Applicability of low-melting-point microcrystalline wax to develop

Transcription

1 1 2 Applicability of low-melting-point microcrystalline wax to develop temperature-sensitive formulations 3 Kohei Matsumoto, Shin-ichiro Kimura, Yasunori Iwao, Shigeru Itai* 4 Department of Pharmaceutical Engineering and Drug Delivery Science, School of 5 Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, , 6 Japan *Corresponding author. s-itai@u-shizuoka-ken.ac.jp; Tel.: ; Fax: Abbreviations: MCW, microcrystalline wax; WM, wax matrix; APAP, acetaminophen, SEM, scanning electron microscope 13 1

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3 16 ABSTRACT 17 Low-melting-point substances are widely used to develop temperature-sensitive formulations. 18 In this study, we focused on microcrystalline wax (MCW) as a low-melting-point substance. 19 We evaluated the drug release behavior of wax matrix (WM) particles using various MCW 20 under various temperature conditions. WM particles containing acetaminophen were prepared 21 using a spray congealing technique. In the dissolution test at 37 C, WM particles containing 22 low-melting-point MCWs whose melting was starting at approx. 40 C (Hi-Mic-1045 or ) released the drug initially followed by the release of only a small amount. On the other 24 hand, in the dissolution test at 20 and 25 C for WM particles containing Hi-Mic-1045 and at 25 20, 25, and 30 C for that containing Hi-Mic-1070, both WM particles showed faster drug 26 release than at 37 C. The characteristic drug release suppression of WM particles containing 27 low-melting-point MCWs at 37 C was thought attributable to MCW melting, as evidenced by 28 differential scanning calorimetry analysis and powder X-ray diffraction analysis. Taken 29 together, low-melting-point MCWs may be applicable to develop implantable 30 temperature-sensitive formulations that drug release is accelerated by cooling at administered 31 site KEYWORDS: Wax matrix, microcrystalline wax, acetaminophen, temperature-sensitive 34 release formulations, spray congealing technique 3

4 35 1. Introduction 36 Various controlled-release formulations have been developed for improving 37 therapeutic efficacy and patients quality of life (Okuda et al., 2014; Sandberg et al., 1988; 38 Sarkar et al., 2017). These formulations were devised to control the release of a drug and thus 39 reduce side effects by maintaining an optimum drug concentration and improve medication 40 adherence by decreasing the number of doses required. 41 Temperature-sensitive formulation is one of the controlled-release formulations. It 42 releases the drug with changes in body temperature due to the surrounding environment or 43 the disease. It is often administered by hypodermic injection for the purpose of local delivery 44 of drugs or sustained drug release (Gou et al., 2008; Kang et al., 2006). Therefore, it is 45 necessary to design the formulation that can be implanted into body. Various 46 temperature-sensitive substances, such as poly-(n-isopropylacrylamide) (polynipaam) (Wu 47 et al., 2005), poly-(ethylene glycol-b-(dl-lactic acid-co-glycolic acid)-ethylene glycol) 48 (PEG PLGA PEG) triblock co-polymers (Jeong et al., 2000), and poly-(ethylene oxide) and 49 poly-(propylene oxide) multiblock copolymers (Sosnik et al., 2005), have been used in 50 temperature-sensitive release formulations. Low-melting-point substances are another type of 51 substances that have been used in these formulations. Specifically, Choi et al (2010) 52 described a system using 1-tetradecanol with a melting point of C and dodecanoic acid 53 with a melting point of C. In this system, melting of these substances as the 4

5 54 temperature rose caused drug release. 55 In this study, we focused on microcrystalline wax (MCW). Since MCW is 56 biodegradable (Hanstveit, 1992), it is safe for both the environment and the human body. It is 57 widely used as a brightening agent and thickening agent in the cosmetic and food industries 58 (Mohamed, 2012). In addition, various formulations using MCW have been reported in the 59 pharmaceutical field (Hasa et al., 2015; Quintavalle et al., 2008; Zhou et al., 1996). 60 Previously, Hasa et al. (2011) described a wax matrix (WM) dosage form containing 61 theophylline and MCW in a seven to three ratio. De Brabander et al. (2000) developed 62 mini-tablets based on a combination of MCW and starch, using ibuprofen as a model drug. In 63 this formulation, drug release rate was easily varied by modifying the ratio of MCW and 64 starch. As a unique feature of MCW, it is a mixture of line, branched, and cyclic alkanes, and 65 exists in various grades depending on ratio of the alkanes (Zaky et al., 2010). Therefore, 66 MCW has a broad endothermic peak in thermal analysis (Petersson et al., 2008) and MCWs 67 show various melting behavior. There are low-melting-point MCWs whose endothermic 68 onset temperature is C in thermal analysis (Dorset, 2000; Liu et al., 2001). Using these 69 MCWs, it seems possible to develop temperature-sensitive formulations which enable to 70 control the drug release around body temperature. However, until now, WM dosage forms 71 have generally been prepared by MCWs with a melting point of approx. 60 C and developed 5

