Recrystallization and Micronization of Camptothecin by the

Oct 7, 2013 - ABSTRACT: Recrystallization and micronization of camptothecin (CPT), a potent anticancer agent, has been performed using the supercritic...
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Recrystallization and Micronization of Camptothecin by the Supercritical Antisolvent Process: Influence of Solvents Guijin Liu, Hongdi Wang, and Yanbin Jiang* School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, China ABSTRACT: Recrystallization and micronization of camptothecin (CPT), a potent anticancer agent, has been performed using the supercritical antisolvent (SAS) process in this study. The effect of different solvents on the morphology, mass median diameter (Dp50), particle size distribution (PSD) and the crystallinity of obtained CPT microparticles was investigated in detail. Five pure solvents, acetic acid, dimethylformamide, chloroform, N-methyl-2-pyrrolidone, dimethyl sulfoxide (DMSO), and a series of solvent mixture of ethanol/DMSO with different volume ratios (R), where R was defined as the ethanol volume to total solvent volume, were selected as solvents. The raw CPT and processed CPT particles were characterized using SEM, laser diffraction particle size analysis, FT-IR spectroscopy, LC−MS, powder XRD, and DSC. The processed CPT microparticles were flake-like and much thinner and more uniform than the lamelliform CPT raw material. The Dp50 of the processed CPT ranged from 0.39 to 2.14 μm, and the PSD of the processed CPT microparticles became much narrower. Solvents with a higher ratio of density and viscosity, lower surface tension, and lower solvation power will form smaller CPT microparticles with lower crystallinity, which helps to improve the solubility of CPT in biological liquids. Furthermore, the addition of ethanol into DMSO improves the properties of DMSO and decreases the CPT solubility, which is beneficial to the acquisition of smaller particles, and the particle size decreased significantly with the increase of R. The chemical structure and crystalline structure of CPT did not change after the SAS process. However, the crystallinity of the obtained CPT microparticles was less than that of raw CPT, and varied for different solvents.

1. INTRODUCTION Camptothecin (CPT), a pentacyclic alkaloid, was first isolated from extracts of the Chinese tree Camptotheca acuminata by Wall and co-workers in 1966.1 Although numerous studies show that CPT is a potent agent against a wide spectrum of human cancer, therapeutic application of unmodified CPT is hindered by its poor solubility and unexpected toxicity.2−6 Because of its poor solubility, CPT was formulated as a watersoluble sodium salt (CPT-Na+) in early clinical trials,7 as shown in Figure 1. However, it is found that CPT-Na+ is inactive and

potential in the recrystallization and micronization of pharmaceuticals because of its unique properties:13−16 pharmaceutical particles with controlled particle size can be prepared and distributed, it is environmentally benign, and has low organic solvent residue and low operating temperature. Recently, micronized CPT was presented by Zhao et al.17 using the SAS process, where dimethyl sulfoxide (DMSO) was used as the solvent, and four factors, namely precipitation temperature, pressure, CPT solution concentration, and flow rate were optimized. Under the optimum conditions, micronized CPT with a mean particle size of 0.25 ± 0.020 μm was obtained, and the results suggested that the micronized CPT had a low level of crystallinity; the quasi-amorphous particles showed an enhanced solubility at about 30 μg/mL. The influence of solvents on the obtained particle properties of CPT, however, was not investigated. Normally, organic solvent is the factor that is more important than any other process variables in determining the particle morphology, particle size, and particle size distribution (PSD) in the SAS process.18−21 Reverchon et al.22,23 listed several organic solvents used in SAS experiments. The commonly used solvents are DMSO, N-methyl-2-pyrrolidone (NMP), ethanol (EtOH), methanol, dichloromethane (DCM); also chloroform (CHF), acetone, isopropyl alcohol, dimethyl formamide (DMF), formic acid, ethyl acetate, and acetic acid (AA) have been used. In some other cases, mixtures of two of these

Figure 1. The structures of CPT (a) and CPT-Na+ (b).

readily binds to human serum albumin, making it inaccessible for cellular uptake. More unfortunately, CPT-Na+ is cleared by the kidneys and causes hemorrhagic cystitis and myelotoxicity, which resulted in suspension of the trials.8,9 To overcome the limitations of solubility for pharmaceuticals, various methods have been investigated. One of the simplest but effective methods to improve the therapeutic efficacy and bioavailability of drugs is to produce micro- or nanoparticles, because the solubility and dissolution rate can be improved due to the decrease in particle size and the resulting increase in the specific surface area.10−12 The supercritical antisolvent (SAS) process has been shown to have great © XXXX American Chemical Society

