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Erythromycin micro-particles produced by supercritical fluid atomization
Powder Technology 141 (2004) 100 – 108 www.elsevier.com/locate/powtec
Erythromycin micro-particles produced by supercritical fluid atomization E. Reverchon *, A. Spada Dipartimento di Ingegneria Chimica e Alimentare, Universita` di Salerno, Via Ponte Don Melillo, 84084, Fisciano (SA), Italy Received 15 January 2003; received in revised form 11 February 2004; accepted 18 February 2004
Abstract Supercritical fluid-assisted atomization has been used to micronize erythromycin in the range of aerosilizable drugs. This process allows a very efficient production of micro-particles due to the release of supercritical CO2 from the inside of the primary droplets formed during the atomization process. The experiments have been performed using three different organic solvents (methanol, ethanol, acetone) and the influence of several process parameters: feed ratio between CO2 and liquid solution, concentration of solute in the liquid solution and precipitation temperature, have been tested. Spherical micrometric and non-coalescing particles have been obtained using methanol and ethanol at various CO2-solution feed ratio and concentrations. In all cases, particles with diameters smaller than 3 Am have been obtained; up to 88% of the particles volume was included in the range from 1 to 3 Am, at the most favourable operating conditions. In the case of acetone, well-defined spherical particles were obtained only when a feed ratio of 1.6 was used, in all the other experiments the particles coalesced in large groups. No degradation occurred to erythromycin as shown by the comparison of the HPLC traces of processed and raw materials. Head space GC analysis revealed a maximum solvent residue in the processed material of 90 ppb. D 2004 Elsevier B.V. All rights reserved. Keywords: Erythromycin; Supercritical fluid; Micro-particles
1. Introduction Lung aerosols are commonly used for asthma therapy, however, some other drug categories have also been proposed for aerosol delivery to the lung. For example, antibiotics to treat bronchopulmonary infections [1] in order to decrease the systemic dosage necessary to achieve effective antibacterial control and limit systemic adverse effects. Aerosolised antibiotics have been demonstrated to be therapeutically effective while causing fewer systemic side effects. Among the others erythromycin has been used to treat some pulmonary infections [2]. However, to obtain an efficient delivery to the deep lung, particles ranging between 1 and 5 Am are required. Some authors proposed an ever more restricted range between 1 and 3 Am as the most effective for this route of administration of drugs [1].
* Corresponding author. Tel.: +39-089-964116; fax: +39-089-964057. E-mail address: [email protected] (E. Reverchon). 0032-5910/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2004.02.017
Traditional micronization techniques, like jet milling, in some cases, cannot produce optimised particles for aerosol delivery. Cohesive powders and materials prone to thermal degradation are examples of materials challenging to micronize. Irregular particles are produced that range, as a rule, between 0.1 and 10 –20 Am, as shown by Reverchon and Della Porta [3] in the case of terbutalin. Therefore, in these cases, only a reduced quantity of the drug is efficiently used. To overcome the limitations of the current micronization techniques, some supercritical based techniques have been proposed [4,5]. These processes can take advantage of some specific properties of gases at supercritical conditions like the modulation of the solvent power, large diffusivity, solventless or organic solvent reduced operation. However, only in some cases, particles falling in the particle size range useful for aerosol delivery were obtained. The better results were obtained using the supercritical antisolvent precipitation [4,5]. Very recently, new supercritical fluids based micronization techniques have been proposed that can give a good control of particle size (PS) and particle size distribution (PSD) in the specific range of lung aerosols. A supercritical
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based atomization method has been proposed by Sievers et al. [6 – 9] and called Carbon dioxide Assisted NebulizationBubble Dryer (CAN-BD). In this process, prior to atomization, supercritical CO2 and the liquid solution are introduced in a near-zero internal volume tee connection (I.V. < 0.1 Al) and, then, in a short capillary (50 – 70 mm long). This device is characterized by a very short residence time (between 0.1 and 0.01 s) and by a very limited possibility of mixing between the fluids. A two-phase mixture is likely to be produced (not a solution) from which the supercritical fluid is released in the liquid jet at the exit of the capillary. An efficient atomization can be obtained, therefore this process has been successfully tested on several compounds, included some drugs, obtaining micro-particles [10]. Another kind of supercritical based processes that shares many similarities with supercritical atomization is the Particles Generation from Gas Saturated Solutions (PGSS) [11 – 14]. In this process, no solvents are used. An adequate heating of the vessel and the introduction of supercritical CO2, are used to induce the melting of the solid substance. Particularly, the glass transition temperature of a polymer can be largely reduced by the presence of CO2. Then, the CO2-containing melt mixture is depressurized and particles are generated by solidification of the material treated. This process has been tested on several polymers and some solid pharmaceutical compounds (nifedipine and felodipine). Irregular particles of several microns in diameter have been obtained. Since, as a rule, drugs are thermally labile compounds, they tend to decompose before the melting temperature has been reached [12,13]. Since supercritical CO2 is very soluble in many liquid solvents, (from 0.1 to 0.7 mole fraction and more) as demonstrated by the large compilation of binary equilibrium data for CO2-organic solvents collected by Ohe [15], it is possible to take advantage of this characteristic by the
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addition of a solubilization device prior to atomization. Thus, the process arrangement developed by our research group [16,17] is based on the use of a packed saturator characterized by a high specific surface and large residence times. In the saturator, supercritical CO2 dissolves in the liquid solution (an organic solvent or water and a solid solute) before the atomization in a thin wall injector. Therefore, a ternary solution of the liquid, CO2 and the solid solute is formed. The atomization is particularly efficient since CO2 is released from the internal of the droplets and enhances their fragmentation (formation of secondary droplets). We named this process Supercritical Assisted Atomization (SAA). This process has been tested on some pharmaceutical compounds, catalyst and superconductor precursors [3,17 – 20], obtaining micrometric and submicrometric particles of controlled size and distribution. The scope of this work is to evaluate the possibility of producing erythromycin micro-particles in the 1 –3 Am range using SAA. We also want to explore the potential of this micronization technique studying the influence on the morphology, particle size and distribution of different liquid solvents (methanol, ethanol and acetone), different precipitation temperatures, feed ratios and concentrations of the liquid solution.
