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Preparation of Finely Dispersed Drugs
Journal of Colloid and Interface Science 250, 503–506 (2002) doi:10.1006/jcis.2001.8137
NOTE Preparation of Finely Dispersed Drugs III. Cyclosporine1,2
2. EXPERIMENTAL Two different precipitation processes are described, which produced dispersions of spherical particles of cyclosporine ranging in diameter from ∼10 nm to several micrometers. This drug is of interest for its immunosuppressive activity in the antirejection of transplanted organs. The effects of several experimental parameters on the average particles size and uniformity have been investigated. Aging of spherical particles resulted in large crystalline-type aggregates. C 2002 Elsevier Science (USA) Key Words: colloidal cyclosporine; cyclosporine; drug precipitation; nanosized cyclosporine.
1. INTRODUCTION The vast majority of synthesized monodispersed matter consists of inorganic particles, ranging in size from several nanometers to several micrometers (1–3). In contrast, only a few such dispersions of pure organic compounds have been reported, such as carotenoids by Horn and associates (4, 5). Of specific interest are uniform nanosized colloidal drugs (6, 7). These materials have been essentially prepared by wet or dry milling processes, resulting in a somewhat broad size distribution. Furthermore, it is not possible to obtain uniform particles of desired shapes by such techniques. Recently, the latter goal was achieved by different precipitation methods. For example, in the case of naproxen, which has an ionizable (hydroxyl) group in its molecule, advantage was taken of the decrease in the solubility of this compound in aqueous solutions when the pH was lowered (8). Alternatively, a drug without ionizable groups, dissolved in a good solvent, can be precipitated by the addition of a miscible nonsolvent, as was done with the corticosteroid budesonide (9). In both described cases the nature of the particles depended strongly on the applied mixing procedures. Furthermore, if an ionizable drug is dissolved in aqueous solutions as anionic species, it can be precipitated in the form of uniform particles by reacting with cations that yield sparingly soluble salts, as was exemplified by calcium and barium naproxenates (8, 10). This work describes the preparation of finely dispersed cyclosporine, which is a member of a family of compounds that possess immunosuppressive activity and is commonly administrated following an organ transplant to prevent rejection. In this specific case, advantage is taken of the much higher solubility of this drug in ethanol than in water. Again, different methods have been used to produce spherical particles of reasonably narrow size distribution over a range of modal diameters.
2.1. Materials Cyclosporine (cyclo[[hydroxy-3-methyl-4(methylamino-2) octenoy-6 (2S, 3R, 4R)-(E)] L-aminobutyryl-N -methyl-glycyl-N -methyl L-leucyl L-valyl N methyl L-leucyl L-alanyl D-alanyl N -methyl L-leucyl N -methyl L-leucyl N methyl L-valyl] and hydroxypropyl cellulose (HPC-SL, average MW 70,000) were obtained from e´ lan pharmaceuticals technologies. Cyclosporine is insoluble in water but dissolves well in ethanol. See Scheme 1.
2.2. Preparation of Particles Stock solutions of cyclosporine in ethanol, freshly prepared at a concentration of 3.2 × 10−2 mol dm−3 , were filtered through 0.22-µm Millipore membranes before use. Nonsolvents used were, just water, or aqueous solutions of a stabilizer or of an electrolyte. 2.2.1. Evaporation method. In this procedure, water (nonsolvent) was rapidly added to an ethanol solution of cyclosporine to yield a mixed solution still below the limit of solubility of the drug. A drop of this solution was deposited on a glass slide and alcohol was allowed to slowly evaporate, which caused cyclosporine to precipitate. The process was continued until dryness. The influence of (a) the initial ratio of volumes of alcohol to water (Ra/w ) ranging from 10 to 1.6, (b) the added hydroxypropyl cellulose, and (c) the presence of NaCl was investigated in some detail. 2.2.2. Precipitation method. In this method, to a solution of cyclosporine in ethanol, water was rapidly added in sufficient amounts to precipitate the drug. The resulting dispersions were immediately frozen in liquid nitrogen to avoid further changes and then freeze-dried and the obtained powder was stored in a capped bottle before further studies. In all precipitation experiments to 2 cm3 of the alcoholic solution of cyclosporine, water was added, the amount of which depended on the concentration of the drug. Thus, to solutions containing 3 × 10−2 and 5 × 10−3 mol dm−3 of the drug, 4 and 8 cm3 of water were added, respectively.
