Pseudomonas aeruginosa

inosa

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Liposomal antibiotic formulations for targeting the lungs in the treatment of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative bacterium that causes serious lung infections in cystic fibrosis, non-cystic fibrosis bronchiectasis, immunocompromised, and mechanically ventilated patients. The arsenal of conventional antipseudomonal antibiotic drugs include the extended-spectrum penicillins, cephalosporins, carbapenems, monobactams, polymyxins, fluoroquinolones, and aminoglycosides but their toxicity and/or increasing antibiotic resistance are of particular concern. Improvement of existing therapies against Pseudomonas aeruginosa infections involves the use of liposomes – artificial phospholipid vesicles that are biocompatible, biodegradable, and nontoxic and able to entrap and carry hydrophilic, hydrophobic, and amphiphilic molecules to the site of action. The goal of developing liposomal antibiotic formulations is to improve their therapeutic efficacy by reducing drug toxicity and/or by enhancing the delivery and retention of antibiotics at the site of infection. The focus of this review is to appraise the current progress of the development and application of liposomal antibiotic delivery systems for the treatment pulmonary infections caused by P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium widespread in nature, living in soil, water, and plants. It usually does not cause infection in healthy people but loss of the integrity of a physical barrier to infection or a deficiency in the immune system can result in serious infections. The lung is one of the most common body sites of P. aeruginosa infection that presents as a spectrum of clinical conditions, ranging from a rapidly acute and/or fatal pneumonia in a neutropenic patient or mechanically ventilated patient, to a chronic destructive lung disease in patients suffering from cystic fibrosis (CF) [1] . Pseudomonas infections are acquired most often in hospitals, where the organism is frequently found in moist areas such as sinks and antiseptic solutions, as well as equipment such as mechanical ventilators, intravenous lines, urinary or dialysis catheters, pacemakers and endoscopes [2] . The pathogenesis of P. aeruginosa infections is complex and depends on numerous toxins or virulence factors. The membrane-

10.4155/TDE.14.13 © 2014 Future Science Ltd

Misagh Alipour1 & Zacharias E Suntres*,2 Centre of Excellence for Gastrointestinal Inflammation & Immunity Research, Department of Pediatrics, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada 2 Northern Ontario School of Medicine, Medical Sciences Division, 955 Oliver Road, Thunder Bay, Ontario, P7B 5E1, Canada *Author for correspondence: Tel.: +1 807 766 7395 [email protected] 1

bound (e.g., lipopolysaccharide, pili, and alginate polysaccharides) and secreted (e.g., neutral and alkaline proteases, elastase, phospholipase C, and a rhamnolipid hemolysin) factors help P. aeruginosa infect the hosts [3] . Another contributing factor to its pathogenesis is the organization of complex communities encased in a polymeric matrix when attached to biotic or abiotic surfaces, known as biofilms. Bioﬁlm formation is a progressive process that involves the transport of microbes to a surface, initial attachment, formation of microcolonies, bioﬁlm maturation and dispersal of single cells from the biofilm. The formation of these surface communities and their resistance to antimicrobial agents and host immune system are the cause of many persistent and chronic infections [4,5] . CF and ventilator-associated pneumonia are some of the diseases that are considerably complicated by the formation of bacterial biofilms. The expression of the virulence factors and biofilm formation by P. aeruginosa are under

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Key Terms Cystic fibrosis: Autosomal recessive disorder caused by a defective cystic fibrosis transmembrane conductance regulator protein that results in ionic imbalance of epithelial secretions in several organ systems, such as the respiratory system, pancreas, gastrointestinal tract and liver. Mucoid bacteria: Aggregates of nonmotile cells expressing surface or secreted polysaccharides. The bacteria express mucoidity once adhered to a biotic or abiotic surface. Antibiotic resistance: Ability of microbes, such as bacteria, viruses, parasites, or fungi, to grow in the presence of a chemical (drug) that would normally kill them or limit their growth.

the regulation of quorum sensing, a chemical communication process that bacteria use to regulate collective behavior (Figure 1) [6–8] . Certain virulence factors are invasive and are injected into the host epithelial cells through a needle complex, the type III secretion system. The effector proteins (ExoU, ExoS, ExoT and ExoY) injected into the cytosol induces cell apoptosis, and disrupts cytoskeletal structure and tight junctions [9,10] . Other secreted toxic proteins such as exotoxin A and exoenzyme S are known to induce necrosis and inhibit protein synthesis [11,12] . The proteases LasA and LasB, alkaline protease and protease IV cleave immunoglobulins, cellular receptors, complement peptides and cytokines, and degrade the extracellular matrix [13–15] . Lipolytic enzymes like lipases (LipA and LipC) and phospholipase C inhibit monocyte chemotaxis, promote inflammation and degrade phosphatidylcholine surfactants in the lung [16,17] . Rhamnolipids disrupt ciliary beating and mucociliary clearance, induce rapid necrosis of polymorphonuclear leukocytes and are involved in biofilm architecture and formation of channels (presumably for the passage of nutrients and waste) [18,19] . Others such as pyocyanin disrupt the calcium and glutathione balance, and the iron siderophore pyoverdine is required for biofilm development [20–22] . The adherence, colonization and infection of P. aeruginosa in the lower respiratory tract is most dramatically exemplified in CF patients where infection with mucoid bacteria strains is common and difficult, if not impossible, to eradicate. In the lungs of CF patients, P. aeruginosa proliferates within biofilms covered with an extracellular charged matrix and patient-excreted polyanionic sputum (a mixture of DNA, filamentous actin and glycoproteins) produced by infiltrating neutrophils and alveolar macrophages (Figure 2) [23,24] . Different classes of cationic antibiotics (e.g., aminoglycosides and polymyxins) with broadspectrum activity against these pathogens bind to the negatively charged polymers impeding their penetra-

