WO2010007604A2

WO2010007604A2 - Inhalable microparticles, and methods for the production thereof

Info

Publication number: WO2010007604A2
Application number: PCT/IE2009/000046
Authority: WO
Inventors: Sally-Ann Cryan; Neeraj Sivadas
Original assignee: Royal College Of Surgeons In Ireland
Priority date: 2008-07-16
Filing date: 2009-07-15
Publication date: 2010-01-21
Also published as: WO2010007604A3

Abstract

A microparticle preparation suitable for pulmonary delivery of an active agent comprises solid particles having a three-dimensional matrix structure and an active agent entrapped and dispersed throughout the three dimensional matrix structure. The particles having a Mass Median Aerodynamic Diameter (MMAD) of from 1 to 5μm, and the three-dimensional matrix comprises a cross-linked polymer susceptible to degradation by human neutrophil elastase. The three dimensional matrix may comprise an interpenetrating network of a polymeric protein and a cross-linked polysaccharide polymer. Methods for producing the microparticles are also described.

Description

INHALABLE MICROPARTICLES, AND METHODS FOR THE PRODUCTION THEREOF

Introduction

The invention relates to inhalable microparticles suitable for pulmonary delivery of an active agent, and methods for the production thereof. The invention also relates to a method of treating or preventing a disease or condition characterized by inflammation of the pulmonary tract or lung.

There is a growing interest in the delivery of drugs to and via the lungs. While improved engineering has led to the development of more efficient devices, limited work has been done to stabilise agents before, during and after delivery, to target the drug to specific areas/cell types or to optimise delivery (particularly important for high cost drugs). There is a growing need for imiovative, safe biopolymers that can be used to control the fate of drugs once inhaled. Responsive polymeric drug delivery systems, capable of adjusting drug release rate in response to a physiological need, offer the advantage over conventional inhalation technology of providing controlled drug administration. Site-specific drug delivery after oral administration of drugs using polymers has become common-place e.g. enteric coated tablets, but little work has been done to-date on harnessing polymeric carriers for site-specific delivery in the lungs after inhalation.

Inhalation is gaining increasing acceptance as a convenient, reproducible, and non-invasive method of drug delivery locally to the lungs or the systemic circulation. Inefficient delivery, poor stability and rapid elimination associated with aerosol delivery of drugs, particularly proteins and genes, has limited clinical development to-date.

WO2007/134245 discloses the use of elastin like polypeptides (ELP) in complex with a therapeutic molecule as a vehicle for drug delivery. ELPs are thermosensitive polymers enabling methods for hyperthermic targeting to specific sites for therapeutic purposes. The ELPs of this application may be conjugated to a small molecule or therapeutic peptide for example Hsp90 antagonist by means of a cleavable linker. WO96/32149 teaches a method for synthesizing dry powder compositions for use in pulmonary drug delivery. The microparticles of this application preferably have a mass median diameter of between l-3μm and an aerosol particle size distribution of 1.5-4.0 μm and can be delivered by means of aerosolization. The proteins employed in this patent are not suitable for targeted and controlled release, and are not cross-linked. Thus, they would a very fast, uncontrolled, release of active agent.

It is an object of the invention to overcome at least one of the above problems.

Statements of Invention

The invention relates to inhalable microparticles that incorporate a polymeric protein that is susceptible to degradation by the enzyme elastase. The microparticles have a three- dimensional matrix structure that incorporates the elastase-sensitive polymer throughout the matrix, or as a coating encapsulating the matrix, or both. An active agent is dispersed throughout, and physically entrapped within, the matrix. The particles are delivered to the lungs generally in the form of an aerosol of respirable microparticles. When the microparticles encounter an area of inflammation, endogenous elastase, which is produced by neutrophils at the site of inflammation, will degrade the elastase sensitive polymeric protein to release the active agent in a targeted manner.

In this specification, the term "polymeric protein that is susceptible to degradation by the enzyme elastase" should be taken to include not only elastase-sensitive polymeric proteins (such as those described herein), but also fragments of such proteins (including peptides and polypeptides), and synthetically derived peptides and polypeptides, that are susceptible to degradation by elastase.

In one aspect, the method of producing microparticles suitable for pulmonary delivery of an active agent comprises the steps of forming a liquid hydrogel by suspending or dissolving a polymeric protein susceptible to proteolysis by elastase in a suitable solvent, the hydrogel further comprising the active agent dispersed throughout the hydrogel, and ideally spray drying the liquid hydrogel to form microparticles, wherein the hydrogel is crosslinked. Crosslinking of the hydrogel prior to spray drying forms a more robust three-dimensional polymeric matrix that prolongs the release of the active agent when the microparticle encounters neutrophil elastase at a site of inflammation. This is apparent from Figures 3-6 below which show a slow gradual release of active agent over a period of 60 to 210 minutes. This is a suitable pharmacokinetic profile for drugs for respiratory conditions, which require a prolonged release of active. Additionally, the fact that the microparticles comprises a matrix which is sensitive to elastase ensures that the release of active is more pronounced at sites of inflammation.

