Tailoring the particle microstructures of gefitinib by supercritical CO2 anti-solvent process

Journal of CO₂ Utilization 20 (2017) 43–51

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Tailoring the particle microstructures of gefitinib by supercritical CO2 antisolvent process Guijin Liua,b, Qing Lina, Yinxia Huanga, Guoqiang Guana, Yanbin Jianga, a b

MARK

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School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, China Honz Pharmaceutical Co., Ltd., Haikou 570311, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Supercritical CO2 Gefitinib Microstructure Polymorph Micronization

Supercritical CO2 anti-solvent (SAS) process is a green and effective method to produce particles with designated microstructures. In this study, the particle microstructures of gefitinib, a potent anticancer agent, are tailored by the SAS process to improve its aqueous solubility. The dichloromethane/ethanol (1:4, v/v) was selected as the suitable solvent from typical solvents used in the SAS process at first. Then, the effects of other SAS operating parameters, i.e., the flow rate of gefitinib solution (F), the concentration of gefitinib in the solution (C), the precipitation pressure (P) and the temperature (T), on the gefitinib particle size were investigated in detail. Lower F, lower C, higher P and suitable T were recommended for the formation of gefitinib particles with small particle size. The properties of the raw material and SAS processed samples of gefitinib were characterized by different methods. The results showed that a new polymorphic form (Form β) of gefitinib, which present different physicochemical properties, i.e., smaller particle size, narrower particle size distribution and higher solubility, with raw gefitinib (Form 1), was captured after the SAS process. The predicted structures of gefitinib crystals, which were consistent with the experiments, were performed from their experimental XRD data by the direct space approach using the Reflex module of Materials Studio. Meanwhile, the SAS processed gefitinib particles showed much higher solubility and faster dissolution rate than that of raw gefitinib, which had the potential to improve its bioavailability and decrease the dose-related adverse effects.

1. Introduction Gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor [1,2], has been approved by FDA in 2015 for the first-line treatment of patients with metastatic non-small cell lung cancer [3]. Gefitinib is a lipophilic dibasic compound with pKa values of 5.28 and 7.17, its chemical structure is shown in Fig. 1. Gefitinib is practically insoluble in aqueous solvents with pH > 7, which limits its oral absorption along the intestine, resulting in a low bioavailability [4,5]. Also, due to its poor aqueous solubility, a high dose is required during clinical utilization of gefitinib, which may lead to the dose-related adverse effects, such as vomiting, diarrhea, nausea, etc [6,7]. Therefore, improving the aqueous solubility is necessary for widening the therapeutic window of gefitinib. To overcome the limitations of solubility for pharmaceuticals, various strategies have been investigated, such as prodrug [8], micronization [9,10], crystal engineering [11], solid dispersions [12] or incorporation formulations [13], etc. For any strategies, the drug solubility and dissolution rate is directly influenced by the particle

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Corresponding author. E-mail address: [email protected] (Y. Jiang).

http://dx.doi.org/10.1016/j.jcou.2017.04.015 Received 22 January 2017; Received in revised form 26 April 2017; Accepted 30 April 2017 Available online 16 May 2017 2212-9820/ © 2017 Elsevier Ltd. All rights reserved.

microstructures, including morphology, particle size, particle size distribution, crystal form, etc. Theoretically, a decrease in particle size will lead to an increase in effective surface area in the diffusion layer, which, in turn, increases the drug dissolution rate [14]. Crystalline polymorphs show significant effects on the solubility and bioavailability of drug products [15,16]. Thus, tailoring the particle microstructures is a fundamental method to improve the therapeutic efficacy and bioavailability of poorly water-soluble drugs. However, major advances in drug manufacture have highlighted the limitations of conventional particle formation processes in fine-tuning the characteristics required, due to the harsh processing conditions and poor properties of products [17]. The conventional techniques also face some problems, e.g., thermal and chemical degradation of products, large amounts of solvent use and residues. Supercritical CO2 antisolvent (SAS) process, as an alternative strategy of traditional technologies [18,19], offers a simpler and better control process for the development and production of nano- or micro- particle drugs, such as spherical microparticles of sulfasalazine [20], indomethacin amorphous solid dispersions [21], rifampicin-loaded submicron-sized parti-

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Table 1 Summary of operating conditions and corresponding results.

