Beclomethasone Microparticles for Wet Inhalation, Produced by

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Ind. Eng. Chem. Res. 2010, 49, 12747–12755

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Beclomethasone Microparticles for Wet Inhalation, Produced by Supercritical Assisted Atomization Ernesto Reverchon,*,† Renata Adami,† Mariarosa Scognamiglio,† Giuseppe Fortunato,‡ and Giovanna Della Porta† Department of Chemical and Food Engineering, UniVersity of Salerno, I-84084, Fisciano (SA), Italy, and Farmabios S.p.a, I-27027, Gropello Cairoli (PV), Italy

Supercritical assisted atomization (SAA) was used to produce micronized particles of beclomethasone dipropionate (BDP) to be used for inhalation therapy. Methanol, acetone, acetone + water 9% v/v, and methanol + water 4% v/v were tested as liquid solvents to explore their influence on particle size and crystalline structure of the precipitated powder. Particles with narrow size distributions (PSD) were obtained by changing the SAA process operating conditions. In particular, using acetone + water 9% v/v with a BDP concentration of 90 mg/mL, crystalline particles ranging between 0.2 and 4.7 µm were produced and 70% by volume of these particles ranged between 1and 3 µm. PSD stability analysis in a wet formulation showed that BDP powder obtained by SAA does not modify over a long period of time. The results obtained on the laboratory scale plant were successfully reproduced on a pilot plant and a further refinement of the operating conditions was performed. About 30 g of BDP microparticles were produced per batch and a recovery of about 93 wt % of the micronized powder with respect to the injected quantity was obtained. The largest volumetric fraction of BDP particles falling in the range 1-3 µm was 83% on the pilot scale. Introduction One of the current methods to deliver corticosteroids to the lung is wet inhalation: the drug is suspended in an aqueous solution, used to form an aerosol that is, then sent to the lung. The formulation is a stabilized water-based suspension and the particle size of the inhalable compound has to range between 1 and 5 µm to allow an efficient delivery to the target. A further improvement of the particle size distribution could be the tailoring of the particles in a more restrictive range of diameters; i.e., between 1 and 3 µm. It can maximize the effective inhaled fraction: the fraction of particles that arrives to lung alveoli with respect to the total inhaled powder.1–3 Corticosteroid particles contained in commercial products, as a rule, fall in the inhalable range for percentages between 15 and 30%. Their particle size distributions (PSD) are also characterized by a long tail of particles with diameters up to 10-20 µm. Another target, for particles used in water based suspensions, is their crystalline form, which should ensure a longer stability and a larger shelf life of the product. However, using aqueous suspensions it is possible to induce crystal growth and solvate or clatrate formation of the micronized drug.4 Using traditional micronization processes it is very difficult to obtain the previously discussed particle size distribution; therefore, alternative processes are currently explored, based on the use of supercritical carbon dioxide (SC-CO2).5–10 One of the most effective processes is Supercritical Assisted Atomization (SAA). SAA is based on the dissolution of SC-CO2 in a solution formed by a liquid solvent and a solid solute, and on its subsequent atomization. During SC-CO2 dissolution, an expanded liquid is formed that is characterized by a reduced viscosity and a lower surface tension with respect to an ordinary liquid. The reduction of these cohesive forces, together with the release of CO2 from the liquid, largely improves the * To whom correspondence should be address. E-mail: ereverchon@ unisa.it. Fax: +39 089 964057. † University of Salerno. ‡ Farmabios S.p.a.

atomization process, with the production of small micronic droplets that become micrometric particles after drying. SAA has been until now successfully applied to the micronization of some pharmaceutical compounds11–14 and of some polymers;15–17 as a rule, spherical, amorphous microparticles were obtained. Indeed, drying of the microdroplets formed during atomization is very fast and does not allow the organization of the solute molecules in the ordinate form characteristic of crystals. Beclomethasone dipropionate (BDP) is a corticosteroid typically used for the treatment of asthma in water-based suspensions18–20 and the commercial formulations based on BDP suffer the limitations previously discussed. Therefore, the scopes of this work are • to apply SAA to the micronization of BPD; • to obtain particle size distributions with the largest possible volumetric percentage in the 1-3 µm range; • to force the SAA process to produce crystalline particles; • to scale-up the process up to the pilot scale. To produce crystalline particles, different precipitation temperatures and different solvent are tested and, for the first time in SAA processing, organic solvent (acetone, methanol)-water mixtures are used. The stability of the SAA-produced BDP powders in the final aqueous formulation is also analyzed. Apparatus, Materials, and Methods Materials. Beclomethasone dipropionate (BDP) purity 98% was given by Farmabios (Pavia, Italy). Methanol, purity of 99.9%, and acetone, purity of 99.9%, HPLC purity, were supplied by Sigma-Aldrich (Milan, Italy). Distilled water was supplied by Carlo Erba Reagenti (Milano, Italy). Dimethylformamide (DMF), purity >98%, polysorbate 20, sorbitan monolaurate, and sodium chloride, purity 99.5%, were supplied by Sigma-Aldrich (Milan, Italy). Carbon dioxide (CO2) and nitrogen (N2) (purity 99%) were purchased from SON (Napoli, Italy). The solubility of BDP in the tested solvents and solvent mixtures is reported in Table 1. The solubility tests were performed by putting 10 mg of BDP in a vial and adding increasing amounts

