials in near future. Other liposomal approaches are currently still in early stages of development. Polymeric NPs Although polymeric NPs favor the advantage of higher physicochemical stability, more modified surface properties and prolonged drug delivery and shelf-life compared to liposomes, only a few polymeric encapsulated antibiotics have recently emerged in the context of pulmonary delivery. Most of these studies were performed in vitro; in vivo applications are still rare (Table 3). For the treatment of CF, it can be distinguished between synthetic polymers such as poly(lactide-co-glycolide) (PLGA), polyacrylates and polyanhydrides and natural polymers such as albumin, gelatin, alginate, collagen and chitosan [67]. In contrast to synthetic non-biodegradable polymers such as polystyrene (PS) or latex, polymers based on organic compounds like PLGA, poly(sebatic acid) (PSA) and poly(lactic acid) (PLA) show excellent biodegradability and biocompatibility, with PLGA being the most intensively investigated polymeric drug delivery systems for different pulmonary applications [67]. The complete disappearance is an intrinsic characteristic of PLGA polymers because of the high water solubility of the monomers. Their rate of degradation can be tailored by molar mass, stereochemistry, crystallinity, the molar ratio of lactic and glycolic acid and the formation of copolymers reaching time frames from days to several months [68]. In addition to the inherent polymer properties, the release profiles and residence time of the biodegradable particles can be adapted by processing and formation of copolymers or addition of additives. Incorporated amphiphilic substances will increase wettability. Formation of highly porous particles will increase the water penetration. NPs consisting of tertiary-amine-modified polyvinyl alcohol (PVA) backbones grafted to PLGA demonstrated a breakdown of the NPs within 4 h [69] for pulmonary applications. Also, diethylaminopropylamine PVA-graftedPLGA formed nanocarrier systems with a rapid rate of degradation [70]. PLGA was considered unsuitable for pulmonary drug delivery, especially in cases where frequent dosing is required [71]. However, for long-term CF therapy, degradation and release profiles over several weeks would be desirable to reduce the number of applications, avoid NP accumulation and improve the patient’s compliance. Nevertheless, safety concerns have to be intensively investigated in clinical trials even when single applications are used. Detailed reviews on preparation, composition and aerodynamic behavior are not in the focus of this review, but can be found in detail elsewhere [61]. Antimicrobial efficacy and mucus penetration of the polymeric systems depend, on the one hand, on NP characteristics such as size, density, morphology, surface charge and mucus adhesion and, on the other hand, on mucus characteristics


4.2

such viscoelasticity. The smaller the NPs, the higher were the diffusional rates in the mucus, with 120 nm being more effective than 270 or 500 nm, owing to the mucus pore size in the range of 100 -- 400 nm [72,73]. Although exhibiting excellent aerodynamics [24], highly porous PLGA and PLA-lysine particles with diameters of > 5 µm and low density (< 0.4 g/cm3) were comparably inefficient in mucus penetration [74]. Electrostatic interactions between NPs and, for example, anionic mucus components such as glycoproteins [75], were reported to be of minor importance compared to steric hindrance. Negatively charged NPs crossed the mucus barrier with comparable [76] or even lower velocity [77] than positive or neutral NPs, respectively. In contrast, hydrophobic interactions between particles and mucin exhibited a more pronounced hindrance of movement [78]. To reduce the steric hindrance by the sticky and viscous mucus, coadministration of mucolytic agents such as N-acetyl cysteine or rhDNase (Pulmozyme) enlarged the pores of the mucosal mesh [77,79]. Accordingly, the mucus penetration properties of polymeric NPs can be improved by surface modifications leading to neutral surface charges and/or hydrophilic surface characteristics. As reported for PSA [80], PS [74] and PLGA [81] NPs (100 - 200 nm), PEG effectively shielded the hydrophobic core, inhibited particle--mucus interactions and stabilized the NPs against agglomeration with increasing molar mass and increasing density of PEG chains [82,83]. PEG is an uncharged hydrophilic polymer that is frequently used in pharmaceutics to increase circulation time and reduce opsonization by alveolar macrophages [84] PEG-coated L-tyrosine polyphosphate NPs (471 to 2891 nm in size) could be uniformly deposited in P. aeruginosa biofilms within 2 to 4 h in an in vitro fluidic model [85], most likely via pores within the biofilm matrix. However, NP sizes > 560 nm were found to be inefficient for penetration of CF mucus. Other coatings such as chitosan [66] or carboxymethyl cellulose [86] that are known to improve biocompability and stabilize the NPs against aggregation have been shown to increase the adhesion of NPs to the mucus and thus seem to be inappropriate for application in CF patients. Experiments with polymer-encapsulated antibiotics are rare and were only performed in vitro so far. Anti-staphylococcal vancomycin was successfully incorporated in pH-responsive surface-charge switching triblock copolymeric NPs made of PLGA, poly-L-histidine (PLH) and PEG. PLH contains imidazole groups that attain protons under acidic conditions (pKa ~ 6.0 -- 6.5). As a consequence, PLH segments get positively charged at the NP surface which facilitates a strong multivalent electrostatic-mediated binding to negatively charged parts of the bacterial cell walls. The triblock-polymer encapsulated vancomycin displayed an up to fourfold increased efficacy against S. aureus compared to free and PLGA-PEG-encapsulated vancomycin [87]. Although the NPs exhibited a suitable size of ~ 200 nm, because of the data regarding surface charge mentioned above, the proof of concept in mucus has still to be provided.