2. Materials and methods 2.1. Experimental apparatus The apparatus used for SAA experiments is schematically reported in Fig. 1. It consists of three feed lines that deliver supercritical CO2, the liquid solution and warm N2, respectively. Three vessels are the major capacities of the plant: saturator (Sa), precipitator (Pr) and condenser (Co).
Fig. 1. Schematic representation of the SAA apparatus. 1) CO 2 cylinder; 2) Liquid solution; 3) N2 cylinder; 4) cooling bath; 5) heating bath; 6) high pressure pumps; 7) dampener; 8) heat exchanger; 9) saturator; 10) precipitor; 11) condenser.
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Liquid CO2, from the high-pressure pump, (Gilson mod. 305) is sent to a heated bath (Forlab, Carlo Erba mod. TR12) and then to the saturator where it solubilizes into the liquid solution. The liquid solution is taken from a graduated glass vessel, pressurized by a high-pressure pump (Gilson mod. 305), heated and sent to the saturator. N2 is taken from a cylinder, heated in an electric heat exchanger (Watlow mod. CBEN 24G6) and then is sent to the precipitator. The saturator (Sa) is a high-pressure vessel (I.V. 50 cm3) loaded with stainless steel perforated saddles, to assure a large surface for the contact between CO2 and the liquid solution and obtain the dissolution of the gaseous stream in the liquid solution. The obtained solid –liquid – gas mixture, at the exit of the saturator is sent to a thin wall stainless steel injector (I.D. 100 Am) to produce a spray of liquid droplets in the precipitator. Residence times in the saturator can vary from several seconds to minutes at the usual process conditions. The precipitator is a stainless steel vessel (I.V. 3 dm3) operating at atmospheric pressure. It receives the flow of heated N2 to favour the evaporation of the liquid droplets. The powder and the gas mixture (CO2/N2/solvent vapour) generated in the precipitator are forced to assume an ordinate motion by a flux conveyor. It consists of a helicoidal stainless steel device that occupies all the horizontal precipitator section. The powder is collected at the bottom of the precipitator on a stainless steel sintered frit (mean pore diameter 0.1 Am), whereas the gaseous stream passes through the frit and reaches a cooled separator where the liquid solvent is condensed. The resulting gas mixture (CO2 and N2) is sent to a dry test meter (Schlumberger, mod. 2000AP LPG G2.5) to measure the overall flow rate. More details on the SAA apparatus have been previously published [16]. 2.2. Materials Erythromycin (ETM, purity 98%) was supplied by ICN Biomedicals (Milano, Italy). Methanol (MeOH, purity 99.5%), ethanol (EtOH, purity 99.8%) and acetone (Ac, purity 99.8%) were supplied by Carlo Erba Reagenti (Italy). CO2 (purity 99.9%) was purchased from SON (Naples, Italy). All compounds were used as received. The approximate solubilities of ETM in MeOH, EtOH and Ac were measured at room temperature and are 65, 65 and 50 mg/ml, respectively. 2.3. Analytical methods Samples of the powder precipitated on the metallic frit were observed by Scanning Electron Microscopy (SEM, ˚ of mod. LEO 420). SEM samples were covered with 250 A gold using a sputter coater (Agar mod. 108A). The PS and the PSD were measured using the Sigma Scan Pro software (Jandel Scientific). SEM images from samples collected at different levels in the precipitator were used in PSD calcu-
lations. About 1000 particles were considered in each calculation of PSD, to obtain results that are representative of the whole precipitate. The untreated ETM consisted of irregular crystal with particle sizes ranging between 8 and 40 Am. Drug degradation was evaluated performing HPLCUVvis (Hewlett-Packard mod. G131-132) analysis on the untreated material and on SAA processed powder. The elution was obtained using a reverse phase C18 column ˚ pore size). The (4.6 250 mm; 5 Am particle size; 80 A column was equilibrated at a flow rate of 1.4 ml/min with a mobile phase consisting of acetonitrile and 0.05M phosphate buffer (pH 6.3, 45:55) [21]. The drug was monitored at 215 nm with a retention time of 2.4 min. All chromatographic analysis were carried out at room temperature. The average column back pressure was of about 200 bar. Solvent residue was measured by a gas chromatograph interfaced with a flame ionization detector (GC-FID) coupled to an head space sampler (Hewlett and Packard mod. 50 SCAN). Solvent residue was separated by using a fusedsilica capillary column (mod. DB-1, J&W, Folsom, CA) 30 m length, 0.25 mm internal diameter, 0.25 Am film thickness. GC conditions were oven temperature of 40 jC for 8 min. The injector was maintained at 180 jC (split mode, ratio 1:1) and helium was used as the carrier gas (7 ml/min). Head space conditions were: equilibration time 60 min at 100 jC, pressurization time 2 min, loop fill time 1 min. Head space samples were prepared in 10 ml vials filled by 1 ml of water in which were suspended samples of ETM processed with MeOH, Ac and EtOH.