10.1021/ie101574z  2010 American Chemical Society Published on Web 11/08/2010

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Table 1. Solubility of BDP in the Tested Solvents and Mixtures at 23 °C (Accuracy (2%) solvent

solubility, mg/mL

acetone methanol acetone-water 9% v/v methanol-water 4% v/v

46 55 95 34

of solvent or solvent mixture until the liquid became clear and the BDP was totally dissolved. The measurements were performed at 23 °C and the accuracy of the measurements is (2%. All materials were used as received. Laboratory-Scale Apparatus. The laboratory apparatus used for SAA experiments mainly consists of 3 feed lines used to deliver SC-CO2, the liquid solution, and an inert gas; and 3 main process vessels: saturator, precipitator, and condenser. The CO2 line consists of liquid CO2 taken from a cylinder and sent to a high-pressure pump (Gilson model 305, Milano, Italy); then CO2 is sent to a heated bath and to the contactor in which it solubilizes into the liquid solution. The liquid solution is taken from a container, pressurized in a high-pressure pump (Gilson model 305, Milano, Italy), heated and sent to the saturator. The inert gas (N2) is taken from a cylinder, sent to a calibrated rotameter and, after heating in a heat exchanger, is sent to the precipitator to assist the evaporation of the liquid solvent. The saturator is a high-pressure vessel (internal volume of 0.05 dm3) loaded with stainless steel perforated saddles. The high surface packing enhances the contact between CO2 and the liquid solution, thus promoting the dissolution of the gaseous stream in the liquid solution, up to near-saturation conditions. Residence times in the saturator from 5 to 10 min are obtained at the commonly adopted process conditions. The solution at the exit of the contactor is sent to a thin wall injector (80 µm internal diameter). The injector produces a spray of liquid droplets in the precipitator, which is a stainless steel vessel operating at near-atmospheric conditions (internal volume 3 dm3). The powder generated by the evaporation of the liquid droplets is collected at the bottom of the precipitator on a stainless steel sintered frit (mean pore diameter of 0.1 µm); whereas the gases are discharged in a cooled condenser with the aim of condensating the liquid solvent. SAA layout and further details on the apparatus have been published elsewhere.12,13 Pilot Plant. The SAA pilot plant is similar to the laboratory one; the main differences between the two apparatuses are, as expected, in the size of the saturator and of the precipitator. The heating system of the precipitation vessel and the injection device also show some differences. The saturator is a thermostatted high-pressure vessel with an internal volume of 0.15 dm3; the precipitator is a cylindrical vessel with an internal volume of 14 dm3. At the exit of the precipitator, the gaseous flow of CO2, N2, and solvent is sent to the condenser, which is a shell and tube heat exchanger that allows the recovery of the solvent from the gas stream. In the case of the laboratory plant, electric band heaters around the vessel are used. For the pilot plant a heated water jacket is used to ensure a better heating efficiency and a smoother temperature distribution in the precipitator. The injection devices used in the two apparatuses differ only for the dimensions; indeed, a 180-µm diameter stainless steel nozzle was used in the pilot plant. Further details on the pilot apparatus have been published elsewhere.11 Experimental Procedures. The saturator provides a large contacting surface and an adequate residence time for the solvent and supercritical CO2 (SC-CO2). Therefore, we can obtain an efficient, continuous solubilization of controlled quantities of SC-CO2 in the liquid solution and we can optimize the mixing

and the residence time of the two fluids. When the right molar fractions are used, the SAA operating conditions are located on the left of the 2-phase region in the pressure-composition diagram of the binary system solvent-SC-CO221 and an expanded liquid is formed. When the formed ternary solution is injected into the precipitator, an enhanced atomization process is obtained. Indeed, the presence of CO2 solubilized in the liquid reduces the viscosity and surface tension of the solution, which are among the cohesive forces of the liquid; therefore, smaller primary droplets are produced than in the classical atomization processes. The atomization mechanism can be described by a two-step process. In the first step, a classical decompressive atomization is obtained due to the pressure drop between the contactor and the precipitator, which is operated near atmospheric pressure. Then a secondary atomization is obtained by the fast release of CO2 from the primary atomized droplets. The resulting secondary droplets are usually very small, their size distribution is in the range 0.3-5 µm or narrower; then they are dried in the precipitator and produce very small and uniform particles. Powder Morphology. Samples of the processed powder were observed by scanning electron microscopy (SEM; LEO 420, USA). Powders were dispersed on a carbon tab previously stuck to an aluminum stub and coated with gold (layer thickness 250 Å) using a sputter coater (model 108A; Agar Scientific, UK). Several SEM photomicrographs from different parts of the precipitation vessel were taken for each run to verify the powder uniformity. Particles Size Distribution. Particle size (PS) and particle size distribution (PSD) were initially measured from SEM photomicrographs using the Sigma Scan Pro Software (release 5.0, Aspire Software International, USA). Approximately 1000 particles were considered in each particle size distribution calculation based on SEM images. Histograms representing the particle size distribution were best fitted using Microcal Origin Software (release 7.0, Microcal Software Inc., USA). Lognormal curves were obtained that give a fair good representation of nonsymmetric distributions. Since BDP crystalline or semicrystalline particles partly lose their spherical shape and tend to coalesce, dynamic laser scattering (DLS) analysis was added to obtain a more objective measurement of PSD. DLS analyses were performed using a Malvern Mastersizer S laser diffractometer (Alfatest s.r.l., Rome, Italy). The smallest particle diameter detectable with this instrument is 0.05 µm. In each measure, 50 mg of BDP microparticles was suspended in 10 mL of distilled water, using 0.08% w/w of Tween 80 as dispersant and, then sonicated before analysis. The PSD of each sample was monitored at fixed time intervals, that is, every 2 min for 20 min. A good reproducibility of the results was obtained: the PSDs obtained using this procedure practically overlapped. Drug Degradation. Drug degradation was evaluated by HPLC-UV/vis (HP model G131-132, Agilent Technologies Mfg. Gmbh & Co. KG, USA) analysis of the untreated material and SAA processed powder. BDP (4-6 mg) was dissolved in acetonitrile and diluted to 10 mL with the mobile phase. The elution was obtained using a stainless steel cartridge packed with octadecysilyl silica gel for chromatography (4 × 250 mm; 5 µm particle size; LiChroCart Spherisorb RP-18). The column was equilibrated for about 10 min at a flow rate of 1.5 mL/min with a mobile phase consisting of water and acetonitrile (ratio 45:55 v/v). The drug was monitored at 238 nm with a retention time of 9.75 min.