Fig. 1. Chemical structure of gefitinib, where carbon is marked in grey, oxygen in red, nitrogen in blue, fluorine in cyan, chlorine in green and hydrogen in white. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Runs

F (mL/min)

C (mg/mL)

P (bar)

T (°C)

Dp50 ± SD* (μm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1.0 0.5 1.5 2.0 2.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

1.0 1.0 1.0 1.0 1.0 0.5 1.5 2.0 2.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

90 90 90 90 90 90 90 90 90 100 110 120 130 90 90 90 90

40 40 40 40 40 40 40 40 40 40 40 40 40 30 35 45 50

1.73 0.60 1.99 2.43 2.51 1.50 1.88 2.11 2.31 1.51 1.30 1.00 0.84 2.34 2.00 1.89 1.99

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.088 0.068 0.091 0.076 0.080 0.061 0.077 0.078 0.081 0.089 0.056 0.067 0.069 0.070 0.066 0.053 0.073

solubility and exhibited dramatic improvement in anti-tumour efficacy [30]. The aim of this work is to tailor the particle microstructures of gefitinib by the SAS process for improving its aqueous solubility. The suitable organic solvent was selected at first. Then, the influences of other SAS operating parameters on particle size were investigated in detail. The properties of the raw material and SAS processed samples of gefitinib were characterized by different methods. The crystal structures were predicted by the molecular simulation. The aqueous solubility of the raw and SAS processed samples of gefitinib in vitro were also evaluated. 2. Materials and methods 2.1. Materials Gefitinib (mass purity fraction > 99%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., China. CO2 (mass purity > 99.9%) was purchased from Guangzhou Shengying Gas Co., Ltd., China. Dichloromethane (DCM), ethanol (EtOH), dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) were analytical grade (Guangdong Guanghua Sci. Tech. Co., Ltd., China). All materials were used directly without further purification. 2.2. Apparatus and procedure An automatic semi-continuous SAS process (SAS50-2-ASSY, Thar Technologies, Inc., USA) is employed to tailor the particle microstructures of gefitinib. The flow diagram is illustrated in Fig. 2, and the operating procedure has been described in detail in our previous work [29,31–33]. In brief, CO2 is liquefied firstly and continuously delivered via a high-pressure pump, where the flow rate of CO2 was established at 20 g/min on the basis of the device capability. Before entering the injector, the stream of CO2 was preheated by a heat exchanger. When reaching the desired temperature (T) and pressure (P), pure solvent is charged at a given flow rate (F) by another high-pressure pump and sprayed into the precipitation vessel through a nozzle (0.5 mm) for 15 min to achieve a quasi-steady state composition of solvent and CO2 in the precipitation vessel. Then, the gefitinib solution is injected instead of pure solvent to produce the gefitinib precipitation. An ultrafiltration membrane (0.22 μm) and a metal filter (5 μm) are located at the bottom of the precipitation vessel for particle collection. At the end of the solution delivery, CO2 is kept flowing for 40 min to remove the residual solvent. After the washing step, the precipitation vessel is depressurized gradually. Finally, the obtained particles are collected from the wall and bottom of the precipitation vessel.

Fig. 2. Flow diagram of the supercritical CO2 antisolvent (SAS) process.

cles [22], cocrystals-pure powders of naproxen and nicotinamide [23], primidone microcrystals [24], et al. The particle microstructures can be tailored by controlling the SAS operating parameters, which indirectly influence the interactions among high-pressure vapor-liquid equilibria, surface tension variations, jet fluid dynamics, mass transfer, nucleation and growth [25]. These published works have demonstrated that SAS process holds great promise in particle design. SAS process can effectively decrease the particle size, or modify the crystal form of the polymorphic drugs, or produce amorphous particles to enhance the dissolution rate and solubility of poorly water-soluble drugs [26–28]. In our previous study, the particle microstructures of 10-hydroxycamptothecin (HCPT) were tailored using the SAS process. The crystal form of raw HCPT was changed from pancake-like form to prismatic form and needle-like form after SAS processing [29], where the needlelike form with a larger surface-to-volume ratio had a dramatically higher 44