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tion into the sputum and biofilms and thus, preventing their contact with the bacteria. Administration of antibiotics via a liposomal delivery system may circumvent this antibiotic–polyanion interaction and improve treatment outcome in pulmonary infections [25,26] . The focus of this review is to summarize the current progress of the development and application of liposomal antibiotic delivery systems for the treatment of pulmonary infections caused by P. aeruginosa. Current treatment of P. aeruginosa lung infections The arsenal of traditional antibiotic drugs with antipseudomonal activity includes the extended-spectrum penicillins, cephalosporins, carbapenems, monobactams, aminoglycosides, fluoroquinolones, and polymyxins (Table 1) [27–29] . Newer strategies under investigation that might be potentially promising in the treatment of P. aeruginosa infections include the development of drugs that inhibit quorum sensing, the use of pilicide compounds that inhibit bacterial adhesion, and the use of mucosal vaccination to effectively protect against P. aeruginosa lung infection [30–33] . Therapeutic management of P. aeruginosa pneumonia is challenging because the high-level resistance of these microorganisms to most classes of antimicrobial agents frequently leads to recurrence of infections or decline in physical health [34] . Choosing adequate antibiotic drugs is crucial in decreasing morbidity and mortality. The use of combination therapies for P. aeruginosa pneumonia is a standard practice in many hospitals, however, the potential increased value of combination therapy over monotherapy remains controversial [34,35] . Possible benefits of combination therapy for P. aeruginosa infections include in vitro antibiotic synergy, prevention of the emergence of bacterial resistance while receiving therapy and improved adequacy of empiric therapy. Unfortunately, the potential disadvantages are also considerable, the most worrisome of which are drug toxicity and the establishment of multidrug-resistant organisms [35] . According to the American Thoracic Society–Infectious Diseases Society of America guidelines, consideration should be given to short-duration (5 days) aminoglycoside therapy, when used in combination with a β-lactam to treat P. aeruginosa pneumonia with de-escalation to monotherapy based on organism culture sensitivity [36] . In the elderly, in addition to making dose modifications because of loss of renal function, it has been suggested that the use of aminoglycosides should be used cautiously, and possibly replaced with a combination of quinolone and a β-lactam, notwithstanding the possible increased pressure for selection of resistance with the latter combination [36] .

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O A

O

O

O

O O

N H N-3-(oxo)-dodecanoyl homoserine lactone (3O-C12-HSL)

Cyclic di-guanylate

OH O

N H

N H 2-heptyl-3-hydroxy-4-quinolone (PQS)

N-butanoyl homoserine lactone (C4-HSL)

B

lasR

lasl

Lasl

Auto-induction

3O-C12-HSL

LasR

Biofilm formation Cytokine modulation IL-8 induction Neutrophil apoptosis

Pseudomonas Rhli

rhlR

rhll

Precursor

RhlR PqsR C4-HSL

LasA protease LasB elastase Alkaline protease Lipase Rhamnolipids Sigma factor RpoS Pyocyanin

Lectins A and B Hydrogen cyanide Exoenzyme S Pyoverdine Swarming Twitching motility PQS

PQS

Auto-induction

LasA protease LasB elastase Alkaline protease Lipase Biofilm formation Exotoxin A Neuraminidase Catalase Superoxide dismutase Amino peptidase Hydrogen cyanide xcp secretion system Swimming Swarming Twitching motility PQS

O

LasA Elastase Rhamnolipid LecA lectin Pyocyanin

Figure 1. The transcriptional regulation of quorum sensing and the expression of virulence factors by Pseudomonas aeruginosa. (A) Acyl homoserine lactone and 2-alkyl-4-quinolone signaling molecules of P. aeruginosa.(B) The two homologs lasI/lasR and rhlI/rhlR regulate gene expression. The LasI, RhlI and PqsR catalyze the formation of autoinducers shown in (A). The freely soluble autoinducers bind to LasR, RhlR and PqsR (transcriptional activator proteins), which regulate virulence factors. Adapted with permission from [8] .

The antipseudomonal penicillins (i.e., ticarcillin, piperacillin), the third and fourth generation cephalosporins (i.e., ceftazidime, cefepime), the monobactams (i.e., aztreonam), and carbapenems (i.e., imipenem, doripenem and biapenem) are β-lactams commonly used to treat P. aeruginosa infections [29,34] . β-Lactam antibiotics bind to the cell wall transpeptidases (penicillin binding proteins), which are involved in the cross-linking of the bacterial cell wall, rendering them unable to perform their role in cell wall synthesis leading to cell death. P. aeruginosa is intrinsically resistant to most β-lactams due to the inducible β-lactamases and the involvement of multidrug efflux systems (e.g., MexAB-OprM) [27–29,34,37] . Fluoroquinolones (i.e., ciprofloxacin, levofloxacin) inhibit DNA synthesis by promoting cleavage of bacterial DNA in the DNA-enzyme complexes of DNA gyrase and type


IV topoisomerase, resulting in rapid bacterial death. Primary intrinsic resistance of the wild-type P. aeruginosa to fluoroquinolones is due to gene mutations in topoisomerase and/or expression of efflux systems MexAB-OprM as well as to MexXY-OprM [29,34,37] . The polymyxins remain the most consistently effective agents against multidrug-resistant P. aeruginosa, however, their use is limited due to adverse effects. The mechanism of action of polymyxins involves an initial stage of interaction with the lipid A of the lipopolysaccharide (LPS), promoting membrane permeabilization and diffusion of polymyxin B through the periplasm to the inner membrane, where it disrupts cellular respiration and results in cell lysis [38] . The most common mechanism of resistance to polymyxin has been shown to arise from modification of LPS lipid A with 4-amino-l-arabinose, a process that has been seen

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Live, dead, mucoid bacterium

DNA, actin in sputum Immune response

ENaC

CFTR Cl-

Na+

Antibiotic susceptibility

Pathogen clearance

Non-antibiotics Antibiotics Liposomes

DNase

OHO ROS

Biofilm formation

Immune response

AIgL

Figure 2. The colonization and treatment of Pseudomonas aeruginosa infection in the lower respiratory tract of cystic fibrosis patients. Mutations in the CFTR protein cause a reduction in chloride and sodium (regulated by the epithelial sodium channel) concentrations. A reduction in mucin hydration and oxygen levels and an ineffective mucociliary clearance of cellular debris from the lungs coupled with anionic sputum buildup (DNA, actin and glycoproteins) favors bacterial colonization. P. aeruginosa proliferates and forms biofilms with extracellular alginate, promoting an overwhelming immune response which further exacerbates inflammatory damage by neutrophils influx. Although, the sputum buildup inactivates antibiotic treatments and inhibits their penetration, nonantibiotic enzymes like DNase and AlgL) could reduce sputum viscosity and breakdown alginate respectively, allowing better conventional and liposomal antibiotic delivery. CFTR: Cystic fibrosis transmembrane conductance regulator; ENaC: Epithelial sodium channel; ROS: Reactive oxygen species.