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Drug Crystallinity. Solid state analysis of BDP samples was performed using an X-ray powder diffractometer (model D8 Advance, Bruker, USA) with a Cu sealed tube source. Samples were placed in the holder and flattened with a glass slide to ensure a good surface texture. The measuring conditions were as follows: Ni-filtered Cu KR radiation, λ ) 1.54 A, 2θ angle ranging from 10 to 65° with a scan rate of 3 s/step and a step size of 0.02°. Calorimetric analysis was performed using a DSC TC11 (Mettler, USA) using Mettler STARe system. Temperature and enthalpy of fusion were calibrated with pure indium standard (melting point 156.6 °C, enthalpy of fusion 28.52 J/g); the enthalpy changes (∆H) were evaluated from the peak areas using the integration program of the TC11 processor. The calculated areas lie within the experimental error ((5%). Powder samples (5 ( 0.5 mg), prepared in duplicates, were accurately weighed, crimped in an aluminum crucible, and heated from 25 to 250 °C at 10 °C/min under a nitrogen purge (50 mL/min). The measurements were performed with no previous thermal treatments to consider the thermal history of the compound. Solvent Residues. Acetone and methanol residues were measured by a gas chromatograph equipped with a flame ionization detector (GC-FID, model 6890 GC-SYSTEM, HP, Agilent Technologies Mfg. Gmbh & Co. KG, USA) Acetone and methanol were separated using a fused-silica capillary column (model Cp Sil 5CB CHROMPACK, 25 m length, 0.53 mm i.d., df 5 µm, Stepbios, Italy). GC conditions were: oven temperature of 60 °C per 5 min and increase to 200 at 20 °C/ min. The injector was maintained at 250 °C, the detector was maintained at 280 °C, and nitrogen was used as the carrier gas (15 mL/min). Samples were prepared in 10-mL vials loaded with 950-1050 mg of drug, 5 mL of internal standard, and diluted to 10 mL with dimethylformamide (DMF). The internal standard was prepared by dissolving 40 mg of absolute ethanol in DMF to make 100 mL, and 2.5 mL have been diluted to 25 mL with DMF. The minimum solvent residue that can be measured using this method is about 2 ppm for acetone and 5 ppm for methanol. Analyses were performed on each batch of processed drug, in triplicates. PSDs Stability in Wet Formulation. Micronized BDP (40 mg) was suspended in a formulation formed by polysorbate 20, sorbitan monolaurate, and sodium chloride in 100 mL of bidistilled water. The suspension was analyzed by DLS, using a Malvern Mastersizer S laser diffractometer. This analysis was performed the first day the suspension was prepared, and then each day during the first week. After this period of time, it was repeated once a week for two months. To investigate any sample degradation, the formulations were submitted weekly to 50 thermal cycles form 2 to 40 °C with the aim of accelerating the degradation processes.2 Experimental Results and Discussion The major SAA process variables are saturator temperature (Tsat), saturator pressure (psat), precipitator temperature (Tprec), precipitator pressure (pprec), solute concentration, and the flow rate ratio between CO2 and the liquid solution (R). Different values of R can be used, but they should be compatible with the formation of a single expanded liquid phase (CO2 + liquid solvent) to improve process performance. This information can be deduced from a phase equilibrium diagram for the system solvent-CO2 when psat and Tsat have been selected.21 In this work (see Table 2) we selected the conditions in the saturator on the basis of previous SAA works in which the same liquid

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Table 2. Summary of the SAA Experiments Performed on the Laboratory Plant test code

solvent

R ) wCO2/wsol C (mg/mL) Tprec (°C)