Suitably, the liquid hydrogel is formed by the interaction of the polymeric proteins in the solvent. Generally, the solvent is aqueous, ideally water. Thus, the polymeric proteins interact to form a three-dimensional matrix structure in the solvent which may take the form of a gel, a colloid, or a micelle, although it will generally be a gel. The amount of polymeric protein employed is controlled to ensure that the hydrogel remains fluid such that it can be processed by spray diying. The actual concentrations of polymeric protein employed will depend on a number of factors, including the choice of protein and the choice of solvent. Generally, the polymeric protein will be employed at between 0.1% and 10%, preferably from 0.5% to 2.0% of the hydrogel (w/w). In another aspect, the method of producing microparticles suitable for pulmonary delivery of an active agent comprises the steps of forming a liquid hydrogel by suspending or dissolving a polymeric material in a suitable solvent, the hydrogel further comprising the active agent dispersed throughout the hydrogel, and spray drying the liquid hydrogel to form microparticles, and coating the microparticles with a polymeric protein susceptible to hydrolysis by elastase. Coating the microparticles may be carried out in any suitable manner such as, for example, absorption or by mixing the microparticles with a solution of the elastase sensitive polymeric protein and re-spray drying the mixture. Other methods of coating the microparticles will be apparent to those skilled in the art. Typically, the polymeric material is not sensitive to degradation by elastase (i.e. PLGA). In one embodiment, the polymeric material is a bioactive material having, for example, anti- inflammatory properties (i.e. hyaluronic acid).

The polymeric protein is typically selected from the group consisting of: gelatin; ovalbumin; and elastin, or salts or derivatives thereof. Typically, the cross-linking agent is selected from the group consisting of: lactic acid; a solution providing calcium ions, for example calcium chloride; and glyoxal. Thus, when the polymeric protein is gelatin or casein, or a derivative thereof, the cross-linking agent is glyoxal. When the polymeric protein is ovalbumin or a derivative thereof, the cross-linking agent is typically lactic acid. When the polymeric protein is elastin or a derivative thereof, the cross-linking agent is typically a calcium ion solution, i.e. calcium chloride.

In a preferred embodiment of the invention, the liquid hydrogel is formed by dissolving or suspending elastin and a gelling agent in a suitable solvent. Typically, the gelling agent is an alginate, or a salt or derivative thereof. Ideally, the solvent is aqueous. Other forms of non- proetin gelling agents well known to those skilled in the art include: sodium hyaluronate, chitosan, chondroitin sulfate, starch, amylose, amylopectin, pectins, heparan sulfate, galactomannans, dextrans, carrageenans, xanthan, inulin, cyclodextrins or a salt or derivative thereof.

The liquid hydrogel is ideally spray dried to form the microparticles. However, it will be appreciated that other methods of forming microparticles may be employed such as, for example, solvent evaporation, coacervation, and the like. The liquid hydrogel is spray dried in conditions to produce inhalable microparticles. In this specification, the term "inhalable microparticles" should be understood to mean microparticles that have a mass median aerodynamic diameter (MMAD) of lμm to 5μm. A method for determining the MMAD of particles is described below.

Typically, the spray drying is carried out through a nozzle having a diameter of 0.3mm and 0.7mm, especially from 0.4mm to 0.6mm. The spray drying is carried out in a heated gas, generally air or an inert gas. The inlet air temperature may be varied between 90° and 190°, and the outlet temperature varied between 30° and 70°.

The invention also relates to microparticles obtainable by a method of the invention.

The invention also provides a microparticle suitable for pulmonary delivery of an active agent and comprising a solid particle having a cross-linked three-dimensional matrix structure formed of a polymeric material, and an active agent entrapped and dispersed throughout the three dimensional matrix structure, the microparticle comprising a polymeric protein susceptible to proteolysis by elastase.

In one embodiment of the invention, the three-dimensional matrix structure is at least partly formed of the polymeric protein. Ideally, the polymeric protein is cross-linked. Ideally, the three-dimensional matrix structure is formed by an elastase-sensitive protein substrate and a non-protein gelling agent. The use of a non-protein gelling agent has surprisingly been found to be advantageous as it reduces the amount of protein in the microparticle and therefore reduces the immunogenicity of the particles. This is evidenced by the data in Table 8 below showing that the amount of IL-8 produced with microparticles comprising a proteinaceous and non-proteinaceous gelling agent is less that those formed with only a proteinacoeus gelling agent. Typically, the non-protein gelling agent is cross-linked, and ideally is a polysaccharide such as, for example, an alginate or an alginate derivative. In a preferred embodiment, the gelling agent in an anti-inflammatory gelling agent such as hyaluronic acid.

