Chitosan and inhalers: a bioadhesive polymer for pulmonary drug delivery

6 Chitosan and inhalers: a bioadhesive polymer for pulmonary drug delivery R. HARRIS, N. ACOSTA and A. HERAS, Complutense University of Madrid, Spain DOI: 10.1533/9780857098696.2.77 Abstract: Pulmonary drug delivery routes present several advantages compared to conventional drug administration, such as fewer systemic side effects than oral or parenteral administration. Many studies show drug delivery systems that are intended for pulmonary administration. However, the time that these systems stay at the mucose surfaces is limited. Mucoadhesive polymers are needed to increase residence time of drugs and therefore to promote absorption through the mucose. Chitosan, a polysaccharide obtained by chitin deacetylation, is used in drug release systems and has mucoadhesion and absorption-promoting properties. There are numerous studies on the use of chitosan drug delivery systems to increase the bioavailability of drugs at the lung mucose. This chapter is a review of literature reports detailing chitosan-based drug delivery systems intended for use in inhalers. The effect of different types of chitosan particulate systems on aerosolization properties will be compared. Key words: chitosan, inhalers, mucoadhesion, nanoparticles, microparticles.

6.1

Introduction

Pulmonary drug delivery has been used for decades to treat respiratory tract diseases, but there is now an increasing interest in the development of drug delivery technologies to treat local and systemic diseases. The delivery of drugs via the lungs is attractive because of the large surface area available for absorption, high vascularization and a thin blood-alveolar barrier. These characteristics facilitate macromolecule transport into systemic circulation and can reduce the administered dose and side effects. On the other hand, lung delivery also avoids gastric and first pass hepatic effect (Brain 2007, Azarmi et al. 2008). A special consideration has to be taken into account here and that is the growing interest in the pharmaceutical industry on the development of biomolecular therapeutics, such as proteins, antibodies and nucleic acids. These biomolecules are large in size and hydrophilic and hence show low membrane permeability. They are also unable to withstand the environment in the gastrointestinal tract where the macromolecule can be degradated by enzymes. Therefore, they present a low absorption and bioavailability when administered orally. This is the reason why most of these macromolecules have been predominantly administered parenterally. The disadvantages of the parenteral route, patients’ discomfort, pain 77 © Woodhead Publishing Limited, 2013

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and non-compliance, show the need for alternative non-invasive routes, such as pulmonary administration. Inhalation is a widely used administration method to deliver drugs to the respiratory epithelium. Among inhalation aerosol formulations that can be used for pulmonary administration of drugs for local or systemic affect, dry powders have shown good stability and bioavailability of the active ingredient compared to liquid formulations (Grainger et al. 2004, Al-Qadi et al. 2012, Sivadas et al. 2008, Learoyd et al. 2008a and 2009, Jafarinejad et al. 2012). Pulmonary administration also presents certain challenges and barriers that can interfere with the inhalation therapy, such as airway geometry and humidity, pulmonary epithelium and the specific defense mechanisms, including the mucociliary escalator, as well as the macrophagic and enzymatic activities (James et al. 2008). Hence, many formulations have been developed to overcome these barriers; formulations with enhanced aerosolization and delivery properties. Among them, the use of mucoadhesive and permeation-enhancing polymers has been widely studied in drug delivery (Huppertz et al. 2002, Hussain et al. 2004, Issa et al. 2005, Cui et al. 2006, Ventura et al. 2008, Zhang et al. 2008, Grenha et al. 2007, Illum et al. 2001, Vllasaliu et al. 2010).