1 Supported by a grant from e ´ lan pharmaceutical technologies, King of Prussia, Pennsylvania. 2 Part II, Ref. (9).
SCHEME 1. 503
Cyclosporine. 0021-9797/02 $35.00
C 2002 Elsevier Science (USA)
All rights reserved.
504
NOTE
FIG. 1. Scanning electron micrographs (SEM) of (a) raw cyclosporine and (b–d) of cyclosporine particles prepared by evaporation. In all cases 2.5 cm3 of ethanol solution containing 3 × 10−2 mol dm−3 of the drug were used to which different amounts of nonsolvents were added as follows: (b) 1.5 cm3 of water (pH 6.5); (c) 0.25 cm3 of water (pH 6.5); and (d) 0.25 cm3 of water (pH 1.5).
NOTE
505
FIG. 2. SEM of cyclosporine particles prepared by evaporation of 2.5 cm3 of a 3 × 10−2 mol dm−3 solution of the drug to which 0.25 cm3 of water (pH 6.5) was added containing (a) 0.1 wt% HPC-CL, (b) 1 mol dm−3 NaCl as described in the text, (c) cyclosporine particles obtained by aging the powder shown in Fig. 1a for 1 month at room temperature, and (d) cyclosporine particles prepared by rapidly adding in an ultrasonic bath 4 cm3 of water (pH 6.5) into 2 cm3 of a 3 × 10−2 mol dm−3 solution of cyclosporine.
506
NOTE
2.3. Particles Characterization The size and the shape of the obtained particles were evaluated by scanning electron microscopy (SEM). Since the silver paint used for the preparation of SEM samples contained acetone, which dissolved cyclosporine, the studs were covered with a carbon film before the solids were deposited. To inspect samples obtained by the evaporation technique, the glass slides were directly inserted into the SEM holder.
3. RESULTS
3.1. Evaporation Method The SEM of the original cyclosporine is reproduced in Fig. 1a, which shows the raw material to be made of irregular and polydispersed particles ranging in size from 1 to 10 µm. In contrast, spherical particles prepared by the described evaporation technique are illustrated in Figs. 1b and 1c. These two samples differ only in the ratio of the solvent to nonsolvent (Ra/w ) used in the experiments. The pH of water in these systems was ∼6.5. It would appear that the particle size distribution narrows with the increase of Ra/w ; in all other evaporation studies this ratio was kept at 10. The acidification of the nonsolvent had a significant effect on the resulting solid drug. As an example, Fig. 1d illustrates a gel-like film with holes obtained upon evaporation of the system, which was prepared by adding an aqueous solution of pH 1.5 to the ethanol solution of the drug. Replacing water with a 1 wt% HPC-SL solution, under otherwise the same conditions, yielded particles of the same morphology but somewhat smaller in size, ranging between 0.25 and 1 µm as displayed in Fig. 2a. The average size could be reduced further by using aqueous electrolyte solutions in the precipitation experiments. Particles obtained when a 1.0 mol dm−3 NaCl aqueous solution was used as a nonsolvent are shown in Fig. 2b. In this case the dried sample was immersed in water overnight to remove the salt, leaving behind a uniform dispersion of nanosized cyclosporine. Figure 2c shows the same sample as described in Fig. 1a after 1 month of aging at room temperature. This process yielded large rectangular particles obtained by aggregation of the original spherical beads.