15

16

[107]

[122] [66]

[87]

[121]

Ceftaroline Ciprofloxacin Clindamycin Colistin Doxycycline Vancomycin 2014

Alg: Alginate; CS: Chitosan; NP: Nanoparticle; PLGA: Poly(lactide-co-glycolide); PLH: Poly(L-histidine); PVA: Polyvinyl alcohol.

µm

In vitro µm µm µm

-- 48 -- 36 -- 51 µm µm -- 37 42 60 48 41 41 33

In vitro In vivo/in vitro 1 -- 5 µm 270 -- 600 nm Tobramycin Tobramycin 2012 2012

PLGA PLGA (50:50) PLGA (25:75) PLGA

In vitro Vancomycin 2012

PLGA-PLH-PEG

~ 200 nm

Encapsulating nanoCipro in large porous PLGA particles resulted in a steady release of ciprofloxacin Vancomycin-encapsulated into PLGA-PLH-PEG NPs bound at bacterial cell walls and displayed an increase in MIC Sustained release of the drug up to 10 days PVA-modified Alg/PLGA NPs reached the deep lung, while CSmodified NPs were found in great amounts in the upper airways Charge of antibiotics was the significant loading factor; release of smaller antibiotics was faster; no significant changes in the MIC but retained efficacy In vitro 10 -- 15 µm Ciprofloxacin 2007

PLGA (50:50)

Study design Particle size Polymer Drugs Year

Table 3. Polymeric drug delivery systems under experimental investigations.


Results

Ref.

M. Klinger et al.

Acetalated-dextran NPs loaded with silver as antimicrobial showed suitable properties for nebulization and improved the efficacy of silver against Gram-positive and Gram-negative bacteria [88]. However, it is unclear if these small (100 -- 250 nm) NPs with negatively charged surface will be able to deposit in deeper lung regions and penetrate the mucus to reach the bacteria. The agglomerated vesicle technology (AVT) offers a possibility to increase the size and the aerodynamic diameter of the particles to improve the inhaled deposition. This method uses specific modification of the polymeric NPs to crosslink the particles into agglomerates which become cleaved in vivo after administration. Such crosslinkage might be performed with all particles containing, for example, carboxyl or amine or sulfhydryl groups. In a recent study, CIP was loaded into modified liposomes with incorporated dithiobenzyl carbamate (DTB-urathene)-linked amine-PEG-1,2-distearoyl-snglycero-3-phosphoethanolamine that could be agglomerated via an thiolyticaly cleavable crosslinker [89]. The particle size could be incereased from 200 nm (parental liposom) to microparticles of 2 -- 31 µm (mean diameter ~ 8 µm) by agglomeration with subsequently improved CIP release kinetics after cysteine-induced cleavage. In healthy airways, the mucus contains many cysteine-rich compounds (mucins) with an adequate redox potential, but in CF these mucines are rare [90]. It is unclear if the lowered pH of the CF mucus will be sufficient to reduce such crosslinkers. However, known mycolytics such as N-acetylcystein (or glutathione), being controversial in CF treament, possess similar redox potential to cysteine and might be suitable reducing agents for such agglomerates at the mucus site. For the administration of polymer-based antibiotics, DPIs benefit the advantage of long-term storage, avoiding instability issues and providing an easy-to-inhale system for the patients. Additionally, excipients such as glucose or lactose dissolve after deposition in the aqueous mucus or lung epithelium releasing small mucus-diffusive NPs. In this context gelatin and poly(butylcyanoacrylate) NPs were dry-powdered with lactose as carrier yielding dry-powders with suitable aerodynamic diameters (large but porous particles) for inhaled lung deposition [91]. Lactose was also successfully used for dry-powdering of tobramycin embedded in PLGA NPs with different modifications: chitosan, PVA and alginate [66]. Despite the enhanced aerodynamics, only PVA-modified NPs reached the deep lung (i.e., alveoli), whereas chitosancoated NPs were found mostly in upper airways (bronchi and bronchioles). Interestingly, incorporation of alginate into the NPs prolonged the release of anti-pseudomonal tobramycin up to 1 month. Alginate is also produced by mucoid pseudomonal biofilms and protects the bacteria against phagocytosis [92]. Thus, it could also have some antiphagocytic effects on the NPs [66]. It would be useful to prove if incorporated alginate will also improve or impede the penetration through biofilms of mucoid P. aeruginosa and to examine in vivo the efficacy.




