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Supercritical antisolvent precipitation of salbutamol microparticles
Powder Technology 114 Ž2001. 17–22 www.elsevier.comrlocaterpowtec
Supercritical antisolvent precipitation of salbutamol microparticles E. Reverchon a,) , G. Della Porta a , P. Pallado b a
Dipartimento di Ingegneria Chimica e Alimentare, UniÕersita` di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy b Via M. RaÕel n. 8, 35132 PadoÕa, Italy Received 30 April 1999; received in revised form 29 February 2000; accepted 22 March 2000
Abstract The micronization of salbutamol has been tested by supercritical antisolvent precipitation to assess the possibility of producing microparticles with controlled particle size and distribution. Various liquid solvents have been used to find the solvent–solute couple suitable for optimum processing; supercritical CO 2 has been used as the antisolvent. Salbutamol was tested using dimethylsulphoxide ŽDMSO., methyl alcohol and ethanol–water mixtures. Only when DMSO was used, was obtained a successful micronization. Various morphologies were observed on varying the precipitation pressure. The influence of liquid solution concentration on the dimensions of salbutamol particles was studied. The most interesting morphology consisted of rod-like particles of controlled length between 1 and 3 mm and diameters between 0.2 and 0.35 mm; these particles were obtained operating at 408C and in the pressure range between 100 and 150 bar. q 2001 Elsevier Science S.A. All rights reserved. Keywords: Supercritical antisolvent; Microparticles; Aerosol; Salbutamol
1. Introduction Aerosol drug administration represents a valuable route to deliver many therapeutic agents. It has the advantage of direct delivery in the case of lung diseases and can also be used for systemic drug delivery since it provides close contact with the blood. However, it requires the use of micrometric drug particles with controlled particle size and particle size distribution ŽPSD.. Aerosols with diameters between 1 and 5 mm are deposited primarily in the tracheobronchial and pulmonary regions whereas, aerosols with diameters less than 1 mm are deposited predominantly in the pulmonary region w1x. However, the optimal particle size of drugs to be used by this route of administration is particularly difficult to obtain by traditional micronization techniques. The most commonly used industrial technique to produce aerosol particles is jet milling. It is difficult to obtain the proper particle size, the PSD is very large and electrostatic and hot spot problems have also to be overcome. Moreover, recovery of the micronized matter is around 90%. Supercritical antisolvent processing techniques have been proposed as an alternative to traditional milling tech)
Corresponding author. Tel.: q39-089-964116; fax: q39-089-964057. E-mail address: [email protected] ŽE. Reverchon..
niques. Some different acronyms have been used to describe these processes w2–6x. We have chosen the acronym SAS ŽSupercritical Anti-Solvent. precipitation since in our opinion is the one that better describes the process used. SAS techniques are based on the use of a supercritical antisolvent ŽCO 2 , as a rule. that is added to the liquid solution and induces the fast precipitation of the solute if this is insoluble in the antisolvent. The proper selection of the liquid solvent is very relevant for a successful SAS processing. The use of different supercritical antisolvents has also been proposed w7x. Until now, the SAS process has been proposed for micronization of some pharmaceutical compounds like insulin, lysozyme and trypsin w2,8,9x, salmeterol xinafoate w6,10x, methyl prednisolone and hydrocortisone acetate w11x and sodium cromoglicate w12x. Micronization of biopolymers that can be used as controlled drug delivery system has also been proposed. For example, poly-L-lactic acid ŽPLLA. has been successfully micronized by various authors w3,13,14x. However, not all particles obtained by many of these authors were in the range of maximum efficiency of aerosol delivery. For example, salmeterol xinafoate particles ranged from 1 to 10 mm, sodium cromoglicate particles ranged from 0.1 to 20 microns w6,10,12x. Other examples of pharmaceutical compounds processed by SAS have recently been reviewed by Subra-
0032-5910r01r$ - see front matter q 2001 Elsevier Science S.A. All rights reserved. PII: S 0 0 3 2 - 5 9 1 0 Ž 0 0 . 0 0 2 5 7 - 6
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maniam et al. w15x. SAS processing has also been proposed for many other kind of materials like colouring matter, superconductors, catalysts and inorganic compounds w7x. However, the quantitative analysis of the effect of SAS process parameters has been performed only in a few cases and general rules to obtain a successful micronization and the proper particle size are still missing. Therefore, a case by case study has to be performed to select the liquid solvent, the solute and the process conditions. In this work, we studied the SAS precipitation of salbutamol, which is a b-adrenergic drug used in the treatment of asthma. This drug is commonly delivered as an aerosol to the lung. The production of microparticles and the influence of SAS process parameters on morphology, particle size and PSD have been studied. Various analytical methods have been used before and after processing to assess if drug was modified by SAS.