6 72 as oral formulations (Hasa et al., 2011; De Brabander et al., 2000), and the implantable 73 formulations using low-melting-point MCWs have not been reported yet. 74 Therefore, the objective of this study was to determine the applicability of 75 low-melting-point MCWs to develop implantable temperature-sensitive formulations. WM 76 particles containing drugs were prepared using MCWs of different grades, and changes in 77 drug release behavior with temperature change were evaluated. 78 6

7 79 2. Materials and Methods Materials 81 Acetaminophen (APAP) was kindly provided by Iwaki Pharmaceutical Co. Ltd. 82 (Shizuoka, Japan), and MCWs (Hi-Mic-1045, 1070, 1080, and 1090) were provided by 83 Nippon Seiro Co. Ltd. (Tokyo, Japan). The appearance of MCWs are shown in Fig Differential scanning calorimetry analysis of MCWs 86 Ten milligrams of each MCW was put into an aluminum pan (P/N SSC000E Open Sample Pan φ5, Seiko Instruments Co. Ltd., Chiba, Japan) and analyzed by differential 88 scanning calorimetry (DSC; DSC7020, Hitachi High-Tech Science Co. Ltd., Tokyo, Japan). 89 The heating rate was 10 C/min and the nitrogen gas flow was 40 ml/min. The sample was 90 heated to C Preparation of MCW particles without APAP 93 MCW particles without APAP were prepared for measurement of powder X-ray 94 diffraction. MCW particles were prepared using a spray congealing technique as described 95 previously (Agata et al., 2011; Nitanai et al., 2012). The schematic view of apparatus used for 96 spray congealing technique is shown in Fig. 2. MCW was melted at 120 C. This solution was 7

8 97 dropped onto a metal disk rotating at approx rpm. The sprayed solution immediately 98 solidified into spherical particles, which were classified to less than 106 µm Preparation of WM particles containing APAP 101 WM particles containing APAP were also prepared using a spray congealing 102 technique. The specifications for the formulations of WM particles are summarized in Table g of MCW was melted at 120 C, the MCW solution was stirred, and 10 g of APAP was 104 added. This solution was stirred until APAP was sufficiently dispersed. This solution was 105 dropped onto a metal disk rotating at approx rpm. The sprayed solution immediately 106 solidified into spherical particles, which were classified to µm Measurement of roundness of WM particles 109 WM particles with a particle size of µm were placed on a glass plate. The 110 particles were photographed through an object lens of 4 magnifications using a digital camera 111 (Nikon D80, Nikon Co. Ltd., Tokyo, Japan) attached to an optical microscope (Olympus Co. 112 Ltd., Tokyo, Japan). The images were monochromatized (pixel value: 0 255) and binarized 113 (pixel threshold: 82) using the image analysis software WinROOF 5.5 (Mitani Co. Ltd., 114 Tokyo, Japan). The equivalent circle diameter and perimeter of the particles were measured 8

9 115 using the processed images. Roundness was calculated according to the following equation The closer the roundness value was to 1, the rounder the particle. Roundness = Equivalent circle diameter π Perimeter Scanning electron microscope observation of WM particles 120 The surface form of WM particles with a particle size of µm was 121 observed using a scanning electron microscope (SEM; Miniscope TM3030, Hitachi 122 High-Technologies Co. Ltd., Tokyo, Japan). The samples were placed on double-sided 123 adhesive tape and sputter coated with platinum under vacuum prior to imaging Drug content ratio 126 Approximately 20 mg of WM particles with a particle size of µm was 127 added to 200 ml of distilled water in a stainless beaker. The solution was stirred while 128 heating. After melting of the WM particles, heating was stopped and the solution was stirred 129 for 10 min. The solution was passed through a membrane filter (pore size: 0.20 µm, Toyo 130 Roshi Co. Ltd., Tokyo, Japan). The amount of APAP into the medium was quantitatively 131 determined via UV absorption (UV-mini, Shimadzu Co. Ltd., Kyoto, Japan) at 243 nm. Drug content ratio was calculated according to the following equation. Drug content ratio (%) = APAP content in WM particles Theoretical APAP content 100 9

