Preparation and Characterization of Amorphous Cefuroxime Axetil

Ind. Eng. Chem. Res. 2006, 45, 8723-8727

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GENERAL RESEARCH Preparation and Characterization of Amorphous Cefuroxime Axetil Drug Nanoparticles with Novel Technology: High-Gravity Antisolvent Precipitation Jian-Feng Chen,*,† Ji-Yao Zhang,†,‡ Zhi-Gang Shen,†,‡ Jie Zhong,† and Jimmy Yun‡ Sin-China Nano Technology Center, Key Laboratory for Nanomaterials, Ministry of Education, Beijing UniVersity of Chemical Technology, Beisanhuan East Road No. 15, Beijing 100029, People’s Republic of China, and Nanomaterials Technology Pte. Ltd., 28 Ayer Rajah Crescent No. 03-03, Singapore 139959, Singapore

Amorphous nanoparticles of cefuroxime axetil (CFA), a kind of poorly water-soluble antibiotic drug, were prepared at massive production rate by a novel continuous process, the high-gravity antisolvent precipitation (HGAP). The produced CFA nanoparticles were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectrophotometry (FTIR), powder X-ray diffraction (XRD), specific surface area analysis (BET), differential scanning calorimetry (DSC), and a dissolution test. The mean particle size of CFA was about 300 nm with a narrow distribution from 100 to 400 nm. The specific surface area reached up to 8.67 m2/g, which was about 4 times higher than that of the commercial spray-dried CFA. And the results of the dissolution test showed that dissolution rate of the former were higher than that of the latter. Hence it is proved the HGAP technique offers a direct and continuous process for mass-production of drug nanoparticles. 1. Introduction Cefuroxime axetil (CFA) is one kind of cephalosporin antibiotic possessing a high activity against a wide spectrum of gram-positive and gram-negative microorganisms. It has been revealed that CFA in amorphous form has a higher bioavailability than that in crystalline form.1 But as a drug of class II (low solubility, high permeability),2 the low solubility/dissolution rate of CFA is still a key factor limiting its oral bioavailability. If its dissolution rate can be enhanced, bioavailability following oral administration of CFA could be significantly improved. A large amount of research has proved that the solubility and dissolution rate of poorly water-soluble drugs could be increased by reducing the particle size to increase the interfacial surface area.3,4 Thus, the study in nanoparticle engineering of hydrophobic drugs has received great attention due to their ability to satisfy the regulatory requirement and also due to their various potential applications. The common way for reducing drug particle size is to comminute larger particles by mechanical disintegration, such as jet-milling,5 media milling,6 and high-pressure homogenization processes.7 The disadvantage of these mechanical comminution processes is that there is only limited opportunity to control the product characteristics, such as size, shape morphology, surface properties, and electrostatic charge.8 Because of the disadvantages of mechanical-comminution methods, other techniques have been developed, including supercritical fluidbased methods,9-13 precipitation-based methods,14-19 and microemulsion techniques,20,21 etc. These methods overcome many limitations of the mechanical process. However, they still have * To whom correspondence should be addressed. Tel.: +86-1064446466. Fax: +86-10-64434784. E-mail: [email protected]. † Beijing University of Chemical Technology. ‡ Nanomaterials Technology Pte. Ltd.

some limitations, such as the difficulty to scale-up and enormous cost of production, which restrict their wider application and further commercialization. The high-gravity technology is a technology usually used in the intensification of multiphase heat and mass transfer. In our previous work, we22 have successfully developed a high-gravity reactive precipitation (HGRP) to synthesize many kinds of inorganic nanoparticles including CaCO3, Al(OH)3, SrCO3, BaTiO3,23 TiO2,24 and ZnS,25 etc. Furthermore, the calcium carbonate nanoparticles (NPCC) synthesized by the HGRP technique were first successfully commercialized in the year of 2000 at an annual capacity of 3000 tons per HGRP reactor in China. Recently, HGRP was also explored to prepare nanoparticles of organic pharmaceutical compounds such as benzoic acid26 and cephradine.27 Commercial amorphous CFA powder is currently prepared by spray-drying technique. The spray-dried CFA particles, however, have a large size with broad particle size distribution (PSD). Furthermore, it may have the problem of decomposition of the drug during the process due to the high temperature. In this study, a novel physical process, the high-gravity antisolvent precipitation (HGAP) method, was proposed to prepare highly pure amorphous CFA nanoparticles, which avoids the chemical reaction and byproduct. Furthermore, it could be expected to have more economic advantages for commercialization compared to other processes. The HGAP process was performed at room temperature without any additives. The produced CFA nanoparticles were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectrophotometry (FTIR), powder X-ray diffraction (XRD), highperformance liquid chromatography (HPLC), specific surface area analysis (BET), differential scanning calorimetry (DSC), and a dissolution test. As a comparison, the commercial spraydried CFA properties were also investigated at the same time.