PLGA was also investigated as excipient in inhalable powders. Here, NPs of the broad range antibiotic CIP (nanoCipro) were encapsulated in large, solid and porous PLGA microparticles, for which oils were used as porogens [93]. These particles showed a prolonged and steady drug release, depending on particle morphology, but the encapsulation efficiency was low due to the zwiterionic properties of CIP at neutral pH. However, it might be interesting to investigate the encapsulation and lung deposition efficiency for other anti-staphylococcal and anti-pseudomonal drugs that are presently administered as powder formulations (aminoglycosides or colistin). Metal NPs The experiences of the use of colloidal magnetic NPs such as iron oxide or gold were mostly focused on lung cancer treatment, whereas reports intending the application as magnetically controllable carrier for antibiotics are still limited [94]. As their major advantage, aerosol deposition can be magnetically targeted to a region of interest in the lung by the presence of a target-directed external magnetic field [95]. The type of aerosolization and the localization of the magnet tip were found to influence the therapeutic efficacy [96]. Using superparamagnetic iron oxide NPs (SPIONs) in dry powder aerosols, it was demonstrated that even with optimized magnet design, the magnetic forces would not be sufficient to reach satisfying guiding, because of their small magnetic moment [96]. In contrast, assembling the SPIONs in aerosol droplets as so-called nanomagnetosol, where the SPIONs are physically connected by a solvent, increased the magnetic moment and improved the magnetic targeting efficacy [96]. The combination of the direct magnetic field with a simultaneous perpendicular, high frequency alternating magnetic field induced vibrations that caused local increase of temperature and facilitated the NP transport through the mucus [97]. Magnetic hyperthermia was shown to be an effective method for decreasing the viscosity of biofilms and mucus and enhancing drug and immune cell penetration to the affected area. Since the bacterial growth is strongly temperaturedependent, hyperthermia can reduce the formation and growth of biofilms. NPs in the superparamagnetic to ferromagnetic size range exhibited excellent heating power. However, the translation of these techniques and instrumentations which are up to now mainly used in animal experiments might be the major challenge. Metal NPs have a controlled size range with favorable low diameters of 5 to 100 nm that facilitates intracellular uptake but promotes aggregation and rapid clearance by macrophages [98,99]. To overcome these issues, surfaces were functionalized by adsorptive or covalent coating with natural and synthetic polymers. Furthermore, the disruption of biofilms and the retardation of S. aureus growth can be controlled by the surface coating of SPIONs, with carboxylate being more effective than functionalization with isocyanate, amine groups or unfunctionalized SPIONs [100]. Magnetite (Fe3O4) was 4.3

preferred over maghemite (Fe2O3) as core material since Fe3 + is naturally internalized by many pathogenic bacteria and triggers targeting. However, the naked iron-containing core might favor the growth of P. aeruginosa and might impair the function of antimicrobial peptides, highlighting the importance of coatings [101]. Antibiotics such as rifampicin can be adsorbed or covalently bound to the metallic core or the polymeric coating [102]. However, studies on antibiotic delivery via metal NPs are rare and preferable and the intrinsic antimicrobial activities of some metals, such as silver or cupper, are under investigation [103]. Coupling of tobramycin and CIP to these particles was recently reported and indicated improved treatment of planktonic P. aeruginosa [104]; however, detailed results regarding the efficacy of these promising particles in mucosal biofilms are pending. 5.