3. Results and discussion The first selection of SAA parameters was based on our previous experience on this process [16 – 20]. The complete set of SAA process parameters includes: saturator pressure and temperature, precipitator temperature, liquid solution concentration, feed ratio CO2/liquid solution (R) and injector diameter. All data related to the SAA experiments performed on ETM have been collected in Table 1. In previous work, it was shown that injector diameters ranging from 80 to 200 Am have only a slight influence on the diameter of the particles, therefore, in this work only one injector 100 Am in diameter was used. Saturator pressure, temperature and composition play an important role in the feasibility of the process since high-pressure vapour – liquid equilibria (VLE) of the ternary solvent –solute– CO2 system are influenced by these parameters. Indeed, they define the possibility of the formation of a ternary homogeneous mixture in the saturator. However, scientific information about these equilibria is scarce and it is only possible to find information about the binary solvent – CO2 system. The information on binary systems was used when available to try to predict the ternary equilibria. Therefore, an empirical basis was employed for the selection of the saturator
E. Reverchon, A. Spada / Powder Technology 141 (2004) 100–108 Table 1 Process data for SAA experiments performed on ETM Solvent
R
Saturator temperature (jC)
Saturator pressure (bar)
1.8 1.8 1.8
80 79 79
90 88 91
80 80 80
Effect of temperature MeOH 40 MeOH 40
1.8 1.8
81 80
90 92
80 118
Effect of feed ratio EtOH 28 EtOH 28 EtOH 28
0.7 1.2 1.8
80 80 79
96 96 98
75 75 75
Effect of concentration EtOH 28 EtOH 35 EtOH 55
1.2 1.2 1.2
79 79 80
96 94 96
75 75 75
Effect of feed ratio Ac 22 Ac 22 Ac 22 Ac 22
0.55 0.98 1.36 1.6
79 79 81 80
79 78 80 80
85 85 85 85
Effect of concentration Ac 22 Ac 35 Ac 50
1.6 1.6 1.6
81 81 80
81 82 80
85 85 85
Effect of MeOH MeOH MeOH
Concentration (mg/ml) concentration 20 40 60
Precipitator temperature (bar)
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taken at the same enlargement (10 K), it is possible to evaluate in a qualitative manner the effect of concentration on the dimensions of particles produced: particle diameter increased with concentration. To transform the qualitative observation into quantitative data, we used the image analysis software described in Materials and methods. Three different SEM images of the same sample were used as rule, measuring more than 1000 particles for each calculation. The results are reported in the form of distributions, expressed in terms of number of particles and particle volume, in Figs. 3 and 4, respectively. Fig. 3 is a differential representation that shows nonsymmetric curves whose mode (the most frequent value of the particle diameter) moves towards larger diameters when solute concentration is increased. It is also noteworthy that the distributions at higher solute concentrations span on a larger diameter interval; i.e., particle size distribution broadens with solute concentration. A relatively large quantity of particles with diameters smaller than 0.5 Am is present, especially at solute concentrations lower than 40 mg/ml.
pressure and temperature. Only the mass feed ratio was varied; CO2 flow rate ranged from 5 to 20 g/min and the solution flow rate ranged from 4 to 24 g/min. The influence of the liquid solution starting concentration is the other parameter that has been studied in this work, since this is perhaps the most relevant process parameter in determining particle size and distribution [17]. In the case of methanol, the effect of precipitation temperature was also tested. 3.1. Erythromycin/methanol 3.1.1. Effect of concentration of the liquid solution MeOH-ETM solutions with concentrations ranging between 20 and 60 mg/ml were tested by SAA operating the saturator at 90 bar (max pressure oscillation F 2 bar) and 80 jC (max temperature oscillation F 1 jC). Precipitator temperature was set at 80 jC and a R = 1.8 was used. ETM particles produced were spherical in all the experiments, with single particles having well-defined boundaries. Two examples, of these micro-particles are reported in Fig. 2a and b. The only difference in process conditions between these two samples is the concentration of the liquid solution that was 20 mg/ml in the top micrograph and 60 mg/ml in the bottom micrograph. Since the two images have been
Fig. 2. (a – b) SEM images of ETM precipitated by SAA from methanol, concentration of the liquid solution are 20 and 60 mg/ml, respectively.
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Several tests were also performed with R ranging between 0.7 and 1.8 at the concentration of the liquid solution of 40 mg/ml and confirmed the results obtained at R = 1.8.
Fig. 3. Number of ETM particles % distributions, produced by SAA from MeOH varying the solute concentration from 20 to 60 mg/ml.
The distributions reported in Fig. 4 are calculated on an integral basis (the contributions of the various particle sizes are added in the diagram) and show the results in terms of volume percentage of the particles produced. This kind of distribution is particularly useful when pharmaceutical products are considered, since it is directly connected (via the density of the compound) to the mass of particles that have a particle size smaller (or larger) than a reference value. For example, if the reference values are between 1.0 and 3.0 Am; i.e., the most restrictive limits for aerosol delivery, all ETM particles produced with MeOH by SAA (Fig. 4) are smaller than 3 Am and only about 12% by volume of those produced at 60 mg/ml have a diameter smaller than 1 Am. Therefore, concentration of the liquid solution of 60 mg/ml gives the best results in terms of the most appropriate particle size distribution for inhalable erythromycin.
Fig. 4. ETM volume % cumulative distributions, produced by SAA from MeOH varying the solute concentration from 20 to 60 mg/ml.
3.1.2. Effect of precipitation temperature Our previous work [17] has shown that an increase of the precipitation temperature can produce smaller particles. Indeed, the increase of temperature reduces the surface tension of the droplets and can produce a more efficient atomization. On the other hand, ETM has a decomposition temperature of about 135 jC and it is possible that decomposition or a change in morphology can occur at process temperatures higher than 80 jC. Therefore, some experiments were performed at the previously described SAA conditions, operating at 40 mg/ml and at a precipitation temperature of 118 jC. Fig. 5 shows a SEM image of ETM particles obtained at 118 jC. The particles maintain a spherical-like shape but are connected by large solid bridges. Fusion and coalescence of the particles occurred at these conditions. Therefore, temperatures lower than 100 jC were always used in this work to avoid the collapse of micro-particles in a network of connected particles. 3.2. Erythromycin/ethanol In this case, saturator pressure and temperature were set at 96 bar (max pressure oscillation F 2 bar) and 80 jC (max temperature oscillation F 1 jC), respectively. The temperature of the precipitation chamber was set at 75 jC. 3.2.1. Effect of feed ratio The influence of the variation of the feed ratio at a fixed value of the concentration of the liquid solution (28 mg/ml) was studied. Tests were performed at values of R ranging between 0.7 and 1.8. In all cases, the observed morphology was represented by spherical particles. Examples of ETM particles precipitated
Fig. 5. SEM image of ETM precipitated by SAA from MeOH at a precipitation temperature of 118 jC. Particles are connected by solid bridges.