2. Materials and methods 2.1. Apparatus The apparatus we used is a continuous co-current precipitator in which the supercritical antisolvent and the liquid solution are separately fed to the top of the chamber and continuously discharged from the bottom. Three highpressure piston pumps ŽGilson, mod. 305. with a pulse dampener ŽGilson, mod. 805. are, respectively, used to deliver the liquid solvent, the liquid solution and the supercritical CO 2 . The pump used for the supercritical solvent was modified to avoid cavitation due to the pumping of a compressible fluid by adding a cooling head and using modified inlet and outlet valves, as suggested by the pump producer. The precipitation chamber was a cylindrical vessel of 500 cm3 I.V. The liquid solvent or the solution was delivered into the precipitation chamber through a 60-mm diameter, 800-mm length, stainless steel nozzle. Supercritical CO 2 was delivered by another inlet point located on the top of the chamber. Before entering the precipitator, CO 2 was heated in a tube section by an electric cable ŽWatlow, mod 62H36ASX. connected to temperature controller ŽWatlow, mod. 920.. The precipitation chamber was also electrically heated by thin band heaters ŽWatlow, mod. STB3J2J1. connected to another controller ŽWatlow, mod. 920.. The pressure in the chamber was measured by a test gauge manometer ŽSalmoiraghi, mod. SC-3200.. It was regulated by a micro-metering valve ŽHoke, mod. 1315G4Y. located at the exit Žbottom. of the chamber. This valve was heated by a cable heater ŽWatlow, mod. 62H24ASX. connected to a controller. A metallic frit placed at the bottom of the chamber was used to collect the precipitated particles. A second collection chamber located downstream of the micro-metering valve was used to recover the liquid solvent. The pressure in this chamber
was regulated by a back-pressure valve ŽTescom, mod. 26-1723-44.. At the exit of the second vessel, a rotameter ŽMatheson, mod. 604. and a wet test meter ŽSim-Brunt, mod. AB-1. were used to measure the CO 2 flow rate and the total quantity of the antisolvent used, respectively. Further details on the apparatus were given elsewhere w16,17x. 2.2. Experimental procedures A typical SAS experiment was started by delivering supercritical CO 2 to the precipitation chamber until the desired pressure was reached. Then, pure liquid solvent was sent through the nozzle to the chamber at a flow rate around 1 mlrmin and SC-CO 2 flow was regulated at 20 grmin. This procedure was aimed at obtaining steady state operating conditions during the solute precipitation. Pure solvent was fed to the chamber for 25 min. Under the condition used, this period should theoretically be sufficient to give an approximately 90% approach to the steady state concentration of liquid solvent in the supercritical CO 2 contained in the chamber, considering the contents of this to be perfectly mixed. The second objective of the pure solvent delivery was to avoid the closure of the nozzle due to the precipitation of solute inside it during the start up procedures. At this point, the flow of the liquid solvent was stopped and the liquid solution was delivered through the nozzle at 1 mlrmin flow rate and SC-CO 2 flow was maintained at 20 grmin. During this period, particles were precipitated on the frit located at the bottom of the chamber and to a minor extent on the walls of the chamber itself. A typical duration of this stage of the process was about 20 min or higher, to allow the collection of solid in a quantity sufficient to perform the analysis of precipitate. The experiment ended when the delivery of the liquid solution to the chamber was interrupted. However, supercritical CO 2 continued to flow for further 90 min to wash the chamber of the residual content of liquid solvent solubilized into the supercritical antisolvent. The washing time of 90 min was calculated as that required to remove 98% of the liquid solvent from the chamber, assuming the contents of this to be perfectly mixed. If the final purge with pure CO 2 is not done, the liquid solvent condenses in droplets during the chamber depressurization and partly solubilizes the powder on the frit modifying its morphology. When the washing process was completed, the CO 2 flow was stopped and the chamber was depressurized down to atmospheric pressure. All the experiments performed in this work were performed at a fixed supercritical CO 2rliquid solution ratio s 20:1 on a mass basis. Liquid solution flow rate was fixed at 1 mlrmin and the precipitator temperature at 408C. The other process parameters, i.e., pressure, solute concentration in the liquid solvent and liquid solvent were varied, one at a time.
E. ReÕerchon et al.r Powder Technology 114 (2001) 17–22
2.3. Materials Dimethylsulphoxide ŽDMSO. Žpurity 99.5%. N-methyl 2-pyrrolidone ŽNMP. Žpurity 99%., methyl alcohol ŽMeOH. Žpurity 99.9%., ethyl-alcohol Ž99.9%. and bi-distilled water were bought from Carlo Erba Reagenti ŽMilan, Italy.. Salbutamol sulphate was supplied by Fidia pharmaceuticals ŽItaly., CO 2 Žpurity 99.9%. was supplied by Societa` Ossigeno Napoli ŽNaples, Italy.. The approximate solubilities of salbutamol in the various solvents tested were: 15 mgrml in DMSO; 12 mgrml in MeOH; 55 mgrml in water, respectively.
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droplet explodes generating particles in the nano- or micron-range. Experimental evidence supporting this explanation of the process has been published recently and SEM images showing largely expanded droplets have been obtained for some compounds w16,17x. The salbutamol received consisted of irregularly shaped particles ranging from about 0.2 to about 60 mm. When we
2.4. Scanning electron microscopy (SEM) Samples of the precipitated powder were observed by a LEO 420 SEM. The samples were fixed by mutual conductive adhesive tape on aluminium stubs and covered with gold-palladium using a sputter coater ŽAgar mod. 108A.. Particle size and PSD were calculated using an image analysis software ŽSigma Scan Pro, Jandel Scientific.. More than 500 particles were considered in each PSD measurement. 2.5. X-ray diffractometry (XRD) X-ray diffraction pattern analysis was performed with a Philips PW 1050 XRD apparatus to ascertain if changes occurred in the physical characteristics of the precipitated powder as a consequence of the SAS process. 2.6. Fourier transform infrared spectrophotometry (FT-IR) FT-IR spectra were recorded with a Bruker IFS66 spectrophotometer in the range 400–4000 cmy1 , using a resolution of 2 cmy1 and 32 scans. Samples were diluted with KBr mixing powder at 1%, dried at 1208C for 30 min and pressed to obtain self-supporting disks.
3. Results and discussion The success of SAS micronization process depends, as in liquid antisolvent precipitation, on the solubility of the liquid solvent in the supercritical antisolvent and on the fact that the solute is not soluble in the antisolvent. However, it also depends on the fast solubilization of the liquid due to the gas-like diffusion characteristic of a supercritical fluids. This last characteristic is fundamental in assuring that very small particles are obtained. A possible explanation of the various stages of the continuous SAS micronization process is: liquid droplets formed at the injector are subject to a very fast expansion by supercritical antisolvent diffusion and when saturation conditions are reached on the droplet surface, an outer AskinB of solute is formed thus creating a balloon-like structure. At the end of the expansion process, the dried
Fig. 1. SEM images of salbutamol balloons precipitated by SAS from DMSO at 95 bar, 408C, 5 mgrml. Ža. Landscape view. Žb. Detailed view of each balloon. They have a diameter of some microns. Žc. Some broken balloons are present.
