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The trachea, the largest airway bifurcates into the bronchi. These bronchi additionally branch into smaller sized bronchioles. The respiratory bronchioles ends with the alveolar sac as indicated in Figure 1. The conducting airways are lined with ciliated columnar epithelium that becomes cuboidal approaching the distal airways. A thin layer of serous fluid covers the lumen of the bronchial airways. A mucosal layer floating on this serous fluid assists in the entrapment of aerolised particles. There is movement of the mucosal layer towards the proximal airways, where it is either swallowed or expectorated (mucociliary clearance). Movement is coordinated by the rhythmic beating of cilia.
Figure 1- Schema of the respiratory system (Learning the Respiratory System, 2008)
Type I pneumocytes which share the basement membrane with the pulmonary capillaries primarily form the alveolar surface. Additionally, the alveolar surface is also composed of type II pneumocytes and macrophages. The role of type II pneumocytes and macrophages is to secrete lung surfactant that prevents alveolar collapse and clearing large particles, respectively. There are approximately 300 million alveoli in the lungs, with a combined surface area that is greater than 100 m2, and with an alveolar epithelium as thin as 0.1 mm. This large surface area, combined with an extremely thin barrier between the pulmonary lumen and the capillaries, creates conditions that are well suited for efficient transfer of materials (Bailey and Berkland, 2009).
The lungs are favourable route for non-invasive drug delivery providing advantages for both systemic and local application. Drug delivery through the pulmonary route has advantage over other delivery routes such as oral and injections. Apart from the pulmonary route being non-invasive, other advantages include the high solute permeability of the lungs, vast surface area for absorption and limited proteolytic activity. Respiratory diseases like asthma or cystic fibrosis are favourably treated by this delivery route where drugs can be targeted locally for action in the lungs. Other potential benefits for target-specific delivery include reduction in overall dosage as well as minimising the side-effects that would result from high levels of systemic drug exposure. Alternatively, systemic drug delivery can be achieved by targeting delivery to the alveolar region where the drug can be absorbed through the thin layer of epithelial cells and into the systemic circulation. This can be desirable to achieve a rapid onset of action, the avoidance of first-pass metabolism or the delivery of biotherapeutics (i.e. peptides and proteins) that cannot be delivered orally (owing to enzymatic degradation and poor intestinal membrane permeability) thus require parenteral delivery. Furthermore, the lungs can be targeted for delivery to specific lung cells such as alveolar macrophages, for treatment of diseases such as tuberculosis.
The process of breathing continuously involves exposing the lungs to materials of various sources and sizes like pollen (20-90 µm), bacteria (0.2-200 µm) and tobacco smoke (0.01-1 µm). These airborne particles deposit along the respiratory tract from the conducting upper airways (with the oropharynx, trachea, main bronchi and terminal bronchioles) down to the respiratory region of the lower airways (with respiratory bronchioles and alveolar sacs). In the upper airways, cilia rapidly clear the particles from within the mucous layer that lines the epithelia to the throat. This site allows the metabolism of the swallowed particles. The pulmonary epithelium is thick (50-60 µm) in the trachea and poses a barrier to absorption. Towards the lower airways, the epithelium of the lung diminishes to a thickness of 0.2 µm in the alveoli. It is in this region that gas exchange occurs and the vast surface area of the alveoli (43-102 m2 in an adult human) provides a highly vascularised expanse with access to the entire systemic circulation. In return, the alveoli are protected by cells of the immune system called the alveolar macrophages. These cells scavenge for foreign materials along the lung surface however allowing particles of extremely small or large sizes to escape phagocytosis. Also, other immune cells like dendritic cells are present throughout the airways where they sample for pathogens and foreign substances (Sung, et al., 2007).
