Frederic Tewes

Krzysztof J Paluch

Lidia Tajber

Karan Gulati

Devesh Kalantri

Carsten Ehrhardt

Anne Marie Healy
email: healyam@tcd.ie

Steroid/mucokinetic hybrid nanoporous microparticles for pulmonary drug delivery

Keywords: Budesonide, Ambroxol, Nanoporous microparticles, Calu-3 cells, Andersen cascade impactor, Dry powder inhaler

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pharmacotherapy of lung diseases often involves treatment with more than one group of therapeutic agents (e.g., bronchodilators, corticosteroids, cromoglycate, and antibiotics). In addition to the oral route, these agents are often delivered by pulmonary inhalation, in order to directly target the lung and increase the therapeutic/systemic side effect index.

The local delivery of drugs to the lung can be obtained by using dry powder inhalers (DPIs), metered dose inhalers (MDIs) or nebulisers. Whenever it is possible, the use of DPIs or MDIs is preferred by patients due to the convenience of use [START_REF] Westerman | Effect of nebulized colistin sulphate and colistin sulphomethate on lung function in patients with cystic fibrosis: A pilot study[END_REF][START_REF] Dolovich | Aerosol drug delivery: Developments in device design and clinical use[END_REF]. The simplest formulation and least problematic, from the point of view of stability, appears to be dry powders due to the presence of the active pharmaceutical ingredient (API) as solid particles and the lack of propellant, in contrast to pressurised MDIs, where the API is dissolved or suspended in the liquefied vehicle [START_REF] Maesen | Formoterol suspension aerosol. Comparison with formoterol solution aerosol for 12 weeks in asthmatic patients[END_REF]. Also, in order to improve the results of pharmacotherapy, increase patient compliance and reduce the time of administration of several APIs, co-formulation of drugs into one product [START_REF] Petraitis | Combination Therapy in Treatment of Experimental Pulmonary Aspergillosis: In Vitro and In Vivo Correlations of the Concentration-and Dose-Dependent Interactions between Anidulafungin and Voriconazole by Bliss Independence Drug Interaction Analysis[END_REF][START_REF] Yang | Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis[END_REF][START_REF] Kumon | Can low-dose combination products for inhalation be formulated in single crystalline particles?[END_REF][START_REF] Traini | In Vitro Investigation of Drug Particulates Interactions and Aerosol Performance of Pressurised Metered Dose Inhalers[END_REF] or co-administration of drugs as a mix of solutions for nebulisation is popular.

However, the latter can bring the risk of physicochemical instability of components when mixed extemporaneously [START_REF] Kamin | Inhalation solutions -which one are allowed to be mixed? Physicochemical compatibility of drug solutions in nebulizers[END_REF].

To optimise the efficacy of DPIs, conventional micronised API carrier-based formulations may be substituted by an optimised API powder having specific particle morphology and designed to provide advantageous flow and aerodynamic properties. For example, tobramycin powder was produced using emulsion-based PulmoSphere technology, producing highly dispersible porous particles [START_REF] Newhouse | Inhalation of a dry powder tobramycin PulmoSphere formulation in healthy volunteers[END_REF]. Excipient-free nanoporous microparticles (NPMPs) [START_REF] Healy | Characterisation of excipient-free nanoporous microparticles (NPMPs) of bendroflumethiazide[END_REF] of budesonide (BUD) [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF] and sodium cromoglycate [START_REF] Nolan | Particle engineering of materials for oral inhalation by dry powder inhalers. II -Sodium cromoglicate[END_REF] prepared by a novel spray drying process had improved in vitro deposition properties compared to non-porous particles, though it remained unknown, if it is possible to produce NPMPs composed of two APIs and still be able to maintain the superior aerodynamic properties.

BUD was previously reported to form NPMPs when processed by spray drying [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF].

BUD is a glucocorticosteroid which, when inhaled, has a dose-dependent anti-inflammatory action in the lung [START_REF] Donnelly | Clinical pharmacokinetics of inhaled budesonide[END_REF]. It acts by binding to glucocorticoid receptors in lung cell cytoplasm [START_REF] Usmani | Glucocorticoid receptor nuclear translocation in airway cells after inhaled combination therapy[END_REF].

Budesonide has been shown to be very effective against the characteristic inflammation of asthma [START_REF] Brand | Inhaled corticosteroids should be the first line of treatment for children with asthma[END_REF]. It is reported to reduce the risk of Chronic Obstructive Pulmonary Disease (COPD) exacerbations in symptomatic COPD patients [START_REF] Alsaeedi | The effects of inhaled corticosteroids in chronic obstructive pulmonary disease: A systematic review of randomized placebo-controlled trials[END_REF]. Budesonide also resulted in an improvement in bronchial hyper-responsiveness and a decrease in cough in cystic fibrosis patient [START_REF] Van Haren | The effects of the inhaled corticosteroid budesonide on lung function and bronchial hyperresponsiveness in adult patients with cystic fibrosis[END_REF].

However, in these two last diseases, efficacy of inhaled corticosteroids can be strongly reduced because of the low diffusion through the viscous surface secretions of the lung [START_REF] Dinwiddie | Anti-inflammatory therapy in cystic fibrosis[END_REF]. In fact, in a number of pulmonary diseases, patients may develop abnormally high viscous thick mucus which is responsible for low diffusion of drugs in the lungs [START_REF] Yang | Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis[END_REF][START_REF] Khanvilkar | Drug transfer through mucus[END_REF][START_REF] Mcgill | Disruption of the Mucus Barrier by Topically Applied Exogenous Particles[END_REF].

To reduce the viscosity of the lung fluid, mucokinetics such as ambroxol hydrochloride (AMB-HCl) are used [START_REF] Paleari | Ambroxol: A multifaceted molecule with additional therapeutic potentials in respiratory disorders of childhood[END_REF]. Ambroxol may provide suitable rheological properties of the airway secretion by acting directly on ciliated epithelial cells [START_REF] Hasegawa | Ambroxol-induced modification of ion transport in human airway Calu-3 epithelia[END_REF]. Ren et al., [START_REF] Ren | Pulmonary selectivity and local pharmacokinetics of ambroxol hydrochloride dry powder inhalation in rat[END_REF] showed that the rat lung epithelial lining fluid (ELF)/plasma AUC 0->10h ratios obtained for ambroxol hydrochloride (AMB-HCl) after dry powder pulmonary administration were 33 to 56 times higher compared to those achieved by intravenous administration. Thus, the co-administration of BUD together with AMB-HCl could possibly improve the diffusion of BUD in the lining fluid of the lung and enhance its bioavailability. For that reason, we developed a combined BUD AMB-HCl formulation as excipient-free NPMPs to be used in DPIs.

Materials and methods.

Materials

Budesonide (BUD) was purchased from Tianjin Tian Mao Technology Development Corp. Ltd., China. Acetonitrile (HPLC grade), ambroxol hydrochloride (AMB-HCl) and ammonium carbonate (NH 4 ) 2 CO 3 (analytical grade) were purchased from Sigma-Aldrich (Dublin, Ireland).

Ultrapure water was produced by a Synergy Ultrapure Water System linked to an Elix Advantage system (Millipore, Carrigtwohill, Ireland). Sodium pyruvate, foetal calf serum (FCS), nonessential amino acids solution (NEAA), minimum essential medium (MEM), glucose, penicillin/streptomycin solution, NaCl, KCl, CaCl 2 , MgCl 2 •6 H 2 O, NaHCO 3 and HEPES were cell culture grade and purchased from Sigma-Aldrich.

Methods

Spray drying

Spray drying experiments were performed based on methods described by Nolan et al. [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF].

Briefly, 1% (w/w) solutions of different BUD:AMB-HCl ratios, with and without (NH 4 ) 2 CO 3 (Table 1), were spray dried using a Büchi B-290 Mini spray dryer (Flawil, Switzerland) from a solvent made of 80% (v/v) ethanol and 20% (v/v) water. The spray dryer was set in an open suction mode using a standard two-fluid nozzle. In all cases the inlet temperature was 78°C; feeding pump was set at 30% (8 mL/min); spraying nitrogen nozzle flow rate was 15 L/min; and nitrogen flowing at 630 L/min was used as the drying gas (aspirator rate 100%). These conditions resulted in outlet temperatures ranging from 42 to 52°C.