PSAA 1 PSAA 2 PSAA 3 PSAA 4 PSAA 5 PSAA 6

methanol

1.8 1.8 1.8 1.8 1.8 1.8

10 25 25 25 25 25

60 69 77 106 112 125

PSAA 7 PSAA 8 PSAA 9

acetone

1.8 1.5 1.2

50 50 50

113 113 112

PSAA 10 acetone-water 9% v/v PSAA 11 PSAA 12

1.5 1.8 2.0

90 90 90

110 111 110

PSAA 13 methanol-water 4% v/v PSAA 14 PSAA 15

1.8 1.8 1.8

10 20 30

100 100 100

solvents were used.11–13,22 If not otherwise stated, we fixed Tsat at 82 °C, psat in the range 80-83 bar, and pprec at atmospheric pressure, and varied the other process variables one at a time. Liquid solvent mixtures have never previously been used in the SAA technique and their use implies the formation in the saturator of a quaternary mixture: solvent 1 + solvent 2 + solute + CO2. The subsequent high pressure vapor liquid equilibria (VLE) could become very complex especially when small quantities of water are present. Indeed, CO2-water mutual solubility is very reduced at the usual SAA mixing conditions.23 However, due to the small percentages of water added to organic solvents, we assumed that the VLEs involved were in first approximation the same as the ones of the organic solvent with CO2. The liquid solvents and the mixtures tested in the present work were methanol, acetone, acetone + water 9% v/v, and methanol + water 4% v/v. The evaporation process of the droplets produced by SAA is usually very fast, due to the small dimensions of the droplets and, as a result, tends to produce amorphous particles. In this work, solvent mixtures have principally been used in the attempt to modify the crystallinity of BDP precipitates: solvents with a lower volatility would increase the evaporation time of the droplets, thus favoring the crystallization of solute.24,25 The solubility of BDP in water alone is practically negligible, but acetone and methanol containing small quantities of water are still good solvents for BDP and evaporation times should be longer due to the presence of water. SAA Experiments Using Methanol. In this series of experiments we fixed R ) 1.8 and the concentration of BDP in the liquid solution to 10 and 25 mg/mL and studied the effect of the precipitation temperature (Tprec) on the morphology and crystallinity of BDP precipitates. The experiments performed using methanol are reported in Table 2. R can largely influence the SAA process performance, since SC-CO2 solubilizes in the liquid solution in the saturator, forming an expanded liquid and the quantity of CO2 in the formed ternary solution, largely influences the properties of the expanded liquid. The larger the CO2 percentage is, the lower are the liquid viscosity and surface tension; and as discussed in the Introduction, this reduction of cohesive forces influences the first step of the atomization process, producing smaller primary droplets. Moreover, a larger quantity of CO2 is released from the primary droplets, enhancing the second atomization step. The experiment performed at Tprec ) 60 °C, C ) 10 mg/mL produced the morphology reported in Figure 1a; i.e., spherical

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Figure 1. SEM photomicrographs of BDP particles obtained by SAA from methanol at (a) 60 °C (PSAA1), (b) 125 °C (PSAA6).

Figure 2. PSD of BDP particles obtained by SAA at different temperatures (tests: PSAA2, PSAA5, PSAA6) in terms of volumetric cumulative curves, calculated using SEM image analysis.

particles that are very similar to those observed in other SAA studies.11–14,22 The size of the obtained particles was in the range 0.1-2 µm with a mean of about 1 µm. The XRPD analyses showed that the particles were amorphous. We want to obtain not only particles of controlled size, but also with a crystal habit similar to the one of the commercial product. Using SAA it has been previously demonstrated that it is possible to obtain crystalline particles of compounds having a simple molecular structure26 since they have very fast crystallization kinetics. In the case of more complex compounds, like BDP, it is possible to increase the precipitation temperature and to use a different solvent whose evaporation times could be longer to induce the crystallization process. In the set of SAA experiments performed using methanol, we increased Tprec in the subsequent experiments (see Table 2) up to 125 °C. From the SEM image reported in Figure 1b, it is possible to observe that the particles maintained a quasi-spherical morphology even at 125 °C. This effect is quantitatively measured in Figure 2 that reports cumulative volumetric distributions of BDP particles and in Table 3 where the range and percentile diameters related to 5, 50, and 95% by volume of the particles obtained (D5, D50,