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tested the powders produced from salbutamolrDMSO solutions, we had the immediate macroscopic evidence that SAS was successful; indeed, the precipitated material was uniformly distributed at the bottom and on the walls of the precipitation vessel. The volume occupied by the processed drug was very large when compared to that of the unprocessed material; i.e., it was largely expanded as the result of the micronization process and of the slow deposition Ždue to the decrease of the apparent density.. We observed very similar macroscopic results every time we performed a successful SAS precipitation w16–18x. Different morphologies were observed as the precipitation pressure was increased from 95 to 150 bar. At 95 bar, we obtained salbutamol as expanded droplets Žballoons. with a diameter of some microns. Examples of this morphology are shown in Figs. 1a, b and c where SEM images are reported. Fig. 1a shows a landscape view of salbutamol balloons, whereas, Fig. 1b shows the details of some particles. Fig. 1c shows some broken balloons demonstrating that they consist of hollow spheres of dried solute and supporting the hypothesis that they have been generated by the rapid Žbut incomplete. expansion of the liquid droplets. The volumetric expansion of the liquid droplets is a measure of the solubility of the liquid solvent in the supercritical CO 2 and it is the main factor that determines the precipitation of the solute since the solvent strength decreases. Its velocity is important in determining the formation of microparticles. A similar morphology was observed at the same SAS conditions, using the same liquid solvent but with superconductor and catalyst precursor compounds w16–19x. The only difference with the previously studied compounds is that, in those experiments, balloons were formed by slightly connected spherical nanoparticles, whereas salbutamolrDMSO balloons are formed by rodlike microparticles and show the tendency to connect together. SAS processing in the range of 110–150 bar produced rod-like particles as the ones reported in Fig. 2 where a
Fig. 2. SEM image of rod-like salbutamol particles precipitated by SAS from DMSO at 150 bar, 408C, 5 mgrml.
Fig. 3. SEM image of star-like salbutamol particles precipitated by SAS from DMSO at 150 bar, 408C, 10 mgrml. At higher concentrations, salbutamol particles tend to form more structured morphologies.
SEM image of salbutamol particles obtained operating at 150 bar, 5 mgrml DMSO is reported. We also observed that rod-like particles tend to organize themselves in a more structured morphology when the concentration of the liquid solution is increased up to values near saturation. In Fig. 3, an example of particles organized in a star-like structure is reported. This result has been obtained operating at the same conditions as in Fig. 2 but at a concentration of 10 mgrml salbutamolrDMSO. From the comparison of Figs. 1 and 2, it seems evident that rod-like particles are the result of the final explosion of the expanded droplets, in accordance to the general explanation of the SAS process proposed at the beginning of this chapter. Another main parameter controlling particle size and distribution was shown to be the concentration of the liquid solution w7x. Therefore, we explored various concentrations of salbutamolrDMSO solutions operating at 150 bar. We performed the analysis of SEM images to obtain the mean particle size and the standard deviation at the various conditions studied. An example of mean particle size and PSD is shown in Fig. 4 for a concentration of 5
Fig. 4. An example of length size distribution of salbutamol particles produced by SAS from DMSO at 150 bar, 408C, 5 mgrml. Mean particle sizes 2.03 mm, standard deviations 0.75 mm.
E. ReÕerchon et al.r Powder Technology 114 (2001) 17–22
Fig. 5. Salbutamol particles length against liquid solution concentration at 150 bar, 408C. The measured standard deviation of the PSD is also reported as vertical bars.
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Fig. 7. Comparison of salbutamol XRD traces before and after SAS processing. Crystalline structure is partly retained because the main peaks are still present in the processed salbutamol trace.
concentration of 10 mgrml in DMSO. The following equation applies for Dae calculation w20x: mgrml salbutamolrDMSO. The length of rods has been used as the characteristic dimension for salbutamol particles. Fig. 4 shows that a very narrow PSD has been obtained. The results obtained at the various concentrations explored have been summarized in Fig. 5 where a diagram of the rod length against the concentration of the liquid mixture is reported. The measured standard deviation of the PSD is also reported in Fig. 5, as vertical bars. The mean length size ranges from about 1 to 3 mm. PSD broadens with rod length. The powders did not show any particular tendency to form aggregates. If aerosol delivery has to be used for such powders, the rod thickness has to be taken into account to calculate the aerodynamic diameter of the powder. Aerodynamic diameter Ž Dae . is defined as the diameter of a sphere of unit density that has the same terminal sedimentation velocity as the particle in question. For very elongated particles, aerodynamic diameter shows a relatively small dependence on the length but is strongly correlated to the short dimension of the particles. In our experiments, we obtained mean rod thicknesses ranging from 0.35 mm at solution concentration of 1 mgrml in DMSO to 0.20 mm at solution
Fig. 6. Comparison of salbutamol FT-IR spectra before and after SAS processing.
Dae s D Ž drd 0 .
1r2
where D is the actual diameter of the sphere, d is the actual density of salbutamol particles and d 0 is the unit density Žgrcm3 .. A problem with this approach is that Dae does not provide a unique correspondence to rate of deposition. Indeed, the impact for particles with the same Dae depends on the flow rate. We measured d for SAS processed salbutamol particles obtaining d s 1.26 grcm3 and calculated D in two different manners: through the diameter of the sphere having the same volume of the particle and through an empirical rule given by Gonda and Hickey w20x, which proposes that for very elongated particles Dae could be two to three times the short dimension of the particle. Dae calculated with the two different procedures ranged between 1.0 and 1.2 mm; therefore, they are within the range of dimensions suitable for aerosol delivery. Traditionally, non-spherical particles have been avoided for potential problems of powder flow. However, some work performed on such particles suggest that the res-
Fig. 8. SEM image of salbutamol precipitated by SAS from MeOH operating at 150 bar, 408C, 3 mgrml. Microparticles are tightly connected.