Scanning electron microscopy (SEM)

Scanning electron micrographs of powder samples were taken using a Tescan Mira XMU (Brno, Czech Republic) SEM. The dry powder samples were fixed on aluminium stubs with doublesided adhesive tape and a 10 nm-thick gold film was sputter coated on the samples, before visualisation. Primary electrons were accelerated under a voltage of 5 kV. Images were formed from the collection of secondary electrons.

Specific surface area

The specific surface area of the samples was determined by the N 2 adsorption B.E.T. multipoint method, with 6 points in the relative pressure range of 0.1-0.3, using a Micromeritics Gemini 2835c (SMS Ltd., London, UK) as previously described [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF]. Samples were prepared by purging under N 2 overnight at 40 °C.

Particle size distribution analysis

The geometric particle size distributions (PSD) were determined by laser diffraction using a Malvern Mastersizer 2000 (Malvern Instruments Ltd. Worcestershire, UK) with the Scirocco 2000 dry powder feeder to disperse the particles. The dispersive air pressure used was 3 bar and vibration feed rate was set to 50%. The PSD of each sample was determined in triplicate.

Differential scanning calorimetry (DSC)

DSC was performed using closed 40 µL-aluminium pans with three vent holes on samples (4 -8 mg) weighed with a MT5 balance (Mettler Toledo, Zürich, Switzerland) [START_REF] Tewes | Development and characterisation of soluble polymeric particles for pulmonary peptide delivery[END_REF]. Samples were run at a heating rate of 10ºC/min under nitrogen purge from 40ºC to 270ºC using a Mettler Toledo DSC 821 e (Switzerland). Mettler Toledo STAR e software was used for analysis of thermal events.

Powder X-Ray Diffraction (XRD)

X-ray powder diffraction measurements were conducted on samples placed in a low background silicon holder, using a Miniflex II desktop X-ray diffractometer (Rigaku, Tokyo, Japan) with a Bragg-Brentano geometry [START_REF] Tewes | Development and characterisation of soluble polymeric particles for pulmonary peptide delivery[END_REF][START_REF] Tewes | Evaluation of HPβCD-PEG microparticles for salmon calcitonin administration via pulmonary delivery[END_REF]. The samples were scanned over a range of 5 -40º 2θ at a step size of 0.05º per second. The X-ray tube composed of copper anode was operated under a voltage of 30 kV and a current of 15 mA, which, after crossing a monochromator, produced copper Kα radiation (λ = 1.542 Ǻ). A scintillation counter detected the X-rays.

Drug quantification

A HPLC method, based on the method described by Nolan et al. [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF] was set up to assay both BUD and AMB-HCl simultaneously. The assay was performed using a LC module I plus (Waters, UK) chromatographic system equipped with a C18, 15-20 µm, 3.9 x 300 mm apolar column (μBondapak TM , Waters, Ireland). Drug elution was detected by measuring the absorbance at 242 nm. Measurements were conducted at room temperature by injecting 50 μL of samples, standards or controls in the mobile phase running at 1.5 mL/min and composed of 55% by volume of 0.05 M CH 3 COOH/Na buffer (pH 4) and 45% of acetonitrile. The running time of the assay was 7 min. A calibration curve for each drug was constructed, allowing the drug concentrations in samples to be calculated. The concentration of the standard solutions of each drug used to construct the calibration curves were in the range 6 μg/mL to 100 μg/mL.

In Vitro Deposition Studies Using the Andersen Cascade Impactor (ACI)

A dry powder inhaler (Cyclohaler ® , N.V. Medicopharma, Zaandam, Netherlands) was filled with a size 3 hard gelatin capsule loaded with 20±2 mg of powder for each test. After inhaler actuation, particle deposition in the device, the induction port, all the stages and the filter was determined by the HPLC assay described above (n=3). The flow rate (60 L/min) used in the ACI.

The time of aspiration was adjusted to obtain 4 L. The total amount of particles with aerodynamic diameters smaller than 5.0 μm was calculated by interpolation from the inverse of the standard normal cumulative mass (mg) distribution less than stated size cut-off against the natural logarithm of the effective cut-off diameter (ECD) of the respective stages. This amount was considered as the fine particle fraction (FPF). The mass median aerodynamic diameter (MMAD) of the particles was defined from a similar plot, but using the cumulative mass fraction (%) against the ECD of the respective stage, as the particle size at which the line crosses the 50% mark.

BUD and AMB transport across Calu-3 cell monolayers

Calu-3 human bronchial epithelial cells were cultured in Eagle's minimum essential medium supplemented with 1 mM of sodium pyruvate, 10% (v/v) foetal calf serum, 1% (v/v) nonessential amino acids, 2.25 g/L glucose and 1% (v/v) penicillin/streptomycin, and were used between passages 38 and 55. Cells were cultured in 75 cm 2 flasks and maintained in a humidified atmosphere of 5% CO 2 and 37 °C. Cell monolayers were obtained by seeding Calu-3 cells (5 × 10 5 cells/cm 2 ) onto polyester Transwell Clear inserts, with 0.4 μm pore diameter and 1.12 cm 2 of surface area (Corning, Netherlands). Growth medium, 1.5 mL on the basal side compartment and 0.5 mL on the apical side, was replaced with fresh medium every other day.

A) A first set of experiments was conducted as follows: After 10 days in culture, the culture medium on the apical side was removed and cells were grown under air-interfaced culture (AIC) conditions for another 10 days. These culture conditions have previously shown to induce the production of mucus by Calu-3 cells [START_REF] Borchard | Transport and local metabolism of budesonide and fluticasone propionate in a human bronchial epithelial cell line (Calu-3)[END_REF][START_REF] Forbes | Human respiratory epithelial cell culture for drug delivery applications[END_REF] with HBSS for 1 h prior to the transport study. For AIC conditions, the basal side of the Calu-3 monolayers were equilibrated with HBSS for 1 h prior to the transport study. For both AIC and LCC conditions, transport studies were initiated by adding to the apical side 0.5 mL of the test solutions containing BUD (20 µg/mL) with or without AMB-HCl (20 µg/mL). After 3 h at 37°C, samples were collected from the acceptor and donor compartments and assayed for BUD and AMB-HCl.

BUD and AMB-HCl were assayed using the chromatographic method described above.

Fluorescein was assayed using a 96-well plate reader (FLUOstar OPTIMA, BMG Labtech, Aylesbury, UK) by measuring the fluorescence intensity at 520 nm after excitation at 492 nm.

Standard solutions of fluorescein were made in KRB with concentration ranging from 0.015 µg/mL to 1 µg/mL. Correlation coefficient of the calibration curves were higher than 0.99.

The apparent permeability (P app ) values in the apical-to-basal direction were calculated using the following equation:

a b b app C t A V C P . . .  (1)
where C b was the solutes concentration and V b the volume (1.5 mL) of buffer in the basal side, t the incubation time (180 min) and A the insert surface area (1.12 cm 2 ). C a was the initial concentration in the apical side.

Statistical data analysis

Data were statistically evaluated by a two-tailed unpaired Student t-test. Significance level was <0.05.

Results

Spray drying of formulations A1-A3 resulted in the production of discrete and non-porous microparticles, having smooth surfaces (Fig. 1). Microparticle morphology depended on the BUD:AMB-HCl ratio. At low AMB-HCl concentrations (A1-A2) the particles were crumpled.

An increase in AMB-HCl concentration (A3) led to particles with more spherical morphology. 1.

Figure 1: SEM of particles made of BUD and AMB-HCl as described in Table

The addition of 15% (w/w) of (NH 4 ) 2 CO 3 altered the morphology of microparticles (Fig. 2). Most particles appeared to be porous with rough surfaces. At a constant (NH 4 ) 2 CO 3 concentration of 15% (w/w) (C1 to C4) an increase in AMB-HCl concentration reduced the number of porous microparticles visible on SEM micrographs. Similarly, the specific surface area measured by N 2 adsorption decreased from 6.50±0.14 m 2 /g for C1 to 2.53±0.05 m 2 /g for C4, respectively. When the content of AMB-HCl was held constant (C2, C5, C6 and C4, C7), a small increase in (NH 4 ) 2 CO 3 concentration (from 15% to 25% (w/w)), increased the number of pores visible by particles (Fig. 2: C2, C5) and increased the specific surface area from 5.67±0.08 m 2 /g for C2 to 7.14±0.17 m 2 /g for C5, respectively. However, a larger increase in (NH 4 ) 2 CO 3 concentration (i.e., 35% w/w) produced hollow particles (Fig. 2: C6 and C7) and reduced the specific surface area to 3.91±0.06 m 2 /g for C6.