Cystic fibrosis (CF) is an autosomal recessive disease which is affects more than 600,000 people globally (Moss, 2002; Pilcer, et al., 2006). CF is a respiratory disease which is characterized by endobronchial infection, exaggerated inflammatory response, progressive airway obstruction, bronchiectasis and eventually respiratory failure (Gibson, et al., 2003; Geller, 2009). CF is associated with mutation of the gene coding for chloride channel protein known as cystic fibrosis transmembrane conductance regulator (CFTR), resulting in covering of lumen of the airways by thick mucus which leads to decrease in mucociliary clearance (Collins, 1992; Moss, 1995). CF sputum contains high amount of glycoproteins and neutrophil derived polyanions including DNA and F-actin, which allows for bacterial growth and makes difficult for host defense and therapeutics to act (Sutherland, 2001; Bucki, et al., 2007; Palmer, et al., 2007; Yang, et al., 2007). The main role of CFTR is that it acts as a chloride channel along with many other regulatory roles like regulation of the outwardly rectifying chloride channel, inhibition of sodium transport through the epithelial sodium channel, regulation of ATP channels, acidification of intracellular organelles, regulation of intracellular vesicle transport and inhibition of endogenous calcium-activated chloride channels. CFTR is also required in bicarbonate-chloride exchange. Lack of bicarbonate secretion results in aggregation and poor solubility of luminal mucin (O'Sullivan, et al., 2009).
The most important bacterial pathogen in cystic fibrosis is Pseudomonas aeruginosa which is found in 54.4% of cystic fibrosis infected patient and in 80% of patient with cystic fibrosis by 18 years of age. After establishment of P.aeruginosa in the lungs of CF patient, the condition can relapse (Geller, 2009). Persistent infection with P.aeruginosa induces generation and secretion of chemotactic cytokines that leads to formation of large numbers of polymorphonuclear cells in the airways. P.aeruginosa releases toxins and elastases which intensify the cycle of inflammation and infection that leads to cleavage of crucial surface markers on polymorphonuclear cells. These polymorphonuclear cells release their own elastases and proteases that worsen injury to any surrounding viable cells (Hartl, et al., 2007). Subsequently, damaged neutrophil products and bacterial exotoxins stimulate inflammation, tissue damage and additional polymorphonuclear cell recruitment. These cells release DNA which leads to increased sputum viscosity. There are various reasons which lead to the growth of P.aeruginosa in the airways of cystic fibrosis patient such as increased bacterial binding to the epithelium, permissive microenvironments within the hypoxic niches of adherent mucous plaques and reduced bacterial clearance via innate immune response. At the beginning, P.aeruginosa develops as non-mucoid strain that can be either elucidated by the host or eliminated with antibiotic treatment. Eventually, P.aeruginosa colonies produce an alginate coat and forms biofilm (O'Sullivan, et al., 2009). There can be an increase in P.aeruginosa growth and biofilm formation caused by mutated CFTR through raising the iron content in the apical domain of epithelia. Moreover, P.aeruginosa develops the quorum sensing (QS) phenomenon by giving rise to signalling molecules (N-acyl homoserine lactones (AHL)) that regulate expression genes which control biofilm formation, thus contributing to tolerance of P.aeruginosa to antimicrobial chemotherapy and host innate system (Halwani, et al., 2009).
Cystic fibrosis airways may also get adversely affected by other pathogens, such as Burkholderia cepacia, Stenotrophomonas maltophilia, meticillin-resistant S aureus (MRSA) and atypical mycobacteria. Various Burkholderia species are transmitted from person to person, resistant to antibiotics and are highly virulent. There is rapid decrease in pulmonary function and increased mortality in patients with infection of B.cepacia complex. Sometimes, infection with B.cepacia complex may cause fatal and invasive bacteraemia - "Cepacia Syndrome". Approximately 15-20% of CF patients carry MRSA in their airways and it is responsible for colonisation which decreases lung function. S.maltophilia has been found in many patients with cystic fibrosis but till now it has not shown any significance decline in pulmonary function or wellbeing. Atypical mycobacteria (Mycobacterium avium complex-72% and Mycobacterium abscessus-16%) are found in airways of some patient with cystic fibrosis but it is unclear about infection caused by it or there is only saprophagous colonisation. Aspergillus fumigatus is a fungus which causes colonisation without invasive infection and also causes intense allergic response known as allergic bronchopulmonary aspergillosis (ABPA). Its occurrence varies geographically and is observed in 1-15% of patients suffering from cystic fibrosis. Various clinical indications of ABPA are pulmonary infiltrates, central bronchiectasis, and wheezing (O'Sullivan, et al., 2009). P.aeruginosa is the predominant pathogen (Figure 2).