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Table 1. Mechanism of action and major resistance mechanism(s) of antipseudomonal antibiotic drugs. Antibiotic class

Mechanism of action Major mechanism(s) of resistance

Penicillins (e.g., ticarcillin, piperacillin)

Interfere with cell wall synthesis

Inactivation by β-lactamases Overexpression of multidrug efflux pump

[29,37,126,127]

Cephalosporins (e.g., ceftazidime, cefepime)

Interfere with cell wall synthesis


[29,37,126,127]

Carbapenems (e.g., imipenem, Interfere with cell doripenem) wall synthesis


[29,37,126,127]

Aminoglycosides (e.g., tobramycin, amikacin)

Interfere with protein synthesis

Inactivation by aminoglycosidemodifying enzymes Ribosome alteration

Fluoroquinolones (e.g., ciprofloxacin)

Inhibit DNA synthesis Topoisomerase mutation Overexpression of multidrug efflux pump

Polymyxins (e.g., polymyxin B, Disrupt membrane colistin) permeability

both in in vitro-selected mutants and in CF isolates [29,34,37–39] . Aminoglycosides bind to the 30S ribosomal subunit and interfere with protein synthesis. Bacterial resistance to aminoglycosides is most frequently associated with the expression of modifying enzymes that can phosphorylate (aminoglycoside phosphoryltransferase), acetylate (aminoglycoside acetyltransferase), or adenylate (aminoglycoside nucleotidyltransferase) them; as well, MexXY-OprM efflux pump is necessary for adaptive resistance of P. aeruginosa to aminoglycosides [29,34,37,40,41] . With respect to CF, individuals with chronic pulmonary infection with P. aeruginosa suffer a more rapid deterioration in lung function, greater morbidity and a shorter life expectancy [42] . There is a window of opportunity to prolong normal pulmonary function and reduce morbidity in CF children by commencing antimicrobial therapy prior to the establishment of mucoid P. aeruginosa infections [27,43] . During early lung infections of children, patients usually receive antibiotics to delay the colonization by susceptible nonmucoid P. aeruginosa [44] . A study evaluating early and aggressive inhaled colistin and oral ciprofloxacin treatments in Denmark revealed that the combination of antibiotics protected the majority of patients from chronic infection with no resistance to colistin and minimal resistance to ciprofloxacin [28] . Another study of patients receiving early aggressive multiple treatments (consisting of oral, intravenous or nebulized antibiotics) showed no significant increase in antibiotic resistance [45] . Although early treatments delay the infection, P. aeruginosa colonization


Ref.

[34,37,40,41]

Modification of lipopolysaccharide lipid A

[29,34,37]

[38,39]

is inevitable as the mucociliary clearance is inhibited and inflammation worsens lung function [46] . As the patients reach adulthood, periods of pathogen-free and mucoid P. aeruginosa exacerbations are observed and aggressive antibiotic regimens using inhalable tobramycin frequently increase lung function and reduce colonization in the respiratory zone [47] . One of the most common class of antibiotics used in CF therapy are the aminoglycosides, in particular tobramycin, delivered either via the inhalation or intravenous routes [48,49] . Clinical trials have shown that tobramycin (28 day cycles of treatment) inhaled by a nebulizer leads to improved lung function, reduced bacterial load, shorter hospital stays and exacerbations, as well as lowered systemic toxicity due to lowered serum levels [48,50,51] . Intravenous administration requires larger and more frequent doses of tobramycin to achieve peak serum levels in order to eradicate bacterial growth because of its rapid distribution and clearance in CF patients [52] and as a consequence, nephrotoxic and ototoxic events are frequently observed [53] . Resistance of P. aeruginosa to antibiotics The increasing antibiotic resistance of P. aeruginosa isolated from Intensive Care Units is of particular concern. In 2013, the Centers for Disease Control and Prevention reported an estimated annual infection rate of 51,000 patients in the USA, with more than 6000 multi-drug resistant P. aeruginosa cases and 400 deaths [54] . Some strains of P. aeruginosa were found to be resistant to all available antibiotics such as ami-


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Review Alipour & Suntres noglycosides, cephalosporins, fluoroquinolones and carbapenems. Today, the resistance to aminoglycosides with antipseudomonal activities, including gentamicin, tobramycin and amikacin, is also all too common throughout all areas of the world. Such resistance is seen particularly in CF and non-CF lung infections, as well as bloodstream, urinary, wound, burn and eye infections [55–59] . The resistance of P. aeruginosa to antibiotics is multifactorial and can be adaptive (activation of resistance in presence and inactivation in absence of antibiotics) [60,61] . Depending on the stage of infection, the pathogens can restrict the uptake of antibiotics by modifying the outer cell membrane and surface characteristics to reduce antibiotic binding and permeability, by inducing enzyme-mediated drug inactivation, by expressing various efflux pumps with wide substrate specificity, and by forming dormant biofilms. It is important to stress that these mechanisms are often present simultaneously, thereby conferring multiresistant phenotypes (Table 1) [60] . P. aeruginosa is naturally resistant to many antibiotics due to the permeability barrier afforded by its Gram-negative outer membrane composed mostly of phospholipids, lipopolysaccharide and proteins, including porins and receptors [62] . The inner face of the outer membrane is composed of a phospholipid layer and the outer layer contains some phospholipid but primarily it is composed of lipopolysaccharide consisting of lipid-A, core polysaccharide, and the O-antigen polysaccharide chains projecting outwards. The lipid portion of LPS serves as the lipid anchor and is commonly composed of fatty acids, sugars and phosphate groups [62,63] . Aminoglycosides and polymyxins interact with lipopolysaccharides changing the permeability of the membrane in order to enter the cell whereas β-lactams and quinolones need to diffuse through certain porin channels. P. aeruginosa expresses mainly specific porins while most bacteria possess several general porins and relatively few specific ones. Unlike the porin protein OmpF in Escherichia coli, the channel formed by OprD is narrower, which causes a lower outer membrane permeability in P. aeruginosa than in E. coli [64] . In addition to carbapenems, OprD acts as a specific channel for basic amino acids and some small peptides [64,65] . Alteration in outer cell membrane and surface characteristics is a remarkable approach utilized by P. aeruginosa to develop resistance against antibiotics. For example, the modifications of the outer membrane components confer resistance to polymyxins, which require binding to specific P. aeruginosa components such as the lipopolysaccharides, are abundant on the bacterium surface [66,67] . Specifically, CF isolated bacterial strains have been described to have distinct lipid A structures from