Figure 2: SEM and specific surface area values of the particles formulated in presence of (NH 4 ) 2 CO 3 as described in Table 1. Error bars in the histogram correspond to the standard deviation of the specific surface area values calculated from 3 measurements.

Selected data of the geometric volume-weighted particles size analysis of the powders formulated using (NH 4 ) 2 CO 3 are displayed in Table 2. The different formulations had similar particle size distributions. However, an increase in AMB-HCl or in (NH 4 ) 2 CO 3 increased the median value of the particle diameter (D 50 ) and widened the range of the particle size distribution, as shown by an increase in the span value. The specific surface area of the powder of formulation C4, calculated from the particle size distribution, considering the particles to be non-porous, was similar to the one calculated from the N 2 adsorption isotherm using the B.E.T. equation. However, B.E.T analysis for C5 gave a specific surface area value which was 2.3 times higher than that calculated from the particle size distribution. This difference can be attributed to the presence of pores in C5 and not in C4.

Table 2: Geometric volume-weighted particle size analysis

Unprocessed materials, i.e., AMB-HCl, (NH 4 ) 2 CO 3 and BUD presented sharp XRD Bragg peaks characteristic of crystalline compounds (Fig. 3). After spray drying, PXRD patterns of pure BUD and AMB-HCl presented a diffuse halo, characteristic of XRD amorphous materials, centred at 15 and 22.5° (2θ), respectively. In the presence of (NH 4 ) 2 CO 3 , one diffraction peak at 2θ = 32.65° appeared in the PXRD pattern of the AMB-HCl/(NH 4 ) 2 CO 3 3/7 (w/w) spray dried system in addition to the diffuse halo (Fig. 3). DSC analysis of unprocessed BUD and AMB-HCl confirmed powders to be crystalline with melting endotherm point onsets at 247°C and 242°C, respectively (Fig. 4). Spray dried BUD and AMB-HCl DSC scans displayed a relaxation endotherm of a glass transition (Tg) with onsets of for BUD and AMB-HCl, respectively (Fig. 4). The melting endotherm observed for DSC crystallised BUD shifted to an onset of 260°C.

Unprocessed and spray dried AMB-HCl presented similar melting endotherms. DSC scans of AMB-HCl/(NH 4 ) 2 CO 3 3/7 (w/w) spray dried system presented four endotherms having the following temperature onsets: 60, 90, 98 and 229°C (Fig. 4).

Figure 5: PXRD patterns of the formulations as described in Table 1.

Formulations made of various BUD:AMB-HCl ratios (A1-A3) presented only a diffuse halo on their PXRD patterns (Fig. 5). The centre of the diffuse halo shifted from 15° (2θ) to higher values as the BUD:AMB-HCl ratio decreased. PXRD patterns of BUD:AMB-HCl formulations spray dried in the presence of (NH 4 ) 2 CO 3 (C1-C8) displayed a diffuse halo that became broader as the BUD:AMB-HCl ratio decreased. Furthermore, a diffraction peak with increasing intensity as the AMB-HCl concentration increased appeared at 2θ = 32.6°. The intensity of this diffraction peak was independent of the (NH 4 ) 2 CO 3 concentration. The thermograms recorded for the formulations A1 to A3 (Fig. 6A) displayed an onset of Tg at around 90°C, followed by one exotherm of crystallisation and two endotherms of melting. The temperature onsets and H of these thermal events changed with the BUD:AMB-HCl ratio as shown in Supplementary data 1.

Supplementary data 1:

Thermal event values for the BUD/AMB-HCl systems obtained by DSC. At low AMB-HCl concentrations (formulation A1 and A2), two distinguishable endotherms (i.e., individual peaks) were observed. As the AMB-HCl concentration increased, the endotherms became closer and were followed by a noisy baseline, which could be attributed to product degradation. Also, DSC analysis of physical mixes of BUD:AMB-HCl in the range of weight ratios 8:2 to 2:8 showed that the materials melted together at a lower temperature than the individual components (Supplementary data 2), demonstrating that they are miscible or partially miscible in the liquid state [START_REF] Caron | A comparison of spray drying and milling in the production of amorphous dispersions of sulfathiazole/polyvinylpyrrolidone and sulfadimidine/ polyvinylpyrrolidone[END_REF].

Supplementary data 2: DSC thermograms of the BUD/AMB-HCl physical mix of the raw materials.

The thermograms of formulations C1 to C8 (Fig. 6B) did not display a visible T g . For all these systems, DSC scans displayed one broad or two merged exotherms above 110°C followed by one or two endotherms. The endotherms were either merged or isolated with their temperature onsets and H varying depending on the BUD:AMB-HCl ratio, as reported in the Supplementary data 2.

Additionally, scans of C1 presented two small endotherms having a T onset at 90 and 98°C.

Results of the particles' content analysis obtained by HPLC are shown in Table 2. For all formulations, the percentage of drugs found was close to that calculated on the basis that all the (NH 4 ) 2 CO 3 was eliminated. Also the relative amount of AMB-HCl plus BUD was close to 100%, suggesting that most of the (NH 4 ) 2 CO 3 was eliminated during the spray drying, and that the particles are mainly composed of BUD and AMB-HCl. Comparable results were obtained when BUD P app value was evaluated after co-processing BUD with AMB-HCl and (NH 4 ) 2 CO 3 (C5). Based on the particle composition analysis, the apical AMB-HCl concentration in this condition was approximately 3.3 μg/mL. Fluorescein P app values showed similar changes as in the case of BUD. In the presence of BUD alone, the fluorescein P app value was 1.4  0.1•10 -7 cm/s and significantly increased to 2.6 ± 0.6•10 -7 cm/s in the presence of formulation C5 (t-test, P < 0.05). AMB-HCl P app values were identical for all the AMB-HCl concentrations used (~1.4 ± 0.2•10 -5 cm/s).

Discussion

In order to increase BUD diffusion within abnormally viscous mucus, the co-administration of BUD and AMB-HCl as a dry powder is of interest. Therefore, our studies aimed to develop highly respirable nanoporous microparticles made of BUD and AMB-HCl. These composite particles were formulated with the aim of enhancing BUD diffusion within thick mucus for a local action and not for a bi-therapy purpose or to enhance BUD systemic absorption. Inhalation therapy results in a high concentration of active drug at the site of action and requires much lower doses than oral administration. As shown by Ren et al., [START_REF] Ren | Pulmonary selectivity and local pharmacokinetics of ambroxol hydrochloride dry powder inhalation in rat[END_REF], this is the case for AMB. Therefore, the drug ratios in the present particle compositions were lower than the doses normally used for the individual drugs when used in oral delivery.

We previously described the formulation of nanoporous microparticles (NPMPs) of pure BUD by spray drying solutions with a solvent made of ethanol and water in a ratio of 80:20 (v/v) [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF].

The BUD NPMPs formulated in this previous study were shown to be optimal for pulmonary administration using a DPI as they had high FPF (74.93.5%) and good MMAD (2.050.10 µm).

However, particles of BUD containing 5 to 20% (w/w) of AMB-HCl prepared in the current work were non-porous and had smooth surfaces (Fig. 1). Particles containing 20 wt% of AMB-HCl (A3) were similar to those obtained by Ren et al., [START_REF] Ren | Influence of formulation and preparation process on ambroxol hydrochloride dry powder inhalation characteristics and aerosolization properties[END_REF] by spray drying an aqueous solution of pure AMB-HCl. The change in BUD particle morphology induced by the addition of AMB-HCl could be due to the slow solidification of AMB-HCl during the solvent evaporation process in the spray dryer. This morphology change was unsatisfactory, as it may adversely affect the particles aerodynamic properties [START_REF] Chow | Particle engineering for pulmonary drug delivery[END_REF][START_REF] Crowder | Fundamental effects of particle morphology on lung delivery: Predictions of Stokes' law and the particular relevance to dry powder inhaler formulation and development[END_REF]. Thus, (NH 4 ) 2 CO 3 was incorporated in the formulation in order to overcome this drawback. In fact, (NH 4 ) 2 CO 3 is commonly used as a blowing agent, [START_REF] Traini | In Vitro Investigation of Drug Particulates Interactions and Aerosol Performance of Pressurised Metered Dose Inhalers[END_REF][START_REF] Edwards | Large porous particles for pulmonary drug delivery[END_REF], pore-forming agent [START_REF] Edwards | Large porous particles for pulmonary drug delivery[END_REF] or process enhancer [START_REF] Healy | Characterisation of excipient-free nanoporous microparticles (NPMPs) of bendroflumethiazide[END_REF][START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF]. This compound decomposes and produces gases during spray drying, thus it is able to create porous or hollow particles.