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Fig. 3. Effects of SAS operating parameters on particle size of gefitinib.

gefitinib was not easy to obtain, the structure was determined from experimental XRD by the module of Reflex [34]. The simulated XRD was compared with the experimental XRD, and molecular arrangement in the crystal structure of gefitinib particles was obtained according to the work of Huang et al. [35]. First, the selected experimental XRD pattern was pretreated and obtained the characteristic peaks by the powder index program, where the unreasonable peaks were manually deleted. The Dicvol 91 method was then selected for indexing. According to the value of the relative figure of merit, the appropriate cell parameters were screened and an empty cell was created. Second, the obtained cell and function profile parameters were refined by using the Pawley method, and the space group was selected from the candidates according to the figure of merit value. Third, after minimization using Discover module, the gefitinib 3D molecule was integrated into the empty cell. The Powder Solve module was applied to solve the structure with Monte Carlo simulated annealing method. The refinement procedure was performed using Rietveld refinement from the powder refinement tool to fit the experimental powder pattern.

2.3. Particle characterization The particle size of gefitinib particles were measured by a laser diffraction particle size analyzer (Mastersizer 2000, Malvern, UK). Before each measurement, the samples were suspended in pure water and stirred ultrasonically for 15 min to disperse effectively. Every measurement was repeated at least three times. The particle size was expressed by the mass median diameter (Dp50) and its standard deviation (SD). The morphology of gefitinib particles was observed by scanning electron microscopy (SEM) (Zeiss MERLIN Field Emission SEM, Carl Zeiss NTS GmbH, Ger.). Before observation, the particles were spread on an aluminium stub using double-sided adhesive carbon tape and then sputter-coated with a thin layer of gold under high vacuum conditions (0.05 mTorr). X-ray diffraction (XRD) patterns of gefitinib particles were attained by an X-ray diffractometer (D8 ADVANCE, Bruker AXS, Ger.) with CuKα radiation generated at 40 mA and 40 kV. All samples were scanned between 5° and 50° (2θ). The diffraction patterns were processed using JADE 5.5 software. Thermogravimetric (TG) and Differential Scanning Calorimetry (DSC) analysis were performed using a STA449C thermal analyzer (Netzsch, Ger.) in N2 atmosphere, and the scanning rate was 10 °C/min in the temperature range of 40 °C–500 °C. Fourier transform infrared (FT-IR) spectra of gefitinib particles 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.

2.5. Solubility measurement The in vitro solubility of gefitinib particles were experimentally measured as follows. Excess sample was added into a tube with 10 mL ultra-pure water (pH 7.0). The tube was kept at 37 °C using a thermostat water bath (THD 0506, Ningbo Tianheng Co., CHN) and stirred at 100 rpm. After 18 h, a small amount of the solution was withdrawn and filtered through a syringe filter with a pore size of 0.22 μm. The dissolved amount of gefitinib in the dissolution medium was detected at 350 nm using a UV/vis spectrophotometer (UV-2450, Shimadzu, Japan). Each measurement was repeated three times, and the average value was calculated.

2.4. Simulation of the crystal structures Materials studio (Accelrys, Inc., USA) was selected to calculate the crystal structures of gefitinib particles. Because the single crystal of 45

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Fig. 4. SEM images of raw gefitinib (a), gefitinib particles obtained in run 2 (b), run 4 (c) and run 12 (d) of Table 1, and their particle size distribution patterns (e).

