Preparation and characterization of azithromycin – Aerosil 200 solid dispersions with enhanced physical stability

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IJP 14738 1–10 International Journal of Pharmaceutics xxx (2015) xxx–xxx

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Pharmaceutical nanotechnology

Preparation and characterization of azithromycin – Aerosil 200 solid dispersions with enhanced physical stability Xuechao Li, Huanhuan Peng, Bin Tian, Jingxin Gou, Qing Yao, Xiaoguang Tao, Haibing He, Yu Zhang, Xing Tang, Cuifang Cai * Department of Pharmaceutics, Shenyang Pharmaceutical University, Shenyang, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 November 2014 Received in revised form 26 January 2015 Accepted 16 March 2015 Available online xxx

The main purpose of this study was to investigate the feasibility of azithromycin (AZI) – Aerosil 200 solid dispersions specifically with high stability under accelerated condition (40
C/75% RH). Ball milling (BM) and hot-melt extrusion (HME) were used to prepare AZI solid dispersions. The physical properties of solid dispersions were evaluated by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). For solid dispersions prepared with both methods, no crystalline of AZI was detected (except for AZI: Aerosil 200 = 75:25) by DSC or PXRD, indicating the amorphous state of AZI in solid dispersions. The FT-IR results demonstrated the loss of crystallization water and the formation of hydrogen bonds between Aerosil 200 and AZI during the preparation of solid dispersions. After 4 weeks storage under accelerated condition, the degree of crystallinity of AZI increased in solid dispersions prepared by BM, whereas for solid dispersions containing AZI, Aerosil 200 and glyceryl behenate (GB) prepared by HME, no crystalline of AZI was identified. This high stability can be attributed to the hydrophobic properties of GB and the presence of hydrogen bonds. Based on the above results, it is inferred the protection of hydrogen bonds between AZI and Aerosil 200 formed during preparation process effectively inhibited the recrystallization of AZI and improved the physical stability of amorphous AZI in the presence of Aerosil 200. ã 2015 Published by Elsevier B.V.

Keywords: Solid dispersion Physical stability Ball milling Hot-melt extrusion Aerosil 200

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1. Introduction The concept of a solid dispersion was first introduced in 1961 (Keiji and Noboru, 1961) and, subsequently, solid dispersions have been studied extensively (Djuris et al., 2013; Ghosh et al., 2011; Yang et al., 2010). In a solid dispersion, the active pharmaceutical ingredient (API) may exist in the following forms: crystal, amorphous and/or a molecular state (Qi et al., 2008). When a given drug is present only in molecular form in the carrier, a thermodynamically stable system is generated which is usually called a solid solution. The dissolution rate of the dissolved drugs is determined by the dissolution rate of the carriers (Christian and Jennifer, 2000). However, the low solubility of most drugs in many carriers restricts the drug loading to a relatively low level. When a given drug is present in an amorphous and/or crystalline state in a

* Corresponding author at: Department of Pharmaceutics, Shenyang Pharmaceutical University 32#, 103 Wenhua Road, Shenyang 110016, China. Tel.: +86 24 2398 4395. E-mail address: [email protected] (C. Cai).

solid dispersion, the recrystallization and Ostwald ripening phenomenon may lead to a lower than expected dissolution rate in vitro and bioavailability in vivo (Fule and Amin, 2014; Sotthivirat et al., 2013). Among these forms, amorphous solid dispersions are of great interest in the pharmaceutical field (Fahr and Liu, 2007; Vasconcelos et al., 2007). However, amorphous solid dispersions are rarely used due to chemical and physical stability issues. Therefore, there is an urgent need to find a promising and convenient method to improve the stability of amorphous solid Q3 dispersions. Acceptable physical stability is one of the basic requirements for an amorphous solid dispersion (Guo et al., 2013). Firstly, it is generally agreed that a high glass transition temperature (50
C higher than the storage temperature) can minimize the molecular mobility of an amorphous solid dispersion (Hancock and Shamblin, Q4 1995). So a novel mixing process called KinetiSol1 Dispersing (KSD) had been used to prepare amorphous solid dispersions without the aid of a plasticizer, thus providing an increased glass transition temperature and an enhanced physical stability (DiNunzio et al., 2010). Secondly, the intermolecular interaction between drugs and polymers, such as hydrogen bonds and acid-base ionic reactions, can

http://dx.doi.org/10.1016/j.ijpharm.2015.03.029 0378-5173/ ã 2015 Published by Elsevier B.V.