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ture due to the partial re-solubilization Žsintering. in the mixture inside the precipitator. We also used 90:10 Žvolume ratio. ethanol–water mixtures at the previously indicated reference conditions. In that case, salbutamol was recovered in the precipitator as a tightly adhered powder on the metallic frit and with a morphology resembling one of the untreated drugs ŽFig. 9.. In this case, we think that ethanol worked as a second Žliquid. antisolvent. Acknowledgements
Fig. 9. SEM image of salbutamol precipitated by SAS from a 90:10 ethyl-alcoholrwater mixture operating at 145 bar, 508C, 10 mgrml concentration in water at the starting conditions. Very large particles are produced.
pirable fraction of a drug may not depend very much on particle shape w21x. Moreover, if the growth takes place preferentially in the longitudinal direction, drug particles can have similar thickness Žas in our case. and a powder with a narrower PSD will be obtained. Therefore, the preparation of salbutamol powders with elongated particles may provide a means to achieve a very selective delivery. However, the possibility of using the SAS produced salbutamol for aerosol delivery requires further studies and clinical tests. The problem of solvent residues is also of interest when pharmaceutical products are considered. Ruchatz et al. w22x, by using the head space gas-chromatography, found solvent residues between 70 and 470 ppm working on biopolymers treated by SAS. We performed some analysis on salbutamol samples before and after processing. FT-IR spectra of rough and SAS processed salbutamol are compared in Fig. 6. Solvent residues were not detectable, i.e., DMSO characteristic FT-IR trace is not detectable, within the limits of this technique and no detectable chemical modifications appeared. XRD spectra were also produced as shown in Fig. 7. Salbutamol partly maintained a crystalline structure; indeed, the main peaks of the XRD spectrum are still present in the SAS processed salbutamol. However, in previous works w2,16–19x, spherical amorphous particles were commonly obtained. This evidence confirms that the formation of rod-like particles depends on the activation of a further growth mechanism in the supercritical mixture inside the precipitator. We also tested other salbutamol–liquid solvent mixtures to assess if other morphologies could be obtained by changing the liquid solvent. We tested salbutamol–MeOH mixtures in supercritical CO 2 obtaining a powder formed by tightly networked particles. An example of this powder is reported in Fig. 8. In this case, probably, salbutamol microparticles were formed in the first stages of the precipitation process, then, they collapsed in a network struc-
The authors acknowledge Dr. Maria Grazia Falivene for her help in performing the SAS experiments. The work was partly financed by Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica ŽMurst. and Consiglio Nazionale delle Ricerche ŽCNR., Rome, Italy. References w1x A.J. Hickey, Pharmaceutical Inhalation Technology, Marcel Dekker, New York, 1992, p. 24. w2x S.D. Yeo, G.B. Lim, P.G. Debenedetti, H. Bernstein, Biotechnol. Bioeng. 41 Ž1993. 341. w3x J. Bleich, B.W. Muller, W. Waßmus, Int. J. Pharm. 97 Ž1993., 111. w4x P.M. Gallagher, M.P. Coffey, V.J. Krukonis, N. Klasutis, Supercritical fluids science and technology, ACS Symp. 406 Ž1989., 334, Series. w5x D.J. Dixon, K.P. Johnston, R.A. Bodmeier, AIChE J. 39 Ž1993. 127. w6x P. York, M.H. Hanna, Int. Patent, C07C215r60, Ž1994.. w7x E. Reverchon, J. Supercrit. Fluids 15 Ž1999. 1. w8x S.D. Yeo, P.G. Debenedetti, S.Y. Patro, T.M. Przybycien, J. Pharm. Sci. 83 Ž1994. 1651. w9x M.A. Winters, B.L. Knutson, P.G. Debenedetti, H.G. Sparks, T.M. Przybycien, J. Pharm. Sci. 85 Ž1996. 586. w10x M.H. Hanna, P. York, D. Rudd, S. Beach, Pharm. Res. 12 Ž1995. S141. w11x W.J. Schimtt, M.C. Salada, G.G. Shook, S.M. Speaker III, AIChE J. 41 Ž1995. 2476. w12x S. Jaarmo, M. Rantakyla, O. Aaltonen, in: K. Arai ŽEd.., Proceedings of the 4th Internat. Symp. on Supercritical Fluids, 1997, pp. 263–267, Sendai, ŽJapan., 11–14 May. w13x J. Bleich, P. Kleinebudde, B.W. Muller, Int. J. Pharm. 106 Ž1994. 77. w14x T.W. Randolph, A.D. Randolph, M. Mebes, S. Yeung, Biotechnol. Prog. 9 Ž1993. 429. w15x B. Subramaniam, R.A. Rajewski, K. Snavel, J. Pharm. Sci. 86 Ž1997. 885. w16x E. Reverchon, S. Della Porta, G. Pace, A Di Trolio, Ind. Eng. Chem. Res. 37 Ž1998., 952. w17x E. Reverchon, G. Della Porta, C. Celano, S. Pace, A. Di Trolio, Mater. Res. 13 Ž2. Ž1998. 284. w18x E. Reverchon, G. Della Porta, D. Sannino, L. Lisi, P. Ciambelli, in: Delmon ŽEd.., 7th Internat. Symp. On Scientific Bases for the Preparation of Heterogeneous Catalysts, Elsevier, 1998, p. 349. w19x E. Reverchon, G. Della Porta, D. Sannino, P. Ciambelli et al., Powder Technol. 102 Ž2. Ž1999. 129. w20x I. Gonda, in: J. Hickey ŽEd.., Pharmaceutical Inhalation Technology, Marcel Dekker, New York, 1992, p. 64. w21x L.W. Wong, N.M. Kassem, D. Ganderton, J. Pharm. Pharmacol. 41 Ž1989. suppl. 24P. w22x F. Ruchatz, P. Kleinbudde, B.W. Muller, J. Pharm. Sci. 86 Ž1. Ž1997. 101.