2.6. Study of in vitro dissolution rates

3. Results and discussion

The in vitro gefitinib dissolution rates were investigated using the dialysis bag diffusion technique [30,36]. Briefly, the raw and micronized gefitinib particles were weighted accurately and dispersed into a dialysis bag with 4 mL phosphate buffer saline (PBS, pH 7.4). Then the end-sealed dialysis bags were incubated in 400 mL of PBS at 37 °C and 100 rpm using a thermostat water bath (THD 0506, Ningbo Tianheng Co., China). At fixed time intervals, 4 mL of release medium was withdrawn and replaced with an equal volume of the fresh PBS. The concentration of each sample was determined by ultraviolet spectrophotometry (UV-2450, SHIMADZU, Japan).

3.1. Selection of solvent Organic solvent is the most important factor in tailoring particle microstructures by the SAS process [37]. The used solvent should dissolve the pharmaceutical agents well, and be miscible with the supercritical CO2 for the SAS process. Based on our previous work [38] and the solubility of gefitinib, three pure solvents (DMSO, DMF and DCM) and mixture solvents of DMSO/EtOH (1/1, v/v), DMF/EtOH (1/ 1, v/v) and DCM/EtOH (1/4, v/v), were selected as the candidate solvents for preliminary study. The other operating conditions were F = 1 mL/min, gefitinib solution concentration (C) = 1 mg/mL, 46

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experimentally that the particle size is the most crucial index affecting the drug solubility and dissolution rate [46]. Thus, the influences of various parameters on particle size of gefitinib were investigated in this study. The experimental design and corresponding results are shown in Table 1, where the level of each factor is selected on the basis of our previous study [29,35] and the results of preliminary experiments. For all of the experiments, gefitinib is completely soluble in the used solvent and the overall molar fraction of CO2 inside the vessel is larger than 0.90. It can be found that the Dp50 of processed gefitinib particles varies between 0.60 and 2.51 μm. Fig. 3 shows the effects of SAS operating parameters on particle size of gefitinib, where Fig. 3(a–d) represent the effects of F, C, P and T on Dp50 of gefitinib respectively. From Fig. 3(a), it can be seen that lower F leads to smaller Dp50. Because lower F means higher antisolvent/ solvent ratio in the precipitation vessel, resulting in a faster supersaturation of the solute occurring, which causes fast nucleation and formation of small particles [27]. Fig. 3(b) shows that the particle size of gefitinib increases with the increase of C. According to the formation mechanism “one droplet one particle” under the critical condition [47], higher C means the droplets contain more gefitinib, supplying sufficient solute for the growth of particles. As a result, bigger size particles are produced at higher C. Fig. 3(c) shows that smaller gefitinib particles are produced at higher P. Once the droplets have been formed inside the precipitation vessel, rapid transfer of CO2 into these droplets and the solvent out of these droplets causes the droplets to expand rapidly. Higher P increases the diffusion driving force and solubility of solvent as the volumetric expansion of the liquid phase [48], which causes significant reduction in partial molar volume and cohesive energy density of the solvent, lowering its solvent power for gefitinib, and substantially causes higher degree of supersaturation, resulting in precipitation of particles with smaller particle size [43,49]. Fig. 3(d) shows that Dp50 decreasing to a minimum at T was about 40 °C. Increasing T beyond this point tends to increase Dp50 by a small amount. Two opposite effects of T on particle size of gefitinib depending on whether a diffusivity rate or saturation effect prevails. Increasing T can enhance the diffusion and mass transfer efficiency between supercritical CO2 and solution droplets, resulting in the formation of small particles. But the solubility of gefitinib increased with increasing T, meaning that saturation is reached slowly and larger particles are formed [50]. The above results indicated that the particle size of gefitinib can be tailored effectively by controlling SAS operating parameters. The SEM images and particle size distribution patterns of typical samples are showed in Fig. 4, which indicate that the morphology and particle size of gefitinib particles change greatly after the SAS process. As shown in Fig. 4(a), the raw gefitinib particles are large blocky crystals with different length and size. While, the SAS processed gefitinib particles become small and much uniform. Fig. 4(e) demonstrates that the gefitinib particles obtained by the SAS process present much smaller particle size and narrower distribution compared with the raw gefitinib particles. The gefitinib sample obtained in run 2 of Table 1 has the smallest Dp50, which was selected as the optimal sample in this study.