Future perspectives

Inhaled antibiotics indicate a higher effectiveness in the lung compared to orally or intravenously administered antibiotics. Besides, side effects are reduced to the local site of action. However, treatment with inhaled antibiotics is limited by several problems: i) suboptimal patient’s compliance due to time required (> 1 h/day); ii) the suboptimal pulmonary delivery due to mucus obstruction of the airways; iii) the occurrence of recalcitrant and persistent bacterial biofilms; and iv) the intracellular hiding of small bacterial colony variants. New promising drug delivery strategies for efficient antibiotic therapy for CF-associated infections have to address these limitations. Inhaled antibiotics to be expected in near future Powders of antibiotics represent a kind of a particular application and have been demonstrated to be superior compared to nebulized solutions of the corresponding substances (tobramycin, aztreonam lysine and colistin). However, only data on anti-pseudomonal antibiotics are available so far. A new inhaled dry powder formulation of anti-staphylococcal vancomycin (AeroVanco by Savara, Inc.) is presently assessed in Phase II trial; thus, an approval for treatment of CF-associated infections is unlikely within the next few years. The dry powder formulation of CIP (PulmoSphere) proved inefficient in CF patients; thus, an approval is presently not conceivable. Vancomycin as inhaled solution might be the next approved anti-staphylococcal treatment; but clinical trials are presently only in Phase II. An approval of liposomally encapsulated amikacin (Arikace, Insmed) seems to be likely since this delivery system is now in advanced clinical trials (Phase III) for application in CF-patients. The inhaled levofloxacin solution (Aeroquin by Aptalis Pharma) might also be approved shortly, but not for CF treatment; the treatment proved ineffective in this cohort. The development of a liposomal colistin formulation seems unlikely because of the liposome-destabilizing properties of polymyxins (see above). 5.1


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Also, metal NPs are unlikely to be shortly approved for therapeutic inhaled applications due to the controversial opinions about their benefits and risks. It has to be mentioned that serious side effects, such as decrease bacterial clearance, inflammation or apoptosis, can be induced by many metals when applied into the lung [95,96]. Further weak points are the slow degradation of the magnetic NPs and accumulation of particles as well as of their by-products in various tissues and organs [105]. Targeted deposition and distribution of new NPs Chronic pulmonary bacterial infections in CF patients are usually accompanied by biofilm formation. These sessile microbial communities are embedded in a self-produced matrix within the mucus. This protective environment confers robust phenotypic resistance to antibiotics leading to hardly eradicable and recurring infections. The main hurdles for drug treatment are: i) penetration barrier due to limited diffusion and sequestration by matrix polymers (polysaccharides block both, cationic and anionic drugs such as colistin, CIP and tobramycin) [106]; ii) specific microenvironments, such as low pH or accumulation of enzymes that inactivate or modify the drug; and iii) decreased bacterial metabolism protecting persister cells from respectively interfering antibiotics [15]. To reach the protected bacteria, new NP-based therapeutics, on one hand, have to meet the aerodynamic requirements (geometric diameters 1 -- 5 µm) for optimal deposition. On the other hand, they should sufficiently penetrate the mucus and bacterial biofilms to ensure efficient and secure transport of the drug to the site of action. These NPs have a size between 100 and 500 nm and will unlikely reach the mucus-embedded bacteria in human lungs, as they will become exhaled or opsonized. Currently, the most favorable method to deposit such NPs is nebulization that is nevertheless related with relative low deposit efficiency. The therapeutic efficiency could be strongly increased using DPI, and some promising methods exist to increase the size and improve the properties of the powder particles, such as dry-powdering using biodegradable excipients that dissolve in the aqueous environment of the mucus or AVT.