and D95) are reported. Data reported in Table 3 can also be interpreted in terms of PSD. The range is the whole interval covered by the diameter of particles: D50 is the mean of the distribution and the difference between D95 and D5 is about two times the standard deviation of the PSD curve. In Table 3, X1-3% is also reported, which represents the percentage by volume of the particles ranging between 1 and 3 µm, previously defined as the most restrictive target for pulmonary delivery. The increase of the mean particle size in these experiments can be attributed to droplet coalescence phenomena. It is particularly worth noting that in PSAA 5 an X1-3 % of 86 is obtained: i.e., 86% by volume of the microparticles produced in this experiment fall within the designated 1-3 µm range. The largest particles were produced during the experiment PSAA6 with a maximum diameter of 7.6 µm. From the point of view of crystallinity, BDP microparticles precipitated by SAA using methanol were practically amorphous at Tprec of 60 and 69 °C, semicrystalline at 112 °C, and crystalline at 125 °C (see analysis in XRPD Figure 3). Correspondingly, the morphology of precipitates modified from perfectly spherical particles (SEM image Figure 1a) to quasi-spherical spheres at 125 °C (see SEM photomicrograph in Figure 1b). Therefore, the increase of the precipitation temperature increased the crystallization rate of BDP microparticles also modifying their shape from the original spherical morphology, originated from the shape of the droplets. The coalescence of microparticles is also evident from the PSDs reported in Table 3. Due to these phenomena, the diameter of the various fractions of the distribution increases with temperature. The inhalable fraction (X1-3%) is very high at precipitation temperatures up to 112 °C (86%), then, it decreases at a minimum of 28% at 125 °C. SAA Experiments Using Acetone. This set of experiments was performed by fixing the concentration of the liquid solution at 50 mg/mL and the precipitation temperature at 112 °C. R was varied from 1.2 to 1.8, which corresponds to different quantities of CO2 dissolved in the liquid solution, as previously discussed. A summary of these experiments is reported in Table 2 and an example of the related SEM images is showed in Figure 4. As expected, since increasing R also increases the quantity of CO2 dissolved in the liquid solution, smaller particles are produced as a result of the enhanced atomization. These results are quantitatively measured in Table 3 and summarized in the diagram in Figure 5, where cumulative curves of the volumetric particle size distributions are indicated. The increase of R tends also to produce BDP microparticles with a less defined spherical shape. This last effect is presently not clear, since the precipitation temperature, as a rule, could be responsible for these modifications; but, in this case, it has not been modified (see Table 2). A maximum X1-3% of 72% has been obtained operating at R ) 1.2 and a very similar value was obtained at R ) 1.5. The particles produced at R ) 1.8 are too small to match the 1-3 µm criterion (see Table 3). In this set of experiments, even if the Tprec was relatively high (112 °C), amorphous BDP particles were always produced, as confirmed by XRPD analysis (Figure 3). This fact can be easily explained: the solvent used (acetone) is very volatile and the evaporation of the droplets is particularly fast, as a consequence BDP molecules do not have time enough to organize in a regular, crystalline form. In conclusion, in the experiments described until now, the SAA process was successful from the point of view of the particle size distribution produced; but, in the case of acetone, process conditions were not found to obtain BDP particles that were also crystalline.

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Table 3. Effect of Different Parameters on the PSD Calculated Using SEM Image Analysis test code

solvent

C ) 25 mg/mL and R ) 1.8 PSAA 2 PSAA 3 PSAA 4 PSAA 5 PSAA 6

methanol

C ) 50 mg/mL and T ) 112 °C PSAA 7 PSAA 8 PSAA 9

acetone

C ) 90 mg/mL and T ) 110 °C PSAA 10 PSAA 11 PSAA 12

acetone-water 9% v/v

T ) 100 °C and R ) 1.8 PSAA 13 PSAA 14 PSAA 15

methanol-water 4% v/v

parameter studied

range (µm)

D5 (µm)

D50 (µm)

D95 (µm)

X1-3 %

0.1-2.5 0.1-2.7 0.3-3.0 0.4-3.5 0.1-7.6

0.6 0.6 0.9 1.0 1.7

1.2 1.3 1.7 1.9 3.7

1.9 2.0 2.4 2.8 6.0

68 70 85 86 28

0.1-1.9 0.2-3.1 0.2-3.2

0.5 0.6 0.7

0.9 1.4 1.5

1.4 2.3 2.6

40 70 72

0.3-4.8 0.2-4.7 0.2-1.7

1.3 0.6 0.4

2.9 1.9 0.9

4.4 3.9 1.4

49 70 49

0.2-1.8 0.3-3.3 0.4-4.9

0.4 0.6 0.8

0.7 1.3 1.7

1.0 2.2 2.9

13 67 74

T(°C) 69 77 105 112 125 R 1.8 1.5 1.2 R 1.5 1.8 2 C (mg/mL) 10 20 30

At this point of the work, to gain more flexibility from the point of view of the crystallization times, we decided to test solvent mixtures: methanol and acetone in presence of small quantities of water. SAA Experiments Using Acetone + Water 9% v/v. Until now SAA experiments using liquid solvent mixtures have never been performed. The high pressure phase equilibria of the system formed in the saturator (solvent + solvent 2 + CO2 + solute)

are not available in the literature and could be very different with respect to those of a single organic solvent in the presence of SC-CO2. BDP solubility in water is practically negligible; but it is known that the addition of small quantities of water in organic solvents can modify the general behavior of the solvent from the point of view of volatility, maintaining a reasonable solvent power toward the compounds to be solubilized. For these reasons, we tested two mixtures of water with the previously used solvents: namely acetone plus water at 9% v/v and methanol plus water at 4% v/v. Water addition should reduce solvent volatility and maintain the solubility of the BDP in the liquid solution, thus modifying the crystallization process. In these experiments we decided to fix all the other SAA process parameters and to vary the value of R as in the case of acetone experiments. A summary of acetone + water experiments is reported in Table 3 and the particle size distributions are reported in tabular form in Table 3. A maximum X1-3% of 70 has been obtained at R ) 1.8. The trend observed in the previous paragraphs, about the influence of R on BDM particle size, is confirmed: R ) 2.0 produced smaller particles (too small

Figure 3. XRPD analyses of BDP particles precipitated by SAA at different temperatures from methanol (tests: PSAA2, PSAA5, PSAA6) and acetone-water 9% v/v (test PSAA11).