Supercritical antisolvent precipitation of salbutamol microparticles E. Reverchon a,) , G. Della Porta a , P. Pallado b a
Dipartimento di Ingegneria Chimica e Alimentare, UniÕersita` di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy b Via M. RaÕel n. 8, 35132 PadoÕa, Italy Received 30 April 1999; received in revised form 29 February 2000; accepted 22 March 2000
Abstract The micronization of salbutamol has been tested by supercritical antisolvent precipitation to assess the possibility of producing microparticles with controlled particle size and distribution. Various liquid solvents have been used to find the solvent–solute couple suitable for optimum processing; supercritical CO 2 has been used as the antisolvent. Salbutamol was tested using dimethylsulphoxide ŽDMSO., methyl alcohol and ethanol–water mixtures. Only when DMSO was used, was obtained a successful micronization. Various morphologies were observed on varying the precipitation pressure. The influence of liquid solution concentration on the dimensions of salbutamol particles was studied. The most interesting morphology consisted of rod-like particles of controlled length between 1 and 3 mm and diameters between 0.2 and 0.35 mm; these particles were obtained operating at 408C and in the pressure range between 100 and 150 bar. q 2001 Elsevier Science S.A. All rights reserved. Keywords: Supercritical antisolvent; Microparticles; Aerosol; Salbutamol
1. Introduction Aerosol drug administration represents a valuable route to deliver many therapeutic agents. It has the advantage of direct delivery in the case of lung diseases and can also be used for systemic drug delivery since it provides close contact with the blood. However, it requires the use of micrometric drug particles with controlled particle size and particle size distribution ŽPSD.. Aerosols with diameters between 1 and 5 mm are deposited primarily in the tracheobronchial and pulmonary regions whereas, aerosols with diameters less than 1 mm are deposited predominantly in the pulmonary region w1x. However, the optimal particle size of drugs to be used by this route of administration is particularly difficult to obtain by traditional micronization techniques. The most commonly used industrial technique to produce aerosol particles is jet milling. It is difficult to obtain the proper particle size, the PSD is very large and electrostatic and hot spot problems have also to be overcome. Moreover, recovery of the micronized matter is around 90%. Supercritical antisolvent processing techniques have been proposed as an alternative to traditional milling tech)
Corresponding author. Tel.: q39-089-964116; fax: q39-089-964057. E-mail address: [email protected] ŽE. Reverchon..
niques. Some different acronyms have been used to describe these processes w2–6x. We have chosen the acronym SAS ŽSupercritical Anti-Solvent. precipitation since in our opinion is the one that better describes the process used. SAS techniques are based on the use of a supercritical antisolvent ŽCO 2 , as a rule. that is added to the liquid solution and induces the fast precipitation of the solute if this is insoluble in the antisolvent. The proper selection of the liquid solvent is very relevant for a successful SAS processing. The use of different supercritical antisolvents has also been proposed w7x. Until now, the SAS process has been proposed for micronization of some pharmaceutical compounds like insulin, lysozyme and trypsin w2,8,9x, salmeterol xinafoate w6,10x, methyl prednisolone and hydrocortisone acetate w11x and sodium cromoglicate w12x. Micronization of biopolymers that can be used as controlled drug delivery system has also been proposed. For example, poly-L-lactic acid ŽPLLA. has been successfully micronized by various authors w3,13,14x. However, not all particles obtained by many of these authors were in the range of maximum efficiency of aerosol delivery. For example, salmeterol xinafoate particles ranged from 1 to 10 mm, sodium cromoglicate particles ranged from 0.1 to 20 microns w6,10,12x. Other examples of pharmaceutical compounds processed by SAS have recently been reviewed by Subra-
0032-5910r01r$ - see front matter q 2001 Elsevier Science S.A. All rights reserved. PII: S 0 0 3 2 - 5 9 1 0 Ž 0 0 . 0 0 2 5 7 - 6
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E. ReÕerchon et al.r Powder Technology 114 (2001) 17–22
maniam et al. w15x. SAS processing has also been proposed for many other kind of materials like colouring matter, superconductors, catalysts and inorganic compounds w7x. However, the quantitative analysis of the effect of SAS process parameters has been performed only in a few cases and general rules to obtain a successful micronization and the proper particle size are still missing. Therefore, a case by case study has to be performed to select the liquid solvent, the solute and the process conditions. In this work, we studied the SAS precipitation of salbutamol, which is a b-adrenergic drug used in the treatment of asthma. This drug is commonly delivered as an aerosol to the lung. The production of microparticles and the influence of SAS process parameters on morphology, particle size and PSD have been studied. Various analytical methods have been used before and after processing to assess if drug was modified by SAS.