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from EtOH are reported in Fig. 6a and b and correspond to the experiments performed at R = 0.7 and 1.8. The quantity of CO2 in the liquid solution, calculated in the hypothesis that all CO2 dissolves in the liquid, in this range of R varies from about 0.4 to 0.63 in terms of mole fraction of CO2 in EtOH. From the same images, it can be qualitatively observed that particle size decreases with increasing R. This observation can be performed in quantitative mode from the diagrams shown in Figs. 7 and 8. The diagram in Fig. 7 is similar to the one proposed in Fig. 3 and shows ETM particle size distributions expressed in terms of the percentage of particles with a given diameter. The mode of these distributions moves to smaller diameters when R increases; a sharpening of the distributions can also been observed. Fig. 8 reports the distributions in terms of particles volume (as in Fig. 4). Also in this case, a decrease of particle size can been observed when R increases. Less than 15% by volume of the particles obtained at R 0.7 shows diameters lower than 1 Am; moreover, all particles are smaller than 2.5
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Fig. 7. Number of ETM particles % distributions, produced by SAA from EtOH varying R from 0.7 to 1.8.
Am. Therefore, at these EtOH operating conditions, the optimum PSD for the most restrictive aerosol delivery has been obtained. A possible explanation of the results obtained at varying R can be proposed in terms of the content of CO2 in the solution obtained in the saturator. To better explain this concept, a simplified representation of the high-pressure ternary system behaviour through the corresponding pseudo-binary system is reported in Fig. 9. The hypothesis made in this case is that the presence of solute does not produce a relevant modification of the VLE diagram with respect to the solvent – CO2 system. If the P-x diagram in Fig. 9 applies, points A, B and C represent the operating point for R values of 0.7, 1.2 and 1.8, respectively. In this hypothesis, all these experiments were carried out in the one phase region (liquid-rich phase) of the diagram with an
Fig. 6. (a – b) SEM images of ETM precipitated by SAA from EtOH at R 0.7 and 1.8.
Fig. 8. ETM volume % cumulative distributions, produced by SAA from EtOH varying R from 0.7 to 1.8.
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increasing content of CO2 in the liquid phase from points A to C. Thus, an increase of R produces an enhancement of the secondary atomization that explains the observed decrease in particle size.
Fig. 9. The hypothesized pressure-composition vapour – liquid equilibria diagram for the system EtOH-CO2 modified by the presence of ETM at 80 jC. Points A, B and C are referred to R values of 0.7, 1.2 and 1.8, respectively.
Fig. 10. (a – b) SEM images of ETM precipitated by SAA from ethanol, concentration of the liquid solution 25 and 55 mg/ml, respectively.
3.2.2. Effect of concentration of the liquid solution Fixing R at 1.2, several experiments were performed at different concentrations of the liquid solution. Two examples of ETM particles obtained at these process conditions are shown in Fig. 10a and b and correspond to tests performed at 25 and 55 mg/ml, respectively. Particle size increases again with concentration. Therefore, these results confirm the trend observed for the SAA of ETM/MeOH and the general trend observed in our previous works [16 – 19]. Comparing PS and PSD of ETM particles obtained using EtOH and MeOH at similar SAA conditions, it was noted that particles obtained from EtOH are slightly smaller.
Fig. 11. (a – b) SEM images of ETM precipitated by SAA from Ac at R 0.98 and 1.6, respectively. Effect of R on the morphology of ETM particles: at R = 0.98 particles are coalescing.
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Head space GC has also been used to measure solvent residues in ETM processed powders. GC-FID head space analyses of SAA processed ETM revealed a MeOH residue of 7 ppb, a EtOH residue of 42 ppb and a Ac residue of 90 ppb. Therefore, in all cases, only parts per billion solvent concentrations were detected that are well below all the solvent residue limits of the European Pharmacopea.
Acknowledgements
Fig. 12. HPLC trace of untreated and SAA processed ETM.
3.3. Erythromycin/acetone 3.3.1. Effect of feed ratio In this case, saturator pressure and temperature were set at 80 bar (max pressure oscillation F 2 bar) and 80 jC (max temperature oscillation F 1 jC). The temperature of the precipitation chamber was maintained at 85 jC. Experiments were performed at R values ranging between 0.55 and 1.6 at a fixed concentration of 22 mg/ml. SAA experiments using Ac produced always coalescing ETM particles except for the experiments performed at R = 1.6, in which spherical non-coalescing particles were obtained. The comparison between the two morphologies is shown in Fig. 11a and b where SEM images of particles produced at R = 0.98 and 1.6 are reported. Due to the difficulties found in SAA processing of ETM with Ac, this experimentation was abandoned. In conclusion the morphology of the particles is the same for MeOH, EtOH and Ac, but for the latter solvent only a value of R was found that allowed successful precipitation. These results confirm that the solvent/ETM system and its interactions with supercritical CO2 in the saturator play a major role in SAA processing; i.e., by changing the solvent, different vapor –liquid equilibria can be obtained that can favour the subsequent atomization.
4. Further characterization of precipitates ETM precipitates were tested by HPLC to assess if any decomposition occurred due to SAA processing. The HPLC method has been discussed in Materials and methods and corresponds to the standard procedure used in the pharmaceutical industry to verify if a drug has been modified as a consequence of intermediate processing. A typical result of HPLC analysis is reported in Fig. 12. The two traces are referred to unprocessed and processed ETM. The characteristic peak of ETM is the same in the two traces, no other peaks, related to decomposition products, are present.