Figure 2 - Age-specific prevalence of airway infections in patients with CF (Gibson, et al., 2003)
Biofilm are complex bacterial communities found attached to biological or inert surfaces and surrounded by bacterially-produced extracellular matrix composed of exo-polysaccharides, proteins and DNA. A biofilm involves sensing and responds to bacterial cell density, nutrient availability and energy source present in the environment, as it develop in a complex and well co-ordinated manner. Biofilm formation is considered as a survival strategy to bacteria (Moreau-Marquis, et al., 2008). The expansion of biofilm occurs by those cells which are involved in the process of biofilm formation as they express enzymes involved in the synthesis and secretion of exo-polysaccharides and proteins. Bacteria become resistant to antibiotics as they start expressing some proteins. This leads to impairment of oxygen and nutrients transportation. Further, there is increase in the number of bacteria which exacerbates the metabolic stress exerted on bacteria. This leads to rupture of the biofilm and released bacteria regain their initial transcriptome, and begin to colonize the support at several other locations. This phenomenon accounts for a decline in the clinical status of the patient. Therefore, a study of bacteria within the biofilm is also required when studying the sensitivity of bacterial strains to various treatments (Nagant, et al., 2010).
Treatment and Prevention of Cystic Fibrosis:
Clearance of airway secretion:
CFTR dysfunction prevents chloride ion transport into the airway lumen, which causes dehydration of the airway surface liquid lining the airway epithelium. Therefore, cilia are not able to function properly leading to a decrease in mucociliary clearance. Moreover, there is a high level release of DNA from degenerating leukocytes due to neutrophilic inflammation and this also leads to increase in mucus viscosity. Current therapies include dornase alfa and hypertonic saline. The role of dornase alfa is to break down DNA and decrease mucus viscosity where as hypertonic saline may improve airway surface hydration (Zemanick, et al., 2010).
There are several classes of mucoactive agents: mucolytics, which disrupt mucus or sputum polymers; ion-transport modifiers, which promote ion and water transport across the epithelium of the airway; mucokinetics, which improve cough mediated clearance by increasing airflow or reducing sputum adhesivity; expectorants, which add water to the airway; and other mucoregulatory agents. Two new treatments currently in clinical trials are discussed below, denufusol and inhaled mannitol.
Denufusol tetrasodium is a P2Y2-receptor agonist and P2Y2 receptors are present on the luminal surface of the airway epithelial cells. They act as ion-transport modifiers, which stimulate chloride ion secretion and inhibit sodium ion absorption, leading to hydration of the airway lumen. They also have secretagogue function i.e., secretion of mucin from submucosal glands and goblet cells (Bye and Elkins, 2007). Nebulised denufusol has demonstrated safety and tolerability in healthy non-smokers, smokers, and patient with CF, while some increase in adverse events and intolerability was noted among CF subjects with lower lung function (<75%) during Phase I and early Phase II studies (Deterding, et al., 2005).
Inhaled mannitol is available as a stable dry powder for inhalation. Its mechanism of action is by creating an osmotic gradient which causes as influx of water into the airway and restoring volume of airway surface liquid (ASL) (Bye and Elkins, 2007). Phase III, multinational randomized trials are ongoing to determine the safety and efficacy of inhaled mannitol in patients with CF.
Inhaled bronchodilators are regularly prescribed for cystic fibrosis patient with atopy or those who develop hyper-reactivity secondary to bronchial damage. Bronchodilator therapy may increase mucociliary transport, exercise tolerance and decrease dyspnoea and inflammatory damage to the airways. The most commonly prescribed inhaled bronchodilators are short acting salbutamol or the long acting salmeterol (Heijerman, et al., 2009).
Transplantation is the final therapeutic option for CF patients with end stage lung disease. It is helpful in selected patient and has the potential to extend and substantially improve quality of life. 5-year survival post transplant for children is less than 50%, with slightly better outcomes in adults (50% of recipients are alive 6 years post transplant) (O'Sullivan and Freedman, 2009).