6.2

Chitosan-based inhaler drug delivery systems

Chitosan is a natural biopolymer that is obtained from chitin, which can be found in crustaceans, insects, molluscs and fungi. Chitin is an important subproduct of various industries such as the fish and beer industries. Both chitin and chitosan have gained much interest recently due to their wide range of applications, especially in the food and biotechnology areas (Okuyama et al. 2000, Aranaz et al. 2009, Paños et al. 2008, Vllasaliu et al. 2010, Harris et al. 2008 and 2011, Miralles et al. 2011). Chitin is formed by β-(1→4) linked 2-acetamide-2-deoxyβ-D-glucose units. Chitosan is obtained by chitin partial deacetylation and therefore it is a copolymer of 2-acetamide-2-deoxy-β-D-glucose and 2-amine-2deoxy-β-D-glucose (Peter 1995). The source of chitin and the method used to obtain chitosan are responsible for the chitosan chain composition and its size. Therefore, the deacetylation degree and the molecular weight of chitosan are the two main parameters that are needed to characterize this polymer and that determine its functional properties. The deacetylation degree is the percentage of free amino groups that are present in the chitosan molecule and it determines its solubility. As a consequence of the N-acetyl group hydrolysis, the solubility of chitosan increases in respect to chitin and it is soluble in acid solutions (pKa 6.5) (Acosta et al. 1993). The presence of protonated amino groups determines the interaction of chitosan with other molecules that are negatively charged. Other physical-chemical characteristics that have to be considered for specific applications of chitosan are its cristallinity and its water, ash and protein content.

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Some of chitosan’s functional properties are: biodegradability, biocompatibility, mucoadhesion, filmogenic, hemostatic, absorption enhancer, antimicrobial activity, anticolesterolemic and antioxidant (Aranaz et al. 2009). These functional properties have promoted its use in areas such as agriculture, the food industry, pharmacy and medicine. Due to its gelling and filmogenic properties, chitosan has been studied in the pharmaceutical area for its potential as a drug delivery system (Illum 1998, Shu and Zhu 2000). Chitosan systems act as vehicles for drug encapsulation, protection and controlled release and also promote drug absorption through epithelium (Vllasaliu et al. 2010, Harris et al. 2008 and 2011).

6.3

The absorption enhancing effect of chitosan

The use of drug absorption enhancers in pharmaceutical formulations has been the object of numerous studies in recent years in order to improve drug release through mucose. Among these enhancers, multifunctional polymers that have mucoadhesive properties, that are not toxic and that are not absorbed have gained most interest. Chitosan has been widely studied as one of these mucoadhesive polymers (Issa et al. 2005, Grenha et al. 2007, Vllasaliu et al. 2010, BernkopSchnürch et al. 2006). Numerous studies have reported the use of chitosan as a drug absorption enhancer. The effect of a high molecular chitosan solution on the transport of insulin through nasal mucose in rats and sheep has been studied. These results were promising and since then many studies on the potential of chitosan to improve peptide drug absorption through mucosa have been reported (Illum et al. 1994). The effect of chitosan on the absorption of drugs is due to a combination of its mucoadhesive properties and its ability to open tight junctions between epithelia cells.

6.3.1 Mucoadhesion Mucoadhesion prolongs the residence time and the contact between membranes and formulations, which allows a sustained drug delivery and reduces the frequency of administration. Many studies describe that the administration of drugs in combination with chitosan increases contact time of chitosan with mucose (Soane et al. 1999). Chitosan mucoadhesive properties are due to the interaction of its protonated amino groups with the mucus layer. Mucus is composed of a glycoprotein, mucine, with negative charges due to the presence of sialic acid residues. The interaction depends on the amount of sialic acid and the deacetylation degree or free amino groups in chitosan. Mucoadhesion properties of chitosan microspheres in rat intestine epithelia showed that mucoadhesiveness increased with the amount of free amino groups in chitosan (He et al. 1998). pH value also affects mucoadhesive

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properties of chitosan, thus chitosan amino groups are positively charged at acid pH (pKa 6.5). It has also been reported that chitosans with longer chains and therefore high molecular weights can promote mucoadhesion (Borchard et al. 1996, Majithiya and Ramchandra 2005) because it enters into the mucine layer more easily.