The authors acknowledge MiUR (Italian Ministry for University and Research) for the financial support (PRIN 2000).
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Erythromycin micro-particles produced by supercritical fluid atomization E. Reverchon *, A. Spada Dipartimento di Ingegneria Chimica e Alimentare, Universita` di Salerno, Via Ponte Don Melillo, 84084, Fisciano (SA), Italy Received 15 January 2003; received in revised form 11 February 2004; accepted 18 February 2004
Abstract Supercritical fluid-assisted atomization has been used to micronize erythromycin in the range of aerosilizable drugs. This process allows a very efficient production of micro-particles due to the release of supercritical CO2 from the inside of the primary droplets formed during the atomization process. The experiments have been performed using three different organic solvents (methanol, ethanol, acetone) and the influence of several process parameters: feed ratio between CO2 and liquid solution, concentration of solute in the liquid solution and precipitation temperature, have been tested. Spherical micrometric and non-coalescing particles have been obtained using methanol and ethanol at various CO2-solution feed ratio and concentrations. In all cases, particles with diameters smaller than 3 Am have been obtained; up to 88% of the particles volume was included in the range from 1 to 3 Am, at the most favourable operating conditions. In the case of acetone, well-defined spherical particles were obtained only when a feed ratio of 1.6 was used, in all the other experiments the particles coalesced in large groups. No degradation occurred to erythromycin as shown by the comparison of the HPLC traces of processed and raw materials. Head space GC analysis revealed a maximum solvent residue in the processed material of 90 ppb. D 2004 Elsevier B.V. All rights reserved. Keywords: Erythromycin; Supercritical fluid; Micro-particles
1. Introduction Lung aerosols are commonly used for asthma therapy, however, some other drug categories have also been proposed for aerosol delivery to the lung. For example, antibiotics to treat bronchopulmonary infections [1] in order to decrease the systemic dosage necessary to achieve effective antibacterial control and limit systemic adverse effects. Aerosolised antibiotics have been demonstrated to be therapeutically effective while causing fewer systemic side effects. Among the others erythromycin has been used to treat some pulmonary infections [2]. However, to obtain an efficient delivery to the deep lung, particles ranging between 1 and 5 Am are required. Some authors proposed an ever more restricted range between 1 and 3 Am as the most effective for this route of administration of drugs [1].
* Corresponding author. Tel.: +39-089-964116; fax: +39-089-964057. E-mail address: [email protected] (E. Reverchon). 0032-5910/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2004.02.017
Traditional micronization techniques, like jet milling, in some cases, cannot produce optimised particles for aerosol delivery. Cohesive powders and materials prone to thermal degradation are examples of materials challenging to micronize. Irregular particles are produced that range, as a rule, between 0.1 and 10 –20 Am, as shown by Reverchon and Della Porta [3] in the case of terbutalin. Therefore, in these cases, only a reduced quantity of the drug is efficiently used. To overcome the limitations of the current micronization techniques, some supercritical based techniques have been proposed [4,5]. These processes can take advantage of some specific properties of gases at supercritical conditions like the modulation of the solvent power, large diffusivity, solventless or organic solvent reduced operation. However, only in some cases, particles falling in the particle size range useful for aerosol delivery were obtained. The better results were obtained using the supercritical antisolvent precipitation [4,5]. Very recently, new supercritical fluids based micronization techniques have been proposed that can give a good control of particle size (PS) and particle size distribution (PSD) in the specific range of lung aerosols. A supercritical
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based atomization method has been proposed by Sievers et al. [6 – 9] and called Carbon dioxide Assisted NebulizationBubble Dryer (CAN-BD). In this process, prior to atomization, supercritical CO2 and the liquid solution are introduced in a near-zero internal volume tee connection (I.V. < 0.1 Al) and, then, in a short capillary (50 – 70 mm long). This device is characterized by a very short residence time (between 0.1 and 0.01 s) and by a very limited possibility of mixing between the fluids. A two-phase mixture is likely to be produced (not a solution) from which the supercritical fluid is released in the liquid jet at the exit of the capillary. An efficient atomization can be obtained, therefore this process has been successfully tested on several compounds, included some drugs, obtaining micro-particles [10]. Another kind of supercritical based processes that shares many similarities with supercritical atomization is the Particles Generation from Gas Saturated Solutions (PGSS) [11 – 14]. In this process, no solvents are used. An adequate heating of the vessel and the introduction of supercritical CO2, are used to induce the melting of the solid substance. Particularly, the glass transition temperature of a polymer can be largely reduced by the presence of CO2. Then, the CO2-containing melt mixture is depressurized and particles are generated by solidification of the material treated. This process has been tested on several polymers and some solid pharmaceutical compounds (nifedipine and felodipine). Irregular particles of several microns in diameter have been obtained. Since, as a rule, drugs are thermally labile compounds, they tend to decompose before the melting temperature has been reached [12,13]. Since supercritical CO2 is very soluble in many liquid solvents, (from 0.1 to 0.7 mole fraction and more) as demonstrated by the large compilation of binary equilibrium data for CO2-organic solvents collected by Ohe [15], it is possible to take advantage of this characteristic by the
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addition of a solubilization device prior to atomization. Thus, the process arrangement developed by our research group [16,17] is based on the use of a packed saturator characterized by a high specific surface and large residence times. In the saturator, supercritical CO2 dissolves in the liquid solution (an organic solvent or water and a solid solute) before the atomization in a thin wall injector. Therefore, a ternary solution of the liquid, CO2 and the solid solute is formed. The atomization is particularly efficient since CO2 is released from the internal of the droplets and enhances their fragmentation (formation of secondary droplets). We named this process Supercritical Assisted Atomization (SAA). This process has been tested on some pharmaceutical compounds, catalyst and superconductor precursors [3,17 – 20], obtaining micrometric and submicrometric particles of controlled size and distribution. The scope of this work is to evaluate the possibility of producing erythromycin micro-particles in the 1 –3 Am range using SAA. We also want to explore the potential of this micronization technique studying the influence on the morphology, particle size and distribution of different liquid solvents (methanol, ethanol and acetone), different precipitation temperatures, feed ratios and concentrations of the liquid solution.