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strains grown in vitro. These resistant strains incorporate 4-aminoarabinose in the lipid A thereby reducing its negative charge, which correlates with reduced binding to cationic antibiotics [67,68] . The development of high carbapenem resistance rates in P. aeruginosa isolates has been attributed partly to genetic events that led to the loss of the outer membrane protein OprD, which regulates the entry of carbapenems [69] . Pre-exposure of P. aeruginosa to β-lactam antibiotics causes the induction of a chromosomally encoded β-lactamase (encoded by the amp C gene; Class C), which can cause enzymatic inactivation of many β-lactams. Clinical failure of ceftazidime, anti-pseudomonal penicillin, or cefotaxime treatment is strongly associated with dysregulation of AmpC enzymes because these drugs all lead to a strong upregulation of the ampC gene [60] . Beta-lactamases are divided into class A, C, and D enzymes, which utilize serine for β-lactam hydrolysis and class B metalloenzymes, which require divalent zinc ions for substrate hydrolysis. A significant number of β-lactamases of all four molecular classes are found in P. aeruginosa [61] . Efflux pumps are transport proteins involved in the extrusion of toxic substrates (including virtually all classes of clinically relevant antibiotics) from within cells into the external environment. There is a handful of efflux pumps (from the resistance-nodulation-cell division family) identified in P. aeruginosa, such as the antibiotic-induced MexXY-OprM and constitutively expressed MexAB-OprM [70,71] . The efflux pump consists of an inner membrane antibiotic-proton antiporter (e.g., MexB or MexY) for trapping the drug, an outer membrane channel (OprM) for discharging of drug and a periplasmic protein (e.g., MexA or MexX), which acts as an adapter between the two proteins [72,73] . The induction of the mexXY or mexAB gene implicates the adaptive resistance of P. aeruginosa to a wide range of inducers such as dyes, cationic antimicrobial peptides, reactive oxygen species and antibiotics (e.g., aminoglycosides, β-lactams, macrolides and fluoroquinolones) that enter the periplasm; the inactivation of one or more efflux pumps increases bacterial sensitivity [74–76] . The formation of biofilm complexes, composed of cells in high metabolic outer regions and low metabolic central regions, is also beneficial to the development of P. aeruginosa resistance [77] . Since most antibiotics exert their effects on active cells, dormant regions of biofilms, comprised of persister cells, promote biofilm survival and recurrent infection [78] . As the bacterial biofilms mature, secretions of exopolysaccharides, such as alginate, inhibit or delay antibiotic diffusion. Alginate serves to protect the bacteria from adversity in its surroundings and also enhances adhesion to solid surfaces [79] . The formation of biofilms tend to decrease



the susceptibility of P. aeruginosa by several orders of magnitude and popular antibiotics, such as tobramycin, are known to mediate biofilm formation and drug resistance in CF infections [80] . Liposomes: an antibiotic drug-delivery system Over the last three decades, a large number of compounds have been examined for their potential to combat the emergence of resilient infectious diseases. However, only a fraction of these compounds are clinically relevant today, and considerable effort continues to be placed in research activities to discover and develop novel drugs with reduced toxicity and increased antibacterial efficacy [81,82] . One of the promising approaches towards the improvement of existing therapies against Gram negative bacterial infections implicates the use of specific carriers or modifiedrelease formulations of antibiotics, which improve the distribution and residence time of antibiotics in different organs, such as the lungs thus, allowing a precise release of the drug at the site of infection. Liposomes, first discovered in 1961 by Dr Bangham at the Babraham Institute in Cambridge (UK), are biocompatible, biodegradable and essentially nontoxic vesicles utilized as drug carriers in drug-delivery systems [83–86] . These spherical vesicles consist of one (unilamellar vesicles) or more phospholipid bilayers (multilamellar vesicles) of natural or synthetic origin enclosing an internal aqueous space. Liposomes are usually within the size range of 20 nm to several micrometers in diameter [83] . The size of liposomes plays an important role in the clearance of liposomes from the circulation. Liposomes have been widely used as pharmaceutical carriers for drugs and genes, in particular for the treatment of cancer [87,88] . Therapeutic applications of liposomal drugs include the US FDA-approved formulations for fungal infections and cancer therapy, with certain liposomal antibiotics for inhalation in the final stages of clinical trials [49,89–92] . The goal of developing liposomal antibiotic formulations is to improve the therapeutic efficacy of drugs by reducing drug toxicity and/or enhancing drug delivery and retention at the site of infection [49,93] . Data from studies examining the potential of liposomes for their use in the therapy of infections indicate that liposomal encapsulation may improve the therapeutic efficacy of antibiotic drugs [49] . The liposomal encapsulation efficiency varies depending on the physicochemical properties of the drug itself, the characteristics of the liposomes (lipid composition, surface charge, size distribution and lamellarity) and the preparation methods [94] . The physicochemical properties of the drug itself, especially solubility and partition coefficient, are impor-


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tant determinants of the extent of its incorporation in liposomes. Hydrophilic drugs are entrapped in the aqueous space, while hydrophobic drugs are incorporated in the lipid bilayers. The water soluble antibiotics gentamicin and ceftriaxone are passively loaded into the liposomal aqueous phase [95] . Ciprofloxacin, a drug that is soluble and charged under acidic and basic conditions, but is neutral and poorly soluble at a physiological pH range, can actively accumulate in neutral liposomes in response to a pH gradient [95,96] . In other cases, it has been shown that the highest encapsulation efficiencies occur when lipids and antibiotics have opposite charges. For example, ticarcillin (a negatively charged β–lactam antibiotic) loaded into cationic liposomes prepared by extrusion technique, increases its entrapment and as well as its killing of P. aeruginosa [97] . Encapsulation efficiency also depends on the characteristics of liposomes and methods of preparation [98] . One of the most commonly used lipids for the preparation of liposomes is the key building block of cell membranes, phosphatidylcholine, which is a neutral phospholipid with fatty acyl chains of varying degrees of saturation and length. Typical methods for preparing liposomes include the sonication (disruption of large multilamellar vesicle suspensions using sonic energy to small unilamellar vesicles) and the extrusion (lipid suspension is forced through a polycarbonate filter with a defined pore size to yield particles having a diameter near the pore size of the filter used) methods [98] . As an example, liposomes composed from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (used to adjust membrane rigidity and stability) had greater entrapment efficiency for polymyxin B when prepared by sonication than extrusion [99] . The difference observed between the two methods of liposomal preparation is attributed to the smaller diameter size and hence, reduced entrapment volume of the extruded liposomes. When liposomes were prepared from another phosphatidylcholine (1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol, the liposomes had similar entrapment efficiencies for polymyxin B, regardless of the method of preparation, an effect attributed to the fluidity state of the liposomal lipid [99] . Experimental & clinical evidence for the use of liposomal antibiotic drugs in P. aeruginosa lung infections In vitro studies for the evaluation of antibiotic properties & mechanism(s) of action of liposomal antibiotic formulations

As resistance to antibiotics is on the rise, it is not surprising that multiple studies have focused on increasing the activity of available antibiotic drugs by means