Please cite this article in press as: Li, X., et al., Preparation and characterization of azithromycin – Aerosil 200 solid dispersions with enhanced physical stability. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.03.029

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X. Li et al. / International Journal of Pharmaceutics xxx (2015) xxx–xxx Table 1 Formulations of solid dispersions.

Fig. 1. The chemical structure of azithromycin.

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not only lower the enthalpy during the mixing process, but also elevate the glass transition temperature of the system, thereby reducing the molecular mobility (Painter and Graf, 1991). Among these methods, KSD requires unique processing equipment and the Q5 formation of a salt bond needs the presence of a carboxyl group and a hydroxyl group in the system, so hydrogen bond is usually used to improve the physical stability of amorphous solid dispersions (Zhang et al., 2013). In this study, AZI was selected as the model drug. As shown in Fig. 1, the hydroxyl group in AZI molecule provides a good opportunity for hydrogen bonding with other excipients. Aerosil 200, acting as a stability enhancer, was introduced to the system to allow hydrogen bonding with AZI. According to the description of Evonik industries, Aerosil 200 is a hydrophilic fumed silica with large specific area (200  25 m2/g, as determined by the BET method). The large number of silanol groups on its surface act as a proton donating or accepting group during the formation of hydrogen bonds (Takeuchi et al., 2004). So we hypothesized that during the molecular-level mixing process, hydrogen bonds may be formed between AZI and Aerosil 200, thereby maintaining the stability of amorphous AZI in the solid dispersion. In order to achieve molecular-level mixing without the aid of an organic reagent, both BM and HME were selected to prepare amorphous AZI solid dispersions. It has been well documented that various kinds of ball milling were used to prepare solid dispersions containing drugs and inorganic materials (such as Aerosil or Neusilin) (Maclean et al., 2011; Gupta et al., 2003; Nakahashi et al., 2014). During the co-milling process, drugs can be mixed with inorganic materials uniformly and molecular interaction may (such as acid-base interactions or hydrogen bonds) occur. HME is a convenient and continuous industrial technique for the preparation of solid dispersions without the presence of an organic solvent (Sarode et al., 2012). During the heating process, crystalline drugs Q6 melt and the thermoplastic polymers become soft. With the high shear stress and kneading effect, each part was mixed uniformly and molecular interactions may also occur (Puncochova et al., 2014). The objective of this study was to investigate whether Aerosil 200 had the stabilizing effect on the amorphous AZI in solid dispersions under accelerated condition. BM and HME were used to prepare the amorphous AZI solid dispersions. All the samples were stored under accelerated condition for 4 weeks. Furthermore, the underlying mechanism was also elucidated based on the results obtained with different samples.

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2. Materials and methods

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2.1. Materials

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Azithromycin dihydrate was purchased from Yidu HEC Biochemical Pharmaceutical Co., Ltd., China. Aerosil 200 was obtained from Evonik Degussa (SEA) Pet. Ltd., Germany. Glyceryl behenate

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Formulation

AZI dihydrate (g)

Glyceryl behenate (g)

Aerosil 200 (g)

A B C D E

50 60 75 50 50

– – – 50 30

50 40 25 – 20

(GB, Compritol1 888 ATO) was provided by Gattefosse Trading Co., Ltd., All the materials used were of analytical or HPLC grade.

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2.2. Methods

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2.2.1. Solid dispersion preparation Two commonly used methods have been followed for preparation of AZI solid dispersions. Using ball milling method, solid dispersions of formulation A–C as shown in Table 1 were produced by a table model ball mill. The sample container was cylindrical in shape and the diameter of mill beads ranged from 1 to 3 cm. After 5 min pre-mixing of all components in a polyethylene bag, the physical mixture was then transferred to the sample container and milled for 24 h at room temperature. For formulation A, samples were taken out at predetermined intervals (0.25, 1, 6, 12 and 24 h) and DSC, PXRD and FT-IR measurements were carried out. The solids was sifted through an 80-mesh sieve and stored in desiccators prior to use. With hot-melt extrusion technique, solid dispersions of formulation D and E were prepared. A co-rotating twin-screw extruder TE20 (Coperion Keya Co., China) was used to prepare the amorphous AZI solid dispersions containing GB. From feeder to die, the barrel can be divided into 4 zones, the temperature of the four zones was set at 140
C and the extrusion rate was set at 3.5 Hz. After cooling at room temperature, all extrudates were collected and milled in a laboratory cutting mill. After grinding, the powder between 80 and 100-mesh was collected and stored in desiccators for further study.