Most of the composite particles made of BUD and AMB-HCl, formulated with (NH 4 ) 2 CO 3 were porous (Fig. 2). The size and number of pores depended on the initial concentration of AMB-HCl and (NH 4 ) 2 CO 3 in the spray dried solution. The increase in porosity and the enlargement of the pore size with the increase in (NH 4 ) 2 CO 3 content may be related to the increase in the amount of CO 2 and NH 3 produced from (NH 4 ) 2 CO 3 [START_REF] Traini | In Vitro Investigation of Drug Particulates Interactions and Aerosol Performance of Pressurised Metered Dose Inhalers[END_REF]. For AMB-HCl contents <15% (w/w) and (NH 4 ) 2 CO 3 contents <25% (w/w), the microparticle morphology was similar to that of the pure BUD particles formulated by Nolan et al. [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF]. However, Nolan et al. showed that (NH 4 ) 2 CO 3

did not have any influence on the pure BUD particle morphology produced in these conditions. In fact, NPMPs of BUD are produced due to the early BUD precipitation in the spray droplet and the formation of gas bubbles do not change their structure [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF]. Thus, the formation of nanopores in the BUD/AMB-HCl composite particles, by the addition of (NH 4 ) 2 CO 3 , should be related to its effect on AMB-HCl.

Additionally to its capacity to decompose into gases upon heating, (NH 4 ) 2 CO 3 dissolved in water produces a solution composed of HCO 3 -, NH 4 + and NH 3 , with a pH around 9.2. AMB is a weak base having a pKa of 8.05 at 25°C [START_REF] Meloun | The thermodynamic dissociation constants of ambroxol, antazoline, naphazoline, oxymetazoline and ranitidine by the regression analysis of spectrophotometric data[END_REF]. As a hydrochloride salt, AMB-HCl can react with the HCO 3 -and NH 3 species leading to the formation of AMB base, NH 4 + , Cl -and H 2 CO 3 as described by the following chemical equilibria.

Using the pKa, the relative concentrations of the different species in solution, and considering the medium as pure water, the percentage of AMB in unionised form (base) calculated ranged from 91.8 to 88% of the whole AMB for C1 and C4, respectively. These calculations were performed considering pure aqueous solutions. However, spray dried solutions were made with 80 %v/v of

𝐴𝑀𝐵𝐻 + , 𝐶𝑙 -+ 𝑁𝐻 3 ↔ 𝐴𝑀𝐵 + 𝑁𝐻 4 + , 𝐶𝑙 - 𝐴𝑀𝐵𝐻 + , 𝐶𝑙 -+ 𝐻𝐶𝑂 3 -, 𝑁𝐻 4 + ↔ 𝐴𝑀𝐵 + 𝑁𝐻 4 + , 𝐶𝑙 -+ 𝐻 2 𝐶𝑂 3
ethanol. Ethanol is an amphiprotic solvent like water, but having a lower dielectric constant [START_REF] Akerlof | Dielectric constants of some organic solvent-water mixtures at various temperatures[END_REF].

The apparent ionization constants (psKa) of base, such as ambroxol, in organic solvent:water mixtures decrease with an increase in organic solvent concentration [START_REF] Takács-Novák | Potentiometric pKa determination of water-insoluble compounds: validation study in methanol/water mixtures[END_REF]. Therefore, in the presence of ethanol the chemical reactions described above should be favoured, leading to the formation of AMB base more than in pure water.

To support this, DSC scans of a AMB-HCl/(NH 4 ) 2 CO 3 3/7 (w/w) spray dried system (Fig. 4) and of the formulation C1 (Fig. 6) showed 4 endotherms. The first endotherm, with a temperature onset of 60°C may correspond to solvent evaporation. As observed by Caira et al., [START_REF] Caira | Thermal and structural properties of ambroxol polymorphs[END_REF] the following two endotherms having a T onset at 90 and 98 o C are attributed to the melting of the AMB base crystallised as form I and form II, respectively. Furthermore, XRD patterns of AMB-HCl and BUD-AMB-HCl formulations spray dried with (NH 4 ) 2 CO 3 presented a Bragg peak of diffraction at 2θ = 32.65° and at 2θ = 22.85° (Fig. 5). These peaks characterised the presence of NH 4 Cl in the particles [START_REF] Carter | The effect of processing parameters on particle size in ammonia-induced precipitation of zirconyl chloride under industrially relevant conditions[END_REF], produced by the reaction of AMB-HCl with (NH 4 ) 2 CO 3 . In fact the melting temperature of NH 4 Cl is 338°C, thus any residual solid NH 4 Cl cannot by eliminated during the spray drying process.

However, the last endotherm observed on DSC scans performed after spray drying AMB-HCl in the presence of (NH 4 ) 2 CO 3 (Fig. 4), having an onset at 229°C, may correspond to the melting of AMB-HCl. This endotherm had a lower temperature onset than the endotherm corresponding to the melting of the pure AMB-HCl, as the melting of AMB-HCl occurred in the liquid AMB base, lowering its chemical potential. This AMB-HCl could result from the re-protonation of AMB base during the spray drying process as described by the following chemical equation.

The re-protonation of AMB base to form AMB-HCl in the BUD/AMB composite formulation 𝐴𝑀𝐵 + 𝑁𝐻 4 + , 𝐶𝑙 -→ 𝑁𝐻 3 ↑ + 𝐴𝑀𝐵𝐻 + , 𝐶𝑙 - made by spray drying solutions containing (NH 4 ) 2 CO 3 was also observed. The endotherms corresponding to AMB base were not observed in the DSC scans recorded for formulations other than C1. Furthermore, DSC scans recorded for some formulations displayed two merged exotherms followed by two endotherms, suggesting the recrystallisation and the subsequent melting of 2 materials (BUD and AMB-HCl). Additionally, the PXRD patterns of pure AMB-HCl spray dried in the presence of (NH 4 ) 2 CO 3 and those of the formulations showed a diffuse halo centred between 2θ = 15-22.5° (Fig. 5) suggesting the presence of amorphous AMB-HCl.

It appears that the presence of AMB base mixed with BUD during the spray drying process was necessary to formulate nanoporous microparticles. AMB base is less polar than the hydrochloride form and should behave similarly to BUD. During the evaporation process in the spray dryer, the water/ethanol proportion in the solvent droplets should increase as ethanol is more volatile than water. This should induce the nanoprecipitation of BUD, as its water solubility is low [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF]. Also, the early BUD nanoprecipitation that occurs in the sprayed droplets, leading to the formation of NPMPs [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF], could also occur with the AMB base and not with the AMB-HCl, explaining the difference in the morphology of the BUD-AMB-HCl particles formulated with and without (NH 4 ) 2 CO 3 . The early nanoprecipitation of AMB base could act as seeds to trigger the AMB-HCl solidification.

The reconversion of AMB base to AMB-HCl salt during the spray drying process is an advantageous point in relation to the solubility and potentially the bioavailability of AMB.

Similarly, BUD and AMB-HCl were amorphous in the particles formulated with or without (NH 4 ) 2 CO 3 , which also may lead to an improvement of their solubility, dissolution rate and thus, diffusion in thick mucus. Amorphous materials can often be considered as undesirable for pharmaceutical development, mainly because of the concern about their physical stability; however, Nolan et al. [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF] demonstrated that amorphous particles of BUD were stable up to 1 year at 25°C and 60% RH.

The aerodynamic properties of various types of BUD-AMB-HCl composite particles tested (A1, C1, C5) were satisfactory. In fact, their MMADs were in the 1-5 μm aerodynamic diameter range, which may lead to deep alveolar lung deposition [START_REF] Westerman | Effect of nebulized colistin sulphate and colistin sulphomethate on lung function in patients with cystic fibrosis: A pilot study[END_REF][START_REF] Patton | Inhaling medicines: Delivering drugs to the body through the lungs[END_REF][START_REF] Edwards | Bioengineering of therapeutic aerosols[END_REF]. These MMADs were in the same range as those of pure BUD NPMP prepared and analysed under similar conditions (using an identical impactor, flow rate and inhaler) by Nolan et al. [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF]. These MMAD values of the BUD-AMB-HCl composite particles were lower than the values measured for the particles made of pure AMB-HCl [START_REF] Ren | Influence of formulation and preparation process on ambroxol hydrochloride dry powder inhalation characteristics and aerosolization properties[END_REF]. Furthermore, the FPF of these composite particles were higher than that obtained for the pure BUD NPMP (68.69 ± 1.33%) prepared by Nolan et al. [START_REF] Nolan | Excipient-free nanoporous microparticles of budesonide for pulmonary delivery[END_REF].