Treatment of pulmonary infections: Antibiotic therapy
The standard treatment for P.aeruginosa endobronchial infections in CF patients includes the administration of two parenteral anti-pseudomonal antibiotics, including a Î²-lactam and an aminoglycoside agent. Antibiotic treatment helps to stabilize lung function and, if possible, restoration of the lost lung functions in cystic fibrosis patients. As parenteral antibacterial regimens are commonly used to treat acute infection in disease like bronchiectasis, pneumonia, cystic fibrosis and chronic obstructive pulmonary disease (COPD), the aerosolized antibiotics have showed to improve lung function, reduce systemic long-term toxicity as well as decrease hospitalization. Moreover, aminoglycoside antibiotics are highly polar and exhibit poor drug penetration into the endobronchial space when administered parenterally. Also these classes of antibiotics have narrow safety margin and may cause severe ototoxicity and nephrotoxicity (Parlati, et al., 2009).
Figure 3 - Structure of Tobramycin
Appearance - White
Solubility - Freely soluble in water, very slightly soluble in ethanol (96%)
By inhalation of nebuliser solution, adult and child over 6 years, 300 mg every 12 hours for 28 days, subsequent courses repeated after 28 days interval without tobramycin nebuliser solution
Bramitob® - Nebuliser solution - 75 mg/ml
Tobi® - Nebuliser solution - 60 mg/ml (BNF, 2009)
The bactericidal activity of tobramycin (O-3-amino-3-deoxy-Î±-D-glucopyranosyl-(1-6)-O-[2,6diamino-2,3,6-trideoxy-Î±-D-ribohexopyranosyl-(1-4)]-2-deoxy-D-streptamine) is accomplished by irreversibly binding to 30S and 50S ribosomal subunits resulting in a defective protein. Tobramycin also has relatively narrow safety margin in comparison to other aminglycoside antibiotics. The therapeutic plasma concentration of tobramycin is in the range of 4-8 mg/L (Feng, et al., 2002). The administration of aminoglycoside by inhalation is an alternative route to deliver high concentration of antibiotics directly to the site of infection while minimizing systemic bioavailability. Pulmonary inhalation is not subjected to first-pass metabolism, in comparison to oral route of drug administration (Pilcer, et al., 2006).
Chemotherapeutic agent can be encapsulated in inert nanoparticles in order to decrease drug toxicity. Liposomes are most commonly used as nanoparticles for encapsulation as they are non-immunogenic and biodegradable (Halwani, et al., 2009).
Liposomes are closed, bilayer-membrane vesicles that have an aqueous centre surrounded by a phospholipid membrane and can be either unilamellar or multilamellar vesicles with a size range from 50 nm to several micrometres. Vesicles formulations are composed of natural or synthetic phospholipids, lipoproteins and cholesterol. Vesicles act as carriers for both hydrophobic and hydrophilic compounds. There are three types of vesicles mainly large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) and small unilamellar vesicles (SUVs). The lipid surface can be use to associate charged drug, and the size of the vesicles extensively influences drug distribution. The physicochemical properties of liposomes can be modified by changing (Figure 4):
â€¢ the types of lipids;
â€¢ the composition and proportions of lipids in the liposomal formulation;
â€¢ the size of the liposome;
â€¢ the charge of the liposomal surface: positive, negative, or neutral;
â€¢ pH sensitivity;
â€¢ temperature sensitivity;
â€¢ the fluidity of the liposomal membrane: rigid and fluid liposomes.
Figure 4 - Properties of liposomes (Drulis-Kawa, et al., 2010)
Common methods of preparation for liposomes are the thin film method technique, reverse phase evaporation method, proliposome method, freeze thawing method, and relatively novel detergent dialyzing method, etc. There are various factors like lipid composition, methods of preparation, charges that drug carries, drug/lipid ratio, electrostatic and hydrophobic forces which affect the characteristics of the subsequent production, such as size, charge, encapsulation efficiency, the amount of drug loaded, release rate and stability (Bi and Zhang, 2007).