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2.2.2. Differential scanning calorimetry (DSC) DSC measurements were carried out using a Thermal Anacyzer60WS (Shimadzu, Japan). Samples approximately 3–5 mg were accurately weighed and put into sealed aluminum pans with a pin hole in the lid. All samples were heated at a heating rate of 10
C/ min in an atmosphere of nitrogen from 30
C to 150
C. The degree of crystallinity (DOC) can be calculated according to the following equation (Chen and Hwang, 1995):

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DOCð%Þ ¼

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DH s  100% DHAZI  W

where, DHs and DHAZI are the melting enthalpy of the test sample and pure AZI, respectively. W is the weight fraction of AZI in the solid dispersions.

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2.2.3. Scanning electron microscopy (SEM) All solid dispersions were examined by scanning electron microscopy (Hitachi SU8010, Japan). The samples were sputtercoated with gold immediately prior to examination. An accelerating voltage of 15 or 20 kV was used.

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2.2.4. Powder X-ray diffraction (PXRD) PXRD was used to examine the crystallinity of AZI in amorphous solid dispersions. All the samples were analyzed using a D/Max2400 diffractometer (Rigaku, Osaka, Japan). These samples were exposed to Cu-Ka radiation and standard runs were performed using a voltage of 56 kV, a current of 182 mA and a scanning rate of 2
/min over a 2u range from 5–45
.

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2.2.5. Fourier transform infrared spectroscopy (FT-IR) FT-IR experiments were also carried out with KBr disk method. The infrared spectra were recorded in the FTIR (Bruker IFS-55, Germany) instrument in the wavelength region between 4000 and 400 cm1. 2.2.6. Thermogravimetric analysis (TGA) Thermogravimetric analysis was carried out using TA-60 (Shimadzu, Japan) with the TA-60WS system. Samples (1–10 mg) were put in alumina crucibles and heating up to 180
C at a rate of 10
C/min under nitrogen purge (40 ml/min).

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2.2.7. Stability test All solid dispersions were placed into open glass vials and exposed to 40
C/75% RH for 4 weeks. After storage, the samples prepared by BM were characterized by DSC and SEM while samples prepared with HME by PXRD and SEM.

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3. Results and discussion

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3.1. Characterization of AZI solid dispersions during ball milling

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Fig. 3. DSC thermograms of AZI dihydrate (a), AZI monohydrate (b), and anhydrous AZI (c).

Fig. 2 shows DSC and PXRD patterns of formulation A milled for 0.25, 1, 6, 12 and 24 h. As shown in the DSC pattern, the endothermic peak of AZI can be observed within 12 h. When the milling time reached 24 h, the endothermic peak disappeared, indicating the complete transformation of crystalline AZI to an amorphous state.

The PXRD results were in agreement with the DSC results. However, two interesting phenomena were observed in the DSC patterns: firstly, a broad endothermic peak around 90
C was observed in curves a and b. This phenomenon may be caused by the presence of disordered AZI (Yoshinobu et al., 1984). During the co-milling process, three phases (phase 1 – crystal structure; phase 2 –

Fig. 2. DSC thermograms (A) and PXRD patterns (B) of formulation A milled for 0.25 h (a), 1 h (b), 6 h (c), 12 h (d), and 24 h (e).

Fig. 4. DSC thermograms (A) and PXRD patterns (B) of pure AZI milled for 0.25 h (a), 1 h (b), 6 h (c), 12 h (d), and 24 h (e).