10 134 This process was repeated for each WM formulation. All values were reported as the mean of 135 three recordings Dissolution test at 20, 25, 30 and 37 C 138 The release behavior of WM particles with a particle size of µm was 139 examined using the paddle method listed in the Japanese Pharmacopoeia 17th Edition (JP th). The test medium was 900 ml distilled water. The medium temperature was set to ± 0.5 C and the paddle speed was 50 rpm. One hundred mg of WM particles (APAP amount 142 was approx. 10 mg) were put into the test solutions. In addition, in order to confirm changes 143 of dissolution behavior with changing temperature, dissolution tests were also conducted at ± 0.5, 25.0 ± 0.5, and 30.0 ± 0.5 C. At each time point (5, 15, 30, 60, 120, 240, 480, 720, , and 1440 min), 5 ml of the test solution was withdrawn and replaced with an equal 146 volume of medium, and the sample was passed through a membrane filter. The amount of 147 APAP released into the medium was quantitatively determined by UV absorption at 243 nm. 148 This process was repeated for each WM formulation. All values were reported as the mean of 149 three recordings Dissolution test at 50 C 10

11 152 The release behavior of WM particles with a particle size of µm was 153 examined using a shaking apparatus (Uni Thermo Shaker NTS-1300, Tokyo Rikakikai Co., 154 Ltd., Tokyo, Japan). The test medium was 100 ml of distilled water and it was poured in 155 Erlenmeyer flask. The medium temperature was set to 50.0 ± 0.5 C and the shaking speed 156 was 100 rpm. Fifty mg of WM particles (APAP amount was approx. 5 mg) were put into the 157 test solutions in Erlenmeyer flask. At each time point (5, 15, 30, 60, 120, 240, 360, 480 and min), 5 ml of the test solution was withdrawn and replaced with an equal volume of 159 medium, and the sample was passed through a membrane filter. The amount of APAP 160 released into the medium was quantitatively determined in the same way as described in 161 section 2.8. This process was repeated for each WM formulation. All values were reported as 162 the mean of three recordings Drug release kinetics 165 The experimental data was evaluated kinetically using the Korsmeyer-Peppas 166 (Korsmeyer et al., 1983) and Higuchi equations (Higuchi, 1961). These equations were fitted 167 to the results of the dissolution tests by a nonlinear least-squares method using the statistics 168 software package Origin 9.1 (Originlab Co. Ltd., Northampton, MA). The 169 Levenberg-Marquardt algorithm was used for nonlinear fitting. 170 Korsmeyer-Peppas equation: Higuchi equation: M M = kt, M < 0.6M M M = kt. 11

12 171 where Mt is the amount of drug released at time t, M is the amount of drug released after an 172 infinite amount of time, k is release rate constant, and n is the release exponent. The 173 coefficient of determination (R 2 ) was used as a measure of goodness of fit of the experimental data. R 2 was calculated according to the following equation: R = 1 (y y ) (y y ) where N is the number of experimental data points, yi is the measured value, y is the 177 estimated value, and y is the mean of measured values. In the Korsmeyer-Peppas equation, 178 a release exponent value of n = 0.5 was considered consistent with diffusion-controlled 179 release behavior (Higuchi equation), a value of n between 0.5 and 1 indicated diffusion and 180 erosion release behavior, and n = 1 was considered consistent with erosion-controlled release 181 behavior (zero-order equation). In addition, the Higuchi equation could express dissolution 182 behavior from an insoluble matrix Powder X-ray diffraction using synchrotron X-rays of MCW particles 185 MCW particles which were prepared in section 2.3 were enclosed into capillaries 186 (Hilgenberg GmbH, Malsfeld, Germany) with diameters of 0.3 mm. Powder X-ray diffraction 187 data were collected at the Aichi synchrotron radiation center BL5S2, which is equipped with 188 a Debye-Sherrer camera and Pilatus 100 K (Rigaku, Tokyo, Japan). The wavelength was set 189 to Å. The diffraction patterns with a 2θ range of were recorded. The 12