Fig. 5. FT-IR spectra of raw gefitinib (a) and gefitinib particles obtained in run 2 of Table 1(b).

P = 90 bar and T = 40 °C. The preliminary experiments were failed when using DMSO, DMF, DMSO/EtOH (1/1, v/v) and DMF/EtOH (1/1, v/v) as solvents, where very little or none particles were obtained in the precipitation vessel. This may be because that the operating conditions near or slightly higher than the mixture critical point of the system of CO2 with these solvents. The precipitation is carried out at subcritical conditions, the formation of particles is controlled by the fluid mechanics and the kinetics of evaporation of the solvent [39]. Moreover, these solvents have good solvation power for gefitinib and high surface tension, hence the difficulties for supersaturation and precipitation of gefitinib from the solution. Fortunately, the experimental phenomena showed that fine gefitinib particles were obtained on the wall and bottom of the precipitation vessel, when using the DCM and DCM/EtOH (1/4, v/v) as solvents. Compared with pure DCM, EtOH has lower toxicity and almost can not dissolve gefitinib. According to the report of Chen et al. [40,41], the application of organic nonsolvent in the SAS process was effective in producing smaller particles and reducing the usage of CO2. Therefore, the DCM/EtOH (1/4, v/v) was selected as the prior solvent in this study. 3.2. Effect of operating parameters on particle size Once the solvent had been screened, the particle microstructures of gefitinib were tailored by controlling the SAS operating parameters, i.e., F, C, P and T. The effect of SAS operating parameters on the obtained particle microstructures has been investigated in many published works [42–45]. Rational explanations and influence mechanisms of operating parameters on particle microstructures have been reported in most experimental studies, which involve complex thermodynamics and fluid dynamics. Unfortunately, a fairly general influence rule for different drugs is not available up to now. It has been observed

Fig. 6. XRD patterns of raw gefitinib (a) and gefitinib particles obtained in run 2 of Table 1(b).

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Fig. 7. DSC, TG and DTG curves of raw gefitinib (a) and gefitinib particles obtained in run 2 of Table 1(b).

XRD characterization were conducted to determine the crystal structure of gefitinib, and the XRD patterns of raw gefitinib and gefitinib particles obtained in run 2 of Table 1 were shown in Fig. 6. Two crystal structures of gefitinib available from the Cambridge Structural Database (CSD) are solvent-free (Form 1) and trihydrate forms [52]. And a metastable polymorph (Form II) was captured in 2014 by Thorat et al. [53] As shown in Fig. 6(a), the characteristic highintensity diffraction peaks of raw gefitinib at the diffraction angles of 2θ = 7.2°, 16.1°, 19.5°, 24.1°, 27.0°, 36° and 44.6° reveal the existence of its natural crystalline form (Form 1). While, the XRD pattern of gefitinib particles obtained in run 2 of Table 1 shows diffraction peaks at 2θ = 6.3°, 13.0°, 14.0° and 26.1° (Fig. 6(b)), which is differ with that of raw gefitinib and other known crystalline forms. This suggested that a novel polymorph of gefitinib (defined as Form β) was perhaps obtained by the SAS process. DSC and TG analysis were performed to further confirm the polymorphs, and the results were shown in Fig. 7. The DSC and TG curves of raw gefitinib is in accordance with the characterization results of Thorat et al. [53]. DSC analysis of raw gefitinib shows a single sharp endotherm centred at 198.7 °C, corresponding to the melting point of the crystals. However, the DSC profile of the SAS processed gefitinib particles exists a small peak at 108.3 °C, before the melting point of 197.9 °C, suggesting a possible polymorphic transformation before melting. To confirm that, the XRD pattern of SAS processed gefitinib particles after storage at 110 °C for 10 h was characterized. As shown in Fig. 8, the XRD pattern shows obvious differences with that before heating (Fig. 6(b)), where both the characteristic diffraction peaks of Form 1 and Form β are existed simultaneously. This result indicates the conversion of the Form β to Form 1 crystals at the transition temperature. Furthermore, the TG and its derivative (DTG) curves of raw gefitinib and SAS processed gefitinib particles in Fig. 7 show a similar onset temperature for degradation around 300 °C and no relative weight loss before this temperature, suggesting that there is no structural water or solvent in them. Thus, it is corroborated by DSC and TG results that the formation of novel polymorph of gefitinib (Form β) after the SAS process, where Form β is solvent-free and not thermally stable. The XRD