10.1021/ie060445h CCC: $33.50 © 2006 American Chemical Society Published on Web 11/09/2006

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Figure 1. Schematic of the high-gravity antisolvent precipitation setup: (1) Casing, (2) packed rotator, (3) motor, (4) liquid distributors, (5) flow meters, (6) seal ring, (7) outlet, (8) pump, and (9, 10) S/AS storage container.

2. Experiments 2.1. Materials. The crystalline cefuroxime axetil and the spray-dried CFA were supplied by NCPC Beta Co., Ltd., China. Acetone and isopropyl ether (A.R. grade) were provided by Beijing Chemical Agent Co. (Beijing). 2.2. Preparation of the Amorphous CFA Nanoparticles by HGAP. The HGAP experiments were conducted using the apparatus shown in Figure 1. The key part of the apparatus is the rotating packed bed (RPB), and the packing is stainless steel wire mesh packing. The detailed structure of RPB could be referenced to our previous work.22 In the typical HGAP experimental run, which was based upon the optimal condition of the preexperiments in the stirred beakerrun, the raw crystalline form of cefuroxime axetil was dissolved in acetone at the concentration of 10% (w/v). The solution was filtrated through 0.45 µm pore size membranes to remove the impure particulates. The solution and the antisolvent, i.e., isopropyl ether, were added into the storage containers 9 and 10, respectively. Then the two liquids were pumped through the liquid distributors 4 into RPB with flow rates of 10 and 200 L/h, separately. Both liquids were sprayed onto the inside edge of the rotator and mixed in the packed-bed zone to yield nanoparticles immediately. The rotating speed of RPB was controlled at 2800 rpm (the high-gravity level is about 438g, “g” being the gravitation of acceleration speed, 9.8 m/s2). The slurry finally left the equipment through the liquid exit for collection and then was filtered. The filter cake was dried in a vacuum oven at around 60 °C for 12 h to yield the drug powder used in this experiment. 2.3. Characterization. 2.3.1. Scanning Electron Microscopy. The morphology, size, and shape of the prepared particles were examined by SEM (S250MK3, Cambridge, U.K.). The samples were prepared by dropping the freshly formed nanoparticle suspension on the glass slide. The solvent was evaporated at room temperature, and the particles were deposited on the surface of the glass. The glass slide with CFA particles was fixed on 13 mm diameter aluminum stubs using double-sided adhesive tape and was sputter-coated with gold in a vacuum for 30 s. The particle size distribution was determined by the IBAS I/II image analyzer system (Germany) via the obtained SEM photographs. Furthermore, the dried CFA powder was also characterized by SEM. 2.3.2 Fourier Transform Infrared Spectrometry. A FTIR spectrometer (60-SXB, Nicolet, USA) was used to define the molecule states with the scan range of 450-4000 cm-1 using a resolution of 4 cm-1 and 16 scans. Samples were diluted with KBr mixing powder and pressed to obtain self-supporting disks. 2.3.3. X-ray Diffraction. Phase identification of CFAs was