Pulmonary surfactants are produced by the alveolar type II cells in the lungs. There are two major pools of surfactants: an intracellular and an extracellular surfactant compartment. The intracellular compartment consists of the lamellar bodies in the alveolar type II cells which functions as storage of surfactant before it is released into the alveolar space. The extracellular compartment is surfactant that is secreted into the alveolar space, and its collection is done by bronchoalveolar lavage.
Pulmonary surfactants are composed of two main components namely, lipids (90%) and surfactant specific proteins (10%). Lipids mainly consist of phospholipids and other lipids are triacylglycerol, cholesterol and free fatty acids. 70-80% of the total amount of lipids contains phosphatidylcholine (PC) and it is present in the saturated form (50-70%) as the dipalmitoylated form (DPPC). Surfactant proteins can be categorised into two groups: hydrophilic surfactant proteins SP-A and SP-D, and the hydrophobic surfactant proteins SP-B and SP-C. These are exclusively associated with lungs. Hydrophilic surfactants main role is the first line defense against inhaled pathogens and specifically SP-A has a regulatory function in the formation of the monolayer that lowers the surface tension (Creuwels, et al., 1997).
The main function of pulmonary surfactant is to maintain the stability of the lung by reducing the surface tension at the air/alveoli interface (Yu, et al., 1999). Lipids can exist in two forms, i.e. either in fluid liquid-crystalline form or solid gel form. At the phase transition temperature (Tm), lipids undergo transition between these two forms. On inspiration, surfactant lipid spreads on the alveolar surface if surfactant film exists in the liquid crystalline form. As the DPPC (Table 1) has a phase transition temperature of 41oC, its film will exist in the gel form at body temperature and hence adsorb slowly to the air-liquid interface. After addition of other lipids like cholesterol into the surface film upon inspiration lower the Tm of the lipid mixture, enabling it to exist in the fluid state at the same body temperature. Therefore, in fluid state these lipids are able to disperse to coat the surface of the expanding fluid layer (Daniels and Orgeig, 2003).
Table 1 - Different lipids and its structure.
Delivery of liposomal antibiotics to lungs
Several antibiotics in healthcare have limited application because of poor bio-distribution, pharmacokinetics and toxicity. Lipid vesicles can be used for encapsulation of drug in order to alter pharmacokinetic and pharmacodynamic properties. There are several advantages of liposomes as antibiotic carriers:
â€¢ Improved pharmacokinetics and biodistribution, decreased toxicity - Liposomes act as a carrier and they help in gradual or sustained release of antibiotics during drug circulation in the body. This will maintain the proper drug concentration for a relatively long term. Encapsulation of drug in liposome vesicles will also improve pharmacokinetics and protect antibiotic against the hydrolytic activity of enzyme, chemical and immunological deactivation (Drulis-Kawa, et al., 2010).
â€¢ Enhanced activity against intracellular pathogens - Intracellular bacterial diseases can be treated by rigid conventional liposome vesicles and PEG-coated vesicles which can improve drug retention in tissues, provides sustained release, decrease toxicity and enhance the concentration at the site of infection. Some studies have demonstrated that application of liposomal forms of rifampicin, isoniazid and clarithromycin considerably enhanced antibacterial efficacy in comparison to free drugs (Drulis-Kawa, et al., 2010).
â€¢ Target selectivity - After intensive research, drug carriers have shown the possibility of targeting liposomes to particular micro-organisms, tissue and organs. Target selectivity of liposomal drug formulation may be achieved by addition of proteins, specific immunoglobulin, specific oligosaccharides chain and construction of thermo-sensitive and pH-sensitive vesicles. The type of interaction with target, i.e. specific and non-specific, depends on the composition of the vesicle surface. Specific targeted liposomes consist of antibodies, proteins, or immunoglobulin fragments which have affinity to specific receptors located on the target surface (infected cells or pathogen). In non-specific targeting of the liposomes, charge of the membrane plays vital role. Cells possessing negatively charged surfaces like eukaryotic and bacterial cells exhibit strong vesicle-cell interaction with positively charged liposomal vesicles (Drulis-Kawa, et al., 2010).