Received: April 13, 2013 Revised: September 11, 2013 Accepted: October 7, 2013

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temperature and pressure was reached, pure solvent was delivered through the nozzle to the precipitation vessel for 15 min to achieve a quasi-steady state composition of solvent and CO2 in the precipitation vessel, then, the injection of the pure solvent was stopped and the CPT solution was injected to produce the CPT precipitation. At the end of the solution delivery, CO2 was kept flowing for 40 min to remove the residual solvent in the precipitation vessel. After the washing step, the precipitation vessel was depressurized gradually to atmospheric pressure. Finally, the obtained particles were collected from the bottom of the precipitation vessel for further characterization analysis. This study focuses on the effect of solvents on micronizing CPT by the SAS process. Other SAS operating parameters, which affect the obtained particle properties, were selected based on preliminary experiments and the results of Zhao et al.;17 that is, pressure = 14 MPa, temperature = 40 °C, CPT solution concentration = 1 mg/mL and feed rate = 0.8 mL/ min, and the steady flow rate of CO2 was established at 20 g/ min to make sure that the overall molar fraction of CO2 inside the vessel was larger than 0.97 for all of the experiments. 2.3. Characterization Methods. The surface morphologies of samples were observed through a scanning electron microscope (SEM) (S-3700N, HITACHI, Japan). Before observation, the particles were spread on an aluminum stub using double-sided adhesive carbon tape, and then sputtercoated with a thin layer of gold under high vacuum conditions (0.05 mTorr). The particle size and PSD of the raw CPT and processed CPT were measured by a laser diffraction particle size analyzer (Mastersizer 2000, Malvern, UK). Before each measurement, the CPT particles were suspended in pure water, then stirred under ultrasound for 15 min in order to effectively disperse. Each measurement was repeated at least three times. The particle size and PSD were expressed by the mass median diameter (Dp50) and its standard deviation (SD). Fourier transform infrared (FT-IR) spectra were obtained by using a FT-IR spectrophotometer (Nicolet Nexus 670, Thermo Electron Corporation, USA). Samples were prepared by dispersing the particles in KBr and pressing the mixture into disc form. The scanning range was 400−4000 cm−1, and the resolution was 4 cm−1. An X-ray diffractometer (D8 ADVANCE, Bruker AXS, German) with Cu-Kα radiation generated at 40 mA and 40 kV, was used for attaining X-ray diffraction (XRD) patterns of products. All samples were scanned between 5° and 50° (2θ). The thermal behavior of samples was measured by a differential scanning calorimeter (DSC) (Q200, TA Instruments, USA). The samples (2−3 mg) were weighed accurately, sealed in aluminum pans. The measurements were carried out over the heating range of 40−300 °C under a nitrogen purge; the temperature heating rate was 10 °C/min at temperature below 240 °C, and then decreased to 2 °C/min. 2.4. Measurement of the Solvation Power of Solvent. The solvation power (SP) of solvent was defined as the solubility of drug in solvent or mixture of solvents, which was measured as follows. Excess CPT was added into a tube with 5 mL of solvent. The tube was kept at 40 °C by a thermostat water bath (THD 0506, China) and stirred at 100 rpm for 6 h and then centrifuged at 2000 rpm (TDL-80-2B, China) for 10 min. After that, 10 μL supernatant was withdrawn and added into 5 mL of DMSO, and the content of CPT was detected

solvents have also been used. According to the drug structure and operating conditions, selecting a suitable solvent is crucial to the SAS process.24 However, the SAS process is extremely complex, since it involves many physical phenomena, such as the phase equilibrium, mass transfer, the fluid mechanics of the mixing between the organic solution and the supercritical antisolvent, as well as kinetics of particle nucleation and growth.25 Therefore, it is difficult to predict and explain how solvents influence the properties of obtained particles. For the formation of amorphous particles, the influence of solvents can be investigated by systematically studying the concurring time scales of jet break-up τjb and interfacial tension degradation τi.19 But, the time scales approach fails for the explanation of some crystallizing SAS systems, especially when the solute has a high solubility in the solvent, such as the paracetamol/EtOH/CO2 system, where paracetamol is the solute, EtOH is the solvent and CO2 is the antisolvent. For such systems, the saturation solubility of the solute in mixtures of solvents and antisolvents is the key parameter for the final particle characteristics.26 The aim of this work is to recrystallize and micronize CPT by the SAS process using different solvents at fixed other process parameters, and the influence of solvents on the morphology, particle size, PSD, and the crystallinity of the processed CPT microparticles by combining the solvent properties with the physical phenomena involved in the SAS process is discussed in detail.