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Review Alipour & Suntres of delivering antibiotics more efficiently to the bacterial infection sites. Such studies have shown that challenge of mucoid and nonmucoid P. aeruginosa strains with liposomal antibiotic formulations enhanced the bacterial killing when compared with the free antibiotic. For example, liposomally encapsulated antibiotics, such as tobramycin [100] , amikacin [26] , gentamicin [101] , clarithromycin [102] , ceftazidime and cefepime [103] , polymyxin B [99] , ciprofloxacin [104] , and meropenem [105,106] improved the antimicrobial efficacy of the antibiotics against P. aeruginosa. Nonantibiotic agents, such as silver ion and tea tree oil when encapsulated in liposomes, have also shown to improve their antimicrobial efficacy against P. aeruginosa [107] . In vitro studies designed to examine the direct interaction between the liposomal antibiotic formulations and bacteria are a first step in screening the antibacterial effectiveness of the drug-loaded delivery systems. There are several bacterial factors that can play an important role in the interaction/fusion of liposomes with bacteria, such as bacterial membrane properties, the presence of divalent cations, bacterial surface pH, and temperature [108] . Also, understanding the importance of the liposomal characteristics (i.e., type of lipid, lipid charge) is crucial in the development of an optimized vesicle that will efficiently fuse with the intact bacteria, deliver its contents into the cytoplasm and kill the bacteria [108] . For instance, Drulis-Kawa et al. have described an 18 kDa protein on the outer membrane of P. aeruginosa, which may be crucial to the fusion with cationic liposomes, suggesting that fusion may be favored by an outermembrane protein [94] . Using a lipid-mixing assay, Ma et al. also showed that fusion with liposomal tobramycin was independent of liposome size and lamellarity, and was enhanced with addition of calcium ions and phosphatidylethanolamine, the fusogenic lipid commonly found in bacterial membranes [108] . Electron transmission microscopic studies from in vitro studies revealed that the penetration of polymyxin B into P. aeruginosa was higher following its administration as a liposomal formulation and more effective in killing bacteria than when it was administered in its free form [99] . Sachetelli et al. by means of using flow cytometry, lipid-mixing assay and transmission electron microscopy showed a fusion process between tobramycin-loaded fluidosomes (liposomes composed of phosphatidylcholine and a negatively charged phosphoglycerol) and bacterial membranes to explain the bactericidal efficacy of fluidosomes [109] . When liposomes fuse with cell membranes, a high concentration of the antibiotic is immediately delivered in the cytoplasm of bacteria, which can potentially suppress the antibacterial

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resistance by overwhelming the efflux pumps of the bacteria [98] . It is believed that the use of antibiotic combinations that are synergistic in vitro against an offending microorganism might be associated with a significantly better clinical response to antibacterial therapy [110] . In vitro studies have shown that the co-encapsulation of agents with antibacterial properties in a liposomal formulation, increased the susceptibility of bacteria when compared with the effects of either antibiotic tested alone, suggesting that the use of ‘multidrug’ liposomes might be advantageous in the treatment of pulmonary infections. Results from in vitro studies have clearly demonstrated the superiority of liposomal aminoglycosides (e.g., tobramycin gentamicin, amikacin) against CF isolates of P. aeruginosa [101,111,112] however, the co-encapsulation of aminoglycosides with the trivalent bismuth or gallium metals has shown superior antipseudomonal properties [113] . Gallium, due to its chemical similarities to iron, can substitute for iron in many biological systems and inhibit iron-dependent processes. Disruption of iron metabolism increases the vulnerability of most infecting bacteria because iron is essential for growth and the functioning of key enzymes, such as those involved in protein and DNA synthesis, electron transport and oxidative stress. Although bismuth salts alone do not contain strong antimicrobial properties against P. aeruginosa, the susceptibility of bacteria can be increased with the chelation of bismuth to a lipophilic thiol [114,115] . Bismuth-dithiols have been shown to interfere with P. aeruginosa adherence to abiotic and biotic surfaces, iron uptake, virulence factors secretions, and also suppress exopolysaccharide expression and biofilm formation. Incorporation of bismuth-ethanedithiol and tobramycin further increases P. aeruginosa susceptibility [116] . Also, the tobramycin bismuthethanedithiol liposomal formulation exhibits a greater ability to attenuate quorum-sensing, virulence factors, and biofilms [25] and electron microscopic studies show an increase in the penetration of the tobramycin into resistant mucoid P. aeruginosa [117] . The advantages of ‘multidrug’ antibiotic liposomes in vivo studies remain to be seen, however, it is conceivable that the concurrent delivery of two or more drugs to bacteria via a liposomal formulation would be more valuable than the concomitant administration of the same drugs. In vitro studies have also be useful to elucidate whether the effectiveness of the liposomal antibiotics can be improved by the co-administration of adjunct agents that would alter the abnormal airway environment resulting from infection and inflammation to more favorable conditions. As indicated previously, P. aeruginosa in the lungs of CF patients reside within biofilms covered with pathogen-generated polysaccha-



rides and patient-excreted polyanions that retard the diffusion of antibiotics through the biofilm matrix and prevent antibiotics from reaching the buried dormant bacteria. Also, the abnormally viscous airway secretions in CF patients, due to the high content of inflammatory by-products such as the neutrophil-derived DNA and filamentous actin, pose a barrier to the effective diffusion of drugs in the CF lung [118] . The nonantibiotic enzymes DNase and alginate lyase reduce the sputum viscosity and the negatively charged alginate levels in biofilms, respectively and improve the efficacy of liposomal aminoglycosides by enhancing their penetration across these barriers [23] . Efficacy of liposomal antibiotics: evidence from the in vivo & clinical studies It has been more than 40 years since it was suggested that liposomes could become effective drug-delivery carriers of antibiotics against drug-resistant intracellular and extracellular pathogens. Unlike the poor stability, systemic toxicity, and inactivation of conventional antibiotics, once administered, liposomes provide a protective barrier to the drugs against the hydrolytic activity of enzymes as well as the chemical and immunological deactivation [106] . Presently, accomplishments in liposomology include the development of nanocarriers for the delivery of antibiotics with reduced toxicity, which not only increase, but also sustain drug levels in circulation and at the site of infection [98,119,120] . Conventional liposomes (first-generation liposomes composed of phospholipids and cholesterol), when delivered intravenously, are rapidly removed from the circulation by the cells of the reticuloendothelial system, particularly in the liver and spleen [120] . Conjugating ‘stealth’ material (e.g., PEG) on the surface of liposomes extends their blood-circulation time while reducing their uptake by the reticuloendothelial system cells [98] . Nevertheless,