2. Materials and methods 2.1. Experimental apparatus The apparatus used for SAA experiments is schematically reported in Fig. 1. It consists of three feed lines that deliver supercritical CO2, the liquid solution and warm N2, respectively. Three vessels are the major capacities of the plant: saturator (Sa), precipitator (Pr) and condenser (Co).
Fig. 1. Schematic representation of the SAA apparatus. 1) CO 2 cylinder; 2) Liquid solution; 3) N2 cylinder; 4) cooling bath; 5) heating bath; 6) high pressure pumps; 7) dampener; 8) heat exchanger; 9) saturator; 10) precipitor; 11) condenser.
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Liquid CO2, from the high-pressure pump, (Gilson mod. 305) is sent to a heated bath (Forlab, Carlo Erba mod. TR12) and then to the saturator where it solubilizes into the liquid solution. The liquid solution is taken from a graduated glass vessel, pressurized by a high-pressure pump (Gilson mod. 305), heated and sent to the saturator. N2 is taken from a cylinder, heated in an electric heat exchanger (Watlow mod. CBEN 24G6) and then is sent to the precipitator. The saturator (Sa) is a high-pressure vessel (I.V. 50 cm3) loaded with stainless steel perforated saddles, to assure a large surface for the contact between CO2 and the liquid solution and obtain the dissolution of the gaseous stream in the liquid solution. The obtained solid –liquid – gas mixture, at the exit of the saturator is sent to a thin wall stainless steel injector (I.D. 100 Am) to produce a spray of liquid droplets in the precipitator. Residence times in the saturator can vary from several seconds to minutes at the usual process conditions. The precipitator is a stainless steel vessel (I.V. 3 dm3) operating at atmospheric pressure. It receives the flow of heated N2 to favour the evaporation of the liquid droplets. The powder and the gas mixture (CO2/N2/solvent vapour) generated in the precipitator are forced to assume an ordinate motion by a flux conveyor. It consists of a helicoidal stainless steel device that occupies all the horizontal precipitator section. The powder is collected at the bottom of the precipitator on a stainless steel sintered frit (mean pore diameter 0.1 Am), whereas the gaseous stream passes through the frit and reaches a cooled separator where the liquid solvent is condensed. The resulting gas mixture (CO2 and N2) is sent to a dry test meter (Schlumberger, mod. 2000AP LPG G2.5) to measure the overall flow rate. More details on the SAA apparatus have been previously published [16]. 2.2. Materials Erythromycin (ETM, purity 98%) was supplied by ICN Biomedicals (Milano, Italy). Methanol (MeOH, purity 99.5%), ethanol (EtOH, purity 99.8%) and acetone (Ac, purity 99.8%) were supplied by Carlo Erba Reagenti (Italy). CO2 (purity 99.9%) was purchased from SON (Naples, Italy). All compounds were used as received. The approximate solubilities of ETM in MeOH, EtOH and Ac were measured at room temperature and are 65, 65 and 50 mg/ml, respectively. 2.3. Analytical methods Samples of the powder precipitated on the metallic frit were observed by Scanning Electron Microscopy (SEM, ˚ of mod. LEO 420). SEM samples were covered with 250 A gold using a sputter coater (Agar mod. 108A). The PS and the PSD were measured using the Sigma Scan Pro software (Jandel Scientific). SEM images from samples collected at different levels in the precipitator were used in PSD calcu-
lations. About 1000 particles were considered in each calculation of PSD, to obtain results that are representative of the whole precipitate. The untreated ETM consisted of irregular crystal with particle sizes ranging between 8 and 40 Am. Drug degradation was evaluated performing HPLCUVvis (Hewlett-Packard mod. G131-132) analysis on the untreated material and on SAA processed powder. The elution was obtained using a reverse phase C18 column ˚ pore size). The (4.6 250 mm; 5 Am particle size; 80 A column was equilibrated at a flow rate of 1.4 ml/min with a mobile phase consisting of acetonitrile and 0.05M phosphate buffer (pH 6.3, 45:55) [21]. The drug was monitored at 215 nm with a retention time of 2.4 min. All chromatographic analysis were carried out at room temperature. The average column back pressure was of about 200 bar. Solvent residue was measured by a gas chromatograph interfaced with a flame ionization detector (GC-FID) coupled to an head space sampler (Hewlett and Packard mod. 50 SCAN). Solvent residue was separated by using a fusedsilica capillary column (mod. DB-1, J&W, Folsom, CA) 30 m length, 0.25 mm internal diameter, 0.25 Am film thickness. GC conditions were oven temperature of 40 jC for 8 min. The injector was maintained at 180 jC (split mode, ratio 1:1) and helium was used as the carrier gas (7 ml/min). Head space conditions were: equilibration time 60 min at 100 jC, pressurization time 2 min, loop fill time 1 min. Head space samples were prepared in 10 ml vials filled by 1 ml of water in which were suspended samples of ETM processed with MeOH, Ac and EtOH.