6.3.2 Widening of tight junctions Epithelium acts as a barrier that separates the organism from the outside of the body. Molecules can move across the epithelium by diffusion or passive transport down the concentration gradient, that is, from the area of higher concentration to the area with the lowest concentration. Passive transport can occur through the cell membrane (transcellular transport) or through adjacent cells (paracellular transport). Solutes can also cross the epithelium by active transport against concentration gradient. This process involves chemical energy. Lipophilic molecules can easily cross the cellular membrane by passive diffusion, but hydrophilic molecules cannot cross the hydrophobic membrane and therefore have to cross the epithelium via the paracellular route. This route is restricted due to the presence of tight junctions, which are complex and dynamic protein structures that form a semipermeable barrier that restricts diffusion depending on the charge and size of solutes. In the epithelium, tight junctions are located between the plasmatic membranes of adjacent cells and they form a continual structure that surrounds the cells completely (Matter and Balda 2003). Tight junctions are formed by a group of transmembrane and cytosolic proteins that not only interact among themselves, but also with the cell membrane and the actin cytoskeleton. Thus they form a system that links the tight junction components with the cytoskeleton (Matter and Balda 2003). The disruption of the cytoskeleton is associated with the opening of the tight junctions and therefore with an increase in paracellular permeability (Ward et al. 2000). The ability of chitosan solution and chitosan nanoparticles to open tight junctions is due to the interactions of the polymer with specific receptors present on the cell surface (Smith et al. 2005). This interaction activates the sign transduction dependent on kinase C protein. The activation of this protein also induces the dissociation of the tight junction proteins in the plasmatic membrane and therefore the loss of tight junctions. Ranaldi et al. (2002) demonstrated that the treatment with chitosan altered the distribution of F-actin in Caco-2 cells. Various studies have shown that chitosan transiently opens cell tight junctions upon incubation with cell lines representative of the respiratory epithelium, such as Calu-3 and A549 (Grenha et al. 2007, Vllasaliu et al. 2010) A decrease in transepithelial electrical resistance (TER) is shown in these studies due to the interaction of the positively charged amino groups of CS with the sialic acid residues present in membranes. This reversible disruption of the tight junctions

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enables greater permeation of the released macromolecule and is dependent on several factors, such as chitosan concentration, Mw and deacetylation degree (Al-Qadi et al. 2012).

6.4

Types of particle systems used in inhalers

The effective delivery of drugs from inhalers depends on the inhaler device used and on the composition of the drug formulation, which controls the physical delivery mechanism and that can be modified to improve the effective dispersion of drug particles or drug carrier particles. Various formulations have been described for the delivery of drug powders by inhalation. Micronization of active drugs is used to reduce particle size of drug powder although the resulting powder size (less than 5 μm) promotes particle aggregation and therefore poor powder flow properties are shown (Steckel and Brandes 2004). In order to improve powder aerosolization, drugs are being mixed with excipients and encapsulated in carrier particles (Larhrib et al. 2003). The production of micro- and nanoparticle-based formulations has gained interest because they present certain advantages, such as the control of drug release and targeting at the tissue and cellular levels. These systems also protect the drug and maintain its stability. Morphology, surface area, particle size and distribution, density and adhesion/cohesion forces all play a part in the effectiveness of the dispersion after inhalation (Azarmi et al. 2008, Learoyd et al. 2008b). Various studies have also shown the importance of surface roughness on the cohesive/ adhesive forces between particles. Rough surfaces improve dispersibility due to a reduction in the attractive forces between individual particles (Steckel and Brandes 2004, Giovagnoli et al. 2007, Islam and Cleary 2012). Successful delivery of inhaled particles is governed by their deposition patterns, which are mainly controlled by particle size and density. In addition, both aerodynamic diameter and particle density are dependent on particle composition (Bosquillon et al. 2001). Particles contained in aerosols should be neither too small, because they would be exhaled, nor too large, because they would stay in the upper airways. For deep lung deposition, it has been reported that dry powders are required to present an aerodynamic diameter less than 5 μm. Depending on the object of the therapy, the deposition of particles should be predominant in different areas of the lung; if the delivery is intended for systemic application, the powder aerodynamic diameter must be modified to allow particles to deposit in the alveolar region where absorption into systemic circulation occurs. It is considered that droplets or particles with an aerodynamic diameter within the range 1–3 μm will present appreciable deposition in the alveolar region, while those with a higher diameter will mainly deposit in the upper regions (Al-Qadi et al. 2011). Small particles in the size range of 0.1–1.0 μm are inhaled in the alveoli, but also exhaled without being deposited significantly (Al-Qadi et al. 2012, Shoyele and Cawthorne 2006).