Please cite this article in press as: Li, X., et al., Preparation and characterization of azithromycin – Aerosil 200 solid dispersions with enhanced physical stability. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.03.029

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disordered structure; phase 3 – adsorbed on the surface of Aerosil 200) may exist in the milled powder simultaneously. The presence of phase 2 during the co-milling process accounts for the presence of the broad endothermic peak around 90
C; Secondly, a little left shift about AZI endothermic peak was noticed after 6 h co-milling. This phenomenon may be brought about by the loss of crystallization water of AZI dihydrate. According to the literature (Gandhi et al., 2002), we can know that AZI hydrates exhibits different endothermic peaks during the DSC analysis. So we hypothesized that during the co-milling process, AZI dihydrate was gradually transformed to anhydrous AZI, leading to the left shift of AZI endothermic peak. In order to confirm this deduction, monohydrate and anhydrous AZI were prepared as described previously (Gandhi et al., 2002) and characterized by DSC and PXRD. DSC thermograms of AZI dihydrate, monohydrate and anhydrous AZI were shown in Fig. 3. The melting point of monohydrate is higher than other two forms of AZI while anhydrous AZI shows the lowest melting point. At the same time, the melting point of anhydrous AZI is in accordance with the shifted melting point in Fig. 2A. Based on Fig. 3, it can be concluded that the left shift of melting point in Fig. 2A is caused by the loss of crystallization water during the co-milling process. However, no difference was observed among the PXRD curves (data not shown). It has been reported in the literature

(Gandhi et al., 2002), the internal crystal structure of AZI dihydrate, monohydrate and anhydrous AZI is the same and the water content is the only difference among three AZI hydrates. The same milling process was also carried out on pure AZI. As shown in Fig. 4, the endothermic peak of AZI can be observed during the entire milling process while diffraction peaks of AZI persisted with a slight reduction in their intensity, indicating the crystalline state of AZI. The PXRD result was in agreement with the DSC result. Two interesting phenomenon were also observed in Fig. 4A. Firstly, no endothermic peak was observed around 90
C compared to Fig. 2, indicating that the presence of phase 2 is due to the addition of Aerosil 200. Secondly, two endothermic peaks were observed in curves c, d and e. This was caused by the presence of anhydrous AZI. This different results between pure AZI and formulation A demonstrated that the driving force of amorphization is the molecular interaction between AZI and Aerosil 200. Considering the chemical structure of AZI and Aerosil 200, the driving force may be the formation of hydrogen bonds during the co-milling process. Fig. 5 shows the FT-IR curves of pure AZI, Aerosil 200 and formulation A milled for different times. Among different samples, great differences have been observed in the O—H stretching vibration region (3400 cm1). Before milling, the presence of the sharp peak (3496.0 cm1) indicates the presence of “tightly bound” water in the

Fig. 5. FT-IR spectra of Aerosil 200 (a), pure AZI (b), and formulation A milled for 0.25 h (c), 1 h (d), 6 h (e), 12 h (f), and 24 h (g).

Please cite this article in press as: Li, X., et al., Preparation and characterization of azithromycin – Aerosil 200 solid dispersions with enhanced physical stability. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.03.029

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Fig. 6. Schematic representation of disruption process of hydrogen bonds between AZI and crystallization water. 211 212 213 214 215 216 217 218 Q8 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258