3. Results and discussion The first selection of SAA parameters was based on our previous experience on this process [16 – 20]. The complete set of SAA process parameters includes: saturator pressure and temperature, precipitator temperature, liquid solution concentration, feed ratio CO2/liquid solution (R) and injector diameter. All data related to the SAA experiments performed on ETM have been collected in Table 1. In previous work, it was shown that injector diameters ranging from 80 to 200 Am have only a slight influence on the diameter of the particles, therefore, in this work only one injector 100 Am in diameter was used. Saturator pressure, temperature and composition play an important role in the feasibility of the process since high-pressure vapour – liquid equilibria (VLE) of the ternary solvent –solute– CO2 system are influenced by these parameters. Indeed, they define the possibility of the formation of a ternary homogeneous mixture in the saturator. However, scientific information about these equilibria is scarce and it is only possible to find information about the binary solvent – CO2 system. The information on binary systems was used when available to try to predict the ternary equilibria. Therefore, an empirical basis was employed for the selection of the saturator
E. Reverchon, A. Spada / Powder Technology 141 (2004) 100–108 Table 1 Process data for SAA experiments performed on ETM Solvent
R
Saturator temperature (jC)
Saturator pressure (bar)
1.8 1.8 1.8
80 79 79
90 88 91
80 80 80
Effect of temperature MeOH 40 MeOH 40
1.8 1.8
81 80
90 92
80 118
Effect of feed ratio EtOH 28 EtOH 28 EtOH 28
0.7 1.2 1.8
80 80 79
96 96 98
75 75 75
Effect of concentration EtOH 28 EtOH 35 EtOH 55
1.2 1.2 1.2
79 79 80
96 94 96
75 75 75
Effect of feed ratio Ac 22 Ac 22 Ac 22 Ac 22
0.55 0.98 1.36 1.6
79 79 81 80
79 78 80 80
85 85 85 85
Effect of concentration Ac 22 Ac 35 Ac 50
1.6 1.6 1.6
81 81 80
81 82 80
85 85 85
Effect of MeOH MeOH MeOH
Concentration (mg/ml) concentration 20 40 60
Precipitator temperature (bar)
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taken at the same enlargement (10 K), it is possible to evaluate in a qualitative manner the effect of concentration on the dimensions of particles produced: particle diameter increased with concentration. To transform the qualitative observation into quantitative data, we used the image analysis software described in Materials and methods. Three different SEM images of the same sample were used as rule, measuring more than 1000 particles for each calculation. The results are reported in the form of distributions, expressed in terms of number of particles and particle volume, in Figs. 3 and 4, respectively. Fig. 3 is a differential representation that shows nonsymmetric curves whose mode (the most frequent value of the particle diameter) moves towards larger diameters when solute concentration is increased. It is also noteworthy that the distributions at higher solute concentrations span on a larger diameter interval; i.e., particle size distribution broadens with solute concentration. A relatively large quantity of particles with diameters smaller than 0.5 Am is present, especially at solute concentrations lower than 40 mg/ml.
pressure and temperature. Only the mass feed ratio was varied; CO2 flow rate ranged from 5 to 20 g/min and the solution flow rate ranged from 4 to 24 g/min. The influence of the liquid solution starting concentration is the other parameter that has been studied in this work, since this is perhaps the most relevant process parameter in determining particle size and distribution [17]. In the case of methanol, the effect of precipitation temperature was also tested. 3.1. Erythromycin/methanol 3.1.1. Effect of concentration of the liquid solution MeOH-ETM solutions with concentrations ranging between 20 and 60 mg/ml were tested by SAA operating the saturator at 90 bar (max pressure oscillation F 2 bar) and 80 jC (max temperature oscillation F 1 jC). Precipitator temperature was set at 80 jC and a R = 1.8 was used. ETM particles produced were spherical in all the experiments, with single particles having well-defined boundaries. Two examples, of these micro-particles are reported in Fig. 2a and b. The only difference in process conditions between these two samples is the concentration of the liquid solution that was 20 mg/ml in the top micrograph and 60 mg/ml in the bottom micrograph. Since the two images have been
Fig. 2. (a – b) SEM images of ETM precipitated by SAA from methanol, concentration of the liquid solution are 20 and 60 mg/ml, respectively.
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Several tests were also performed with R ranging between 0.7 and 1.8 at the concentration of the liquid solution of 40 mg/ml and confirmed the results obtained at R = 1.8.
Fig. 3. Number of ETM particles % distributions, produced by SAA from MeOH varying the solute concentration from 20 to 60 mg/ml.
The distributions reported in Fig. 4 are calculated on an integral basis (the contributions of the various particle sizes are added in the diagram) and show the results in terms of volume percentage of the particles produced. This kind of distribution is particularly useful when pharmaceutical products are considered, since it is directly connected (via the density of the compound) to the mass of particles that have a particle size smaller (or larger) than a reference value. For example, if the reference values are between 1.0 and 3.0 Am; i.e., the most restrictive limits for aerosol delivery, all ETM particles produced with MeOH by SAA (Fig. 4) are smaller than 3 Am and only about 12% by volume of those produced at 60 mg/ml have a diameter smaller than 1 Am. Therefore, concentration of the liquid solution of 60 mg/ml gives the best results in terms of the most appropriate particle size distribution for inhalable erythromycin.
Fig. 4. ETM volume % cumulative distributions, produced by SAA from MeOH varying the solute concentration from 20 to 60 mg/ml.
3.1.2. Effect of precipitation temperature Our previous work [17] has shown that an increase of the precipitation temperature can produce smaller particles. Indeed, the increase of temperature reduces the surface tension of the droplets and can produce a more efficient atomization. On the other hand, ETM has a decomposition temperature of about 135 jC and it is possible that decomposition or a change in morphology can occur at process temperatures higher than 80 jC. Therefore, some experiments were performed at the previously described SAA conditions, operating at 40 mg/ml and at a precipitation temperature of 118 jC. Fig. 5 shows a SEM image of ETM particles obtained at 118 jC. The particles maintain a spherical-like shape but are connected by large solid bridges. Fusion and coalescence of the particles occurred at these conditions. Therefore, temperatures lower than 100 jC were always used in this work to avoid the collapse of micro-particles in a network of connected particles. 3.2. Erythromycin/ethanol In this case, saturator pressure and temperature were set at 96 bar (max pressure oscillation F 2 bar) and 80 jC (max temperature oscillation F 1 jC), respectively. The temperature of the precipitation chamber was set at 75 jC. 3.2.1. Effect of feed ratio The influence of the variation of the feed ratio at a fixed value of the concentration of the liquid solution (28 mg/ml) was studied. Tests were performed at values of R ranging between 0.7 and 1.8. In all cases, the observed morphology was represented by spherical particles. Examples of ETM particles precipitated
Fig. 5. SEM image of ETM precipitated by SAA from MeOH at a precipitation temperature of 118 jC. Particles are connected by solid bridges.