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There is an increased interest in polymeric nanoparticles for lung delivery. Compared to microparticles, nanoparticles have the ability to diffuse through the mucus layer and translocate through the alveolar epithelium by endocytosis (Yang et al. 2008). Nevertheless, the use of nanoparticles alone for inhalation has encountered some obstacles, including the high aggregation of nanoparticles and exhalation of them (Jafarinejad et al. 2012). Various studies have been reported in the literature of nanoparticles that are encapsulated in microparticles because this improves handling and aerosolization performance. A higher stability is achieved and better aerodynamic properties have been shown, which result in efficient lung delivery. In order to enhance the retention of the nanocarrier in the lung and to prevent rapid elimination by ciliary movement, much attention is given to mucoadhesive formulations (Makhlof et al. 2010). Also, the use of carriers such as lactose and mannitol seems to be necessary to optimize alveolar deposition and to avoid aggregation of the nanoparticles (Jafarinejad et al. 2012). Chitosan is attractive for transmucosal drug delivery due to its low toxicity, biodegradability, biocompatibility (Grenha et al. 2007) and mucoadhesivity, as well as for its role as a macromolecule permeation enhancement (Vllasaliu et al. 2010, Issa et al. 2005). Therefore, this polymer is being extensively studied for the development of micro and nanocarriers for pulmonary drug delivery. Furthermore, it has also been used as a dispersibility enhancer in dry powders (Al-Qadi et al. 2012, Li and Birchall 2006).

6.5

Inhaler formulations based on chitosan and chitosan derivatives

6.5.1 Chitosan particulate systems used in inhalers Many studies can be found in the literature on inhalers where chitosan or chitosan derivatives are used in the drug delivery formulations and where this polymer can be found forming part of micro and nanoparticles, on its own or in combination with other polymers. Various types of inhalers are available and some are more adequate than others when needing to deliver small particle formulations. Dry powder inhalers (DPI) deliver drugs to the lung as dry powder formulations. On the other hand, in pressurized metered-dose inhaler (pMDI) formulations, the active ingredient is either suspended or dissolved in a hydrofluoroalkane propellant (HFA). Studies found in the literature report the use of formulations based on chitosan prepared to be aerosolized through DPIs or pMDIs. Chitosan microspheres and nanoparticles have been obtained by different methods for this purpose. In many of these studies the active ingredient is a macromolecule that needs protection so as not to be degraded. The most recent studies found are the ones reported in this chapter. Microspheres have been obtained by spray-drying using a range of biopolymers including sodium alginate, chitosan, gelatin, hydroxypropyl cellulose,

