Cyclodextrins in the production of large porous particles: Development of dry powders for the sustained release of insulin to the lungs

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Abstract

The aim of this work was to develop dry powders intended for insulin pulmonary delivery. To this purpose, large porous particles (LPP) made of poly(lactide-co-glycolide) (PLGA) were produced by the double emulsion-solvent evaporation technique. Hydroxypropyl-β-cyclodextrin (HPβCD), also known as absorption enhancer for pulmonary protein delivery, was tested as aid excipient to optimize the aerodynamic behaviour of the microparticles. Several microsphere formulations, differing in HPβCD and insulin loadings, were produced and their properties compared. A contemporary release of insulin and HPβCD from the system can be achieved by selecting appropriate formulation conditions. HPβCD-containing LPP with flow properties and dimensions suitable for aerosolization and deposition in deep regions of the lung following inhalation were produced. In conclusion, the developed system turns to be of great potential for the combined delivery of the protein and the adsorption promoter in the respiratory tract.

Introduction

The unique features of the lung, namely its large surface area, high permeability and wide blood supply, make pulmonary route an attractive non-invasive way for the systemic administration of peptides and proteins. To date, several protein-based drugs, such as insulin, human growth hormone, calcitonin and deslorelin, have been reported to reach the systemic circulation following aerosol administration (Agu et al., 2001). Advanced dry powder inhalers (DPIs), somewhat overcoming solubility, bioavailability and stability issues related to protein delivery by means of classical metered-dose inhalers (MDIs), have further increased research attention on this matter (Newman and Busse, 2002). Nevertheless, efficient pulmonary delivery through the newest DPIs requires drug powders with well-defined bulk properties (e.g. particle size, density, and surface area), which affect particle flow, handling and dispersibility and, therefore, its likelihood of depositing in the desired region of the lungs (Johnson, 1997, Van Campen and Venthoye, 2002). Recent data indicate that major improvements in aerosol particle performance may be achieved by lowering particle mass density (<0.4 g/ml) and increasing particle geometric size (10–20 μm). Large porous particles (LPP), by virtue of their porosity, display an aerodynamic diameter much lower than geometric one, facilitating their deep lung deposition (Edwards et al., 1998).

Poly(lactide-co-glycolide) (PLGA) and polylactide particles have been already used for drug delivery to the lungs. In case of lung infections, such as tuberculosis, particles with mean diameters of 1–3 μm have been produced to target the resident alveolar macrophages (O’Hara and Hickey, 2000, Suarez et al., 2001). On the contrary, LPP of PLGA, due to their large size, have been demonstrated to escape macrophage uptake and permit an efficient delivery of inhaled insulin into the systemic circulation for long periods of time (Edwards et al., 1997). More recently, LPP of PLGA produced by supercritical fluids have been further shown to sustain desorelin delivery to the deep lung (Koushik et al., 2004). Nevertheless, the great challenge for researchers remains the full optimisation of the delivery system, that is the achievement of all those particle properties – good encapsulation efficiency, prevention of protein degradation, predictable release of the drug – required for therapeutic applications.

Porous PLGA microparticles are obtained when double emulsion-solvent evaporation technique is employed and differences in the osmotic pressure between the internal and the external aqueous phases of the emulsion are generated (Pistel and Kissel, 2000, Ravivarapu et al., 2000, De Rosa et al., 2002). The use of salts, such as calcium and sodium chloride, in the internal water phase has been reported to induce this process and enable the formation of highly porous particles. The use of this formulation strategy to obtain protein-loaded LPP of PLGA, indeed, can be dangerous due to the fact that salts can cause protein precipitation/aggregation/inactivation as a function of salt type and concentration used (Pean et al., 1998, Perez and Griebenow, 2003). In particular, a destabilizing effect of sodium chloride on insulin has been observed, leading to enhanced protein fibrillation (Brange et al., 1997). Thus, the selection of alternative osmotic agents, which can act as aid-excipients in producing porous particles, without altering insulin integrity, becomes a key challenge.

Cyclodextrins (CD) are well known molecular entities used as pharmaceutical excipients mainly to solubilize and stabilize drugs through complexation. CD derivatives, such as hydroxypropyl-β-cyclodextrin (HPβCD), also display osmotic properties in aqueous solutions depending on their chemical structure and total degree of substitution (Zannou et al., 2001). CD incorporation in PLGA microspheres has been attempted to control release rate of low molecular weight drug zolpidem (Trapani et al., 2003). On the other hand, the use of CD into PLGA microparticles for the controlled release of proteins has been mainly considered as a way to stabilize the encapsulated macromolecule improving its therapeutic efficacy (Morlock et al., 1997, Meinel et al., 2001, Murillo et al., 2002) and in some cases modulate the release features of the particles (Quaglia et al., 2003, De Rosa et al., 2005). Nevertheless, it has been demonstrated that HPβCD can also promote insulin pulmonary adsorption by a direct disruption effect on alveolar epithelial membrane (Shao et al., 1992).

In this work, HPβCD was used to produce LPP of PLGA intended for pulmonary delivery of insulin. Several microsphere formulations, differing in HPβCD and insulin loadings, were produced and their properties compared. The aerodynamic behavior of the powders was investigated in depth in order to highlight their potential for in vivo applications.

Section snippets

Materials

Poly(d,l-lactide-co-glycolide) (50:50) (PLGA) (Resomer RG 504 H; Mw 41.9 kDa; inherent viscosity 0.5 dl/g) was purchased from Boehringer Ingelheim (Germany). Insulin from bovine pancreas, Trizma base (TRIS), trifluoroacetic acid (TFA), sodium carbonate and sodium azide were obtained from Sigma Chemical Co. (USA). Hydroxypropyl-β-cyclodextrin (Mw 1380 Da, molar substitution 0.6) (HPβCD), phenolphthalein, polysorbate 80, and polyvinylalcohol (PVA, Mowiol® 40–88) were purchased from Aldrich (USA).

Results and discussion

The aim of this study was to investigate the potential of HPβCD in producing LPP of PLGA for the pulmonary delivery of insulin and understand how HPβCD incorporation affects the properties of the particles. Insulin was entrapped within the particles by double emulsion-solvent evaporation technique alone or with HPβCD at two different mole ratios (Table 1).

Conclusions

This study has demonstrated that HPβCD can be used as an excipient to design LPP of PLGA intended for insulin pulmonary delivery. Combination of HPβCD with insulin is essential to elicit microsphere surface porosity due to osmotic phenomena. HPβCD concentration in the formula appears crucial in determining the release properties and aerodynamic behaviour of insulin-containing particles. HPβCD-containing LPP showed flow properties and dimensions suitable for aerosolization and deposition in deep

Acknowledgement

The financial support of Italian Ministry of University and Research (PRIN 2004) is gratefully acknowledged.

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