13 190 samples were heated at 10 C/min with nitrogen gas flow, and the exposure time was 18 s. 191 Based on the intensity of the peak (2θ = approx. 14 ) at room temperature (27 C), the ratio of 192 intensity of the peak at each temperature was determined. The sigmoid equation shown below 193 was fitted to the results of the ratio of intensity of the peak at each temperature by a nonlinear 194 least-squares method using the statistics software Origin 9.1. The Levenberg-Marquardt algorithm was used for nonlinear fitting. y (T) = a 1 + exp ( k(t b)) where y (T) is the ratio of the peak intensity at each temperature to that of the peak at room 198 temperature; T is the temperature; k, a, and b are the parameters of the sigmoid equation. 13

14 Results and Discussion Differential scanning calorimetry analysis of MCWs 201 The result of differential scanning calorimetry analysis of MCWs are shown in Fig MCWs showed a broad endothermic peak, due to the fact that MCW is made up of a 203 mixture of various alkanes. In addition, the endothermic peak shifted toward higher 204 temperatures in the following order: Hi-Mic-1045, 1070, 1080 and The endothermic 205 onset temperature of Hi-Mic-1045 and 1070 was approx. 40 C. Thus, it was considered that 206 Hi-Mic-1045 and Hi-Mic-1070 could be used for temperature-sensitive formulations as a 207 low-melting-point substance. Hi-Mic-1080 and 1090 were used for subsequent evaluation as 208 a comparison of low- melting- point MCWs (Hi-Mic-1045 and 1070) Physical properties of WM particles 211 Ii is known that particle properties are important factors which affect the drug 212 release behavior (Champion et al., 2007). Roundness and drug content ratio were evaluated as 213 physical properties of WM particles (Table 2). In all formulations, roundness showed high 214 value and spherical particles were observed in SEM images (Fig. 4). In addition, drug content 215 ratio was close to the theoretical value in all formulations. It revealed that uniform WM 216 particles could be designed in all formulations. Therefore, in the subsequent evaluation, it is 217 not necessary to consider the effect of the particle properties on drug release behaviors. 14

15 Drug release profiles at 37 C 220 In order to confirm the drug release behavior under temperature condition close to 221 body temperature, the dissolution test was conducted at 37 C. The results of the dissolution 222 test of F1, F2, F3, and F4 at 37 C are shown in Fig. 5. In F1 and F2, although a drug release 223 was observed initially, almost no drug release was observed after 120 min. Drug release in 224 these formulations was delayed compared with that of F3 and F4. In order to evaluate these 225 drug release behaviors in detail, the Korsmeyer-Peppas equation and Higuchi equation were 226 fitted to the results of these dissolution tests. The results of fitting are shown in Table 3. In F1 227 and F2, the values of n in Korsmeyer-Peppas equation were significantly lower than 0.5. In 228 this case, it was not possible to estimate the dissolution behavior from the Korsmeyer-Peppas 229 equation. In contrast, in F3 and F4, the values of n in the Korsmeyer-Peppas equation were 230 close to 0.5 at 37 C, which possibly suggested diffusion-controlled release behavior. Thus, 231 the Higuchi equation was fitted to the results of these dissolution tests. In F3 and F4, the 232 values of R 2 in the Higuchi equation were greater than Together with the results of the 233 Korsmeyer-Peppas equation, it was revealed that the dissolution behavior of F3 and F4 at C showed diffusion-controlled release behavior Drug release profiles at 20, 25 and 30 C 15