For the same initial amount of AMB-HCl of 5% (w/w), the addition of (NH 4 ) 2 CO 3 decreased the MMAD and significantly increased the FPF from 72.3±2.5% (A1) to 84.0±2.6% (C1) (Fig. 7).

Thus, the formulation of nanoporous particles improved the particles' aerodynamic properties.

An increase in the AMB-HCl content to 14% (w/w) (C5) led to an increase in the MMAD, although it remained in the favourable 1 to 5 μm diameter range. Interestingly, the FPF remained high (74.0±2.5%) which could be attributed to the low GSD (1.79±0.03) of this system. This narrow aerodynamic particles diameter distribution may presumably result in a good reproducibility in the dose administered to the patient, which is one of the weak points of drug administration by DPI [START_REF] Westerman | Effect of nebulized colistin sulphate and colistin sulphomethate on lung function in patients with cystic fibrosis: A pilot study[END_REF]. Also, the FPF of the BUD-AMB-HCl composite particles was higher than that reported by Ren et al. [START_REF] Ren | Influence of formulation and preparation process on ambroxol hydrochloride dry powder inhalation characteristics and aerosolization properties[END_REF], who obtained a FPF of 30.93% for particles composed of AMB-HCl/leucine/mannitol in a 2.5/0.5/1 weight ratio and made by spray drying.

In a number of pulmonary diseases, such as cystic fibrosis, patients develop abnormally thick mucus which is responsible for the low diffusion and poor efficacy of drugs [START_REF] Yang | Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis[END_REF][START_REF] Dinwiddie | Anti-inflammatory therapy in cystic fibrosis[END_REF][START_REF] Khanvilkar | Drug transfer through mucus[END_REF]. The coadministration of BUD together with AMB-HCl, a mucokinetic, could improve BUD diffusion within the lung and thus increase BUD efficacy.

AMB-HCl is a mucokinetic and influences the rheological properties of the airway fluid by acting directly on epithelial cells [START_REF] Paleari | Ambroxol: A multifaceted molecule with additional therapeutic potentials in respiratory disorders of childhood[END_REF], for example by modulating their ions secretion [START_REF] Hasegawa | Ambroxol-induced modification of ion transport in human airway Calu-3 epithelia[END_REF][START_REF] Yamada | Action of N-acylated ambroxol derivatives on secretion of chloride ions in human airway epithelia[END_REF].

To assess the effect of AMB on BUD diffusion within mucus, different transport experiments across Calu-3 cell AIC cultured monolayers, a model of mucus-producing epithelium [START_REF] Borchard | Transport and local metabolism of budesonide and fluticasone propionate in a human bronchial epithelial cell line (Calu-3)[END_REF][START_REF] Forbes | Human respiratory epithelial cell culture for drug delivery applications[END_REF],

were carried out (Fig. 8 and Fig. 9). In fact, AMB was previously shown to act on the ions secretion of Calu-3 cell monolayers, controlling the mucus viscosity [START_REF] Hasegawa | Ambroxol-induced modification of ion transport in human airway Calu-3 epithelia[END_REF]. Albeit Calu-3 secretions are probably different from the highly viscous mucus in cystic fibrosis or COPD, measures of BUD permeability could be used as an indirect evaluation of its diffusion within mucus, if the diffusion within the mucus is the limiting factor to the transport across the monolayer. This model was also used to determine if AMB can enhance BUD diffusion through mucus when coadministered. AMB apical-to-basal P app values across Calu-3 cell AIC cultured monolayers (1.4 ± 0.1•10 -5 cm/s) were constant for the different AMB-HCl concentrations tested (3, 6 and 20 μg/mL). This value was close to P app values found for AMB transport across the Caco-2 cell monolayers (i.e., 3.8 -4.6•10 -5 cm/s) [START_REF] Štětinová | Transepithelial transport of ambroxol hydrochloride across human intestinal Caco-2 cell monolayers[END_REF], a model in which a mucus layer is absent [START_REF] Meaney | Mucus as a barrier to the permeability of hydrophilic and lipophilic compounds in the absence and presence of sodium taurocholate micellar systems using cell culture models[END_REF]. The authors of this study reported that the permeability of AMB was independent of concentration over a wide concentration range (38 -380 μg/mL) [START_REF] Štětinová | Transepithelial transport of ambroxol hydrochloride across human intestinal Caco-2 cell monolayers[END_REF]. The consistency of AMB P app values at different concentrations, cell type and the presence of mucus could be due to the relatively high permeability of the drug (i.e., > 1•10 -5 cm/s), making it less sensitive to the viscosity change and suggesting that the AMB diffusion is not limiting its transport across the cell monolayer.

To determine if the mucus is limiting the BUD transport across Calu-3 cell monolayers, BUD permeability was measured across AIC cultured and LCC cultured Calu-3 cell monolayers. In this latter condition, the mucus is highly diluted and cells produce less glycoprotein compared to AIC grown cells [START_REF] Grainger | Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier[END_REF]. In LCC conditions, a BUD apical-to-basal P app of 9.3 ± 0.4•10 -6 cm/s was determined, which was close to the value of 15•10 -6 cm/s measured in previous transport studies across LCC cultured Calu-3 [START_REF] Forbes | Human respiratory epithelial cell culture for drug delivery applications[END_REF]. In AIC conditions (BUD C a = 20 μg/mL; t = 180 min), BUD apical-to-basal P app values (i.e., 4.5 ± 0.3•10 -6 to 6.8 ± 0.1•10 -6 cm/s) were significantly (P <0.001) lower than in LCC conditions and similar to those reported by Borchard et al. [START_REF] Borchard | Transport and local metabolism of budesonide and fluticasone propionate in a human bronchial epithelial cell line (Calu-3)[END_REF] (i.e., 5.33 ± 0.95•10 -6 cm/s) under comparable conditions (BUD C a = 6.5 -21.5 μg/mL; t = 120 min).

Grainger et al. [START_REF] Grainger | Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier[END_REF] have shown that the Calu-3 membrane integrity was higher when grown under LCC conditions compared to AIC conditions. For example, cells cultured using LCC exhibited higher expression levels of ZO-1 protein than the AIC counterpart and the P app value of the fluorescein was significantly lower across LCC than AIC cultured monolayers treated similarly [START_REF] Grainger | Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier[END_REF]. Also, the decrease in BUD apical-to-basal P app observed in the presence versus the absence of mucus suggests that the diffusion of BUD within this mucus limits its transport across Calu-3 and that the permeability change can be attributed to a change in BUD diffusion.

The addition of AMB-HCl at 20 μg/mL or at 3 μg/mL, when co-formulated by spray drying with BUD, significantly increased the P app of BUD across non-washed AIC cultured Calu-3 cell monolayers. The effect of AMB-HCl was also observed from the fluorescein P app , which was significantly increased in the presence of AMB in comparison to its value measured in the presence of BUD alone. To verify that the increase in BUD permeability observed in the presence of AMB across AIC cultured Calu-3 monolayers was due to a decrease in mucus viscosity and not to an alteration of the membrane integrity caused by the AMB, BUD permeability across LCC grown monolayer was also determined in the presence of AMB. No significant differences were observed for the BUD permeability across LCC grown Calu-3 monolayers in the presence versus the absence of AMB (Fig. 9). This demonstrated that the AMB was able to increase the BUD diffusion within the Calu-3 mucus. Therefore, BUD efficacy should be increased after inhalation of these BUD-AMB hybrid particles in patients having abnormally viscous mucus such as in the case of cystic fibrosis. However, it must be noted that the significant increase of BUD permeability induced by the AMB was only 1.3-fold. In order to increase this gain in BUD diffusion, other mucoactive agents which act quickly and directly on the mucus, such as mucolytics (N-acetylcysteine) could by investigated and combined with BUD to formulate hybrid particles.