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from EtOH are reported in Fig. 6a and b and correspond to the experiments performed at R = 0.7 and 1.8. The quantity of CO2 in the liquid solution, calculated in the hypothesis that all CO2 dissolves in the liquid, in this range of R varies from about 0.4 to 0.63 in terms of mole fraction of CO2 in EtOH. From the same images, it can be qualitatively observed that particle size decreases with increasing R. This observation can be performed in quantitative mode from the diagrams shown in Figs. 7 and 8. The diagram in Fig. 7 is similar to the one proposed in Fig. 3 and shows ETM particle size distributions expressed in terms of the percentage of particles with a given diameter. The mode of these distributions moves to smaller diameters when R increases; a sharpening of the distributions can also been observed. Fig. 8 reports the distributions in terms of particles volume (as in Fig. 4). Also in this case, a decrease of particle size can been observed when R increases. Less than 15% by volume of the particles obtained at R 0.7 shows diameters lower than 1 Am; moreover, all particles are smaller than 2.5
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Fig. 7. Number of ETM particles % distributions, produced by SAA from EtOH varying R from 0.7 to 1.8.
Am. Therefore, at these EtOH operating conditions, the optimum PSD for the most restrictive aerosol delivery has been obtained. A possible explanation of the results obtained at varying R can be proposed in terms of the content of CO2 in the solution obtained in the saturator. To better explain this concept, a simplified representation of the high-pressure ternary system behaviour through the corresponding pseudo-binary system is reported in Fig. 9. The hypothesis made in this case is that the presence of solute does not produce a relevant modification of the VLE diagram with respect to the solvent – CO2 system. If the P-x diagram in Fig. 9 applies, points A, B and C represent the operating point for R values of 0.7, 1.2 and 1.8, respectively. In this hypothesis, all these experiments were carried out in the one phase region (liquid-rich phase) of the diagram with an
Fig. 6. (a – b) SEM images of ETM precipitated by SAA from EtOH at R 0.7 and 1.8.
Fig. 8. ETM volume % cumulative distributions, produced by SAA from EtOH varying R from 0.7 to 1.8.
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increasing content of CO2 in the liquid phase from points A to C. Thus, an increase of R produces an enhancement of the secondary atomization that explains the observed decrease in particle size.
Fig. 9. The hypothesized pressure-composition vapour – liquid equilibria diagram for the system EtOH-CO2 modified by the presence of ETM at 80 jC. Points A, B and C are referred to R values of 0.7, 1.2 and 1.8, respectively.
Fig. 10. (a – b) SEM images of ETM precipitated by SAA from ethanol, concentration of the liquid solution 25 and 55 mg/ml, respectively.
3.2.2. Effect of concentration of the liquid solution Fixing R at 1.2, several experiments were performed at different concentrations of the liquid solution. Two examples of ETM particles obtained at these process conditions are shown in Fig. 10a and b and correspond to tests performed at 25 and 55 mg/ml, respectively. Particle size increases again with concentration. Therefore, these results confirm the trend observed for the SAA of ETM/MeOH and the general trend observed in our previous works [16 – 19]. Comparing PS and PSD of ETM particles obtained using EtOH and MeOH at similar SAA conditions, it was noted that particles obtained from EtOH are slightly smaller.
Fig. 11. (a – b) SEM images of ETM precipitated by SAA from Ac at R 0.98 and 1.6, respectively. Effect of R on the morphology of ETM particles: at R = 0.98 particles are coalescing.
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Head space GC has also been used to measure solvent residues in ETM processed powders. GC-FID head space analyses of SAA processed ETM revealed a MeOH residue of 7 ppb, a EtOH residue of 42 ppb and a Ac residue of 90 ppb. Therefore, in all cases, only parts per billion solvent concentrations were detected that are well below all the solvent residue limits of the European Pharmacopea.
Acknowledgements
Fig. 12. HPLC trace of untreated and SAA processed ETM.
3.3. Erythromycin/acetone 3.3.1. Effect of feed ratio In this case, saturator pressure and temperature were set at 80 bar (max pressure oscillation F 2 bar) and 80 jC (max temperature oscillation F 1 jC). The temperature of the precipitation chamber was maintained at 85 jC. Experiments were performed at R values ranging between 0.55 and 1.6 at a fixed concentration of 22 mg/ml. SAA experiments using Ac produced always coalescing ETM particles except for the experiments performed at R = 1.6, in which spherical non-coalescing particles were obtained. The comparison between the two morphologies is shown in Fig. 11a and b where SEM images of particles produced at R = 0.98 and 1.6 are reported. Due to the difficulties found in SAA processing of ETM with Ac, this experimentation was abandoned. In conclusion the morphology of the particles is the same for MeOH, EtOH and Ac, but for the latter solvent only a value of R was found that allowed successful precipitation. These results confirm that the solvent/ETM system and its interactions with supercritical CO2 in the saturator play a major role in SAA processing; i.e., by changing the solvent, different vapor –liquid equilibria can be obtained that can favour the subsequent atomization.
4. Further characterization of precipitates ETM precipitates were tested by HPLC to assess if any decomposition occurred due to SAA processing. The HPLC method has been discussed in Materials and methods and corresponds to the standard procedure used in the pharmaceutical industry to verify if a drug has been modified as a consequence of intermediate processing. A typical result of HPLC analysis is reported in Fig. 12. The two traces are referred to unprocessed and processed ETM. The characteristic peak of ETM is the same in the two traces, no other peaks, related to decomposition products, are present.
The authors acknowledge MiUR (Italian Ministry for University and Research) for the financial support (PRIN 2000).
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