Fig. 8. The XRD pattern of gefitinib particles obtained in run 2 of Table 1 after storage at 110 °C for 10 h. *The characteristic diffraction peaks of Form 1, **the characteristic diffraction peaks of Form β.

After that, its particle properties were compared with that of raw gefitinib. 3.3. Properties of the gefitinib particles FT-IR spectroscopy was carried out to determine the change of chemical structure of gefitinib before and after the SAS process. Fig. 5 displays the FT-IR spectra of the raw gefitinib and SAS processed gefitinib sample. The gefitinib spectrum exhibits strong absorption bands at 1625 cm−1 (C]C, C]N), 1500 cm−1 (HC]CH, aryl), 1110 cm−1 (CeO) and 1028 cm−1 (CeF), which is in accordance with the previous report [51]. Moreover, it can be found that there is no significant difference between the spectra of the raw gefitinib and the SAS processed gefitinib samples, which indicates that the chemical structure of processed gefitinib particles is the same as that of raw gefitinib. While, there are some minor differences around the wavenumber of 830 cm−1, 1040 cm−1 and 1130 cm−1, which are possibly caused by the different crystal structure of gefitinib.

Fig. 9. The XRD patterns (a), DSC, TG and DTG curves (b) of gefitinib particles obtained in run 12 of Table 1.

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Fig. 10. Comparison of calculated and experimental XRD patterns (a, b) and three-dimensional crystal structures (c, d) of gefitinib samples.

Table 2 Crystal lattice parameters of Gefitinib. Cell parameters

Form 1

Form β

Chemical formula Formula weight Sample type Crystal system Space group a/Å b/Å c/Å α/(°) β/(°) γ/(°) V/(Å3) Z Rwp, Rp

C22H24ClFN4O3 446.9 Powder Triclinic P-1 7.88 9.70 12.54 93.51 97.36 101.70 1043.67 2 0.247, 0.178

C22H24ClFN4O3 446.9 Powder Tetragonal P-4 8.88 8.88 13.68 90 90 90 1080.17 4 0.175, 0.159 Fig. 11. Solubility of raw gefitinib and typical SAS processed gefitinib particles in purity water (pH = 7) at 37 °C.

patterns, DSC, TG and DTG curves of another sample obtained in run 12 of Table 1 were shown in Fig. 9, which were almost consistent with that of run 2. This indicates that Form β can be obtained under other operating parameters by the SAS process.

XRD patterns are consistent with the experimental XRD patterns, although the characteristic peak intensities and positions exhibited few differences between the two patterns. Thus, it further confirms that gefitinib has different polymorphs, and new gefitinib polymorphs could be obtained by the SAS process. Fig. 10(c, d) show the final simulated three-dimensional (3-D) crystal structures of Form 1 (raw gefitinib) and Form β (run 2). the relative calculated crystal lattice parameters are listed in Table 2. The simulation results show that the crystal of Form 1 belongs to the triclinic system and space group P-1 with 2 molecules in a unit cell and the crystal of Form β belongs to the tetragonal system and space group P-4 with 4 molecules in a unit cell.