Review

systemic delivery of liposomal antibiotics for the treatment of pulmonary infections has several other limitations including their low accumulation in the lungs, and possible adverse side effects in other organs and tissues. Besides the invasive intravenous route, the inhalation route is becoming a more attractive approach for the administration of antibiotics, given the convenience to the patient and the possibility to administer antibiotics directly to the lung. Delivery of liposomal antibiotics directly to the lungs enables extremely high concentrations of antibiotics to be reached directly at the site of infection potentially overcoming adaptive resistance and limiting their leakage into the bloodstream and distribution to other healthy organs thus, avoiding the potential for cumulative systemic toxicities [121] . The effectiveness of liposomal antibiotics (aminoglycosides, fluoroquinolones) as dry powders (using an inhaler), or in aqueous aerosol form (using a nebulizer) in animals with lung infections, is undoubtedly revolutionizing the management of P. aeruginosa patients [91,100,122,123] . Such studies have been valuable for paving the way to study the safety, efficacy or pulmonary deposition of nebulized liposomal amikacin and inhaled liposomal ciprofloxacin in clinical trials (Table 2) for the treatment of both CF and non-CF bronchiectasis [90,124,125] . Most studies regarding liposomal antibiotics as a potential treatment of pulmonary P. aeruginosa infections deal with the β-lactams, aminoglycosides quinolones, and polypeptides. Certain studies focus on improving the pharmacokinetics and reducing toxicity of antibiotics, while others are designed to demonstrate an enhanced antibacterial activity. In this section, we are providing an overview of the outcomes from animal studies and/or clinical trials set out to examine the effectiveness of these liposomal antibiotic formulations as potential treatments against P. aeruginosa induced lung infections.

Table 2. Liposome-entrapped antibiotics presently in clinical trial studies for the treatment of Pseudomonas aeruginosa lung infections. Product name (manufacturer)

Lipid composition

Administration route

Target

Status

Ref.

DPPC: Chol

Inhalation

CF Phase III Non-CF bronchiectasis

HSPC: Chol

Inhalation

CF Phase III Non-CF bronchiectasis

Amikacin Arikace™ (Insmed†)

[135]

Ciprofloxacin Lipoquin Pulmaquin® (Aradigm‡)

[143]

VA, USA. CA, USA. Chol: Cholesterol; CF: Cystic fibrosis; DPPC: Dipalmitoyl phosphatidylcholine; HSPC: Hydrogenated soy phosphatidylcholine.

† ‡



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Review Alipour & Suntres Effectiveness of liposomal β-lactams in in vivo studies β–lactams are a family of broad spectrum antibiotics that include the bicyclic penicillins, cephalosporins and carbapenems, as well as the monocyclic nocardicins and monobactams, which are regularly used against Grampositive and Gram-negative pathogens [126] . Their extensive availability and use in hospitals and the community has created major environmental pressures for bacteria to evolve towards resistance. Bacterial resistance against penicillins, cephalosporins, monobactams and carbapenems is most often mediated by β-lactamases, which have emerged and evolved rapidly in bacteria, prompting the production of β–lactamase resistant β-lactams [127,128] . Liposomes have been studied as a means to deliver β-lactams, as well as to protect them from β-lactamases [126] . In a study examining the therapeutic efficacy of liposomal cefoperazone against P. aeruginosa in a granulocytopenic mouse model of acute lung infection, it was demonstrated that intraperitoneal administration of liposomal cefoperazone (prepared by the dehydration– rehydration method) was more effective than cefoperazone alone in preventing death of granulocytopenic mice from lethal pulmonary challenge with P. aeruginosa and the bacterial infection was cleared faster from the lungs of mice treated with liposomal cefoperazone; the halflife of free cefoperazone in the lungs following intraperitoneal administration of the liposomal drug was significantly lengthened (13 vs 261 min) [129] . While there are limited in vivo studies examining the efficacy of liposomal β-lactams by inhalation against P. aeruginosa lung infections, much more research effort is encouraged to assess the role of liposomes as a drug-delivery system in restoring the β-lactams’ activity by avoiding resistance mechanisms and helping to get the drug to the site of infection. Effectiveness of liposomal aminoglycosides in animal & human studies The efficacy of liposomal aminoglycosides has been studied for more than a decade in preclinical models of CF infection [26,122,124,130,131] . It has been shown that the intratracheal administration of the liposomal tobramycin is safe, nonimmunogenic, enhances biodistribution and elevates concentration of the antibiotic in the lungs, reduces systemic toxicity (lowered serum levels), and lowers P. aeruginosa counts when compared with conventional tobramycin [122,130,131] . Recently, the intratracheal administration of a tobramycin and bismuth–ethanedithiol liposomal formulation (prepared by a modified dehydration–rehydration method), was studied in an in vivo murine lung infection, showing a reduction in bacterial load [132] .

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Preclinical studies have demonstrated the ability of inhaled liposomal amikacin to penetrate expectorated CF sputum with the sustained release of amikacin from liposomes significantly lessening bacterial counts in a rat infection model [26] . These promising results have warranted the entry of liposomal amikacin (marketed as Arikace™ by Insmed; VA, USA) into clinical trials. Phase I/II clinical trials have shown that the administration of 500 mg liposomal amikacin using a nebulizer once daily for 14 days was safe and well tolerated [124,125] . Insmed completed Phase I/II clinical trials of Arikace, in a nebulized formulation [133] prepared from neutral unmodified liposomes (DPPC:cholesterol). These studies determined the safety, tolerability, pharmacokinetics and pharmacodynamics of Arikace (28 days; up to 560 mg daily Arikace inhalation) in CF patients [134] . CF patients receiving Arikace had reduced P. aeruginosa in sputum and increased forced expiratory volume. Phase III clinical trials have been initiated to determine the effectiveness of Arikace in the treatment of chronic lung infections caused by P. aeruginosa in CF patients and to compare it with TOBI, an inhalation antibiotic formulation of tobramycin already available for use [135] . Effectiveness of liposomal ciprofloxacin in animal & human studies Fluoroquinolones are a class of broad-spectrum antibiotics commonly used to treat lung infections in those with CF. Anti-pseudomonal properties are used commonly in P. aeruginosa eradication regimens, the treatment of mild exacerbations in those chronically infected with P. aeruginosa and for the treatment of infections with other bacteria, including Stenotrophomonas maltophilia. Oral or intravenously administered fluoroquinolones have been associated with improvements in lung function and clinical score; however, in the majority of these studies, a sustained effect is not seen once the antibiotic course is completed suggesting that treatment may be sub-optimal and concerns about side effects remain [136,137] . Inhaled versions of ciprofloxacin have shown good tolerability and microbiological efficacy in preliminary studies [138] . The advantages of inhaled therapy are; increased antibiotic concentration at the site of infection, enhanced bacterial killing, and reduction in need for intravenous and prolonged oral therapy to reduce systemic toxicity [139] . Liposomal ciprofloxacin (prepared using negatively charged lipids) delivered either by inhalation or intravenous routes have shown great promise in the treatment of P. aeruginosa lung infections as evidenced by the increased concentrations of ciprofloxacin in the lungs, alveolar macrophages with reduced distribution into the circulatory system and potent activity against lipopolysaccharide-induced pneumonia [140,141] .