conducted using an X-ray diffractometer (Shimadzu XRD-6000) with Cu KR radiation at a scanning speed of 0.05°/min. 2.3.4. Differential Scanning Calorimetry. Powder samples of about 5 mg in loosely covered aluminum pans were analyzed by differential scanning calorimeter (Pyris 1, Perkin-Elmer, USA) at a heating rate of 10 °C/min. A dry nitrogen purge of 25 mL/min was employed, and samples were heated form 40 to 160 °C. Calibration of the instrument with respect to temperature and enthalpy was achieved using a high-purity standard of indium. 2.3.5. Specific Surface Area. The specific surface area was determined using the nitrogen gas absorption method. Calculation is based on the BET equation. A surface area analyzer (ASAP 2010, Micromeritics, USA) was used. A known amount of powder was loaded into a Quantachrome sample cell and degassed for at least 3 h prior to analysis. 2.3.6. High-Performance Liquid Chromatography. HPLC (Prostar, Varian Inc., USA) was used to measure the drug purity with a 250 mm long C-18 column (ODS, 5 µm). According to the pharmacopoeia of the People’s Republic of China (2005), the mobile phase was a solution of methanol/(0.2 M aqueous NH4H2PO4) (600:400, v/v), and the detection wavelength was 278 nm. The flow rate was about 1 mL/min. The standard curve, whose equation is shown as eq 1, was linear (r ) 0.999 87) in the range from 5 to 50 µg‚mL-1.

y ) 3.577 × 10-6x - 0.066

(1)

where y is the concentration of CFA, µg‚mL-1, and x is the peak area. 2.3.7. Dissolution Test. Dissolution testing for the CFA samples was carried out using a dissolution apparatus (D-800LS, Tianjin, CN) following the USP apparatus II (paddle) method. Paddle speed and bath temperature were set at 100 rpm and 37.0 ( 0.5 °C, respectively. A 20 mg amount of CFA was placed into vessels containing 900 mL of 0.1 M HCl solution, which included 0.1% (w/v) sodium dodecyl sulfate (SDS). A 5 mL aliquot of sample was withdrawn at specific intervals. These samples were filtered through a 0.22 µm membrane. The concentration of samples was analyzed in an ultraviolet spectrophotometer (UV-3000, Shimadzu, Japan) at 278 nm. The dissolution test of each sample was carried out three times. The result showed the average value. 3. Results and Discussion The SEM image and the particle size distribution of the amorphous CFA nanoparticles prepared by the HGAP method, which was obtained from freshly formed suspension, are shown in Figure 2. It can be seen that the CFA particles were spherical and the mean particle size was 305 nm with narrow distribution from 100 to 400 nm. As a comparison, the SEM image and particle size distribution of commercial CFA particles prepared by traditional spray-drying process are also shown in Figure 2. It was obvious that the spray-dried CFA particles were hollow spheres with the mean particle size of 15 µm and the size range was from about 2 to 30 µm. The dried CFA powder was also characterized by SEM. As shown in Figure 3, the CFA particles after drying had some agglomeration or aggregation. It indicated that the process of drying influenced the particles dispersibility. In principle,22 in order to obtain nanoparticles and controlled particle size distribution (PSD) in the preparation process, uniform spatial distribution of the concentration and supersaturation must be created and kept in the vessel before nucleation.

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Figure 4. FT-IR spectra of various CFA: (a) HGAP nanosized CFA; (b) raw CFA.

Figure 2. SEM images and PSD of various CFA: (a) HGAP nanosized CFA (from freshly formed suspension); (b) spray-dried CFA; (c) PSD of CFA (9, HGAP nanosized CFA (from freshly formed suspension); 2, spraydried CFA).

Figure 5. X-ray diffraction patterns of various CFA: (a) HGAP nanosized CFA; (b) spray-dried CFA; (c) raw CFA.

Figure 3. SEM image of the nanosized CFA particles after being dried.

So intensification of micromixing to reach the region of tm < τ should be taken, where tm is the characteristic time of micromixing for species reaching a maximum mixed state at the molecular level, and τ is the induction of nucleation. In the HGAP process, the fluids going through the packing of RPB were spread or split into very fine droplets, thread, and thin film under the high-gravity field. The rate of mass transfer between a gas and liquid or liquid and liquid in a RPB was 1-3 orders of magnitude larger than that in a conventional stirred tank reactor.22 This intensifies micromixing between the fluid elements in RPB and the tm could decrease to about 100 µs.28 Generally, in typical low viscous solution, the τ is around 5-50 ms.22 Therefore, the theoretical requirement of tm < τ could be met in RPB. This favored the generation of high and uniformly supersaturated concentrations of the product in the antisolvent precipitation process before nucleation. As a result, the drug nanoparticles with narrow PSD were obtained. The molecular structures of the raw and nanosized CFA particles were studied by means of FT-IR. Figure 4 shows the FT-IR spectrum of the CFA in the range of 500-4000 cm-1. The spectra of the above two CFA particles were characterized by the NH, NH2 complex (3210-3480 cm-1), β-lactam (1782 cm-1), acetate (1760 cm-1), 4-ester group (1720 cm-1), and 7-amido (1676 and 1534 cm-1). The identical FT-IR spectra curves between the raw CFA and nanosized CFA suggested that there was no chemical structure change in the CFA