crystal lattice (Bettinetti et al.,1999) and the sharp peak at 3561.5 cm1 demonstrates the presence of two crystalline water in AZI (Gandhi et al., 2002). However, as the milling time increased, the intensity of the two peaks decreased, indicating the presence of “loosely bound” water. When the milling time reached 24 h, these two peaks disappeared completely and instead, a broad band was observed at 3444.5 cm1. The disappearance of the two peaks indicated the loss of crystallization water and the red shift of the broad band from (3496.0 cm1 to 3444.5 cm1) suggested the formation of hydrogen bonding between AZI and Aerosil 200. This phenomenon also demonstrates that the left shift of AZI endothermic peak during the milling process is caused by the loss of crystallization water. In addition, for the stretching vibration peak of carbonyl group (nC¼O), an obvious difference was also observed. Before milling, the stretching vibration peak (nC¼O) was sharp and intense and the wavenumber was 1720.9 cm1. After 12 h and 24 h milling, the wavenumber of the peak (nC¼O) was 1730.0 cm1 and 1730.6 cm1, respectively. The intensity of the peak (nC¼O) also decreased. The blue shift in the C¼O region (1720.0 cm1 to 1730.6 cm1) indicates the disruption of hydrogen boning between the crystalline water and AZI (Kauss et al., 2013). Based on the DSC and FT-IR results, this proposed process was schematically presented in Fig. 6. During the co-milling process, the hydrogen bonds between AZI and the crystallization water was disrupted. When the milling time was extended, the “loosely bound” water was excluded from the AZI molecule lattice. Simultaneously, large number of silanol groups on the surface of Aerosil 200 provide a favorable environment to form hydrogen bonds with AZI, and the hydrogen bonds were formed between AZI and Aerosil 200, which promoted the transformation of crystalline AZI to an amorphous state. From the above, it is feasible to prepare amorphous AZI solid dispersions through ball milling. In order to evaluate the maximal drug loading of amorphous AZI – Aerosil 200 solid dispersions, formulations A–C were milled for 24 h to prepare amorphous AZI solid dispersions. As shown in Fig. 7, no endothermic peak was found in curves b and c, indicating the amorphization of AZI during the co-milling process. Regarding formulation C, the endothermic peak of AZI was witnessed in curve d, indicating residual crystalline AZI in the solid dispersion. Based on the enthalpy of fusion, the calculated degree of crystallinity is 0.73% in the solid dispersion (formulation C). Hence, the highest drug loading of AZI – Aerosil 200 amorphous solid dispersions prepared by ball milling is 60–75%. The high drug loading is one of the most outstanding advantages for AZI – Aerosil 200 solid dispersions.

retardant material with a hydrophilic–lipophilic balance value of 2. In the DSC analysis, a sharp endothermic peak was observed at about 70
C (Brubach et al., 2007). Due to the low melting point of GB, crystalline AZI may dissolve in the molten GB during the heating process. As an alternative, PXRD was utilized to characterize the physical state of AZI in the solid dispersions. Fig. 8 shows the PXRD patterns of the solid dispersions prepared by HME. Regarding the freshly prepared solid dispersions, only GB diffraction peaks were observed in curves c and d, indicating the crystalline state of GB and the amorphous state of AZI in the solid dispersions. Based on Fig. 8, it was verified that amorphous AZI solid dispersions were successfully produced by HME at 140
C. Fig. 9 shows the FT-IR results of solid dispersions prepared by HME. For the carbonyl group, the stretching vibration peaks (nC¼O) of GB and AZI are at 1740 cm1 and 1721.2 cm1, respectively. Two peaks can be seen clearly in curves d and e, indicating that no interaction occurred between the different components in the physical mixture. However, for solid dispersions, only one stretching vibration peak (nC¼O) was observed. As we know, the disruption of hydrogen bonds between crystalline water and AZI can lead to a blue shift of the stretching vibration peak (nC¼O) and the stretching vibration peak (nC¼O) of AZI may be concealed by the stretching vibration peak (nC¼O) of GB. As a result, only one stretching vibration peak (nC¼O) was observed in the FT-IR spectrum. In the case of the O—H stretching vibration region, the peaks (3561.0 and 3494.5 cm1) disappeared in curves f and g, indicating the loss of crystalline water during the HME process. As known in ball milling process, the formation of hydrogen bonds between AZI and Aerosil 200 led to a red shift of O—H stretching vibration region from (3496.0 cm1 to 3444.5 cm1). Coincidentally, the O—H stretching vibration region of GB was around

3.2. Characterization of AZI solid dispersions during the hot-melt extrusion process Formulations D and E were extruded at 140
C in order to prepare solid dispersions containing GB. As we know, GB is a waxy

Fig. 7. DSC thermograms of solid dispersions freshly prepared by ball milling: pure AZI (a), formulation A (b), formulation B (c), and formulation C (d).

Please cite this article in press as: Li, X., et al., Preparation and characterization of azithromycin – Aerosil 200 solid dispersions with enhanced physical stability. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.03.029

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Fig. 8. PXRD patterns of solid dispersions prepared by HME: pure GB (a), pure AZI (b), formulation D (c), and formulation E (d).

3447.8 cm1. Consequently, the shifted O—H stretching vibration region of AZI may be concealed by the O—H stretching vibration region of GB, which generates only a broad band in the O—H stretching vibration region observed in curve g.