Bakker-Woudenberg et al. examined the activity of ciprofloxacin-loaded long-circulating negatively charged liposomes (liposome surfaces coated with PEG) in a P. aeruginosa infection model [142] and concluded that conventional ciprofloxacin alone, (twice daily for 7 days) was not effective in an acute infection model, but ciprofloxacin-loaded long-circulating negatively charged liposomes along with the conventional form (one injection on the first day) was 100% effective. Recently, Aradigm Corporation (CA, USA) introduced liposomal ciprofloxacin formulations for the treatment of CF (Lipoquin™ or ARD-3100) as a rapid-release formulation and the treatment of non-CF bronchiectasis (Pulmaquin™ or ARD-3150) as a slowrelease formulation [143] . Lipoquin consists of ciprofloxacin active-loaded in a liposomal formulation, while Pulmaquin is a dual release ciprofloxacin for inhalation formulation containing Lipoquin mixed with a solution of free ciprofloxacin for inhalation in order to provide an initially high peak of ciprofloxacin in the lung [49] . Preclinical studies have confirmed favorable pharmacokinetic characteristics of inhaled liposomal ciprofloxacin with the liposomal component having a lung clearance half-life after inhalation of approximately 12 h (compared with approximately 1 h for free ciprofloxacin) supporting once-daily dosing. Clinical trials studying safety, tolerability and pharmacokinetics with increasing doses (450, 300 or 150 mg; 14 or 28 days) showed that the formulation was tolerable, increased forced expiratory volume, lowered ciprofloxacin levels in plasma, elevated ciprofloxacin levels in sputum and reduced P. aeruginosa density [89,92] . In addition, in in vitro and ex vivo models, liposomal ciprofloxacin formulation has shown a slow-release rate and low absorption in rat lungs [144] . In a clinical Phase IIa trial involving 22 adult CF patients, Lipoquin was shown to be safe and well-tolerated when administered once-daily for 14 days at a 300 mg dose [49] . Further Phase II clinical trials to assess the safety and efficacy of liposomal ciprofloxacin in adults with CF and non-CF bronchiectasis and ciprofloxacin-sensitive P. aeruginosa have shown that once-daily inhaled formulations demonstrated strong antipseudomonal microbiological efficacy, was well tolerated and delayed P. aeruginosa exacerbation [90] . Effectiveness of liposomal peptide antibiotics in in vivo studies Polymyxin B (colistin) has bactericidal action against almost all Gram-negative bacilli. It is a cyclic-polycationic peptide antibiotic that binds to anionic lipids and leading to permeability changes in the outer membrane resulting in bacterial cell death. Their nephrotoxic and neurotoxic side effects limited their use; however, in the last decade the emergence of multidrug-resistant Gram-


Review

negative bacteria led to the reintroduction of polymyxins into clinical practice [145] . Incorporation of antibiotics in liposomes is known to enhance their antibacterial efficacy while minimizing their toxic effects [99,146] . Polymyxin B-loaded liposomes administered by intratracheal instillation or intravenous administration increased drug concentrations in the lungs of the rodents, reduced proinflammatory markers and significantly reduced the P. aeruginosa infection in comparison to conventional polymyxin B [146] . In addition, the absence of measurable quantities of polymyxin B in the kidneys and serum of animals treated intratracheally with liposomal polymyxin B suggests that most of the liposomal polymyxin B did not escape into the general circulation, or it was present below the level of detection, in either case, decreasing the risk of systemic effects and nephrotoxicity [146] . Preliminary biodistribution studies in mice following intravenous administration of liposomal polymyxin B suggested that the drug exposures achieved in renal tissues were lower with liposomes compared with the standard solution [147] . Delivery of polymyxin B as a liposomal formulation can be effective in the treatment of pulmonary infection with P. aeruginosa by enhancing retention of the antibiotic in the lung while at the same time decreasing its systemic exposure and adverse effects. Liposomal delivery of newer compounds The shortage of new antibiotics combined with the increasing number of antibiotic-resistant bacteria constitutes a worrying threat for the population worldwide and a critical challenge for healthcare institutions. A better understanding of bacterial growth, metabolism and virulence has offered several potential targets for nonantibiotic antimicrobial therapies, such as targeting adhesion, communication, toxins, virulence factors, direct bacterial killing by bacteriophages and vaccine strategies [148] . The search for newer compounds with antibacterial activity has focused on the isolation and identification of plant-derived materials that can have their activity enhanced by entrapment in liposomes. An increase in the penetration of plant-derived materials into bacteria cells and their nonsensitivity to degradation by bacterial enzymes may explain the mechanism of the enhanced antimicrobial activities of these liposomal formulations [149,150] . For example, encapsulation of cyanidinum chloride in liposomes by the extrusion technique demonstrated efficacy against a resistant strain of P. aeruginosa ATCC 15692 in an in vivo skin infection [151] . A cyanidinum ion is a hydrolysis product from cyanidin salts and a flavonoid occurring in many red berries [151] . Photodynamic inactivation (PDI) of bacteria is a promising approach for combating the increasing emergence of antibiotic resistance in pathogenic bacteria [152] . PDI involves the use of certain dyes, termed photosensi-