molecular with reprecipitation of CFA particles caused by the HGAP process. XRD was performed to investigate the effect of the HGAP on the crystallinity of CFA. The XRD pattern of the raw CFA (Figure 5) exhibits intense crystalline peaks between 5 and 50°. This proves that the raw CFA was highly crystallized. However, only one broad and diffuse maxima peak was detected in the pattern of the nanosized CFA, which indicated that the nanosized CFA particles prepared by the HGAP process were in the completely amorphous form. In the HGAP process, the high nucleation rate was induced by the high supersaturation resulting in the precipitation of amorphous CFA instead of the crystalline form. This was consistent with Ostwald’s law of stage,29 “when leaving an unstable state, a system does not seek out the most stable state, rather the nearest metastable state which can be reached with loss of free energy.” It was analogous for the spray-dried CFA particles. The physical state of CFA samples was also investigated by differential scanning calormetry. The DSC scan of the raw CFA (Figure 6) shows two sharp melting endotherm peaks at 126.67 and 180.33 °C, and the melting enthalpies were 34.225 and 42.496 J/g. It showed that the raw CFA was not only crystalline form but also polymorphous. For the spray-dried and the nanosized CFA powder, the endotherm occurred at a lower and broader temperature stage. Furthermore, the ∆H of 5.353 and 6.593 J/g of the nanosized and the spray-dried CFA were lower than that of the crystalline form. It was indicated that the HGAP nanosized sample and the spray-dried sample were at higher energy state, i.e., amorphous state. Such reduction of crystallinity might be expected to enhance the bioavailability of the waterinsoluble drug.30 The difference of the powders produced by the various processes was further characterized by surface area measurement. The HGAP nanosized particles had a specific surface area

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particles, which also contributes to the increase of the dissolution rate

hH ) k(L1/2/V1/2)

(3)

where hH is the hydrodynamic boundary layer thickness, k denotes a constant, L is the length of the surface in the direction of flow, and V is the relative velocity of the flowing liquid against the flat surface. Therefore, the HGAP method is a promising approach to enhance the solubility of poorly water-soluble drugs by decreasing particle size. Figure 6. DSC of various CFA: (a) HGAP nanosized CFA; (b) spraydried CFA; (c) raw CFA.

Figure 7. Dissolution profiles for (9) HGAP nanosized CFA and (b) spraydried CFA.

of 8.67 m2/g, which was significantly greater than that of the spray-dried particles, 1.86 m2/g. It was due to the particle size of the HGAP nanosized powder being significantly smaller than that of the latter one. The purity of the CFA nanoparticles was also measured by HPLC. The result shows that the purity of the nanosized CFA was 99.5%, which was higher than the 98.2% of the raw CFA and the 98.5% of the commercial spray-dried product. It was due to that there were no other additives in the HGAP process. Furthermore, the HGAP was a reprecipitation process, just like the recrystallization process, which resulted in the purity of the final product being higher than that of raw material. Dissolution profiles of the commercial CFA and the CFA nanoparticles are compared in Figure 7. At 37 °C, the dissolution rate of the nanosized CFA was increased to 78% after 30 min, while only 51% of the commercial spray-dried CFA dissolved at that time. After 80 min, all of the nanosized CFA particles had been completely dissolved, but there was still 40% of spraydried CFA un-dissolved. This shows that the amorphous CFA nanoparticles dissolved significantly faster than that of the commercial spray-dried CFA. it can be explained by the NoyesWhitney equation:31

dm/dt ) (DA/h)(Cs - C)

(2)

where dm/dt is the rate of dissolution, D is the diffusion coefficient, A is the surface area, h is the diffusion layer thickness, Cs is the saturation concentration, and C is the bulk concentration. The particle size of the nanosized CFA was induced to greatly increase the specific surface area, which offered significant potential for enhancing the dissolution rate of CFA. Furthermore, according to the Prandtl equation (eq 3),3 a decrease in the particle size resulted in a thinner hydrodynamic layer around