16 237 Since the characteristic drug release suppression seen in F1 and F2 were thought to 238 be caused due to MCW melting, we conducted the dissolution tests at 20, 25, and 30 C. The 239 results of the dissolution tests for all formulations at 20, 25, 30, and 37 C are shown in Fig Similar drug release suppression of F1 was seen at 30 C and 37 C, whereas, interestingly, the 241 characteristic drug release suppression was not observed at 20 and 25 C, and drug release 242 was faster than that at 37 C (Fig. 6a). In addition, the characteristic drug release suppression 243 of F2 was not also observed at 20, 25 and 30 C, and the drug releases were also faster than 244 that at 37 C (Fig. 6b). In contrast, in F3 and F4, the dissolution behavior at 20, 25, and 30 C 245 was similar to that at 37 C and the drug release rate decreased with decreasing temperature 246 (Fig. 6c and d). The results of the Korsmeyer-Peppas equation and Higuchi equation fitting at , 25 and 30 C are shown in Table 4. In F1, the values of n in Korsmeyer-Peppas equation 248 were significantly lower than 0.5 at 30 C. On the other hand, at 20 and 25 C, the values of n 249 were close to 0.5 and the values of R 2 in Higuchi equation were higher than It was 250 shown that the dissolution behavior of F1 was diffusion-controlled release at 20 and 25 C. In 251 F2, at 20, 25 and 30 C, the values of n in the Korsmeyer-Peppas equation were all close to and the values of R 2 in Higuchi equation were higher than These data revealed that 253 the dissolution behavior of F2 was diffusion-controlled release behavior at 20, 25 and 30 C. 254 In F3 and F4, under temperature conditions at C, the values of n in the 255 Korsmeyer-Peppas equation were close to 0.5 and the values of R 2 in Higuchi equation were 16

17 256 higher than These data suggested that the dissolution behavior of F3 and F4 was 257 diffusion-controlled release behavior at C Estimate of the mechanism of the characteristic drug release suppression 260 To elucidate the change in dissolution behaviors of the WM particles when the 261 temperature was changed, the relationship between physical properties of MCW and 262 dissolution behavior of MCW was evaluated. The changes in crystallinity of MCW under 263 increasing temperature conditions were investigated in detail. The results of powder X-ray 264 diffraction of each MCW under various temperature conditions are shown in Fig. 7. At 27 C, 265 MCW had a characteristic peak (2θ = approx. 14 ). The intensity of this peak decreased with 266 increasing temperature. In other words, melting the MCW appeared to decrease the 267 crystallinity. Based on the intensity of this peak at 27 C, the ratio of intensity of the peak at 268 each temperature (y (T)) was determined. The crystal peak area ratio (2θ = approx. 14 ) at 269 each temperature to that at 27 C is shown in Fig. 8. As the temperature increased, the values 270 of y (T) decreased and they decreased in order of MCW having endothermic peak at lower 271 temperature. In addition, the sigmoid equation was fitted to the results and estimated values 272 of k, a and b for the sigmoid equation of each MCW are shown in Table 5. Using the sigmoid 273 equation, the crystal peak area ratio (y (T)) of each MCW at 30, 37, and 50 C, calculated and 274 the results were shown in Table 6. At 30 C, the y (T) value of Hi-Mic-1045 decreased to

18 275 As the temperature increased to 37 C, the y (T) of Hi-Mic-1045 and 1070 decreased to and 0.89, respectively. In contrast, in Hi-Mic-1080 and 1090, a drastic change of the values of 277 y (T) was not observed when temperature rose from 27 to 37 C. Combined with the results of 278 the dissolution test, it appears that the characteristic drug release suppression occurred at the 279 temperature conditions where the values of y (T) decreased below approximately Thus, we hypothesized that characteristic drug release suppression was due to 281 partial melting of MCW. In order to confirm whether this phenomenon occurs when using the 282 high-melting-point MCW, the dissolution test using the other MCWs at 50 C was conducted, 283 where the value of y (T) decreased to 0.76 in Hi-Mic-1080 and a drastic change of the value 284 of y (T) was not observed (y (T) = 0.94) in Hi-Mic The results of dissolution tests at C are shown in Fig. 9. The characteristic drug release suppression was observed in F3 as 286 well as in F1 and F2. The results of Korsmeyer-Peppas equation and Higuchi equation 287 fittings at 50 C are shown in Table 7. In F1, F2, and F3, the values of n in Korsmeyer-Peppas 288 equation were significantly lower than 0.5. However, in F4, the value of n in 289 Korsmeyer-Peppas equation was close to 0.5 and the value of R 2 in the Higuchi equation was 290 greater than In other words, the dissolution behavior of F4 at 50 C was 291 diffusion-controlled release behavior, as with at 37 C. These results showed the generality of 292 the correlation between the melting of MCW and the characteristic drug release suppression. 293 The results of the evaluation of the dissolution behavior and crystallinity of MCW 18