2. EXPERIMENTAL SECTION 2.1. Materials. CPT (mass purity >99%) was purchased from Hubei Kangbaotai Fine Chemical Co., Ltd., China. CO2 (mass purity >99.9%) was purchased from Guangzhou Shengying Gas Co., Ltd., China. DMSO, DMF, NMP, CHF, AA, and EtOH with mass purity >99.5% were supplied by Guangdong Guanghua Sci. Tech. Co., Ltd., China, as well as the standard phosphate buffer saline (PBS, pH 6.86). All materials were used directly without further purification. 2.2. Apparatus and Procedure. An automatic semicontinuous SAS process (SAS50-2-ASSY, Thar Technologies, Inc., USA) was employed to carry out the micronization experiments. The flow diagram was illustrated in Figure 2, and the operating procedure was described in detail in our previous work.13,27,28 In brief, CO2 was liquefied first and continuously introduced into the precipitation vessel. When the desired

Figure 2. Flow diagram of the supercritical antisolvent (SAS) process. B

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Table 1. Properties of Solvents and Experimental Results for CPT after the SAS Treatment Using Different Solvents solvent and its properties expt. no. raw CPT 1 2 3 4 5 6 7 8 9 10 11

solvent AA DMF CHF NMP DMSO EtOH/DMSO EtOH/DMSO EtOH/DMSO EtOH/DMSO EtOH/DMSO EtOH/DMSO

R

0.2 0.3 0.4 0.5 0.6 0.7

ρ/μ (kg·m−3·cP−1)

results

σ × 102 (N·m−1)

1018 1409 3096 727 716 805 837 866 886 888 872

2.53 3.32 2.47 4.05 4.09 3.61 3.41 3.20 3.00 2.80 2.60

SP (mg/mL) 7.59 9.25 3.19 16.60 13.50 8.48 8.19 4.56 4.47 3.67 2.59

Dp50 ± SD (μm) 3.05 2.14 0.75 0.44 0.87 1.18 0.60 0.57 0.48 0.43 0.39 0.44

± ± ± ± ± ± ± ± ± ± ± ±

0.87 0.63 0.10 0.08 0.14 0.22 0.09 0.05 0.06 0.04 0.05 0.05

Figure no. of SEM Figure Figure Figure Figure Figure Figure

3a 3b 3c 3d 3e 3f

Figure 3g Figure 3h Figure 3i

Figure 3. SEM images for raw CPT and typical processed CPT using different solvents: (a) raw CPT; (b) no. 1; (c) no. 2; (d) no. 3; (e) no. 4; (f) no. 5; (g) no. 7; (h) no. 9; (i) no. 11.

with the supercritical fluid. On the basis of preliminary experiments, five pure solvents (AA, DMF, CHF, NMP, and DMSO) were selected as solvents. When the solution concentration is at 1 mg/mL, CPT is completely soluble in these five solvents. According to the report of Chen et al.,29,30 the application of organic nonsolvent in the SAS process was effective in producing smaller particles and reducing the usage of CO2. In this study, EtOH, which is one of the organic solvents with lowest toxicity and almost cannot dissolve CPT, was selected as a nonsolvent and added into the CPT solution of DMSO to study the effect of EtOH volume ratios (R) on the micronization of CPT by the SAS process, where R was defined as the ethanol volume to total solvent volume, and calculated as follow:

using an UV−vis spectrophotometer (UV-2450, Shimadzu, Japan) by measuring the absorbance at a wavelength of 366 nm. 2.5. Solubility Study in Vitro. The in vitro solubility of raw CPT and the SAS processed CPT microparticles were experimentally measured as follows. Excess sample was added into a tube with 10 mL of PBS (pH 6.86). The tube was kept at 37 °C and stirred at 100 rpm. After 18 h, a small amount of the solution was withdrawn and filtered through syringe filter with pore size of 0.22 μm. The dissolved amount of CPT in the dissolution medium was detected using an UV−vis spectrophotometer (UV-2450, Shimadzu, Japan). Every measurement was repeated three times and the data was expressed as a mean value ± SD.