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3.3. Stabilization effect of Aerosil 200 on AZI solid dispersions

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After 4 weeks storage under accelerated condition, solid dispersions prepared by ball milling were characterized by DSC and SEM. As shown in Fig. 10, the endothermic peak of AZI in curves a, b and c indicated the recrystallization of AZI during storage. Based on the enthalpy of fusion, the degrees of crystallinity were calculated and for formulation A–C were 0.38%, 0.43% and 1.17%, respectively. The physical stability was improved with an increase of Aerosil 200 in the solid dispersions, but crystalline AZI was also detected in the solid dispersion (formulation A) after 4 weeks storage under accelerated condition. Taking formulation C as an example, SEM experiments were carried out. As shown in Fig. 11, coarse AZI particles were mostly in cubic shape with smooth surfaces. A loose network structure can

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Fig. 9. FT-IR spectra of pure GB (a), pure AZI (b), Aerosil 200 (c), physical mixture of formulation D (d), physical mixture of formulation E (e), solid dispersion of formulation D (f) and solid dispersion of formulation E (g).

Please cite this article in press as: Li, X., et al., Preparation and characterization of azithromycin – Aerosil 200 solid dispersions with enhanced physical stability. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.03.029

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Fig. 10. DSC thermograms of solid dispersions after storage for 4 weeks under 40
C/75% RH: formulation A (a), formulation B (b) and formulation C (c). 308 309 310 311 312 313 314 315 316 317 318 319 320

be seen in pattern B, indicating the linking of Aerosil 200 particles. After co-milling, the AZI/Aerosil 200 particles tended to be irregular in shape with bumpy surfaces. The interspaces of Aerosil 200 were filled with AZI molecules. After 4 weeks storage under accelerated conditions, distinct protuberances were observed on the surface of the milled powder. The protuberances were spherical in shape and showed different characteristics compared with coarse AZI. The particle size of the protuberances was around 50 nm, which was much smaller than the coarse AZI. In line with the DSC results, it is inferred that a crystallization process occurred during the 4 week storage period. As seen in Fig. 11D, AZI crystals were largely distributed on the surface of the milled powder. Generally, the high humidity (75%

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RH) is the main reason for the recrystallization of amorphous AZI. Moreover, it has been reported that the adsorbed water molecules have a great influence on the physical stability of amorphous solid dispersions (Guo et al., 2013). The plasticizing effect of adsorbed water in amorphous solid dispersions can significantly lower the glass transition temperature of most amorphous drugs, leading to an increased molecular mobility. For amorphous AZI solid dispersions prepared by ball milling, the large specific area of the milled powder not only provides a good opportunity for the adsorption of water molecules, but also results in a higher water intrusion. On the other hand, the hygroscopic properties of anhydrous AZI also promote the adsorption of water molecules (Timoumi et al., 2014). As a result, the recrystallization process happened on the surface of milled powder. The increased molecular mobility and the intrusion of water molecules account for the recrystallization of AZI during storage. After 4 weeks storage under accelerated condition, PXRD experiments were also carried out to study the physical state of AZI solid dispersions prepared by HME. As shown in Fig. 12, with the combination of AZI and GB in the solid dispersion, the diffraction peaks of AZI at 7.214
, 9.842
and 19.328
were seen clearly in curve b, indicating the appearance of crystalline AZI in the solid dispersion after 4 weeks storage (40
C/75% RH). On the other hand, with addition of Aerosil 200 in the formulation, only GB diffraction peaks were observed in curve d, indicating an improved physical stability under accelerated condition. The significant difference between the two solid dispersions demonstrated that the presence of Aerosil 200 in the formulations can improve the physical stability of amorphous AZI under accelerated conditions. Although crystalline AZI was observed by PXRD in the solid dispersion (formulation D), no obvious difference was observed

Fig. 11. SEM images of coarse AZI (A), Aerosil 200 (B) and solid dispersion of formulation C prepared by BM before (C) and after storage under 40
C/75% RH for 4 weeks (D).