3.2. Precipitation Method The precipitation of the drug dissolved in ethanol by the addition of water resulted, as a rule, in spherical particles of rather narrow size distribution. Specifically, Fig. 2d displays the dispersion prepared by rapidly adding, in an ultrasonic bath, 4 cm3 of water into a test tube containing 2 cm3 of a 3 × 10−2 mol dm−3 solution of cyclosporine. The average diameter of the obtained particles is ∼1 µm. Analogous experiments were carried out with a lower drug concentration, requiring a larger amount of water to induce precipitation, but essentially the same kind of particles were produced. Other intermediate concentrations yielded similar results. One experimental parameter of importance was the rate of addition of the nonsolvent. If the latter was slow, the particle size distribution was rather broad.
In this study strictly physical methods were used to precipitate the drug in colloidal forms. Different procedures yielded, as a rule, spherical particles, but the size and uniformity were affected in different degrees by several experimental conditions. Thus, faster mixing in the precipitation method was conducive to more uniform dispersions. Somewhat surprisingly, the added stabilizer (HPCSL) had little effect on the products. In contrast, the ionic strength was a critical parameter. The presence of NaCl in the system resulted in much smaller particles. Obviously, under these conditions a larger number of nuclei are formed, most likely due to a lower solubility of the drug caused by the high ionic strength. In contrast, the gel formation in acidic media may be explained by the protonation of amido groups in cyclosporine which would greatly affect the solubility of the drug in alcohol and possibly cause interactions of the biomolecules themselves, resulting in a network. The aggregation process upon aging, which produced crystal-like large particles, has been observed in numerous examples as a main process in the formation of uniform inorganic particles (11). It is noteworthy that the same kind of mechanism seems to be operational in organic systems. Finally, it was also hoped that the morphology of precipitated drug particles might be related to the shape of the constituent molecules. However, this has certainly not been the case with the drug used in this study. There are a number of additional experiments one may wish to carry out with the described systems. However, the exorbitant cost of cyclosporine limited the amount of work that could be carried out.
ACKNOWLEDGMENT The authors appreciate useful comments by Dr. Niels Ryde, e´ lan pharmaceutical technologies.
REFERENCES 1. Matijevi´c, E., Chem. Mater. 5, 412 (1993). 2. Matijevi´c, E., Langmuir 10, 8 (1994). 3. “Fine Particles, Synthesis, Characterization and Mechanisms of Growth” (T. Sugimoto, Ed.). Dekker, New York, 2000. 4. Horn, D., Angew. Makromol. Chem. 166/167, 139 (1989). 5. Horn, D., and L¨uddecke, E., in “Fine Particles Science and Technology,” (E. Palizzetti, Ed.). NATO Asi. Ser. 3; Vol. 3, p. 761. 1996, Kleuver. 6. Liversidge, G. G., and Cundy, K. C., Int. J. Pharm. 125, 91 (1995). 7. Liversidge, G. G., and Conzentino, P., Int. J. Pharm. 125, 309 (1995). 8. Pozarnsky, G. A., and Matijevi´c, E., Colloids Surf. (A) 125, 47 (1997). 9. Ruch, F., and Matijevi´c, E., J. Colloid Interface Sci. 229, 207 (2000). 10. Goia, C., and Matijevi´c, E., J. Colloid Interface Sci. 206, 583 (1998). 11. Privman, V., Goia, D. V., Park, J., and Matijevi´c, E., J. Colloid Interface Sci. 213, 583 (1999). Laurent Joguet Egon Matijevi´c3 Center for Advanced Material Processing (CAMP) Clarkson University Potsdam, New York 13699-5814
4. DISCUSSION Many colloidal systems consisting of inorganic particles of well-defined size, composition, and stability have been extensively described. (1, 2) This investigation clearly indicates the possibility of achieving well-defined colloid dispersions of organic compounds, which are of significant theoretical and practical interests.