Conclusions

With the aim of enhancing budesonide efficacy in the lung of patients suffering from abnormally thick mucus, we developed budesonide-ambroxol hydrochloride hybrid nanoporous microparticles for pulmonary administration by powder inhalation. These hybrid particles were formulated by using (NH 4 ) 2 CO 3 to obtain nanopores. They showed excellent aerodynamic properties with MMAD ranging from 1.87 to 3.18 µm and FPF higher than 72%. These particles were able to significantly improve budesonide apparent permeability in an in vitro model of mucus-producing respiratory epithelium, and should be able to enhance budesonide diffusion within the abnormally thick mucus of cystic fibrosis patients. The formulation of hybrid nanoporous microparticles containing a mucokinetic, such as ambroxol hydrochloride, could also be of interest for other drugs such as antibiotics, in order to treat bacteria protected in thick mucus or biofilms. Figure 4: PXRD patterns of the formulation as described in Table 1. ***

After 3 h

 3 at 37°C, samples were collected from the acceptor and donor compartments and assayed for BUD, AMB-HCl and fluorescein content. B) A second set of experiments was performed as follows: 6 Transwell Clear filters were cultured under liquid-covered culture (LCC) conditions for 15 days. Six additional filter inserts were cultured in AIC conditions for 12 days after 3 days in LCC conditions. Transport experiments were carried out in Hank's balanced salt solution (HBSS) supplemented with 5 mM HEPES. For LCC conditions, basal and apical sides of the Calu-3 monolayers were equilibrated

Figure 3 :

 3 Figure 3: PXRD patterns of the raw and spray dried materials.

Figure 4 :

 4 Figure 4: DSC thermograms of the unprocessed and spray dried materials. Insert in the

Figure 6 :

 6 Figure 6: Thermograms of the powders formulated without (A) and with (B) (NH 4 ) 2 CO 3 .

Figure 7 :

 7 Figure 7: Cumulative particles mass (%) versus the effective cut-off diameter (ECD) of each ACI stages.

Figure 8 :Figure 9 :

 89 Figure 8: BUD, AMB-HCl and fluorescein apical-to-basal P app values measured across Calu-3 cell monolayers. Under all conditions the apical BUD concentration was 20 μg/mL. (n = 3, t-test * P < 0.05).

Figure 2 :

 2 Figure 2: PXRD patterns of the raw and spray dried materials.

Figure 3 :

 3 Figure 3: DSC thermograms of the unprocessed and spray dried materials. Insert in the figure is a magnified view of the spray dried AMB-HCl thermogram in the region of the Tg.

Figure 6 :

 6 Figure 6: Thermograms of the powders formulated without (A) and with (B) (NH 4 ) 2 CO 3 .

Figure 7 :

 7 Figure 7: Cumulative particles mass (%) versus the effective cut-off diameter (ECD) of each ACI stages.

Figure 8 :Figure 9 :

 89 Figure 8: BUD, AMB-HCl and fluorescein apical-to-basal P app values measured across Calu-3 cell monolayer cultured at an air-liquid interface. Under all conditions apical BUD concentration was 20 μg/mL. The values are means ± SD (n=3, t-test * P<0.05)

Figure(s)Figure 1 :

 1 Figure(s)

Figure 2 :Figure 3 :

 23 Figure 2: SEM and specific surface area values of the particles formulated in presence of (NH 4 ) 2 CO 3 as described in Table 1. Error bars in the histogram correspond to the standard deviation of the specific surface area values calculated from 3 measurements

Figure 4 :Figure 5 :

 45 Figure 4: DSC thermograms of the unprocessed and spray dried materials. Insert in the

Figure 6 :

 6 Figure 6: Thermograms of the powders formulated without (A) and with (B) (NH 4 ) 2 CO 3 .

Figure 7 :Figure 8 :Figure 9 :

 789 Figure 7: Cumulative particles mass (%) versus the effective cut-off diameter (ECD) of

Table 1 : Total content of solids materials (% w/w) used to prepare the feed solutions (1 % w/v).

 1

				Solid content % (w/w)
	Compounds			With (NH 4 ) 2 CO 3		Without (NH 4 ) 2 CO 3
		C1	C2 C3 C4 C5 C6 C7 C8 A1 A2 A3
	BUD	80	75	70 65 65 55 45	33	95 90 80
	AMB-HCl	5	10	15 20 10 10 20	33	5	10 20
	(NH 4 ) 2 CO 3	15	15	15 15 25 35 35	33	0	0	0

Table 3 :

 3 Measured and theoretical content of BUD and AMB-HCl in the spray dried particles (n=3 ± SD). Theoretical contents of drugs in the different formulations were calculated on the basis that: (a) all or (b) none of the (NH 4 ) 2 CO 3 was eliminated during the spray drying process. NH 4 ) 2 CO 3 concentration (C5) produced particles with higher MMAD (3.18±0.28 μm) but having a narrow diameter distribution (GSD of 1.79±0.03) and high FPF (74.0±2.5%).

	NA (Non Applicable)
			BUD			AMB-HCl
		Theo. cont.	Theo. cont.	Measured	Theo. conc.	Theo. conc.	Measured
		(w/w) (a)	(w/w) (b)	conc. (w/w)	(w/w) (a)	(w/w) (b)	conc. (w/w)
	C1	94	80	96.83±2.31	6	5	6.10±0.15
	C2	88	75	87.42±3.91	12	10	11.55±0.55
	C4	76	65	81.66±2.24	24	20	24.25±1.37
	C5	86	65	89.73±3.81	14	10	14.53± 0.21
	C6	84	55	82.97±2.61	15	10	14.35±0.67
	C7	69	45	71.19±2.28	30	20	28.08±0.52
	A3	NA	80	82.2±1.5	NA	20	17.8±1.5
	A2	NA	90	94.6±1.8	NA	10	5.7±2.9
	A1	NA	95	97.1±1.9	NA	5	3.7±1.2
	Cumulative mass depositions of selected formulations versus ECD of each ACI stage are shown
	in Fig. 7. Formulation A1 was composed of particles with MMAD value corresponding to high
	lung alveoli delivery efficiency (2.2±0.21 μm) and had a high FPF (72.3±2.5%). The addition of
	15% (w/w) of (NH 4 )

2 CO 3 (C1) significantly reduced the MMAD to 1.87±0.22 μm and significantly increased the FPF (84.0±2.6%) (t-test, p<0.05). A further increase in AMB-HCl and (

Table 1 : Total content of solids materials (% w/w) used to prepare the feed solutions (1 % w/v).

 1

	Table(s)
			Solid content % (w/w)
	Compounds		With (NH 4 ) 2 CO 3	Without (NH 4 ) 2 CO 3
		C1 C2 C3 C4 C5 C6 C7 C8 A1 A2 A3
	BUD	80	75 70 65 65 55 45 33 95 90 80
	AMB-HCl	5	10 15 20 10 10 20 33	5	10 20
	(NH 4 ) 2 CO 3	15	15 15 15 25 35 35 33	0	0	0

Table 2 :

 2 Geometric volume-weighted particles size analysis

				Specific Surface Area of
		D 50 (µm)	SPAN	particles calculated as smooth spheres (m 2 /g)
	C1	2.4±0.3	1.75±0.05	3.19±0.31
	C2	2.6±0.2	1.70±0.1	2.94±0.28
	C4	3.0±0.3	2.20±0.15	2.52±0.34
	C5	2.6±0.3	1.67±0.09	3.07±0.30
	C6	2.8±0.4	1.70±0.15	3.06±0.35
	C7	4.6±1.1	2.90±0.62	1.98±0.46

Table 3 :

 3 Measured and theoretical content of BUD and AMB-HCl in the spray dried particles (n=3 ± SD). Theoretical contents of drugs in the different formulations were calculated on the basis that: (a) all or (b) none of the (NH 4 ) 2 CO 3 was eliminated during the spray drying process. NA (Non Applicable)

			BUD			AMB-HCl
		Theo. cont.	Theo. cont.	Measured	Theo. conc.	Theo. conc.	Measured
		(w/w) (a)	(w/w) (b)	conc. (w/w)	(w/w) (a)	(w/w) (b)	conc. (w/w)
	C1	94	80	96.83±2.31	6	5	6.10±0.15
	C2	88	75	87.42±3.91	12	10	11.55±0.55
	C4	76	65	81.66±2.24	24	20	24.25±1.37
	C5	86	65	89.73±3.81	14	10	14.53± 0.21
	C6	84	55	82.97±2.61	15	10	14.35±0.67
	C7	69	45	71.19±2.28	30	20	28.08±0.52
	A3	NA	80	82.2±1.5	NA	20	17.8±1.5
	A2	NA	90	94.6±1.8	NA	10	5.7±2.9
	A1	NA	95	97.1±1.9	NA	5	3.7±1.2

SEM of particles made of BUD and AMB-HCl as described in Table 1. Figure 2: SEM and specific surface area values of the particles formulated in presence of (NH 4 ) 2 CO 3 as described in Table 1. Error bars in the histogram correspond to the standard deviation of the specific surface area values calculated from 3 measurements.