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Fig. 12. PXRD patterns of solid dispersions prepared by HME: formulation D before (a) and after accelerated test (b), formulation E before (c) and after accelerated test (d). 352 353 354 355 356 357 358 359 360 361 362 363 364

between SEM image A and B (Fig. 13). This may be due to the following two reasons: firstly, according to the intensity of the Q9 diffraction peaks in Fig. 11, the crystalline AZI may not be large enough to be observed by SEM; secondly, the high temperature (40
C) may be the main reason for the physical aging of the solid dispersion (formulation D) because the hydrophobic properties of GB can hinder the entry of water molecules during storage. The crystalline AZI may mostly be present in the interior of the solid dispersions. These two reasons may explain the absence of crystalline AZI in the SEM images. In order to confirm our deduction, TGA experiments were carried out to measure the water uptake of solid dispersions during the 4 weeks storage under accelerated condition. To our surprise, a

high water uptake (1.007%) was observed for solid dispersion (formulation E) prepared by HME, while solid dispersion (formulation C) prepared by BM showed a relative low water uptake (0.708%) in Fig. 14. The TGA results demonstrated that the impact of water uptake on the physical stability of solid dispersions prepared by BM is severe. Although the water uptake of solid dispersions prepared by HME is high, the hydrophobic properties of GB may hinder the intrusion of water molecules. The concept of water uptake is not equivalent to water intrusion for solid dispersions prepared by HME. Regarding the solid dispersions prepared by HME, AZI existed in an amorphous state in solid dispersions initially. For the solid dispersion containing AZI and GB (formulation D), the amorphous

Fig. 13. SEM images of solid dispersions prepared by HME: formulation D before (A) and after accelerated test (B), formulation E before (C) and after accelerated test (D).

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combination of the hydrophobic property of GB, the interaction of AZI and Aerosil 200 was protected and high stability was achieved in the accelerated test. Also, further studies will be required in order to fully understand the stabilizing mechanism.

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Acknowledgements

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426 This work was supported by National 973 Project of China (2015CB932100). Financial support fromNational Natural Science Q10 427 428 Foundation of China (No.81102399, No.81402858) is also acknowl429 edged.

References

Fig. 14. TGA thermograms of solid dispersions: formulation C before (a) and after accelerated test (b), formulation E before (c) and after accelerated test (d). 378

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AZI was in a thermodynamic unstable state. The excess energy in the system (high enthalpy of amorphous AZI) would lead to the conversion of amorphous AZI to crystalline AZI, until reaching a thermodynamic equilibrium. At the same time, the relatively high temperature (40
C) also increases the mobility of AZI molecules. As a result, diffraction peaks of AZI were observed in Fig. 12, indicating the recrystallization of amorphous AZI under accelerated condition. In the case of the solid dispersion containing AZI, GB and Aerosil 200 (formulation E), the existence of hydrogen bonds between AZI and Aerosil 200 acts as a competing mechanism for the physical aging of amorphous AZI. With the formation of hydrogen bonds, the amorphous AZI becomes relatively stable in the solid dispersion. These results confirmed that physical aging is the predominant mechanism for solid dispersion (formulation D) during the 4 weeks storage period. Meanwhile, for the ternary solid dispersion containing AZI, GB and Aerosil 200, the existence of hydrogen bonds between AZI and Aerosil 200 is a competing mechanism for the physical aging process. In the presence of hydrogen bonds, the amorphous AZI becomes relatively stable in the solid dispersion (formulation E). To sum up, for solid dispersions prepared by ball milling, the physical instability may be explained by the intrusion of water molecules and the high molecular mobility of AZI. The crystals on the surface of the power also confirmed this. In order to exclude the effect of water molecules on the physical stability of amorphous AZI solid dispersions, a waxy material, GB (Compritol 888 ATO), was introduced into the amorphous AZI solid dispersions. In the case of the solid dispersion containing AZI and GB, the high temperature (40
C) is the predominant factor affecting the stability of the solid dispersion. Regarding solid dispersion containing AZI, GB and Aerosil 200, the lower molecular mobility and hydrogen bonds may account for its high physical stability.

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4. Conclusions

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In conclusion, this research confirmed that it is feasible to improve the physical stability of AZI amorphous solid dispersion by molecular interaction with carriers as Aerosil 200 and the protection of this interaction by hydrophobic agent. Using ball milling method, the crystal structure of AZI during milling process was monitored and the crystal water loss was observed. With addition of Aerosil 200, during co-milling process amorphous AZI was detected and formation of hydrogen bonds between them was confirmed. However, under accelerated condition, the recrystallization of AZI was detected. Using hot-melt technique, with the

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