419

Review Alipour & Suntres tizers, which are able to store the absorbed energy in longlived electronic states upon light activation with appropriate wavelengths and thus, make these states available for chemical activation of the immediate surroundings. The interaction with molecular oxygen leads to different, very reactive and thus, cytotoxic oxygen species, which damage irreversible the pathogens during illumination [153] . In a study examining strategies to improve PDI efficiency on bacteria, a generation II photosensitizer (temoporfin) was incorporated into liposomes, followed by conjugation with a specific lectin (wheat germ agglutinin; WGA) on the liposomal surface. Fluorescence microscopy revealed that temoporfin was delivered to P. aeruginosa, while flow cytometry demonstrated that WGA-modified liposomes delivered more temoporfin to bacteria in vitro compared with nonmodified liposomes. Consequently, the WGA-modified liposomes significantly enhanced the PDI of P. aeruginosa [154] . Cationic antimicrobial peptides, an innate line of defense against pathogens, have also shown increased activity when coupled to liposomes. Using site-specific attachment of cationic antimicrobial peptides to polymerized liposomes, increases antibacterial activity in absence of increased cytotoxicity to human corneal epithelial cells [155] . To combat multidrug resistance attributed to the production of efflux pumps, novel anionic liposomes bearing anti-oprM phosphorothioate oligodeoxynucleotide (oprM is involved in drug extrusion) have shown to reduce oprM expression and increase susceptibility to antibiotics [156] . Designing liposomal vaccines for the induction of a humoral response to pilin have also shown limited success with intranasal vaccination resulting in IgA and/or IgG production directed against an immunogenic peptide of P. aeruginosa [157] . Conclusion There is a serious and growing problem of antibiotic resistance in P. aeruginosa to conventional antibiotics. Also, the toxicity and adverse effects of some antibiotics to healthy tissues poses a significant constraint in their use. To deal with these issues, research activities are directed in the improvement of existing strategies and search for new molecules that promise to be more effective and more resilient. Among the most promising opportunities for enhancing efficacy, as well as reducing toxicity, of antibiotics for the treatment of lung P. aeruginosa infections is the exploitation of drug-delivery systems such as liposomes. Liposomes have been studied as potential nanocarriers of therapeutics to cells, macrophages, fungi and bacteria. They can be prepared from natural and synthetic phospholipids that are biocompatible, biodegradable, and nonimmunogenic and practically do not

420


cause toxic effects or antigenic reactions. The ability for precise adjustments of liposome parameters such as size, charge, lipid composition, and the conjugation of ligands coupled with the more efficient and safer loading techniques position liposomes favorably to revolutionize antibiotic delivery. In vitro studies show that challenge of mucoid and nonmucoid P. aeruginosa strains with liposomal formulations loaded with antipseudomonal antibiotics enhanced the bacterial killing when compared with their corresponding conventional form. Administration of liposomal antibiotic formulations to animals with P. aeruginosa lung infection enhances the antibacterial efficacy while minimizing the toxic effects of the conventional antibiotics. Clinical trials are currently underway to assess the safety and efficacy of liposomal ciprofloxacin or amikacin formulations in the treatment of P. aeruginosa lung infections. Future perspective The discovery and development of new antibiotics that are urgently required to prevent the growing health threat posed by P. aeruginosa and other resistant pathogenic microorganisms is disappointing. Treatment of P. aeruginosa lung infections, particularly in patients with chronic infections, involves the modification of existing antibiotic therapy protocols that involve prevention or eradication of the infection. Still, current antibiotic therapy is barely able to eliminate P. aeruginosa colonization and no preventative measures have been found to be effective. The clinical use of most conventional antibiotics is limited by the low concentrations of drugs delivered to the lungs, the involvement of intrinsic and adaptive resistance of bacteria, and/or the occurrence of toxicity in healthy organs and tissues. Inhalational administration of antibiotics directly to the lung requires lower doses, which often result in reduced systemic adverse effects. Also, liposomes can change the pharmacokinetic parameters of these encapsulated antibiotics by enhancing their retention and concentration at the site of infection and/or by reducing their toxicity. The extensive research efforts carried out over the last few decades have transformed liposomal drug delivery from a concept to clinical applications. Currently there are a few liposome-based drugs approved for clinical use and more are in various stages of clinical trials offering the potential to enhance the therapeutic index of drugs, such as those used for the treatment of cancer, fungal infections, and Hepatitis A. The Phase II and III clinical trials for aerosolized liposomal amikacin or ciprofloxacin for the treatment of P. aeruginosa lung infections in CF and non-CF bronchiectasis patients are evidence that demonstrate that in the next few years the liposome-based antibiotic-delivery systems will prove to be remarkable treatment alternatives for infectious dis-



eases. For P. aeruginosa lung infections, the inhalational route of administration of liposomal antibiotic formulations has the potential to increase patient compliance, reduce the duration of the antibiotic treatment and possibly decrease the likelihood of bacterial resistance. The unique properties of liposomes will provide many opportunities to develop a wide range of liposomes to offer a clinically proven, biocompatible versatile platform for the enhancement of pharmacological efficacy of antibiotics. It is anticipated that in the next few years, liposomes may be used to deliver a combination of antibiotic agents (i.e., aminoglycoside and β-lactam) or an antibacterial agent and an inhibitor of a major resistance mechanism (i.e., β-lactam and β-lactamase inhibitor). Also, liposomes that specifically target bacterial cells may be engineered by attaching amino acid fragments,

Review

such as antibodies or proteins or appropriate fragments that target specific sites in bacteria. It is envisioned that the future of liposomal antibiotic development is bright with a number of these lipid-based products proving to be remarkable treatment alternatives in the treatment of P. aeruginosa lung infections. Financial & competing interest disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert t-estimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive Summary Background • Pseudomonas aeruginosa is a Gram-negative bacterium that can cause serious lung infections in cystic fibrosis, non-cystic fibrosis bronchiectasis, immunocompromised and mechanically ventilated patients.

Current treatment of P. aeruginosa lung infections • Therapeutic management of P. aeruginosa lung infections is challenging because the high-level resistance of these microorganisms to most classes of antimicrobial agents often leads to clinical failure.

Liposomes: an antibiotic drug-delivery system • Liposomes are biocompatible, biodegradable and essentially nontoxic vesicles utilized as drug carriers in drugdelivery systems. • Development of liposomal antibiotic formulations improves the therapeutic efficacy of drugs by reducing drug toxicity and/or enhancing drug delivery at the site of infection.

Experimental & clinical evidence for the use of liposomal antibiotic drugs in P. aeruginosa lung infections • In vitro studies show that challenge of mucoid and nonmucoid P. aeruginosa strains with liposomal formulations loaded with antibiotics (tobramycin, amikacin, gentamicin, clarithromycin, ceftazidime, cefepime, polymyxin B, ciprofloxacin and meropenem) enhanced the bacterial killing when compared with their corresponding conventional form. • Administration of liposomal antibiotic formulations to animals with a P. aeruginosa lung infection enhances the antibacterial efficacy while minimizing the toxic effects of the conventional antibiotics. • Clinical trials are currently underway to assess the safety and efficacy of liposomal ciprofloxacin or amikacin formulations in the treatment of P. aeruginosa lung infections.

Conclusion • The future of liposomal antibiotic development is promising with a number of these lipid-based products evolving to be remarkable treatment alternatives for the treatment of P. aeruginosa lung infections.

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