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poly(lactide-co-glycolide), sodium hyaluronate and ovoalbumin as potential inhaled protein carriers. In order to determine the aerosolization efficiency, microparticles blended with mannitol were aerosolized through a DPI (Sivadas et al. 2008). In this type of study, aerosolization properties are usually measured using a cascade impactor which measures aerodynamic particle size distribution (and is composed of various stages) where the particles deposit after inhalation depending on their aerodynamic diameter, with larger particles being retained in the earlier stages of the impactor (from the induction port to stage 2), while the smaller particles find their way to the lower stages of the impactor (stages 3 to 7). Fine particle fraction (FPF) is an important parameter that is calculated in these studies. A higher FPF denotes greater deposition of the formulation in the deep lung. In this study, 83% and 95% of the loaded dose was recovered. The FPF was highest in the case of hydroxypropyl cellulose particles (26.1%) and lowest with ovalbumin particles (11.9%). The other polymers produced FPF values between 14% and 21%. In the specific case of chitosan microspheres the FPF was 15.4%. The experimental mass median aerodynamic diameter (MMAD) values were larger than the theoretical aerodynamic diameter and ranged between 2.9 and 4.7 μm possibly due to particle aggregation. Chitosan microspheres showed a value of 3.9 μm. The corticosteroid beclometasone dipropionate was incorporated into a spraydried formulation containing chitosan and an aerosolization enhancer (leucine) to develop potentially respirable powders for local pulmonary drug delivery (Learoyd et al. 2008b). Spray-dried powders tend to be cohesive and therefore are poorly dispersed during aerosolization (Gilani et al. 2004). Leucine has been demonstrated to increase the respirable fraction of particles in dry powder formulations (Rabbani and Seville 2005). In this work the effect of different molecular weight chitosans on the release of the drug from the powders and the aerosolization properties of the spray-dried powders were investigated in vitro. All the powder formulations tested showed a deposition of less than 10% in the throat region, suggesting that inhalation of these powders would be associated with limited oropharyngeal deposition. The control powder containing beclometasone dipropionate, leucine and lactose showed low deposition in stages 1 and 2 and 70% deposition in stage 3 or below. A significantly lower deposition in the lower stages of the MSLI was observed in the case of chitosan powders. The results showed a significantly lower FPF for all molecular weight chitosan powders than the control powder. Therefore, although the inclusion of chitosan would provide modified release properties to the formulation, its aerosolization properties did not improve with respect to the control. Regarding the MMAD of the spray-dried powders, it was smaller than the physical diameter of the particles measured by laser diffraction. This shows the low density and high dispersibility of these powders and is probably due to their high leucine content. In a second study, the same authors obtained spray-dried formulations with different molecular weight chitosans and it was demonstrated that with an

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appropriate molecular weight chitosan it was possible to sustain the release of terbutaline sulfate (Learoyd et al. 2008a). In this case they also used leucine in the formulation as a dispersibility enhancer to generate highly dispersible powders. As in the previous study, aerosolization properties of the spray-dried powders were investigated. All the formulations that they obtained showed high dispersibility and therefore good aerodynamic properties. The deposition pattern of the spraydried powders showed a very low deposition in the inhaler and throat regions. Although the inclusion of chitosan in the formulation significantly increased the deposition of powder in the earlier stages compared to the control powder, a significant deposition was shown at the lower stages. They also found differences depending on the molecular weight of chitosan. The FPF of the low molecular weight chitosan powder was significantly higher than that of the medium and high molecular weight chitosan powders. The MMAD of the spray-dried powders was similar to the theoretical estimates of aerodynamic diameter which suggests that particles did not aggregate during aerosolization. This may be due to the high proportion of leucine in these powders, although researchers have shown that chitosan can enhance the dispersibility of spray-dried powders (Li and Birchall 2006). Chitosan is probably also modifying the surface of the powder particles, decreasing interparticulate cohesion and therefore improving powder dispersibility. Crosslinked chitosan nanoparticles were prepared for delivery from pMDIs (Sharma et al. 2012). Polyethylene glycol (PEG) was used since it acts as a polymeric surfactant and helps to reduce the cohesive interactive forces between particles that are suspended in a fluorinated solvent. The physical stability of nanoparticles after dispersion in propellant HFA-227 was studied. While chitosan nanoparticles without PEG aggregated, the inclusion of PEG 1000 greatly improved the nanoparticle physicochemical characteristics. It provided steric stabilization and minimized particle interactions. Long-term stability, dispersibility and ease of redispersion of HFA-based pMDI formulations are key parameters for the quality of an inhalation product. The crosslinked chitosan-PEG 1000 particle formulation produced a more uniform size distribution with an adequate volume median diameter size for deep lung alveolar delivery (1.53 μm). In this study chitosan was fluorescently labeled to quantify its concentrations after aerosolization and deposition in all stages. Among the formulations that they tested with different molecular weight PEG, chitosan-PEG 1000 nanoparticles showed the highest FPF of 34% with a MMAD of 4.92 μm. Commercially-available pMDI products deliver 30% of the total emitted dose to the lungs (Rau 2005) and the following studies mentioned have reported similar results. An FPF of 31.5% was reported for semi-interpenetrating polymeric network microspheres loaded with bovine serum albumin delivered from a DPI (El-Sherbiny and Smyth 2010). An FPF of 45% for insulin loaded nanoparticles was reported for nanoparticles from an HFA-134a-based pMDI system (Nyambura et al. 2009). Other published studies report the entrapment of loaded nanoparticles in microparticles in order to maintain the physical stability of the formulation. In