3. RESULTS AND DISCUSSION 3.1. Operating Parameters and Summary of Experimental Results. For the SAS process, there is a limitation in the choice of solvents, because of the pharmaceutical agents must be soluble in a suitable organic solvent that is miscible

R= C

VEtOH VEtOH + VDMSO

(1)

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Where VEtOH and VDMSO is the volume of the corresponding solvent, and measured at room temperature (25 °C) before mixing, respectively. The major focus in this study is to investigate the effect of different solvents on the morphology, particle size, PSD, and the crystallinity of the processed CPT while fixed other process parameters. The properties of selected solvents at 40 °C were listed in Table 1. For single solvents, density (ρ) and viscosity (μ) were taken from the Perry’s Chemical Engineers Handbook,31 and surface tensions (σ) were evaluated using the Parachor method.32 For EtOH/DMSO, ρ, μ, and σ were obtained from the literature.33,34 It is worth mentioning that EtOH has lower ρ and μ than DMSO; thus, when compared with pure DMSO, the addition of EtOH to the solvent mixture causes both ρ and μ to decrease, but ρ/μ increases when R < 0.7 and then decreases at R = 0.7 because of the different ρ and μ of the solvent mixtures. The experimental results are listed in Table 1 and discussed as follows. 3.2. Effect of Solvents on Morphology, Dp50 and PSD of the Processed CPT. The SEM images of raw CPT and SAS processed CPT microparticles were shown in Figure 3, the raw CPT particles were irregular lamelliform crystals with different length and size, while the processed CPT particles were flakelike and thinner and more uniform than the raw CPT particles. For different solvents, the morphologies of processed CPT particles, mainly reflected in the thickness and size, were quite different. The CPT particles obtained by using AA, NMP, and DMSO were relatively integrated and largely flake-like, as Figure 3b,e,f shows, while, the CPT particles obtained by using DMF and EtOH/DMSO (R = 0.3) were small and imperfectly flake-like, as Figure 3c,g shows, but, as Figure 3d,h,i shows, the CPT particles obtained by using CHF and EtOH/DMSO (R = 0.5 and 0.7) were much smaller, thinner, and irregular, and showed a tendency to form aggregates because of smaller particle size with higher surface activity. Also it can be seen from Figure 3f−i that R had a great effect on the particle morphologies, the higher R is, the thinner and smaller the obtained CPT particles are. When DMSO is used as a solvent, it is worth mentioning that, different CPT morphologies, for example, spherical, irregularly spherical, and plate-like were observed by Zhao et al.;17 however, the morphology of the CPT particle obtained in this study was flake-like. The differences of the obtained CPT morphologies were mainly caused by differences of SAS equipment and operating conditions. The Dp50 of raw CPT and SAS processed CPT particles under various solvents was summarized in Table 1, it showed that the Dp50 of processed CPT particles was quite different for the different solvents, the maximum Dp50 of micronized CPT was 2.14 ± 0.63 μm, and the minimum was 0.39 ± 0.05 μm. It was also found that the SD of the particle size decreased significantly after the SAS process, which indicated that a narrower PSD was obtained after the SAS treatment. A graphical comparison for the PSD of the typical CPT samples was shown in Figure 4, which indicated that the Dp50 of processed CPT particles was much smaller than that of the raw CPT particles, and the PSD became narrower. Furthermore, the addition of EtOH into DMSO decreased the Dp50 and narrowed the PSD of the processed CPT particles shown in Figure 4d,e. From the experimental results, it can be concluded that the solvents and solvent ratios have a great effect on the morphology, Dp50, and PSD of micronized particles using the

Figure 4. The PSD of raw CPT and typical processed CPT using different solvents: (a) raw CPT; (b) no. 1; (c) no. 3; (d) no. 5; (e) no. 9.