Figure 4. SEM photomicrographs of BDP particles obtained by SAA from acetone at 50 mg/mL, 112 °C, and feed ratio R ) 1.2 (PSAA9).

Figure 5. PSD of BDP particles obtained by SAA with acetone, at different feed ratios (tests: PSAA7, PSAA8, PSAA9) in terms of volumetric cumulative curves, obtained using SEM image analysis.

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with respect to our target) whereas R ) 1.5 produced larger particles, thus reducing their percentage in the required 1-3 µm range. It is interesting to note that, when we compare these experiments with the ones performed using acetone alone, the particles produced at the same values of R are larger. This result is due to the fact that in the case of acetone + water mixtures a higher BDP concentration has been used and to the presence of water. Both these parameters increase the surface tension of the liquid solution, thus producing larger droplets by atomization. However, the main reason for the use of this solvent mixture was the attempt to obtain crystalline particles. From this point of view, the experiments were successful: SAA precipitation of BDP from acetone-water mixtures produced substantially crystalline powders, as demonstrated by the XRPD reported in Figure 3 and related to the experiments performed at R ) 1.8. This result confirms the hypothesis that the presence of water can modify the precipitation behavior of BDP. Indeed, powders precipitated using acetone alone were in all cases amorphous; whereas, the presence of water at 9% v/v produced in all cases crystalline powders, even if the precipitation temperature used in this last case was slightly lower than that in the experiments performed using pure acetone. This could be expected, because water as solvent favors the formation of crystals during SAA process, especially in the case of simple molecules such as NaCl.26 Also the morphology of BDP particles obtained in these experiments reflected the superposition of the crystallization on the drying process: the particles are less defined than in the previous cases: the spherical morphology is partly lost due to the onset of crystallization during the drying process, as in the case of methanol at high precipitation temperatures. It is worth noting that PSD results have been obtained using DLS analysis that is particularly useful when irregular particles have to be analyzed. It calculates the equivalent diameter of the particles giving an objective PSD measurement even for particles that seem somewhat coalescing in SEM images. SAA Experiments Using Methanol + Water 4%. In the case of the experiments using methanol plus water 4% v/v we focused our attention on the influence of the concentration of BDP in the liquid solution, maintaining the same value of R (1.8) used in the experiments performed using methanol alone. An intermediate value of the precipitation temperature (100 °C) was used, if compared to the experiments performed with methanol, but lower with respect to the experiments performed using acetone. The morphology of BDP particles confirmed the tendency observed in the case of acetone + water experiments: the particles showed a less defined spherical morphology, as show by SEM images in Figure 6 and Figure 7. An increase of particle size was observed when BDP concentration in the liquid solution was increased (see Figure 8). Particle size data in Table 3 show that the most appropriate particle size distribution was obtained at a concentration of BDP of 30 mg/mL, with an X1-3% of about 74. The most important information obtained in the last set of experiments is that the presence of water in the liquid solution favors the crystallization process. Indeed, crystalline particles have been produced at lower precipitation temperatures than using methanol alone and at all the concentrations tested. Further Analysis of BDP Particles. DSC analysis was also systematically performed on SAA processed BDP particles and confirmed XRPD results from the point of view of crystallinity. HPLC analysis was also performed to control if SAA processing induced BDP degradation; but, in all cases we found total

Figure 6. PSD of BDP particles obtained by SAA at different feed ratios (tests: PSAA10, PSAA11, PSAA12) in terms of volumetric cumulative curves, calculated using SEM image analysis.

Figure 7. SEM photomicrographs of BDP particles obtained by SAA from methanol + water 4% v/v (PSAA13).

Figure 8. PSD of BDP particles obtained by SAA at different concentrations of BDP in methanol + water 4% v/v (tests: PSAA13, PSAA14, PSAA15) in terms of volumetric cumulative curves, calculated using SEM image analysis.

impurities lower than 0.35% and substantially related to beclomethasone derivatives, as in the starting material. Also solvent residues analysis was performed. BDP processed SAA showed a maximum solvent residue of 44 ppm for acetone and 36 ppm for methanol based experiments.