Supplementary material

Click here to download Supplementary material: Supplementary AMB-BUD.docx


Goulven Guillou
email: goulven.guillou@univ-brest.fr

Jean-Philippe Babau
email: jean-philippe.babau@univ-brest.fr

IMOCA: a Model-Based Code Generator for the Development of Multi-Platform Marine Embedded Systems

Keywords: sofware architecture, control, code generation

Process control systems embedded in disturbed environments are usually developed case by case for specific deployment platforms and their behaviours closely depend on the characteristics of the environment. The obtained code is not portable and not reconfigurable. In order to help the software development of such applications, IMOCA offers architectural modelisation tools. The associated code generator allows to product adaptative and reconfigurable code for a simulator as well as embedded code for various platforms. This approach has been tested on NXT bricks, Arduino boards and Armadeus boards.

Introduction

Embedded systems in an unpredictable and disturbed environment, like underwater control systems, have to take into account various situations by considering different strategies. Their development requires large parameters configuration in order to ensure safety and efficiency of the controlled system.

The configurable parameters are used to characterize the context (environment interpretation and its evolution), the execution platform and the control part of the system. The tuning of the parameters is based on simulations for cost reasons and on real testing for safety reason. At the end, the system has to be equipped with adaptation and/or learning abilities to adjust some parameters in-line. Therefore, there is a strong need for online and offline tuning tools. Unfortunately in industry, the development of the software for such systems is mainly focused on the code efficiency. The produced embedded code is dedicated to a given platform for a specific application in a specific context. The obtained code is difficult to maintain and to adapt for new applications and new contexts. Portability and reusability are limited.

To tackle these limitations, we propose in [START_REF] Guillou | IMOCA : une architecture à base de modes de fonctionnement pour une application de contrôle dans un environnement incertain[END_REF] a model-based software architecture integrating high adaptive capabilities, the IMOCA approach. Once the architecture model is established, a key point is then the code generation. In this paper, we present the implementation of the model-based code generator from the architecture model IMOCA. Even if the deployed code remains specific, we propose generic models of code generation to allow:

the control of quality and efficiency of the generated code: the code is optimized for static parts and modular for adaptive part; the integration, and so the reuse, of existing specific legacy code is facilitated: domain functions such as control law, communication protocol, acquisition policy can be easily integrated; the code generation for different target platforms: the architecture model and most of the code generator is independent of a given platform, specific aspects are encapsulated in a platform abstraction view; the model and the code generator integrate testing, adaptation and tuning facilities: design facilitates declaration of adaptive parameters and functions, parameters can be tested with a generated simulator, code integrates reconfiguration capabilities for adaptive parts.

The generator incorporates tuning aspects and takes into account various platforms, including testing platforms. The simulator is written in Java, while the generated embedded code is written in C language (or family of C language). To generate optimized embedded code, the code generator is based on the principles presented in [START_REF] Navas | Reconciling run-time evolution and resourceconstrained embedded systems through a component-based development framework[END_REF].The models and tools have been tested on NXT bricks, Arduino boards and Armadeus boards for simple control applications, sand yacht control and autonomous sailing boat VAIMOS control. After a presentation of the IMOCA architecture model, we present the structure and the underlying principles of the code generator while using it with two applications on two different execution platforms.

Related Works

Component-based approaches [START_REF] Szyperski | Component Software : Beyond Object-Oriented Programming[END_REF] for the conception of the embedded systems are relatively classical and allow to deal with the sofware complexity [START_REF] Crnkovic | Component-based software engineering for embedded systems[END_REF] and the well-known separation of concerns in Model Driven Architecture (MDA) terminology [START_REF] He | Think : View-based support of non-functional properties in embedded systems[END_REF]. In particular, software evolution ability in order to take into account either the platform maintenance or system behavior adaptation or parameters tuning [START_REF] Chapin | Types of software evolution and software maintenance[END_REF] can be viewed as software flexibility [START_REF] Mathieu | Flex-eWare : a Flexible MDE-based Solution for Designing and Implementing Embedded Distributed Systems[END_REF]. Generally, efforts to increase the flexibility lead to conflicts with respect to the material resources of the execution platform [START_REF] Navas | Minimal yet effective reconfiguration infrastructures in component-based embedded systems[END_REF]. In [START_REF] Borde | Mode-based reconfiguration of critical software component architectures[END_REF] binding each component to an adaptation policy allows software evolution, but this solution assumes that the evolutions are predictable. [START_REF] Navas | Reconciling run-time evolution and resourceconstrained embedded systems through a component-based development framework[END_REF] tries to conciliate the software evolution at run-time with the hard resource constraints of the embedded platforms. Relying on this work, we focus on the conception of domain-specific models of components (we focus on a specific domain without modeling specific features of the components), then we generate optimized code which embeds the necessary elements for the reconfiguration [START_REF] Navas | Reconciling run-time evolution and resourceconstrained embedded systems through a component-based development framework[END_REF].

3 The Architecture Model IMOCA IMOCA for archItecture for MOde Control Adaptation is an architecture model dedicated to the development of process control systems embedded in a disturbed environment. This architecture is composed of three layers called Target, Interpretation and Control (see figure 1). Target with its Actuators and Sensors is a platform-specific model of I/O. Control uses Data (an ideal view of the environment) to compute ideal Commands to act on the environment. The Interpretation layer realizes the adaptation between the Target layer and the Control layer by linking Sensors and Actuators on the one hand, and Data and Commands on the other hand. In this way Control and Target are independent like in SAIA [START_REF] Deantoni | A MDA-based approach for real time embedded systems simulation[END_REF] and this allows the development independently of specific sensors and actuators technologies. This independence is important in the context of embedded systems because material platforms may be various and may evolved (change or add a sensor for example). The Controller is composed of three sub-controllers. The ReactiveController uses Data to compute a Command based on a control law. The ExpertController is in charge of defining the current control law. It is based on a finite state automaton that manages running modes. Each state is associated with a Mode which is itself associated with a control law. A state change is linked to a change of state of the environment (a function of Data which returns a boolean). Finally, an AdaptativeController adjusts different parameters of the control law with respect to a look-up table in which appear all the possible Configuration. Based on this three collaborating layers, the Controller allows to answer the three following requirements: controlling the process with the ReactiveController by applying an adapted control law thanks to the ExpertController, and finally, adjusting the control laws with respect to the context in order to keep a high quality of control with the AdaptativeController.

Data

Target Control

Real

Interpretation

Code Generation

The model of the software architecture design is an instance of the meta-model IMOCA, expressed using ecore. The code generator leans on Acceleo technology which allows to define code generation principles through an Ecore meta-model.

Parameters configuration

A software application based on the architecture model IMOCA integrates variability to adapt the system to a specific context or a specific platform. Variability is first implemented through parameters to characterize, for example, a threshold in a filter adapter, or a coefficient for a control law. Second, the ExpertController automaton defines a dynamic behavior through running modes. To tune parameters and modes, we propose to generate simulation tools to evaluate the impact of different parameter values and specific actions on the system.

In this paper, we target the generation of an adaptive and reconfigurable embedded code. "Adaptive code" means the application is able to take into account the evolution of its environment through the adaptativeController. For each environment context, specific values of parameters are defined at design time using the simulation. Then, the adaptativeController adapts online the values depending on the context. "Reconfigurable code" means that the embedded values of the parameters can be modified online without the need to recompile the code. The reconfiguration capability concerns parameters only, the architecture of the application cannot be modified online.

In this version, a Java simulator is generated. The generation of this simulator is based on high-level data (sensors and actuators are not considered here) and includes all the controllers. The designer can test different control laws and parameter setting through a dedicated generated User Interface. Each Data can be controlled and each new Command is printed on a control screen. To view the real effect of the controller, is is necessary to implement an environment simulator for the system itself (as in [START_REF] Deantoni | A MDA-based approach for real time embedded systems simulation[END_REF]). The latter must be connected to the commands sent by the generated simulator.