Received September 20, 2001; accepted November 24, 2001; published online May 15, 2002
3
To whom correspondence should be addressed.
NOTE Preparation of Finely Dispersed Drugs III. Cyclosporine1,2
2. EXPERIMENTAL Two different precipitation processes are described, which produced dispersions of spherical particles of cyclosporine ranging in diameter from ∼10 nm to several micrometers. This drug is of interest for its immunosuppressive activity in the antirejection of transplanted organs. The effects of several experimental parameters on the average particles size and uniformity have been investigated. Aging of spherical particles resulted in large crystalline-type aggregates. C 2002 Elsevier Science (USA) Key Words: colloidal cyclosporine; cyclosporine; drug precipitation; nanosized cyclosporine.
1. INTRODUCTION The vast majority of synthesized monodispersed matter consists of inorganic particles, ranging in size from several nanometers to several micrometers (1–3). In contrast, only a few such dispersions of pure organic compounds have been reported, such as carotenoids by Horn and associates (4, 5). Of specific interest are uniform nanosized colloidal drugs (6, 7). These materials have been essentially prepared by wet or dry milling processes, resulting in a somewhat broad size distribution. Furthermore, it is not possible to obtain uniform particles of desired shapes by such techniques. Recently, the latter goal was achieved by different precipitation methods. For example, in the case of naproxen, which has an ionizable (hydroxyl) group in its molecule, advantage was taken of the decrease in the solubility of this compound in aqueous solutions when the pH was lowered (8). Alternatively, a drug without ionizable groups, dissolved in a good solvent, can be precipitated by the addition of a miscible nonsolvent, as was done with the corticosteroid budesonide (9). In both described cases the nature of the particles depended strongly on the applied mixing procedures. Furthermore, if an ionizable drug is dissolved in aqueous solutions as anionic species, it can be precipitated in the form of uniform particles by reacting with cations that yield sparingly soluble salts, as was exemplified by calcium and barium naproxenates (8, 10). This work describes the preparation of finely dispersed cyclosporine, which is a member of a family of compounds that possess immunosuppressive activity and is commonly administrated following an organ transplant to prevent rejection. In this specific case, advantage is taken of the much higher solubility of this drug in ethanol than in water. Again, different methods have been used to produce spherical particles of reasonably narrow size distribution over a range of modal diameters.
2.1. Materials Cyclosporine (cyclo[[hydroxy-3-methyl-4(methylamino-2) octenoy-6 (2S, 3R, 4R)-(E)] L-aminobutyryl-N -methyl-glycyl-N -methyl L-leucyl L-valyl N methyl L-leucyl L-alanyl D-alanyl N -methyl L-leucyl N -methyl L-leucyl N methyl L-valyl] and hydroxypropyl cellulose (HPC-SL, average MW 70,000) were obtained from e´ lan pharmaceuticals technologies. Cyclosporine is insoluble in water but dissolves well in ethanol. See Scheme 1.
2.2. Preparation of Particles Stock solutions of cyclosporine in ethanol, freshly prepared at a concentration of 3.2 × 10−2 mol dm−3 , were filtered through 0.22-µm Millipore membranes before use. Nonsolvents used were, just water, or aqueous solutions of a stabilizer or of an electrolyte. 2.2.1. Evaporation method. In this procedure, water (nonsolvent) was rapidly added to an ethanol solution of cyclosporine to yield a mixed solution still below the limit of solubility of the drug. A drop of this solution was deposited on a glass slide and alcohol was allowed to slowly evaporate, which caused cyclosporine to precipitate. The process was continued until dryness. The influence of (a) the initial ratio of volumes of alcohol to water (Ra/w ) ranging from 10 to 1.6, (b) the added hydroxypropyl cellulose, and (c) the presence of NaCl was investigated in some detail. 2.2.2. Precipitation method. In this method, to a solution of cyclosporine in ethanol, water was rapidly added in sufficient amounts to precipitate the drug. The resulting dispersions were immediately frozen in liquid nitrogen to avoid further changes and then freeze-dried and the obtained powder was stored in a capped bottle before further studies. In all precipitation experiments to 2 cm3 of the alcoholic solution of cyclosporine, water was added, the amount of which depended on the concentration of the drug. Thus, to solutions containing 3 × 10−2 and 5 × 10−3 mol dm−3 of the drug, 4 and 8 cm3 of water were added, respectively.