SAS process. For different solvents and solvent ratios, the physical properties of solvent, the solubility of drug in the solvent, and the interaction between drug and solvent are different. Figure 5 shows the relationship between solvent properties (ρ/μ, σ, and SP) and the corresponding Dp50 of processed CPT; generally, it can be found that solvents with higher ρ/μ, lower σ, and lower SP are more apt to form smaller CPT particles. Reverchon et al.18 found that when a pure solvent was injected into supercritical CO2, three different phenomena can be observed using Mie- and Rayleigh scattering images: (1) jet break-up into rather large droplets (drops), (2) jet atomization into small droplets, and (3) “gas-plume” like mixing, when no droplets are formed. Higher ρ/μ means the higher Reynolds of solution at the nozzle exit, and low σ of the pure liquid shortens the elapsed time of the interface between injected solution and bulk CO2. These are beneficial to the formation of small droplets or “gas-plume”. When droplets are formed, mass transport of CO2 into the droplet and solvent evaporation into the bulk CO2 are the two phenomena that characterize the SAS process. Smaller droplets provide a larger mass transfer surface between the liquid and the gaseous phase, resulting in a faster supersaturation of the solute occurring and less time for the particle growth, which benefits the droplets to form small particles. Meanwhile, the lower SP means a higher saturation ratio of the solution at the same CPT concentration; that is, the supersaturation occurs more rapidly and at a higher level in the SAS process, which produces a much larger number of nucleuses and reduces the particle size significantly.26,29,30 Therefore, the Dp50 of processed CPT particles obtained by using CHF and DMF were much smaller than that obtained by pure DMSO and NMP. However, although AA has a relative high ρ/μ, low σ, and low SP, the Dp50 of processed CPT particles obtained by using AA was the largest. Compared to other solvents, the −COOH group of AA has more powerful noncovalent interaction with the −OH of CPT, which is dominated by hydrogen bonding. The strong interaction between AA and CPT affects the mass transfer of AA to supercritical CO2, it suggests a long time to nucleate, few nuclei for solute and slow growth of nuclei, which results in large CPT particles. Compared with DMSO, EtOH (ρ/μ = 953 kg·m−3·cP−1, σ = 2.03 × 10−2 N·m−1) has higher ρ/μ and lower σ. Thus, the properties of single DMSO can be improved to produce fine CPT particles by the addition of EtOH. More importantly, the solubility of CPT in the liquid phase is greatly decreased by the D

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Figure 6. The influence of R on Dp50.

Figure 7. FT-IR spectra for raw CPT and typical processed CPT using different solvents: (a) raw CPT; (b) no. 1; (c) no. 3; (d) no. 5; (e) no. 9.

be found that there was no significant difference between the spectra of the raw CPT and the SAS processed CPT particles using various solvents, which indicates that the chemical structure of processed CPT microparticles is the same as that of raw CPT. Figure 8 showed the LC−MS spectra of unprocessed and processed CPT. After SAS processed, CPT exhibits an unchanged molecular weight (349.1), it confirms that the SAS process has not induced the degradation of CPT, and the processed CPT microparticles do not have a solvate form. 3.3.2. Result of XRD. Figure 9 showed the XRD results of typical CPT samples. The characteristic high-intensity diffraction peaks of raw CPT at the diffraction angles of 2θ = 8.9°, 13.3°, 17.8°, and 25.6° represent the existence of its natural crystalline form. However, the processed CPT presented a diffractogram with similar diffraction angles but less peak intensity compared with that of raw CPT, it suggests that CPT particles after SAS processing are less crystalline, because the precipitation process in supercritical conditions is too fast to organize the solute in a regular crystalline form during the recrystallization.30 On the basis of the intensity of the relevant peaks, the crystallinity of processed CPT microparticles is also different for different solvents. 3.3.3. Result of DSC. To further investigate crystalline structure and crystallinity of particles, DSC analysis was performed. Figure 10 showed the DSC curves of raw CPT and typical processed samples. The raw CPT particles had a sharp peak at 255 °C and a weak peak at 249 °C. After SAS processing, microparticles obtained by using CHF and EtOH/

Figure 5. The influence of solvent properties on Dp50.