3.4. Predictions of the crystal structure Computer simulation is an important prediction method for study of drug polymorph. In this work, the crystal structures were illustrated by molecular simulation based on the experimental XRD results using the direct space approach in conjunction with the Monte Carlo method, followed by Rietveld refinement. The calculated and experimental XRD patterns of Form 1 (raw gefitinib) and Form β (run 2), as well as their differences, are shown in Fig. 10(a, b). It indicates that the calculated 49

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microstructures of gefitinib by the SAS process can enhance its aqueous solubility and dissolution rate, which is expected to be favorable for improving the drug potency, reducing its toxicity and side-effects. Acknowledgements Financial support from the National Natural Science Foundation of China (Nos. 21276091, 21476086, 91434126) and Guangdong Natural Science Foundation (No. 2014A030312007) is greatly appreciated. References [1] J.A. Engelman, K. Zejnullahu, T. Mitsudomi, Y. Song, C. Hyland, J.O. Park, N. Lindeman, C.-M. Gale, X. Zhao, J. Christensen, T. Kosaka, A.J. Holmes, A.M. Rogers, F. Cappuzzo, T. Mok, C. Lee, B.E. Johnson, L.C. Cantley, P.A. Jänne, MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling, Science 316 (2007) 1039–1043. [2] C. Gridelli, F. De Marinis, M. Di Maio, D. Cortinovis, F. Cappuzzo, T. 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Subra-Paternault, Challenge of the supercritical antisolvent technique SAS to prepare cocrystal-pure powders of naproxen-nicotinamide, Chem. Eng. J. 303 (2016) 238–251. [24] H.-H. Chen, C.-S. Su, Recrystallizing primidone through supercritical antisolvent precipitation, Org. Process. Res. Dev. 20 (2016) 878–887. [25] E. Reverchon, I. De Marco, Mechanisms controlling supercritical antisolvent precipitate morphology, Chem. Eng. J. 169 (2011) 358–370.

Fig. 12. Dissolution profile of raw gefitinib and gefitinib particles obtained in run 2 of Table 1 in PBS (pH = 7.4) at 37 °C.

3.5. Results of the solubility and dissolution rate study in vitro Numerous studies show that particle size has a close correlation with solubility for poorly water-soluble drugs [9]. It is well known that the solubility of crystalline materials becomes enhanced when the particle size is reduced to submicron levels, due to the Gibbs-Thomson effect (Eq. (1))[54].

ln

2Mγ s (r ) = s* vRTρr

(1)

where s(r) is the solubility of particles of radius r, s* is the normal equilibrium solubility of the substance, R is the gas constant, T is the absolute temperature, ρ is the density of the solid, M is the molar mass, γ is the interfacial tension and ν represents the number of separate species in solution phase formed from one mole of solute. Fig. 11 shows the solubility of the raw gefitinib and typical SAS processed gefitinib particles in pure water (pH = 7). It indicates that the SAS processed gefitinib samples have relatively higher solubility (≥6.49 μg/mL) than that of the raw gefitinib sample (2.55 μg/mL). And the SAS processed gefitinib sample obtained in run 2 of Table 1 shows higher solubility than that of other samples, due to its much smaller particle size. Moreover, the SAS treated gefitinib particles yield a much higher dissolution rate than that of raw gefitinib particles (Fig. 12). According to Noyes-Whitney equation [14], particle size reduction leads to an increase in effective surface area in the diffusion layer, which, in turn, increases the drug dissolution rate. The increased solubility and dissolution rate of the SAS processed gefitinib particles has the potential to improve its bioavailability and decrease the doserelated adverse effects. 4. Conclusion Gefitinib particles with designated microstructures were successfully produced by the SAS process using DCM/EtOH (1/4, v/v) as solvent in this study. Smaller gefitinib particles with narrower distribution are obtained after the SAS process, where the particle size of gefitinib can be tailored effectively by controlling the SAS operating parameters. FT-IR analyses show that there is no significant change in the chemical structure of the processed gefitinib particles. XRD, DSC and TG results indicate that a new polymorph of gefitinib (Form β) is generated from Form 1 of raw gefitinib after the SAS process. Form β is solvent-free and not thermally stable, which can convert itself again into Form 1 when the T higher than its transition point (around 110 °C). The simulation results show that Form 1 and Form β belong to the triclinic system and tetragonal system respectively. The results of in vitro solubility and dissolution rate test indicate that tailoring particle 50

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