1 Supported by a grant from e ´ lan pharmaceutical technologies, King of Prussia, Pennsylvania. 2 Part II, Ref. (9).
SCHEME 1. 503
Cyclosporine. 0021-9797/02 $35.00
C 2002 Elsevier Science (USA)
All rights reserved.
504
NOTE
FIG. 1. Scanning electron micrographs (SEM) of (a) raw cyclosporine and (b–d) of cyclosporine particles prepared by evaporation. In all cases 2.5 cm3 of ethanol solution containing 3 × 10−2 mol dm−3 of the drug were used to which different amounts of nonsolvents were added as follows: (b) 1.5 cm3 of water (pH 6.5); (c) 0.25 cm3 of water (pH 6.5); and (d) 0.25 cm3 of water (pH 1.5).
NOTE
505
FIG. 2. SEM of cyclosporine particles prepared by evaporation of 2.5 cm3 of a 3 × 10−2 mol dm−3 solution of the drug to which 0.25 cm3 of water (pH 6.5) was added containing (a) 0.1 wt% HPC-CL, (b) 1 mol dm−3 NaCl as described in the text, (c) cyclosporine particles obtained by aging the powder shown in Fig. 1a for 1 month at room temperature, and (d) cyclosporine particles prepared by rapidly adding in an ultrasonic bath 4 cm3 of water (pH 6.5) into 2 cm3 of a 3 × 10−2 mol dm−3 solution of cyclosporine.
506
NOTE
2.3. Particles Characterization The size and the shape of the obtained particles were evaluated by scanning electron microscopy (SEM). Since the silver paint used for the preparation of SEM samples contained acetone, which dissolved cyclosporine, the studs were covered with a carbon film before the solids were deposited. To inspect samples obtained by the evaporation technique, the glass slides were directly inserted into the SEM holder.
3. RESULTS
3.1. Evaporation Method The SEM of the original cyclosporine is reproduced in Fig. 1a, which shows the raw material to be made of irregular and polydispersed particles ranging in size from 1 to 10 µm. In contrast, spherical particles prepared by the described evaporation technique are illustrated in Figs. 1b and 1c. These two samples differ only in the ratio of the solvent to nonsolvent (Ra/w ) used in the experiments. The pH of water in these systems was ∼6.5. It would appear that the particle size distribution narrows with the increase of Ra/w ; in all other evaporation studies this ratio was kept at 10. The acidification of the nonsolvent had a significant effect on the resulting solid drug. As an example, Fig. 1d illustrates a gel-like film with holes obtained upon evaporation of the system, which was prepared by adding an aqueous solution of pH 1.5 to the ethanol solution of the drug. Replacing water with a 1 wt% HPC-SL solution, under otherwise the same conditions, yielded particles of the same morphology but somewhat smaller in size, ranging between 0.25 and 1 µm as displayed in Fig. 2a. The average size could be reduced further by using aqueous electrolyte solutions in the precipitation experiments. Particles obtained when a 1.0 mol dm−3 NaCl aqueous solution was used as a nonsolvent are shown in Fig. 2b. In this case the dried sample was immersed in water overnight to remove the salt, leaving behind a uniform dispersion of nanosized cyclosporine. Figure 2c shows the same sample as described in Fig. 1a after 1 month of aging at room temperature. This process yielded large rectangular particles obtained by aggregation of the original spherical beads.