addition of EtOH, because the CPT is almost insoluble in EtOH. Thus, the mixture solvent of EtOH/DMSO is more suitable to obtain small particles than pure DMSO. Moreover, for the same final concentration of CPT solution, the increase in the volume ratio of EtOH in EtOH/DMSO will increase the saturation ratio of the CPT solution, and this helps to produce smaller particles. The influence of R on Dp50 was shown in Figure 6, which indicated that R had a great effect on the particle size and PSD. The Dp50 decreased with the increase of R, especially when the R < 0.5. 3.3. Results of Characterizations. 3.3.1. Result of FT-IR and LC−MS. FT-IR measurements were carried out to obtain information of the change of chemical structure after the SAS process. Figure 7 showed the FT-IR spectra of typical CPT samples. In these spectra, the peaks at 1040 cm−1 are due to the O−H stretching vibration, the characteristic bands at 1740 cm−1 are attributed to stretching vibration of ester and lactone carbonyl group, and the peaks at 1157 cm−1 are caused by the C−O−C asymmetry stretching vibration. From Figure 7, it can E

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Figure 8. LC−MS spectra for raw CPT and typical processed CPT using different solvents: (a) raw CPT; (b) no. 1; (c) no. 3; (d) no. 5; (e) no. 9.

lower melting temperature is mainly caused by the slow heating rate, 2 °C/min. For the particles produced by using CHF as solvent, only a weak peak at 249 °C was observed, as Figure 10c shows. Combined with its XRD pattern shown in Figure 9c, it can be concluded that the peak at 249 °C corresponds to the melting of amorphous particles. Therefore, the existence of this peak in other DSC curves means the presence of a certain amount of quasi-amorphous particles.35 For the particles produced by using AA and DMSO as solvent, the endothermic peak was shifted to 252 °C, as Figure 10b,d shows. This decrease in melting temperature is due to an increase in the available surface for thermal transfer and a decrease in CPT crystallinity.15 The increase in the width of the peak also indicates the existence of imperfect and irregular crystals, which is a natural consequence of the fast crystallization rates of SAS precipitations.35 While, for the particles produced by using EtOH/DMSO (R = 0.5) as solvent, a sharp endothermic peak at 250 °C was observed from Figure 10e. It showed that relative to pure DMSO, the addition of EtOH into DMSO decreased the melting temperature of CPT particles and the width of the peak, for the decrease in Dp50 and PSD of obtained CPT particles. Furthermore, a combination of the XRD pattern and the DSC curves shown in Figure 10d,e imply that the addition of EtOH into DMSO decreased the crystallinity of obtained CPT particles. 3.3.4. Summary of the Characterization Results. From the characterization results, it can be concluded that the solvents and solvent ratios do not change the chemical structure and crystalline structure of CPT, but affect the crystallinity of processed CPT particles. The CPT particles obtained by using CHF and EtOH/DMSO (R = 0.5) almost transformed into amorphous particles, and imperfect and irregular crystals existed in the CPT microparticles obtained by using other solvents. Solvents that have higher ρ/μ, lower σ, lower SP, and weaker interaction with CPT are more effective in improving the mass transfer between the liquid and the gaseous phase. This enhancement on the mass transfer shortens the time of the

Figure 9. XRD patterns for raw CPT and typical processed CPT using different solvents: (a) raw CPT; (b) no. 1; (c) no. 3; (d) no. 5; (e) no. 9.

Figure 10. DSC curves for raw CPT and typical processed CPT using different solvents: (a) raw CPT; (b) no. 1; (c) no. 3; (d) no. 5; (e) no. 9.

DMSO (R = 0.5) had a weak endothermic peak at 249−250 °C, and particles obtained by using other solvents had a wide endothermic peak between 249 and 255 °C. The reported melting temperature of CPT is between 264− 267 °C.1 In this study, however, the raw CPT showed a sharp endothermic melting peak at 255 °C, as Figure 10a shows. The F

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study, the residual CHF in the processed CPT particles was measured by a gas chromatograph (GC−MS 4000, Varian, USA).13 The relationship between the peak area (Y) and the CHF concentration (x) is Y = 3286x + 9.556 (R2 = 0.995). According to the standard curves, the residual CHF is 386 ppm, which is higher than the ICH limits. For the SAS process, although the residual organic solvent can be controlled by adjusting the CO2 washing time, the lower limit of ICH class II is still difficult to reach. Thus, CHF is less suitable than EtOH/ DMSO mixtures for micronizing CPT, and solvents with lower toxicity are recommended for the SAS process.