In an alternative embodiment, the polymeric protein forms a coating on the microparticle. In this regard, the three-dimensional matrix structure may be formed of a polymeric material that is not sensitive to degradation by elastase (i.e. PLGA). The polymeric material may have anti-inflammatory properties (i.e. hyaluronic acid or derivatives thereof).

Suitably, the polymeric protein is selected from the group consisting of: gelatin; ovalbumin; casein; and elastin, or salts or derivatives thereof.

In one embodiment of the invention, the microparticles have a Mass Median Aerodynamic Diameter (MMAD) of from 2μm to 5μm. Suitably, the microparticles have a density of 0.05 to 0.3 g/cm³.

Ideally, at least 10%, 15%, 25%, 40%, 50%, 60% or 70% of the microparticle consists of the polymeric protein (w/w).

The active agent may be any substance or compound, including a pharmaceutically active agent, an imaging dye, a flavoring agent, a coloring agent, a perfume, a detergent, and a cosmetic. In cases where the microparticles are employed as a means of pulmonary delivery, the active agent is generally a pharmaceutically active agent such as, for example, an antiinflammatory agent.

The invention also relates to an inhalable microparticle composition comprising a preparation of microparticles according to the invention.

The invention also relates to a method of treatment, prevention, or management, of a pulmonary disease or condition characterized by inflammation of epithelial cells in the pulmonary tract or lungs, the method comprising a step of administering an inhalable microparticle composition according to the invention to an individual in need thereof.

Suitably, the inhalable microparticle composition is administered in the form of an aerosol.

Typically, the disease or condition is selected from the group consisting of: asthma; chronic obstructive pulmonary disorder; cystic fibrosis; cough; infection; pneumonia; bronchopulmonary dysplasia; bronchitis; and tuberculosis.

Brief description of the Figures

Figure 1: Sites of pulmonary inflammation are characterised by the aberrant presence of neutrophils and thus neutrophil elastase. Polymers that are sensitive to the presence of elastase can provide targeted sites for specific drug delivery.

Figure 2: An illustration of the Franz Diffusion Cell used as an in vitro representation of the pulmonary system.

Figure 3: Respirable fraction (RF) of microparticles evaluated with Twin stage impinger. Particles of gelatin, casein and ovalbumin were blended with a coarse carrier, mannitol to improve flow. No carrier was used for alginate-elastin particles. The values are represented as mean±S.D. (n= 3).

Figure 4: Protein release profile from Ovalbumin microparticles in the presence or absence of elastase. Ovalbumin microparticles loaded with BSA-FITC were prepared as described in the Method section "Ovalbumin microparticle preparation." Degradation of the microparticles in the presence or absence of the elastase enzyme was assessed over time by spectrophotometrically monitoring the percentage of BSA-FITC released, relative to control. The microparticles that degraded in the presence of elastase showed a statistically significant increase in the rate of release of BSA-FITC relative to the sample of microparticles not exposed to elastase. Differences between treatment and control groups were compared by two-tailed Student's t test. P values of <0.05 were considered statistically significant. Each time point represents mean + SD, (n = T).

Figure 5: Protein release profile from Gelatin microparticles in the presence or absence of elastase. Gelatin microparticles were prepared with BSA-FITC as described in the Method section entitled "Gelatin microparticle preparation." Degradation of the microparticles was assessed over time by spectrophotometrically monitoring the percentage of BSA-FITC released (relative to control) in the presence and absence of the elastase enzyme. The microparticles that degraded in the presence of elastase showed a statistically significant increase in the rate of release of BSA-FITC relative to the microparticles in the sample not exposed to elastase. Differences between treatment and control groups were compared by two-tailed Student's t test. P values of <0.05 were considered statistically significant. Each time point represents mean + SD, (n = 2).

Figure 6: Protein release profile from elastase sensitive microparticles in the presence or absence of elastase. Protein release profiles from elastase sensitive microparticles in the presence (dotted lines) and absence (full lines) of elastase in phosphate buffer (pH 7.4). Gelatin (■), Casein (A), Ovalbumin (♦). Data shown as mean ± standard deviation (n=3).

Figure 7: Protein release profile from Alginate microparticles in the presence or absence of elastase. Alginate microparticles were prepared with BSA-FITC as described in the Method section "Alginate microparticle preparation". Degradation of the microparticles was assessed over time by spectrophotometrically monitoring the percentage of BSA-FITC released (relative to control) in the presence and absence of the elastase enzyme. Differences between treatment and control groups were compared by two-tailed Student's t test. P values of <0.05 were considered statistically significant. Each time point represents mean + SD, (n = 3).