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these formulations microparticles act as a shell that should disintegrate when in contact with an aqueous fluid. The following studies are an example of this type of formulation. A pulmonary delivery system for antibiotics, in this case tobramycin, was developed (Ungaro et al. 2012). First, PLGA-based nanoparticles were prepared by adding other polymers such as chitosan and PVA. This would provide the particles with the desired surface charge, mucoadhesiveness, size and release properties. The resulting nanoparticles were engineered into fluorescent micronsized dry powders by spray-drying using lactose as carrier. The effect of nanoparticle composition on the in vitro and in vivo aerosolization properties was studied. These properties were tested in vitro by delivering the formulation and studying the deposition pattern of the powder. Both types of microparticles (chitosan and PVA nanoembebbed microparticles) showed very good flow and aerosolization properties with nearly 100% of the capsule content being emitted during aerosolization. Microparticles with chitosan showed a MMAD of 5.7 μm and a FPF of 38%. Again, due to the challenges that direct delivery of nanoparticles for pulmonary administration poses, microencapsulated insulin-loaded chitosan nanoparticles were investigated (Al-Qadi et al. 2012). In this study, ionic gelation was used to obtain nanoparticles and this was combined with spray-drying to obtain microspheres. First, nanoparticles were prepared by ionic gelation and then were resuspended in mannitol and spray-dried. Aerodynamic diameters were obtained and the aerosol delivery was tested in vivo by intratracheal administration of insulin-loaded formulations in rats. Both the formulations tested, with two different molecular weight chitosans, showed diameters which fell within the range recommended for optimal alveolar delivery (between 1 and 5 μm). Their results show that microspheres with insulin-loaded nanoparticles induced a significant reduction in plasma glucose levels compared to controls (insulin solution and insulin-loaded nanoparticles). The hypoglycemic effect was more pronounced and prolonged in the case of nanoparticles entrapped in microspheres than for the controls. This was confirmed by encapsulating FITC-BSA in nanoparticles and administering it to rats to study lung deposition. Images of lung slices showed that chitosan nanoparticles were delivered to the deep lung. The authors attribute these results to a possible retention of the nanoparticles at the absorption site due to an interaction of the positive particle surface charge with the negatively charged sialic acid group of mucin in the mucous layer, allowing drug release in the lung over a prolonged period of time (Vllasaliu et al. 2010, Takeuchi et al. 2001). As mentioned in Chapter 3, chitosan is known to be mucoadhesive and to open tight junctions, which minimizes particle mucociliary clearance and enhances macromolecule absorption. Itraconazole-loaded nanoparticles were prepared by ionic gelation of chitosan and TPP and then spray-dried with lactose, mannitol and/or leucine (Jafarinejad et al. 2012). The addition of low quantities of lactose and mannitol improved the