19 294 suggested the following mechanism for the characteristic drug release suppression of F1 and 295 F2 at 37 C. First, APAP localized near the surface of the WM particles was released until min. From the SEM image, it was confirmed that the formation of pores appeared to be due 297 to APAP released (Fig. 10A). Next, the shape of the WM particles was gradually transformed 298 via the melting of MCW, and the pores generated by APAP-release were transformed. At this 299 point, the SEM image showed the reconstruction of the surface (Fig. 10B). This 300 transformation may have blocked the solution conveyance pathway, which would have 301 inhibited the release of APAP encapsulated inside WM particles. According to the report of 302 Choi et al (2010), the drastic drug release was caused by complete melting of the 303 low-melting-point base with increasing temperature; however, in this study, the drug release 304 was suppressed with increasing temperature. It was considered that MCW has a broad 305 endothermic peak, and it didn t completely melt (partially melted) at the temperature 306 condition where the characteristic drug release suppression occurred. Partially melted MCW 307 caused transformation of WM particles and the characteristic drug release suppression was 308 thought to occur in this study. It might be necessary for further investigation of the carbon 309 number and composition of included alkanes of MCWs using gas chromatography to 310 elucidate relationship between MCWs physicochemical properties and drug release 311 behaviors

20 Conclusions 314 The objective of this study was to determine the applicability of MCW to 315 temperature-sensitive formulations. We evaluated the relationship between temperature and 316 drug release behavior of WM particles using low-melting-point MCW. In the dissolution test 317 at 37 C, F1 and F2 didn t show diffusion-controlled release behavior and released the drug 318 initially followed by the release of only a small amount. In order to confirm changes of 319 dissolution behavior of the WM particles with changing temperature, dissolution tests were 320 conducted at 20, 25, and 30 C. The dissolution behaviors of F1 at 20 and 25 C and that of F2 321 at 20, 25, and 30 C showed diffusion-controlled release behavior, and faster drug release than 322 at 37 C. In addition, when the dissolution test at 50 C was conducted, the characteristic drug 323 release suppression occurred in F3, as well as in F1 and F2. The results of thermal analysis 324 and crystallinity studies suggested that the characteristic drug release suppression occurred at 325 the temperature at which the MCW started melting. Shape change due to melting of MCW 326 was presumed to be the cause of the drug release suppression. 327 Therefore, this study revealed that the difference in melting behavior of MCW 328 influences the drug release behavior of WM particles. In particular, in the formulations using 329 Hi-Mic-1045 and 1070, drug release behavior was changed under temperature conditions 330 close to body temperature. These formulations may be applicable to temperature-sensitive 331 controlled-release formulations which inhibit drug release at body temperature and cause 20

21 332 drug release by cooling from outside the body. Specifically, it is conceivable to develop WM 333 particles which can be implanted into body by, for example, hypodermic injection. The 334 knowledge obtained in this study will promote the versatility of MCW and is important 335 fundamental knowledge that will enable the development of the formulations as described 336 above

22 338 Acknowledgements 339 The authors thank the following companies for kindly providing reagents used in 340 this study: Nippon Seiro Co. Ltd., and Iwaki Seiyaku Co., Ltd. The synchrotron radiation 341 experiment at BL5S2 at Aichi SR was performed with the approval of the Aichi Synchrotron 342 Radiation Center, Aichi Science & Technology Foundation, Aichi, Japan (Proposal Nos )

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27 414 TABLES 415 Table 1. Formulations of WM particles. Formulation APAP (%) MCW (%) Hi-Mic-1045 Hi-Mic-1070 Hi-Mic-1080 Hi-Mic-1090 F F F F Table 2. Physical properties of WM particles. Each data represents the mean ± S. D. 418 (Roundness: n = 30, Drug content ratio: n = 3) Formulation Roundness Drug content ratio (%) F1 (1045) 0.95 ± ± 6.0 F2 (1070) 0.94 ± ± 4.7 F3 (1080) 0.95 ± ± 0.8 F4 (1090) 0.95 ± ±