Figure 8: Protein release profile from Elastin-Alginate sensitive microparticles in the presence or absence of elastase. Elastin-Alginate microparticles were prepared with BSA- FITC by the method previously described. Degradation of the microspheres was assessed over time by spectrophotometrically monitoring the percentage of BSA-FITC released (relative to control) in the presence and absence of the elastase enzyme. There was a statistically significant difference in the rate of release of BSA-FITC from the microparticle samples exposed to elastase relative to those that were not. Differences between treatment and control groups were compared by two-tailed Student's t test. P values of <0.05 were considered statistically significant. Each time point represents mean + SD, (n = 3).

Figure 9: Protein release profile from Elastin-Alginate sensitive microparticles in the presence or absence of elastase. Protein release profiles from alginate-elastin microparticles in the presence (•) and absence (o) of elastase in phosphate buffer (pH 7.4). Full lines: no mock sputum; dotted lines: with mock sputum. Data shown as mean ± standard deviation, n

=_).

Figure 10: Cross section of a microparticle.

Figure H: Scanning electron micrographs of BSA loaded microparticles. Alginate- elastin(A) at 30,00OX magnification; (B) and (C) at 80,000X magnification. Crosslinked microparticles of gelatin (D), casein (E), ovalbumin (F).

Table 1 - release level BSA-FITC from microparticle samples exposed to elastase relative to control

Detailed Description of the Invention Ovalbumin microparticle preparation

Ovalbumin (1% w/v) was dissolved in deionised water and mixed with an aqueous solution of BSA-FITC (0.01% w/v) at a ratio of 100:1. The crosslinking agent lactic acid (0.25%v/v) was added to the solution with mixing. The final solution was spray dried according to parameters outlined in Table 2.

Gelatin microparticle preparation

Gelatin (l%w/v) was dissolved in deionised water and aqueous glyoxal (40% v/v) was added to the solution at (0.5% v/v). This was mixed with an aqueous solution of BSA-FITC such that the ratio of Gelatin: BSA-FITC was 100:1. The final solution was then spray dried according to the parameters listed in Table 2.

Alginate microparticle preparation

BSA-FITC was dissolved in water and mixed with an aqueous solution of sodium alginate such that the concentration of polymer in water was l%w/v. The BSA:Polymer ratio was 1 : 100 and the final solution was spray dried according to the parameters listed in Table 2.

Alginate-Elastin microparticle preparation

Alginate (0.3%w/v) was dissolved in deionised water. Elastin (0.05%w/v) and BSA-FITC (0.003%w/v) were added to this solution and stirred to dissolve. The solution was then centrifuged at 6500rpm for 10 minutes while 10ml of CaCl₂ solution (1OmM Ca⁺²) was added drop wise at constant rate. The resulting mixture was spray dried according to the parameters outlined in Table 2.

Casein microparticle preparation

BSA-FITC was dissolved in water and mixed with an aqueous solution of sodium caseinate.

Glyoxal solution (40%w/w in water), as the cross linking agent, was added at 0.9%w/w relative to casein. The ratio of casein: BSA-FITC was 100:1. The resulting mixture was spray dried according to the parameters outlined in Table 2.

Spray Drying

A laboratory standard Buchi 190 spray dryer was used throughout with a nozzle size of

0.5mm. The parameters described in Table 2 indicate the specific requirements for each polymer. Table 2 - spray drying parameters

Particle Size Analysis The size of the microparticles was determined by laser diffraction (Malvern Mastersizer 2000, Malvern Instruments Ltd., Malvern, UK).

The microparticles (~25mg) were suspended in ethanol (Reagent grade, 5ml) in a test tube and sonicated by means of bath sonication at (320 W, Branson Ultrasonic, Danbury, CT) for one minute. The resultant solution was analysed by laser diffraction (Malvern Mastersizer 2000, Malvern Instruments Ltd, Malvern, UK) and the volume mean geometric diameters (d₅o) of the microparticles were calculated and are shown below in Table 3

Table 3 —particle size analysis

Percentage Protein Encapsulation Efficiency

To determine the encapsulation efficiency (%EE) of BSA-FITC, known amounts (~10mg) of microparticles were completely dissolved in 5ml of deionized water by magnetic stirring for 4 hours. For alginate-elastin particles, an aqueous solution of sodium citrate (0.1M) was used instead of deionized water. .

% EE was determined for each polymer and is shown in Table 4. The protein content was analysed by UV spectrophotometry at 495nm.

Table 4 -percentage protein encapsulation efficiency

In vitro release study to monitor the effect ofelastase on the breakdown of the microparticles All microparticles were incubated with porcine pancreatic elastase (PPE) to evaluate elastase induced degradation and its effect on the release of BSA-FITC. The solution chamber of the Franz Diffusion Cell (Figure 2) was filled to contain (10ml volume) of deionised water.