2. Materials and methods 2.1. Apparatus The apparatus we used is a continuous co-current precipitator in which the supercritical antisolvent and the liquid solution are separately fed to the top of the chamber and continuously discharged from the bottom. Three highpressure piston pumps ŽGilson, mod. 305. with a pulse dampener ŽGilson, mod. 805. are, respectively, used to deliver the liquid solvent, the liquid solution and the supercritical CO 2 . The pump used for the supercritical solvent was modified to avoid cavitation due to the pumping of a compressible fluid by adding a cooling head and using modified inlet and outlet valves, as suggested by the pump producer. The precipitation chamber was a cylindrical vessel of 500 cm3 I.V. The liquid solvent or the solution was delivered into the precipitation chamber through a 60-mm diameter, 800-mm length, stainless steel nozzle. Supercritical CO 2 was delivered by another inlet point located on the top of the chamber. Before entering the precipitator, CO 2 was heated in a tube section by an electric cable ŽWatlow, mod 62H36ASX. connected to temperature controller ŽWatlow, mod. 920.. The precipitation chamber was also electrically heated by thin band heaters ŽWatlow, mod. STB3J2J1. connected to another controller ŽWatlow, mod. 920.. The pressure in the chamber was measured by a test gauge manometer ŽSalmoiraghi, mod. SC-3200.. It was regulated by a micro-metering valve ŽHoke, mod. 1315G4Y. located at the exit Žbottom. of the chamber. This valve was heated by a cable heater ŽWatlow, mod. 62H24ASX. connected to a controller. A metallic frit placed at the bottom of the chamber was used to collect the precipitated particles. A second collection chamber located downstream of the micro-metering valve was used to recover the liquid solvent. The pressure in this chamber
was regulated by a back-pressure valve ŽTescom, mod. 26-1723-44.. At the exit of the second vessel, a rotameter ŽMatheson, mod. 604. and a wet test meter ŽSim-Brunt, mod. AB-1. were used to measure the CO 2 flow rate and the total quantity of the antisolvent used, respectively. Further details on the apparatus were given elsewhere w16,17x. 2.2. Experimental procedures A typical SAS experiment was started by delivering supercritical CO 2 to the precipitation chamber until the desired pressure was reached. Then, pure liquid solvent was sent through the nozzle to the chamber at a flow rate around 1 mlrmin and SC-CO 2 flow was regulated at 20 grmin. This procedure was aimed at obtaining steady state operating conditions during the solute precipitation. Pure solvent was fed to the chamber for 25 min. Under the condition used, this period should theoretically be sufficient to give an approximately 90% approach to the steady state concentration of liquid solvent in the supercritical CO 2 contained in the chamber, considering the contents of this to be perfectly mixed. The second objective of the pure solvent delivery was to avoid the closure of the nozzle due to the precipitation of solute inside it during the start up procedures. At this point, the flow of the liquid solvent was stopped and the liquid solution was delivered through the nozzle at 1 mlrmin flow rate and SC-CO 2 flow was maintained at 20 grmin. During this period, particles were precipitated on the frit located at the bottom of the chamber and to a minor extent on the walls of the chamber itself. A typical duration of this stage of the process was about 20 min or higher, to allow the collection of solid in a quantity sufficient to perform the analysis of precipitate. The experiment ended when the delivery of the liquid solution to the chamber was interrupted. However, supercritical CO 2 continued to flow for further 90 min to wash the chamber of the residual content of liquid solvent solubilized into the supercritical antisolvent. The washing time of 90 min was calculated as that required to remove 98% of the liquid solvent from the chamber, assuming the contents of this to be perfectly mixed. If the final purge with pure CO 2 is not done, the liquid solvent condenses in droplets during the chamber depressurization and partly solubilizes the powder on the frit modifying its morphology. When the washing process was completed, the CO 2 flow was stopped and the chamber was depressurized down to atmospheric pressure. All the experiments performed in this work were performed at a fixed supercritical CO 2rliquid solution ratio s 20:1 on a mass basis. Liquid solution flow rate was fixed at 1 mlrmin and the precipitator temperature at 408C. The other process parameters, i.e., pressure, solute concentration in the liquid solvent and liquid solvent were varied, one at a time.
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2.3. Materials Dimethylsulphoxide ŽDMSO. Žpurity 99.5%. N-methyl 2-pyrrolidone ŽNMP. Žpurity 99%., methyl alcohol ŽMeOH. Žpurity 99.9%., ethyl-alcohol Ž99.9%. and bi-distilled water were bought from Carlo Erba Reagenti ŽMilan, Italy.. Salbutamol sulphate was supplied by Fidia pharmaceuticals ŽItaly., CO 2 Žpurity 99.9%. was supplied by Societa` Ossigeno Napoli ŽNaples, Italy.. The approximate solubilities of salbutamol in the various solvents tested were: 15 mgrml in DMSO; 12 mgrml in MeOH; 55 mgrml in water, respectively.
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droplet explodes generating particles in the nano- or micron-range. Experimental evidence supporting this explanation of the process has been published recently and SEM images showing largely expanded droplets have been obtained for some compounds w16,17x. The salbutamol received consisted of irregularly shaped particles ranging from about 0.2 to about 60 mm. When we
2.4. Scanning electron microscopy (SEM) Samples of the precipitated powder were observed by a LEO 420 SEM. The samples were fixed by mutual conductive adhesive tape on aluminium stubs and covered with gold-palladium using a sputter coater ŽAgar mod. 108A.. Particle size and PSD were calculated using an image analysis software ŽSigma Scan Pro, Jandel Scientific.. More than 500 particles were considered in each PSD measurement. 2.5. X-ray diffractometry (XRD) X-ray diffraction pattern analysis was performed with a Philips PW 1050 XRD apparatus to ascertain if changes occurred in the physical characteristics of the precipitated powder as a consequence of the SAS process. 2.6. Fourier transform infrared spectrophotometry (FT-IR) FT-IR spectra were recorded with a Bruker IFS66 spectrophotometer in the range 400–4000 cmy1 , using a resolution of 2 cmy1 and 32 scans. Samples were diluted with KBr mixing powder at 1%, dried at 1208C for 30 min and pressed to obtain self-supporting disks.
3. Results and discussion The success of SAS micronization process depends, as in liquid antisolvent precipitation, on the solubility of the liquid solvent in the supercritical antisolvent and on the fact that the solute is not soluble in the antisolvent. However, it also depends on the fast solubilization of the liquid due to the gas-like diffusion characteristic of a supercritical fluids. This last characteristic is fundamental in assuring that very small particles are obtained. A possible explanation of the various stages of the continuous SAS micronization process is: liquid droplets formed at the injector are subject to a very fast expansion by supercritical antisolvent diffusion and when saturation conditions are reached on the droplet surface, an outer AskinB of solute is formed thus creating a balloon-like structure. At the end of the expansion process, the dried
Fig. 1. SEM images of salbutamol balloons precipitated by SAS from DMSO at 95 bar, 408C, 5 mgrml. Ža. Landscape view. Žb. Detailed view of each balloon. They have a diameter of some microns. Žc. Some broken balloons are present.