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aerosolization capability of nanoparticles and leucine increased the emitted dose percentage of the aerosolized particles. Of the formulations tested, nanoparticles with mannitol and leucine showed the highest fine particle fraction (42.9%). Due to the difficulty of stabilizing particle dispersions in low dielectric hydrofluoroalkane propellants, polymeric nanocarriers have been entrapped within microparticles. These microparticles represent a shell that could be well solvated by the propellant and that could provide physical stability to the formulation (Bharatwaj et al. 2010). The shell was designed so that it maintained the system’s integrity while dispersed in the propellant, but so that it would disintegrate when in contact with an aqueous fluid. The shell was composed of oligo(lactide)-grafted-chitosan. The aim of this work was to efficiently deliver polymeric nanocarriers to the lung and show the potential of the formulation in the treatment of Chlamydia pneumoniae related infections. The nanocarriers were prepared by emulsion solvent evaporation using a fluorescent marker to follow their fate in vitro. They studied the aerosol characteristics of the corresponding nanocarrier pMDI formulation and the ability of the formulations to target chlamydial inclusion membranes in airway epithelial cells. Entrapped nanocarriers showed a higher stability. Therefore, this could be a strategy to disperse nanocarriers in propellant-based pMDIs. An eight-stage Anderson Cascade Impactor was used to determine the aerosol characteristics of the formulations. The respirable fraction and the fine particle fraction were lower for nanocarriers alone (20.1% and 10.5% respectively) than for core-shell nanocarriers (72% and 55% respectively), therefore showing that the formulation with core shell particles has better aerosol characteristics compared to nanocarriers alone. This formulation also showed a mass median aerodynamic diameter within the desirable range for pMDIs. Comparing these results with other pMDI formulations, the FPF of 55% obtained by Bharatwaj et al. (2010) is comparable to the FPF of Ventolin HFA®, a commercial formulation for asthma and chronic obstructive pulmonary disease that has a reported FPF of 46% (Bharatwaj et al. 2010). One promising research area is the delivery of genes to the lungs via oral inhalation. There are many examples of pulmonary diseases that could potentially be treated by gene therapy and these genes could be targeted to the lungs via oral inhalation. A pMDI system for the oral inhalation of genes for the treatment of pulmonary diseases has been proposed (Conti et al. 2012). Core-shell particles loaded with chitosan-DNA nanoparticles were obtained. The aerosol properties of the chitosanDNA core-shell particles and chitosan-DNA nanoparticles alone in pMDI formulations were determined. The aerosol characteristics of formulations containing chitosan-DNA nanoparticles entrapped within a biodegradable shell were significantly better than those of polyplexes alone. The FPF was lower for formulations prepared with nanoparticles alone (30.2%) than for core-shell formulations (56.6%). Therefore, the core-shell formulations are performing significantly better than those without the co-oligomer shell. Nanoparticles alone

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were entrapped mostly in the induction port, stage 0 and filter, showing a very poor mass distribution along the other stages, possibly due to particle aggregation in the propellant, because of the absence of a stabilizing shell. Formulations prepared with core-shell particles showed a much better distribution of the DNA on the different stages, and significantly less DNA entrapment in the induction port. The MMAD of the chitosan-DNA core-shell particles fell between 1 to 5 μm. Other studies on DNA encapsulation have been reported. Mohri et al. (2010) prepared a dry plasmid DNA powder with chitosan by spray-freeze drying and Mohajel et al. (2012) obtained microparticles by spray-drying containing polyplexes, which were prepared by binding plasmid with low molecular weight chitosan. No aerosolization studies were performed in any of these two cases. Pulmonary immunization has also gained increased interest as an alternative route for administration of vaccines. Parenteral administration is usually required because most antigens are macromolecules and are unstable in the gastrointestinal tract. Due to the drawbacks that parenteral administration has, there is a need for non-invasive vaccines. The extensive dendritic cell network present in the respiratory epithelium and the macrophages network have an important role in generating systemic and local immune responses. The pathogen-specific secretory IgA antibodies in the respiratory tract are also important for the prevention and control of local infections (Amidi et al. 2007, Sou et al. 2011). To overcome the barriers that antigens can encounter in the pulmonary tract, which have been described previously in this chapter, mucoadhesive antigen-containing particles are being studied. The modification of chitosan to improve its already existing properties is gradually gaining interest because chitosan is subjected to aggregation and loss of surface positive charge at physiological pH values (chitosan’s pKa varies between 5.5 and 6.5). N-Trimethyl chitosan chloride microparticles loaded with diphtheria toxoid were obtained by a supercritical fluid spraying process (Amidi et al. 2007). Results showed high FPF values and higher specific antibody levels against diphtheria toxoid than those obtained with conventional vaccines which were used as a control. Thiolated chitosan, for example, improves mucoadhesive properties of chitosan because the thiol groups form covalent disulfide bonds with thiol groups present in cysteine sub-domains in mucus (Leitner et al. 2003). In order to encapsulate calcitonin in nanoparticles, a glycol chitosan and a glycol chitosan-thioglycolic acid polymer conjugate were synthesized (Makhlof et al. 2010). No in vitro aerosolization properties were measured in this study. After intratracheal administration of nanoparticle formulations with fluorescence-labeled polymers in rats, the association of nanoparticles to the lung tissue was shown to be higher when thiol groups were attached. The blood calcium level was also measured in rats after formulation administration, and reduced blood calcemia was observed. Therefore, the formulation showed a higher permeability of lung epithelium to