For alginate, ovalbumin and gelatin, lOmg of microparticles were deposited on the cellulose acetate membrane that separated the donor and receiver compartments of the cells. 500μl of 0.1M PBS (pH 7.4) containing 100μg/ml PPE was added to the upper chamber containing the microparticles. For control experiments, 500 μl of buffer without elastase was added.

For Alginate-Elastin microparticles, ~5mg of microparticles were deposited on the cellulose acetate membrane that separated the donor and receiver compartments. 50μl of 0.1M PBS (pH 7.4) containing 100μg/ml PPE was added to the upper chamber containing the particles. For control experiments, 50 μl of 0.1M PBS (without elastase) was added.

Time point zero was when the PBS (with or without elastase) was added on the microparticles in the upper chamber. Samples (250μl) were withdrawn from the lower compartment of the Franz Diffusion Cell at regular time points (either every 15 or 30 minutes) and analysed spectrophotometrically at 495nm for the presence of BSA-FITC. The Ovalbumin and Gelatin experiments were repeated in duplicate. The Alginate and Alginate- Elastin studies were repeated in triplicate.

Determination of aerosolization efficiency

To estimate the aerosolization efficiency of the particles, the Twin Stage Impinger (TSI) was used. This involves determining the quantity of microparticles deposited in the 2 stages of the impinger. For gelatin, casein and ovalbumin microparticles, this would involve hydrolyzing the particles to extract the encapsulated BSA-FITC and then measuring the fluorescence. Since both the polymeric carrier and the model drug (BSA-FITC) are proteins, hydrolyzing the carrier would also degrade BSA leading to erroneous results. To avoid this, batches containing sodium fluorescein (Na-FIu) (instead of BSA-FITC) were prepared for gelatin, casein and ovalbumin. Particles of alginate-elastin were used as before. D-Mannitol (Mannidex^®, Cerestar, Belgium) was used as a carrier for ovalbumin, casein and gelatine particles. Alginate-elastin particles had better dispersivity and were therefore used without a carrier. Mannitol was sieved to give particles in the range 60 to 125 μm. It was then mixed with the microparticles at a ratio of 24:1 (mannitol: microparticles). To achieve uniform distribution, the blend was passed thrice through a #125μm sieve. 30ml of DI water was added to Stage 2 of the device. 7ml was put into Stage 1. About 25mg of the blend was loaded into a Diskhaler^® and aerosolized by drawing air through the TSI at a flow rate of όOlmin^"1 for 5 seconds. TMs procedure was repeated 4 times for each polymer to facilitate quantification of Na-FIu. After inspiration, the apparatus was dismantled and each stage along with the device were washed with appropriate volumes of DI water and collected separately. Three independent experiments were conducted for the blend. The collected samples were freeze dried to remove water. In case of gelatin, ovalbumin and casein, the dried mass was digested in 0.1 M HCl by stirring overnight to enable hydrolysis. After neutralization with 0.1M NaOH, the samples were filtered and estimated for the content of Na-FIu. The fluorescence of flu-Na was measured using a fluorescence plate reader (Wallac Victor, Perkin Elmer, Cambridge, United Kingdom) at excitation and emission wavelengths of 488 and 530nm respectively. In case of alginate-elastin particles, the freeze dried mass was completely dissolved in aqueous solution of sodium citrate (0.1M) by magnetic stirring for 4 hours. The solutions were then analysed for BSA content using by UV spectrophotometry at 495nm. Size, density and calculated aerodynamic diameter (d_aer) of polymeric microparticles (mean±SD, n=3)

Table 5 — aerosolization efficiency of microparticles

The Twin Impinger described as Apparatus A in the European Pharmacopoeia was used to determine the respirable fraction. This is the fraction of the inhaled dose that reaches the lower bronchioles and alveolar regions of the lung. Table 6 shows the respirable fraction for the microparticles. % RF was highest in case of alginate-elastin particles (40.3%) even though no coarse carrier was used during aerosolization. Between gelatin, casein and ovalbumin, the RF was lowest in case of ovalbumin (26.3%).

Deposition in an twin stage impinger after aerosolisation of microparticles from a DPI at 60 1/min (mean±SD, n=3)

Table 6 - respirable fraction of microparticles

* Particles aerosolized as such (without blending with mannitol)