â€¢ Enhanced activity against extracellular pathogens, in particular to overcome bacterial drug resistance - There are many studies describing lipid formulations, drug distribution and vesicle-bacterium interactions leading to enhancement of antimicrobial drug activity against most common extracellular bacteria, such as P.aeruginosa, K.pneumonia, E.coli, S.aureus, and Acinetobacter species. Encapsulation of antibiotics like aminoglycoside and fluoroquinolones were selected (Table 2) (Drulis-Kawa, et al., 2010).
Table 2 - Extracellular bacterial eradication by using liposomal aminoglycosides and quinolones (Drulis-Kawa, et al., 2010).
Burkholderia cepacia; Burkholderia cenocepacia.
Nanoparticles for the purpose of drug delivery are defined as sub-micron (<1 µm) colloidal particles. This includes nanocapsules in which the drug is confined to an aqueous or oily core surrounded by a shell like wall and monolithic nanoparticles in which drug is dissolved, adsorbed or dispersed throughout the matrix (Gelperina, et al., 2005).
Nano-carrier systems offer many advantage in pulmonary delivery:
1) The potential to achieve relatively uniform distribution of drug dose among the alveoli;
2) An achievement of enhanced solubility of the drug than its own aqueous solubility;
3) The sustained-release of drug which consequently reduces the dosing frequency;
4) Suitability for delivery of macromolecules;
5) Decreased incidence of side effects;
6) Improved patient compliance; and
7) The potential of drug internalization by cells.
Nano-carrier systems for pulmonary delivery include:
Polymeric nanoparticles are used in pulmonary delivery system to carry drug molecules, to control drug release and to protect drug from degradation. Therapeutically used polymeric nanoparticles are poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (¥-caprolactone) (PCL), alginic acid, proticle, chitosan and gelatin. These are composed of biocompatible and biodegradable materials. They are extensively studied using various important pulmonary drugs like anti-tuberculosis drugs, anti-asthmatic drugs, anticancer drugs and pulmonary hypertension drugs because of their sustained release properties, surface modification capability and biocompatibility (Mansou, et al., 2009).
Solid Lipid Nanoparticles (SLNs) are made from solid lipids (i.e. lipids solid at room temperature), surfactant(s) and water. There are many advantages of SLNs for the release of drugs in the lung such as prolonged release, control of the release profile and faster in vivo degradation compared to particles made from PLA and PLGA. In comparison to other particles made up from polymeric materials, they have high tolerability in the lungs. Pulmonary delivery of SLNs using physiological lipids has a better toxicological profile as compared to polymer based system, because physiological lipids have little or no cytotoxicity. Pulmonary delivery of SLN formulation like aqueous suspension or dry powder formulation is feasible using nebulisers and dry powder inhalers (Mansou, et al., 2009).
Dendrimer are polymers which have hyper-branched structures, with layered architectures. The research in dendrimer-mediated drug delivery has mainly been focused on the delivery of DNA drugs into the cell nucleus for gene or antisense therapy, and many studies has been reported on the possible use of dendrimer as non-viral gene transfer agent. Several studies have been published regarding pulmonary applications of dendrimer as systemic delivery carriers for macromolecules (Mansou, et al., 2009).
Pulmonary delivery devices
Pulmonary delivery devices can be divided into three categories: dry powder inhalers (DPIs), pressurized metered-dose inhalers (pMDIs) and nebulisers. Each category has got strengths and weakness. These devices should generate an aerosol of suitable size, in the range of 0.5-5 µm, and provide reproducible drug dosing. The ideal pulmonary device should be simple, inexpensive, convenient and portable. In addition it should also protect the physical and chemical stability of the drug formulation.