5.2

Targeted antibiotic release Another interesting concept for targeted drug delivery are carriers that are degraded under specific conditions or by bacterial species. Drug carriers would, thus, persist in the mucus, traversing mucus-plugged bronchi and bronchioles, and drug release would only be initiated in the presence of specific bacteria or bacterial enzymes or metabolites in infected regions. First steps in this direction have been taken by coupling species-specific antibodies to the NP surface or by agglomeration of the particles via a cleavable crosslinker. Additionally, a pH drift within biofilms and the CF mucus provides a basis for further concepts of pH-controlled drug delivery systems. A combination of time- and pH-dependent 5.3

18

release mechanisms might be a promising option for inhaled antibiotic therapies. Drug-optimized nanocarriers The efficiency of encapsulation, stability of the particle and tailored release of a drug also strongly depend on the chemical interaction of the drug and the encapsulating material. The lipid composition seems to be decisive for the stability of the particle and for antimicrobial efficacy. Lipophilic moieties of amphiphilic antibiotic, in particular in the case of polymyxins, are assumed to destabilize the lipid bilayer leading to uncontrolled release of the drug. In this regard, more rigid liposomal particles with increased content of solid lipids, such as cholesterol, might be more convenient. Lipophilic antibiotics might be even more suitable for encapsulation in polymeric NPs, like recently shown for PLGA particles [107]. In this work, six different antibiotic classes, including colistin, CIP and vancomycin, were efficiently encapsulated in PLGA. The carrier exhibited a sustained release of the drug for 20 days (CIP) to 40 days (vancomycin) in vitro. However, it is questionable if these results can be transferred to patients with the permanent mucus turnover. Many antibiotics are modified or degraded when exposed to bacteria and their environment. The porous structure of polymeric NPs that is commonly permeable for aqueous compounds offers little protection against small molecules and ions that induce modifications of some sensitive antibiotics (e.g., aminoglycosides). Thus, the impermeability of liposomes for hydrophilic and charged molecules, even as small as hydrogen ions, represents ideal packaging for such antibiotics or an outer coating for other nanocarriers loaded with sensitive antibiotics. 5.4

Antibiotic-specific release kinetics The main requirements for efficient antibiotic therapy of CFassociated infections are enhanced penetration of the therapeutic agent into the mucus and into biofilm structures, followed by a sufficient release of soluble antibiotics in an adequate concentration within the infected mucus compartment. Thus, the drug-release profile of the deposited antibiotic particles is an important factor for determining efficacy. Some polymeric NP compositions tend to quickly release the encapsulated drugs early after nebulization (‘initial burst’) that in consequence will result in high antibiotic concentrations in the central bronchi, but does not achieve sufficient concentrations in the mucus-plugged bronchi or the peripheral bronchioles. The initial burst depends on the interaction of the polymeric compound and the drug and is associated with drug ‘loosely’ adsorbed to the NP surface [108]. Emulsification techniques or surface coatings can increase the encapsulation efficiency reducing the timely drug lost. Aminoglycosides, fluoroquinolones, polymyxins and vancomycin act concentration-dependently. For aminoglycosides, the bactericidal effect can be expressed as the ratio of Cmax: MIC, where Cmax stands for the maximum concentration of 5.5




the antibiotic and MIC for the minimum inhibitory concentration of the pathogen. Therefore, the particles encapsulating tobramycin or amikacin should release an effective dose at the site of action promptly after deposition. An effective dose is usually several times higher than the MIC. In addition, the mucosal and biofilm barriers have to be considered: bacteria in biofilms exhibit some log higher minimal biofilm eradicating concentrations compared to MIC. On the other hand, for fluoroquinolones, polymyxins and vancomycin, the efficacy is related to the ratio AUC:MIC, where AUC represents the area under the curve of the pharmacokinetic plot (antibiotic concentration over the time) in the body compartment. Thus, the release of the antibiotics should be high right from the outset but preferable also remains high. Most of the experimental studies aimed at a sustained release, but in the design of new delivery systems, stronger emphasis should be placed on the therapeutically relevant pharmacodynamics of a given drug. Translocation through epithelium and toxicity Velocity of mucociliary clearance for NPs trapped in the pulmonary mucus layer was shown to be independent of size, shape, charge and surface properties of the particles [109]. However, the toxicological response and uptake into epithelial cells in vitro turned out to strongly depend on the surface charge and shape of the particles [110]. In contrast to more spherical nanomaterials, nanotubes and nanofibers have been proved to induce toxic effects. They have been shown to cause sustained inflammatory response and to migrate into subpleural tissue leading to lesions and fibrosis [111]. Liposomes are nearly spherical and composed of natural or nature-like compounds, therefore causing fewer side effects. But toxicity of less-compatible particle formulations, such as metals or polymers, can be reduced as well by neutral and nature-like coatings. Such coatings can in addition enlarge particle and alter its properties, such as mucus adhesiveness, mucociliary clearance and cell or tissue penetration. An efficient blockage of the translocation through the epithelium is one of the aims of micro- and nanoparticulate inhaled applications. Advanced delivery systems should therefore provide a high and effective local concentration of the antibiotic, but at the same time should avoid increased systemic concentrations leading to toxic effects on organs. 5.6

6.