Toxicity Assay Cellular viability after exposure to the microparticles was assessed using the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay. Calu-3 cells were plated at a density of (3 x 10⁴ cells/well) in a 96 well plate and cultured at 37⁰C and 5% CO₂. After 24 hours in culture, the cells were exposed to a suspension of the microparticles (lOOμl/well) that was made from a stock solution of lmg/lml microparticles in growth media (1:1 mixture of Ham's F12:Dulbecco's modified Eagle's medium containing 200 units/ml Penicillin G Sodium and 200μg/ml Streptomycin Sulfate.). The cells were incubated for a further 24 hours at 37⁰C and 5% CO₂ At the end of this period, the supernatant was aspirated. To each well, 100 μl of growth media÷ 20 μl of a 5mg/ml solution of MTT was added. The cells were incubated for 4h at 37°C and 5% CO₂. The supernatant was then removed and the formazan crystals were dissolved in 100 μl of SDS (10% w/v SDS in 0.0 IN HCl) through further incubation at 37°C for 24 hours. The absorbance of each well was read on a Wallac microplate reader at 572nm. The spectrophotometer was calibrated to zero absorbance using SDS solution containing no cells. The relative cell viability (%) related to control wells containing cell culture medium without microparticles was calculated by [A]_test/[A]_COntroi x 100, where [A]test is the absorbance of the test sample, [A]_controi is the absorbance of the control sample.

Table 7 - % cell viability

Method for Papp measurement

Calu-3 bronchial epithelial cells were seeded on to Transwell clear polyester inserts at a seeding density of 0.5 x 10⁶ cells/cm² and cultured at an air-liquid interface. 12-13 days after seeding, particles were aerosolised onto the cell monolayers using a custom made device. The Transwell^® filter inserts were placed into new wells containing 1.5ml of bicarbonated Krebs- Ringer (KRB) solution in the basolateral compartment. 0.5ml of a 50μM Sodium fluorescein (Na-flu) solution in KRB was added to the apical compartment of each well. lOOul samples were taken at predetermined intervals up to 4hours from the basolateral compartment and replaced with an equal amount of fresh buffer. The fluorescence of Na-flu was measured in 96 well plates using a fluorescence plate reader (Wallac Victor, Perkin Elmer, Cambridge, United Kingdom) at excitation and emission wavelengths of 488 and 530nm respectively. The apparent permeability coefficient values were calculated using the following equation:

where Q: cumulative amount of Na-flu permeated across the cell monolayers over time t A: surface area of transwells (1.12 cm²); C₀: initial Na-flu concentration in the apical chamber.

Method for IL-8 measurement Basolateral media collected after 4 hours exposure to the microparticles was analysed for IL- 8 levels using ELISA MAX™ Kit (Biolegend, Inc., San Diego, CA, USA). The assay detection limit was 30pg/ml. The media collected was diluted.

Table 8 - immunogenicity of microparticles in Calu-3 cells: P_app of Fluorescein Sodium and cytokine release (IL-8)

* Mean±SD (n=4), Significantly different compared to control (p<0.05), ND: Not determined

Coated core microparticle preparation -double emulsion method The double emulsion method is used to prepare drug-loaded microparticles suitable for inhalation. A range of elastase-resistant polymers including for example sodium alginate, chitosan, HPC, sodium hyaluraonte and PLGA are used to prepare core elastase-resistant microparticles in the inhalable size range. As an example the method for preparing PLGA microparticles in the inhalable size range is given below:

Poly (lactic-co-glycolic acid) polymer (PLGA) was dissolved in an organic phase. A solution of 2.5% poly vinyl-alcohol (PVA) (alternatives include Polaxamer, Tween) was then added to create a primary emulsion (W₁Zo), which was homogenised or subjected to probe sonication. A secondary emulsion was then prepared by adding the Wi/o to a larger volume of aqueous phase surfactant (w₂) creating the wj/o/w₂ double emulsion that is subjected to homogenisation. Operating at a speed (rpm) from 6,500 - 21,500 rpm homogenisation it is possible to create microparticles ranging from 0.5 μm - 15μm. The microparticles are mechanically stirred to allow for the organic phase to evaporate in the fume hood to facilitate solvent extraction. The particles were centrifuged and washed in water to remove residual surfactant.

Spray-drying method

Drug loaded core microparticles may also be prepared by spray-drying. A range of elastase- resistant polymers including for example sodium alginate, chitosan, HPC, sodium hyaluraonte and PLGA may be used to prepare microparticles in the inhalable size range by spray-drying. The methods for each of these examples are given below. The feed solution for each polymer was prepared as described below:

• Sodium Alginate: BSA-FITC was dissolved in water and mixed with an aqueous solution of sodium alginate such that the concentration of polymer in water was l%w/v. BSA:Polymer ratio was 1:100. • Chitosan: BSA-FITC was dissolved in water. Chitosan was dissolved in 1% v/v Acetic acid. The two solutions were mixed such that the polymer concentration was 0.5%w/v and the BSA:Polymer ratio was 1:100.

• Hydroxypropyl Cellulose (HPC): BSA-FITC was dissolved in water and mixed with an aqueous solution of HPC-L (Protein: Polymer = 1:100). The polymer concentration was

0.5% w/v.