Dry powder inhalers (DPIs) are devices through which a dry powder formulation of an active drug is delivered for local or systemic effect via the pulmonary route (Figure 5). DPIs are useful for delivering drug directly into the deep lungs utilizing the patient's inhalation and it depends on the integration between powder formulations and the device performance (Peart and Clarke, 2001). DPIs are drug powder filled in hard capsules or foil blister discs, or in an inhalation device and produce clouds of aerosols by breath actuation (Bi and Zhang, 2007). Dry powders are formulated as carrier based interactive mixtures with micronized drug particles adhered onto the surface of large lactose carriers or as loose agglomerates of micronized drug particles with aerodynamic particle sizes of less than 5 µm. Drug particles are separated from the carrier or de-agglomerates drug particles when the dry powder formulations are aerosolized through a DPI device, and the dose is delivered into the patient's deep lungs. There are various factors which influences the performance of the system such as particle size and flow property, drug-carrier adhesion, formulation, respiratory flow rate and design of DPI devices.
There is a range of DPI devices available on the market like single or multiple dose devices, breath activated and power driven; though development of new novel devices continues as design of the devices affects the DPI performance. DPI can be classified into three categories i.e., the first generation DPI were breath activated single unit dose (capsule) like Rotahaler® and Spinhaler® and the problem associated with delivery of drugs were particle size and de-agglomeration of drug-carrier agglomerates or drug-carrier mixtures delivered by patient's inspiratory flow. The second generation DPIs are multidose (measures dose from a powder reservoir) or multi-unit dose (pre-metered dose into disks, blisters, dimples, strips and tubes) which probably ensure reproducibility of the formulation. The third generation DPIs are also called active devices and they disperse drugs from the formulation by motor driven impellers, use electronic vibration or employ compressed gas. This device is respiratory force independent dosing precision and reproducible aerosol production. Essential components of the DPI are drug holder, mouth piece, the air inlet and the de-agglomeration compartment and they are designed in such a way that it helps in producing sufficient turbulence, de-agglomeration of particles and particle-particle collisions to detached drug particles from the carrier surface. The majority of DPI devices are primed by pressing (Rotahaler®), rotating (Twisthaler®), sliding (Spinhaler®) or piercing (Handihaler®) to prepare the dose for fluidization with tangential flow of air during patient inspiration (Islam and Gladki, 2008).
Figure 5 - Photographs of some currently available DPI devices: (A) AerolizerTM, (B) EasyhalerTM, (C) TurbohalerTM, (D) DiskhalerTM, (E) NovolizerTM, (F) RotahalerTM (G) ClickhalerTM, (H) MAGhalerTM, (I) SpinhalerTM, (J) HandihalerTM (Islam and Gladki, 2008).
Liposomal DPI formulation are prepared by encapsulation of drug into liposome which are homogenized or dispersed into carrier and converted into DPI by spray drying or freeze drying. On breathe actuation, drug encapsulated liposome get rehydrated and releases drug into the lung (Chougule, et al., 2007).
For therapeutic aerosol delivery, pressurized metered-dose inhalers (pMDIs) are the most common devices prescribed (Figure 6). pMDIs are used to administer anti-cholinergics, bronchodilators, steroids and anti-inflammatory agents. pMDIs comprise a pressurised canister containing a mixture of surfactants, preservatives, propellant and flavouring agent, with approximately 1% of active drug in the total content (Fink, 2000). In pMDIs, the drug is either suspended or dissolved in a propellant that is pressurized until it liquefies in a canister. The liquefied propellant acts in two ways, mainly as a source of energy for expelling the formulation from the valve in the form of evaporating droplets and as a dispersion medium for the drug and other excipients. Mostly two groups of propellants are used namely, Chlorofluorocarbon (CFC) and Hydrofluoroalkane (HFA). CFC-based pMDIs contains a combination of propellant like liquefied low boiling point propellant, CFC 12 (dichlorodifluoromethane) and liquefied higher boiling point propellant, CFC 11 (trichlorofluoromethane) or CFC 114 (dichlorotetrafluoromethane). However, CFC based pMDIs are little used because of ozone depletion in the upper atmosphere. HFA propellant (replacements for CFC) are considered as established excipient eg., HFA 134a (1,1,1,2-tetrafluoroethane) and HFA 227 (heptafluoropropane) (Pilcer and Amighi, 2010).