Expert opinion

Currently, the gap between the rapidly increasing antimicrobial resistance in CF patients with frequent exposure to antibiotics and the lack of novel antibiotic classes capable of counteracting this trend is widening. An introduction of novel antibiotics that could resolve this problem on the market seems unlikely within the next years. Innovative delivery systems that maintain antimicrobial efficacy of available

antibiotics represent a short-term but promising solution for the vulnerable cohort of patients with CF. Lipid-based systems, such as liposomes and SLMs, are intensively investigated for the controlled pulmonary release of hydrophilic and lipophilic drugs in CF treatment; hence, their clinical evaluation is further advanced compared to polymer-based carriers. Although the latter have not reached the developmental status of their lipid counterparts, significant progress has been made in recent years regarding the design of biocompatible, biodegradable and efficient systems. One of the most promising and most reported organic polymers in this context is PLGA. This organic polymer has already been approved by the FDA for various applications and might accomplish the requirements in terms of a facilitated respiratory uptake of antibiotic carriers in CF patients. PLGA is highly biocompatible and completely biodegradable without known significant toxic effects. It can be easily modified to improve the encapsulation efficacy, aerosolization and penetration properties or to optimize the drug release from days to months. PEG-modified PLGA NPs have been shown to increase mucus penetration into plugged peripheral bronchi/bronchioles and to sufficiently target bacterial biofilms with sustained release of the antibiotic. A multidrug therapy of P. aeruginosa infections is common for CF patients. In PLGA, various classes of antibiotics can be efficiently encapsulated and, thus, combined antibiotic therapy might even be achieved in one particle. In contrast, there is no evidence that combinations of CF-recommended antibiotic classes can be efficiently incorporated into liposomes. Besides antipseudomonal antibiotics, anti-staphylococcal vancomycin was also efficiently loaded into PLGA and exhibited a sustained release. Thus, usage of organic polymers offers a potential forceful antibiotic efficacy against recalcitrant infections and against a variety of pathogens. The optimized and prolonged drug release might also render the annoyingly frequent and time-consuming inhalations unnecessary and improve the life quality of the CF patients. However, more research -- particularly in vivo -- is needed to explore potential long-term toxicity. As targeted drug delivery systems provide improved efficacy against pathogens, they may also prevent accumulation of bacterial resistance mechanisms but there is still the risk in selecting for highly resistant clones. Besides resistant bacteria, other microorganisms, for example, fungi that are increasingly found in CF lung might fill the resulting niche. Currently, these questions have been poorly addressed and should be taken into account in future clinical studies.

Acknowledgments The authors thank Margrit Leitner, University Hospital Jena, Centre for Sepsis Control and Care, for proofreading the manuscript. M Klinger-Strobel and C Lautenschla¨ger have equally contributed to this manuscript.


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Declaration of interest This work was supported by grants from the German Ministry of Education and Research (BMBF), grant numbers 01KI1204 and 01EO1002, as well as by the Deutsche Bibliography

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Affiliation Mareike Klinger-Strobel1,2, Christian Lautenschla¨ger2,3, Dagmar Fischer4, Jochen G Mainz5, Tony Bruns2,3, Lorena Tuchscherr6 & Mathias W Pletz1,2, Oliwia Makarewicz†1,2 † Author for correspondence 1 Jena University Hospital, Center for Infectious Diseases and Infection Control, Erlanger Allee 101, 07740 Jena, Germany Tel: +49 3641 9324227; E-mail: [email protected] 2 Jena University Hospital, CSCC-Center for Sepsis Control and Care, Erlanger Allee 101, 07740 Jena, Germany 3 Jena University Hospital, Department of Internal Medicine IV, Erlanger Allee 101, 07740 Jena, Germany 4 Friedrich-Schiller University Jena, Institute of Pharmacy, Department of Pharmaceutical Technology, Otto-Schott-Strasse 41, 07745 Jena, Germany 5 Jena University Hospital, Cystic Fibrosis Center, Pediatric Pulmonology, Kochstrasse 2, 07745 Jena, Germany 6 Jena University Hospital, Institute of Medical Microbiology, Erlanger Allee 101, 07740 Jena, Germany