• Sodium Hyaluronate: BSA-FITC was dissolved in water and mixed with an aqueous solution of Sodium Hyaluronate (BSA-FITC: Sodium Hyaluronate = 1:100). The polymer concentration was 0.3%w/v. • Poly (lactide-co-glycolide): A w/o emulsion was prepared. Briefly, 50 mg BSA was dissolved in ImI of water containing 0.1%w/v of the surfactant, Tween 20. PLGA (Resormer® RG504H) was dissolved in 50ml of Dichloromethane. The aqueous phase was added to the organic phase drop wise under homogenisation at 9500 rpm using an Ika Homogeniser (Werke, Germany). The resulting emulsion was spray dried.

The drying parameters optimised for each polymer are provided in Table 7. Spray drying was done through a 0.5mm nozzle using a laboratory Buchi 190 spray dryer (Buchi, Flawil, Switzerland). The recovered particles were stored in a desiccator at 4°C until further use.

Table 9 — spray drying parameters double emulsion

Coating methods The core drug-loaded microparticles described above may be coated with elastase-sensitive polymers including elastin, ovalbumin and gelatin. Three methods are used to coat microparticles with these polymers including stirring, chemical conjugation or spray-drying.

• Stirring involves dissolving the protein coating in a solvent that will not dissolve the micro-particle. The micro-particles prepared by one of the methods described above are gently stirred in the presence of the protein to allow adsorption to occur and then separated from unadsorbed protein by centrifugation. The polymer coating is subsequently cross-linked using a cross-linking agent e.g. glutaraldehyde, transglutaminase, sucrose. • Spray drying is also used to coat particles with elastase-sensitive proteins. The core micro-particles are prepared by one of the methods described above and are then suspended in a solution of the proteins. This suspension is then spray-dried to produce a coated micro-particle. The protein coating must subsequently be cross-linked using a cross-linking agent e.g. glutaraldehyde, transglutaminase, sucrose. • For some core micro-particles chemical conjugation is used to enhance coating with the proteins. For example for PLGA core microparticles, chemical conjugation involves forming a covalent bond between the elastase sensitive protein and the PLGA using a cross-linking agent such as 1,1' -carbonyldiimidazole (CDI) or EDC (1 -Ethyl-3-[3-dimethylaminopropyl]carbodiirnide Hydrochloride).

The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.

Claims

1. A microparticle preparation suitable for pulmonary delivery of an active agent and comprising solid particles having a three-dimensional matrix structure and an active agent entrapped and dispersed throughout the three dimensional matrix structure, the particles having a Mass Median Aerodynamic Diameter (MMAD) of from 1 to 5μm, wherein the three-dimensional matrix comprises a cross-linked polymer susceptible to degradation by human neutrophil elastase.

2. A microparticle preparation as claimed in Claim 1 in which the polymer is a protein susceptible to degradation to human neutrophil elastase.

3. A microparticle preparation as claimed in Claim 1 in which the three dimensional matrix structure consists essentially of the polymeric protein.

4. A microparticle preparation as claimed in Claim 1, 2 or 3 in which polymeric protein is selected from the group consisting of: gelatin; ovalbumin; and elastin, or salts or derivatives thereof.

5. A microparticle preparation as claimed in any preceding Claim in which the solid microparticles have a MMAD of from 3 to 5μm.

6. A microparticle preparation as claimed in Claim 5 in which the solid microparticles have a MMAD of from 3 to 4μm.

7. A microparticle preparation as claimed in Claim 5 in which the solid microparticles have a MMAD of from 4 to 5μm.

8. A microparticle preparation as claimed in any preceding Claim in which the solid particles are spray-dried particles.

9. A microparticle preparation as claimed in any preceding Claim in which the three- dimensional matrix structure comprises an interpenetrating network formed of the polymeric protein and a cross-linked non-protein gelling agent.

1

10. A microparticle preparation as claimed in Claim 9 in which the non-protein gelling agent is a polysaccharide.

11. A microparticle preparation as claimed in Claim 10 in which the polysaccharide gelling agent is selected from the group consisting of: alginate; and alginate derivatives.

12. A microparticle preparation as claimed in Claim 11 in which the polymeric protein is elastase and the polysaccharide gelling agent is alginate, or an alginate derivative.

13. A microparticle preparation as claimed in Claim 10 in which the polysaccharide gelling agent is an anti-inflammatory polysaccharide gelling agent.

14. A microparticle preparation as claimed in Claim 13 in which the polysaccharide gelling agent is a hyaluronic acid.

15. A microparticle preparation as claimed in any of Claims 9 to 14 in which the ratio of polymeric protein to non-protein gelling agent is from 5:100 to 30:100 (w/w).

16. A microparticle preparation as claimed in any of Claims 9 to 14 in which the ratio of polymeric protein to non-protein gelling agent is from 10:100 to 20: 100 (w/w).