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tested the powders produced from salbutamolrDMSO solutions, we had the immediate macroscopic evidence that SAS was successful; indeed, the precipitated material was uniformly distributed at the bottom and on the walls of the precipitation vessel. The volume occupied by the processed drug was very large when compared to that of the unprocessed material; i.e., it was largely expanded as the result of the micronization process and of the slow deposition Ždue to the decrease of the apparent density.. We observed very similar macroscopic results every time we performed a successful SAS precipitation w16–18x. Different morphologies were observed as the precipitation pressure was increased from 95 to 150 bar. At 95 bar, we obtained salbutamol as expanded droplets Žballoons. with a diameter of some microns. Examples of this morphology are shown in Figs. 1a, b and c where SEM images are reported. Fig. 1a shows a landscape view of salbutamol balloons, whereas, Fig. 1b shows the details of some particles. Fig. 1c shows some broken balloons demonstrating that they consist of hollow spheres of dried solute and supporting the hypothesis that they have been generated by the rapid Žbut incomplete. expansion of the liquid droplets. The volumetric expansion of the liquid droplets is a measure of the solubility of the liquid solvent in the supercritical CO 2 and it is the main factor that determines the precipitation of the solute since the solvent strength decreases. Its velocity is important in determining the formation of microparticles. A similar morphology was observed at the same SAS conditions, using the same liquid solvent but with superconductor and catalyst precursor compounds w16–19x. The only difference with the previously studied compounds is that, in those experiments, balloons were formed by slightly connected spherical nanoparticles, whereas salbutamolrDMSO balloons are formed by rodlike microparticles and show the tendency to connect together. SAS processing in the range of 110–150 bar produced rod-like particles as the ones reported in Fig. 2 where a
Fig. 2. SEM image of rod-like salbutamol particles precipitated by SAS from DMSO at 150 bar, 408C, 5 mgrml.
Fig. 3. SEM image of star-like salbutamol particles precipitated by SAS from DMSO at 150 bar, 408C, 10 mgrml. At higher concentrations, salbutamol particles tend to form more structured morphologies.
SEM image of salbutamol particles obtained operating at 150 bar, 5 mgrml DMSO is reported. We also observed that rod-like particles tend to organize themselves in a more structured morphology when the concentration of the liquid solution is increased up to values near saturation. In Fig. 3, an example of particles organized in a star-like structure is reported. This result has been obtained operating at the same conditions as in Fig. 2 but at a concentration of 10 mgrml salbutamolrDMSO. From the comparison of Figs. 1 and 2, it seems evident that rod-like particles are the result of the final explosion of the expanded droplets, in accordance to the general explanation of the SAS process proposed at the beginning of this chapter. Another main parameter controlling particle size and distribution was shown to be the concentration of the liquid solution w7x. Therefore, we explored various concentrations of salbutamolrDMSO solutions operating at 150 bar. We performed the analysis of SEM images to obtain the mean particle size and the standard deviation at the various conditions studied. An example of mean particle size and PSD is shown in Fig. 4 for a concentration of 5
Fig. 4. An example of length size distribution of salbutamol particles produced by SAS from DMSO at 150 bar, 408C, 5 mgrml. Mean particle sizes 2.03 mm, standard deviations 0.75 mm.
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Fig. 5. Salbutamol particles length against liquid solution concentration at 150 bar, 408C. The measured standard deviation of the PSD is also reported as vertical bars.
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Fig. 7. Comparison of salbutamol XRD traces before and after SAS processing. Crystalline structure is partly retained because the main peaks are still present in the processed salbutamol trace.
concentration of 10 mgrml in DMSO. The following equation applies for Dae calculation w20x: mgrml salbutamolrDMSO. The length of rods has been used as the characteristic dimension for salbutamol particles. Fig. 4 shows that a very narrow PSD has been obtained. The results obtained at the various concentrations explored have been summarized in Fig. 5 where a diagram of the rod length against the concentration of the liquid mixture is reported. The measured standard deviation of the PSD is also reported in Fig. 5, as vertical bars. The mean length size ranges from about 1 to 3 mm. PSD broadens with rod length. The powders did not show any particular tendency to form aggregates. If aerosol delivery has to be used for such powders, the rod thickness has to be taken into account to calculate the aerodynamic diameter of the powder. Aerodynamic diameter Ž Dae . is defined as the diameter of a sphere of unit density that has the same terminal sedimentation velocity as the particle in question. For very elongated particles, aerodynamic diameter shows a relatively small dependence on the length but is strongly correlated to the short dimension of the particles. In our experiments, we obtained mean rod thicknesses ranging from 0.35 mm at solution concentration of 1 mgrml in DMSO to 0.20 mm at solution
Fig. 6. Comparison of salbutamol FT-IR spectra before and after SAS processing.
Dae s D Ž drd 0 .
1r2
where D is the actual diameter of the sphere, d is the actual density of salbutamol particles and d 0 is the unit density Žgrcm3 .. A problem with this approach is that Dae does not provide a unique correspondence to rate of deposition. Indeed, the impact for particles with the same Dae depends on the flow rate. We measured d for SAS processed salbutamol particles obtaining d s 1.26 grcm3 and calculated D in two different manners: through the diameter of the sphere having the same volume of the particle and through an empirical rule given by Gonda and Hickey w20x, which proposes that for very elongated particles Dae could be two to three times the short dimension of the particle. Dae calculated with the two different procedures ranged between 1.0 and 1.2 mm; therefore, they are within the range of dimensions suitable for aerosol delivery. Traditionally, non-spherical particles have been avoided for potential problems of powder flow. However, some work performed on such particles suggest that the res-
Fig. 8. SEM image of salbutamol precipitated by SAS from MeOH operating at 150 bar, 408C, 3 mgrml. Microparticles are tightly connected.