28 420 Table 3. Estimated values of k and n in the Korsmeyer-Peppas equation and Higuchi 421 equations for each formulation at 37 C. Each point represents the mean ± S. E. Formulation Korsmeyer-Peppas equation Higuchi equation k (min -n ) n R 2 k (min -1/2 ) R 2 F1 (1045) 1.87 ± ± ± F2 (1070) 3.61 ± ± ± F3 (1080) 2.42 ± ± ± F4 (1090) 2.05 ± ± ±

29 423 Table 4. Estimated values of k and n in the Korsmeyer-Peppas and Higuchi equations for 424 each formulation at 20, 25, and 30 C. Each point represents the mean ± S. E. Formulation Temperature Korsmeyer-Peppas equation Higuchi equation k (min -n ) n R 2 k (min -1/2 ) R 2 20 C 0.93 ± ± ± F1 (1045) 25 C 2.24 ± ± ± C 2.07 ± ± ± C 0.90 ± ± ± F2 (1070) 25 C 1.57 ± ± ± C 1.85 ± ± ± C 0.80 ± ± ± F3 (1080) 25 C 1.54 ± ± ± C 2.10 ± ± ± C 0.75 ± ± ± F4 (1090) 25 C 1.65 ± ± ± C 1.90 ± ± ±

30 426 Table 5. Estimated values of k, a, and b for the sigmoid equation of crystal peak area ratio for 427 each MCW. Each data represents the mean ± S. E. MCW k ( C -1 ) a b ( C) Hi-Mic ± ± ± 3.6 Hi-Mic ± ± ± 0.5 Hi-Mic ± ± ± 0.4 Hi-Mic ± ± ± Table 6. Changes in crystallinity of MCW with increasing temperature. The crystal peak area 430 ratio (y (T)) at 30, 37, and 50 C were calculated using the sigmoid equation. MCW y (30) y (37) y (50) Hi-Mic Hi-Mic Hi-Mic Hi-Mic

31 432 Table 7. Estimated values of k and n in the Korsmeyer-Peppas and Higuchi equations for 433 each formulation at 50 C. Each point represents the mean ± S. E. Formulation Korsmeyer-Peppas equation Higuchi equation k (min -n ) n R 2 k (min -1/2 ) R 2 F1 (1045) 6.79 ± ± ± F2 (1070) 3.61 ± ± ± F3 (1080) 1.94 ± ± ± F4 (1090) 3.87 ± ± ±

32 435 FIGURES 436 Figure 1. Appearance of microcrystalline wax (MCW): (a) Hi-Mic-1045; (b) Hi-Mic-1070; 437 (c) Hi-Mic-1080; (d) Hi-Mic Figure 2. Spray congealing apparatus for preparing particles Figure 3. Differential scanning calorimetry curves of microcrystalline wax (MCW): (a) 442 Hi-Mic-1045; (b) Hi-Mic-1070; (c) Hi-Mic-1080; (d) Hi-Mic Figure 4. Scanning electron microscopy images of wax matrix particles: (a) F1; (b) F2; (c) 445 F3; (d) F4. Observations were carried out at 300 magnifications Figure 5. Drug release profiles of wax matrix particles (F1, F2, F3, and F4) at 37 ± 0.5 C. 448 Each data point represents the mean ± S. E. (n = 3) Figure 6. Drug release profiles of wax matrix particles at various temperatures: (a) F1; (b) 451 F2; (c) F3; (d) F4. Temperatures: 20 ± 0.5, 25 ± 0.5 and 30 ± 0.5 C. Each data point 452 represents the mean ± S. E. (n = 3)

33 454 Figure 7. Powder X-ray diffraction patterns of microcrystalline wax (MCW) at 27, 30, 37, 50, and 100 C: (a) Hi-Mic-1045; (b) Hi-Mic-1070; (c) Hi-Mic-1080; (d) Hi-Mic Figure 8. The ratio of the crystal peak area (2θ = 14 ) at each temperature to that at 27 C Figure 9. Drug release profiles of wax matrix particles (F1, F2, F3, and F4) at 50 ± 0.5 C. 460 Each data point represents the mean ± S. D. (n = 3) Figure 10. Scanning electron microscopy images of F1 after the dissolution test: (A) after min; (B) after 1440 min. Observations were carried out at 300 magnifications

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