4. Conclusion The novel high-gravity antisolvent precipitation (HGAP) method was developed to successfully prepare amorphous CFA nanoparticles without any additives. The mean particle size of the amorphous CFA nanoparticles was about 300 nm with a narrow PSD. The specific surface area reached up to 8.67 m2/ g. The small particle size and large specific surface area of amorphous CFA nanoparticles led to the dissolution rate being significantly enhanced. As a result, the HGAP technique offered a direct and continuous process to massively produce drug nanoparticles with a simple physical reprecipitation process. Furthermore, the HGAP process was easy to scale-up for industrial application. Since June 2005, a HGAP product line of 40 tons per annum of amorphous nanosized CFA, which was designed and built according to the GMP principles, has been in operation at Beta Co., Ltd., of North China Pharmaceutical Group Corp. Acknowledgment This work was supported by the NSF of China (Grant No. 20325621) and the “863” plan of China (Grant No. 2004AA218042). The authors also thank Beta Co., Ltd., of North China Pharmaceutical Group Corp. for supplying the crystalline form and spray-dried CFA materials. Literature Cited (1) Crisp, H. A.; Clayton, J. C. Amorphous form of cefuroxime ester. U.S. Pat. 4562181, 1985. (2) Kanfer, I. Report on the international workshop on the biopharmaceutics classification system (BCS): Scientific and regulatory aspects in practice. J. Pharm. Pharm. Sci. 2000, 5, 1. (3) Mosharrafand, M.; Nystro¨m, C. The effect of particle size and shape on the surface specific dissolution rate of microsized practically insoluble drugs. Int. J. Pharm. 1995, 122, 35. (4) Mu¨ller, R. H.; Jacobs, C.; Kayser, O. Nanosuspensions as particulate drug formulations in therapy. Rationale for development and what we can expect for the future. AdV. Drug DeliVery ReV. 2001, 47, 3. (5) Midoux, N.; Hosˇek, P.; Pailleres, L.; Authelin, J. R. Micronization of pharmaceutical substances in a spiral jet mill. Powder Technol. 1999, 104, 113. (6) Merisko, L. E.; Liversidge, G. G.; Cooper, E. R. Nanosizing: A formulation approach for poorly-water-soluble compounds. Eur. J. Pharmacol. Sci. 2003, 18, 113. (7) Mu¨llerand, R. H.; Peters, K. Nanosuspensions for the formulation of poorly soluble drugs I. Preparation by a size-reduction technique. Int. J. Pharm. 1998, 160, 229. (8) Rasenackand, N.; Mu¨ller, B. W. Micron-size drug particles: Common and novel micronization techniques. Pharm. DeV. Technol. 2004, 9, 1. (9) Reverchon, E. Supercritical antisolvent precipitation of micro- and nano-particles. J. Supercrit. Fluid 1999, 15, 1. (10) Reverchonand, E.; Porta, G. D. Production of antibiotic micro- and nano-particles by supercritical antisolvent precipitation. Powder Technol. 1999, 106, 23.