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macromolecules compared to the intestinal barrier (Makhlof et al. 2010). Both formulations also prolonged the hypocalcemic effect of calcitonin.

6.5.2 Clinical trials In Europe, no clinical trials on chitosan inhaler formulations have been reported (www.clinicaltrialsregister.eu). On the webpage that specializes in clinical trials (www.clinicaltrials.gov) there are no reported clinical studies on chitosan inhaler formulations. At present, only two clinical trials are reported on chitin microparticles for the treatment of allergic rhinitis: 1. Phase I/IIa study on chitin microparticles in subjects suffering from allergic rhinitis (NCT00443495). This study started in October 2006, has been completed and was last updated in March, 2007. The principal researcher of the study is Steven J. Warrington from Hammersmith Medicines Research. The information is provided by CMP Therapeutics Ltd, the sponsor. Chitin microparticles act as an immunenhancer which improves immune function by stimulating macrophages and other cells such as T-helper lymphocytes (Th cells). The primary purpose of the study is to demonstrate safety in a first into man study on 24 human volunteers. The secondary objective is to demonstrate efficacy by choosing subjects that demonstrate a response to a nasal allergen challenge using grass pollen. No study results were posted on the website (www. clinicaltrials.gov). 2. Immunological effects from intranasal chitin micro-particles (INCA) (NCT01508039). The start date was April 2011, it has been completed and was last updated on January 25, 2012. The principal investigator of the study is Torben Sisgsgaard from Aarhus University, School of Public Health, Department of Environmental and Occupational Medicine. The information was provided by University of Aarhus, and CMP Therapeutics Ltd was a collaborator. The purpose of the study is to investigate whether chitin can affect healthy adults, enhancing the immune response to infection, and to investigate whether chitin influence on the nasal mucosa is well tolerated and make sure there is no inflammation, as it has been seen when exposed to endotoxin. No results were posted on the website (www.clinicaltrials.gov) for this study.

6.6

Conclusions

The engineering of inhalable particles using biocompatible polymers has improved drug stability, promoted controlled release and enhanced drug absorption via the lungs. Many of the formulations that have been studied are not commercialized.

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Many factors could be responsible for this, such as the lack of comparative data on polymer influence on the aerodynamic properties, the lack of licensed excipients for inhalation, or concerns regarding the safety and clearance of these polymers from the lungs. Nevertheless, the use of bioadhesive and absorptionenhancing polymers, such as chitosan and its derivatives, widens the range of possible drug administration routes, which seems necessary for certain types of drugs. Therefore, many more studies need to be done on formulations that are based on these polymers in order to state which specific chitosan/s would be the best for pulmonary administration.

6.7

References

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