17. A microparticle preparation as claimed in any preceding Claim in which the active agent is selected from the group consisting of: a pharmaceutically active agent; a diagnostic agent; and a dye.

18. A microparticle preparation as claimed in Claim 17 in which the active agent is a pharmaceutically active agent for the treatment of a disease or condition characterized by inflammation of the pulmonary tract or lung.

19. A medicament comprising a microparticle preparation as claimed in any preceding

Claim.

20. Use of a microparticle preparation of any of Claims 1 to 18 in the manufacture of a medicament for the treatment of a disease or condition characterized by inflammation of the respiratory tract or lung.

21. A method of treating a disease or condition characterized by inflammation of the respiratory tract or lung in an individual in need thereof, the method comprising administering a microparticle preparation of any of Claims 1 to 18 to the individual in the form of an aerosol delivered to the respiratory tract and/or lungs.

22. A method for the controlled and targeted delivery of an active agent to a locus in the respiratory tract or lung characterized by inflammation, the method comprising the steps of administering a microparticle preparation of any of Claims 1 to 18 to the individual in the form of an aerosol delivered to the respiratory tract and/or lungs, wherein endogenous elastase present at the loci in the respiratory tract or lung characterized by inflammation cause a controlled and targeted release of the active agent from the microparticle due to controlled degradation of the elastase sensitive polymer.

23. A use of Claim 20, or a method of Claim 21 or 22, in which the disease or condition characterized by inflammation of the respiratory tract or lung is selected from the group consisting of: asthma; chronic obstructive pulmonary disorder; cystic fibrosis; cough; infection; pneumonia; bronchopulmonary dysplasia; bronchitis; and tuberculosis.

24. A method of producing microparticles suitable for pulmonary delivery of an active agent, the method comprising the steps of forming a liquid hydrogel by suspending or dissolving a polymer susceptible to proteolysis by elastase in a suitable solvent, the hydrogel further comprising the active agent dispersed throughout the hydrogel, and spray drying the liquid hydrogel to form microparticles having a MMAD of from 1 to 5μm, wherein the liquid hydrogel is cross-linked.

25. A method as claimed in Claim 24 in which the polymer susceptible to proteolysis by elastase is a protein.

26. A method as claimed in Claim 25 in which polymeric protein is selected from the group consisting of: gelatin; ovalbumin; casein; and elastin, or salts or derivatives thereof.

27. A method as claimed in Claim 25 or 26 in which the hydrogel is cross-linked by cross-linking of the polymeric protein.

28. A method as claimed in Claim 27 in which the polymeric protein is gelatin, wherein the cross-linking agent is glyoxal.

29. A method as claimed in Claim 27 in which the polymeric protein is ovalbumin, wherein the cross-linking agent is lactic acid.

30. A method as claimed in Claim 27 in which the polymeric protein is casein, wherein the cross-linking agent is glyoxal.

31. A method as claimed in Claim 27 in which the polymeric protein is elastin, wherein the cross-linking agent is calcium chloride.

32. A method as claimed in any of Claims 24 to 31 in which the liquid hydrogel comprises an interpenetrating network formed of the polymeric protein and a nonprotein gelling agent, wherein the non-protein gelling agent is cross-linked.

33. A method as claimed in Claim 32 in which the liquid hydrogel is formed by dissolving or suspending the polymeric protein and the non-protein gelling agent in a suitable solvent, and centrifuging the mixture while adding a cross-linking agent.

34. A method as claimed in Claim 32 or 33 in which the non-protein gelling agent is a polysaccharide.

35. A method as claimed in Claim 34 in which the polysaccharide gelling agent is selected from the group consisting of: alginate; and alginate derivatives.

36. A method as claimed in any of Claims 32 to 35 in which the polymeric protein is elastase and the polysaccharide gelling agent is alginate, or an alginate derivative.

37. A method as claimed in Claim 36 in which the cross-linking agent is a calcium ion solution such as calcium chloride.

38. A method as claimed in Claim 34 in which the polysaccharide gelling agent is an anti- inflammatory polysaccharide gelling agent.

39. A method as claimed in Claim 38 in which the polysaccharide gelling agent is a hyaluronic acid.

40. A method as claimed in any of Claims 24 to 39 in which the polymeric protein comprises between 0.05 and 10% of the hydrogel (w/w).

41. A method as claimed in any of Claims 24 to 40 in which the spray drying is carried out through a nozzle having a diameter of 0.3mm and 0.7mm.

42. A method as claimed in any of Claims 24 to 40 in which the spray drying is carried out through a nozzle having a diameter of especially from 0.4mm to 0.6mm.

43. A method as claimed in any of Claims 24 to 42 in which the inlet air temperature of the spray dryer is from 90° and 190°, and the outlet temperature is from 30° and 70°