3.2. Precipitation Method The precipitation of the drug dissolved in ethanol by the addition of water resulted, as a rule, in spherical particles of rather narrow size distribution. Specifically, Fig. 2d displays the dispersion prepared by rapidly adding, in an ultrasonic bath, 4 cm3 of water into a test tube containing 2 cm3 of a 3 × 10−2 mol dm−3 solution of cyclosporine. The average diameter of the obtained particles is ∼1 µm. Analogous experiments were carried out with a lower drug concentration, requiring a larger amount of water to induce precipitation, but essentially the same kind of particles were produced. Other intermediate concentrations yielded similar results. One experimental parameter of importance was the rate of addition of the nonsolvent. If the latter was slow, the particle size distribution was rather broad.
In this study strictly physical methods were used to precipitate the drug in colloidal forms. Different procedures yielded, as a rule, spherical particles, but the size and uniformity were affected in different degrees by several experimental conditions. Thus, faster mixing in the precipitation method was conducive to more uniform dispersions. Somewhat surprisingly, the added stabilizer (HPCSL) had little effect on the products. In contrast, the ionic strength was a critical parameter. The presence of NaCl in the system resulted in much smaller particles. Obviously, under these conditions a larger number of nuclei are formed, most likely due to a lower solubility of the drug caused by the high ionic strength. In contrast, the gel formation in acidic media may be explained by the protonation of amido groups in cyclosporine which would greatly affect the solubility of the drug in alcohol and possibly cause interactions of the biomolecules themselves, resulting in a network. The aggregation process upon aging, which produced crystal-like large particles, has been observed in numerous examples as a main process in the formation of uniform inorganic particles (11). It is noteworthy that the same kind of mechanism seems to be operational in organic systems. Finally, it was also hoped that the morphology of precipitated drug particles might be related to the shape of the constituent molecules. However, this has certainly not been the case with the drug used in this study. There are a number of additional experiments one may wish to carry out with the described systems. However, the exorbitant cost of cyclosporine limited the amount of work that could be carried out.
ACKNOWLEDGMENT The authors appreciate useful comments by Dr. Niels Ryde, e´ lan pharmaceutical technologies.
REFERENCES 1. Matijevi´c, E., Chem. Mater. 5, 412 (1993). 2. Matijevi´c, E., Langmuir 10, 8 (1994). 3. “Fine Particles, Synthesis, Characterization and Mechanisms of Growth” (T. Sugimoto, Ed.). Dekker, New York, 2000. 4. Horn, D., Angew. Makromol. Chem. 166/167, 139 (1989). 5. Horn, D., and L¨uddecke, E., in “Fine Particles Science and Technology,” (E. Palizzetti, Ed.). NATO Asi. Ser. 3; Vol. 3, p. 761. 1996, Kleuver. 6. Liversidge, G. G., and Cundy, K. C., Int. J. Pharm. 125, 91 (1995). 7. Liversidge, G. G., and Conzentino, P., Int. J. Pharm. 125, 309 (1995). 8. Pozarnsky, G. A., and Matijevi´c, E., Colloids Surf. (A) 125, 47 (1997). 9. Ruch, F., and Matijevi´c, E., J. Colloid Interface Sci. 229, 207 (2000). 10. Goia, C., and Matijevi´c, E., J. Colloid Interface Sci. 206, 583 (1998). 11. Privman, V., Goia, D. V., Park, J., and Matijevi´c, E., J. Colloid Interface Sci. 213, 583 (1999). Laurent Joguet Egon Matijevi´c3 Center for Advanced Material Processing (CAMP) Clarkson University Potsdam, New York 13699-5814
4. DISCUSSION Many colloidal systems consisting of inorganic particles of well-defined size, composition, and stability have been extensively described. (1, 2) This investigation clearly indicates the possibility of achieving well-defined colloid dispersions of organic compounds, which are of significant theoretical and practical interests.
Received September 20, 2001; accepted November 24, 2001; published online May 15, 2002
3
To whom correspondence should be addressed.