Ind. Eng. Chem. Res., Vol. 45, No. 25, 2006 8727 (11) Charoenchaitrakool, M.; Dehghani, F.; Foster, N. R. Micronization by rapid expansion of supercritical solutions to enhance the dissolution rates of poorly water-soluble pharmaceuticals. Ind. Eng. Chem. Res. 2000, 39, 4794. (12) Mezianiand, M. J.; Sun, Y. P. Protein-conjugated nanoparticles from rapid expansion of supercritical fluid solution into aqueous solution. J. Am. Chem. Soc. 2003, 125, 8015. (13) Tu¨rk, M.; Hils, P.; Helfgen, B.; Schaber, K.; Martin, H. J.; Wahl, M. A. Micronization of pharmaceutical substances by the rapid expansion of supercritical solutions (RESS): A promising method to improve bioavailability of poorly soluble pharmaceutical agents. J. Supercrit. Fluid 2002, 22, 75. (14) Rogers, T. L.; Gillespie, I. B.; Hitt, J. E.; Fransen, K. L.; Crowl, C. A.; Tucker, C. J.; Kupperblatt, G. B.; Becker, J. N.; Wilson, D. L.; Todd, C. Development and characterization of a scalable controlled precipitation process to enhance the dissolution of poorly water-soluble drugs. Pharm. Res. 2004, 21, 2048. (15) Rasenackand, N.; Mu¨ller, B. W. Dissolution rate enhancement by in situ micronization of poorly water-soluble drugs. Pharm. Res. 2002, 19, 1894. (16) Bilati, U.; Alle´mann, E.; Doelker, E. Development of a nanoprecipitation method intended for the entrapment of hydrophilic drugs into nanoparticles. Eur. J. Pharmacol. Sci. 2005, 24, 67. (17) Ruch, F.; Matijevic´, E. Preparation of micrometer Ssize Budesonide particles by precipitation. J. Colloid Interface Sci. 2000, 229, 207. (18) Sarkari, M.; Brown, J.; Chen, X.; Swinnea, S.; Williams, R. O., III; Johnston, K. P. Enhanced drug dissolution using evaporative precipitation into aqueous solution. Int. J. Pharm. 2002, 243, 17. (19) Hua, J.; Johnston, K. P.; Williams, R. O., III Spray freezing into liquid (SFL) particle engineering technology to enhance dissolution of poorly water-soluble drugs: Organic solvent versus organic/aqueous co-solvent systems. Eur. J. Pharmacol. Sci. 2003, 20, 295. (20) Trotta, M.; Gallarate, M.; Carlotti, M. E.; Morel, S. Preparation of griseofulvin nanoparticles from water-dilutable microemulsions. Int. J. Pharm. 2003, 254, 235. (21) Debuigne, F.; Cuisenaire, J.; Jeunieau, L.; Masereel, B.; Nagy, J. B. Synthesis of nimesulide nanoparticles in the microemulsion epikuron/ isopropyl myristate/water/n-butanol (or Isopropanol). J. Colloid Interface Sci. 2001, 243, 90.

(22) Chen, J. F.; Wang, Y. H.; Guo, F.; Wang, X. M.; Zheng, C. Synthesis of nanoparticles with novel technology: High-gravity reactive precipitation. Ind. Eng. Chem. Res. 2000, 39, 948. (23) Shen, Z. G.; Chen, J. F.; Yun, J. Preparation and characterizations of uniform nanosized BaTiO3 crystallites by the high-gravity reactive precipitation method. J. Cryst. Growth 2004, 267, 325. (24) Chen, J. F.; Shao, L.; Zhang, C. G.; Chen, J. M.; Chu, G. W. Preparation of TiO2 nanoparticles by a rotating packed bed reactor. J. Mater. Sci. Lett. 2003, 22, 437. (25) Chen. J. F.; Li, Y. L.; Wang, Y. H.; Yun, J.; Cao, D. P. Preparation and characterization of zinc sulfide nanoparticles under high-gravity environment. Mater. Res. Bull. 2004, 39, 185. (26) Chen, J. F.; Zhou, M. Y.; Shao, L.; Wang, Y. H.; Yun, J. Feasibility of preparing nanodrugs by high-gravity reactive precipitation. Int. J. Pharm. 2004, 269, 267. (27) Zhong, J.; Shen, Z. G.; Yang, Y.; Chen, J. F. Preparation and characterization of uniform nanosized cephradine by combination of reactive precipitation and liquid anti-solvent precipitation under high gravity environment. Int. J. Pharm. 2005, 301, 286. (28) Yang, H. J.; Chu, G. W.; Zhang, J. W.; Shen, Z. G.; Chen, J. F. Micromixing efficiency in a rotating packed bed: Experiments and simulation. Ind. Eng. Chem. Res. 2005, 44, 7730. (29) Rodrı´guez, H. N.; Murphy, D. Significance of controlling crystallization mechanisms and kinetics in pharmaceutical systems. J. Pharm. Sci. 1999, 88, 651. (30) Chen, X.; Young, T. J.; Sarkari, M.; Williams, R. O., III Preparation of cyclosporine A nanoparticles by evaporative precipitation into aqueous solution. Int. J. Pharm. 2002, 242, 3. (31) Horn, D.; Rieger, J. Organic nanoparticles in aqueous phase. Angew. Chem., Int. Ed. 2001, 40, 4330.

ReceiVed for reView April 10, 2006 ReVised manuscript receiVed August 11, 2006 Accepted September 20, 2006 IE060445H