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ture due to the partial re-solubilization Žsintering. in the mixture inside the precipitator. We also used 90:10 Žvolume ratio. ethanol–water mixtures at the previously indicated reference conditions. In that case, salbutamol was recovered in the precipitator as a tightly adhered powder on the metallic frit and with a morphology resembling one of the untreated drugs ŽFig. 9.. In this case, we think that ethanol worked as a second Žliquid. antisolvent. Acknowledgements
Fig. 9. SEM image of salbutamol precipitated by SAS from a 90:10 ethyl-alcoholrwater mixture operating at 145 bar, 508C, 10 mgrml concentration in water at the starting conditions. Very large particles are produced.
pirable fraction of a drug may not depend very much on particle shape w21x. Moreover, if the growth takes place preferentially in the longitudinal direction, drug particles can have similar thickness Žas in our case. and a powder with a narrower PSD will be obtained. Therefore, the preparation of salbutamol powders with elongated particles may provide a means to achieve a very selective delivery. However, the possibility of using the SAS produced salbutamol for aerosol delivery requires further studies and clinical tests. The problem of solvent residues is also of interest when pharmaceutical products are considered. Ruchatz et al. w22x, by using the head space gas-chromatography, found solvent residues between 70 and 470 ppm working on biopolymers treated by SAS. We performed some analysis on salbutamol samples before and after processing. FT-IR spectra of rough and SAS processed salbutamol are compared in Fig. 6. Solvent residues were not detectable, i.e., DMSO characteristic FT-IR trace is not detectable, within the limits of this technique and no detectable chemical modifications appeared. XRD spectra were also produced as shown in Fig. 7. Salbutamol partly maintained a crystalline structure; indeed, the main peaks of the XRD spectrum are still present in the SAS processed salbutamol. However, in previous works w2,16–19x, spherical amorphous particles were commonly obtained. This evidence confirms that the formation of rod-like particles depends on the activation of a further growth mechanism in the supercritical mixture inside the precipitator. We also tested other salbutamol–liquid solvent mixtures to assess if other morphologies could be obtained by changing the liquid solvent. We tested salbutamol–MeOH mixtures in supercritical CO 2 obtaining a powder formed by tightly networked particles. An example of this powder is reported in Fig. 8. In this case, probably, salbutamol microparticles were formed in the first stages of the precipitation process, then, they collapsed in a network struc-
The authors acknowledge Dr. Maria Grazia Falivene for her help in performing the SAS experiments. The work was partly financed by Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica ŽMurst. and Consiglio Nazionale delle Ricerche ŽCNR., Rome, Italy. References w1x A.J. Hickey, Pharmaceutical Inhalation Technology, Marcel Dekker, New York, 1992, p. 24. w2x S.D. Yeo, G.B. Lim, P.G. Debenedetti, H. Bernstein, Biotechnol. Bioeng. 41 Ž1993. 341. w3x J. Bleich, B.W. Muller, W. Waßmus, Int. J. Pharm. 97 Ž1993., 111. w4x P.M. Gallagher, M.P. Coffey, V.J. Krukonis, N. Klasutis, Supercritical fluids science and technology, ACS Symp. 406 Ž1989., 334, Series. w5x D.J. Dixon, K.P. Johnston, R.A. Bodmeier, AIChE J. 39 Ž1993. 127. w6x P. York, M.H. Hanna, Int. Patent, C07C215r60, Ž1994.. w7x E. Reverchon, J. Supercrit. Fluids 15 Ž1999. 1. w8x S.D. Yeo, P.G. Debenedetti, S.Y. Patro, T.M. Przybycien, J. Pharm. Sci. 83 Ž1994. 1651. w9x M.A. Winters, B.L. Knutson, P.G. Debenedetti, H.G. Sparks, T.M. Przybycien, J. Pharm. Sci. 85 Ž1996. 586. w10x M.H. Hanna, P. York, D. Rudd, S. Beach, Pharm. Res. 12 Ž1995. S141. w11x W.J. Schimtt, M.C. Salada, G.G. Shook, S.M. Speaker III, AIChE J. 41 Ž1995. 2476. w12x S. Jaarmo, M. Rantakyla, O. Aaltonen, in: K. Arai ŽEd.., Proceedings of the 4th Internat. Symp. on Supercritical Fluids, 1997, pp. 263–267, Sendai, ŽJapan., 11–14 May. w13x J. Bleich, P. Kleinebudde, B.W. Muller, Int. J. Pharm. 106 Ž1994. 77. w14x T.W. Randolph, A.D. Randolph, M. Mebes, S. Yeung, Biotechnol. Prog. 9 Ž1993. 429. w15x B. Subramaniam, R.A. Rajewski, K. Snavel, J. Pharm. Sci. 86 Ž1997. 885. w16x E. Reverchon, S. Della Porta, G. Pace, A Di Trolio, Ind. Eng. Chem. Res. 37 Ž1998., 952. w17x E. Reverchon, G. Della Porta, C. Celano, S. Pace, A. Di Trolio, Mater. Res. 13 Ž2. Ž1998. 284. w18x E. Reverchon, G. Della Porta, D. Sannino, L. Lisi, P. Ciambelli, in: Delmon ŽEd.., 7th Internat. Symp. On Scientific Bases for the Preparation of Heterogeneous Catalysts, Elsevier, 1998, p. 349. w19x E. Reverchon, G. Della Porta, D. Sannino, P. Ciambelli et al., Powder Technol. 102 Ž2. Ž1999. 129. w20x I. Gonda, in: J. Hickey ŽEd.., Pharmaceutical Inhalation Technology, Marcel Dekker, New York, 1992, p. 64. w21x L.W. Wong, N.M. Kassem, D. Ganderton, J. Pharm. Pharmacol. 41 Ž1989. suppl. 24P. w22x F. Ruchatz, P. Kleinbudde, B.W. Muller, J. Pharm. Sci. 86 Ž1. Ž1997. 101.