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showed an oscillation of (0.14 µm around the mean value, without any definite trend in increase or decrease of the particles’ diameter; i.e., the mean diameter oscillated randomly around the values indicated. This observation means that the variations observed in particle size distributions mainly depend on the experimental error connected to the single DLS measurement of the sample. To compare the particles produced by SAA with those usually used in the commercial formulation, we performed the stability test also on a commercial sample of micronized BDP given by Farmabios. The PSD measurements were performed using the same procedure as described above. The curves obtained by DLS show the same trend of the ones obtained for SAA micronized particles and the same stability with time. The commercial sample showed a larger PSD and an X1-3% of about 35, due to the different micronization technique used. However, also in this case DLS data oscillated randomly, for example D50 showed an oscillation of (0.10 µm, confirming that these oscillations are due to the experimental error. Experiments Performed on the Pilot Plant. Pilot-scale experiments are an important step to confirm the laboratory results. To obtain the same results using different scale apparatuses, the geometric scale-up of the process volumes is generally not enough. The key step is the identification of the proper dimensionless parameters that govern the process and to replicate their values on the different scales. In a previous paper,11 we demonstrated that the reproduction of SAA experiments, once pressure, temperature, and concentration are fixed, depends on the residence time in the saturator and on two dimensionless parameters connected to the atomization process: the Reynolds number (Re) and the Ohnesorge number (Oh).27,28 Reynolds number is related to the ratio between inertial and cohesive forces in the liquid jet and Ohnesorge number correlates Weber and Reynolds number, where the Weber number is the ratio between kinetic and capillary energy in the liquid jet. A residence time of some minutes in the saturator assures the attainment of quasi-equilibrium conditions for the dissolution of CO2 in the liquid solution. The operation at the same value of Re and Oh can ensure the reproducibility of SAA experiments also on different scales. The experiments replicated on the pilot scale were performed using the acetone/water 9% mixture starting from the process conditions tested in PSAA11, using the same values of Re. Acetone/water experiments were previously indicated as the best compromise among processability, dimension of particles, and crystallinity of BDP powders. Also methanol/water 4% experiments were successful, but they were not selected for pilot scale experiments for two main reasons: BDP solubility in this mixture is lower than in acetone/water 9%, and methanol residues, though lower than the current pharmacopeia limits, are, however, referred to a more toxic solvent. In Table 4 a summary of pilot scale experiments is reported. Performing pilot scale experiments we also tried to further optimize the results; therefore, we performed the experiments not only at the previous laboratory-scale tested process conditions, but we tried to operate at lower precipitation temperatures

Figure 9. Volumetric cumulative curves of the PSD stability test of BDP particles obtained using acetone-water 9% v/v (PSAA11) calculated by DLS analysis. The various lines are related to the replication of the measurement at different times.

In conclusion of this part of the work, the SAA experiments were successful in the case of solvent + water mixtures either for the control of particle size or control of the crystallinity of the processed material. Moreover, no degradation products were found, thus SAA processing has not modified the pharmaceutical compound. PSD Stability in the Wet Formulation. To perform these tests, we selected the BDP particles obtained using acetone-water 9% v/v, considered the best result in terms of PSD, crystallinity, and toxicity of the solvent residues. The behavior of SAAproduced BDP microparticles in an aqueous environment, since they are intended for wet inhalation application, and the length of their shelf life in water is a very relevant parameter for their commercial application. The problem might be very relevant if in the compound presents also non structural water: it is possible that the particles aggregate in the wet formulation. The powder used is related to the test PSAA11. We replicated this experiment and the powder obtained presented the same PSD characteristics, confirming the reproducibility of the results previously obtained. To perform the stability tests in the aqueous environment, we used the formulation indicated in the methods section. PSD measurements by DLS were repeated each day during the first week of testing; then the test was repeated once a week for two months. The temperature-cycling experiments used in the two months should conventionally simulate two years of shelf life for the product.22 Some of the resulting volumetric integral particle size distributions obtained during these tests are summarized in Figure 9. DLS measurements showed very similar results during the whole duration of the tests and there is not a defined time in the curves obtained. For example, D50

Table 4. Summary of Pilot Scale SAA Experiments Using Acetone-Water 9% V/V As Solvent test code

solvent

R ) wCO2/wsol

C (mg/mL)

Tprec (°C)

processed g

lost g

recovery wt%

PSAA 16 PSAA 17 PSAA 18 PSAA 19 PSAA 20 PSAA 21 PSAA 22

acetone-water 9% v/v

1.8 1.5 1.5 1.4 1.5 1.5 1.5

70 50 25 25 50 50 50

100 90 90 90 70 70 70

5 10 10 10 10 10 30

2.4 3.2 2.5 3.1 2.0 1.5 2.0

56 70 75 70 80 85 93

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Figure 10. SEM photomicrographs of BDP particles obtained by SAA from acetone-water 9% v/v using the pilot plant (PSAA20).

to reduce the possible stress on the processed material and modified R and C values to obtain this result. A SEM image of the BDP particles obtained in these experiments is reported in Figure 10; as in the previous experiments, irregular particles are produced by crystallization phenomena superimposed to droplets drying. In particular, looking at PSD calculated by DLS, the optimum operating conditions were obtained in the experiments PSAA20-22 where we obtained crystalline BDP powders at a concentration of drug in solution of 50 mg/ mL, precipitation temperature of 70 °C (whereas in laboratory experiments 100 °C were used) with a D50 of 1.64 µm and an X1-3% value of 83. This percentage is much better than the maximum value obtained in the previous laboratory scale experiments. Performing the experiments on the pilot plant the yield of the process was also measured by weighing the powder recovered at the end of each experiment (Table 4). The unrecovered material depends on the powder stuck onto the filter put at the bottom of the precipitator and on the difficulty in the complete recovery of the material on the walls of the precipitator. No leakage of solid through the filter was observed. When we increased the quantity of compound injected in each experiment, we observed that the loss of material was practically constant; i.e., the quantity of BDP lost on the walls of the precipitator does not depend on the quantity injected. For example, in the experiment PSAA16, 5 g of BDP was used, with a recovery of 56%; in the experiments from PSAA17 to PSAA21, 10 g of BDP was injected and a recovery between 75 and 85 wt % was obtained. Increasing the quantity of BDP injected, the product recovery increased up to 93 wt % (PSAA22), since the quantity of material lost ranges from 1.5 to 2.5 g, independently from the quantity of BDP used in the experiment. We also replicated, on pilot-scale produced BDP powders, all the analyses performed on SAA laboratory scale and found that BDP particles were crystalline and particle size distributions were reproducible. No degradation products increase was observed and acetone residues similar to the laboratory products were detected. Conclusions Though SAA-produced microparticles for large molecules tend to be amorphous, in this work crystalline BDP microparticles were obtained using higher precipitation temperatures and different solvents and solvent mixtures. However, SAA precipitation of BDP at higher temperatures tended also to produce larger particles due to coalescence phenomena; therefore, the process based on solvent mixture acetone-water was selected.