Taking into Account the Behavior

The architecture model IMOCA is a declarative model. It allows to focus on specific features of the target application (activation periods, number of operating modes, ...). However, IMOCA is not a programming language, the expected behavior is not described explicitly. The expected behavior is implemented by the code generator, following the IMOCA semantic.

For this purpose we distinguish two parts. The former corresponds to the operational semantic of IMOCA which has been given in the previous section. The code generator is directly in charge of this part. The behavior is expressed in a simple C code (no pointer) to be reusable in different languages. The latter is specific to application-dependent part and is encapsulated in domain-specific libraries. The code generator is in charge to produce glue code to link these two parts via a simple library integration.

According to these principles, the ExpertController and the adaptiveController automata are generated together with a general controller and the code dedicated to Data. For the Sensors, the Interpreters and the ReactiveController, only the declaration and glue code is generated. To complete the code, the user provides specific libraries to deal with sensors acquisition (filters, ...), actuators management, Interpreter and control laws.

For example, the following code defines the ExpertController of a mini sand yacht and is, in fact, the implementation of a finite state automaton. Each state change is bound to the evaluation of the data theta which represents the heeling of the sand-yacht in degrees. For information, here we have three states (states 1, 2 and 3) respectively corresponding to a normal state, a (excessive) heel to starboard and a heel to port.

#ifndef EXPERT #define EXPERT

/**

Domain-Specific Code Integration

As previously said, generated code must be completed by adding domain-specific code. Since many dedicated libraries exist, the idea is to generate code in a way that allows a smooth integration. As in a component-based approach [START_REF] He | Think : View-based support of non-functional properties in embedded systems[END_REF], IMOCA produces a set of signatures of functions for all relevant components (sensors, actuators, filters and reactive controller). Thus, the designer has to provide the set of corresponding implementations (user code) while respecting the static typing. In addition, we use the properties of Acceleo to add, in the generated code, portions of customizable code. For this purpose, a special place is pointed by a commentary and is available for the user (see below). By default, a code is provided which can be modified and completed. It should be noted that subsequent generations of code take into account these changes (automatically performed by Acceleo). This default code calls a user function for each domainspecific component, user function that we have to implement.

Taking into Account Platforms

In order to address various execution platforms, we need to generate specific code for each of them. However, a significant part of the code generator must remain generic and independent of the target language. For example, the behavior of the ExpertController is independant of the target language.

To address this requirement, the code generator is based on three software layers. The first one is concerned with generating the specific behavior related to IMOCA. It can generate imperative code or object-oriented code. We add a parameter to each generative function to define the expected code (imperative or object-oriented). The second layer is based on the first one and is specific to the target language (Java or C for example). It is in charge of generating the files respecting the specific language features for the declaration of files, classes or functions. Currently, the specificities of the executive are included in the second layer via the definition and the call of tasks. The last layer is in charge of general services.

For the first version of the code generator, the target language is based on the basic constructs of C language (assignment, control structures). Pointers are not used. Thus, we can rely on this layer to generate C, C++, Java code or any C-like language.

We have used the code generator for two applications deployed on different platforms. The former is a NXT Lego brick equipped with a motor and a touch sensor. We just control the speed of rotation of the motor by using the touch sensor. The latter is a mini sand yacht with an Arduino board Mega and an inertial motion unit (IMU). We have to try to keep the course and, in the same time, to avoid the capsize of the vehicle due to the wind action.

At the level of second layer, the first part of the code presented figure 2 allows to generate the file Input.nxc whereas figure 3 presents the same thing for the simulator (some lines of commentary have been removed).

We generate NXC source code for the NXT platform. NXC means Not eXactly C, a C-like language with some specific features. Generated files have an extension of nxc (here the files are Input.nxc and Output.nxc) and we retrieve the preprocessor invocations like in C. We include only implementation files, there is no interface file with an extension of h. Each data needs a declaration, the definition of their attributes (value, frequency, format ...) and of their access methods.

The equivalent Acceleo code to generate the simulator (see figure 3) produces Java code. A Data is viewed as a high-level data, that is to say here as a class. However calls like [generateWriteDefinition(data,0)/] remains identical to those used to generate code for the NXT brick.

In the first layer, Acceleo modules are parameterized by the type of the used language (0 for object-oriented programming, and 1 for imperative programming). Figure 4 shows this case.

This leads for the case of the NXT brick (the commentaries have been removed) to: void InitControls() { InitGo(); Init_Stop(); } whereas the generated Java code is: public void InitControls() { myReactiveController.InitGo(); myReactiveController.Init_Stop(); }

Structure of the Code Generator

The code generator is modular with a set of specific modules for each component of IMOCA. For the first and second layers, a code generation module is proposed for each modeled entity (Sensors, ExpertController, ...). Utilities modules are used to complete the code generator in order to factorize and simplify common pieces of code.

For the first layer (behavior), simple components (ReactiveController, Sensor, Actuator and Interpreter) are built according to the same principle. A class (or a structure in the case of imperative code) is generated which manages objects that contain a value, an initialization function, a configuration function, a getter, a setter and specific executive functions (a "run" for each filter and for each control law of reactive controller). The other control components are generated through a specific function which implements automata. Other components implement a service layer, a main program (the main) and communication tools.

Figure 5 shows a piece of Acceleo code which stands in the first layer of the generator and concerns the Data. To assist testing, the generated code is also modular. For object-oriented language, a class is generated for each element (each sensor, each data, each controller, ...). For an imperative language, a file is generated for each element. Thus, the structure helps on unit testing activity.

Reconfiguration

The code generator generates reconfigurable code in the sense that parameter values must be changed online. To prepare the code generation, each parameter can be recorded as reconfigurable (the value of the property IsControllable which is false by default, is set to true). If at least one reconfigurable parameter exists, a client interface (currently in Java) is generated to allow the tuning of the values of reconfigurable parameters. In the embedded code, a server task retrieves the changes and modifies online the corresponding parameters (this modification is done via a call to the corresponding setter). This tool is especially useful during prototyping phase. The behavior of the system can be tested without having to stop and recompile the whole application [START_REF] Navas | Reconciling run-time evolution and resourceconstrained embedded systems through a component-based development framework[END_REF].

Conclusion and Future Works

This paper presents an approach to generate adaptive and reconfigurable embedded code based on the architecture model IMOCA. The code is designed for process control systems in disturbed environments and can be generated for multiple platforms. A Java simulator is generated to assist the user in the tuning of control laws. The case of an NXT robot and a mini sand-yacht equipped with an Arduino board have been used for experiments.

This work is in progress and we will extend it to include many other aspects of the code generation. We would like addressing other platforms and trying to optimize the code to take into account the limited memory resource and the limited computing power of some platforms. We also seek to control the consumption of autonomous systems, like drones, by using control policies based on energy criteria. Finally, we seek to enrich and automate the integration of existing specific libraries in order to deal with other application areas.

The aim of this work is to provide a comprehensive model-based environment for the development of code generators for architectures IMOCA.

Fig. 1 .

 1 Fig. 1. Principles of IMOCA approach

  bool UpdateBoolSensorTouch(){ bool aBoolSensorTouch; // Start of user code for ReadBoolSensorTouch definition aBoolSensorTouch=UserReadBoolSensorTouch(touchPort); // End of user code boolSensorTouch = aBoolSensorTouch; return aBoolSensorTouch; } If the user keeps the call of the specific function, he must provide a function that respects the signature required by the code generator to the specific library: bool UserReadBoolSensorTouch(int port) { return ((Sensor(port)==0)) ; }

[Fig. 2 .

 2 Fig. 2. Fragment of the generateInputOutputNXC.mtl Acceleo module

Fig. 4 .

 4 Fig. 4. Acceleo template for generating object-oriented and imperative code

Fig. 5 .

 5 Fig. 5. Fragment of the generateDataUtils.mtl Acceleo module

  3. Fragment of the generateInputOutputJava.mtl module

	[template public generateInitControlsDefinition
	(aSystem : System, lang : Integer)]
	void InitControls() {
	// [protected ('for Init Adapters Definition')]
	// user code
	// [/protected]
	[for(control : Control | aSystem.modes.command)]
	[if (lang=0)]myReactiveController.[/if]
	Init[control.name/]();
	[/for]
	}
	[/template]