Figure 6 - Cross-sectional of pMDIs (http://emedicine.medscape.com/article/1413366-media)
The HFA propellants have different physical properties and are incompatible with some valve components and have extremely poor solvent properties. This property helps in preventing dissolution of small drug particles, but there is also drawback of solubility of surface active agents which does not provide physical stability of drug particles in suspension. There are various approaches to solve the problem of drug-particle instability like developing new specific surface active agents, co-solvents such as ethanol, reducing the interfacial tension by modifying the particle surface properties and particle engineering to generate more HFA compatible material. Ethanol is required as a co-solvent in HFA based system for their surfactants. Various surfactants are used in order to dissolve partially soluble drugs, in lubricating metering mechanism and in dispersion of suspended drug particles, for eg., sorbitan trioleate (SPAN 85), oleic acid and soya lecithin. To enhance chemical stability of the formulation, chelating agents (EDTA) or antioxidants (ascorbic acid) are used. Flavouring agent and sweeteners are added in order to mask the unpleasant taste of the formulation (Pilcer and Amighi, 2010).
There are three types of devices used in nebulisation: air jet, ultrasonic and mesh nebulisers and are available in the market. Nebulisers can be used to conduct relatively large volumes of water compatible liposomal suspensions for inhalation, which means by using nebuliser, liposome can be directly aerosolized with no additional fabrication. The jet nebuliser utilizes high velocity compressed gas (e.g., nitrogen, oxygen, and air) through a narrow hole and generates particles from the drug containing solution or suspension from one or more capillaries mainly by momentum transfer (Figure 7). The first generated aerosol droplets which are small enough can easily leave nebuliser. The remaining large droplets impact on the baffles or the walls of the nebuliser chamber and then are recycled into the reservoir fluid. The liquid mass returns to the reservoir and is re-nebulised (Bi and Zhang, 2007). The ultrasonic nebuliser utilizes a high frequency vibrating plate to provide the energy needed to break the fluid into small particles. It works on the principle that by means of transducer, high frequency sound waves can break up water into aerosol particles. The frequency (usually 1-3 MHz) of the vibrating piezoelectric crystal decides the droplet size of the given drug formulation. About 70% of the particles produced are present in the size ranging from 1 to 5 µm. However, heat resulting from frictional forces induced by movement of the transducing crystal may be harmful to thermolabile formulations (Pilcer and Amighi, 2010). Ultrasonic nebulisers are not generally suitable for delivery of liposomes and suspensions (Ghazanfari et al., 2007).
Jet Nebuliser 1.jpg
Figure 7 - Jet nebuliser schematic (http://emedicine.medscape.com/article/1413366-media)
Nebulisers have the advantage over DPIs and pMDIs that the drug formulation may be inhaled during normal tidal breathing through a mouth piece or face mask. Therefore, it can be used to deliver aersolised drug to patients, such as children, the elderly and patients suffering from arthritis, who experience difficulties using other devices. In comparison to DPIs and pMDIs, nebulisers can deliver relatively large volumes of drug solutions and suspensions to the lungs. Drugs which are not conveniently formulated into DPIs or pMDIs and whose therapeutic dose is large are used in nebuliser (McCallion, et al., 1996).
Recently, a third type of nebuliser has been commercialised, called vibrating-mesh nebulisers. These may overcome the drawbacks of air-jet and ultrasonic nebuliser. This device has perforated plates which vibrate in order to produce aerosols and do not heat the fluid during atomisation. These devices are suitable for delivery of liposomes, suspensions and nucleic acids. They are divided into passively and actively vibrating mesh devices. Passively vibrating-mesh devices contain a perforated plate with 6000 tapered holes, approximately 3 µm in diameter. Passive vibration is induced in the perforated plate by a vibrating piezoelectric crystal attached to a transducer horn in front of it, which results into extrusion of fluid through the holes and generation of the aerosols. Actively vibrating-mesh devices contain a "micropump" system which comprises an aerosol generator consisting of a plate with up to 1000 dome-shaped apertures and a vibrating element which contracts and expands on application of an electric current. This results in upward and downward movements of the mesh by a few micrometers, extruding the fluid and generating the aerosols (Ghazanfari et al., 2007). Some of the marketed nebulised products are given in Table 3.
Table 3 - Marketed nebulised product (Pilcer and Amighi, 2010).
Nebuliser mkted prep.JPG