Pilot-scale SAA experiments confirmed the scalability of the BDP SAA micronization and were also used as an opportunity for a further improvement of laboratory-scale results The product with the required PSD and crystallinity was recovered up to 93% wt. The volumetric fraction of BDP particles falling in the range 1-3 µm was 83% on the pilot scale, which represents large improvement with respect to the 35% by volume measured for the commercial sample. In the formulation for wet inhalation, a stable PSD was maintained for more than 2 months. A further development of the SAA process will be to produce micronized compounds at GMP and sterile conditions to allow pharmaceutical experimentation. A plant operating according to SAA process and GMP standards was constructed by Prometeo Consortium (Lugano, Switzerland) to produce GMP microparticles. Acknowledgment We gratefully acknowledge Dr. Alessandro Di Giacomo and Dr. Rossella Morrone for help in performing the experiments. The MiUR (Italian Ministry of Scientific Research) is acknowledged for the partial financial support. Literature Cited (1) Gonda, I. Targeting by deposition. In Pharmaceutical Inhalation Aerosol Technology; Hickey, A. J., Ed.; Marcel Dekker: New York, 1992. (2) Johnson, M. A. The Aerosol Handbook; Wayne E. Dorland, Co.: Mendham, NJ, 1982. (3) Hickey, J. Inhalation Aerosols: Physical and Biological Basis for Therapy; Informa Healthcare: London, 1996. (4) Atkins, P.; Barker, N.; MAthiens, D. The design and development of drug inhalation delivery systems. In Pharmaceutical Inhalation Aerosol Technology; Hickey, A. J., Ed.;, Marcel Dekker: New York, 1992. (5) Reverchon, E.; Adami, R. Nanomaterials and supercritical fluids. J. Supercrit. Fluids 2006, 37, 1. (6) Shariati, A.; Peters, C. J. Recent developments in particle design using supercritical fluids. Curr. Opin. Solid State Mater. Sci. 2003, 7, 371. (7) York, P. Strategies for particle design using supercritical fluid technologies. Pharm. Sci. Technol. Today 1999, 2, 430. (8) Reverchon, E.; Torino, E.; Dowy, S.; Braeuer, A.; Leipertz, A. Interactions of phase equilibria, jet fluid dynamics and mass transfer during supercritical antisolvent micronization. Chem. Eng. J. 2010, 156, 446. (9) Sievers, R. E.; Quinn, B. P.; Cape, S. P.; Searles, J. A.; Braun, C. S.; Bhagwat, P.; Rebits, L. G.; McAdams, D. H.; Burger, J. L.; Best, J. A.; Lindsay, L.; Hernandez, M. T.; Kisich, K. O.; Iacovangelo, T.; Kristensen, D.; Chen, D. Near-critical fluid micronization of stabilized vaccines, antibiotics and anti-virals. J. Supercrit. Fluids 2007, 42, 385. (10) Wang, Q.; Guan, Y.-X.; Yao, S.-J.; Zhu, Z.-Q. Microparticle formation of sodium cellulose sulfate using supercritical fluid assisted atomization introduced by hydrodynamic cavitation mixer. Chem. Eng. J. 2010, 159, 220. (11) Reverchon, E.; Adami, R.; Caputo, G. Supercritical assisted atomization: Performance comparison between laboratory and pilot scale. J. Supercrit. Fluids 2006, 37, 298. (12) Reverchon, E.; Adami, R.; Caputo, G. Production of cromolyn sodium microparticles for aerosol delivery by supercritical assisted atomization. AAPS Pharmscitech 2007, 8, 272. (13) Adami, R.; Sesti Osséo, L.; Reverchon, E. Micronization of Lysozyme by Supercritical Assisted Atomization. Biotechnol. Bioeng. 2009, 104, 1162. (14) Cai, M.-Q.; Guan, Y.-X.; Yao, S.-J.; Zhu, Z.-Q. Supercritical fluid assisted atomization introduced by hydrodynamic cavitation mixer (SAAHCM) for micronization of levofloxacin hydrochloride. J. Supercrit. Fluids 2008, 43, 524. (15) Reverchon, E.; Adami, R.; Cardea, S.; Della Porta, G. Supercritical fluids processing of polymers for pharmaceutical and medical applications. J. Supercrit. Fluids 2009, 47, 484. (16) Reverchon, E.; Antonacci, A. Chitosan Microparticles Production by Supercritical Fluid Processing. Ind. Eng. Chem. Res. 2006, 45, 5722. (17) Reverchon, E.; Antonacci, A. Polymer microparticles production by supercritical assisted atomization. J. Supercrit. Fluids 2007, 39, 444.

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ReceiVed for reView July 23, 2010 ReVised manuscript receiVed October 4, 2010 Accepted October 12, 2010 IE101574Z