solute to organize in a regular crystalline form during the recrystallization, which is prior to the formation of particles with less crystallinity. The decreased crystallinity of processed CPT would promote its solubility, because the shifting of physical form from crystalline to amorphous would result in an excess free energy and entropy, and this inherent thermodynamic instability would favor an increase in the dissolution rate of a drug powder.15,30 3.4. Results of the Solubility Study in Vitro. The solubility of raw CPT and processed CPT in PBS (pH 6.86) was shown in Figure 11, it indicates that the raw CPT has a low

4. CONCLUSION CPT particles dissolved in various solvents were recrystallized and micronized in this study using the SAS technology. Depending on the solvents used, the processed CPT microparticles showed a variety of morphology, Dp50, and PSD. After the SAS treatment, the Dp50 of processed CPT particles was much smaller, and the PSD became narrower; the maximum Dp50 of micronized CPT was 2.14 ± 0.63 μm and the minimum was 0.39 ± 0.05 μm. Solvents with higher ρ/μ, lower σ, lower SP, and weaker interaction with drugs are more apt to form smaller and thinner particles. Smaller particles can be obtained by using EtOH/ DMSO, because the addition of EtOH improved the properties of single DMSO and decreased the solubility of CPT in the solution, and the particle size decreased significantly with increasing volume ratio of EtOH. FT-IR and LC−MS analyses showed that the chemical structure of processed CPT particles under various solvents was the same as that of the raw CPT. XRD and DSC results indicated that the crystallinity of obtained CPT particles was less than that of original CPT and varied for different solvents. Results of the solubility study in vitro proved that the solubility of CPT could be effectively increased by the SAS micronization, and CPT particles obtained by using CHF and EtOH/DMSO (R ≥ 0.5) have higher solubility than those obtained by using other solvents. But the results of solvent residue analysis indicated that CHF was less suitable than EtOH/DMSO mixtures for micronizing CPT, and solvents with lower toxicity are recommended for the SAS process.

Figure 11. Solubility of raw CPT and processed CPT in PBS (pH = 6.86).

solubility in PBS (2.5 ± 0.4 μg/mL) and its solubility can be effectively increased by the SAS micronization. The increase in solubility of processed CPT mainly attributes to the increasing specific surface area with the reduction of particle size and the decreased crystallinity. Also it can be found that the solubility of the micronized CPT particles using different solvents were quite different, the maximum solubility of micronized CPT was 12.3 ± 0.9 μg/mL, and the minimum was 6.2 ± 0.5 μg/mL. And the micronized CPT particles obtained by using CHF and EtOH/DMSO (R ≥ 0.5) have higher solubility than that obtained by using other solvents; that is, the particles with smaller particle size and less crystalline have higher solubility. 3.5. Solvent Residue Analysis for the Optimal Samples. The problem of solvent residue is also under consideration in pharmaceutical products. According to the above results, CPT particles obtained by using CHF and EtOH/DMSO (R ≥ 0.5) have smaller particle size and are less crystalline, resulting in higher solubility than that obtained by using other solvents. Thus CHF and EtOH/DMSO (R ≥ 0.5) are more suitable as solvents for micronizing CPT, and the samples produced by using CHF and EtOH/DMSO (R ≥ 0.5) were selected as the optimal samples for solvent residue analysis. DMSO and EtOH belong to ICH class III solvents with low toxicity; the result of Zhao et al.17 found that the residual DMSO content in micronized CPT is less than the limit set by the ICH of 5000 ppm for class III solvents. Furthermore, the elimination of EtOH is much easier than DMSO by supercritical CO2 because of its higher volatility, and the content of residual DMSO can be decreased by the addition of EtOH. Thus, it can be deduced that the residual DMSO and EtOH content are less than the limit of ICH class III for using EtOH/DMSO to micronized CPT. However, CHF belongs to ICH class II solvents with high toxicity, and its content should be less than 60 ppm. In this

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-20-8711-2051. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial supports from National Natural Science Foundation of China (Nos. 21076084, 21276091), Guangdong Provincial Science & Technology Project (No. 2011B050400013) and Ph.D. Programs Foundation of Ministry of Education of China (No. 20120172110010) are greatly appreciated.

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REFERENCES

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dx.doi.org/10.1021/ie401173g | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX